The functional property of royal jelly 10-hydroxy-2-decenoic acid as a melanogenesis inhibitor.

PubMed

Peng, Chi-Chung; Sun, Hui-Tzu; Lin, I-Ping; Kuo, Ping-Chung; Li, Jen-Chieh

2017-08-09

It has been reported that royal jelly would reduce melanin synthesis and inhibit the expression of melanogensis related proteins and genes. In this study, we evaluate the anti-melanogenic and depigmenting activity of 10-hydroxy-2-decenoic acid (10-HDA) from royal jelly of Apis mellifera. In this study, we assesses the 10-HDA whitening activity in comparison with the changes in the intracellular tyrosinase activity, melanin content and melanin production related protein levles in B16F1 melanoma cells after treating with 10-HDA. Furthermore, the skin whitening effect was evaluated by applying a cream product containing with 0.5%, 1% and 2% of 10-HDA onto the skin of mice (C57BL/6 J) for 3 week to observe the effect of DL*-values. The results showed that 10-HDA inhibited the MITF protein expression (IC50 0.86 mM) in B16F1 melanoma cells. Western blot analysis revealed that 10-HDA inhibited the activity of tyrosinase and the expression of tyrosinase-related protein 1 (TRP-1), TRP-2, and microphthalmia-associated transcription factor (MITF) in B16F1 melanoma cells. In addition, the 10-HDA was applied on the skin of mice show significantly increased the average skin-whitening index (L value). The validation data indicated the potential of 10-HDA for use in suppressing skin pigmentation. The 10-HDA is proposed as a candidate to inhibit melanogenesis, thus it could be developed as cosmetics skin care products.

The pink-eyed dilution locus controls the biogenesis of melanosomes and levels of melanosomal proteins in the eye.

PubMed

Orlow, S J; Brilliant, M H

1999-02-01

The pink-eyed dilution (p) locus is known to control the quantity of melanin pigment made within melanocytes and retinal pigment epithelium (RPE) in the eye. We have examined the effects of several mutant allele combinations at the murine p locus on the number and morphology of melanosomes in choroidal melanocytes and RPE cells as well as on the levels of four proteins known to be present within melanosomes: tyrosinase, tyrosinase-related proteins 1 and 2 (TRP-1 and TRP-2) and lysosome-associated membrane protein-1 (LAMP-1). By electron microscopy, we observed a modest diminution in the size and number of choroidal melanosomes in pbs/pJ mice but a more dramatic decrease in the RPE in comparison with wild-type P/P mice. By contrast, a drastic reduction in melanosome size and number was present in the choroid and RPE of pun/pun and p6H/pcp mice, and in the RPE of p6H/pcp mice, melanosomes were essentially undetectable. In wild-type mice, levels of tyrosinase, TRP-1 and TRP-2 were high at birth and showed a second peak of expression at 10-14 days of age, declining to undetectable levels by 42 days. All three mutant allele combinations reduced the levels of these melanosomal proteins with the relative severity of effects being p6H/pcp>pun/pun>pbs/pJ. In the null p6H/pcp mice, levels of these proteins were extremely low at birth, no postnatal peak was observed, and levels declined to undetectable by 14 days. Levels of LAMP-1 in wild-type mice rose initially and then declined whereas in the mutant mice, levels decreased gradually from birth. Higher levels of LAMP-1 were observed in each of the mutants than in the wild-type mice at 21 days of age. Our results demonstrate that mutations at the p locus affect the size, number, shape and contents of melanosomes, implicating the p gene product in the normal biogenesis of this organelle. Copyright 1999 Academic Press.

Effects of Ganodermanondiol, a New Melanogenesis Inhibitor from the Medicinal Mushroom Ganoderma lucidum

PubMed Central

Kim, Ji-Woong; Kim, Hong-Il; Kim, Jong-Hyeon; Kwon, O-Chul; Son, Eun-Suk; Lee, Chang-Soo; Park, Young-Jin

2016-01-01

Ganoderma lucidum, a species of the Basidiomycetes class, has been attracting international attention owing to its wide variety of biological activities and great potential as an ingredient in skin care cosmetics including “skin-whitening” products. However, there is little information available on its inhibitory effect against tyrosinase activity. Therefore, the objectives of this study were to investigate the chemical composition of G. lucidum and its inhibitory effects on melanogenesis. We isolated the active compound from G. lucidum using ethanol extraction and ethyl acetate fractionation. In addition, we assayed its inhibitory effects on tyrosinase activity and melanin biosynthesis in B16F10 melanoma cells. In this study, we identified a bioactive compound, ganodermanondiol, which inhibits the activity and expression of cellular tyrosinase and the expression of tyrosinase-related protein-1 (TRP-1), TRP-2, and microphthalmia-associated transcription factor (MITF), thereby decreasing melanin production. Furthermore, ganodermanondiol also affected the mitogen-activated protein kinase (MAPK) cascade and cyclic adenosine monophosphate (cAMP)-dependent signaling pathway, which are involved in the melanogenesis of B16F10 melanoma cells. The finding that ganodermanondiol from G. lucidum exerts an inhibitory effect on tyrosinase will contribute to the use of this mushroom in the preparation of skin care products in the future. PMID:27801787

Effects of Ganodermanondiol, a New Melanogenesis Inhibitor from the Medicinal Mushroom Ganoderma lucidum.

PubMed

Kim, Ji-Woong; Kim, Hong-Il; Kim, Jong-Hyeon; Kwon, O-Chul; Son, Eun-Suk; Lee, Chang-Soo; Park, Young-Jin

2016-10-27

Ganoderma lucidum , a species of the Basidiomycetes class, has been attracting international attention owing to its wide variety of biological activities and great potential as an ingredient in skin care cosmetics including "skin-whitening" products. However, there is little information available on its inhibitory effect against tyrosinase activity. Therefore, the objectives of this study were to investigate the chemical composition of G. lucidum and its inhibitory effects on melanogenesis. We isolated the active compound from G. lucidum using ethanol extraction and ethyl acetate fractionation. In addition, we assayed its inhibitory effects on tyrosinase activity and melanin biosynthesis in B16F10 melanoma cells. In this study, we identified a bioactive compound, ganodermanondiol, which inhibits the activity and expression of cellular tyrosinase and the expression of tyrosinase-related protein-1 (TRP-1), TRP-2, and microphthalmia-associated transcription factor (MITF), thereby decreasing melanin production. Furthermore, ganodermanondiol also affected the mitogen-activated protein kinase (MAPK) cascade and cyclic adenosine monophosphate (cAMP)-dependent signaling pathway, which are involved in the melanogenesis of B16F10 melanoma cells. The finding that ganodermanondiol from G. lucidum exerts an inhibitory effect on tyrosinase will contribute to the use of this mushroom in the preparation of skin care products in the future.

N-(4-methoxyphenyl) caffeamide-induced melanogenesis inhibition mechanisms.

PubMed

Kuo, Yueh-Hsiung; Chen, Chien-Chia; Wu, Po-Yuan; Wu, Chin-Sheng; Sung, Ping-Jyun; Lin, Chien-Yih; Chiang, Hsiu-Mei

2017-01-23

The derivative of caffeamide exhibits antioxidant and antityrosinase activity. The activity and mechanism of N-(4-methoxyphenyl) caffeamide (K36E) on melanogenesis was investigated. B16F0 cells were treated with various concentrations of K36E; the melanin contents and related signal transduction were studied. Western blotting assay was applied to determine the protein expression, and spectrophotometry was performed to identify the tyrosinase activity and melanin content. Our results indicated that K36E reduced α-melanocyte-stimulating hormone (α-MSH)-induced melanin content and tyrosinase activity in B16F0 cells. In addition, K36E inhibited the expression of phospho-cyclic adenosine monophosphate (cAMP)-response element-binding protein, microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein-1 (TRP-1). K36E activated the phosphorylation of protein kinase B (AKT) and glycogen synthase kinase 3 beta (GSK3β), leading to the inhibition of MITF transcription activity. K36E attenuated α-MSH induced cAMP pathways, contributing to hypopigmentation. K36E regulated melanin synthesis through reducing the expression of downstream proteins including p-CREB, p-AKT, p-GSK3β, tyrosinase, and TRP-1, and activated the transcription factor, MITF. K36E may have the potential to be developed as a skin whitening agent.

The functional property of egg yolk phosvitin as a melanogenesis inhibitor.

PubMed

Jung, Samooel; Kim, Dong Hee; Son, Jun Ho; Nam, Kichang; Ahn, Dong Uk; Jo, Cheorun

2012-12-01

Phosvitin is a phosphoglycoprotein present in egg yolk. More than half of the amino acids in phosvitin molecule are serine, of which >90% are phosphorylated. Therefore, phosvitin has a strong metal binding capability. The aim of this study was to investigate the effect of phosvitin on the inhibition of melanogenesis in melanoma cells. The results showed that phosvitin inhibited the activity of mushroom tyrosinase. Addition of phosvitin at a concentration of 50μg/ml, to B16F10 melanoma cells inhibited tyrosinase activity by approximately 42% and melanin synthesis by 17% compared to those in a control without phosvitin. Phosvitin inhibited the expression of tyrosinase, tyrosinase-related protein 1 (TRP-1), TRP-2, and microphthalmia-associated transcription factor (MITF) in B16F10 melanoma cells. In addition, phosvitin reduced the cellular cAMP concentration in B16F10 melanoma cells. These results indicate that phosvitin has the potential to be used as a melanogenesis inhibitor in the food and cosmetics industry. Copyright © 2012 Elsevier Ltd. All rights reserved.

Inhibitory Effect of Dried Pomegranate Concentration Powder on Melanogenesis in B16F10 Melanoma Cells; Involvement of p38 and PKA Signaling Pathways

PubMed Central

Kang, Su Jin; Choi, Beom Rak; Lee, Eun Kyoung; Kim, Seung Hee; Yi, Hae Yeon; Park, Hye Rim; Song, Chang Hyun; Lee, Young Joon; Ku, Sae Kwang

2015-01-01

Plants rich in antioxidant substances may be useful for preventing skin aging. Pomegranates, containing flavonoids and other polyphenolic compounds, are widely consumed due to their beneficial properties. We examined the underlying mechanisms of dried pomegranate concentrate powder (PCP) on melanin synthesis in B16F10 melanoma cells. The antioxidant effects of PCP were determined by measuring free radical scavenging capacity and transcript levels of antioxidant enzymes. To explore the inhibitory effects of PCP on melanin synthesis, we measured tyrosinase activity and melanin content in α-melanocyte stimulating hormone (α-MSH)-stimulated B16F10 cells. In addition, the levels of tyrosinase-related protein-1 (TRP-1), TRP-2, tyrosinase, and microphthalmia-associated transcription factor (MITF) expression were determined by Western blotting. Changes in the phosphorylation status of protein kinase A (PKA), cAMP response element-binding protein (CREB), mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K), serine/threonine kinase Akt, and glycogen kinase 3β (GSK3β) were also examined. The free radical scavenging activity of PCP increased in a dose-dependent manner. In PCP-treated B16F10 cells, transcript levels of glutathione peroxidase-1 (GPx-1) were increased compared with α-MSH-stimulated cells. In addition, PCP led to the down-regulation of phospho-p38, phospho-PKA, phospho-CREB, phospho-GSK3β, MITF, and TRP-1 compared with α-MSH-stimulated B16F10 cells. We believe this effect may be associated with PCP activity, which leads to the inhibition of melanin production and tyrosinase activity. These results suggest that PCP decreases tyrosinase activity and melanin production via inactivation of the p38 and PKA signaling pathways, and subsequently decreases phosphorylation of CREB, MITF, and melanogenic enzymes. These observations provided new insights on the molecular mechanisms of the skin-whitening property of PCP. PMID:26473849

Emodin isolated from Polygoni Multiflori Ramulus inhibits melanogenesis through the liver X receptor-mediated pathway.

PubMed

Kim, Mi Ok; Park, Yong Seek; Nho, Youn Hwa; Yun, Seok Kyun; Kim, Youngsoo; Jung, Eunsun; Paik, Jean Kyung; Kim, Minhee; Cho, Il-Hoon; Lee, Jongsung

2016-04-25

Melanogenesis is a physiological process that results in the synthesis of melanin pigments, which play a crucial protective role against skin photocarcinogenesis. We investigated the effects of a Polygoni Multiflori Ramulus extract on melanogenesis and isolated emodin from Polygoni Multiflori as an active compound. In addition, the possible mechanisms of action were examined. We found that emodin inhibited both melanin content and tyrosinase activity concentration and time dependently. Tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 mRNA levels decreased following emodin treatment. However, while the mRNA levels of microphthalmia-associated transcription factor (MITF) were not affected by emodin, emodin reduced MITF protein levels. Furthermore, expression of the liver X-receptor (LXR) α gene, but not the LXR β gene was upregulated by emodin. Moreover, emodin regulated melanogenesis by promoting degradation of the MITF protein by upregulating the LXR α gene. The emodin effects on MITF was found to be mediated by phosphorylation of p42/44 MAPK. Taken together, these findings indicate that the inhibition of melanogenesis by emodin occurs through reduced MITF protein expression, which is mediated by upregulation of the LXR α gene and suggest that emodin may be useful as a hyperpigmentation inhibitor. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Mechanistic studies of anti-hyperpigmentary compounds: elucidating their inhibitory and regulatory actions.

PubMed

Lam, Rosanna Y Y; Lin, Zhi-Xiu; Sviderskaya, Elena V; Cheng, Christopher H K

2014-08-21

Searching for depigmenting agents from natural sources has become a new direction in the cosmetic industry as natural products are generally perceived as relatively safer. In our previous study, selected Chinese medicines traditionally used to treat hyperpigmentation were tested for anti-hyperpigmentary effects using a melan-a cell culture model. Among the tested chemical compounds, 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol were found to possess hypopigmentary effects. Western blot analysis, reverse transcriptase polymerase chain reaction (RT-PCR), cyclic adenosine monophosphate (cAMP) assay, protein kinase A (PKA) activity assay, tyrosinase inhibition assay and lipid peroxidation inhibition assay were performed to reveal the underlying cellular and molecular mechanisms of the hypopigmentary effects. 4-Ethylresorcinol and 4-ethylphenol attenuated mRNA and protein expression of tyrosinase-related protein (TRP)-2, and possessed antioxidative effect by inhibiting lipid peroxidation. 1-Tetradecanol was able to attenuate protein expression of tyrosinase. The hypopigmentary actions of 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol were associated with regulating downstream proteins along the PKA pathway. 4-Ethylresorcinol was more effective in inhibiting melanin synthesis when compared to 4-ethylphenol and 1-tetradecanol.

Hydroalcoholic extract of Rhodiola rosea L. (Crassulaceae) and its hydrolysate inhibit melanogenesis in B16F0 cells by regulating the CREB/MITF/tyrosinase pathway.

PubMed

Chiang, Hsiu-Mei; Chien, Yin-Chih; Wu, Chieh-Hsi; Kuo, Yueh-Hsiung; Wu, Wan-Chen; Pan, Yu-Yun; Su, Yu-Han; Wen, Kuo-Ching

2014-03-01

We investigated the effects of an aqueous alcohol extract of Rhodiola rosea (R. rosea) and its hydrolysate on melanin synthesis and the mechanisms mediating the activity. The ratio of tyrosol to salidroside was 2.3 in hydroalcoholic extract, and 51.0 in hydrolysate. We found that R. rosea extract and its hydrolysate inhibited melanin synthesis and tyrosinase activity in mouse melanoma cells (B16F0 cells). R. rosea extract also inhibited gene and protein expression of melanocortin 1 receptor (MC1R) and inhibited c-AMP response element binding protein (CREB) phosphorylation, suppressed the activation of AKT and glycogen synthase kinase-3 beta (GSK3β), and inhibited the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase-related protein 1 (TRP-1). R. rosea hydrolysate inhibited the phosphorylation of CREB, the activation of AKT and GSK3β, and the expression of MITF and tyrosinase. Our results suggest that R. rosea extract is a novel tyrosinase inhibitor and that it exerts its effects by regulating the CREB/MITF/tyrosinase pathway in B16F0. Further in vivo studies are needed to determine the effectiveness of R. rosea extract as a skin whitening agent. Copyright © 2014 Elsevier Ltd. All rights reserved.

Aqueous fraction from Cuscuta japonica seed suppresses melanin synthesis through inhibition of the p38 mitogen-activated protein kinase signaling pathway in B16F10 cells.

PubMed

Jang, Ji Yeon; Kim, Ha Neui; Kim, Yu Ri; Choi, Yung Hyun; Kim, Byung Woo; Shin, Hwa Kyoung; Choi, Byung Tae

2012-05-07

Semen cuscutae has been used traditionally to treat pimples and alleviate freckles and melasma in Korea. The present study aimed to investigate the inhibitory effect of Cuscuta japonica Choisy seeds on alpha-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis. The aqueous fraction from Semen cuscutae (AFSC) was used to determine anti-melanogenic effects by examination of cellular melanin contents, tyrosinase activity assay, cAMP assay and Western blot analysis for melanin synthesis-related signaling proteins in B16F10 mouse melanoma cells. AFSC markedly inhibited α-MSH-induced melanin synthesis and tyrosinase activity, and also decreased α-MSH-induced expression of microphthalmia-associated transcription factor (MITF) and tyrosinase-related proteins (TRPs). Moreover, AFSC significantly decreased the level of phosphorylated p38 mitogen-activated protein kinase (MAPK) signaling through the down-regulation of α-MSH-induced cAMP. Furthermore, we confirmed that the specific inhibitor of p38 MAPK (SB203580)-mediated suppressed melanin synthesis and tyrosinase activity was further attenuated by AFSC. AFSC also further decreased SB203580-mediated suppression of MITF and TRP expression. These results indicate that AFSC inhibits p38 MAPK phosphorylation with suppressed cAMP levels and subsequently down-regulate MITF and TRP expression, which results in a marked reduction of melanin synthesis and tyrosinase activity in α-MSH-stimulated B16F10 cells. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Agmatine modulates melanogenesis via MITF signaling pathway.

PubMed

Kwon, Eun-Jeong; Kim, Moon-Moo

2017-01-01

Agmatine contained in soybean is also found in Manaca, an anti-aging plant, inhabited in Amazon and induces vasodilation by the promotion of NO synthesis in blood vessel. However, the research of agmatine on melanin synthesis related to hair greying is lacking. The aim of this study was to investigate the melanogenic effect of agmatine via regulation of MITF signaling pathway in B16F1 cells. It was determined whether agmatine regulates melanin synthesis at cellular level in addition to the effect of agmatine on mushroom tyrosinase in vitro in the presence of different concentrations of agmatine. Furthermore, the effect of agmatine on the protein expressions of tyrosinase, TRP-1, TRP-2, BMP-4, BMP-6, C-KIT, p-p38, MITF and C-FOS were examined by western blot analysis. In addition, immunofluorescence staining was carried out to visualize the location of MITF expression in cell. Agmatine at 256μM or more increased melanin synthesis as well as tyrosinase activity. Moreover, whereas agmatine increased the expression levels of TRP-1, BMP-6, p-p38 and MITF, it reduced the expression level of BMP-4. It was also found that agmatine enhanced the expression level of MITF in nucleus. These results suggest that agmatine could induce melanin synthesis though the regulation of MITF transcription factor via BMP-6/p38 signaling pathway. Copyright © 2016 Elsevier B.V. All rights reserved.

Inhibition of melanogenesis and antioxidant properties of Magnolia grandiflora L. flower extract

PubMed Central

2012-01-01

Background Magnolia grandiflora L. flower is wildly used in Asian as a traditional herbal medication. The purpose of the study was to investigate the antimelanogenic and antioxidant properties of Magnolia grandiflora L. flower extract. In the study, the inhibitory effects of M. grandiflora L. flower extract on mushroom tyrosinase, B16F10 intracellular tyrosinase activity and melanin content were determined spectrophotometrically. Meanwhile, the antioxidative capacity of the flower extract was also investigated. Results Our results revealed that M. grandiflora L. flower extract inhibit mushroom tyrosinase activity (IC50 =11.1%; v/v), the flower extract also effectively suppressed intracellular tyrosinase activity (IC50 = 13.6%; v/v) and decreased the amount of melanin (IC50 = 25.6%; v/v) in a dose-dependent manner in B16F10 cells. Protein expression level of tyrosinase and tyrosinase-related protein 1 (TRP-1) were also decreased by the flower extract. Additionally, antioxidant capacities such as ABTS+ free radical scavenging activity, reducing capacity and total phenolic content of the flower extract were increased in a dose-dependent pattern. Conclusions Our results concluded that M. grandiflora L. flower extract decreased the expression of tyrosinase and TRP-1, and then inhibited melanogenesis in B16F10 cells. The flower extract also show antioxidant capacities and depleted cellular reactive oxygen species (ROS). Hence, M. grandiflora L. flower extract could be applied as a type of dermatological whitening agent in skin care products. PMID:22672352

Inhibition of melanogenesis and antioxidant properties of Magnolia grandiflora L. flower extract.

PubMed

Huang, Huey-Chun; Hsieh, Wan-Yu; Niu, Yu-Lin; Chang, Tsong-Min

2012-06-06

Magnolia grandiflora L. flower is wildly used in Asian as a traditional herbal medication. The purpose of the study was to investigate the antimelanogenic and antioxidant properties of Magnolia grandiflora L. flower extract. In the study, the inhibitory effects of M. grandiflora L. flower extract on mushroom tyrosinase, B16F10 intracellular tyrosinase activity and melanin content were determined spectrophotometrically. Meanwhile, the antioxidative capacity of the flower extract was also investigated. Our results revealed that M. grandiflora L. flower extract inhibit mushroom tyrosinase activity (IC(50) = 11.1%; v/v), the flower extract also effectively suppressed intracellular tyrosinase activity (IC(50) = 13.6%; v/v) and decreased the amount of melanin (IC(50) = 25.6%; v/v) in a dose-dependent manner in B16F10 cells. Protein expression level of tyrosinase and tyrosinase-related protein 1 (TRP-1) were also decreased by the flower extract. Additionally, antioxidant capacities such as ABTS(+) free radical scavenging activity, reducing capacity and total phenolic content of the flower extract were increased in a dose-dependent pattern. Our results concluded that M. grandiflora L. flower extract decreased the expression of tyrosinase and TRP-1, and then inhibited melanogenesis in B16F10 cells. The flower extract also show antioxidant capacities and depleted cellular reactive oxygen species (ROS). Hence, M. grandiflora L. flower extract could be applied as a type of dermatological whitening agent in skin care products.

Artemisia asiatica ethanol extract exhibits anti-photoaging activity.

PubMed

Jeong, Deok; Lee, Jongsung; Jeong, Seong-Gu; Hong, Yo Han; Yoo, Sulgi; Han, Sang Yun; Kim, Ji Hye; Kim, Sunggyu; Kim, Jin Sic; Chung, Young Soo; Kim, Jong-Hoon; Yi, Young-Su; Cho, Jae Youl

2018-06-28

Artemisia asiatica Nakai is a traditional herbal plant that has long been used in anti-inflammatory, anti-infective and skin protective remedies. In this study, traditionally known skin-protective activity of Artemisia asiatica Nakai was examined with its ethanol extract (Aa-EE) under various photoaging conditions using skin-originated cells, and the underlying mechanism was also examined using various types of cells. Effects of Aa-EE on cell viability, photocytotoxicity, and expression of matrix metalloproteinases (MMPs), cyclooxygenase (COX)-2, and moisturizing factors were measured in B16F10, HEK293, NIH3T3, and HaCaT cells under untreated and ultraviolet B (UVB)-irradiation conditions. Anti-melanogenic effect of Aa-EE was also examined by measuring both melanin content in B16F10 cells and tyrosinase activity. Anti-photoaging mechanism of Aa-EE was explored by determining the activation levels of signaling molecules by immunoblotting analysis. Aa-EE protected HaCaT cells from UVB irradiation-induced death. Aa-EE increased the expression of a type 1 pro-collagen gene and decreased the expression of matrix metalloproteinases, and COX-2 in NIH3T3 cells induced by UVB. Aa-EE increased the expression of transglutamase-1, hyaluronic acid synthase (HAS)-2, and HAS-3 in HaCaT cells and decreased the production of melanin in α-melanocyte-stimulating hormone-stimulated B16F10 cells by suppressing tyrosinase activity and the expression of tyrosinase, microphthalmia-associated transcription factor, tyrosinase-related protein (TRP)-1 and TRP-2. The results suggest that Aa-EE could be skin-protective remedy with anti-photoaging, anti-apoptotic, skin remodeling, moisturizing, and anti-melanogenesis properties. Copyright © 2018 Elsevier B.V. All rights reserved.

Cosmetic applications of glucitol-core containing gallotannins from a proprietary phenolic-enriched red maple (Acer rubrum) leaves extract: inhibition of melanogenesis via down-regulation of tyrosinase and melanogenic gene expression in B16F10 melanoma cells.

PubMed

Ma, Hang; Xu, Jialin; DaSilva, Nicholas A; Wang, Ling; Wei, Zhengxi; Guo, Liangran; Johnson, Shelby L; Lu, Wei; Xu, Jun; Gu, Qiong; Seeram, Navindra P

2017-05-01

The red maple (Acer rubrum) is a rich source of phenolic compounds which possess galloyl groups attached to different positions of a 1,5-anhydro-D-glucitol core. While these glucitol-core containing gallotannins (GCGs) have reported anti-oxidant and anti-glycative effects, they have not yet been evaluated for their cosmetic applications. Herein, the anti-tyrosinase and anti-melanogenic effects of a proprietary phenolic-enriched red maple leaves extract [Maplifa ™ ; contains ca. 45% ginnalin A (GA) along with other GCGs] were investigated using enzyme and cellular assays. The GCGs showed anti-tyrosinase activity with IC 50 values ranging from 101.4 to 1047.3 μM and their mechanism of tyrosinase inhibition (using GA as a representative GCG) was evaluated by chelating and computational/modeling studies. GA reduced melanin content in murine melanoma B16F10 cells by 79.1 and 56.7% (at non-toxic concentrations of 25 and 50 μM, respectively), and its mechanisms of anti-melanogenic effects were evaluated by using methods including fluorescent probe (DCF-DA), real-time PCR, and western blot experiments. These data indicated that GA was able to: (1) reduce the levels of reactive oxygen species, (2) down-regulate the expression of MITF, TYR, TRP-1, and TRP-2 gene levels in a time-dependent manner, and (3) significantly reduce protein expression of the TRP-2 gene. Therefore, the anti-melanogenic effects of red maple GCGs warrant further investigation of this proprietary natural product extract for potential cosmetic applications.

Tangeretin triggers melanogenesis through the activation of melanogenic signaling proteins and sustained extracellular signal- regulated kinase in B16/F10 murine melanoma cells.

PubMed

Yoon, Hoon Seok; Ko, Hee-Chul; Kim, Sang Suk; Park, Kyung Jin; An, Hyun Joo; Choi, Young Hun; Kim, Se-Jae; Lee, Nam-Ho; Hyun, Chang-Gu

2015-03-01

In order to test the effectiveness of tangeretin at ameliorating melanoma and melanoma-associated depigmentation, western blotting was used to assess the melanin content of treated melanoma cells. Tangeretin, a 4',5,6,7,8-pentamethoxyflavone, was found to trigger intracellular melanin production in a concentration-dependent manner in B16/F10 murine melanoma cells. Melanin content increased 1.74-fold in response to treatment with 25 μM of tangeretin, compared to that in non-treated cells. Examination of melanogenic protein expression showed that tyrosinase, tyrosinase-related protein (TRP)-1, and extracellular signal-regulated kinase (ERK) 1/2 levels increased in a dose-dependent manner. Furthermore, the expression of cyclic adenosine monophosphate response element binding protein (CREB) and microphthalmia transcription factor (MITF) was increased by tangeretin in 1 h and 4 h, respectively. Tangeretin- upregulated melanogenesis was suppressed by ERK 1/2 inhibitor and not by ERK1 inhibitor. These results suggest that tangeretin has therapeutic potential for melanoma and melanoma-associated depigmentation because it can induce hyperpigmentation through the activation of melanogenic signaling proteins and initiation of sustained ERK2 expression.

FGF21 regulates melanogenesis in alpaca melanocytes via ERK1/2-mediated MITF downregulation.

PubMed

Wang, Ruiwei; Chen, Tianzhi; Zhao, Bingling; Fan, Ruiwen; Ji, Kaiyuan; Yu, Xiuju; Wang, Xianjun; Dong, Changsheng

2017-08-19

Fibroblast growth factor 21 (FGF21) is known as a metabolic regulator to regulate the metabolism of glucose and lipids. However, the underlying mechanism of FGF21 on melanin synthesis remains unknown. Therefore, the current study investigates the effect of FGF21 on melanogenesis in alpaca melanocytes. We transfected the FGF21 into alpaca melanocytes, then detected the melanin contents, protein and mRNA levels of pigmentation-related genes in order to determine the melanogenesis-regulating pathway of FGF21. The results showed that FGF21 overexpression suppressed melanogenesis and decreased the expression of the major target genes termed microphthalmia-associated transcription factor (MITF) and its downstream genes, including tyrosinase (TYR) and tyrosinase-related protein 2 (TRP2). However FGF21 increased the expression of phospho-extracellular signal-regulated kinase (p-Erk1/2). In contrast, FGF21-siRNA, a small interference RNA mediating FGF21 silencing, abolished the inhibition of melanogenesis. Altogether, FGF21 may decrease melanogenesis in alpaca melanocytes via ERK activation and subsequent MITF downregulation, which is then followed by the suppression of melanogenic enzymes and melanin production. Copyright © 2017. Published by Elsevier Inc.

Functionality study of santalin as tyrosinase inhibitor: A potential depigmentation agent.

PubMed

Hridya, Hemachandran; Amrita, Anantharaman; Mohan, Sankari; Gopalakrishnan, Mohan; Dakshinamurthy, Thirumal Kumar; Doss, George Priya; Siva, Ramamoorthy

2016-05-01

Excessive melanin production leads to hyperpigmentation disorders which results in distressing aesthetic values. Though there are some synthetic depigmentation agents available it has been reported to possess cytotoxic and mutagenic effects. Hence there is a need for the development of safe and non toxic natural tyrosinase inhibitors. Here we report the role of santalin, the chief constituent of Pterocarpus santalinus in inhibition of tyrosinase and melanin synthesis. Santalin inhibited tyrosinase activity dose dependently. Inhibitory kinetic studies revealed mixed type of inhibition with reversible mechanism. Santalin was found to interact with the fluorophore amino acid residue of tyrosinase. Analysis of circular dichroism spectra showed the binding of santalin to tyrosinase which induced the loss of secondary helical structure. Molecular docking result suggested that santalin interact with the catalytic core of tyrosinase through strong hydrogen and hydrophobic bonding. The results of in vitro studies showed santalin inhibited melanogenesis through down regulation of MITF, tyrosinase, TRP-1 and TRP-2 without any cytotoxic effects towards B16F0 melanoma cells. Therefore, our results suggested that santalin possesses anti-tyrosinase activity, which could be utilized as a safe depigmentation agent in the cosmetic field for the treatment of hyperpigmentation disorder. Copyright © 2016 Elsevier B.V. All rights reserved.

Melanogenesis inhibition in mice using a low-fluence 1064-nm Q-switched neodymium-doped yttrium aluminum garnet laser: a pilot study.

PubMed

Nam, Jae-Hui; Min, Joon Hong; Kim, Wang-Kyun; Yim, Sunmin; Kim, Won-Serk

2017-07-01

A low-fluence 1064-nm Q-switched neodymium-doped yttrium aluminum garnet laser, or laser toning, has yielded favorable outcomes in various benign pigmented disorders. However, the exact mechanism of action of laser toning has not been fully elucidated. We sought to determine the inhibitory effect of laser toning on melanogenesis and to assess how laser passes influence the outcomes. To produce perceptible pigmentation, nine HRM-2 melanin-possessing hairless mice were treated with ultraviolet (UV) B radiation on the dorsal skin. This was followed by zero, two, four, or six passes of laser toning twice in 2 weeks on each designated quadrant. The spectrophotometric values and pigmentation-related protein expressions were measured. Pigment changes were found in the mice skin using the Fontana-Masson stain for histopathological analysis. Four- and six-pass laser toning significantly improved the lightness compared to that in the unirradiated control (p < 0.002). The Fontana-Masson stain showed that melanin was considerably decreased in laser-irradiated skin. As the number of laser passes increased, the expression of tyrosinase decreased (p < 0.008). The following parameters also decreased in proportion to the number of laser passes: MITF, TRP-1, TRP-2, p-ERK, and p-Akt. In contrast, TGF-β increased in proportion to the number of laser passes. However, the changes in these six proteins were not statistically significant. Our study demonstrates that laser toning improves skin pigmentation with increased number of passes in a dose-dependent manner. This effect is mediated by tyrosinase inhibition.

Liver X receptor activation inhibits melanogenesis through the acceleration of ERK-mediated MITF degradation.

PubMed

Lee, Chang Seok; Park, Miyoung; Han, Jiwon; Lee, Ji-Hae; Bae, Il-Hong; Choi, Hyunjung; Son, Eui Dong; Park, Young-Ho; Lim, Kyung-Min

2013-04-01

Liver X receptors (LXRs) are nuclear receptors that act as ligand-activated transcription factors regulating lipid metabolism and inflammation. In the skin, activation of LXRs stimulates differentiation of keratinocytes and augments lipid synthesis in sebocytes. However, the function of LXRs in melanocytes remains largely unknown. We investigated whether LXR activation would affect melanogenesis. In human primary melanocytes, MNT-1, and B16 melanoma cells, TO901317, a synthetic LXR ligand, inhibited melanogenesis. Small interfering RNA (siRNA) experiments revealed the dominant role of LXRβ in TO901317-mediated antimelanogenesis. Enzymatic activities of tyrosinase were unaffected, but the expression of tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2 was suppressed by TO901317. Expressions of microphthalmia-associated transcription factor (MITF), a master transcriptional regulator of melanogenesis, and cAMP-responsive element-binding activation were not affected. It is noteworthy that the degradation of MITF was accelerated by TO901317. Extracellular signal-regulated kinase (ERK) contributed to TO901317-induced antimelanogenesis, which was evidenced by recovery of melanogenesis with ERK inhibitor. Other LXR ligands, 22(R)-hydroxycholesterol (22(R)HC) and GW3965, also activated ERK and suppressed melanogenesis. The intermediary role of Ras was confirmed in TO901317-induced ERK phosphorylation. Finally, antimelanogenic effects of TO901317 were confirmed in vivo in UVB-tanning model in brown guinea pigs, providing a previously unreported line of evidence that LXRs may be important targets for antimelanogenesis.

Dual inhibition of γ-oryzanol on cellular melanogenesis: inhibition of tyrosinase activity and reduction of melanogenic gene expression by a protein kinase A-dependent mechanism.

PubMed

Jun, Hee-jin; Lee, Ji Hae; Cho, Bo-Ram; Seo, Woo-Duck; Kang, Hang-Won; Kim, Dong-Woo; Cho, Kang-Jin; Lee, Sung-Joon

2012-10-26

The in vitro effects on melanogenesis of γ-oryzanol (1), a rice bran-derived phytosterol, were investigated. The melanin content in B16F1 cells was significantly and dose-dependently reduced (-13% and -28% at 3 and 30 μM, respectively). Tyrosinase enzyme activity was inhibited by 1 both in a cell-free assay and when analyzed based on the measurement of cellular tyrosinase activity. Transcriptome analysis was performed to investigate the biological pathways altered by 1, and it was found that gene expression involving protein kinase A (PKA) signaling was markedly altered. Subsequent analyses revealed that 1 stimulation in B16 cells reduced cytosolic cAMP concentrations, PKA activity (-13% for cAMP levels and -40% for PKA activity), and phosphorylation of the cAMP-response element binding protein (-57%), which, in turn, downregulated the expression of microphthalmia-associated transcription factor (MITF; -59% for mRNA and -64% for protein), a key melanogenic gene transcription factor. Accordingly, tyrosinase-related protein 1 (TRP-1; -69% for mRNA and -82% for protein) and dopachrome tautomerase (-51% for mRNA and -92% for protein) in 1-stimulated B16F1 cells were also downregulated. These results suggest that 1 has dual inhibitory activities for cellular melanogenesis by inhibiting tyrosinase enzyme activity and reducing MITF and target genes in the PKA-dependent pathway.

Triterpene glycosides and other polar constituents of shea (Vitellaria paradoxa) kernels and their bioactivities.

PubMed

Zhang, Jie; Kurita, Masahiro; Shinozaki, Takuro; Ukiya, Motohiko; Yasukawa, Ken; Shimizu, Naoto; Tokuda, Harukuni; Masters, Eliot T; Akihisa, Momoko; Akihisa, Toshihiro

2014-12-01

The MeOH extract of defatted shea (Vitellaria paradoxa; Sapotaceae) kernels was investigated for its constituents, and fifteen oleanane-type triterpene acids and glycosides, two steroid glucosides, two pentane-2,4-diol glucosides, seven phenolic compounds, and three sugars, were isolated. The structures of five triterpene glycosides were elucidated on the basis of spectroscopic and chemical methods. Upon evaluation of the bioactivity of the isolated compounds, it was found that some or most of the compounds have potent or moderate inhibitory activities against the following: melanogenesis in B16 melanoma cells induced by α-melanocyte-stimulating hormone (α-MSH); generation of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, against Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-teradecanoylphorbol 13-acetate (TPA) in Raji cells; t TPA-induced inflammation in mice, and proliferation of one or more of HL-60, A549, AZ521, and SK-BR-3 human cancer cell lines, respectively. Western blot analysis established that paradoxoside E inhibits melanogenesis by regulation of expression of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein-1 (TRP-1) and TRP-2. In addition, tieghemelin A was demonstrated to exhibit cytotoxic activity against A549 cells (IC50 13.5 μM) mainly due to induction of apoptosis by flow cytometry. The extract of defatted shea kernels and its constituents may be, therefore, valuable as potential antioxidant, anti-inflammatory, skin-whitening, chemopreventive, and anticancer agents. Copyright © 2014 Elsevier Ltd. All rights reserved.

The lactoferricin B-derived peptide, LfB17-34, induces melanogenesis in B16F10 cells.

PubMed

Huang, Hsiu-Chin; Lin, Hsuan; Huang, Min-Chuan

2017-03-01

Lactoferricin B (LfcinB), a peptide of bovine lactoferrin (LfB), exhibits multiple biological functions, including antimicrobial, antiviral, antioxidant and immunomodulatory activities. However, the role of LfcinB-related peptides in melanogenesis remains unclear. In this study, a set of five LfcinB-related peptides was examined. We found that LfB17‑34, an 18-mer LfcinB-derived peptide, increased melanogenesis in B16F10 melanoma cells without significantly affecting cell viability. LfB17‑34 increased in vitro tyrosinase activity and melanin content in a dose-dependent manner. The results of RT-qPCR and western blot analyses showed that LfB17‑34 increased the mRNA and protein expression of tyrosinase and tyrosinase-related protein 1 (Trp1). Moreover, LfB17‑34 inhibited the phosphorylation of MAPK/Erk, but not p38 and Akt, and constitutively active MEK was able to reverse the LfB17-34-enhanced pigmentation, melanin content, and tyrosinase activity, suggesting a role of Erk signaling in the process of LfB17‑34-mediated pigmentation. Taken together, these results suggest that LfB17‑34 induces melanogenesis in B16F10 cells primarily through increased tyrosinase expression and activity and that LfB17‑34 could be further developed for the treatment of hypopigmentation disorders.

The lactoferricin B-derived peptide, LfB17-34, induces melanogenesis in B16F10 cells

PubMed Central

Huang, Hsiu-Chin; Lin, Hsuan; Huang, Min-Chuan

2017-01-01

Lactoferricin B (LfcinB), a peptide of bovine lactoferrin (LfB), exhibits multiple biological functions, including antimicrobial, antiviral, antioxidant and immuno-modulatory activities. However, the role of LfcinB-related peptides in melanogenesis remains unclear. In this study, a set of five LfcinB-related peptides was examined. We found that LfB17-34, an 18-mer LfcinB-derived peptide, increased melanogenesis in B16F10 melanoma cells without significantly affecting cell viability. LfB17-34 increased in vitro tyrosinase activity and melanin content in a dose-dependent manner. The results of RT-qPCR and western blot analyses showed that LfB17-34 increased the mRNA and protein expression of tyrosinase and tyrosinase-related protein 1 (Trp1). Moreover, LfB17-34 inhibited the phosphorylation of MAPK/Erk, but not p38 and Akt, and constitutively active MEK was able to reverse the LfB17-34-enhanced pigmentation, melanin content, and tyrosinase activity, suggesting a role of Erk signaling in the process of LfB17-34-mediated pigmentation. Taken together, these results suggest that LfB17-34 induces melanogenesis in B16F10 cells primarily through increased tyrosinase expression and activity and that LfB17-34 could be further developed for the treatment of hypopigmentation disorders. PMID:28204812

Antioxidative and melanogenesis-inhibitory activities of caffeoylquinic acids and other compounds from moxa.

PubMed

Akihisa, Toshihiro; Kawashima, Kohta; Orido, Masashi; Akazawa, Hiroyuki; Matsumoto, Masahiro; Yamamoto, Ayako; Ogihara, Eri; Fukatsu, Makoto; Tokuda, Harukuni; Fuji, Jizaemon

2013-03-01

The MeOH extract of moxa, the processed leaves of Artemisia princeps PAMP. (Asteraceae), exhibited potent 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity and melanogenesis-inhibitory activity in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16 melanoma cells. Eight caffeoylquinic acids, 1 and 6-12, five flavonoids, 13-17, two benzoic acid derivatives, 18 and 19, three coumarin derivatives, 20-22, four steroids, 23-26, and six triterpenoids, 27-32, were isolated from the MeOH extract. Upon evaluation of compounds 1, 6-23, and four semisynthetic caffeoylquinic acid esters, 2-5, for their DPPH radical-scavenging activity, 15 compounds, 1-13, 17, and 19, showed potent activities (IC(50) 3.1-16.8 μM). The 15 compounds exhibited, moreover, potent inhibitory activities (51.1-92.5% inhibition) against peroxidation of linoleic acid emulsion at 10 μg/ml concentration. In addition, when 27 compounds, 1-8, 10, 12, 13, 15-18, 20-25, and 27-32, were evaluated for their inhibitory activity against melanogenesis in α-MSH-stimulated B16 melanoma cells, five caffeoylquinic acids, i.e., chlorogenic acid (1), ethyl chlorogenate (3), propyl chlorogenate (4), isopropyl chlorogenate (5), and butyl chlorogenate (6), along with homoorientin (17) and vanillic acid (18), exhibited inhibitory activities with 33-62% reduction of melanin content at 100 μM concentration with no or almost no toxicity to the cells (89-114% of cell viability at 100 μM). Western blot analysis showed that compound 6 reduced the protein levels of microphtalmia-associated transcription factor (MITF), tyrosinase, tyrosine-related protein 1 (TRP-1), and TRP-2 mostly in a concentration-dependent manner, suggesting that this compound inhibits melanogenesis on α-MSH-stimulated B16 melanoma cells by, at least in part, inhibiting the expression of MITF, followed by decreasing the expression of tyrosinase, TRP-1, and TRP-2. Furthermore, four compounds, 13, 15, 16, and 30, exhibited cytotoxicities against HL60 human leukemia cell line (IC(50) 7.0-11.1 μM), and nine compounds, 14-16, 23, 26-28, 31, and 32, showed inhibitory effects (IC(50) 272-382 mol ratio/32 pmol 12-O-tetradecanoylphohrbol-13-acetate (TPA)) against Epstein-Barr virus early antigen (EBV-EA) activation induced by TPA in Raji cells. Copyright © 2013 Verlag Helvetica Chimica Acta AG, Zürich.

Regeneration of Murine Hair Follicles is Inhibited by Low-Dose-Rate Gamma Irradiation.

PubMed

Sugaya, Kimihiko; Hirobe, Tomohisa; Ishihara, Yoshie; Inoue, Sonoe

2016-10-01

To determine whether the effects of low-dose-rate gamma (γ) irradiation are identifiable in the regeneration of murine hair follicles, we irradiated whole bodies of C57BL/10JHir mice in the first telogen phase of the hair cycle with 137 Cs γ-rays. The mice were examined for effects on hair follicles, including number, morphology, and pigmentation in the second anagen phase. Effects of γ-radiation on melanocyte stem cells were also investigated by the indirect immunolabeling of tyrosinase-related protein 2 (TRP2). Irradiated skin showed a decrease in hair follicle density and the induction of curved hair follicles along with the presence of white hairs and hypopigmented hair bulbs. There was a small, but not significant, change in the number of TRP2-positive melanocyte stem cells in the hair bulge region of the irradiated skin. These results suggest that low-dose rate γ-irradiation does not deplete melanocyte stem cells, but can damage stem cells and progenitors for both keratinocytes and melanocytes, thereby affecting the structure and pigmentation of regenerated hair follicles in the 2 nd anagen phase.

Norartocarpetin from a folk medicine Artocarpus communis plays a melanogenesis inhibitor without cytotoxicity in B16F10 cell and skin irritation in mice

PubMed Central

2013-01-01

Background Many natural products used in preventive medicine have also been developed as cosmeceutical ingredients in skin care products, such as Scutellaria baicalensis and Gardenia jasminoides. Norartocarpetin is one of the antioxidant and antityrosinase activity compound in Artocarpus communis; however, the cytotoxicity, skin irritation and antimelanogenesis mechanisms of norartocarpetin have not been investigated yet. Methods In the present study, cell viability in vitro and skin irritation in vivo are used to determine the safety of norartocarpetin. The melanogenesis inhibition of norartocarpetin was determined by cellular melanin content and tyrosinase in B16F10 melanoma cell. Moreover, we examined the related-melanogenesis protein by western blot analysis for elucidating the antimelanogenesis mechanism of norartocarpin. Results The result of the present study demonstrated that norartocarpetin not only present non-cytotoxic in B16F10 and human fibroblast cells but also non-skin irritation in mice. Moreover, our result also first found that norartocarpetin downregulated phospho-cAMP response element-binding (phospho-CREB) and microphthalmia-associated transcription factor (MITF) expression, which in turn decreased both synthesis of tyrosinases (TRP-1 and TRP-2) and cellular melanin content. This process is dependent on norartocarpetin phosphorylation by mitogen-activated protein kinases such as phospho-JNK and phospho-p38, and it results in decreased melanogenesis. Conclusion The present study suggests that norartocarpetin could be used as a whitening agent in medicine and/or cosmetic industry and need further clinical study. PMID:24325567

Optimization of extraction conditions for osthol, a melanogenesis inhibitor from Cnidium monnieri fruits.

PubMed

Beom Kim, Seon; Kim, CheongTaek; Liu, Qing; Hee Jo, Yang; Joo Choi, Hak; Hwang, Bang Yeon; Kyum Kim, Sang; Kyeong Lee, Mi

2016-08-01

Coumarin derivatives have been reported to inhibit melanin biosynthesis. The melanogenesis inhibitory activity of osthol, a major coumarin of the fruits of Cnidium monnieri Cusson (Umbelliferae), and optimized extraction conditions for the maximum yield from the isolation of osthol from C. monnieri fruits were investigated. B16F10 melanomas were treated with osthol at concentration of 1, 3, and 10 μM for 72 h. The expression of melanogenesis genes, such as tyrosinase, TRP-1, and TRP-2 was also assessed. For optimization, extraction factors such as extraction solvent, extraction time, and sample/solvent ratio were tested and optimized for maximum yield of osthol using response surface methodology with the Box-Behnken design (BBD). Osthol inhibits melanin content in B16F10 melanoma cells with an IC50 value of 4.9 μM. The melanogenesis inhibitory activity of osthol was achieved not by direct inhibition of tyrosinase activity but by inhibiting melanogenic enzyme expressions, such as tyrosinase, TRP-1, and TRP-2. The optimal condition was obtained as a sample/solvent ratio, 1500 mg/10 ml; an extraction time 30.3 min; and a methanol concentration of 97.7%. The osthol yield under optimal conditions was found to be 15.0 mg/g dried samples, which were well matched with the predicted value of 14.9 mg/g dried samples. These results will provide useful information about optimized extraction conditions for the development of osthol as cosmetic therapeutics to reduce skin hyperpigmentation.

Tyrosol and its analogues inhibit alpha-melanocyte-stimulating hormone induced melanogenesis.

PubMed

Wen, Kuo-Ching; Chang, Chih-Shiang; Chien, Yin-Chih; Wang, Hsiao-Wen; Wu, Wan-Chen; Wu, Chin-Sheng; Chiang, Hsiu-Mei

2013-11-28

Melanin is responsible for skin color and plays a major role in defending against harmful external factors such as ultraviolet (UV) irradiation. Tyrosinase is responsible for the critical steps of melanogenesis, including the rate-limiting step of tyrosine hydroxylation. The mechanisms of action of skin hypopigmenting agents are thought to be based on the ability of a given agent to inhibit the activity of tyrosinase and, hence, down regulate melanin synthesis. Tyrosol and its glycoside, salidroside, are active components of Rhodiola rosea, and in our preliminary study we found that Rhodiola rosea extract inhibited melanogenesis. In this study, we examined the effects of tyrosol and its analogues on melanin synthesis. We found that treatment of B16F0 cells to tyrosol (1), 4-hydroxyphenylacetic acid (5), 3-hydroxyphenylacetic acid (6), 2-hydroxyphenylacetic acid (7), or salidroside (11) resulted in a reduction in melanin content and inhibition of tyrosinase activity as well as its expression. Tyrosol (1), 4-hydroxyphenylacetic acid (5) and 2-hydroxyphenylacetic acid (7) suppressed MC1R expression. Tyrosol (1), 4-hydroxyphenylacetic acid (5), 3-hydroxyphenylacetic acid (6), and 2-hydroxyphenylacetic acid (7) inhibited α-MSH induced TRP-1 expression, but salidroside (11) did not. All the compounds did not affect MITF and TRP-2 expression. Furthermore, we found that the cell viability of tyrosol (1), 4-hydroxyphenylacetic acid (5), 3-hydroxyphenylacetic acid (6), and 2-hydroxyphenylacetic acid (7) at concentrations below 4 mM and salidroside (11) at concentrations below 0.5 mM were higher than 90%. The compounds exhibited metal-coordinating interactions with copper ion in molecular docking with tyrosinase. Our results suggest that tyrosol, 4-hydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 2-hydroxyphenylacetic acid, and salidroside are potential hypopigmenting agents.

Tyrosol and Its Analogues Inhibit Alpha-Melanocyte-Stimulating Hormone Induced Melanogenesis

PubMed Central

Wen, Kuo-Ching; Chang, Chih-Shiang; Chien, Yin-Chih; Wang, Hsiao-Wen; Wu, Wan-Chen; Wu, Chin-Sheng; Chiang, Hsiu-Mei

2013-01-01

Melanin is responsible for skin color and plays a major role in defending against harmful external factors such as ultraviolet (UV) irradiation. Tyrosinase is responsible for the critical steps of melanogenesis, including the rate-limiting step of tyrosine hydroxylation. The mechanisms of action of skin hypopigmenting agents are thought to be based on the ability of a given agent to inhibit the activity of tyrosinase and, hence, down regulate melanin synthesis. Tyrosol and its glycoside, salidroside, are active components of Rhodiola rosea, and in our preliminary study we found that Rhodiola rosea extract inhibited melanogenesis. In this study, we examined the effects of tyrosol and its analogues on melanin synthesis. We found that treatment of B16F0 cells to tyrosol (1), 4-hydroxyphenylacetic acid (5), 3-hydroxyphenylacetic acid (6), 2-hydroxyphenylacetic acid (7), or salidroside (11) resulted in a reduction in melanin content and inhibition of tyrosinase activity as well as its expression. Tyrosol (1), 4-hydroxyphenylacetic acid (5) and 2-hydroxyphenylacetic acid (7) suppressed MC1R expression. Tyrosol (1), 4-hydroxyphenylacetic acid (5), 3-hydroxyphenylacetic acid (6), and 2-hydroxyphenylacetic acid (7) inhibited α-MSH induced TRP-1 expression, but salidroside (11) did not. All the compounds did not affect MITF and TRP-2 expression. Furthermore, we found that the cell viability of tyrosol (1), 4-hydroxyphenylacetic acid (5), 3-hydroxyphenylacetic acid (6), and 2-hydroxyphenylacetic acid (7) at concentrations below 4 mM and salidroside (11) at concentrations below 0.5 mM were higher than 90%. The compounds exhibited metal-coordinating interactions with copper ion in molecular docking with tyrosinase. Our results suggest that tyrosol, 4-hydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 2-hydroxyphenylacetic acid, and salidroside are potential hypopigmenting agents. PMID:24287915

The Effect of Butin on the Vitiligo Mouse Model Induced by Hydroquinone.

PubMed

Huo, Shi-Xia; Wang, Qiong; Liu, Xin-Min; Ge, Chun-Hui; Gao, Li; Peng, Xiao-Ming; Yan, Ming

2017-05-01

Vernonia anthelmintica (L.) Willd has been traditionally used in the treatment of vitiligo in Uyghur medicine. This study used butin, the main component of V. anthelmintica, to study the influence on hydroquinone-induced vitiligo in mice. The animals were randomly divided into six groups: control, model, 8-methoxypsoralen (8-MOP, 4.25 mg/kg), and butin (0.425, 4.25, and 42.5 mg/kg) groups. The number of melanin-containing hair follicles, basal layer melanocytes, melanin-containing epidermal cells, the expression of tyrosinase (TYR) and tyrosinase-related protein-1 (TRP-1), the malondialdehyde (MDA), and cholinesterase (CHE) activity in serum were measured. Our results indicated that compared with the model group, the melanin-containing hair follicles, the expression of TYR and TRP-1 increased, the activity of CHE decreased after treatment with 8-MOP and all doses of butin (p < 0.05, p < 0.01), the basal layer melanocytes and melanin-containing epidermal cells increased significantly after treatment with butin 4.25 and 42.5 mg/kg (p < 0.05, p < 0.01), and the MDA activity decreased after using butin 4.25 and 42.5 mg/kg and 8-MOP (p < 0.05, p < 0.01). Our results support the use of butin on vitiligo, and its possible mechanisms may be related to increase the TYR and TRP-1 protein expression and decrease the activity of MDA and CHE in hydroquinone-induced vitiligo model in mice. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Depigmenting effect of argan press-cake extract through the down-regulation of Mitf and melanogenic enzymes expression in B16 murine melanoma cells.

PubMed

Bourhim, Thouria; Villareal, Myra O; Gadhi, Chemseddoha; Hafidi, Abdellatif; Isoda, Hiroko

2018-06-26

Oil extraction from the kernels of Argania spinosa (L.) Skeels (Sapotaceae), an endemic tree of Morocco, produces argan press-cake (APC) used as a shampoo and to treat sprains, scabies, and for healing wounds. We have previously reported that argan oil has antimelanogenesis effect. Here, we determined if the by-product, APC, has melanogenesis regulatory effect using B16 murine melanoma cells. The effect of APC ethanol extract on cell proliferation and melanin content of B16 cells were measured, and to elucidate the mechanism involved, the expression level of melanogenic enzymes tyrosinase (TYR), dopachrome tautomerase (DCT), and tyrosinase-related protein 1 (TRP1) were determined and mRNA expression level of microphthalmia- associated transcription factor (Mitf) and Tyr genes were quantified. APC ethanol extract showed a significant melanin biosynthesis inhibitory effect on B16 cells in a time-dependent manner without cytotoxicity, which could be due to the decreased expression of TYR, TRP1, and DCT in a time-dependent manner. APC extract down regulated Mitf and Tyr. Decreased TRP1 and DCT levels could be due to post-translational modifications. These results suggest that APC extract may be used as a new source of natural whitening products and may be introduced as an important pharmacological agent for the treatment of hyperpigmentation disorders.

Membrane-Associated Transporter Protein (MATP) Regulates Melanosomal pH and Influences Tyrosinase Activity

PubMed Central

Bin, Bum-Ho; Bhin, Jinhyuk; Yang, Seung Ha; Shin, Misun; Nam, Yeon-Ju; Choi, Dong-Hwa; Shin, Dong Wook; Lee, Ai-Young; Hwang, Daehee; Cho, Eun-Gyung; Lee, Tae Ryong

2015-01-01

The SLC45A2 gene encodes a Membrane-Associated Transporter Protein (MATP). Mutations of this gene cause oculocutaneous albinism type 4 (OCA4). However, the molecular mechanism of its action in melanogenesis has not been elucidated. Here, we discuss the role of MATP in melanin production. The SLC45A2 gene is highly enriched in human melanocytes and melanoma cell lines, and its protein, MATP, is located in melanosomes. The knockdown of MATP using siRNAs reduced melanin content and tyrosinase activity without any morphological change in melanosomes or the expression of melanogenesis-related proteins. Interestingly, the knockdown of MATP significantly lowered the melanosomal pH, as verified through DAMP analysis, suggesting that MATP regulates melanosomal pH and therefore affects tyrosinase activity. Finally, we found that the reduction of tyrosinase activity associated with the knockdown of MATP was readily recovered by copper treatment in the in vitro L-DOPA oxidase activity assay of tyrosinase. Considering that copper is an important element for tyrosinase activity and that its binding to tyrosinase depends on melanosomal pH, MATP may play an important role in regulating tyrosinase activity via controlling melanosomal pH. PMID:26057890

[6]-Shogaol Inhibits α-MSH-Induced Melanogenesis through the Acceleration of ERK and PI3K/Akt-Mediated MITF Degradation

PubMed Central

Huang, Huey-Chun; Chang, Shu-Jen; Wu, Chia-Yin; Ke, Hui-Ju; Chang, Tsong-Min

2014-01-01

[6]-Shogaol is the main biologically active component of ginger. Previous reports showed that [6]-shogaol has several pharmacological characteristics, such as antioxidative, anti-inflammatory, antimicrobial, and anticarcinogenic properties. However, the effects of [6]-shogaol on melanogenesis remain to be elucidated. The study aimed to evaluate the potential skin whitening mechanisms of [6]-shogaol. The effects of [6]-shogaol on cell viability, melanin content, tyrosinase activity, and the expression of the tyrosinase and microphthalmia-associated transcription factor (MITF) were measured. The results revealed that [6]-shogaol effectively suppresses tyrosinase activity and the amount of melanin and that those effects are more pronounced than those of arbutin. It was also found that [6]-shogaol decreased the protein expression levels of tyrosinase-related protein 1 (TRP-1) and microphthalmia-associated transcriptional factor (MITF). In addition, the MITF mRNA levels were also effectively decreased in the presence of 20 μM [6]-shogaol. The degradation of MITF protein was inhibited by the MEK 1-inhibitor (U0126) or phosphatidylinositol-3-kinase inhibitor (PI3K inhibitor) (LY294002). Further immunofluorescence staining assay implied the involvement of the proteasome in the downregulation of MITF by [6]-shogaol. Our confocal assay results also confirmed that [6]-shogaol inhibited α-melanocyte stimulating hormone- (α-MSH-) induced melanogenesis through the acceleration of extracellular responsive kinase (ERK) and phosphatidylinositol-3-kinase- (PI3K/Akt-) mediated MITF degradation. PMID:25045707

[6]-Shogaol inhibits α-MSH-induced melanogenesis through the acceleration of ERK and PI3K/Akt-mediated MITF degradation.

PubMed

Huang, Huey-Chun; Chang, Shu-Jen; Wu, Chia-Yin; Ke, Hui-Ju; Chang, Tsong-Min

2014-01-01

[6]-Shogaol is the main biologically active component of ginger. Previous reports showed that [6]-shogaol has several pharmacological characteristics, such as antioxidative, anti-inflammatory, antimicrobial, and anticarcinogenic properties. However, the effects of [6]-shogaol on melanogenesis remain to be elucidated. The study aimed to evaluate the potential skin whitening mechanisms of [6]-shogaol. The effects of [6]-shogaol on cell viability, melanin content, tyrosinase activity, and the expression of the tyrosinase and microphthalmia-associated transcription factor (MITF) were measured. The results revealed that [6]-shogaol effectively suppresses tyrosinase activity and the amount of melanin and that those effects are more pronounced than those of arbutin. It was also found that [6]-shogaol decreased the protein expression levels of tyrosinase-related protein 1 (TRP-1) and microphthalmia-associated transcriptional factor (MITF). In addition, the MITF mRNA levels were also effectively decreased in the presence of 20 μM [6]-shogaol. The degradation of MITF protein was inhibited by the MEK 1-inhibitor (U0126) or phosphatidylinositol-3-kinase inhibitor (PI3K inhibitor) (LY294002). Further immunofluorescence staining assay implied the involvement of the proteasome in the downregulation of MITF by [6]-shogaol. Our confocal assay results also confirmed that [6]-shogaol inhibited α-melanocyte stimulating hormone- (α-MSH-) induced melanogenesis through the acceleration of extracellular responsive kinase (ERK) and phosphatidylinositol-3-kinase- (PI3K/Akt-) mediated MITF degradation.

5-HT1A/1B Receptors as Targets for Optimizing Pigmentary Responses in C57BL/6 Mouse Skin to Stress

PubMed Central

Wu, Hua-Li; Pang, Si-Lin; Liu, Qiong-Zhen; Wang, Qian; Cai, Min-Xuan; Shang, Jing

2014-01-01

Stress has been reported to induce alterations of skin pigmentary response. Acute stress is associated with increased turnover of serotonin (5-hydroxytryptamine; 5-HT) whereas chronic stress causes a decrease. 5-HT receptors have been detected in pigment cells, indicating their role in skin pigmentation. To ascertain the precise role of 5-HT in stress-induced pigmentary responses, C57BL/6 mice were subjected to chronic restraint stress and chronic unpredictable mild stress (CRS and CUMS, two models of chronic stress) for 21 days, finally resulting in abnormal pigmentary responses. Subsequently, stressed mice were characterized by the absence of a black pigment in dorsal coat. The down-regulation of tyrosinase (TYR) and tyrosinase-related proteins (TRP1 and TRP2) expression in stressed skin was accompanied by reduced levels of 5-HT and decreased expression of 5-HT receptor (5-HTR) system. In both murine B16F10 melanoma cells and normal human melanocytes (NHMCs), 5-HT had a stimulatory effect on melanin production, dendricity and migration. When treated with 5-HT in cultured hair follicles (HFs), the increased expression of melanogenesis-related genes and the activation of 5-HT1A, 1B and 7 receptors also occurred. The serum obtained from stressed mice showed significantly decreased tyrosinase activity in NHMCs compared to that from nonstressed mice. The decrease in tyrosinase activity was further augmented in the presence of 5-HTR1A, 1B and 7 antagonists, WAY100635, SB216641 and SB269970. In vivo, stressed mice received 5-HT precursor 5-hydroxy-l-tryptophan (5-HTP), a member of the class of selective serotonin reuptake inhibitors (fluoxetine; FX) and 5-HTR1A/1B agonists (8-OH-DPAT/CP94253), finally contributing to the normalization of pigmentary responses. Taken together, these data strongly suggest that the serotoninergic system plays an important role in the regulation of stress-induced depigmentation, which can be mediated by 5-HT1A/1B receptors. 5-HT and 5-HTR1A/1B may constitute novel targets for therapy of skin hypopigmentation disorders, especially those worsened with stress. PMID:24586946

Melanoma cells present high levels of HLA-A2-tyrosinase in association with instability and aberrant intracellular processing of tyrosinase.

PubMed

Michaeli, Yael; Sinik, Keren; Haus-Cohen, Maya; Reiter, Yoram

2012-04-01

Short-lived protein translation products are proposed to be a major source of substrates for major histocompatibility complex (MHC) class I antigen processing and presentation; however, a direct link between protein stability and the presentation level of MHC class I-peptide complexes has not been made. We have recently discovered that the peptide Tyr((369-377)) , derived from the tyrosinase protein is highly presented by HLA-A2 on the surface of melanoma cells. To examine the molecular mechanisms responsible for this presentation, we compared characteristics of tyrosinase in melanoma cells lines that present high or low levels of HLA-A2-Tyr((369-377)) complexes. We found no correlation between mRNA levels and the levels of HLA-A2-Tyr((369-377)) presentation. Co-localization experiments revealed that, in cell lines presenting low levels of HLA-A2-Tyr((369-377)) complexes, tyrosinase co-localizes with LAMP-1, a melanosome marker, whereas in cell lines presenting high HLA-A2-Tyr((369-377)) levels, tyrosinase localizes to the endoplasmic reticulum. We also observed differences in tyrosinase molecular weight and glycosylation composition as well as major differences in protein stability (t(1/2) ). By stabilizing the tyrosinase protein, we observed a dramatic decrease in HLA-A2-tyrosinase presentation. Our findings suggest that aberrant processing and instability of tyrosinase are responsible for the high presentation of HLA-A2-Tyr((369-377)) complexes and thus shed new light on the relationship between intracellular processing, stability of proteins, and MHC-restricted peptide presentation. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

N-terminal fatty acylated His-dPhe-Arg-Trp-NH(2) tetrapeptides: influence of fatty acid chain length on potency and selectivity at the mouse melanocortin receptors and human melanocytes.

PubMed

Todorovic, Aleksandar; Holder, Jerry Ryan; Bauzo, Rayna M; Scott, Joseph Walker; Kavanagh, Renny; Abdel-Malek, Zalfa; Haskell-Luevano, Carrie

2005-05-05

The melanocortin system is involved in the regulation of a diverse number of physiologically important pathways including pigmentation, feeding behavior, weight and energy homeostasis, inflammation, and sexual function. All the endogenous melanocortin agonist ligands possess the conserved His-Phe-Arg-Trp tetrapeptide sequence that is postulated to be important for melanocortin receptor molecular recognition and stimulation. Previous studies by our laboratory resulted in the discovery that increasing alkyl chain length at the N-terminal "capping" region of the His-dPhe-Arg-Trp-NH(2) tetrapeptide resulted in a 100-fold increased melanocortin receptor agonist potency. This study was undertaken to systematically evaluate the pharmacological effects of increasing N-capping alkyl chain length of the CH(3)(CH(2))(n)CO-His-dPhe-Arg-Trp-NH(2) (n = 6-16) tetrapeptide template. Twelve analogues were synthesized and pharmacologically characterized at the mouse melanocortin receptors MC1R and MC3R-MC5R and human melanocytes known to express the MC1R. These peptides demonstrated melanocortin receptor selectivity profiles different from those of previously published tetrapeptides. The most notable results of enhanced ligand potency (20- to 200-fold) and receptor selectivity were observed at the MC1R. Tetrapeptides that possessed greater than nine alkyl groups were superior to alpha-MSH in terms of the stimulation of human melanocyte tyrosinase activity. Additionally, the n-pentadecanoyl derivative had a residual effect on tyrosinase activity that existed for at least 4 days after the peptide was removed from the human melanocyte culture medium. These data demonstrate the utility, potency, and residual effect of melanocortin tetrapeptides by adding N-terminal fatty acid moieties.

Heterologous expression of tyrosinase recapitulates the misprocessing and mistrafficking in oculocutaneous albinism type 2: effects of altering intracellular pH and pink-eyed dilution gene expression.

PubMed

Ni-Komatsu, Li; Orlow, Seth J

2006-03-01

The processing and trafficking of tyrosinase, a melanosomal protein essential for pigmentation, was investigated in a human epithelial 293 cell line that stably expresses the protein. The effects of the pink-eyed dilution (p) gene product, in which mutations result in oculocutaneous albinism type 2 (OCA2), on the processing and trafficking of tyrosinase in this cell line were studied. The majority of tyrosinase was retained in the endoplasmic reticulum-Golgi intermediate compartment and the early Golgi compartment in the 293 cells expressing the protein. Coexpression of p could partially correct the mistrafficking of tyrosinase in 293 cells. Tyrosinase was targeted to the late endosomal and lysosomal compartments after treatment of the cells with compounds that correct the tyrosinase mistrafficking in albino melanocytes, most likely through altering intracellular pH, while the substrate tyrosine had no effect on the processing of tyrosinase. Remarkably, this heterologous expression system recapitulates the defective processing and mistrafficking of tyrosinase observed in OCA2 albino melanocytes and certain amelanotic melanoma cells. Coexpression of other melanosomal proteins in this heterologous system may further aid our understanding of the details of normal and pathologic processing of melanosomal proteins.

New Whitening Constituents from Taiwan-Native Pyracantha koidzumii: Structures and Tyrosinase Inhibitory Analysis in Human Epidermal Melanocytes.

PubMed

Lin, Rong-Dih; Chen, Mei-Chuan; Liu, Yan-Ling; Lin, Yi-Tzu; Lu, Mei-Kuang; Hsu, Feng-Lin; Lee, Mei-Hsien

2015-12-02

Nontoxic natural products useful in skin care cosmetics are of considerable interest. Tyrosinase is a rate-limiting enzyme for which its inhibitor is useful in developing whitening cosmetics. Pyracantha koidzumii (Hayata) Rehder is an endemic species in Taiwan that exhibits tyrosinase-inhibitory activity. To find new active natural compounds from P. koidzumii, we performed bioguided isolation and studied the related activity in human epidermal melanocytes. In total, 13 compounds were identified from P. koidzumii in the present study, including two new compounds, 3,6-dihydroxy-2,4-dimethoxy-dibenzofuran (9) and 3,4-dihydroxy-5-methoxybiphenyl-2'-O-β-d-glucopyranoside (13), as well as 11 known compounds. The new compound 13 exhibited maximum potency in inhibiting cellular tyrosinase activity, the protein expression of cellular tyrosinase and tyrosinase-related protein-2, as well as the mRNA expression of Paired box 3 and microphthalmia-associated transcription factor in a concentration-dependent manner. In the enzyme kinetic assay, the new compound 13 acted as an uncompetitive mixed-type inhibitor against the substrate l-3,4-dihydroxyphenylalanine and had a Km value against this substrate of 0.262 mM, as calculated using the Lineweaver-Burk plots. Taken together, our findings show compound 13 exhibits tyrosinase inhibition in human melanocytes and compound 13 may be a potential candidate for use in cosmetics.

New Whitening Constituents from Taiwan-Native Pyracantha koidzumii: Structures and Tyrosinase Inhibitory Analysis in Human Epidermal Melanocytes

PubMed Central

Lin, Rong-Dih; Chen, Mei-Chuan; Liu, Yan-Ling; Lin, Yi-Tzu; Lu, Mei-Kuang; Hsu, Feng-Lin; Lee, Mei-Hsien

2015-01-01

Nontoxic natural products useful in skin care cosmetics are of considerable interest. Tyrosinase is a rate-limiting enzyme for which its inhibitor is useful in developing whitening cosmetics. Pyracantha koidzumii (Hayata) Rehder is an endemic species in Taiwan that exhibits tyrosinase-inhibitory activity. To find new active natural compounds from P. koidzumii, we performed bioguided isolation and studied the related activity in human epidermal melanocytes. In total, 13 compounds were identified from P. koidzumii in the present study, including two new compounds, 3,6-dihydroxy-2,4-dimethoxy-dibenzofuran (9) and 3,4-dihydroxy-5-methoxybiphenyl-2ʹ-O-β-d-glucopyranoside (13), as well as 11 known compounds. The new compound 13 exhibited maximum potency in inhibiting cellular tyrosinase activity, the protein expression of cellular tyrosinase and tyrosinase-related protein-2, as well as the mRNA expression of Paired box 3 and microphthalmia-associated transcription factor in a concentration-dependent manner. In the enzyme kinetic assay, the new compound 13 acted as an uncompetitive mixed-type inhibitor against the substrate l-3,4-dihydroxyphenylalanine and had a Km value against this substrate of 0.262 mM, as calculated using the Lineweaver–Burk plots. Taken together, our findings show compound 13 exhibits tyrosinase inhibition in human melanocytes and compound 13 may be a potential candidate for use in cosmetics. PMID:26633381

Melanogenesis inhibitory activity of a 7-O-9'-linked neolignan from Alpinia galanga fruit.

PubMed

Manse, Yoshiaki; Ninomiya, Kiyofumi; Nishi, Ryosuke; Kamei, Iyori; Katsuyama, Yushi; Imagawa, Takahito; Chaipech, Saowanee; Muraoka, Osamu; Morikawa, Toshio

2016-12-01

An aqueous acetone extract from the fruit of Alpinia galanga (Zingiberaceae) demonstrated inhibitory effects on melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells (IC 50 =7.3μg/mL). Through bioassay-guided separation of the extract, a new 7-O-9'-linked neolignan, named galanganol D diacetate (1), was isolated along with 16 known compounds including 14 phenylpropanoids (2-15). The structure of 1, including its absolute stereochemistry in the C-7 position, was elucidated by means of extensive NMR analysis and total synthesis. Among the isolates, 1 (IC 50 =2.5μM), 1'S-1'-acetoxychavicol acetate (2, 5.0μM), and 1'S-1'-acetoxyeugenol acetate (3, 5.6μM) exhibited a relatively potent inhibitory effect without notable cytotoxicity at effective concentrations. The following structural requirements were suggested to enhance the inhibitory activity of phenylpropanoids on melanogenesis: (i) compounds with 4-acetoxy group exhibit higher activity than those with 4-hydroxy group; (ii) 3-methoxy group dose not affect the activity; (iii) acetylation of the 1'-hydroxy moiety enhances the activity; and (iv) phenylpropanoid dimers with the 7-O-9'-linked neolignan skeleton exhibited higher activity than those with the corresponding monomer. Their respective enantiomers [1' (IC 50 =1.9μM) and 2' (4.5μM)] and racemic mixtures [(±)-1 (2.2μM) and (±)-2 (4.4μM)] were found to exhibit melanogenesis inhibitory activities equivalent to those of the naturally occurring optical active compounds (1 and 2). Furthermore, the active compounds 1-3 inhibited tyrosinase, tyrosine-related protein (TRP)-1, and TRP-2 mRNA expressions, which could be the mechanism of melanogenesis inhibitory activity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Resistance to ursolic acid-induced apoptosis through involvement of melanogenesis and COX-2/PGE{sub 2} pathways in human M4Beu melanoma cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hassan, Lama; Pinon, Aline; Limami, Youness

Melanoma is one of the most aggressive forms of cancer with a continuously growing incidence worldwide and is usually resistant to chemotherapy agents, which is due in part to a strong resistance to apoptosis. Previously, we had showed that B16-F0 murine melanoma cells undergoing apoptosis are able to delay their own death induced by ursolic acid (UA), a natural pentacyclic triterpenoid compound. We had demonstrated that tyrosinase and TRP-1 up-regulation in apoptotic cells and the subsequent production of melanin were implicated in an apoptosis resistance mechanism. Several resistance mechanisms to apoptosis have been characterized in melanoma such as hyperactivation ofmore » DNA repair mechanisms, drug efflux systems, and reinforcement of survival signals (PI3K/Akt, NF-κB and Raf/MAPK pathways). Otherwise, other mechanisms of apoptosis resistance involving different proteins, such as cyclooxygenase-2 (COX-2), have been described in many cancer types. By using a strategy of specific inhibition of each ways, we suggested that there was an interaction between melanogenesis and COX-2/PGE{sub 2} pathway. This was characterized by analyzing the COX-2 expression and activity, the expression of tyrosinase and melanin production. Furthermore, we showed that anti-proliferative and proapoptotic effects of UA were mediated through modulation of multiple signaling pathways including Akt and ERK-1/2 proteins. Our study not only uncovers underlying molecular mechanisms of UA action in human melanoma cancer cells but also suggest its great potential as an adjuvant in treatment and cancer prevention.« less

Inhibition of Melanogenesis by Gallic Acid: Possible Involvement of the PI3K/Akt, MEK/ERK and Wnt/β-Catenin Signaling Pathways in B16F10 Cells

PubMed Central

Su, Tzu-Rong; Lin, Jen-Jie; Tsai, Chi-Chu; Huang, Tsu-Kei; Yang, Zih-Yan; Wu, Ming-O; Zheng, Yu-Qing; Su, Ching-Chyuan; Wu, Yu-Jen

2013-01-01

Gallic acid is one of the major flavonoids found in plants. It acts as an antioxidant, and seems to have anti-inflammatory, anti-viral, and anti-cancer properties. In this study, we investigated the effects of gallic acid on melanogenesis, including the activation of melanogenesis signaling pathways. Gallic acid significantly inhibited both melanin synthesis and tyrosinase activity in a dose- and time-dependent manner, and decreased the expression of melanogenesis-related proteins, such as microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP1), and dopachrome tautomerase (Dct). In addition, gallic acid also acts by phosphorylating and activating melanogenesis inhibitory proteins such as Akt and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK). Using inhibitors against PI3K/Akt (LY294002) or MEK/ERK-specific (PD98059), the hypopigmentation effect was suppressed, and the gallic acid-initiated activation of MEK/ERK and PI3K/Akt was also revoked. Gallic acid also increased GSK3β and p-β-catenin expression but down-regulated p-GSK3β. Moreover, GSK3β-specific inhibitor (SB216763) restored gallic acid-induced melanin reduction. These results suggest that activation of the MEK/ERK, PI3K/Akt, and inhibition of Wnt/β-catenin signaling pathways is involved in the melanogenesis signaling cascade, and that activation by gallic acid reduces melanin synthesis via down-regulation of MITF and its downstream signaling pathway. In conclusion, gallic acid may be a potentially agent for the treatment of certain skin conditions. PMID:24129178

Resveratrol-Enriched Rice Down-Regulates Melanin Synthesis in UVB-Induced Guinea Pigs Epidermal Skin Tissue

PubMed Central

Lee, Taek Hwan; Seo, Jae Ok; Do, Moon Ho; Ji, Eunhee; Baek, So-Hyeon; Kim, Sun Yeou

2014-01-01

Synthetic compounds that are used in the clinic to regulate skin hyperpigmentation, such as arbutin, hydroquinone, and kojic acid, are only moderately effective. But, their use is limited by side effects. As part of an effort to overcome the limitations, we developed resveratrol-enriched rice (RR) using genetic engineering technique. Each of resveratrol and rice has been reported to produce anti-melanogenic effects. Therefore, we hypothesized that RR would show more anti-melanogenic effects than those of resveratrol or rice alone. Anti-melanogenic effect of RR was done by using melan-a mouse melanocytes. The depigmenting efficacy was then observed following topical application of the RR to UVB-stimulated hyperpigmented dorsal skin of guinea pigs. Treatment with RR extract resulted a 21.4 ± 0.7% decrease in tyrosinase expression at melan-a cells. Colorimetric analysis showed a significantly lower depigmenting value by day 9 following treatment with RR in UVB-irradiated guinea pigs the dorsal skin (p<0.01), indicating that RR produced a depigmentation effect. By staining with Fontana-Masson stain, we found that the RR-treated group had more effect histopathologically in epidermal melanin production than resveratrol or rice alone-treated group. RR was associated with reduction in the levels of microphthalmia-associated transcription factor (MITF), and downregulation of tyrosinase and tyrosinase-related protein (TRP-2) expression, leading to inhibit epidermal melanin production by western blot analysis. This study suggests that the resveratrol-enriched rice may be a promising candidate in regulating skin pigmentation with UVB exposure. PMID:25414774

BLOC-2, AP-3, and AP-1 Proteins Function in Concert with Rab38 and Rab32 Proteins to Mediate Protein Trafficking to Lysosome-related Organelles*

PubMed Central

Bultema, Jarred J.; Ambrosio, Andrea L.; Burek, Carolyn L.; Di Pietro, Santiago M.

2012-01-01

Lysosome-related organelles (LROs) are synthesized in specialized cell types where they largely coexist with conventional lysosomes. Most of the known cellular transport machinery involved in biogenesis are ubiquitously expressed and shared between lysosomes and LROs. Examples of common components are the adaptor protein complex-3 (AP-3) and biogenesis of lysosome-related organelle complex (BLOC)-2. These protein complexes control sorting and transport of newly synthesized integral membrane proteins from early endosomes to both lysosomes and LROs such as the melanosome. However, it is unknown what factors cooperate with the ubiquitous transport machinery to mediate transport to LROs in specialized cells. Focusing on the melanosome, we show that the ubiquitous machinery interacts with cell type-specific Rab proteins, Rab38 and Rab32, to facilitate transport to the maturing organelle. BLOC-2, AP-3, and AP-1 coimmunoprecipitated with Rab38 and Rab32 from MNT-1 melanocytic cell extracts. BLOC-2, AP-3, AP-1, and clathrin partially colocalized with Rab38 and Rab32 by confocal immunofluorescence microscopy in MNT-1 cells. Rab38- and Rab32-deficient MNT-1 cells displayed abnormal trafficking and steady state levels of known cargoes of the BLOC-2, AP-3, and AP-1 pathways, the melanin-synthesizing enzymes tyrosinase and tyrosinase-related protein-1. These observations support the idea that Rab38 and Rab32 are the specific factors that direct the ubiquitous machinery to mediate transport from early endosomes to maturing LROs. Additionally, analysis of tyrosinase-related protein-2 and total melanin production indicates that Rab32 has unique functions that cannot be carried out by Rab38 in melanosome biogenesis. PMID:22511774

BLOC-2, AP-3, and AP-1 proteins function in concert with Rab38 and Rab32 proteins to mediate protein trafficking to lysosome-related organelles.

PubMed

Bultema, Jarred J; Ambrosio, Andrea L; Burek, Carolyn L; Di Pietro, Santiago M

2012-06-01

Lysosome-related organelles (LROs) are synthesized in specialized cell types where they largely coexist with conventional lysosomes. Most of the known cellular transport machinery involved in biogenesis are ubiquitously expressed and shared between lysosomes and LROs. Examples of common components are the adaptor protein complex-3 (AP-3) and biogenesis of lysosome-related organelle complex (BLOC)-2. These protein complexes control sorting and transport of newly synthesized integral membrane proteins from early endosomes to both lysosomes and LROs such as the melanosome. However, it is unknown what factors cooperate with the ubiquitous transport machinery to mediate transport to LROs in specialized cells. Focusing on the melanosome, we show that the ubiquitous machinery interacts with cell type-specific Rab proteins, Rab38 and Rab32, to facilitate transport to the maturing organelle. BLOC-2, AP-3, and AP-1 coimmunoprecipitated with Rab38 and Rab32 from MNT-1 melanocytic cell extracts. BLOC-2, AP-3, AP-1, and clathrin partially colocalized with Rab38 and Rab32 by confocal immunofluorescence microscopy in MNT-1 cells. Rab38- and Rab32-deficient MNT-1 cells displayed abnormal trafficking and steady state levels of known cargoes of the BLOC-2, AP-3, and AP-1 pathways, the melanin-synthesizing enzymes tyrosinase and tyrosinase-related protein-1. These observations support the idea that Rab38 and Rab32 are the specific factors that direct the ubiquitous machinery to mediate transport from early endosomes to maturing LROs. Additionally, analysis of tyrosinase-related protein-2 and total melanin production indicates that Rab32 has unique functions that cannot be carried out by Rab38 in melanosome biogenesis.

Aminotryptophan-containing barstar: structure--function tradeoff in protein design and engineering with an expanded genetic code.

PubMed

Rubini, Marina; Lepthien, Sandra; Golbik, Ralph; Budisa, Nediljko

2006-07-01

The indole ring of the canonical amino acid tryptophan (Trp) possesses distinguished features, such as sterical bulk, hydrophobicity and the nitrogen atom which is capable of acting as a hydrogen bond donor. The introduction of an amino group into the indole moiety of Trp yields the structural analogs 4-aminotryptophan ((4-NH(2))Trp) and 5-aminotryptophan ((5-NH(2))Trp). Their hydrophobicity and spectral properties are substantially different when compared to those of Trp. They resemble the purine bases of DNA and share their capacity for pH-sensitive intramolecular charge transfer. The Trp --> aminotryptophan substitution in proteins during ribosomal translation is expected to result in related protein variants that acquire these features. These expectations have been fulfilled by incorporating (4-NH(2))Trp and (5-NH(2))Trp into barstar, an intracellular inhibitor of the ribonuclease barnase from Bacillus amyloliquefaciens. The crystal structure of (4-NH(2))Trp-barstar is similar to that of the parent protein, whereas its spectral and thermodynamic behavior is found to be remarkably different. The T(m) value of (4-NH(2))Trp- and (5-NH(2))Trp-barstar is lowered by about 20 degrees Celsius, and they exhibit a strongly reduced unfolding cooperativity and substantial loss of free energy in folding. Furthermore, folding kinetic study of (4-NH(2))Trp-barstar revealed that the denatured state is even preferred over native one. The combination of structural and thermodynamic analyses clearly shows how structures of substituted barstar display a typical structure-function tradeoff: the acquirement of unique pH-sensitive charge transfer as a novel function is achieved at the expense of protein stability. These findings provide a new insight into the evolution of the amino acid repertoire of the universal genetic code and highlight possible problems regarding protein engineering and design by using an expanded genetic code.

Quercetin-3-O-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranoside suppresses melanin synthesis by augmenting p38 MAPK and CREB signaling pathways and subsequent cAMP down-regulation in murine melanoma cells

PubMed Central

Jung, Hyun Gug; Kim, Han Hyuk; Paul, Souren; Jang, Jae Yoon; Cho, Yong Hun; Kim, Hyeon Jeong; Yu, Jae Myo; Lee, Eun Su; An, Bong Jeun; Kang, Sun Chul; Bang, Byung Ho

2015-01-01

In this study, the effect of purified quercetin-3-O-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosid (QCGG) on melanogenesis was investigated. QCGG was isolated from the calyx of a traditional Korean medicinal herb, Persimmon (Diospyros kaki). The hypopigmentation effects of QCGG were determined by examination of cellular melanin contents, tyrosinase activity assay, cAMP assay, and Western blotting of α-MSH-stimulated B16F10 mouse melanoma cells. Our results showed that QCGG inhibited both melanin synthesis and tyrosinase activity in a concentration-dependent manner as well as significantly reduced the expression of melanogenic proteins such as microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1, tyrosinase-related protein-2, and tyrosinase. Moreover, QCGG inhibited intracellular cAMP levels, cAMP response element-binding protein (CREB), and p38 MAPK expression in α-MSH-stimulated B16F10 cells. Taken together, the suppressive effects of QCGG on melanogenesis may involve down-regulation of MITF and its downstream signaling pathway via phosphorylation of p38 MAPK and CREB along with reduced cAMP levels. These results indicate that QCGG reduced melanin synthesis by reducing expression of tyrosine and tyrosine-related proteins via extracellular signal-related protein kinase (ERK) activation, followed by down-regulation of CREB, p38, and MITF. PMID:26586997

Quercetin-3-O-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranoside suppresses melanin synthesis by augmenting p38 MAPK and CREB signaling pathways and subsequent cAMP down-regulation in murine melanoma cells.

PubMed

Jung, Hyun Gug; Kim, Han Hyuk; Paul, Souren; Jang, Jae Yoon; Cho, Yong Hun; Kim, Hyeon Jeong; Yu, Jae Myo; Lee, Eun Su; An, Bong Jeun; Kang, Sun Chul; Bang, Byung Ho

2015-11-01

In this study, the effect of purified quercetin-3-O-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosid (QCGG) on melanogenesis was investigated. QCGG was isolated from the calyx of a traditional Korean medicinal herb, Persimmon (Diospyros kaki). The hypopigmentation effects of QCGG were determined by examination of cellular melanin contents, tyrosinase activity assay, cAMP assay, and Western blotting of α-MSH-stimulated B16F10 mouse melanoma cells. Our results showed that QCGG inhibited both melanin synthesis and tyrosinase activity in a concentration-dependent manner as well as significantly reduced the expression of melanogenic proteins such as microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1, tyrosinase-related protein-2, and tyrosinase. Moreover, QCGG inhibited intracellular cAMP levels, cAMP response element-binding protein (CREB), and p38 MAPK expression in α-MSH-stimulated B16F10 cells. Taken together, the suppressive effects of QCGG on melanogenesis may involve down-regulation of MITF and its downstream signaling pathway via phosphorylation of p38 MAPK and CREB along with reduced cAMP levels. These results indicate that QCGG reduced melanin synthesis by reducing expression of tyrosine and tyrosine-related proteins via extracellular signal-related protein kinase (ERK) activation, followed by down-regulation of CREB, p38, and MITF.

Antioxidant and Anti-tyrosinase Activities of Phenolic Extracts from Rape Bee Pollen and Inhibitory Melanogenesis by cAMP/MITF/TYR Pathway in B16 Mouse Melanoma Cells.

PubMed

Sun, Liping; Guo, Yan; Zhang, Yanxin; Zhuang, Yongliang

2017-01-01

Rape bee pollen possesses many nutritional and therapeutic properties because of its abundant nutrimental and bioactive components. In this study, free (FPE) and bound (BPE) phenolic extracts of rape bee pollen were obtained, phenolic and flavonoid contents were determined, and composition of phenolic acids was analyzed. In vitro antioxidant and anti-tyrosinase (TYR) activities of FPE and BPE were compared, and inhibitory melanogenesis of FPE was further evaluated. Results showed FPE and BPE contain total phenolic contents of 11.76 and 0.81 mg gallic acid equivalents/g dry weight (DW) and total flavonoid contents of 19.24 and 3.65 mg rutin equivalents/g DW, respectively. Phenolic profiling showed FPE and BPE fractions contained 12 and 9 phenolic acids, respectively. FPE contained the highest rutin content of 774.87 μg/g. FPE and BPE showed the high antioxidant properties in vitro and high inhibitory activities for mushroom TYR. Higher activities of FPE than those of BPE can be attributed to difference in their phenolic compositions. Inhibitory melanogenesis activities of FPE against B16 were further evaluated. Results showed suppressed intracellular TYR activity, reduced melanin content, and promoted glutathione synthesis ( p < 0.05) in FPE-treated cells. FPE reduced mRNA expression of TYR, TYR-related protein (TRP)-1 and TRP-2, and significantly suppressed cyclic adenosine monophosphate (cAMP) levels through down-regulation of melanocortin 1 receptor gene expression ( p < 0.05). FPE reduced mRNA expression of microphthalmia-associated transcription factor (MITF), significantly inhibiting intracellular melanin synthesis ( p < 0.05). Hence, FPE regulates melanogenesis of B16 cells involved in cAMP/MITF/TYR pathway. These results revealed that FPE can be used as pharmaceutical agents and cosmetics to protect cells from abnormal melanogenesis.

The expression of KRT2 and its effect on melanogenesis in alpaca skins.

PubMed

Cui, Yucong; Song, Yajun; Geng, Qingling; Ding, Zengfeng; Qin, Yilong; Fan, Ruiwen; Dong, Changsheng; Geng, Jianjun

2016-06-01

In order to investigate the effects of the keratin 2 (KRT2) on alpaca melanocyte in vivo and vitro, the immunohistochemistry (IHC), quantitative real-time PCR (qPCR), Western blot, and alpaca melanocytes transfection methods were used. The results showed that mRNA and protein expression of KRT2 was highly expressed in brown skin in comparison with that in white skin. Moreover, we found that KRT2 was expressed in alpaca melanocytes in vitro by immunocytochemistry. After transfection with KRT2 in alpaca melanocytes, the relative mRNA and protein expression of KRT2, microphthalmia-associtated transcription factor (MITF), tyrosinase (TYR) and tyrosinase-related protein 1 (TYRP1) in alpaca skin melanocytes was increased with significant differences; a further result was the increase of melanin production. The results suggested that KRT2 functions in alpaca hair color formation, which offered an essential theoretical basis for further exploration of the role of melanogenesis. Copyright © 2016 Elsevier GmbH. All rights reserved.

5-Fluoroindole Resistance Identifies Tryptophan Synthase Beta Subunit Mutants in Arabidopsis Thaliana

PubMed Central

Barczak, A. J.; Zhao, J.; Pruitt, K. D.; Last, R. L.

1995-01-01

A study of the biochemical genetics of the Arabidopsis thaliana tryptophan synthase beta subunit was initiated by characterization of mutants resistant to the inhibitor 5-fluoroindole. Thirteen recessive mutations were recovered that are allelic to trp2-1, a mutation in the more highly expressed of duplicate tryptophan synthase beta subunit genes (TSB1). Ten of these mutations (trp2-2 through trp2-11) cause a tryptophan requirement (auxotrophs), whereas three (trp2-100 through trp2-102) remain tryptophan prototrophs. The mutations cause a variety of changes in tryptophan synthase beta expression. For example, two mutations (trp2-5 and trp2-8) cause dramatically reduced accumulation of TSB mRNA and immunologically detectable protein, whereas trp2-10 is associated with increased mRNA and protein. A correlation exists between the quantity of mutant beta and wild-type alpha subunit levels in the trp2 mutant plants, suggesting that the synthesis of these proteins is coordinated or that the quantity or structure of the beta subunit influences the stability of the alpha protein. The level of immunologically detectable anthranilate synthase alpha subunit protein is increased in the trp2 mutants, suggesting the possibility of regulation of anthranilate synthase levels in response to tryptophan limitation. PMID:7635295

Molecular Cloning and Characteristic Features of a Novel Extracellular Tyrosinase from Aspergillus niger PA2.

PubMed

Agarwal, Pragati; Singh, Jyoti; Singh, R P

2017-05-01

Aspergillus niger PA2, a novel strain isolated from waste effluents of food industry, is a potential extracellular tyrosinase producer. Enzyme activity and L-DOPA production were maximum when glucose and peptone were employed as C source and nitrogen source respectively in the medium and enhanced notably when the copper was supplemented, thus depicting the significance of copper in tyrosinase activity. Tyrosinase-encoding gene from the fungus was cloned, and amplification of the tyrosinase gene yielded a 1127-bp DNA fragment and 374 amino acid residue long product that encoded for a predicted protein of 42.3 kDa with an isoelectric point of 4.8. Primary sequence analysis of A. niger PA2 tyrosinase had shown that it had approximately 99% identity with that of A. niger CBS 513.88, which was further confirmed by phylogenetic analysis. The inferred amino acid sequence of A. niger tyrosinase contained two putative copper-binding sites comprising of six histidines, a characteristic feature for type-3 copper proteins, which were highly conserved in all tyrosinases throughout the Aspergillus species. When superimposed onto the tertiary structure of A. oryzae tyrosinase, the conserved residues from both the organisms occupied same spatial positions to provide a di-copper-binding peptide groove.

Inhibitory effect of N-adamantyl-3,4-dihydroxybenzamide on melanogenesis in melan-a cells and brown guinea pigs.

PubMed

Rho, Ho Sik; Ahn, Soo Mi; Hwang, Jae Sung

2011-04-01

To find novel depigmenting agents, a new synthetic compound, N-adamantyl-3,4-dihydroxybenzamide (NADB) was produced and the effects on melanogenesis were investigated. Our results showed that NADB reduced melanin synthesis in a dose-dependent manner in melan-a cells. Tyrosinase activity was also reduced by NADB treatment in melan-a cells. However, NADB did not inhibit tyrosinase activity directly in a cell-free system. Treatment of melan-a cells with NADB caused a marked decrease in protein and mRNA levels of tyrosinase along with tyrosinase-related protein 1 and dopachrome tautomerase. To determine whether NADB reduces skin pigmentation, the dorsal skin of brown guinea pigs was shaved and irradiated with UV for 3 weeks using a solar simulator. Then NADB (2 or 1% in propylene glycol:ethanol:water = 5:3:2) was applied topically twice daily for 4 weeks. Visual inspection and Fontana-Masson staining both demonstrated that NADB resulted in lower skin pigmentation and total epidermal melanin in comparison to vehicle-treated areas. These findings suggest that NADB is useful in the treatment of hyperpigmentation.

TRP channel proteins and signal transduction.

PubMed

Minke, Baruch; Cook, Boaz

2002-04-01

TRP channel proteins constitute a large and diverse family of proteins that are expressed in many tissues and cell types. This family was designated TRP because of a spontaneously occurring Drosophila mutant lacking TRP that responded to a continuous light with a transient receptor potential (hence TRP). In addition to responses to light, TRPs mediate responses to nerve growth factor, pheromones, olfaction, mechanical, chemical, temperature, pH, osmolarity, vasorelaxation of blood vessels, and metabolic stress. Furthermore, mutations in several members of TRP-related channel proteins are responsible for several diseases, such as several tumors and neurodegenerative disorders. TRP-related channel proteins are found in a variety of organisms, tissues, and cell types, including nonexcitable, smooth muscle, and neuronal cells. The large functional diversity of TRPs is also reflected in their diverse permeability to ions, although, in general, they are classified as nonselective cationic channels. The molecular domains that are conserved in all members of the TRP family constitute parts of the transmembrane domains and in most members also the ankyrin-like repeats at the NH2 terminal of the protein and a "TRP domain" at the COOH terminal, which is a highly conserved 25-amino acid stretch with still unknown function. All of the above features suggest that members of the TRP family are "special assignment" channels, which are recruited to diverse signaling pathways. The channels' roles and characteristics such as gating mechanism, regulation, and permeability are determined by evolution according to the specific functional requirements.

Alternol inhibits the proliferation and induces the differentiation of the mouse melanoma B16F0 cell line.

PubMed

Wang, Caixia; Xu, Wenjuan; Hao, Wenjin; Wang, Bingsheng; Zheng, Qiusheng

2016-08-01

High malignant potential and low susceptibility to treatment are characteristics of malignant melanoma. Alternol, a novel compound purified from microbial fermentation products obtained from the bark of the yew tree, exhibits a variety of antitumor activities. Based on these findings, the aim of the present study was to extend the knowledge on the antineoplastic effect of alternol in the mouse melanoma B16F0 cell line. Alternol significantly inhibited the proliferation and colony formation of B16F0 cells in a dose-dependent manner as detected by MTT and soft agar colony formation assays. NaOH alkaline lysis and oxidation of Dopa indicated that alternol enhanced the melanin content and tyrosinase activity of the B16F0 cells and results also showed a dose‑response relationship. Morphologic changes accompanied by extended dendrites were discovered in the B16F0 cells after treatment with alternol. Furthermore, the mRNA levels of tyrosinase, Trp1 and Trp2 were increased by alternol. Our results confirmed that alternol possesses marked antineoplastic properties against melanoma cells, indicating that this microbial fermentation product is a promising agent for the differentiation therapy of cancer. The inhibition of cell proliferation and colony formation by alternol was associated with both cytotoxicity and induction of differentiation.

Tryptophan metabolism, disposition and utilization in pregnancy.

PubMed

Badawy, Abdulla A-B

2015-09-17

Tryptophan (Trp) requirements in pregnancy are several-fold: (1) the need for increased protein synthesis by mother and for fetal growth and development; (2) serotonin (5-HT) for signalling pathways; (3) kynurenic acid (KA) for neuronal protection; (4) quinolinic acid (QA) for NAD(+) synthesis (5) other kynurenines (Ks) for suppressing fetal rejection. These goals could not be achieved if maternal plasma [Trp] is depleted. Although plasma total (free + albumin-bound) Trp is decreased in pregnancy, free Trp is elevated. The above requirements are best expressed in terms of a Trp utilization concept. Briefly, Trp is utilized as follows: (1) In early and mid-pregnancy, emphasis is on increased maternal Trp availability to meet the demand for protein synthesis and fetal development, most probably mediated by maternal liver Trp 2,3-dioxygenase (TDO) inhibition by progesterone and oestrogens. (2) In mid- and late pregnancy, Trp availability is maintained and enhanced by the release of albumin-bound Trp by albumin depletion and non-esterified fatty acid (NEFA) elevation, leading to increased flux of Trp down the K pathway to elevate immunosuppressive Ks. An excessive release of free Trp could undermine pregnancy by abolishing T-cell suppression by Ks. Detailed assessment of parameters of Trp metabolism and disposition and related measures (free and total Trp, albumin, NEFA, K and its metabolites and pro- and anti-inflammatory cytokines in maternal blood and, where appropriate, placental and fetal material) in normal and abnormal pregnancies may establish missing gaps in our knowledge of the Trp status in pregnancy and help identify appropriate intervention strategies. © 2015 Authors.

Tryptophan metabolism, disposition and utilization in pregnancy

PubMed Central

Badawy, Abdulla A.-B.

2015-01-01

Tryptophan (Trp) requirements in pregnancy are several-fold: (1) the need for increased protein synthesis by mother and for fetal growth and development; (2) serotonin (5-HT) for signalling pathways; (3) kynurenic acid (KA) for neuronal protection; (4) quinolinic acid (QA) for NAD+ synthesis (5) other kynurenines (Ks) for suppressing fetal rejection. These goals could not be achieved if maternal plasma [Trp] is depleted. Although plasma total (free + albumin-bound) Trp is decreased in pregnancy, free Trp is elevated. The above requirements are best expressed in terms of a Trp utilization concept. Briefly, Trp is utilized as follows: (1) In early and mid-pregnancy, emphasis is on increased maternal Trp availability to meet the demand for protein synthesis and fetal development, most probably mediated by maternal liver Trp 2,3-dioxygenase (TDO) inhibition by progesterone and oestrogens. (2) In mid- and late pregnancy, Trp availability is maintained and enhanced by the release of albumin-bound Trp by albumin depletion and non-esterified fatty acid (NEFA) elevation, leading to increased flux of Trp down the K pathway to elevate immunosuppressive Ks. An excessive release of free Trp could undermine pregnancy by abolishing T-cell suppression by Ks. Detailed assessment of parameters of Trp metabolism and disposition and related measures (free and total Trp, albumin, NEFA, K and its metabolites and pro- and anti-inflammatory cytokines in maternal blood and, where appropriate, placental and fetal material) in normal and abnormal pregnancies may establish missing gaps in our knowledge of the Trp status in pregnancy and help identify appropriate intervention strategies. PMID:26381576

Secretion of the Streptomyces tyrosinase is mediated through its trans-activator protein, MelC1.

PubMed

Leu, W M; Chen, L Y; Liaw, L L; Lee, Y H

1992-10-05

The tyrosinase of Streptomyces antibioticus is encoded by the second open reading frame, melC2 of the melanin operon (melC). The upstream open reading frame melC1 specifies a 146-amino acid protein with a typical NH2-terminal signal-peptide characteristic of a secretory protein. The MelC1 protein is involved in the transfer of copper ion to apotyrosinase MelC2 via binary complex formation (Lee, Y.-H. W., Chen, B.-F., Wu, S.-Y., Leu, W.-M., Lin, J.-J., Chen, C. W., and Lo, S. J. (1988) Gene (Amst.) 65, 71-81; Chen, L.-Y., Leu, W.-M., Wang, K.-T., and Lee, Y.-H.W. (1992) J. Biol. Chem. 267, 20100-20107). To investigate whether the export of tyrosinase is also dependent on MelC1, a mutational study of its signal-peptide sequence was performed. Four different mutants were obtained. Mutation at the positively charged region (mutant M-6LE, Arg6-Arg7----Leu6-Glu7) or the hydrophobic region (mutant M-16D, Val16----Asp16) led to Mel- phenotypes. These lesions caused a severe 7-10-fold reduction of the export of both the MelC1 and MelC2 proteins and a concomitant accumulation of the two proteins in the cytosolic fraction. The cell-associated tyrosinase activity in M-6LE but not in the M-16D mutant was dramatically reduced to 4% of the activity found in the wild type strain, suggesting that the basic NH2 terminus of MelC1 is also important for the trans-activation function of this protein. Nevertheless, the defects on the trans-activation and/or secretory functions of MelC1 in mutants M-6LE and M-16D are not due to the impairment of the formation of the MelC1.MelC2 complex. The translation of melanin operon genes in these two mutants also decreased. In contrast, the tyrosinase activity and the secretion of MelC2 were not affected if the mutations occurred at the putative cleavage site of the signal peptidase (e.g. mutant M-29SM, Arg29-Ala30----Ser29-Met30 or mutant 29-SMG, Arg29-Ala30-Asp31----Ser29-Med30-Gly31+ ++). Additionally, tyrosinase activity and its export were abolished in a MelC1-negative mutant, M-950. Taken together, these results demonstrate that a functional MelC1 is essential for tyrosinase secretion and activity. Furthermore, the results suggest that like other secretory proteins, basic and hydrophobic residues in the MelC1 signal sequence are an important feature of the signal-peptide and play a pivotal role in the secretion of both the MelC1 and MelC2 proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

The etiology of oculocutaneous albinism (OCA) type II: the pink protein modulates the processing and transport of tyrosinase.

PubMed

Toyofuku, Kazutomo; Valencia, Julio C; Kushimoto, Tsuneto; Costin, Gertrude-E; Virador, Victoria M; Vieira, Wilfred D; Ferrans, Victor J; Hearing, Vincent J

2002-06-01

Oculocutaneous albinism (OCA) is caused by reduced or deficient melanin pigmentation in the skin, hair, and eyes. OCA has different phenotypes resulting from mutations in distinct pigmentation genes involved in melanogenesis. OCA type 2 (OCA2), the most common form of OCA, is an autosomal recessive disorder caused by mutations in the P gene, the function(s) of which is controversial. In order to elucidate the mechanism(s) involved in OCA2, our group used several antibodies specific for various melanosomal proteins (tyrosinase, Tyrp1, Dct, Pmel17 and HMB45), including a specific set of polyclonal antibodies against the p protein. We used confocal immunohistochemistry to compare the processing and distribution of those melanosomal proteins in wild type (melan-a) and in p mutant (melan-p1) melanocytes. Our results indicate that the melanin content of melan-p1 melanocytes was less than 50% that of wild type melan-a melanocytes. In contrast, the tyrosinase activities were similar in extracts of wild type and p mutant melanocytes. Confocal microscopy studies and pulse-chase analyses showed altered processing and sorting of tyrosinase, which is released from melan-p1 cells to the medium. Processing and sorting of Tyrp1 was also altered to some extent. However, Dct and Pmel17 expression and subcellular localization were similar in melan-a and in melan-p1 melanocytes. In melan-a cells, the p protein showed mainly a perinuclear pattern with some staining in the cytoplasm where some co-localization with HMB45 antibody was observed. These findings suggest that the p protein plays a major role in modulating the intracellular transport of tyrosinase and a minor role for Tyrp1, but is not critically involved in the transport of Dct and Pmel17. This study provides a basis to understand the relationship of the p protein with tyrosinase function and melanin synthesis, and also provides a rational approach to unveil the consequences of P gene mutations in the pathogenesis of OCA2.

Characterization of two trpE genes encoding anthranilate synthase {alpha}-subunit in Azospirillum brasilense

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ge Shimei; Xie Baoen; Chen Sanfeng

2006-03-10

The previous report from our laboratory has recently identified a new trpE gene (termed trpE {sub 2}) which exists independently in Azospirillum brasilense Yu62. In this study, amplification of trpE(G) (termed trpE {sub 1}(G) here) confirmed that there are two copies of trpE gene, one trpE being fused into trpG while the other trpE existed independently. This is First report to suggest that two copies of the trpE gene exist in this bacterium. Comparison of the nucleotide sequence demonstrated that putative leader peptide, terminator, and anti-terminator were found upstream of trpE {sub 1}(G) while these sequence features did not existmore » in front of trpE {sub 2}. The {beta}-galactosidase activity of an A. brasilense strain carrying a trpE {sub 2}-lacZ fusion remained constant at different tryptophan concentrations, but the {beta}-galactosidase activity of the same strain carrying a trpE {sub 1}(G)-lacZ fusion decreased as the tryptophan concentration increased. These data suggest that the expression of trpE {sub 1}(G) is regulated at the transcriptional level by attenuation while trpE {sub 2} is constantly expressed. The anthranilate synthase assays with trpE {sub 1}(G){sup -} and trpE {sub 2} {sup -} mutants demonstrated that TrpE{sub 1}(G) fusion protein is feedback inhibited by tryptophan while TrpE{sub 2} protein is not. We also found that both trpE {sub 1}(G) and trpE {sub 2} gene products were involved in IAA synthesis.« less

Functions of Adaptor Protein (AP)-3 and AP-1 in Tyrosinase Sorting from Endosomes to MelanosomesD⃞

PubMed Central

Theos, Alexander C.; Tenza, Danièle; Martina, José A.; Hurbain, Ilse; Peden, Andrew A.; Sviderskaya, Elena V.; Stewart, Abigail; Robinson, Margaret S.; Bennett, Dorothy C.; Cutler, Daniel F.; Bonifacino, Juan S.; Marks, Michael S.; Raposo, Graça

2005-01-01

Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies. PMID:16162817

(Z)-5-(2,4-Dihydroxybenzylidene)thiazolidine-2,4-dione Prevents UVB-Induced Melanogenesis and Wrinkle Formation through Suppressing Oxidative Stress in HRM-2 Hairless Mice

PubMed Central

Lee, Bonggi; Moon, Kyoung Mi; Kim, Seong Jin; Kim, So Hee; Kim, Dae Hyun; An, Hye Jin; Jeong, Ji Won; Kim, Ye Ra; Son, Sujin; Kim, Min Jo; Chung, Ki Wung; Lee, Eun Kyeong; Chun, Pusoon; Ha, Young Mi; Kim, Min-Sun; Mo, Sang Hyun; Moon, Hyung Ryong; Chung, Hae Young

2016-01-01

Background. Uncontrolled melanogenesis and wrinkle formation are an indication of photoaging. Our previous studies demonstrated that (Z)-5-(2,4-dihydroxybenzylidene)thiazolidine-2,4-dione (MHY498) inhibited tyrosinase activity and melanogenesis in vitro. Objective. To examine in vivo effects of MHY498 as an antiaging compound on UVB-induced melanogenesis and wrinkle formation, we topically applied MHY498 on dorsal skin of HRM-2 hairless mice. Methods. Using histological analysis, we evaluated effects of MHY498 on melanogenesis and wrinkle formation after UVB exposure. In addition, related molecular signaling pathways were examined using western blotting, fluorometric assay, and enzyme-linked immunosorbent assay. Results. MHY498 suppressed UVB-induced melanogenesis by inhibiting phosphorylation of CREB and translocation of MITF protein into the nucleus, which are key factors for tyrosinase expression. Consistently, tyrosinase protein levels were notably reduced in the dorsal skin of the hairless mice by MHY498 treatment. Furthermore, MHY498 inhibited UVB-induced wrinkle formation and collagen fiber destruction by increasing type 1 procollagen concentration and decreasing protein expression levels of MMPs, which play an essential role in collagen fiber degradation. As a mechanism, MHY498 notably ameliorated UVB-induced oxidative stress and NF-κB activation in the dermal skin of the hairless mice. Conclusion. Our study suggests that MHY498 can be used as a therapeutic or cosmetic agent for preventing uncontrolled melanogenesis and wrinkle formation. PMID:27242917

A conserved WW domain-like motif regulates invariant chain-dependent cell-surface transport of the NKG2D ligand ULBP2.

PubMed

Uhlenbrock, Franziska; van Andel, Esther; Andresen, Lars; Skov, Søren

2015-08-01

Malignant cells expressing NKG2D ligands on their cell surface can be directly sensed and killed by NKG2D-bearing lymphocytes. To ensure this immune recognition, accumulating evidence suggests that NKG2D ligands are trafficed via alternative pathways to the cell surface. We have previously shown that the NKG2D ligand ULBP2 traffics over an invariant chain (Ii)-dependent pathway to the cell surface. This study set out to elucidate how Ii regulates ULBP2 cell-surface transport: We discovered conserved tryptophan (Trp) residues in the primary protein sequence of ULBP1-6 but not in the related MICA/B. Substitution of Trp to alanine resulted in cell-surface inhibition of ULBP2 in different cancer cell lines. Moreover, the mutated ULBP2 constructs were retained and not degraded inside the cell, indicating a crucial role of this conserved Trp-motif in trafficking. Finally, overexpression of Ii increased surface expression of wt ULBP2 while Trp-mutants could not be expressed, proposing that this Trp-motif is required for an Ii-dependent cell-surface transport of ULBP2. Aberrant soluble ULBP2 is immunosuppressive. Thus, targeting a distinct protein module on the ULBP2 sequence could counteract this abnormal expression of ULBP2. Copyright © 2015 Elsevier Ltd. All rights reserved.

The depigmenting effect of natural resorcinol type polyphenols Kuwanon O and Sanggenon T from the roots of morus australis.

PubMed

Hu, Shuting; Zheng, Zongping; Chen, Feng; Wang, Mingfu

2017-01-04

Morus australis, one of the major Morus species growing in East Asia, is rich in phenolic compounds. The extract of M. australis has been used as skin whitening components for a long period. The action mechanisms of its principal constituents are still unclear. This study aims to evaluate the skin lightening effects of phenolic compounds extracted from the root of M. australis in different melanocyte systems and artificial skin models. The depigmenting effect of resorcinol type polyphenols (RTPs) from the root extract of M. australis was evaluated in murine b16 and melan-a cell lines using a combined sulforhodamine B assay. Tyrosinase activity and the expression of melanogenesis proteins were evaluated for the mechanism study. The artificial skin model is used as a replacement of the animal test. Only Kuwanon O and Sanggenon T were found to have significant depigmenting effects in both murine b16 and melan-a cell lines. Their depigmenting mechanisms are slightly different in the two cell systems. In b16 cells, Kuwanon O and Sanggenon T, together with the other two RTPs, induced post-transcriptional degradations of MITF without suppressing its mRNA expression, leading to significant decreases of TRP-1 and TRP-2 production. While in melan-a cells, the levels of tyrosinase families were suppressed via MITF downregulation at both transcription and translation level by RTPs, with Kuwanon O inducing the greatest suppression. Further evaluations in artificial skin model demonstrated the outstanding depigmenting effects of Kuwanon O and Sanggenon T. Kuwanon O and Sanggenon T from M.australis root extract are two potential skin whitening ingredients. To screen resorcinol flavonone derivatives with an isoprenyl group in the Diels-Alder substituent might be an option for the search of potent hypopigmenting agents from plants. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Pigmented-MDCK (P-MDCK) Cell Line with Tunable Melanin Expression: An In Vitro Model for the Outer Blood-Retinal-Barrier

PubMed Central

Kadam, Rajendra S.; Scheinman, Robert. I.; Kompella, Uday B.

2013-01-01

Purpose Retinal pigment epithelium, which forms the outer blood-retinal-barrier, is a critical barrier for transport of drugs to the retina. The purpose of this study was to develop a pigmented MDCK (P-MDCK) cell line as a rapidly established in vitro model for the outer blood-retinal-barrier to assess the influence of melanin pigment on solute permeability. Methods A melanin synthesizing P-MDCK cell line was developed by lentiviral transduction of human tyrosinase and p-protein genes in MDCK (NBL-2) cells. Melanin content, tyrosinase activity (conversion of L-dopa to dopachrome), and transepithelial electrical resistance (TEER) were measured. Expression of tyrosinase protein and p-protein in P-MDCK cells was confirmed by confocal microscopy. Effect of L-tyrosine (0 to 2 mM) in culture medium on melanin synthesis in P-MDCK cells was evaluated. Cell uptake and transepithelial transport of pigment-binding chloroquine (Log D = 1.59) and a negative control salicylic acid (Log D = −1.14) were investigated. Results P-MDCK cells expressed tyrosinase and p-protein. Tyrosinase activity was 4.5 fold higher in P-MDCK cells as compared to wild-type MDCK cells. The transepithelial electrical resistance stabilized by day 4 in both cell types, with the TEER being 871 ± 30 and 876 ± 53 Ω.cm2 for P-MDCK and wild-type cells, respectively. Melanin content in P-MDCK cells depended on the concentration of L-tyrosine in culture medium, and increased from 3 to 54 µg/mg protein with an increase in L-tyrosine content from 0 to 2 mM. When the cells were grown in 2 mM L-tyrosine, uptake of chloroquine was 2.3 fold higher and the transepithelial transport was 2.2 fold lower in P-MDCK cells when compared to wild-type MDCK cells. No significant difference was observed for both cell uptake and transport of salicylic acid. Conclusions We developed a P-MDCK cell line with tunable melanin synthesis as a rapidly developing surrogate for retinal pigment epithelium. PMID:23003570

Synthesis, computational studies and enzyme inhibitory kinetics of substituted methyl[2-(4-dimethylamino-benzylidene)-hydrazono)-4-oxo-thiazolidin-5-ylidene]acetates as mushroom tyrosinase inhibitors.

PubMed

Channar, Pervaiz Ali; Saeed, Aamer; Larik, Fayaz Ali; Rafiq, Muhammad; Ashraf, Zaman; Jabeen, Farukh; Fattah, Tanzeela Abdul

2017-11-01

The present article describes the synthesis and enzyme inhibitory kinetics of methyl[2-(arylmethylene-hydrazono)-4-oxo-thiazolidin-5-ylidene]acetates 5a-j as mushroom tyrosinase inhibitors. The title compounds were synthesized via cyclocondensation of thiosemicarbazones 3a-j with dimethyl but-2-ynedioate (DMAD) 4 in good yields under solvent-free conditions. The synthesized compounds were evaluated for their potential to inhibit the activity of mushroom tyrosinase. It was unveiled that compounds 5i showed excellent enzyme inhibitory activity with IC 50 3.17µM while IC 50 of standard kojic acid is 15.91µM. The presence of heterocyclic pyridine ring in compound 5i play important role in enzyme inhibitory activity as rest of the functional groups are common in all synthesized compounds. The enzyme inhibitory kinetics of the most potent derivative 5i determined by Lineweaver-Burk plots and Dixon plots showed that it is non-competitive inhibitor with Ki value 1.5µM. It was further investigated that the wet lab results are in good agreement with the computational results. The molecular docking of the synthesized compounds was performed against tyrosinase protein (PDBID 2Y9X) to delineate ligand-protein interactions at molecular level. The docking results showed that the major interacting residues are His244, His85, His263, Val 283, His 296, Asn260, Val248, His260, His261 and Phe264 which are located in active binding site of the protein. The molecular modeling demonstrates that the oxygen atom of the compound 5i coordinated with the key residues in the active site of mushroom tyrosinase contribute significantly against inhibitory ability and diminishing the human melanin synthesis. These results evident that compound 5i is a lead structure in developing most potent mushroom tyrosinase inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Replication-Competent Foamy Virus Vaccine Vectors as Novel Epitope Scaffolds for Immunotherapy

PubMed Central

Lei, Janet; Osen, Wolfram; Gardyan, Adriane; Hotz-Wagenblatt, Agnes; Wei, Guochao; Gissmann, Lutz; Eichmüller, Stefan; Löchelt, Martin

2015-01-01

The use of whole viruses as antigen scaffolds is a recent development in vaccination that improves immunogenicity without the need for additional adjuvants. Previous studies highlighted the potential of foamy viruses (FVs) in prophylactic vaccination and gene therapy. Replication-competent FVs can trigger immune signaling and integrate into the host genome, resulting in persistent antigen expression and a robust immune response. Here, we explored feline foamy virus (FFV) proteins as scaffolds for therapeutic B and T cell epitope delivery in vitro. Infection- and cancer-related B and T cell epitopes were grafted into FFV Gag, Env, or Bet by residue replacement, either at sites of high local sequence homology between the epitope and the host protein or in regions known to tolerate sequence alterations. Modified proviruses were evaluated in vitro for protein steady state levels, particle release, and virus titer in permissive cells. Modification of Gag and Env was mostly detrimental to their function. As anticipated, modification of Bet had no impact on virion release and affected virus titers of only some recombinants. Further evaluation of Bet as an epitope carrier was performed using T cell epitopes from the model antigen chicken ovalbumin (OVA), human tyrosinase-related protein 2 (TRP-2), and oncoprotein E7 of human papillomavirus type 16 (HPV16E7). Transfection of murine cells with constructs encoding Bet-epitope chimeric proteins led to efficient MHC-I-restricted epitope presentation as confirmed by interferon-gamma enzyme-linked immunospot assays using epitope-specific cytotoxic T lymphocyte (CTL) lines. FFV infection-mediated transduction of cells with epitope-carrying Bet also induced T-cell responses, albeit with reduced efficacy, in a process independent from the presence of free peptides. We show that primate FV Bet is also a promising T cell epitope carrier for clinical translation. The data demonstrate the utility of replication-competent and -attenuated FVs as antigen carriers in immunotherapy. PMID:26397953

Alternative SAIL-Trp for robust aromatic signal assignment and determination of the χ(2) conformation by intra-residue NOEs.

PubMed

Miyanoiri, Yohei; Takeda, Mitsuhiro; Jee, JunGoo; Ono, Akira M; Okuma, Kosuke; Terauchi, Tsutomu; Kainosho, Masatsune

2011-12-01

Tryptophan (Trp) residues are frequently found in the hydrophobic cores of proteins, and therefore, their side-chain conformations, especially the precise locations of the bulky indole rings, are critical for determining structures by NMR. However, when analyzing [U-(13)C,(15)N]-proteins, the observation and assignment of the ring signals are often hampered by excessive overlaps and tight spin couplings. These difficulties have been greatly alleviated by using stereo-array isotope labeled (SAIL) proteins, which are composed of isotope-labeled amino acids optimized for unambiguous side-chain NMR assignment, exclusively through the (13)C-(13)C and (13)C-(1)H spin coupling networks (Kainosho et al. in Nature 440:52-57, 2006). In this paper, we propose an alternative type of SAIL-Trp with the [ζ2,ζ3-(2)H(2); δ1,ε3,η2-(13)C(3); ε1-(15)N]-indole ring ([(12)C (γ,) ( 12) C(ε2)] SAIL-Trp), which provides a more robust way to correlate the (1)H(β), (1)H(α), and (1)H(N) to the (1)H(δ1) and (1)H(ε3) through the intra-residue NOEs. The assignment of the (1)H(δ1)/(13)C(δ1) and (1)H(ε3)/(13)C(ε3) signals can thus be transferred to the (1)H(ε1)/(15)N(ε1) and (1)H(η2)/(13)C(η2) signals, as with the previous type of SAIL-Trp, which has an extra (13)C at the C(γ) of the ring. By taking advantage of the stereospecific deuteration of one of the prochiral β-methylene protons, which was (1)H(β2) in this experiment, one can determine the side-chain conformation of the Trp residue including the χ(2) angle, which is especially important for Trp residues, as they can adopt three preferred conformations. We demonstrated the usefulness of [(12)C(γ),(12)C(ε2)] SAIL-Trp for the 12 kDa DNA binding domain of mouse c-Myb protein (Myb-R2R3), which contains six Trp residues.

Safety assessment, biological effects, and mechanisms of Myrica rubra fruit extract for anti-melanogenesis, anti-oxidation, and free radical scavenging abilities on melanoma cells.

PubMed

Juang, Lih-Jeng; Gao, Xiang-Yu; Mai, Shou-Ting; Lee, Cheng-Hung; Lee, Ming-Chung; Yao, Chao-Ling

2018-02-20

Currently, the cosmetic and medical industries are paying considerable attention to solve or prevent skin damage or diseases, such as hyperpigmentation and oxidation and free radical damage. In this study, the effective compounds in Myrica rubra fruit were extracted and studied the biological effects of these M. rubra fruit extracts. In this study, we extracted M. rubra fruit using solutions with various ratios of water to ethanol (100:0, 50:50, 5:95) and studied the anti-melanogenesis, anti-oxidation and radical scavenging effects of these M. rubra fruit extracts on two melanoma cell lines: mouse melanoma (B16-F0) and human melanoma (A2058). The cytotoxicity, melanin synthesis, mushroom and cellular tyrosinase activities, enzyme kinetics, melanogenesis-related gene expression, melanogenesis-related protein secretion, radical DPPH scavenging activity and ROS inhibition after treatment with M. rubra fruit extracts were determined. The results showed that the water extract of M. rubra fruit was less cytotoxic to the melanoma cell lines, effectively inhibited melanin synthesis and tyrosinase activity and down-regulated the gene expression and protein secretion of MITF and TRP-1. In addition, the M. rubra fruit extracts also showed the abilities to scavenge DPPH free radicals and suppress ROS production. Finally, the effective compounds in the water extract were Myricetin-O-deoxyhexoside, Quercetin-O-deoxyhexoside, and Kaempferol-O-hexoside determined by LC/MS/MS assay. Overall, the water extract of M. rubra fruit is a safe and effective melanin inhibitor and anti-oxidant and can be applied widely in the fields of cosmetics and medicine. © 2018 Wiley Periodicals, Inc.

PGE2 is a UVR-inducible autocrine factor for human melanocytes that stimulates tyrosinase activation

PubMed Central

Starner, Renny J.; McClelland, Lindy; Abdel-Malek, Zalfa; Fricke, Alex; Scott, Glynis

2013-01-01

Melanocyte proliferation, dendrite formation, and pigmentation are controlled by paracrine factors, particularly following exposure to ultraviolet radiation (UVR). Little is known about autocrine factors for melanocytes. Prostaglandins activate signaling pathways involved in growth, differentiation and apoptosis. Prostaglandin E2 (PGE2) is the most abundant prostaglandin released by keratinocytes following UVR, and stimulates the formation of dendrites in melanocytes. Synthesis of PGE2 is controlled by cPLA2, which releases arachidonic acid from membranes, and COX-2 and prostaglandin E2 synthases (PGES), which convert arachidonic acid to PGH2 and PGH2 to PGE2, respectively. In this report we show that multiple irradiations of human melanocytes with UVR stimulates tyrosinase activity, independent of expression of a functional melanocortin 1 receptor, suggesting the presence of a non-melanocortin autocrine factor. Irradiation of melanocytes activated cPLA2, the rate-limiting step in eicosanoid synthesis, and stimulated PGE2 secretion. PGE2 increased cAMP production, tyrosinase activity and proliferation in melanocytes. PGE2 binds to four distinct G-protein coupled receptors (EP1–4). We show that EP4 receptor signaling stimulates cAMP production in melanocytes. Conversely, stimulation of the EP3 receptor lowered basal cAMP levels. These data suggest that relative levels or activity of these receptors controls effects of PGE2 on cAMP in melanocytes. The data are the first to identify PGE2 as an UVR-inducible autocrine factor for melanocytes that stimulates tyrosinase activity and proliferation, and to show that EP3 and EP4 receptor signaling have opposing effects on cAMP production, a critical signaling pathway that regulates proliferation and melanogenesis in melanocytes. PMID:20500768

Syndecan-2 regulates melanin synthesis via protein kinase C βII-mediated tyrosinase activation.

PubMed

Jung, Hyejung; Chung, Heesung; Chang, Sung Eun; Choi, Sora; Han, Inn-Oc; Kang, Duk-Hee; Oh, Eok-Soo

2014-05-01

Syndecan-2, a transmembrane heparan sulfate proteoglycan that is highly expressed in melanoma cells, regulates melanoma cell functions (e.g. migration). Since melanoma is a malignant tumor of melanocytes, which largely function to synthesize melanin, we investigated the possible involvement of syndecan-2 in melanogenesis. Syndecan-2 expression was increased in human skin melanoma tissues compared with normal skin. In both mouse and human melanoma cells, siRNA-mediated knockdown of syndecan-2 was associated with reduced melanin synthesis, whereas overexpression of syndecan-2 increased melanin synthesis. Similar effects were also detected in human primary epidermal melanocytes. Syndecan-2 expression did not affect the expression of tyrosinase, a key enzyme in melanin synthesis, but instead enhanced the enzymatic activity of tyrosinase by increasing the membrane and melanosome localization of its regulator, protein kinase CβII. Furthermore, UVB caused increased syndecan-2 expression, and this up-regulation of syndecan-2 was required for UVB-induced melanin synthesis. Taken together, these data suggest that syndecan-2 regulates melanin synthesis and could be a potential therapeutic target for treating melanin-associated diseases. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Functional evaluation of tryptophans in glycolipid binding and membrane interaction by HET-C2, a fungal glycolipid transfer protein.

PubMed

Kenoth, Roopa; Zou, Xianqiong; Simanshu, Dhirendra K; Pike, Helen M; Malinina, Lucy; Patel, Dinshaw J; Brown, Rhoderick E; Kamlekar, Ravi Kanth

2018-05-01

HET-C2 is a fungal glycolipid transfer protein (GLTP) that uses an evolutionarily-modified GLTP-fold to achieve more focused transfer specificity for simple neutral glycosphingolipids than mammalian GLTPs. Only one of HET-C2's two Trp residues is topologically identical to the three Trp residues of mammalian GLTP. Here, we provide the first assessment of the functional roles of HET-C2 Trp residues in glycolipid binding and membrane interaction. Point mutants HET-C2 W208F , HET-C2 W208A and HET-C2 F149Y all retained >90% activity and 80-90% intrinsic Trp fluorescence intensity; whereas HET-C2 F149A transfer activity decreased to ~55% but displayed ~120% intrinsic Trp emission intensity. Thus, neither W208 nor F149 is absolutely essential for activity and most Trp emission intensity (~85-90%) originates from Trp109. This conclusion was supported by HET-C2 W109Y/F149Y which displayed ~8% intrinsic Trp intensity and was nearly inactive. Incubation of the HET-C2 mutants with 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles containing different monoglycosylceramides or presented by lipid ethanol-injection decreased Trp fluorescence intensity and blue-shifted the Trp λ max by differing amounts compared to wtHET-C2. With HET-C2 mutants for Trp208, the emission intensity decreases (~30-40%) and λ max blue-shifts (~12nm) were more dramatic than for wtHET-C2 or F149 mutants and closely resembled human GLTP. When Trp109 was mutated, the glycolipid induced changes in HET-C2 emission intensity and λ max blue-shift were nearly nonexistent. Our findings indicate that the HET-C2 Trp λ max blue-shift is diagnostic for glycolipid binding; whereas the emission intensity decrease reflects higher environmental polarity encountered upon nonspecific interaction with phosphocholine headgroups comprising the membrane interface and specific interaction with the hydrated glycolipid sugar. Copyright © 2018 Elsevier B.V. All rights reserved.

Comparison of stochastic optimization methods for all-atom folding of the Trp-Cage protein.

PubMed

Schug, Alexander; Herges, Thomas; Verma, Abhinav; Lee, Kyu Hwan; Wenzel, Wolfgang

2005-12-09

The performances of three different stochastic optimization methods for all-atom protein structure prediction are investigated and compared. We use the recently developed all-atom free-energy force field (PFF01), which was demonstrated to correctly predict the native conformation of several proteins as the global optimum of the free energy surface. The trp-cage protein (PDB-code 1L2Y) is folded with the stochastic tunneling method, a modified parallel tempering method, and the basin-hopping technique. All the methods correctly identify the native conformation, and their relative efficiency is discussed.

Functional alterations due to amino acid changes and evolutionary comparative analysis of ARPKD and ADPKD genes.

PubMed

Edrees, Burhan M; Athar, Mohammad; Abduljaleel, Zainularifeen; Al-Allaf, Faisal A; Taher, Mohiuddin M; Khan, Wajahatullah; Bouazzaoui, Abdellatif; Al-Harbi, Naffaa; Safar, Ramzia; Al-Edressi, Howaida; Alansary, Khawala; Anazi, Abulkareem; Altayeb, Naji; Ahmed, Muawia A

2016-12-01

A targeted customized sequencing of genes implicated in autosomal recessive polycystic kidney disease (ARPKD) phenotype was performed to identify candidate variants using the Ion torrent PGM next-generation sequencing. The results identified four potential pathogenic variants in PKHD1 gene [c.4870C > T, p.(Arg1624Trp), c.5725C > T, p.(Arg1909Trp), c.1736C > T, p.(Thr579Met) and c.10628T > G, p.(Leu3543Trp)] among 12 out of 18 samples. However, one variant c.4870C > T, p.(Arg1624Trp) was common among eight patients. Some patient samples also showed few variants in autosomal dominant polycystic kidney disease (ADPKD) disease causing genes PKD1 and PKD2 such as c.12433G > A, p.(Val4145Ile) and c.1445T > G, p.(Phe482Cys), respectively. All causative variants were validated by capillary sequencing and confirmed the presence of a novel homozygous variant c.10628T > G, p.(Leu3543Trp) in a male proband. We have recently published the results of these studies (Edrees et al., 2016). Here we report for the first time the effect of the common mutation p.(Arg1624Trp) found in eight samples on the protein structure and function due to the specific amino acid changes of PKHD1 protein using molecular dynamics simulations. The computational approaches provide tool predict the phenotypic effect of variant on the structure and function of the altered protein. The structural analysis with the common mutation p.(Arg1624Trp) in the native and mutant modeled protein were also studied for solvent accessibility, secondary structure and stabilizing residues to find out the stability of the protein between wild type and mutant forms. Furthermore, comparative genomics and evolutionary analyses of variants observed in PKHD1 , PKD1 , and PKD2 genes were also performed in some mammalian species including human to understand the complexity of genomes among closely related mammalian species. Taken together, the results revealed that the evolutionary comparative analyses and characterization of PKHD1 , PKD1 , and PKD2 genes among various related and unrelated mammalian species will provide important insights into their evolutionary process and understanding for further disease characterization and management.

Macrocyclic peptides decrease c-Myc protein levels and reduce prostate cancer cell growth.

PubMed

Mukhopadhyay, Archana; Hanold, Laura E; Thayele Purayil, Hamsa; Gisemba, Solomon A; Senadheera, Sanjeewa N; Aldrich, Jane V

2017-08-03

The oncoprotein c-Myc is often overexpressed in cancer cells, and the stability of this protein has major significance in deciding the fate of a cell. Thus, targeting c-Myc levels is an attractive approach for developing therapeutic agents for cancer treatment. In this study, we report the anti-cancer activity of the macrocyclic peptides [D-Trp]CJ-15,208 (cyclo[Phe-D-Pro-Phe-D-Trp]) and the natural product CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]). [D-Trp]CJ-15,208 reduced c-Myc protein levels in prostate cancer cells and decreased cell proliferation with IC 50 values ranging from 2.0 to 16 µM in multiple PC cell lines. [D-Trp]CJ-15,208 induced early and late apoptosis in PC-3 cells following 48 hours treatment, and growth arrest in the G2 cell cycle phase following both 24 and 48 hours treatment. Down regulation of c-Myc in PC-3 cells resulted in loss of sensitivity to [D-Trp]CJ-15,208 treatment, while overexpression of c-Myc in HEK-293 cells imparted sensitivity of these cells to [D-Trp]CJ-15,208 treatment. This macrocyclic tetrapeptide also regulated PP2A by reducing the levels of its phosphorylated form which regulates the stability of cellular c-Myc protein. Thus [D-Trp]CJ-15,208 represents a new lead compound for the potential development of an effective treatment of prostate cancer.

CD4 T cell-mediated masking effects of the immunogenicity of tumor-associated antigens are qualitatively and quantitatively different depending on the individual antigens.

PubMed

Okano, Shinji; Matsumoto, Yoshihiro; Yoshiya, Shohei; Yamashita, Yo-ichi; Harimoto, Norifumi; Ikegami, Toru; Shirabe, Ken; Harada, Mamoru; Yoshikai, Yasunobu; Maehara, Yoshihiko

2013-01-01

The use of cancer immunotherapy as part of multidisciplinary therapies for cancer is a promising strategy for the cure of advanced cancer patients. In cancer immunotherapy, the effective priming of tumor-associated antigen (TAA)-specific CD8+ T cells is essential, and therefore, the appropriate selection of the best peptide for targeting the cancer is a most important concern. One criticism in the selection of a TAA is the immunogenicity of the TAA, the vaccination of which effectively elicits clinical responses. However, the critical basic immunological factors that affect the differences in the immunogenicity of TAAs remain to be elucidated. Here we found that CD4 T-cell responses suppressed the immunogenicity of the concomitant TAA in a murine melanoma model in which intratumoral activated dendritic therapy (ITADT) was used for treatment of the established cancer, and we observed that the antitumor effects were largely dependent on the CD8 T-cell response. CD4 T-cell depletion simply enhanced the tyrosinase-related protein (TRP)-2(180-188) peptide-specific cytotoxic T-cell (CTL) responses, and CD4 T-cell depletion provided immunogenicity for mgp100(25-33) peptide, to which a CTL response could not be detected at all in CD4 T-cell-intact mice in the early therapeutic phase. Further, the mgp100(25-33) peptide-specific CTL response again became undetectable after the recovery of CD4 T cells in previously CD4-depleted, tumor-eradicated mice, whereas the TRP-2(180-188) peptide-specific CTL response was still much stronger in CD4-depleted mice than in CD4-intact mice. These findings suggest that the CD4 T cell-mediated masking effects of the immunogenicity of tumor-associated antigens are qualitatively and quantitatively different depending on the individual antigens.

Fluorescence kinetics of Trp-Trp dipeptide and its derivatives in water via ultrafast fluorescence spectroscopy.

PubMed

Jia, Menghui; Yi, Hua; Chang, Mengfang; Cao, Xiaodan; Li, Lei; Zhou, Zhongneng; Pan, Haifeng; Chen, Yan; Zhang, Sanjun; Xu, Jianhua

2015-08-01

Ultrafast fluorescence dynamics of Tryptophan-Tryptophan (Trp-Trp/Trp2) dipeptide and its derivatives in water have been investigated using a picosecond resolved time correlated single photon counting (TCSPC) apparatus together with a femtosecond resolved upconversion spectrophotofluorometer. The fluorescence decay profiles at multiple wavelengths were fitted by a global analysis technique. Nanosecond fluorescence kinetics of Trp2, N-tert-butyl carbonyl oxygen-N'-aldehyde group-l-tryptophan-l-tryptophan (NBTrp2), l-tryptophan-l-tryptophan methyl ester (Trp2Me), and N-acetyl-l-tryptophan-l-tryptophan methyl ester (NATrp2Me) exhibit multi-exponential decays with the average lifetimes of 1.99, 3.04, 0.72 and 1.22ns, respectively. Due to the intramolecular interaction between two Trp residues, the "water relaxation" lifetime was observed around 4ps, and it is noticed that Trp2 and its derivatives also exhibit a new decay with a lifetime of ∼100ps, while single-Trp fluorescence decay in dipeptides/proteins shows 20-30ps. The intramolecular interaction lifetime constants of Trp2, NBTrp2, Trp2Me and NATrp2Me were then calculated to be 3.64, 0.93, 11.52 and 2.40ns, respectively. Candidate mechanisms (including heterogeneity, solvent relaxation, quasi static self-quenching or ET/PT quenching) have been discussed. Copyright © 2015. Published by Elsevier B.V.

Structure-activity relationships of the unique and potent agouti-related protein (AGRP)-melanocortin chimeric Tyr-c[beta-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template.

PubMed

Wilczynski, Andrzej; Wilson, Krista R; Scott, Joseph W; Edison, Arthur S; Haskell-Luevano, Carrie

2005-04-21

The melanocortin receptor system consists of endogenous agonists, antagonists, G-protein coupled receptors, and auxiliary proteins that are involved in the regulation of complex physiological functions such as energy and weight homeostasis, feeding behavior, inflammation, sexual function, pigmentation, and exocrine gland function. Herein, we report the structure-activity relationship (SAR) of a new chimeric hAGRP-melanocortin agonist peptide template Tyr-c[beta-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) that was characterized using amino acids previously reported in other melanocortin agonist templates. Twenty peptides were examined in this study, and six peptides were selected for (1)H NMR and computer-assisted molecular modeling structural analysis. The most notable results include the identification that modification of the chimeric template at the His position with Pro and Phe resulted in ligands that were nM mouse melanocortin-3 receptor (mMC3R) antagonists and nM mouse melanocortin-4 receptor (mMC4R) agonists. The peptides Tyr-c[beta-Asp-His-DPhe-Ala-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) and Tyr-c[beta-Asp-His-DNal(1')-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) resulted in 730- and 560-fold, respectively, mMC4R versus mMC3R selective agonists that also possessed nM agonist potency at the mMC1R and mMC5R. Structural studies identified a reverse turn occurring in the His-DPhe-Arg-Trp domain, with subtle differences observed that may account for the differences in melanocortin receptor pharmacology. Specifically, a gamma-turn secondary structure involving the DPhe(4) in the central position of the Tyr-c[beta-Asp-Phe-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) peptide may differentiate the mixed mMC3R antagonist and mMC4R agonist pharmacology.

Crystallization and preliminary X-ray crystallographic analysis of latent isoform PPO4 mushroom (Agaricus bisporus) tyrosinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mauracher, Stephan Gerhard; Molitor, Christian; Al-Oweini, Rami

Polyphenol oxidase 4 (PPO4) from the natural source A. bisporus was crystallized in its latent precursor form (pro-tyrosinase; Ser2–Thr565) using the 6-tungstotellurate(VI) salt Na{sub 6}[TeW{sub 6}O{sub 24}]·22H{sub 2}O as a crystallization additive. Tyrosinase exhibits catalytic activity for the ortho-hydroxylation of monophenols to diphenols as well as their subsequent oxidation to quinones. Owing to polymerization of these quinones, brown-coloured high-molecular-weight compounds called melanins are generated. The latent precursor form of polyphenol oxidase 4, one of the six tyrosinase isoforms from Agaricus bisporus, was purified to homogeneity and crystallized. The obtained crystals belonged to space group C121 (two molecules per asymmetric unit)more » and diffracted to 2.78 Å resolution. The protein only formed crystals under low-salt conditions using the 6-tungstotellurate(VI) salt Na{sub 6}[TeW{sub 6}O{sub 24}]·22H{sub 2}O as a co-crystallization agent.« less

Design, synthesis, and antimelanogenic effects of (2-substituted phenyl-1,3-dithiolan-4-yl)methanol derivatives

PubMed Central

Kim, Do Hyun; Kim, Su Jeong; Ullah, Sultan; Yun, Hwi Young; Chun, Pusoon; Moon, Hyung Ryong

2017-01-01

The authors designed and synthesized 17 (2-substituted phenyl-1,3-dithiolan-4-yl) methanol (PDTM) derivatives to find a new chemical scaffold, showing excellent tyrosinase-inhibitory activity. Their tyrosinase-inhibitory activities were evaluated against mushroom tyrosinase at 50 μM, and five of the PDTM derivatives (PDTM3, PDTM7–PDTM9, and PDTM13) were found to inhibit mushroom tyrosinase more than kojic acid or arbutin, the positive controls. Of seventeen PDTMs, PDTM3 (half-maximal inhibitory concentration 13.94±1.76 μM), with a 2,4-dihydroxyphenyl moiety, exhibited greatest inhibitory effects (kojic acid half-maximal inhibitory concentration 18.86±2.14 μM). Interestingly, PDTM compounds with no hydroxyl group, PDTM7–PDTM9, also had stronger inhibitory activities than kojic acid. In silico studies of interactions between tyrosinase and the five PDTMs suggested their binding affinities were closely related to their tyrosinase-inhibitory activities. Cell-based experiments performed using B16F10 mouse-skin melanoma cells showed that PDTM3 effectively inhibited melanogenesis and cellular tyrosinase activity. A cell-viability study conducted using B16F10 cells indicated that the antimelanogenic effect of PDTM3 was not attributable to its cytotoxicity. Kinetic studies showed PDTM3 competitively inhibited tyrosinase, indicating binding to the tyrosinase-active site. We found that PDTM3 with a new chemical scaffold could be a promising candidate for skin-whitening agents, and that the 1,3-dithiolane ring could be used as a chemical scaffold for potent tyrosinase inhibition. PMID:28352157

Photosensitive TRPs.

PubMed

Hardie, Roger C

2014-01-01

The Drosophila "transient receptor potential" channel is the prototypical TRP channel, belonging to and defining the TRPC subfamily. Together with a second TRPC channel, trp-like (TRPL), TRP mediates the transducer current in the fly's photoreceptors. TRP and TRPL are also implicated in olfaction and Malpighian tubule function. In photoreceptors, TRP and TRPL are localised in the ~30,000 packed microvilli that form the photosensitive "rhabdomere"-a light-guiding rod, housing rhodopsin and the rest of the phototransduction machinery. TRP (but not TRPL) is assembled into multimolecular signalling complexes by a PDZ-domain scaffolding protein (INAD). TRPL (but not TRP) undergoes light-regulated translocation between cell body and rhabdomere. TRP and TRPL are also found in photoreceptor synapses where they may play a role in synaptic transmission. Like other TRPC channels, TRP and TRPL are activated by a G protein-coupled phospholipase C (PLCβ4) cascade. Although still debated, recent evidence indicates the channels can be activated by a combination of PIP2 depletion and protons released by the PLC reaction. PIP2 depletion may act mechanically as membrane area is reduced by cleavage of PIP2's bulky inositol headgroup. TRP, which dominates the light-sensitive current, is Ca(2+) selective (P Ca:P Cs >50:1), whilst TRPL has a modest Ca(2+) permeability (P Ca:P Cs ~5:1). Ca(2+) influx via the channels has profound positive and negative feedback roles, required for the rapid response kinetics, with Ca(2+) rapidly facilitating TRP (but not TRPL) and also inhibiting both channels. In trp mutants, stimulation by light results in rapid depletion of microvillar PIP2 due to lack of Ca(2+) influx required to inhibit PLC. This accounts for the "transient receptor potential" phenotype that gives the family its name and, over a period of days, leads to light-dependent retinal degeneration. Gain-of-function trp mutants with uncontrolled Ca(2+) influx also undergo retinal degeneration due to Ca(2+) cytotoxicity. In vertebrate retina, mice knockout studies suggest that TRPC6 and TRPC7 mediate a PLCβ4-activated transducer current in intrinsically photosensitive retinal ganglion cells, expressing melanopsin. TRPA1 has been implicated as a "photo-sensing" TRP channel in human melanocytes and light-sensitive neurons in the body wall of Drosophila.

What do we know about the transient receptor potential vanilloid 2 (TRPV2) ion channel?

PubMed

Perálvarez-Marín, Alex; Doñate-Macian, Pau; Gaudet, Rachelle

2013-11-01

Transient receptor potential (TRP) ion channels are emerging as a new set of membrane proteins involved in a vast array of cellular processes and regulated by a large number of physical and chemical stimuli, which involves them with sensory cell physiology. The vanilloid TRP subfamily (TRPV) named after the vanilloid receptor 1 (TRPV1) consists of six members, and at least four of them (TRPV1-TRPV4) have been related to thermal sensation. One of the least characterized members of the TRP subfamily is TRPV2. Although initially characterized as a noxious heat sensor, TRPV2 now seems to have little to do with temperature sensing but a much more complex physiological profile. Here we review the available information and research progress on the structure, physiology and pharmacology of TRPV2 in an attempt to shed some light on the physiological and pharmacological deorphanization of TRPV2. © 2013 FEBS.

What do we know about the Transient Receptor Potential Vanilloid 2 (TRPV2) ion channel?

PubMed Central

Perálvarez-Marín, Alex; Doñate-Macian, Pau; Gaudet, Rachelle

2013-01-01

Transient receptor potential (TRP) ion channels are emerging as a new set of membrane proteins involved in a vast array of cellular processes and regulated by a large number of physical and chemical stimuli, which involves them with sensory cell physiology. The vanilloid TRP subfamily (TRPV) named after the vanilloid receptor 1 (TRPV1) consists of six members, and at least four of them (TRPV1-TRPV4) have been related to thermal sensation. One of the least characterized members of the TRP subfamily is TRPV2. Although initially characterized as a noxious heat sensor, TRPV2 now seems to have little to do with temperature sensing, but a much more complex physiological profile. Here we review the available information and research progress on the structure, physiology and pharmacology of TRPV2 in an attempt to shed some light on the physiological and pharmacological deorphanization of TRPV2. PMID:23615321

TRPs as chemosensors (ROS, RNS, RCS, gasotransmitters).

PubMed

Shimizu, Shunichi; Takahashi, Nobuaki; Mori, Yasuo

2014-01-01

The transient receptor potential (trp) gene superfamily encodes TRP proteins that act as multimodal sensor cation channels for a wide variety of stimuli from outside and inside the cell. Upon chemical or physical stimulation of cells, TRP channels transduce electrical and/or Ca(2+) signals via their cation channel activities. These functional features of TRP channels allow the body to react and adapt to different forms of environmental changes. Indeed, members of one class of TRP channels have emerged as sensors of reactive oxygen species (ROS), reactive nitrogen species (RNS), reactive carbonyl species (RCS), and gaseous messenger molecules including molecular oxygen (O2), hydrogen sulfide (H2S), and carbon dioxide (CO2). Hydrogen peroxide (H2O2), an ROS, triggers the production of ADP-ribose, which binds and activates TRPM2. In addition to TRPM2, TRPC5, TRPV1, and TRPA1 are also activated by H2O2 via modification of cysteine (Cys) free sulfhydryl groups. Nitric oxide (NO), a vasoactive gaseous molecule, regulates TRP channels directly via Cys S-nitrosylation or indirectly via cyclic GMP (cGMP)/protein kinase G (PKG)-dependent phosphorylation. Anoxia induced by O2-glucose deprivation and severe hypoxia activates TRPM7 and TRPC6, respectively, whereas TRPA1 serves as a sensor of mild hypoxia and hyperoxia in vagal and sensory neurons. TRPA1 also detects other gaseous molecules, such as hydrogen sulfide (H2S) and carbon dioxide (CO2). In this review, we highlight our current knowledge of TRP channels as chemosensors for ROS, RNS, RCS, and gaseous molecules and discuss their functional impacts on physiological and pathological events.

Structural domains required for channel function of the mouse transient receptor potential protein homologue TRP1beta.

PubMed

Engelke, Michael; Friedrich, Olaf; Budde, Petra; Schäfer, Christina; Niemann, Ursula; Zitt, Christof; Jüngling, Eberhard; Rocks, Oliver; Lückhoff, Andreas; Frey, Jürgen

2002-07-17

Transient receptor potential proteins (TRP) are supposed to participate in the formation of store-operated Ca(2+) influx channels by co-assembly. However, little is known which domains facilitate the interaction of subunits. Contribution of the N-terminal coiled-coil domain and ankyrin-like repeats and the putative pore region of the mouse TRP1beta (mTRP1beta) variant to the formation of functional cation channels were analyzed following overexpression in HEK293 (human embryonic kidney) cells. MTRP1beta expressing cells exhibited enhanced Ca(2+) influx and enhanced whole-cell membrane currents compared to mTRP1beta deletion mutants. Using a yeast two-hybrid assay only the coiled-coil domain facilitated homodimerization of the N-terminus. These results suggest that the N-terminus of mTRP1beta is required for structural organization thus forming functional channels.

Acrylonitrile Quenching of Trp Phosphorescence in Proteins: A Probe of the Internal Flexibility of the Globular Fold

PubMed Central

Strambini, Giovanni B.; Gonnelli, Margherita

2010-01-01

Quenching of Trp phosphorescence in proteins by diffusion of solutes of various molecular sizes unveils the frequency-amplitude of structural fluctuations. To cover the sizes gap between O2 and acrylamide, we examined the potential of acrylonitrile to probe conformational flexibility of proteins. The distance dependence of the through-space acrylonitrile quenching rate was determined in a glass at 77 K, with the indole analog 2-(3-indoyl) ethyl phenyl ketone. Intensity and decay kinetics data were fitted to a rate, k(r) = k0 exp[−(r − r0)/re], with an attenuation length re = 0.03 nm and a contact rate k0 = 3.6 × 1010 s−1. At ambient temperature, the bimolecular quenching rate constant (kq) was determined for a series of proteins, appositely selected to test the importance of factors such as the degree of Trp burial and structural rigidity. Relative to kq = 1.9 × 109 M−1s−1 for free Trp in water, in proteins kq ranged from 6.5 × 106 M−1s−1 for superficial sites to 1.3 × 102 M−1s−1 for deep cores. The short-range nature of the interaction and the direct correlation between kq and structural flexibility attest that in the microsecond-second timescale of phosphorescence acrylonitrile readily penetrates even compact protein cores and exhibits significant sensitivity to variations in dynamical structure of the globular fold. PMID:20682273

Effect of the tyrosinase inhibitor (S)-N-trans-feruloyloctopamine from garlic skin on tyrosinase gene expression and melanine accumulation in melanoma cells.

PubMed

Wu, Yan; Wu, Zheng-Rong; Chen, Peng; Yang-Li; Deng, Wan-Rong; Wang, You-Quan; Li, Hong-Yu

2015-04-01

In our searching for novel tyrosinase inhibitors from natural sources, (S)-N-trans-feruloyloctopamine isolated from garlic skin was found to be a potential mushroom tyrosinase inhibitor. Here, we examined the effects of the potential tyrosinase inhibitor in B16F10 cells on intracellular melanin contents, cytotoxicity, and the signaling mechanism involved in the expression of tyrosinase. The results showed the inhibitor displayed little or no cytotoxicity at all concentrations examined and decreased the relative melanin contents in a dose-dependent manner in the α-MSH-stimulated B16F10 cells. Real-time PCR and Western blot analysis showed that it inhibits melanogenesis signaling by down-regulates mRNA and protein expression levels of tyrosinase, which leads to a lower melanin contents. These results suggested that (S)-N-trans-feruloyloctopamine was an ideal tyrosinase inhibitor, and could be used in food and medical industry. Copyright © 2015 Elsevier Ltd. All rights reserved.

Requirement for Innate Immunity and CD90+ NK1.1− Lymphocytes to Treat Established Melanoma with Chemo-Immunotherapy

PubMed Central

Moskalenko, Marina; Pan, Michael; Fu, Yichun; de Moll, Ellen H.; Hashimoto, Daigo; Mortha, Arthur; Leboeuf, Marylene; Jayaraman, Padmini; Bernardo, Sebastian; Sikora, Andrew G.; Wolchok, Jedd; Bhardwaj, Nina; Merad, Miriam; Saenger, Yvonne

2015-01-01

We sought to define cellular immune mechanisms of synergy between tumor-antigen–targeted monoclonal antibodies and chemotherapy. Established B16 melanoma in mice was treated with cytotoxic doses of cyclophosphamide in combination with an antibody targeting tyrosinase-related protein 1 (αTRP1), a native melanoma differentiation antigen. We find that Fcγ receptors are required for efficacy, showing that antitumor activity of combination therapy is immune mediated. Rag1−/− mice deficient in adaptive immunity are able to clear tumors, and thus innate immunity is sufficient for efficacy. Furthermore, previously treated wild-type mice are not significantly protected against tumor reinduction, as compared with mice inoculated with irradiated B16 alone, consistent with a primarily innate immune mechanism of action of chemo-immunotherapy. In contrast, mice deficient in both classical natural killer (NK) lymphocytes and nonclassical innate lymphocytes (ILC) due to deletion of the IL2 receptor common gamma chain IL2γc−/−) are refractory to chemo-immunotherapy. Classical NK lymphocytes are not critical for treatment, as depletion of NK1.1+ cells does not impair antitumor effect. Depletion of CD90+NK1.1− lymphocytes, however, both diminishes therapeutic benefit and decreases accumulation of macrophages within the tumor. Tumor clearance during combination chemo-immunotherapy with monoclonal antibodies against native antigen is mediated by the innate immune system. We highlight a novel potential role for CD90+NK1.1− ILCs in chemo-immunotherapy. PMID:25600438

Antioxidative and Anti-Melanogenic Activities of Bamboo Stems (Phyllostachys nigra variety henosis) via PKA/CREB-Mediated MITF Downregulation in B16F10 Melanoma Cells.

PubMed

Choi, Moon-Hee; Jo, Han-Gyo; Yang, Ji Hye; Ki, Sung Hwan; Shin, Hyun-Jae

2018-01-30

Phyllostachys nigra var. henosis, a domestic bamboo species, has been attracting much attention; its bioactive compounds (especially in the leaf) show antioxidant, anti-inflammatory, and anti-obesity activities. Little information is available on the antioxidative and anti-melanogenetic activities of the bioactive compounds in bamboo stems. The anti-melanogenic and antioxidative activities of the EtOAc fraction (PN3) of a P. nigra stem extract were investigated in a cell-free system and in B16F10 melanoma cells. PN3 consisted of a mixture of flavonoids, such as catechin, chlorogenic acid, caffeic acid, and p -coumaric acid. The antioxidant activity (2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS)), and hydroxyl radical scavenging) was evaluated, as well as the inhibition of reactive oxygen species (ROS) produced by the Fenton reaction. PN3 showed in vitro tyrosinase inhibition activity with the half maximal inbihitory concentration (IC 50 ) values of 240 μg/mL, and in vivo cytotoxic concentration ranges > 100 μg/mL. The protein expression levels and mRNA transcription levels of TYR , TRP-1 , and MITF were decreased in a dose-dependent manner by the treatment with PN3. PN3 interfered with the phosphorylation of intracellular protein kinase A (PKA)/cAMP response element-binding protein (CREB), demonstrating potent anti-melanogenic effects. PN3 could inhibit PKA/CREB and the subsequent degradation of microphthalmia-associated transcription factor (MITF), resulting in the suppression of melanogenic enzymes and melanin production, probably because of the presence of flavonoid compounds. These properties make it a candidate as an additive to whitening cosmetics.

Antioxidative and Anti-Melanogenic Activities of Bamboo Stems (Phyllostachys nigra variety henosis) via PKA/CREB-Mediated MITF Downregulation in B16F10 Melanoma Cells

PubMed Central

Choi, Moon-Hee; Jo, Han-Gyo; Yang, Ji Hye; Ki, Sung Hwan

2018-01-01

Phyllostachys nigra var. henosis, a domestic bamboo species, has been attracting much attention; its bioactive compounds (especially in the leaf) show antioxidant, anti-inflammatory, and anti-obesity activities. Little information is available on the antioxidative and anti-melanogenetic activities of the bioactive compounds in bamboo stems. The anti-melanogenic and antioxidative activities of the EtOAc fraction (PN3) of a P. nigra stem extract were investigated in a cell-free system and in B16F10 melanoma cells. PN3 consisted of a mixture of flavonoids, such as catechin, chlorogenic acid, caffeic acid, and p-coumaric acid. The antioxidant activity (2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS)), and hydroxyl radical scavenging) was evaluated, as well as the inhibition of reactive oxygen species (ROS) produced by the Fenton reaction. PN3 showed in vitro tyrosinase inhibition activity with the half maximal inbihitory concentration (IC50) values of 240 μg/mL, and in vivo cytotoxic concentration ranges > 100 μg/mL. The protein expression levels and mRNA transcription levels of TYR, TRP-1, and MITF were decreased in a dose-dependent manner by the treatment with PN3. PN3 interfered with the phosphorylation of intracellular protein kinase A (PKA)/cAMP response element-binding protein (CREB), demonstrating potent anti-melanogenic effects. PN3 could inhibit PKA/CREB and the subsequent degradation of microphthalmia-associated transcription factor (MITF), resulting in the suppression of melanogenic enzymes and melanin production, probably because of the presence of flavonoid compounds. These properties make it a candidate as an additive to whitening cosmetics. PMID:29385729

A tryptophan-rich motif in the human parainfluenza virus type 2 V protein is critical for the blockade of toll-like receptor 7 (TLR7)- and TLR9-dependent signaling.

PubMed

Kitagawa, Yoshinori; Yamaguchi, Mayu; Zhou, Min; Komatsu, Takayuki; Nishio, Machiko; Sugiyama, Tsuyoshi; Takeuchi, Kenji; Itoh, Masae; Gotoh, Bin

2011-05-01

Plasmacytoid dendritic cells (pDCs) do not produce alpha interferon (IFN-α) unless viruses cause a systemic infection or overcome the first-line defense provided by conventional DCs and macrophages. We show here that even paramyxoviruses, whose infections are restricted to the respiratory tract, have a V protein able to prevent Toll-like receptor 7 (TLR7)- and TLR9-dependent IFN-α induction specific to pDCs. Mutational analysis of human parainfluenza virus type 2 demonstrates that the second Trp residue of the Trp-rich motif (Trp-X(3)-Trp-X(9)-Trp) in the C-terminal domain unique to V, a determinant for IRF7 binding, is critical for the blockade of TLR7/9-dependent signaling.

A transthyretin-related protein is functionally expressed in Herbaspirillum seropedicae.

PubMed

Matiollo, Camila; Vernal, Javier; Ecco, Gabriela; Bertoldo, Jean Borges; Razzera, Guilherme; de Souza, Emanuel M; Pedrosa, Fábio O; Terenzi, Hernán

2009-10-02

Transthyretin-related proteins (TRPs) constitute a family of proteins structurally related to transthyretin (TTR) and are found in a large range of bacterial, fungal, plant, invertebrate, and vertebrate species. However, it was recently recognized that both prokaryotic and eukaryotic members of this family are not functionally related to transthyretins. TRPs are in fact involved in the purine catabolic pathway and function as hydroxyisourate hydrolases. An open reading frame encoding a protein similar to the Escherichia coli TRP was identified in Herbaspirillum seropedicae genome (Hs_TRP). It was cloned, overexpressed in E. coli, and purified to homogeneity. Mass spectrometry data confirmed the identity of this protein, and circular dichroism spectrum indicated a predominance of beta-sheet structure, as expected for a TRP. We have demonstrated that Hs_TRP is a 5-hydroxyisourate hydrolase and by site-directed mutagenesis the importance of three conserved catalytic residues for Hs_TRP activity was further confirmed. The production of large quantities of this recombinant protein opens up the possibility of obtaining its 3D-structure and will help further investigations into purine catabolism.

Analysis of DNA-binding sites on Mhr1, a yeast mitochondrial ATP-independent homologous pairing protein.

PubMed

Masuda, Tokiha; Ling, Feng; Shibata, Takehiko; Mikawa, Tsutomu

2010-03-01

The Mhr1 protein is necessary for mtDNA homologous recombination in Saccharomyces cerevisiae. Homologous pairing (HP) is an essential reaction during homologous recombination, and is generally catalyzed by the RecA/Rad51 family of proteins in an ATP-dependent manner. Mhr1 catalyzes HP through a mechanism similar, at the DNA level, to that of the RecA/Rad51 proteins, but without utilizing ATP. However, it has no sequence homology with the RecA/Rad51 family proteins or with other ATP-independent HP proteins, and exhibits different requirements for DNA topology. We are interested in the structural features of the functional domains of Mhr1. In this study, we employed the native fluorescence of Mhr1's Trp residues to examine the energy transfer from the Trp residues to etheno-modified ssDNA bound to Mhr1. Our results showed that two of the seven Trp residues (Trp71 and Trp165) are spatially close to the bound DNA. A systematic analysis of mutant Mhr1 proteins revealed that Asp69 is involved in Mg(2+)-dependent DNA binding, and that multiple Lys and Arg residues located around Trp71 and Trp165 are involved in the DNA-binding activity of Mhr1. In addition, in vivo complementation analyses showed that a region around Trp165 is important for the maintenance of mtDNA. On the basis of these results, we discuss the function of the region surrounding Trp165.

Chimeric NDP-MSH and MTII melanocortin peptides with agouti-related protein (AGRP) Arg-Phe-Phe amino acids possess agonist melanocortin receptor activity.

PubMed

Joseph, Christine G; Wilczynski, Andrzej; Holder, Jerry R; Xiang, Zhimin; Bauzo, Rayna M; Scott, Joseph W; Haskell-Luevano, Carrie

2003-12-01

Agouti-related protein (AGRP) is one of only two known endogenous antagonists of G-protein coupled receptors (GPCRs). Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis, regulation of feeding behavior, and obesity. Alpha-melanocyte stimulating hormone (alpha-MSH) is one of the known endogenous agonists for these receptors. It has been hypothesized that the Arg-Phe-Phe (111-113) human AGRP amino acids may be mimicking the melanocortin agonist Phe-Arg-Trp (7-9) residue interactions with the melanocortin receptors that are important for both receptor molecular recognition and stimulation. To test this hypothesis, we generated thirteen chimeric peptide ligands based upon the melanocortin agonist peptides NDP-MSH (Ac-Ser-Tyr-Ser-Nle4-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2) and MTII (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2). In these chimeric ligands, the agonist DPhe-Arg-Trp amino acids were replaced by the AGRP Arg-Phe-Phe residues, and resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3-5Rs), supporting the hypothesis that the AGRP antagonist ligand Arg-Phe-Phe residues mimic the agonist Phe-Arg-Trp amino acids. Interestingly, the Ac-Ser-Tyr-Ser-Nle4-Glu-His-Arg-DPhe-Phe-Gly-Lys-Pro-Val-NH2 peptide possessed 7 nM mMC1R agonist potency, and is 850-fold selective for the mMC1R versus the mMC3R, 2300-fold selective for the mMC1R versus the mMC4R, and 60-fold selective for the MC1R versus the mMC5R, resulting in the discovery of a new peptide template for the design of melanocortin receptor selective ligands.

Inhibitory effects of 1-O-methyl-fructofuranose from Schisandra chinensis fruit on melanogenesis in B16F0 melanoma cells.

PubMed

Oh, Eun Young; Jang, Ji Yeon; Choi, Yung Hyun; Choi, Young Whan; Choi, Byung Tae

2010-10-28

1-O-methyl-fructofuranose (1-O-MFF) from the fruit of Schisandra chinensis is a traditional Korean medicinal herb that has a variety of beneficial properties. The effect of purified 1-O-MFF on melanogenesis including the activation of related signaling pathways was investigated. The inhibitory activities of 1-O-MFF were examined by melanin synthesis, tyrosinase activity assay, Western blot and flow cytometric analyses in B16F0 mouse melanoma cells. 1-O-MFF significantly inhibited both melanin synthesis and tyrosinase activity in a concentration-dependent manner, and reduced the expression of melanogenic proteins including microphthalmia-associated transcription factor (MITF), tyrosinase and tyrosinase-related protein-1. 1-O-MFF phosphorylated and activated melanogenesis inhibitory proteins such as mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and Akt. Flow cytometry confirmed that 1-O-MFF phosphorylated ERK and Akt proteins and recovered partially phosphorylated forms in cells treated with the MEK/ERK inhibitor compound PD98059 and the phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor compound LY294002. The suppressive effects of 1-O-MFF on melanogenesis may involve down-regulation of MITF and its downstream signal pathway via the activation of MEK/ERK or PI3K/Akt. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Probing the active site tryptophan of Staphylococcus aureus thioredoxin with an analog

PubMed Central

Englert, Markus; Nakamura, Akiyoshi; Wang, Yane-Shih; Eiler, Daniel; Söll, Dieter; Guo, Li-Tao

2015-01-01

Genetically encoded non-canonical amino acids are powerful tools of protein research and engineering; in particular they allow substitution of individual chemical groups or atoms in a protein of interest. One such amino acid is the tryptophan (Trp) analog 3-benzothienyl-l-alanine (Bta) with an imino-to-sulfur substitution in the five-membered ring. Unlike Trp, Bta is not capable of forming a hydrogen bond, but preserves other properties of a Trp residue. Here we present a pyrrolysyl-tRNA synthetase-derived, engineered enzyme BtaRS that enables efficient and site-specific Bta incorporation into proteins of interest in vivo. Furthermore, we report a 2.1 Å-resolution crystal structure of a BtaRS•Bta complex to show how BtaRS discriminates Bta from canonical amino acids, including Trp. To show utility in protein mutagenesis, we used BtaRS to introduce Bta to replace the Trp28 residue in the active site of Staphylococcus aureus thioredoxin. This experiment showed that not the hydrogen bond between residues Trp28 and Asp58, but the bulky aromatic side chain of Trp28 is important for active site maintenance. Collectively, our study provides a new and robust tool for checking the function of Trp in proteins. PMID:26582921

Interaction of singlet oxygen with bovine serum albumin and the role of the protein nano-compartmentalization.

PubMed

Giménez, Rodrigo E; Vargová, Veronika; Rey, Valentina; Turbay, M Beatriz Espeche; Abatedaga, Inés; Morán Vieyra, Faustino E; Paz Zanini, Verónica I; Mecchia Ortiz, Juan H; Katz, Néstor E; Ostatná, Veronika; Borsarelli, Claudio D

2016-05-01

Singlet molecular oxygen ((1)O2) contributes to protein damage triggering biophysical and biochemical changes that can be related with aging and oxidative stress. Serum albumins, such as bovine serum albumin (BSA), are abundant proteins in blood plasma with different biological functions. This paper presents a kinetic and spectroscopic study of the (1)O2-mediated oxidation of BSA using the tris(2,2'-bipyridine)ruthenium(II) cation [Ru(bpy)3](2+) as sensitizer. BSA quenches efficiently (1)O2 with a total (chemical+physical interaction) rate constant kt(BSA)=7.3(±0.4)×10(8)M(-1)s(-1), where the chemical pathway represented 37% of the interaction. This efficient quenching by BSA indicates the participation of several reactive residues. MALDI-TOF MS analysis of intact BSA confirmed that after oxidation by (1)O2, the mass protein increased the equivalent of 13 oxygen atoms. Time-resolved emission spectra analysis of BSA established that Trp residues were oxidized to N'-formylkynurenine, being the solvent-accessible W134 preferentially oxidized by (1)O2 as compared with the buried W213. MS confirmed oxidation of at least two Tyr residues to form dihydroxyphenylalanine, with a global reactivity towards (1)O2 six-times lower than for Trp residues. Despite the lack of MS evidences, kinetic and chemical analysis also suggested that residues other than Trp and Tyr, e.g. Met, must react with (1)O2. Modeling of the 3D-structure of BSA indicated that the oxidation pattern involves a random distribution of (1)O2 into BSA; allowing also the interaction of (1)O2 with buried residues by its diffusion from the bulk solvent through interconnected internal hydrophilic and hydrophobic grooves. Copyright © 2016 Elsevier Inc. All rights reserved.

Time-resolved fluorescence of thioredoxin single-tryptophan mutants: modeling experimental results with minimum perturbation mapping

NASA Astrophysics Data System (ADS)

Silva, Norberto D., Jr.; Haydock, Christopher; Prendergast, Franklyn G.

1994-08-01

The time-resolved fluorescence decay of single tryptophan (Trp) proteins is typically described using either a distribution of lifetimes or a sum of two or more exponential terms. A possible interpretation for this fluorescence decay heterogeneity is the existence of different isomeric conformations of Trp about its (chi) +1) and (chi) +2) dihedral angles. Are multiple Trp conformations compatible with the remainder of the protein in its crystallographic configuration or do they require repacking of neighbor side chains? It is conceivable that isomers of the neighbor side chains interconvert slowly on the fluorescence timescale and contribute additional lifetime components to the fluorescence intensity. We have explored this possibility by performing minimum perturbation mapping simulations of Trp 28 and Trp 31 in thioredoxin (TRX) using CHARMm 22. Mappings of Trp 29 and Trp 31 give the TRX Trp residue energy landscape as a function of (chi) +1) and (chi) +2) dihedral angles. Time-resolved fluorescence intensity and anisotropy decay of mutant TRX (W28F and W31F) are measured and interpreted in light of the above simulations. Relevant observables, like order parameters and isomerization rates, can be derived from the minimum perturbation maps and compared with experiment.

Promotion of tyrosinase folding in COS 7 cells by calnexin.

PubMed

Toyofuku, K; Wada, I; Hirosaki, K; Park, J S; Hori, Y; Jimbow, K

1999-01-01

To understand the process of expression of tyrosinase, a key enzyme of melanogenesis, we examined its maturation in the endoplasmic reticulum (ER) by using a heterogeneous expression system. When human tyrosinase cDNA was introduced into COS 7 cells, tyrosinase activity was minimally detected. Immunofluorescence study revealed that tyrosinase was immunolocalized in the nuclear rim, the reticular network, and the punctuated structures. Because a cytoplasmic tail of tyrosinase-gene family protein functions as a lysosomal targeting signal in non-melanocytic cells, and immature and/or misfolded molecules are selectively retained in the ER, the observed localization suggested the inefficient maturation in the COS 7 cells. We thus examined if supplementation of calnexin, a membrane-bound chaperone with affinity for oligosaccharide-processing intermediates containing monoglucose, could improve the process. As expected, the activity was enhanced approximately 2-fold by co-transfection of cDNA encoding calnexin. In contrast, co-transfection of the cytosolic tail-free calnexin, which inhibits calnexin function by allowing premature egress of its ligands from the ER, suppressed expression of this enhanced tyrosinase activity. When alpha-glucosidase activity, which is required for calnexin function, was inhibited by castanospermine (CST) treatment, expression of tyrosinase activity was completely abolished. To confirm the direct involvement of calnexin in tyrosinase maturation, the interaction of calnexin with tyrosinase was examined. Immunoprecipitation of calnexin from extracts of [35S]methionine labeled cells with anti-calnexin antibody revealed that the association is highest immediately after the pulse and that nascent tyrosinase is gradually dissociated upon chase. The association was completely inhibited when CST was included in the medium. Hence, we suggest that the proper folding of tyrosinase is largely dependent on its direct interaction with calnexin for the determined duration in the ER.

Effects of granulocyte-macrophage colony-stimulating factor and foreign helper protein as immunologic adjuvants on the T-cell response to vaccination with tyrosinase peptides.

PubMed

Scheibenbogen, Carmen; Schadendorf, Dirk; Bechrakis, Nikolaos E; Nagorsen, Dirk; Hofmann, Udo; Servetopoulou, Fotini; Letsch, Anne; Philipp, Armin; Foerster, Michael H; Schmittel, Alexander; Thiel, Eckhard; Keilholz, Ulrich

2003-03-20

Immunologic adjuvants are used to augment the immunogenicity of MHC class I-restricted peptide vaccines, but this effect has rarely been systematically evaluated in a clinical trial. We have investigated, in a phase I study, whether addition of the 2 adjuvants GM-CSF and KLH can enhance the T-cell response to MHC class I peptide vaccines. Forty-three high-risk melanoma patients who were clinically free of disease received 6 vaccinations with MHC class I-restricted tyrosinase peptides alone, with either GM-CSF or KLH or with a combination of both adjuvants. The primary end point was induction of tyrosinase-specific T cells, and serial T-cell monitoring was performed in unstimulated peripheral blood samples before and after the second, fourth and sixth vaccinations by ELISPOT assay. Tyrosinase-specific IFN-gamma-producing T cells were detected as early as 2 weeks after the second vaccination in 5 of 9 patients vaccinated with tyrosinase peptides in combination with GM-CSF and KLH but not in any patient vaccinated with tyrosinase peptides without adjuvants or in combination with either adjuvant alone. After 6 vaccinations, tyrosinase-specific T cells were found in patients immunized with peptides either without adjuvants (3 of 9 patients) or in combination with the single adjuvant GM-CSF (4 of 9 patients) but not with KLH (0 of 10 patients). Our results suggest that addition of either GM-CSF or KLH as a single adjuvant has little impact on the immunogenicity of tyrosinase peptides. The combined application of GM-CSF and KLH was associated with early induction of T-cell responses. Copyright 2003 Wiley-Liss, Inc.

Structural insight with mutational impact on tyrosinase and PKC-β interaction from Homo sapiens: Molecular modeling and docking studies for melanogenesis, albinism and increased risk for melanoma.

PubMed

Banerjee, Arundhati; Ray, Sujay

2016-10-30

Human tyrosinase, is an important protein for biosynthetic pathway of melanin. It was studied to be phosphorylated and activated by protein kinase-C, β-subunit (PKC-β) through earlier experimentations with in vivo evidences. Documentation documents that mutation in two essentially vital serine residues in C-terminal end of tyrosinase leads to albinism. Due to the deficiency of protective shield like enzyme; melanin, albinos are at an increased peril for melanoma and other skin cancers. So, computational and residue-level insight including a mutational exploration with evolutionary importance into this mechanism lies obligatory for future pathological and therapeutic developments. Therefore, functional tertiary models of the relevant proteins were analyzed after satisfying their stereo-chemical features. Evolutionarily paramount residues for the activation of tyrosinase were perceived via multiple sequence alignment phenomena. Mutant-type tyrosinase protein (S98A and S102A) was thereby modeled, maintaining the wild-type proteins' functionality. Furthermore, this present comparative study discloses the variation in the stable residual participation (for mutant-type and wild-type tyrosinase-PKCβ complex). Mainly, an increased number of polar negatively charged residues from the wild-type tyrosinase participated with PKC-β, predominantly. Fascinatingly supported by evaluation of statistical significances, mutation even led to a destabilizing impact in tyrosinase accompanied by conformational switches with a helix-to-coil transition in the mutated protein. Even the allosteric sites in the protein got poorly hampered upon mutation leading to weaker tendency for binding partners to interact. Copyright © 2016 Elsevier B.V. All rights reserved.

Role of TRP Channels in Dinoflagellate Mechanotransduction.

PubMed

Lindström, J B; Pierce, N T; Latz, M I

2017-10-01

Transient receptor potential (TRP) ion channels are common components of mechanosensing pathways, mainly described in mammals and other multicellular organisms. To gain insight into the evolutionary origins of eukaryotic mechanosensory proteins, we investigated the involvement of TRP channels in mechanosensing in a unicellular eukaryotic protist, the dinoflagellate Lingulodinium polyedra. BLASTP analysis of the protein sequences predicted from the L. polyedra transcriptome revealed six sequences with high similarity to human TRPM2, TRPM8, TRPML2, TRPP1, and TRPP2; and characteristic TRP domains were identified in all sequences. In a phylogenetic tree including all mammalian TRP subfamilies and TRP channel sequences from unicellular and multicellular organisms, the L. polyedra sequences grouped with the TRPM, TPPML, and TRPP clades. In pharmacological experiments, we used the intrinsic bioluminescence of L. polyedra as a reporter of mechanoresponsivity. Capsaicin and RN1734, agonists of mammalian TRPV, and arachidonic acid, an agonist of mammalian TRPV, TRPA, TRPM, and Drosophila TRP, all stimulated bioluminescence in L. polyedra. Mechanical stimulation of bioluminescence, but not capsaicin-stimulated bioluminescence, was inhibited by gadolinium (Gd 3+ ), a general inhibitor of mechanosensitive ion channels, and the phospholipase C (PLC) inhibitor U73122. These pharmacological results are consistent with the involvement of TRP-like channels in mechanosensing by L. polyedra. The TRP channels do not appear to be mechanoreceptors but rather are components of the mechanotransduction signaling pathway and may be activated via a PLC-dependent mechanism. The presence and function of TRP channels in a dinoflagellate emphasize the evolutionary conservation of both the channel structures and their functions.

Tyrosinase-catalyzed hydroxylation of hydroquinone, a depigmenting agent, to hydroxyhydroquinone: A kinetic study.

PubMed

García-Molina, María del Mar; Muñoz Muñoz, Jose Luis; Martinez-Ortiz, Francisco; Martinez, José Rodriguez; García-Ruiz, Pedro Antonio; Rodriguez-López, José Neptuno; García-Cánovas, Francisco

2014-07-01

Hydroquinone (HQ) is used as a depigmenting agent. In this work we demonstrate that tyrosinase hydroxylates HQ to 2-hydroxyhydroquinone (HHQ). Oxy-tyrosinase hydroxylates HQ to HHQ forming the complex met-tyrosinase-HHQ, which can evolve in two different ways, forming deoxy-tyrosinase and p-hydroxy-o-quinone, which rapidly isomerizes to 2-hydroxy-p-benzoquinone or on the other way generating met-tyrosinase and HHQ. In the latter case, HHQ is rapidly oxidized by oxygen to generate 2-hydroxy-p-benzoquinone, and therefore, it cannot close the enzyme catalytic cycle for the lack of reductant (HHQ). However, in the presence of hydrogen peroxide, met-tyrosinase (inactive on hydroquinone) is transformed into oxy-tyrosinase, which is active on HQ. Similarly, in the presence of ascorbic acid, HQ is transformed into 2-hydroxy-p-benzoquinone by the action of tyrosinase; however, in this case, ascorbic acid reduces met-tyrosinase to deoxy-tyrosinase, which after binding to oxygen, originates oxy-tyrosinase. This enzymatic form is now capable of reacting with HQ to generate p-hydroxy-o-quinone, which rapidly isomerizes to 2-hydroxy-p-benzoquinone. The formation of HHQ during the action of tyrosinase on HQ is demonstrated by means of high performance liquid chromatography mass spectrometry (HPLC-MS) by using hydrogen peroxide and high ascorbic acid concentrations. We propose a kinetic mechanism for the tyrosinase oxidation of HQ which allows us the kinetic characterization of the process. A possible explanation of the cytotoxic effect of HQ is discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

Studies on limiting essential amino acids in grieves as source of dietary protein for rainbow trout (Oncorhynchus mykiss).

PubMed

Pfeffer, E; Mandel, S; Rodehutscord, M

1994-01-01

Diets were computed to contain equal concentrations of digestible crude protein either of wheat gluten (diet 1) or of grieves (diets 2-8). Per kg dry diet, 41 g crystalline amino acids were supplemented. All diets contained at least 1.2 g Lys per MJ digestible energy (DE). In diet 2, ratios of Met + Cys, Trp, Leu, Ile and Phe to Lys were about equal to those in diet 1. In each of diets 3-7, one of the respective amino acids, in diet 8 all five were replaced by Glu in the supplemented mixture of amino acids. Each diet was fed to triplicate groups of 20 trout during a trial lasting 66 days. Trout fed the diet containing wheat gluten consumed more dry matter and showed higher growth rates as well as higher protein contents in their gained body mass than trout fed diets based on grieves. Supplementing Met plus Trp significantly improved dry matter intake, growth rate and protein content of gain, though not to the level of trout fed the wheat gluten diet, whereas Leu, Ile and Phe showed no such effect. When grieves were not supplemented with both Met and Trp, gain in body mass contained significantly more lipids. DE required per kg gain by trout fed wheat gluten, grieves + Met + Trp or grieves without supplementation of Met and Trp was 20.1, 21.2 and 29.9 MJ, respectively.

Towards an Integrative Understanding of tRNA Aminoacylation–Diet–Host–Gut Microbiome Interactions in Neurodegeneration

PubMed Central

Paley, Elena L.

2018-01-01

Transgenic mice used for Alzheimer’s disease (AD) preclinical experiments do not recapitulate the human disease. In our models, the dietary tryptophan metabolite tryptamine produced by human gut microbiome induces tryptophanyl-tRNA synthetase (TrpRS) deficiency with consequent neurodegeneration in cells and mice. Dietary supplements, antibiotics and certain drugs increase tryptamine content in vivo. TrpRS catalyzes tryptophan attachment to tRNAtrp at initial step of protein biosynthesis. Tryptamine that easily crosses the blood–brain barrier induces vasculopathies, neurodegeneration and cell death via TrpRS competitive inhibition. TrpRS inhibitor tryptophanol produced by gut microbiome also induces neurodegeneration. TrpRS inhibition by tryptamine and its metabolites preventing tryptophan incorporation into proteins lead to protein biosynthesis impairment. Tryptophan, a least amino acid in food and proteins that cannot be synthesized by humans competes with frequent amino acids for the transport from blood to brain. Tryptophan is a vulnerable amino acid, which can be easily lost to protein biosynthesis. Some proteins marking neurodegenerative pathology, such as tau lack tryptophan. TrpRS exists in cytoplasmic (WARS) and mitochondrial (WARS2) forms. Pathogenic gene variants of both forms cause TrpRS deficiency with consequent intellectual and motor disabilities in humans. The diminished tryptophan-dependent protein biosynthesis in AD patients is a proof of our model-based disease concept. PMID:29587458

BLOC-1 Interacts with BLOC-2 and the AP-3 Complex to Facilitate Protein Trafficking on Endosomes

PubMed Central

Di Pietro, Santiago M.; Falcón-Pérez, Juan M.; Tenza, Danièle; Setty, Subba R.G.; Marks, Michael S.; Raposo, Graça

2006-01-01

The adaptor protein (AP)-3 complex is a component of the cellular machinery that controls protein sorting from endosomes to lysosomes and specialized related organelles such as melanosomes. Mutations in an AP-3 subunit underlie a form of Hermansky-Pudlak syndrome (HPS), a disorder characterized by abnormalities in lysosome-related organelles. HPS in humans can also be caused by mutations in genes encoding subunits of three complexes of unclear function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2, and -3. Here, we report that BLOC-1 interacts physically and functionally with AP-3 to facilitate the trafficking of a known AP-3 cargo, CD63, and of tyrosinase-related protein 1 (Tyrp1), a melanosomal membrane protein previously thought to traffic only independently of AP-3. BLOC-1 also interacts with BLOC-2 to facilitate Tyrp1 trafficking by a mechanism apparently independent of AP-3 function. Both BLOC-1 and -2 localize mainly to early endosome-associated tubules as determined by immunoelectron microscopy. These findings support the idea that BLOC-1 and -2 represent hitherto unknown components of the endosomal protein trafficking machinery. PMID:16837549

Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major.

PubMed

Aoki, Juliana Ide; Coelho, Adriano Cappellazzo; Muxel, Sandra Marcia; Zampieri, Ricardo Andrade; Sanchez, Eduardo Milton Ramos; Nerland, Audun Helge; Floeter-Winter, Lucile Maria; Cotrim, Paulo Cesar

2016-09-01

Tubercidin (TUB) is a toxic adenosine analog with potential antiparasitic activity against Leishmania, with mechanism of action and resistance that are not completely understood. For understanding the mechanisms of action and identifying the potential metabolic pathways affected by this drug, we employed in this study an overexpression/selection approach using TUB for the identification of potential targets, as well as, drug resistance genes in L. major. Although, TUB is toxic to the mammalian host, these findings can provide evidences for a rational drug design based on purine pathway against leishmaniasis. After transfection of a cosmid genomic library into L. major Friedlin (LmjF) parasites and application of the overexpression/selection method, we identified two cosmids (cosTUB1 and cosTU2) containing two different loci capable of conferring significant levels of TUB resistance. In the cosTUB1 contained a gene encoding NUPM1-like protein, which has been previously described as associated with TUB resistance in L. amazonensis. In the cosTUB2 we identified and characterized a gene encoding a 63 kDa protein that we denoted as tubercidin-resistance protein (TRP). Functional analysis revealed that the transfectants were less susceptible to TUB than LmjF parasites or those transfected with the control vector. In addition, the trp mRNA and protein levels in cosTUB2 transfectants were higher than LmjF. TRP immunolocalization revealed that it was co-localized to the endoplasmic reticulum (ER), a cellular compartment with many functions. In silico predictions indicated that TRP contains only a hypothetical transmembrane domain. Thus, it is likely that TRP is a lumen protein involved in multidrug efflux transport that may be involved in the purine metabolic pathway. This study demonstrated for the first time that TRP is associated with TUB resistance in Leishmania. The next challenge is to determine how TRP mediates TUB resistance and whether purine metabolism is affected by this protein in the parasite. Finally, these findings may be helpful for the development of alternative anti-leishmanial drugs that target purine pathway.

Equilibrium folding of pro-HlyA from Escherichia coli reveals a stable calcium ion dependent folding intermediate.

PubMed

Thomas, Sabrina; Bakkes, Patrick J; Smits, Sander H J; Schmitt, Lutz

2014-09-01

HlyA from Escherichia coli is a member of the repeats in toxin (RTX) protein family, produced by a wide range of Gram-negative bacteria and secreted by a dedicated Type 1 Secretion System (T1SS). RTX proteins are thought to be secreted in an unfolded conformation and to fold upon secretion by Ca(2+) binding. However, the exact mechanism of secretion, ion binding and folding to the correct native state remains largely unknown. In this study we provide an easy protocol for high-level pro-HlyA purification from E. coli. Equilibrium folding studies, using intrinsic tryptophan fluorescence, revealed the well-known fact that Ca(2+) is essential for stability as well as correct folding of the whole protein. In the absence of Ca(2+), pro-HlyA adopts a non-native conformation. Such molecules could however be rescued by Ca(2+) addition, indicating that these are not dead-end species and that Ca(2+) drives pro-HlyA folding. More importantly, pro-HlyA unfolded via a two-state mechanism, whereas folding was a three-state process. The latter is indicative of the presence of a stable folding intermediate. Analysis of deletion and Trp mutants revealed that the first folding transition, at 6-7M urea, relates to Ca(2+) dependent structural changes at the extreme C-terminus of pro-HlyA, sensed exclusively by Trp914. Since all Trp residues of HlyA are located outside the RTX domain, our results demonstrate that Ca(2+) induced folding is not restricted to the RTX domain. Taken together, Ca(2+) binding to the pro-HlyA RTX domain is required to drive the folding of the entire protein to its native conformation. Copyright © 2014 Elsevier B.V. All rights reserved.

Utilisation of the isobole methodology to study dietary peptide-drug and peptide-peptide interactive effects on dipeptidyl peptidase IV (DPP-IV) inhibition.

PubMed

Nongonierma, Alice B; FitzGerald, Richard J

2015-01-01

Inhibition of dipeptidyl peptidase-IV (DPP-IV) is used as a means to regulate post-prandial serum glucose in type 2 diabetics. The effect of drug (Sitagliptin®)/peptide and binary peptide mixtures on DPP-IV inhibition was studied using an isobole approach. Five peptides (Ile-Pro-Ile-Gln-Tyr, Trp-Lys, Trp-Pro, Trp-Arg and Trp-Leu), having DPP-IV half maximum inhibitory concentration values (IC₅₀)<60 μM and reported to act through different inhibition mechanisms, were investigated. The dose response relationship of Sitagliptin : peptide (1:0, 0:1, 1:852, 1:426 and 1:1704 on a molar basis) and binary Ile-Pro-Ile-Gln-Tyr : peptide (1:0, 0:1, 1:1, 1:2 and 2:1 on a molar basis) mixtures for DPP-IV inhibition was characterised. Isobolographic analysis showed, in most instances, an additive effect on DPP-IV inhibition. However, a synergistic effect was observed with two Sitagliptin:Ile-Pro-Ile-Gln-Tyr (1:426 and 1:852) mixtures and an antagonistic effect was seen with one Sitagliptin : Trp-Pro (1:852) mixture, and three binary peptide mixtures (Ile-Pro-Ile-Gln-Tyr : Trp-Lys (1:1 and 2:1) and Ile-Pro-Ile-Gln-Tyr:Trp-Leu (1:2)). The results show that Sitagliptin and food protein-derived peptides can interact, thereby enhancing overall DPP-IV inhibition. Combination of Sitagliptin with food protein-derived peptides may help in reducing drug dosage and possible associated side-effects.

Restricted HIV-1 Env glycan engagement by lectin-reengineered DAVEI protein chimera is sufficient for lytic inactivation of the virus

PubMed Central

Parajuli, Bibek; Acharya, Kriti; Bach, Harry C.; Parajuli, Bijay; Zhang, Shiyu; Smith, Amos B.; Abrams, Cameron F.; Chaiken, Irwin

2018-01-01

We previously reported a first-generation recombinant DAVEI construct, a dual action virus entry inhibitor composed of cyanovirin-N (CVN) fused to a membrane proximal external region or its derivative peptide Trp3. DAVEI exhibits potent and irreversible inactivation of HIV-1 (human immunodeficiency virus) viruses by dual engagement of gp120 and gp41. However, the promiscuity of CVN to associate with multiple glycosylation sites in gp120 and its multivalency limit current understanding of the molecular arrangement of the DAVEI molecules on trimeric spike. Here, we constructed and investigated the virolytic function of second-generation DAVEI molecules using a simpler lectin, microvirin (MVN). MVN is a monovalent lectin with a single glycan-binding site in gp120, is structurally similar to CVN and exhibits no toxicity or mitogenicity, both of which are liabilities with CVN. We found that, like CVN-DAVEI-L2-3Trp (peptide sequence DKWASLWNW), MVN-DAVEI2-3Trp exploits a similar mechanism of action for inducing HIV-1 lytic inactivation, but by more selective gp120 glycan engagement. By sequence redesign, we significantly increased the potency of MVN-DAVEI2-3Trp protein. Unlike CVN-DAVEI2-3Trp, re-engineered MVN-DAVEI2-3Trp(Q81K/M83R) virolytic activity and its interaction with gp120 were both competed by 2G12 antibody. That the lectin domain in DAVEIs can utilize MVN without loss of virolytic function argues that restricted HIV-1 Env (envelope glycoprotein) glycan engagement is sufficient for virolysis. It also shows that DAVEI lectin multivalent binding with gp120 is not required for virolysis. MVN-DAVEI2-3Trp(Q81K/M83R) provides an improved tool to elucidate productive molecular arrangements of Env-DAVEI enabling virolysis and also opens the way to form DAVEI fusions made up of gp120-binding small molecules linked to Trp3 peptide. PMID:29343613

The peripheral clock regulates human pigmentation.

PubMed

Hardman, Jonathan A; Tobin, Desmond J; Haslam, Iain S; Farjo, Nilofer; Farjo, Bessam; Al-Nuaimi, Yusur; Grimaldi, Benedetto; Paus, Ralf

2015-04-01

Although the regulation of pigmentation is well characterized, it remains unclear whether cell-autonomous controls regulate the cyclic on-off switching of pigmentation in the hair follicle (HF). As human HFs and epidermal melanocytes express clock genes and proteins, and given that core clock genes (PER1, BMAL1) modulate human HF cycling, we investigated whether peripheral clock activity influences human HF pigmentation. We found that silencing BMAL1 or PER1 in human HFs increased HF melanin content. Furthermore, tyrosinase expression and activity, as well as TYRP1 and TYRP2 mRNA levels, gp100 protein expression, melanocyte dendricity, and the number gp100+ HF melanocytes, were all significantly increased in BMAL1 and/or PER1-silenced HFs. BMAL1 or PER1 silencing also increased epidermal melanin content, gp100 protein expression, and tyrosinase activity in human skin. These effects reflect direct modulation of melanocytes, as BMAL1 and/or PER1 silencing in isolated melanocytes increased tyrosinase activity and TYRP1/2 expression. Mechanistically, BMAL1 knockdown reduces PER1 transcription, and PER1 silencing induces phosphorylation of the master regulator of melanogenesis, microphthalmia-associated transcription factor, thus stimulating human melanogenesis and melanocyte activity in situ and in vitro. Therefore, the molecular clock operates as a cell-autonomous modulator of human pigmentation and may be targeted for future therapeutic strategies.

Structural and functional analysis of PucM, a hydrolase in the ureide pathway and a member of the transthyretin-related protein family

PubMed Central

Jung, Du-Kyo; Lee, Youra; Park, Sung Goo; Park, Byoung Chul; Kim, Ghyung-Hwa; Rhee, Sangkee

2006-01-01

The ureide pathway, which produces ureides from uric acid, is an essential purine catabolic process for storing and transporting the nitrogen fixed in leguminous plants and some bacteria. PucM from Bacillus subtilis was recently characterized and found to catalyze the second reaction of the pathway, hydrolyzing 5-hydroxyisourate (HIU), a product of uricase in the first step. PucM has 121 amino acid residues and shows high sequence similarity to the functionally unrelated protein transthyretin (TTR), a thyroid hormone-binding protein. Therefore, PucM belongs to the TTR-related proteins (TRP) family. The crystal structures of PucM at 2.0 Å and its complexes with the substrate analogs 8-azaxanthine and 5,6-diaminouracil reveal that even with their overall structure similarity, homotetrameric PucM and TTR are completely different, both in their electrostatic potential and in the size of the active sites located at the dimeric interface. Nevertheless, the absolutely conserved residues across the TRP family, including His-14, Arg-49, His-105, and the C-terminal Tyr-118–Arg-119–Gly-120–Ser-121, indeed form the active site of PucM. Based on the results of site-directed mutagenesis of these residues, we propose a possible mechanism for HIU hydrolysis. The PucM structure determined for the TRP family leads to the conclusion that diverse members of the TRP family would function similarly to PucM as HIU hydrolase. PMID:16782815

TRP channels: sensors and transducers of gasotransmitter signals

PubMed Central

Takahashi, Nobuaki; Kozai, Daisuke; Mori, Yasuo

2012-01-01

The transient receptor potential (trp) gene superfamily encodes cation channels that act as multimodal sensors for a wide variety of stimuli from outside and inside the cell. Upon sensing, they transduce electrical and Ca2+ signals via their cation channel activities. These functional features of TRP channels allow the body to react and adapt to different forms of environmental changes. Indeed, members of one class of TRP channels have emerged as sensors of gaseous messenger molecules that control various cellular processes. Nitric oxide (NO), a vasoactive gaseous molecule, regulates TRP channels directly via cysteine (Cys) S-nitrosylation or indirectly via cyclic GMP (cGMP)/protein kinase G (PKG)-dependent phosphorylation. Recent studies have revealed that changes in the availability of molecular oxygen (O2) also control the activation of TRP channels. Anoxia induced by O2-glucose deprivation and severe hypoxia (1% O2) activates TRPM7 and TRPC6, respectively, whereas TRPA1 has recently been identified as a novel sensor of hyperoxia and mild hypoxia (15% O2) in vagal and sensory neurons. TRPA1 also detects other gaseous molecules such as hydrogen sulfide (H2S) and carbon dioxide (CO2). In this review, we focus on how signaling by gaseous molecules is sensed and integrated by TRP channels. PMID:22934072

Physiological factors that regulate skin pigmentation

PubMed Central

Yamaguchi, Yuji; Hearing, Vincent J.

2009-01-01

More than 150 genes have been identified that affect skin color either directly or indirectly, and we review current understanding of physiological factors that regulate skin pigmentation. We focus on melanosome biogenesis, transport and transfer, melanogenic regulators in melanocytes and factors derived from keratinocytes, fibroblasts, endothelial cells, hormones, inflammatory cells and nerves. Enzymatic components of melanosomes include tyrosinase, tyrosinase-related protein 1 and dopachrome tautomerase, which depend on the functions of OA1, P, MATP, ATP7A and BLOC-1 to synthesize eumelanins and pheomelanins. The main structural component of melanosomes is Pmel17/gp100/Silv, whose sorting involves adaptor protein 1A (AP1A), AP1B, AP2 and spectrin, as well as a chaperone-like component, MART-1. During their maturation, melanosomes move from the perinuclear area toward the plasma membrane. Microtubules, dynein, kinesin, actin filaments, Rab27a, melanophilin, myosin Va and Slp2-a are involved in melanosome transport. Foxn1 and p53 up-regulate skin pigmentation via bFGF and POMC derivatives including α-MSH and ACTH, respectively. Other critical factors that affect skin pigmentation include MC1R, CREB, ASP, MITF, PAX3, SOX9/10, LEF-1/TCF, PAR-2, DKK1, SCF, HGF, GM-CSF, endothelin-1, prostaglandins, leukotrienes, thromboxanes, neurotrophins and neuropeptides. UV radiation up-regulates most factors that increase melanogenesis. Further studies will elucidate the currently unknown functions of many other pigment genes/proteins. PMID:19449448

[6]-Shogaol inhibits melanogenesis in B16 mouse melanoma cells through activation of the ERK pathway

PubMed Central

Yao, Cheng; Oh, Jang-hee; Oh, Inn Gyung; Park, Chi-hyun; Chung, Jin Ho

2013-01-01

Aim: To investigate the effect of [6]-shogaol, an active ingredient in ginger, on melanogenesis and the underlying mechanisms. Methods: B16F10 mouse melanoma cells were tested. Cell viability was determined with the MTT assay. Melanin content and tyrosinase activity were analyzed with a spectrophotometer. The protein expression of tyrosinase and microphthalmia associated transcription factor (MITF), as well as phosphorylated or total ERK1/2 and Akt were measured using Western blot. Results: Treatment of the cells with [6]-shogaol (1, 5, 10 μmol/L) reduced the melanin content in a concentration-dependent manner. [6]-Shogaol (5 and 10 μmol/L) significantly decreased the intracellular tyrosinase activity, and markedly suppressed the expression levels of tyrosinase and MITF proteins in the cells. Furthermore, [6]-shogaol (10 μmol/L) activated ERK, which was known to negatively regulate melanin synthesis in these cells. Pretreatment with the specific ERK pathway inhibitor PD98059 (20 μmol/L) greatly attenuated the inhibition of melanin synthesis by [6]-shogaol (10 μmol/L). Conclusion: The results demonstrate that [6]-shogaol inhibits melanogenesis in B16F10 mouse melanoma cells via activating the ERK pathway. PMID:23123645

[6]-Shogaol inhibits melanogenesis in B16 mouse melanoma cells through activation of the ERK pathway.

PubMed

Yao, Cheng; Oh, Jang-hee; Oh, Inn Gyung; Park, Chi-hyun; Chung, Jin Ho

2013-02-01

To investigate the effect of [6]-shogaol, an active ingredient in ginger, on melanogenesis and the underlying mechanisms. B16F10 mouse melanoma cells were tested. Cell viability was determined with the MTT assay. Melanin content and tyrosinase activity were analyzed with a spectrophotometer. The protein expression of tyrosinase and microphthalmia associated transcription factor (MITF), as well as phosphorylated or total ERK1/2 and Akt were measured using Western blot. Treatment of the cells with [6]-shogaol (1, 5, 10 μmol/L) reduced the melanin content in a concentration-dependent manner. [6]-Shogaol (5 and 10 μmol/L) significantly decreased the intracellular tyrosinase activity, and markedly suppressed the expression levels of tyrosinase and MITF proteins in the cells. Furthermore, [6]-shogaol (10 μmol/L) activated ERK, which was known to negatively regulate melanin synthesis in these cells. Pretreatment with the specific ERK pathway inhibitor PD98059 (20 μmol/L) greatly attenuated the inhibition of melanin synthesis by [6]-shogaol (10 μmol/L). The results demonstrate that [6]-shogaol inhibits melanogenesis in B16F10 mouse melanoma cells via activating the ERK pathway.

Purification and characterization of RNA allied extracellular tyrosinase from Aspergillus species.

PubMed

Inamdar, Shrirang; Joshi, Swati; Bapat, Vishwas; Jadhav, Jyoti

2014-02-01

Production of L-DOPA, an anti-Parkinson's drug, using biological sources is widely studied in which tyrosinase is known to play a vital role. Tyrosinase is an omnipresent type 3 copper enzyme participating in many essential biological functions. Understanding properties of tyrosinase is essential for developing useful tyrosinase-based applications. Hence, extracellular tyrosinase from Aspergillus flavus UWFP 570 was purified using ammonium sulphate precipitation and DEAE ion exchange chromatography up to 8.3-fold. Purified protein was a riboprotein in nature containing significant amount of RNA which was confirmed colorimetrically and by electrophoresis. Removal of RNA reduced the activity and altered the conformation of tyrosinase as suggested by spectroflurometric results. Optimum pH and temperature of this 140 kDa protein were 7 and 40 °C, respectively. Copper sulphate and magnesium chloride enhanced the activity whereas in contrast FeCl₃ inhibited the activity completely. Purified tyrosinase transformed L-tyrosine (5 mM) to L-DOPA within 5 h.

Surface display of bacterial tyrosinase on spores of Bacillus subtilis using CotE as an anchor protein.

PubMed

Hosseini-Abari, Afrouzossadat; Kim, Byung-Gee; Lee, Sang-Hyuk; Emtiazi, Giti; Kim, Wooil; Kim, June-Hyung

2016-12-01

Tyrosinases, copper-containing monooxygenases, are widely used enzymes for industrial, medical, and environmental applications. We report the first functional surface display of Bacillus megaterium tyrosinase on Bacillus subtilis spores using CotE as an anchor protein. Flow Cytometry was used to verify surface expression of tyrosinase on the purified spores. Moreover, tyrosinase activity of the displayed enzyme on B. subtilis spores was monitored in the presence of L-tyrosine (substrate) and CuSO 4 (inducer). The stability of the spore-displayed tyrosinase was then evaluated after 15 days maintenance of the spores at room temperature, and no significant decrease in the enzyme activity was observed. In addition, the tyrosinase-expressing spores could be repeatedly used with 62% retained enzymatic activity after six times washing with Tris-HCl buffer. This genetically immobilized tyrosinase on the spores would make a new advance in industrial, medical, and environmental applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

l-tyrosine induces melanocyte differentiation in novel pink-eyed dilution castaneus mouse mutant showing age-related pigmentation.

PubMed

Hirobe, Tomohisa; Ishikawa, Akira

2015-12-01

The mouse pink-eyed dilution (oculocutaneous albinism II; p/Oca2(p)) locus is known to control tyrosinase activity, melanin content, and melanosome development in melanocytes. Pink-eyed dilution castaneus (p(cas)/Oca2(p-cas)) is a novel mutant allele on mouse chromosome 7 that arose spontaneously in Indonesian wild mice, Mus musculus castaneus. Mice homozygous for Oca2(p-cas) usually exhibit pink eyes and beige-colored coat on nonagouti C57BL/6 (B6) background. Recently, a novel spontaneous mutation occurred in the progeny between this mutant and B6 mice. The eyes of this novel mutant progressively become black from pink and the coat becomes dark gray from beige with aging. The aim of this study is to clarify whatever differences exist in melanocyte proliferation and differentiation between the ordinary (pink-eyed) and novel (black-eyed) mutant using serum-free primary culture system. The characteristics of melanocyte proliferation and differentiation were investigated by serum-free primary culture system using melanocyte-proliferation medium (MDMD). The proliferation of melanoblasts in MDMD did not differ between the two mice. However, when the epidermal cell suspensions were cultured with MDMD supplemented with l-tyrosine (Tyr), the differentiation of black-eyed melanocytes was greatly induced in a concentration-dependent manner compared with pink-eyed melanocytes. Immunocytochemistry demonstrated that the expression of tyrosinase and tyrosinase-related protein-1 (Tyrp1) was greatly induced or stimulated both in pink-eyed and black-eyed melanocytes, whereas the expression of microphthalmia-associated transcription factor (Mitf) was stimulated only in black-eyed melanocytes. These results suggest that the age-related coat darkening in black-eyed mutant may be caused by the increased ability of melanocyte differentiation dependent on l-Tyr through the upregulation of tyrosinase, Tyrp1, and Mitf. This mutant mouse may be useful for animal model to clarify the mechanisms of age-related pigmentation in human skin, such as melasma and solar lentigines. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A monomeric TIM-barrel structure from Pyrococcus furiosus is optimized for extreme temperatures.

PubMed

Repo, Heidi; Oeemig, Jesper S; Djupsjöbacka, Janica; Iwaï, Hideo; Heikinheimo, Pirkko

2012-11-01

The structure of phosphoribosyl anthranilate isomerase (TrpF) from the hyperthermophilic archaeon Pyrococcus furiosus (PfTrpF) has been determined at 1.75 Å resolution. The PfTrpF structure has a monomeric TIM-barrel fold which differs from the dimeric structures of two other known thermophilic TrpF proteins. A comparison of the PfTrpF structure with the two known bacterial thermophilic TrpF structures and the structure of a related mesophilic protein from Escherichia coli (EcTrpF) is presented. The thermophilic TrpF structures contain a higher proportion of ion pairs and charged residues compared with the mesophilic EcTrpF. These residues contribute to the closure of the central barrel and the stabilization of the barrel and the surrounding α-helices. In the monomeric PfTrpF conserved structural water molecules are mostly absent; instead, the structural waters are replaced by direct side-chain-main-chain interactions. As a consequence of these combined mechanisms, the P. furiosus enzyme is a thermodynamically stable and entropically optimized monomeric TIM-barrel enzyme which defines a good framework for further protein engineering for industrial applications.

Design, synthesis and bioevaluation of novel umbelliferone analogues as potential mushroom tyrosinase inhibitors.

PubMed

Ashraf, Zaman; Rafiq, Muhammad; Seo, Sung-Yum; Babar, Mustafeez Mujtaba; Zaidi, Najam-Us-Sahar Sadaf

2015-12-01

A series of umbelliferone analogues were synthesized and their inhibitory effects on the DPPH and mushroom tyrosinase were evaluated. The results showed that some of the synthesized compounds exhibited significant mushroom tyrosinase inhibitory activities. Especially, 2-oxo-2-[(2-oxo-2H-chromen-7-yl)oxy]ethyl-2,4-dihydroxybenzoate (4e) bearing 2,4-dihydroxy substituted phenyl ring exhibited the most potent tyrosinase inhibitory activity with IC50 value 8.96 µM and IC50 value of kojic acid is 16.69. The inhibition mechanism analyzed by Lineweaver-Burk plots revealed that the type of inhibition of compound 4e on tyrosinase was non-competitive. The docking study against tyrosinase enzyme was also performed to determine the binding affinity of the compounds. The compounds 4c and 4e showed the highest binding affinity with active binding site of tyrosinase. The initial structure activity relationships (SARs) analysis suggested that further development of such compounds might be of interest. The statistics of our results endorses that compounds 4c and 4e may serve as a structural template for the design and development of novel tyrosinase inhibitors.

Biology and genetics of oculocutaneous albinism and vitiligo - common pigmentation disorders in southern Africa.

PubMed

Manga, Prashiela; Kerr, Robyn; Ramsay, Michèle; Kromberg, Jennifer G R

2013-07-29

Pigmentation disorders span the genetic spectrum from single-gene autosomal recessive disorders such as oculocutaneous albinism (OCA), the autosomal dominant disorder piebaldism to X-linked ocular albinism and multifactorial vitiligo. OCA connotes a group of disorders that result in hypopigmented skin due to decreased melanin production in melanocytes and loss of visual acuity. There are four non-syndromic forms, OCA1-4, which are classified based on the gene that is mutated (tyrosinase, OCA2, tyrosinase-related protein 1 and SLC45A2, respectively). Despite the fact that multiple genes account for the various forms of OCA, the phenotypes of all four forms result from disruption in the maturation and trafficking of the enzyme tyrosinase. OCA2 is the most prevalent autosomal recessive disorder among southern African blacks, affecting 1/3 900 individuals; while OCA3, although rare, is most prevalent in southern Africa. Another common pigmentation disorder in southern Africa is vitiligo, which affects 1 - 2% of people worldwide. Vitiligo is a complex, acquired disorder in which melanocytes are destroyed due to an autoimmune response. The aetiology underlying this disorder is poorly understood, although recent genetic association studies have begun to shed light on the contributing factors. Pigmentation disorders have significant psychosocial implications and co-morbidities, yet therapies are still lacking. Recent progress in our understanding of the pathobiology of both albinism and vitiligo may herald novel treatment strategies for these disorders. 

The Rise of Radicals in Bioinorganic Chemistry.

PubMed

Gray, Harry B; Winkler, Jay R

2016-10-01

Prior to 1950, the consensus was that biological transformations occurred in two-electron steps, thereby avoiding the generation of free radicals. Dramatic advances in spectroscopy, biochemistry, and molecular biology have led to the realization that protein-based radicals participate in a vast array of vital biological mechanisms. Redox processes involving high-potential intermediates formed in reactions with O 2 are particularly susceptible to radical formation. Clusters of tyrosine (Tyr) and tryptophan (Trp) residues have been found in many O 2 -reactive enzymes, raising the possibility that they play an antioxidant protective role. In blue copper proteins with plastocyanin-like domains, Tyr/Trp clusters are uncommon in the low-potential single-domain electron-transfer proteins and in the two-domain copper nitrite reductases. The two-domain muticopper oxidases, however, exhibit clusters of Tyr and Trp residues near the trinuclear copper active site where O 2 is reduced. These clusters may play a protective role to ensure that reactive oxygen species are not liberated during O 2 reduction.

In vivo imaging of inducible tyrosinase gene expression with an ultrasound array-based photoacoustic system

NASA Astrophysics Data System (ADS)

Harrison, Tyler; Paproski, Robert J.; Zemp, Roger J.

2012-02-01

Tyrosinase, a key enzyme in the production of melanin, has shown promise as a reporter of genetic activity. While green fluorescent protein has been used extensively in this capacity, it is limited in its ability to provide information deep in tissue at a reasonable resolution. As melanin is a strong absorber of light, it is possible to image gene expression using tyrosinase with photoacoustic imaging technologies, resulting in excellent resolutions at multiple-centimeter depths. While our previous work has focused on creating and imaging MCF-7 cells with doxycycline-controlled tyrosinase expression, we have now established the viability of these cells in a murine model. Using an array-based photoacoustic imaging system with 5 MHz center frequency, we capture interleaved ultrasound and photoacoustic images of tyrosinase-expressing MCF-7 tumors both in a tissue mimicking phantom, and in vivo. Images of both the tyrosinase-expressing tumor and a control tumor are presented as both coregistered ultrasound-photoacoustic B-scan images and 3-dimensional photoacoustic volumes created by mechanically scanning the transducer. We find that the tyrosinase-expressing tumor is visible with a signal level 12dB greater than that of the control tumor in vivo. Phantom studies with excised tumors show that the tyrosinase-expressing tumor is visible at depths in excess of 2cm, and have suggested that our imaging system is sensitive to a transfection rate of less than 1%.

Identification of two novel mutations in the SLC45A2 gene in a Hungarian pedigree affected by unusual OCA type 4.

PubMed

Tóth, Lola; Fábos, Beáta; Farkas, Katalin; Sulák, Adrienn; Tripolszki, Kornélia; Széll, Márta; Nagy, Nikoletta

2017-03-15

Oculocutaneous albinism (OCA) is a clinically and genetically heterogenic group of pigmentation abnormalities. OCA type IV (OCA4, OMIM 606574) develops due to homozygous or compound heterozygous mutations in the solute carrier family 45, member 2 (SLC45A2) gene. This gene encodes a membrane-associated transport protein, which regulates tyrosinase activity and, thus, melanin content by changing melanosomal pH and disrupting the incorporation of copper into tyrosinase. Here we report two Hungarian siblings affected by an unusual OCA4 phenotype. After genomic DNA was isolated from peripheral blood of the patients, the coding regions of the SLC45A2 gene were sequenced. In silico tools were applied to identify the functional impact of the newly detected mutations. Direct sequencing of the SLC45A2 gene revealed two novel, heterozygous mutations, one missense (c.1226G > A, p.Gly409Asp) and one nonsense (c.1459C > T, p.Gln437*), which were present in both patients, suggesting the mutations were compound heterozygous. In silico tools suggest that these variations are disease causing mutations. The newly identified mutations may affect the transmembrane domains of the protein, and could impair transport function, resulting in decreases in both melanosomal pH and tyrosinase activity. Our study provides expands on the mutation spectrum of the SLC45A2 gene and the genetic background of OCA4.

Inhibition of melanogenesis by β-caryophyllene from lime mint essential oil in mouse B16 melanoma cells.

PubMed

Yang, C-H; Huang, Y-C; Tsai, M-L; Cheng, C-Y; Liu, L-L; Yen, Y-W; Chen, W-L

2015-10-01

Volatile essential oils of mint species are used for cosmetics and in skin care products. In this study, we evaluated the main chemical components of the lime mint and the anti-melanogenic properties of its main components. The essential oil was analysed by gas chromatography-mass spectrometry (GC/MS). The anti-melanogenic effects of mint essential oil and β-caryophyllene were investigated in B16F10 murine melanoma cells. The main components of lime mint essential oil were found to be D-limonene (41.10%), D-carvone (8.58%), δ-selinene (6.73%) and β-caryophyllene (6.24%). The lime mint essential oil reduced melanin production in a dose-dependent manner in murine B16F10 cells. β-Caryophyllene, one of the main compounds in lime mint essential oil, could reduce melanogenesis by down-regulating the expression of MITF, TRP-1, TRP-2 and tyrosinase, resulting in a decrease in melanin content decrease. These results reveal that lime mint essential oil and β-caryophyllene are considered to be valuable as potential skin-whitening agents. © 2015 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

(2E,5E)-2,5-Bis(3-hydroxy-4-methoxybenzylidene) cyclopentanone Exerts Anti-Melanogenesis and Anti-Wrinkle Activities in B16F10 Melanoma and Hs27 Fibroblast Cells.

PubMed

Jung, Hee Jin; Lee, A Kyoung; Park, Yeo Jin; Lee, Sanggwon; Kang, Dongwan; Jung, Young Suk; Chung, Hae Young; Moon, Hyung Ryong

2018-06-11

Ultraviolet (UV) radiation exposure is the primary cause of extrinsic skin aging, which results in skin hyperpigmentation and wrinkling. In this study, we investigated the whitening effect of (2 E ,5 E )-2,5-bis(3-hydroxy-4-methoxybenzylidene)cyclopentanone (BHCP) on B16F10 melanoma and its anti-wrinkle activity on Hs27 fibroblasts cells. BHCP was found to potently inhibit tyrosinase, with 50% inhibition concentration (IC 50 ) values of 1.10 µM and 8.18 µM for monophenolase (l-tyrosine) and diphenolase (l-DOPA), and the enzyme kinetics study revealed that BHCP is a competitive-type tyrosinase inhibitor. Furthermore, BHCP significantly inhibited melanin content and cellular tyrosinase activity, and downregulated the levels of microphthalmia-associated transcription factor (MITF), phosphorylated levels of cAMP response element-binding (CREB) protein, and tyrosinase in α-melanocyte stimulating hormone (α-MSH)-induced B16F10 melanoma cells. Moreover, BHCP inhibited the phosphorylation of p65 and expression of matrix metalloproteinases (MMP-1, MMP-9, MMP-12, and MMP-13) in Hs27 fibroblasts stimulated with UV radiation. Therefore, our results demonstrate that BHCP may be a good candidate for the development of therapeutic agents for diseases associated with hyperpigmentation and wrinkling.

Role of Tryptophan Side Chain Dynamics on the Trp-Cage Mini-Protein Folding Studied by Molecular Dynamics Simulations

PubMed Central

Kannan, Srinivasaraghavan; Zacharias, Martin

2014-01-01

The 20 residue Trp-cage mini-protein is one of smallest proteins that adopt a stable folded structure containing also well-defined secondary structure elements. The hydrophobic core is arranged around a single central Trp residue. Despite several experimental and simulation studies the detailed folding mechanism of the Trp-cage protein is still not completely understood. Starting from fully extended as well as from partially folded Trp-cage structures a series of molecular dynamics simulations in explicit solvent and using four different force fields was performed. All simulations resulted in rapid collapse of the protein to on average relatively compact states. The simulations indicate a significant dependence of the speed of folding to near-native states on the side chain rotamer state of the central Trp residue. Whereas the majority of intermediate start structures with the central Trp side chain in a near-native rotameric state folded successfully within less than 100 ns only a fraction of start structures reached near-native folded states with an initially non-native Trp side chain rotamer state. Weak restraining of the Trp side chain dihedral angles to the state in the folded protein resulted in significant acceleration of the folding both starting from fully extended or intermediate conformations. The results indicate that the side chain conformation of the central Trp residue can create a significant barrier for controlling transitions to a near native folded structure. Similar mechanisms might be of importance for the folding of other protein structures. PMID:24563686

The effects of IGF1 on the melanogenesis in alpaca melanocytes in vitro.

PubMed

Hu, Shuaipeng; Liu, Yu; Yang, Shanshan; Ji, Kaiyuan; Liu, Xuexian; Zhang, Junzhen; Fan, Ruiwen; Dong, Changsheng

2016-09-01

In order to investigate the effects of the insulin-like growth factor 1(IGF-1) on alpaca melanocyte in vitro, different dosees of IGF1 (0, 10, 20, 40 ng/ml) were added in the medium of alpaca melanocyte. The RTCA machine was used to monitor the proliferation, quantitative real-time PCR, and western blot to test the relative gene expression, ELISA to test cAMP production, and spectrum method to test the melanin production. The results showed that compared to the normal melanocyte, the proliferation of melanocytes was increased within 60 h following adding IGF1. It also showed that cAMP content produced by melanocytes was increased, microphthalmia-associtated transcription factor (MITF), tyrosinase (TYR) and tyrosinase-related protein 2 (TYRP2) expression was increased, and melanin production with most obvious change in 10 ng/ml supplementary group, when compared with the control group. The results suggested that IGF1 with the dose of 10 ng/ml had the important effects on the melanogenesis in alpaca melanocyte by the cAMP pathway.

Suberoylanilide hydroxamic acid sensitizes neuroblastoma to paclitaxel by inhibiting thioredoxin-related protein 14-mediated autophagy.

PubMed

Zhen, Zijun; Yang, Kaibin; Ye, Litong; You, Zhiyao; Chen, Rirong; Liu, Ying; He, Youjian

2017-07-01

Paclitaxel is not as effective for neuroblastoma as most of the front-line chemotherapeutics due to drug resistance. This study explored the regulatory mechanism of paclitaxel-associated autophagy and potential solutions to paclitaxel resistance in neuroblastoma. The formation of autophagic vesicles was detected by scanning transmission electron microscopy and flow cytometry. The autophagy-associated proteins were assessed by western blot. Autophagy was induced and the autophagy-associated proteins LC3-I, LC3-II, Beclin 1, and thioredoxin-related protein 14 (TRP14), were found to be upregulated in neuroblastoma cells that were exposed to paclitaxel. The inhibition of Beclin 1 or TRP14 by siRNA increased the sensitivity of the tumor cells to paclitaxel. In addition, Beclin 1-mediated autophagy was regulated by TRP14. Furthermore, the TRP14 inhibitor suberoylanilide hydroxamic acid (SAHA) downregulated paclitaxel-induced autophagy and enhanced the anticancer effects of paclitaxel in normal control cancer cells but not in cells with upregulated Beclin 1 and TRP14 expression. Our findings showed that paclitaxel-induced autophagy in neuroblastoma cells was regulated by TRP14 and that SAHA could sensitize neuroblastoma cells to paclitaxel by specifically inhibiting TRP14. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Inhibitory effects of autolysate of Leuconostoc mesenteroides isolated from kimoto on melanogenesis.

PubMed

Kondo, Sayo; Takahashi, Toshinari; Yoshida, Kazutoshi; Mizoguchi, Haruhiko

2012-10-01

We investigated the inhibitory effects of the autolysate of Leuconostoc mesenteroides, a lactic acid bacterium isolated from sake mash, on melanogenesis in B16F0 murine melanoma cells and a human skin model. The autolysate: induced a decrease in melanin content in B16F0 murine melanoma cells and a 17%, 36%, 41% and 58% decrease in the human skin model by the application of 0.125, 1.25, 6.25, and 12.5 mg/tissue in total; decreased tyrosinase activity to 71%, 46% and 29% of control in B16F0 cells with 0.1, 0.2 and 0.4 mg/ml-medium respectively, but did not inhibit tyrosinase activity under cell-free conditions; decreased amount of tyrosinase in a dose-dependent manner from 74% with 0.1 mg/ml to 33% with 0.4 mg/ml; and decreased amount of tyrosinase mRNA to 80-90% with 0.2-0.4 mg/ml-medium. As the decrease in tyrosinase mRNA levels could not fully account for the reduction in protein, we suggest that the autolysate had post-transcriptional effects rather than transcription inhibition. Our results indicate that the autolysate of L. mesenteroides has potential therapeutic use as an effective anti-melanogenic agent. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Structural and biophysical investigation of the interaction of a mutant Grb2 SH2 domain (W121G) with its cognate phosphopeptide.

PubMed

Papaioannou, Danai; Geibel, Sebastian; Kunze, Micha B A; Kay, Christopher W M; Waksman, Gabriel

2016-03-01

The adaptor protein Grb2 is a key element of mitogenetically important signaling pathways. With its SH2 domain it binds to upstream targets while its SH3 domains bind to downstream proteins thereby relaying signals from the cell membranes to the nucleus. The Grb2 SH2 domain binds to its targets by recognizing a phosphotyrosine (pY) in a pYxNx peptide motif, requiring an Asn at the +2 position C-terminal to the pY with the residue either side of this Asn being hydrophobic. Structural analysis of the Grb2 SH2 domain in complex with its cognate peptide has shown that the peptide adopts a unique β-turn conformation, unlike the extended conformation that phosphopeptides adopt when bound to other SH2 domains. TrpEF1 (W121) is believed to force the peptide into this unusual conformation conferring this unique specificity to the Grb2 SH2 domain. Using X-ray crystallography, electron paramagnetic resonance (EPR) spectroscopy, and isothermal titration calorimetry (ITC), we describe here a series of experiments that explore the role of TrpEF1 in determining the specificity of the Grb2 SH2 domain. Our results demonstrate that the ligand does not adopt a pre-organized structure before binding to the SH2 domain, rather it is the interaction between the two that imposes the hairpin loop to the peptide. Furthermore, we find that the peptide adopts a similar structure when bound to both the wild-type Grb2 SH2 domain and a TrpEF1Gly mutant. This suggests that TrpEF1 is not the determining factor for the conformation of the phosphopeptide. © 2015 The Protein Society.

Structure-based function prediction of the expanding mollusk tyrosinase family

NASA Astrophysics Data System (ADS)

Huang, Ronglian; Li, Li; Zhang, Guofan

2017-11-01

Tyrosinase (Ty) is a common enzyme found in many different animal groups. In our previous study, genome sequencing revealed that the Ty family is expanded in the Pacific oyster ( Crassostrea gigas). Here, we examine the larger number of Ty family members in the Pacific oyster by high-level structure prediction to obtain more information about their function and evolution, especially the unknown role in biomineralization. We verified 12 Ty gene sequences from Crassostrea gigas genome and Pinctada fucata martensii transcriptome. By using phylogenetic analysis of these Tys with functionally known Tys from other molluscan species, eight subgroups were identified (CgTy_s1, CgTy_s2, MolTy_s1, MolTy-s2, MolTy-s3, PinTy-s1, PinTy-s2 and PviTy). Structural data and surface pockets of the dinuclear copper center in the eight subgroups of molluscan Ty were obtained using the latest versions of prediction online servers. Structural comparison with other Ty proteins from the protein databank revealed functionally important residues (HA1, HA2, HA3, HB1, HB2, HB3, Z1-Z9) and their location within these protein structures. The structural and chemical features of these pockets which may related to the substrate binding showed considerable variability among mollusks, which undoubtedly defines Ty substrate binding. Finally, we discuss the potential driving forces of Ty family evolution in mollusks. Based on these observations, we conclude that the Ty family has rapidly evolved as a consequence of substrate adaptation in mollusks.

Structural basis for the substrate selectivity of a HAD phosphatase from Thermococcus onnurineus NA1.

PubMed

Ngo, Tri Duc; Van Le, Binh; Subramani, Vinod Kumar; Thi Nguyen, Chi My; Lee, Hyun Sook; Cho, Yona; Kim, Kyeong Kyu; Hwang, Hye-Yeon

2015-05-22

Proteins in the haloalkaloic acid dehalogenase (HAD) superfamily, which is one of the largest enzyme families, is generally composed of a catalytic core domain and a cap domain. Although proteins in this family show broad substrate specificities, the mechanisms of their substrate recognition are not well understood. In this study, we identified a new substrate binding motif of HAD proteins from structural and functional analyses, and propose that this motif might be crucial for interacting with hydrophobic rings of substrates. The crystal structure of TON_0338, one of the 17 putative HAD proteins identified in a hyperthermophilic archaeon, Thermococcus onnurineus NA1, was determined as an apo-form at 2.0 Å resolution. In addition, we determined the crystal structure TON_0338 in complex with Mg(2+) or N-cyclohexyl-2-aminoethanesulfonic acid (CHES) at 1.7 Å resolution. Examination of the apo-form and CHES-bound structures revealed that CHES is sandwiched between Trp58 and Trp61, suggesting that this Trp sandwich might function as a substrate recognition motif. In the phosphatase assay, TON_0338 was shown to have high activity for flavin mononucleotide (FMN), and the docking analysis suggested that the flavin of FMN may interact with Trp58 and Trp61 in a way similar to that observed in the crystal structure. Moreover, the replacement of these tryptophan residues significantly reduced the phosphatase activity for FMN. Our results suggest that WxxW may function as a substrate binding motif in HAD proteins, and expand the diversity of their substrate recognition mode. Copyright © 2015 Elsevier Inc. All rights reserved.

Evaluation of the Antioxidant Activities and Tyrosinase Inhibitory Property from Mycelium Culture Extracts

PubMed Central

Park, Ki Moon; Kwon, Kyung Min; Lee, Seung Ho

2015-01-01

Since mushrooms have many bioactive components, they have been used as components in folk medicine. Because mycelium has an advantage when it comes to large-scale production, this study aimed to evaluate the antioxidant properties and anti-tyrosinase activity from 55 mycelia in culture media. Relatively high 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging capacity was detected from the ethanol extract of culture media including mycelium (EECiM) of Morchella esculenta var. esculenta (MEVE), Auricularia polytricha (APO), Tremella aurantia (TAU), Volvariella bombycina (VBO), and Oudemansiella sp. (Osp), which also showed strong reducing power and inhibitory activity in relation to the thiobarbituric acid (TBA) value. On the other hand, relatively high tyrosinase inhibitory activity was detected in Inonotus mikadoi (IMI), Coriolus versicolor (CVE), Volvariella volvacea (VVO), Panellus serotinus (PSE), Auricularia auricula (AAU), and Fomitopsis sp. (Fsp). Interestingly, the APO EECiM exhibited the highest DPPH radical scavenging rate (77.5 ± 4.3%) and reducing power (1.18 ± 0.041), while the highest inhibitory power of the TBA value and antityrosinase activity were detected in that of TAU (64.5 ± 4.1%) and IMI (46.0 ± 7.5%), respectively. Overall, our study suggested potential candidates for EECiMs that exhibited powerful antioxidant and tyrosinase inhibitory properties and might be used as natural antioxidant tyrosinase inhibitor. PMID:26345142

Unveiling the water-associated conformational mobility in the active site of ascorbate peroxidase.

PubMed

Chao, Wei-Chih; Lin, Li-Ju; Lu, Jyh-Feng; Wang, Jinn-Shyan; Lin, Tzu-Chieh; Chen, Yi-Han; Chen, Yi-Ting; Yang, Hsiao-Ching; Chou, Pi-Tai

2018-03-01

We carried out comprehensive spectroscopic studies of wild type and mutants of ascorbate peroxidase (APX) to gain understanding of the conformational mobility of the active site. In this approach, three unnatural tryptophans were applied to replace the distal tryptophan (W41) in an aim to probe polarity/water environment near the edge of the heme-containing active site. 7-azatryptophan ((7-aza)Trp) is sensitive to environment polarity, while 2,7-azatryptophan ((2,7-aza)Trp) and 2,6-diazatryptophan ((2,6-aza)Trp) undergo excited-state water-catalyzed double and triple proton transfer, respectively, and are sensitive to the water network. The combination of their absorption, emission bands and the associated relaxation dynamics of these fluorescence probes, together with the Soret-band difference absorption and resonance Raman spectroscopy, lead us to unveil the water associated conformational mobility in the active site of APX. The results are suggestive of the existence of equilibrium between two different environments surrounding W41 in APX, i.e., the water-rich and water-scant forms with distinct fluorescence relaxation. Our results thus demonstrate for the first time the power of integrating multiple sensors (7-aza)Trp, (2,7-aza)Trp and (2,6-aza)Trp in probing the water environment of a specifically targeted Trp in proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Tyrosinase-catalyzed site-specific immobilization of engineered C-phycocyanin to surface

PubMed Central

Faccio, Greta; Kämpf, Michael M.; Piatti, Chiara; Thöny-Meyer, Linda; Richter, Michael

2014-01-01

Enzymatic crosslinking of proteins is often limited by the steric availability of the target residues, as of tyrosyl side chains in the case of tyrosinase. Carrying an N-terminal peptide-tag containing two tyrosine residues, the fluorescent protein C-phycocyanin HisCPC from Synechocystis sp. PCC6803 was crosslinked to fluorescent high-molecular weight forms with tyrosinase. Crosslinking with tyrosinase in the presence of L-tyrosine produced non fluorescent high-molecular weight products. Incubated in the presence of tyrosinase, HisCPC could also be immobilized to amino-modified polystyrene beads thus conferring a blue fluorescence. Crosslinking and immobilization were site-specific as both processes required the presence of the N-terminal peptide in HisCPC. PMID:24947668

Effects of ligand binding on the conformation and internal dynamics in specific regions of porcine pancreatic phospholipase A2 with tryptophan as a probe: a study combining time-resolved fluorescence spectroscopy and site-directed mutagenesis (same as p. 100)

NASA Astrophysics Data System (ADS)

Kuipers, Oscar; Vincent, Michel; Brochon, Jean-Claude; Verheij, Bert; de Haas, Gerard; Gallay, Jacques

1990-05-01

Exploration of the effect of ligand-protein interactions on conformational substates and internal dynamics in different regions of phospholipase A2 from porcine pancreas (PLA2), was performed by combining site-directed mutagenesis and time-resolved fluorescence measurements. The single tryptophan residue (Trp-3) in the wild type protein was replaced by a phenylalanine residue, whereafter Trp was substituted either for leucine-31 ,located in the calcium binding loop, or for phenylalanine-94, located at the "back side" of the enzyme, in a-helix E (Dijkstra et al., J. Mol. Biol., 147, 97-123, 1981). Analyses by the Maximum Entropy Method (MIEM) of the total fluorescence intensity decays, provide in each case a distribution of separate lifetime classes, which can be interpreted as reflecting the existence of discrete conformational substates in slow exchange with respect to the time-scale of the decay kinetics. The fluorescence decay of the W94 mutant is dominated by an extremely short excited state lifetime of ~60 ps, probably arising from the presence of two proximate disulfide bridges. Time-resolved fluorescence anisotropy studies show that the Trp residue near the NH2 terminus (Trp-3) undergoes a more limited rotational motion than the Trp-3 1 located in the calcium binding loop. The widest angular rotation is observed at position 94, in a-helix E. Calcium binding displays the strongest influence on the lifetime distribution of Trp-31: a major local conformation corresponding to a lifetime class with a barycenter value of ~5.5 ns and contributing to ~50% of the decay is selected. The conformations giving rise to the short lifetimes ((tau)1 and (tau)2 lifetime classes) become less important. The contribution of the third lifetime class (c3) stays at a constant value of 30%. In the presence of calcium, the amplitude of motion is wider than without the ion. There is virtually no effect of calcium binding on the lifetime distribution of the Trp residue at the 3 or the 94 position. Binding of the monomeric substrate analog n-dodecylphosphocholine (C12PN) in the presence of calcium hardly affects neither the Trp-3 excited state population distribution, nor its rotational dynamics. The binding of C12PN monomers to the W31 mutant further increases the contribution of the t4lifetime class at the expense of c2. A more restricted rotation of the Trp-31 residue is also induced. The binding of the micellar substrate analog n-hexadecylphosphocholine (C16PN) in the presence of calcium is very efficient in modifying the lifetime distribution of Trp-3. Essentially, one major broad lifetime population (centered at ~2.6 ns) is revealed by MEM analysis of the total intensity decay. The internal motion is slowed down and the angle of rotation is much smaller in this conformation. Neither the excited state lifetime distribution of Trp-31 nor its dynamics are affected by micelle binding relative to monomer binding. In conclusion, by placing a single Tip-residue at strategic positions along the peptide chain of PLA2, relevant to the binding of biological ligands, an excellent model system for the study of selective perturbations of conformational substates and internal dynamics is provided.

Lineage-specific expansion and loss of tyrosinase genes across platyhelminths and their induction profiles in the carcinogenic oriental liver fluke, Clonorchis sinensis.

PubMed

Kim, Seon-Hee; Bae, Young-An

2017-09-01

Tyrosinase provides an essential activity during egg production in diverse platyhelminths by mediating sclerotization of eggshells. In this study, we investigated the genomic and evolutionary features of tyrosinases in parasitic platyhelminths whose genomic information is available. A pair of paralogous tyrosinases was detected in most trematodes, whereas they were lost in cyclophyllidean cestodes. A pseudophyllidean cestode displaying egg biology similar to that of trematodes possessed an orthologous gene. Interestingly, one of the paralogous tyrosinases appeared to have been multiplied into three copies in Clonorchis sinensis and Opisthorchis viverrini. In addition, a fifth tyrosinase gene that was minimally transcribed through all developmental stages was further detected in these opisthorchiid genomes. Phylogenetic analyses demonstrated that the tyrosinase gene has undergone duplication at least three times in platyhelminths. The additional opisthorchiid gene arose from the first duplication. A paralogous copy generated from these gene duplications, except for the last one, seemed to be lost in the major neodermatans lineages. In C. sinensis, tyrosinase gene expressions were initiated following sexual maturation and the levels were significantly enhanced by the presence of O2 and bile. Taken together, our data suggest that tyrosinase has evolved lineage-specifically across platyhelminths related to its copy number and induction mechanism.

Physiological factors that regulate skin pigmentation.

PubMed

Yamaguchi, Yuji; Hearing, Vincent J

2009-01-01

More than 150 genes have been identified that affect skin color either directly or indirectly, and we review current understanding of physiological factors that regulate skin pigmentation. We focus on melanosome biogenesis, transport and transfer, melanogenic regulators in melanocytes, and factors derived from keratinocytes, fibroblasts, endothelial cells, hormones, inflammatory cells, and nerves. Enzymatic components of melanosomes include tyrosinase, tyrosinase-related protein 1, and dopachrome tautomerase, which depend on the functions of OA1, P, MATP, ATP7A, and BLOC-1 to synthesize eumelanins and pheomelanins. The main structural component of melanosomes is Pmel17/gp100/Silv, whose sorting involves adaptor protein 1A (AP1A), AP1B, AP2, and spectrin, as well as a chaperone-like component, MART-1. During their maturation, melanosomes move from the perinuclear area toward the plasma membrane. Microtubules, dynein, kinesin, actin filaments, Rab27a, melanophilin, myosin Va, and Slp2-a are involved in melanosome transport. Foxn1 and p53 up-regulate skin pigmentation via bFGF and POMC derivatives including alpha-MSH and ACTH, respectively. Other critical factors that affect skin pigmentation include MC1R, CREB, ASP, MITF, PAX3, SOX9/10, LEF-1/TCF, PAR-2, DKK1, SCF, HGF, GM-CSF, endothelin-1, prostaglandins, leukotrienes, thromboxanes, neurotrophins, and neuropeptides. UV radiation up-regulates most factors that increase melanogenesis. Further studies will elucidate the currently unknown functions of many other pigment genes/proteins. (c) 2009 International Union of Biochemistry and Molecular Biology, Inc.

Mutation in transforming growth factor beta induced protein associated with granular corneal dystrophy type 1 reduces the proteolytic susceptibility through local structural stabilization.

PubMed

Underhaug, Jarl; Koldsø, Heidi; Runager, Kasper; Nielsen, Jakob Toudahl; Sørensen, Charlotte S; Kristensen, Torsten; Otzen, Daniel E; Karring, Henrik; Malmendal, Anders; Schiøtt, Birgit; Enghild, Jan J; Nielsen, Niels Chr

2013-12-01

Hereditary mutations in the transforming growth factor beta induced (TGFBI) gene cause phenotypically distinct corneal dystrophies characterized by protein deposition in cornea. We show here that the Arg555Trp mutant of the fourth fasciclin 1 (FAS1-4) domain of the protein (TGFBIp/keratoepithelin/βig-h3), associated with granular corneal dystrophy type 1, is significantly less susceptible to proteolysis by thermolysin and trypsin than the WT domain. High-resolution liquid-state NMR of the WT and Arg555Trp mutant FAS1-4 domains revealed very similar structures except for the region around position 555. The Arg555Trp substitution causes Trp555 to be buried in an otherwise empty hydrophobic cavity of the FAS1-4 domain. The first thermolysin cleavage in the core of the FAS1-4 domain occurs on the N-terminal side of Leu558 adjacent to the Arg555 mutation. MD simulations indicated that the C-terminal end of helix α3' containing this cleavage site is less flexible in the mutant domain, explaining the observed proteolytic resistance. This structural change also alters the electrostatic properties, which may explain increased propensity of the mutant to aggregate in vitro with 2,2,2-trifluoroethanol. Based on our results we propose that the Arg555Trp mutation disrupts the normal degradation/turnover of corneal TGFBIp, leading to accumulation and increased propensity to aggregate through electrostatic interactions. © 2013.

Mutation in Transforming Growth Factor Beta Induced protein associated with Granular Corneal Dystrophy Type 1 Reduces the Proteolytic Susceptibility through Local Structural Stabilization#

PubMed Central

Underhaug, Jarl; Koldsø, Heidi; Runager, Kasper; Nielsen, Jakob Toudahl; Sørensen, Charlotte S.; Kristensen, Torsten; Otzen, Daniel E.; Karring, Henrik; Malmendal, Anders; Schiøtt, Birgit; Enghild, Jan J.; Nielsen, Niels Chr.

2014-01-01

Hereditary mutations in the transforming growth factor beta induced (TGFBI) gene cause phenotypically distinct corneal dystrophies characterized by protein deposition in cornea. We show here that the Arg555Trp mutant of the fourth fasciclin 1 (FAS1-4) domain of the protein (TGFBIp/keratoepithelin/βig-h3), associated with granular corneal dystrophy type 1, is significantly less susceptible to proteolysis by thermolysin and trypsin than the WT domain. High-resolution liquid-state NMR of the WT and Arg555Trp mutant FAS1-4 domains revealed very similar structures except for the region around position 555. The Arg555Trp substitution causes Trp555 to be buried in an otherwise empty hydrophobic cavity of the FAS1-4 domain. The first thermolysin cleavage in the core of the FAS1-4 domain occurs on the N-terminal side of Leu558 adjacent to the Arg555 mutation. MD simulations indicated that the C-terminal end of helix α3′ containing this cleavage site is less flexible in the mutant domain, explaining the observed proteolytic resistance. This structural change also alters the electrostatic properties, which may explain increased propensity of the mutant to aggregate in vitro with 2,2,2-trifluoroethanol. Based on our results we propose that the Arg555Trp mutation disrupts the normal degradation/turnover of corneal TGFBIp, leading to accumulation and increased propensity to aggregate through electrostatic interactions. PMID:24129074

A novel melanin inhibitor: hydroperoxy traxastane-type triterpene from flowers of Arnica montana.

PubMed

Maeda, Kazuhisa; Naitou, Tomoko; Umishio, Kenichi; Fukuhara, Tadao; Motoyama, Akira

2007-05-01

We isolated a novel inhibitor of melanin biosynthesis from the flowers of Arnica montana L. (Compositae), and identified it as a traxastane-type triterpene (3beta,16beta-dihydroxy-21alpha-hydroperoxy-20(30)-taraxastene) [1] by means of 1D or 2D-NMR and liquid chromatography/high-resolution mass spectrometry (LC-HR-MS). Compound [1] at the concentration of 0.53 muM completely inhibited melanin accumulation in cultured B16 melanoma cells. It is one of the most potent among known plant inhibitors of melanin biosynthesis in cultured cells, being 50 times more potent than 4-methoxyphenol, which is used as an anti-pigmentation agent. Its mechanism of action is considered to involve inhibition of transcriptional factor MITF-M (melanocyte-type isoform of microphthalmia-associated transcription factor), which would lead to a decrease of tyrosinase and related genes. We confirmed that compound [1] decreased the protein levels of tyrosinase and its related proteins in B16 melanoma cells. Further study revealed that a similar hydroperoxy triterpene also suppressed the melanin pigment accumulation of B16 melanoma cells. These results indicate that the hydroperoxy group may play an important role in the suppression of the melanin accumulation by compound [1].

A brief history of trp: commentary and personal perspective.

PubMed

Hardie, Roger C

2011-05-01

The history of the discovery of the transient receptor potential (TRP) cation channel superfamily began in 1969 with Cosens and Manning's isolation of the Drosophila transient receptor potential mutant, in which the photoreceptor response decays during continuous illumination. Early studies from Minke found that the elementary light response was unaffected in trp mutants, and he attributed the defect to an intermediate stage of phototransduction. Montell and Rubin cloned the trp gene in 1989: they recognised it as a transmembrane protein, but also concluded that it did not encode the light-sensitive channels. In 1991, Minke and Selinger proposed that TRP represented a Ca2+ transporter required for refilling intracellular InsP3-sensitive Ca2+ stores, in turn required for activation of the light-sensitive channels. Also in 1991, after developing a photoreceptor patch clamp preparation, I showed that the light-sensitive channels themselves were highly permeable to Ca2+, questioning the need for such a dedicated Ca2+ transporter. In 1992, in collaboration with Minke, I resolved this paradox by showing there were two classes of light-sensitive channels, one highly Ca2+ permeable and eliminated in trp mutants. This represented the first and compelling evidence that TRP represented a light-sensitive channel and was supported by the cloning of the second light-sensitive channel, TRPL, by Kelly's lab. Three years later, in 1995, the labs of Montell and Birnbaumer independently cloned TRPC1, the first of 29 vertebrate TRP isoforms distributed amongst seven subfamilies.

Novel synthetic kojic acid-methimazole derivatives inhibit mushroom tyrosinase and melanogenesis.

PubMed

Chen, Ming-Jen; Hung, Chih-Chuan; Chen, Yan-Ru; Lai, Shih-Ting; Chan, Chin-Feng

2016-12-01

In this study, two kojic acid-methimazole (2-mercapto-1-methylimidazole, MMI, 1) derivatives, 5-hydroxy-2-{[(1-methyl-1H-imidazol-2-yl)thio]methyl}-4H-pyran-4-one (compound 4) and 5-methoxy-2-{[(1-methyl-1H-imidazol-2-yl)thio]methyl}-4H-pyran-4-one (compound 5), were synthesized to examine their inhibitory kinetics on mushroom tyrosinase. Compound 4 exhibited a potent inhibitory effect on monophenolase activity in a dose-dependent manner, with an IC 50 value of 0.03 mM. On diphenolase activity, compound 4 exhibited a less inhibitory effect (IC 50  = 1.29 mM) but was stronger than kojic acid (IC 50  = 1.80 mM). Kinetic analysis indicated that compound 4 was both as a noncompetitive monophenolase and diphenolase inhibitor. By contrast, compound 5 exhibited no inhibitory effects on mushroom tyrosinase activity. The IC 50 value of compound 4 for the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was 4.09 mM, being much higher than the IC 50 of compound 4 for inhibiting the tyrosinase activity. The results indicated that the antioxidant activity of compound 4 may be partly related to the potent inhibitory effect on mushroom tyrosinase. Compound 4 also exerted a potent inhibitory effect on intracellular melanin formation in B16/F10 murine melanoma cells, and caused no cytotoxicity. Furthermore, compound 4 induced no adverse effects on the Hen's egg test-chorioallantoic membrane (HET-CAM). Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Interaction between G Protein-Coupled Receptor 143 and Tyrosinase: Implications for Understanding Ocular Albinism Type 1.

PubMed

De Filippo, Elisabetta; Schiedel, Anke C; Manga, Prashiela

2017-02-01

Developmental eye defects in X-linked ocular albinism type 1 are caused by G-protein coupled receptor 143 (GPR143) mutations. Mutations result in dysfunctional melanosome biogenesis and macromelanosome formation in pigment cells, including melanocytes and retinal pigment epithelium. GPR143, primarily expressed in pigment cells, localizes exclusively to endolysosomal and melanosomal membranes unlike most G protein-coupled receptors, which localize to the plasma membrane. There is some debate regarding GPR143 function and elucidating the role of this receptor may be instrumental for understanding neurogenesis during eye development and for devising therapies for ocular albinism type I. Many G protein-coupled receptors require association with other proteins to function. These G protein-coupled receptor-interacting proteins also facilitate fine-tuning of receptor activity and tissue specificity. We therefore investigated potential GPR143 interaction partners, with a focus on the melanogenic enzyme tyrosinase. GPR143 coimmunoprecipitated with tyrosinase, while confocal microscopy demonstrated colocalization of the proteins. Furthermore, tyrosinase localized to the plasma membrane when coexpressed with a GPR143 trafficking mutant. The physical interaction between the proteins was confirmed using fluorescence resonance energy transfer. This interaction may be required in order for GPR143 to function as a monitor of melanosome maturation. Identifying tyrosinase as a potential GPR143 binding protein opens new avenues for investigating the mechanisms that regulate pigmentation and neurogenesis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Apolipoprotein C-III Nanodiscs Studied by Site-Specific Tryptophan Fluorescence.

PubMed

Brisbois, Chase A; Lee, Jennifer C

2016-09-06

Apolipoprotein C-III (ApoC-III) is found on high-density lipoproteins (HDLs) and remodels 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles into HDL-like particles known as nanodiscs. Using single-Trp-containing ApoC-III mutants, we have studied local side chain environments and interactions in nanodiscs at positions W42, W54, and W65. Using transmission electron microscopy and circular dichroism spectroscopy, nanodiscs were characterized at the ultrastructural and secondary conformational levels, respectively. Nearly identical particles (15 ± 2 nm) were produced from all proteins containing approximately 25 ± 4 proteins per particle with an average helicity of 45-51% per protein. Distinct residue-specific fluorescence properties were observed with W54 residing in the most hydrophobic environment followed by W42 and W65. Interestingly, time-resolved anisotropy measurements revealed that Trp side chain mobility is uncorrelated to the polarity of its surroundings. W54 is the most mobile compared to W65 and W42, which are more immobile in a nanodisc-bound state. On the basis of Trp spectral comparisons of ApoC-III in micellar and vesicle environments, ApoC-III binding within nanodiscs more closely resembles a bilayer-bound state. Despite the nanodiscs being structurally similar, we found marked differences during nanodisc formation by the Trp variants as a function of temperature, with W42 behaving the most like the wild-type protein. Our data suggest that despite the modest mutations of Trp to Phe at two of the three native sites, the interfacial location of W42 is important for lipid binding and nanodisc assembly, which may be biologically meaningful as of the three Trp residues, only W42 is invariant among mammals.

Effect of tryptophan-rich egg protein hydrolysate on brain tryptophan availability, stress and performance.

PubMed

Markus, C Rob; Verschoor, E; Firk, C; Kloek, J; Gerhardt, C C

2010-10-01

Reduced brain serotonin function is involved in stress-related disturbances and may particularly occur under chronic stress. Although serotonin production directly depends on the availability of its plasma dietary amino acid precursor tryptophan (TRP), previously described effects of tryptophan-rich food sources on stress-related behavior are rather modest. Recently, an egg protein hydrolysate (EPH) was developed that showed a much greater effect on brain TRP availability than pure TRP and other TRP-food sources and therefore may be more effective for performance under stress. The aim of the present study was to investigate the effects of EPH compared to placebo protein on plasma amino acids, stress coping and performance in subjects with high and low chronic stress vulnerabilities. In a placebo-controlled, double-blind, crossover study, 17 participants with high and 18 participants with low chronic stress vulnerabilities were monitored for mood and performance under acute stress exposure either following intake of EPH or placebo. EPH significantly increased plasma TRP availability for uptake into the brain, decreased depressive mood in all subjects and improved perceptual-motor and vigilance performance only in low chronic stress-vulnerable subjects. The acute use of a TRP-rich egg protein hydrolysate (EPH) is an adequate method to increase plasma TRP for uptake into the brain and may be beneficial for perceptual-motor and vigilance performance in healthy volunteers. Copyright © 2010 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Influence of the ionic liquid [C4mpy][Tf2N] on the structure of the miniprotein Trp-cage.

PubMed

Baker, Joseph L; Furbish, Jeffrey; Lindberg, Gerrick E

2015-11-01

We examine the effect of the ionic liquid [C4mpy][Tf2N] on the structure of the miniprotein Trp-cage and contrast these results with the behavior of Trp-cage in water. We find the ionic liquid has a dramatic effect on Trp-cage, though many similarities with aqueous Trp-cage are observed. We assess Trp-cage folding by monitoring root mean square deviation from the crystallographic structure, radius of gyration, proline cis/trans isomerization state, protein secondary structure, amino acid contact formation and distance, and native and non-native contact formation. Starting from an unfolded configuration, Trp-cage folds in water at 298 K in less than 500 ns of simulation, but has very little mobility in the ionic liquid at the same temperature, which can be ascribed to the higher ionic liquid viscosity. At 365 K, the mobility of the ionic liquid is increased and initial stages of Trp-cage folding are observed, however Trp-cage does not reach the native folded state in 2 μs of simulation in the ionic liquid. Therefore, in addition to conventional molecular dynamics, we also employ scaled molecular dynamics to expedite sampling, and we demonstrate that Trp-cage in the ionic liquid does closely approach the aqueous folded state. Interestingly, while the reduced mobility of the ionic liquid is found to restrict Trp-cage motion, the ionic liquid does facilitate proline cis/trans isomerization events that are not seen in our aqueous simulations. Copyright © 2015 Elsevier Inc. All rights reserved.

Heat sensitization in skin and muscle nociceptors expressing distinct combinations of TRPV1 and TRPV2 protein.

PubMed

Rau, K K; Jiang, N; Johnson, R D; Cooper, B Y

2007-04-01

Recordings were made from small and medium diameter dorsal root ganglia (DRG) neurons that expressed transient receptor potential (TRP) proteins. Physiologically characterized skin nociceptors expressed either TRPV1 (type 2) or TRPV2 (type 4) in isolation. Other nociceptors co-expressed both TRP proteins and innervated deep tissue sites (gastrocnemius muscle, distal colon; type 5, type 8) and skin (type 8). Subpopulations of myelinated (type 8) and unmyelinated (type 5) nociceptors co-expressed both TRPs. Cells that expressed TRPV1 were excellent transducers of intense heat. Proportional inward currents were obtained from a threshold of approximately 46.5 to approximately 56 degrees C. In contrast, cells expressing TRPV2 alone (52 degrees C threshold) did not reliably transduce the intensity of thermal events. Studies were undertaken to assess the capacity of skin and deep nociceptors to exhibit sensitization to repeated intense thermal stimuli [heat-heat sensitization (HHS)]. Only nociceptors that expressed TRPV2, alone or in combination with TRPV1, exhibited HHS. HHS was shown to be Ca(2+) dependent in either case. Intracellular Ca(2+) dependent pathways to HHS varied with the pattern of TRP protein expression. Cells co-expressing both TRPs modulated heat reactivity through serine/threonine phosphorylation or PLA(2)-dependent pathways. Cells expressing only TRPV2 may have relied on tyrosine kinases for HHS. We conclude that heat sensitization in deep and superficial capsaicin and capsaicin-insensitive C and Adelta nociceptors varies with the distribution of TRPV1 and TRPV2 proteins. The expression pattern of these proteins are specific to subclasses of physiologically identified C and A fiber nociceptors with highly restricted tissue targets.

Effects of ligand binding on the conformation and internal dynamics in specific regions of porcine pancreatic phospholipase A2 with tryptophan as a probe: a study combinging time-resolved fluorescence spectroscopy and site-directed mutagenesis (same as p. 628)

NASA Astrophysics Data System (ADS)

Kuipers, Oscar; Vincent, Michel; Brochon, Jean-Claude; Verheij, Bert; de Haas, Gerard; Gallay, Jacques

1990-05-01

Exploration of the effect of ligand-protein interactions on conformational substates and internal dynamics in different regions of phospholipase A2 from porcine pancreas (PLA2), was performed by combining site-directed mutagenesis and time-resolved fluorescence measurements. The single tryptophan residue (Trp-3) in the wild type protein was replaced by a phenylalanine residue, whereafter Tip was substituted either for leucine-3 1 ,located in the calcium binding ioop, or for phenylalanine-94, located at the "back side" of the enzyme, in a-helix E (Dijkstra et al., J. Mol. Biol., 147, 97-123, 1981). Analyses by the Maximum Entropy Method (MEM) of the total fluorescence intensity decays, provide in each case a distribution of separate lifetime classes, which can be interpreted as reflecting the existence of discrete conformational substates in slow exchange with respect to the time-scale of the decay kinetics. The fluorescence decay of the W94 mutant is. dominated by an extremely short excited state lifetime of ~60 ps, probably arising from the presence of two proximate disulfide bridges. Time-resolved fluorescence anisotropy studies show that the Trp residue near the NH2 terminus (Trp-3) undergoes a more limited rotational motion than the Trp-3 1 located in the calcium binding loop. The widest angular rotation is observed at position 94, in a-helix E. Calcium binding displays the strongest influence on the lifetime distribution of Trp-3 1: a major local conformation corresponding to a lifetime class with a barycenter value of -5.5 ns and contributing to ~50% of the decay is selected. The conformations giving rise to the short lifetimes (τ1 and τ2 lifetime classes) become less important. The contribution of the third lifetime class (c3) stays at a constant value of 30%. In the presence of calcium, the amplitude of motion is wider than without the ion. There is virtually no effect of calcium binding on the lifetime distribution of the Trp residue at the 3 or the 94 position. Binding of the monomeric substrate analog n-dodecylphosphocholine (C12PN) in the presence of calcium hardly affects neither the Trp-3 excited state population distribution, nor its rotational dynamics. The binding of C12PN monomers to the W31 mutant further increases the contribution of the τ4 lifetime class at the expense of c2. A more restricted rotation of the Trp-3 1 residue is also induced. The binding of the micellar substrate analog n-hexadecylphosphocholine (C16PN) in the presence of calcium is very efficient in modifying the lifetime distribution of Trp-3. Essentially, one major broad lifetime population (centered at ~2.6 ns) is revealed by MEM analysis of the total intensity decay. The internal motion is slowed down and the angle of rotation is much smaller in this conformation. Neither the excited state lifetime distribution of Trp-31 nor its dynamics are affected by micelle binding relative to monomer binding. In conclusion, by placing a single Tip-residue at strategic positions along the peptide chain of PLA2, relevant to the binding of biological ligands, an excellent model system for the study of selective perturbations of conformational substates and internal dynamics is provided.

Role of TRP ion channels in cancer and tumorigenesis.

PubMed

Shapovalov, George; Ritaine, Abigael; Skryma, Roman; Prevarskaya, Natalia

2016-05-01

Transient receptor potential (TRP) channels are recently identified proteins that form a versatile family of ion channels, the majority of which are calcium permeable and exhibit complex regulatory patterns with sensitivity to multiple environmental factors. While this sensitivity has captured early attention, leading to recognition of TRP channels as environmental and chemical sensors, many later studies concentrated on the regulation of intracellular calcium by TRP channels. Due to mutations, dysregulation of ion channel gating or expression levels, normal spatiotemporal patterns of local Ca(2+) distribution become distorted. This causes deregulation of downstream effectors sensitive to changes in Ca(2+) homeostasis that, in turn, promotes pathophysiological cancer hallmarks, such as enhanced survival, proliferation and invasion. These observations give rise to the appreciation of the important contributions that TRP channels make to many cellular processes controlling cell fate and positioning these channels as important players in cancer regulation. This review discusses the accumulated scientific knowledge focused on TRP channel involvement in regulation of cell fate in various transformed tissues.

MHY884, a newly synthesized tyrosinase inhibitor, suppresses UVB-induced activation of NF-κB signaling pathway through the downregulation of oxidative stress.

PubMed

Choi, Yeon Ja; Uehara, Yohei; Park, Ji Young; Kim, Seong Jin; Kim, So Ra; Lee, Hee Won; Moon, Hyung Ryong; Chung, Hae Young

2014-03-01

The skin is the primary target of prolonged and repeated ultraviolet (UVB) irradiation which induces cutaneous inflammation and pigmentation. Nuclear factor κB (NF-κB) is the major factor mediating UVB-induced inflammatory responses through the expression of various proinflammatory proteins such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). We have previously reported that the synthetic novel compound 4-(5-chloro-2,3-dihydrobenzo[d]thiazol-2-yl)-2,6-dimethoxyphenol (MHY884) strongly suppressed tyrosinase activity and melanin synthesis in B16F10 melanoma cells. In the present study, we investigated the effect of MHY884 on the inhibition of UVB-induced NF-κB activation and its proinflammatory downstream proteins through the suppression of oxidative stress in an in vivo model of photoaging. Generation of reactive oxygen species (ROS) and peroxynitrite was measured in vitro and in B16F10 melanoma cells to verify the scavenging activity of MHY884. MHY884 suppressed oxidative stress both in vitro and in the melanoma cells in a dose-dependent manner. Next, melanin-possessing hairless mice were pre-treated with MHY884 and then irradiated with UVB repeatedly. Topical application of MHY884 attenuated UVB-induced oxidative stress, resulting in reduced NF-κB activity. Pre-treatment with MHY884 inhibited Akt and IκB kinase α/β signaling pathways, leading to decreased translocation and phosphorylation of p65, a subunit of NF-κB. This result correlated with the expression levels of iNOS and COX-2 in the skin of MHY884-treated mice. Thus, the novel tyrosinase inhibitor MHY884 suppressed NF-κB activation signaling pathway by scavenging UVB-induced oxidative stress. The discovery of MHY884, a novel tyrosinase inhibitor that targets NF-κB signaling, is significant, because this compound is a promising protective agent against UVB-induced skin damage. Copyright © 2014 Elsevier Ltd. All rights reserved.

Metabolic activation of 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine and DNA adduct formation depends on p53: Studies in Trp53(+/+),Trp53(+/-) and Trp53(-/-) mice.

PubMed

Krais, Annette M; Speksnijder, Ewoud N; Melis, Joost P M; Singh, Rajinder; Caldwell, Anna; Gamboa da Costa, Gonçalo; Luijten, Mirjam; Phillips, David H; Arlt, Volker M

2016-02-15

The expression of the tumor suppressor p53 can influence the bioactivation of, and DNA damage induced by, the environmental carcinogen benzo[a]pyrene, indicating a role for p53 in its cytochrome P450 (CYP)-mediated biotransformation. The carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which is formed during the cooking of food, is also metabolically activated by CYP enzymes, particularly CYP1A2. We investigated the potential role of p53 in PhIP metabolism in vivo by treating Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice with a single oral dose of 50 mg/kg body weight PhIP. N-(Deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP-C8-dG) levels in DNA, measured by liquid chromatography-tandem mass spectrometry, were significantly lower in liver, colon, forestomach and glandular stomach of Trp53(-/-) mice compared to Trp53(+/+) mice. Lower PhIP-DNA adduct levels in the livers of Trp53(-/-) mice correlated with lower Cyp1a2 enzyme activity (measured by methoxyresorufin-O-demethylase activity) in these animals. Interestingly, PhIP-DNA adduct levels were significantly higher in kidney and bladder of Trp53(-/-) mice compared to Trp53(+/+) mice, which was accompanied by higher sulfotransferase (Sult) 1a1 protein levels and increased Sult1a1 enzyme activity (measured by 2-naphthylsulfate formation from 2-naphthol) in kidneys of these animals. Our study demonstrates a role for p53 in the metabolism of PhIP in vivo, extending previous results on a novel role for p53 in xenobiotic metabolism. Our results also indicate that the impact of p53 on PhIP biotransformation is tissue-dependent and that in addition to Cyp1a enzymes, Sult1a1 can contribute to PhIP-DNA adduct formation. © 2015 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.

Design, synthesis, and anti-melanogenic effects of (E)-2-benzoyl-3-(substituted phenyl)acrylonitriles

PubMed Central

Yun, Hwi Young; Kim, Do Hyun; Son, Sujin; Ullah, Sultan; Kim, Seong Jin; Kim, Yeon-Jeong; Yoo, Jin-Wook; Jung, Yunjin; Chun, Pusoon; Moon, Hyung Ryong

2015-01-01

Background Tyrosinase is the most prominent target for inhibitors of hyperpigmentation because it plays a critical role in melaninogenesis. Although many tyrosinase inhibitors have been identified, from both natural and synthetic sources, there remains a considerable demand for novel tyrosinase inhibitors that are safer and more effective. Methods (E)-2-Benzoyl-3-(substituted phenyl)acrylonitriles (BPA analogs) with a linear β-phenyl-α,β-unsaturated carbonyl scaffold were designed and synthesized as potential tyrosinase inhibitors. We evaluated their effects on cellular tyrosinase activity and melanin biosynthesis in murine B16F10 melanoma cells and their ability to inhibit mushroom tyrosinase activity. Results BPA analogs exhibited inhibitory activity against mushroom tyrosinase. In particular, BPA13 significantly suppressed melanin biosynthesis and inhibited cellular tyrosinase activity in B16F10 cells in a dose-dependent manner. A docking study revealed that BPA13 had higher binding affinity for tyrosinase than kojic acid. Conclusion BPA13, which possesses a linear β-phenyl-α,β-unsaturated carbonyl scaffold, is a potential candidate skin-whitening agent and treatment for diseases associated with hyperpigmentation. PMID:26347064

Activation of calcium-sensing receptor increases TRPC3 expression in rat cardiomyocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feng, Shan-Li; Sun, Ming-Rui; Li, Ting-Ting

Research highlights: {yields} Calcium-sensing receptor (CaR) activation stimulates TRP channels. {yields} CaR promoted transient receptor potential C3 (TRPC3) expression. {yields} Adult rat ventricular myocytes display capacitative calcium entry (CCE), which was operated by TRPCs. {yields} TRPC channels activation induced by CaR activator sustained the increased [Ca{sup 2+}]{sub i} to evoke cardiomyocytes apoptosis. -- Abstract: Transient receptor potential (TRP) channels are expressed in cardiomyocytes, which gate a type of influx of extracellular calcium, the capacitative calcium entry. TRP channels play a role in mediating Ca{sup 2+} overload in the heart. Calcium-sensing receptors (CaR) are also expressed in rat cardiac tissue andmore » promote the apoptosis of cardiomyocytes by Ca{sup 2+} overload. However, data about the link between CaR and TRP channels in rat heart are few. In this study, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were used to examine the expression of the TRP canonical proteins TRPC1 and TRPC3 in adult and neonatal rat cardiomyocytes. Laser scan confocal microscopy was used to detect intracellular [Ca{sup 2+}]{sub i} levels in isolated adult rat ventricular myocytes. The results showed that, in adult rat cardiomyocytes, the depletion of Ca{sup 2+} stores in the endoplasmic/sarcoplasmic reticulum (ER/SR) by thapsigargin induced a transient increase in [Ca{sup 2+}]{sub i} in the absence of [Ca{sup 2+}]{sub o} and the subsequent restoration of [Ca{sup 2+}]{sub o} sustained the increased [Ca{sup 2+}]{sub i} for a few minutes, whereas, the persisting elevation of [Ca{sup 2+}]{sub i} was reduced in the presence of the TRPC inhibitor SKF96365. The stimulation of CaR by its activator gadolinium chloride (GdCl{sub 3}) or spermine also resulted in the same effect and the duration of [Ca{sup 2+}]{sub i} increase was also shortened in the absence of [Ca{sup 2+}]{sub o}. In adult and neonatal rat cardiomyocytes, GdCl{sub 3} increased the expression of TRPC3 mRNA and protein, which were reversed by SKF96365 but not by inhibitors of the L-type channels and the Na{sup +}/Ca{sup 2+} exchangers. However, GdCl{sub 3} had no obvious effect on the expression of TRPC1 protein. These results suggested that CaR stimulation induced activation of TRP channels and promoted the expression of TRPC3, but not TRPC1, that sustained the increased [Ca{sup 2+}]{sub i}.« less

Anti-Melanogenic Properties of Greek Plants. A Novel Depigmenting Agent from Morus alba Wood.

PubMed

Chaita, Eliza; Lambrinidis, George; Cheimonidi, Christina; Agalou, Adamantia; Beis, Dimitris; Trougakos, Ioannis; Mikros, Emmanuel; Skaltsounis, Alexios-Leandros; Aligiannis, Nektarios

2017-03-23

In therapeutic interventions associated with melanin hyperpigmentation, tyrosinase is regarded as a target enzyme as it catalyzes the rate-limiting steps in mammalian melanogenesis. Since many known agents have been proven to be toxic, there has been increasing impetus to identify alternative tyrosinase inhibitors, especially from natural sources. In this study, we investigated 900 extracts from Greek plants for potential tyrosinase inhibitive properties. Among the five most potent extracts, the methanol extract of Morus alba wood (MAM) demonstrated a significant reduction in intracellular tyrosinase and melanin content in B16F10 melanoma cells. Bioassay-guided isolation led to the acquisition of twelve compounds: oxyresveratrol (1), kuwanon C (2), mulberroside A (3), resorcinol (4), dihydrooxyresveratol (5), trans-dihydromorin (6), 2,4,3'-trihydroxydihydrostilbene (7), kuwanon H (8), 2,4-dihydroxybenzaldehyde (9), morusin (10), moracin M (11) and kuwanon G (12). Among these, 2,4,3'-trihydroxydihydrostilbene (7) is isolated for the first time from Morus alba and constitutes a novel potent tyrosinase inhibitor (IC50 0.8 ± 0.15). We report here for the first time dihydrooxyresveratrol (5) as a potent natural tyrosinase inhibitor (IC50 0.3 ± 0.05). Computational docking analysis indicated the binding modes of six tyrosinase inhibitors with the aminoacids of the active centre of tyrosinase. Finally, we found both MAM extract and compounds 1, 6 and 7 to significantly suppress in vivo melanogenesis during zebrafish embryogenesis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu Yanning; Beijing Center for Diseases Prevention and Control, 16 Hepingli Middle Street, Dongcheng District, Beijing 100013; Neo, T.L.

SARS-CoV entry is mediated by spike glycoprotein. During the viral and host cellular membrane fusion, HR1 and HR2 form 6-helix bundle, positioning the fusion peptide closely to the C-terminal region of ectodomain to drive apposition and subsequent membrane fusion. Connecting to the HR2 region is a Trp-rich region which is absolutely conserved in members of coronaviruses. To investigate the importance of Trp-rich region in SARS-CoV entry, we produced different mutated S proteins using Alanine scan strategy. SARS-CoV pseudotyped with mutated S protein was used to measure viral infectivity. To restore the aromaticity of Ala-mutants, we performed rescue experiments using phenylalaninemore » substitutions. Our results show that individually substituted Ala-mutants substantially decrease infectivity by >90%, global Ala-mutants totally abrogated infectivity. In contrast, Phe-substituted mutants are able to restore 10-25% infectivity comparing to the wild-type. The results suggest that the Trp-rich region of S protein is essential for SARS-CoV infectivity.« less

Uterine deletion of Trp53 compromises antioxidant responses in mouse decidua

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burnum, Kristin E.; Hirota, Yasushi; Baker, Erin Shammel

2012-09-01

Preterm birth is a global health issue impacting both mothers and children. However, the etiology of preterm birth is not clearly understood. From our recent finding that premature decidual senescence with terminal differentiation is a cause of preterm birth in mice with uterine Trp53 deletion, encoding p53 protein, led us to explore other potential factors that are related to preterm birth. Utilizing proteomics approaches, here we show that 183 candidate proteins cause significant changes in decidua with Trp53 deletion as compared to normal decidua. Functional categorization of these proteins unveiled new pathways that are influenced by p53. In particular, downregulationmore » of a cluster of antioxidant proteins in p53 deficient decidua suggests that increased oxidative stress could be one cause of preterm birth in mice with uterine deletion of Trp53.« less

Activation of neurokinin-1 receptor by substance P inhibits melanogenesis in B16-F10 melanoma cells.

PubMed

Ping, Fengfeng; Shang, Jing; Zhou, Jia; Song, Jing; Zhang, Luyong

2012-12-01

Skin pigmentation plays a number of valuable roles and its regulation is a complex process that is controlled by different factors. Substance P (SP) regulates many biological functions, including neurogenic inflammation, pain, and stress. However, to date, the regulatory role of SP in the control of melanogenesis has not been elucidated. The present study was designed to investigate the effects of SP on melanogenesis and to elucidate its underlying mechanism(s). After treatment for 48 h in mouse B16-F10 melanoma cells, SP (1 and 10nM) significantly down-regulated tyrosinase activity and melanin content. Importantly, western blot analysis revealed the presence of neurokinin-1 receptor (NK-1 R) in B16-F10 cells and the activation of it after SP treatment. It was also found that preincubation with NK-1 receptor antagonist Spantide I could partially reversed SP-induced down-regulations of tyrosinase activity, melanin content and the expression of tyrosinase and tyrosinase-related protein 1. Furthermore, SP could remarkably inhibit the expressions of microphtalmia-associated transcription factor (MITF) and p-p38 MAPK and stimulated p-p70 S6K1. These effects could also be partially reversed by the pretreatment with Spantide I. These results collectively suggested that SP inhibited melanogenesis in B16-F10 cells, which might be attributed to the fact that SP induces the activation of NK-1 receptor, stimulates the phosphorylation of p70 S6K1 and inhibits that of p38 MAPK, decreases the tyrosinase and tyrosinase-related protein 1 expression through MITF, finally resulting in the suppression of melanogenesis. These observations in vitro indicated that the regulation of the SP/NK-1 receptor system might be a useful novel management for skin pigmentation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Biological activity and molecular docking studies of curcumin-related α,β-unsaturated carbonyl-based synthetic compounds as anticancer agents and mushroom tyrosinase inhibitors.

PubMed

Bukhari, Syed Nasir Abbas; Jantan, Ibrahim; Unsal Tan, Oya; Sher, Muhammad; Naeem-Ul-Hassan, M; Qin, Hua-Li

2014-06-18

Hyperpigmentation in human skin and enzymatic browning in fruits, which are caused by tyrosinase enzyme, are not desirable. Investigations in the discovery of tyrosinase enzyme inhibitors and search for improved cytotoxic agents continue to be an important line in drug discovery and development. In present work, a new series of 30 compounds bearing α,β-unsaturated carbonyl moiety was designed and synthesized following curcumin as model. All compounds were evaluated for their effects on human cancer cell lines and mushroom tyrosinase enzyme. Moreover, the structure-activity relationships of these compounds are also explained. Molecular modeling studies of these new compounds were carried out to explore interactions with tyrosinase enzyme. Synthetic curcumin-like compounds (2a-b) were identified as potent anticancer agents with 81-82% cytotoxicity. Five of these newly synthesized compounds (1a, 8a-b, 10a-b) emerged to be the potent inhibitors of mushroom tyrosinase, providing further insight into designing compounds useful in fields of food, health, and agriculture.

International Union of Basic and Clinical Pharmacology. LXXVI. Current Progress in the Mammalian TRP Ion Channel Family

PubMed Central

Wu, Long-Jun; Sweet, Tara-Beth

2010-01-01

Transient receptor potential (TRP) channels are a large family of ion channel proteins, surpassed in number in mammals only by voltage-gated potassium channels. TRP channels are activated and regulated through strikingly diverse mechanisms, making them suitable candidates for cellular sensors. They respond to environmental stimuli such as temperature, pH, osmolarity, pheromones, taste, and plant compounds, and intracellular stimuli such as Ca2+ and phosphatidylinositol signal transduction pathways. However, it is still largely unknown how TRP channels are activated in vivo. Despite the uncertainties, emerging evidence using TRP channel knockout mice indicates that these channels have broad function in physiology. Here we review the recent progress on the physiology, pharmacology and pathophysiological function of mammalian TRP channels. PMID:20716668

Menadione (Vitamin K3) decreases melanin synthesis through ERK activation in Mel-Ab cells.

PubMed

Kim, Eun-Hyun; Kim, Myo-Kyoung; Yun, Hye-Young; Baek, Kwang Jin; Kwon, Nyoun Soo; Park, Kyoung-Chan; Kim, Dong-Seok

2013-10-15

Menadione is a synthetic vitamin K3 derivative. Here, we examined the effects of menadione on melanogenesis and its related signaling pathways. Our results showed that melanin content was significantly reduced after menadione treatment in a dose-dependent manner. However, menadione treatment did not reduce tyrosinase activity directly. Wnt signaling is known to play a major role in the control of melanin synthesis. Thus, we tested the effects of menadione treatment on GSK3β and β-catenin signaling, but found that menadione did not influence either of these signaling pathways. We also investigated changes in the phosphorylation of ERK, which is related to melanin regulation. These results indicated that menadione treatment led to the phosphorylation of ERK. Additionally, menadione treatment reduced both MITF and tyrosinase protein levels. Treatment with PD98059, a specific ERK pathway inhibitor, restored menadione-induced melanin reduction and also prevented MITF and tyrosinase downregulation by menadione. These results suggest that the hypopigmentary action of menadione is due to MITF and tyrosinase downregulation by ERK activation. © 2013 Elsevier B.V. All rights reserved.

Role of TRP channels in the cardiovascular system

PubMed Central

Yue, Zhichao; Xie, Jia; Yu, Albert S.; Stock, Jonathan; Du, Jianyang

2014-01-01

The transient receptor potential (TRP) superfamily consists of a large number of nonselective cation channels with variable degree of Ca2+-permeability. The 28 mammalian TRP channel proteins can be grouped into six subfamilies: canonical, vanilloid, melastatin, ankyrin, polycystic, and mucolipin TRPs. The majority of these TRP channels are expressed in different cell types including both excitable and nonexcitable cells of the cardiovascular system. Unlike voltage-gated ion channels, TRP channels do not have a typical voltage sensor, but instead can sense a variety of other stimuli including pressure, shear stress, mechanical stretch, oxidative stress, lipid environment alterations, hypertrophic signals, and inflammation products. By integrating multiple stimuli and transducing their activity to downstream cellular signal pathways via Ca2+ entry and/or membrane depolarization, TRP channels play an essential role in regulating fundamental cell functions such as contraction, relaxation, proliferation, differentiation, and cell death. With the use of targeted deletion and transgenic mouse models, recent studies have revealed that TRP channels are involved in numerous cellular functions and play an important role in the pathophysiology of many diseases in the cardiovascular system. Moreover, several TRP channels are involved in inherited diseases of the cardiovascular system. This review presents an overview of current knowledge concerning the physiological functions of TRP channels in the cardiovascular system and their contributions to cardiovascular diseases. Ultimately, TRP channels may become potential therapeutic targets for cardiovascular diseases. PMID:25416190

Role of TRP channels in the cardiovascular system.

PubMed

Yue, Zhichao; Xie, Jia; Yu, Albert S; Stock, Jonathan; Du, Jianyang; Yue, Lixia

2015-02-01

The transient receptor potential (TRP) superfamily consists of a large number of nonselective cation channels with variable degree of Ca(2+)-permeability. The 28 mammalian TRP channel proteins can be grouped into six subfamilies: canonical, vanilloid, melastatin, ankyrin, polycystic, and mucolipin TRPs. The majority of these TRP channels are expressed in different cell types including both excitable and nonexcitable cells of the cardiovascular system. Unlike voltage-gated ion channels, TRP channels do not have a typical voltage sensor, but instead can sense a variety of other stimuli including pressure, shear stress, mechanical stretch, oxidative stress, lipid environment alterations, hypertrophic signals, and inflammation products. By integrating multiple stimuli and transducing their activity to downstream cellular signal pathways via Ca(2+) entry and/or membrane depolarization, TRP channels play an essential role in regulating fundamental cell functions such as contraction, relaxation, proliferation, differentiation, and cell death. With the use of targeted deletion and transgenic mouse models, recent studies have revealed that TRP channels are involved in numerous cellular functions and play an important role in the pathophysiology of many diseases in the cardiovascular system. Moreover, several TRP channels are involved in inherited diseases of the cardiovascular system. This review presents an overview of current knowledge concerning the physiological functions of TRP channels in the cardiovascular system and their contributions to cardiovascular diseases. Ultimately, TRP channels may become potential therapeutic targets for cardiovascular diseases. Copyright © 2015 the American Physiological Society.

Mutagenesis of the C2 domain of protein kinase C-alpha. Differential roles of Ca2+ ligands and membrane binding residues.

PubMed

Medkova, M; Cho, W

1998-07-10

The C2 domains of conventional protein kinase C (PKC) have been implicated in their Ca2+-dependent membrane binding. The C2 domain of PKC-alpha contains several Ca2+ ligands that bind multiple Ca2+ ions and other putative membrane binding residues. To understand the roles of individual Ca2+ ligands and protein-bound Ca2+ ions in the membrane binding and activation of PKC-alpha, we mutated five putative Ca2+ ligands (D187N, D193N, D246N, D248N, and D254N) and measured the effects of mutations on vesicle binding, enzyme activity, and monolayer penetration of PKC-alpha. Altered properties of these mutants indicate that individual Ca2+ ions and their ligands have different roles in the membrane binding and activation of PKC-alpha. The binding of Ca2+ to Asp187, Asp193, and Asp246 of PKC-alpha is important for the initial binding of protein to membrane surfaces. On the other hand, the binding of another Ca2+ to Asp187, Asp246, Asp248, and Asp254 induces the conformational change of PKC-alpha, which in turn triggers its membrane penetration and activation. Among these Ca2+ ligands, Asp246 was shown to be most essential for both membrane binding and activation of PKC-alpha, presumably due to its coordination to multiple Ca2+ ions. Furthermore, to identify the residues in the C2 domain that are involved in membrane binding of PKC-alpha, we mutated four putative membrane binding residues (Trp245, Trp247, Arg249, and Arg252). Membrane binding and enzymatic properties of two double-site mutants (W245A/W247A and R249A/R252A) indicate that Arg249 and Arg252 are involved in electrostatic interactions of PKC-alpha with anionic membranes, whereas Trp245 and Trp247 participate in its penetration into membranes and resulting hydrophobic interactions. Taken together, these studies provide the first experimental evidence for the role of C2 domain of conventional PKC as a membrane docking unit as well as a module that triggers conformational changes to activate the protein.

Nrf2 inhibits oxaliplatin-induced peripheral neuropathy via protection of mitochondrial function.

PubMed

Yang, Yang; Luo, Lan; Cai, Xueting; Fang, Yuan; Wang, Jiaqi; Chen, Gang; Yang, Jie; Zhou, Qian; Sun, Xiaoyan; Cheng, Xiaolan; Yan, Huaijiang; Lu, Wuguang; Hu, Chunping; Cao, Peng

2018-05-20

Oxaliplatin-induced peripheral neuropathy (OIPN) is a severe, dose-limiting toxicity associated with cancer chemotherapy. The efficacy of antioxidant administration in OIPN is debatable, as the promising preliminary results obtained with a number of antioxidants have not been confirmed in larger clinical trials. Besides its antioxidant activity, the transcription factor, nuclear factor-erythroid 2 (NF-E2) p45-related factor 2 (Nrf2) plays a crucial role in the maintenance of mitochondrial homeostasis, and mitochondrial dysfunction is a key contributor to OIPN. Here, we have investigated the protective properties of Nrf2 in OIPN. Nrf2 -/- mice displayed severe mechanical allodynia and cold sensitivity and thus experienced increased peripheral nervous system injury compared to Nrf2 +/+ mice. Furthermore, Nrf2 knockout aggravated oxaliplatin-induced reactive oxygen species production, decreased the mitochondrial membrane potential, led to abnormal intracellular calcium levels, and induced cytochrome c-related apoptosis and overexpression of the TRP protein family. Sulforaphane-induced activation of the Nrf2 signaling pathway alleviated morphological alterations, mitochondrial dysfunction in dorsal root ganglion neurons, and nociceptive sensations in mice. Our findings reveal that Nrf2 may play a critical role in ameliorating OIPN, through protection of mitochondrial function by alleviating oxidative stress and inhibiting TRP protein family expression. This suggests that pharmacological or therapeutic activation of Nrf2 may be used to prevent or slow down the progression of OIPN. Copyright © 2018 Elsevier Inc. All rights reserved.

Spectroscopic evidence for an engineered, catalytically active Trp radical that creates the unique reactivity of lignin peroxidase.

PubMed

Smith, Andrew T; Doyle, Wendy A; Dorlet, Pierre; Ivancich, Anabella

2009-09-22

The surface oxidation site (Trp-171) in lignin peroxidase (LiP) required for the reaction with veratryl alcohol a high-redox-potential (1.4 V) substrate, was engineered into Coprinus cinereus peroxidase (CiP) by introducing a Trp residue into a heme peroxidase that has similar protein fold but lacks this activity. To create the catalytic activity toward veratryl alcohol in CiP, it was necessary to reproduce the Trp site and its negatively charged microenvironment by means of a triple mutation. The resulting D179W+R258E+R272D variant was characterized by multifrequency EPR spectroscopy. The spectra unequivocally showed that a new Trp radical [g values of g(x) = 2.0035(5), g(y) = 2.0027(5), and g(z) = 2.0022(1)] was formed after the [Fe(IV)=O Por(*+)] intermediate, as a result of intramolecular electron transfer between Trp-179 and the porphyrin. Also, the EPR characterization crucially showed that [Fe(IV)=O Trp-179(*)] was the reactive intermediate with veratryl alcohol. Accordingly, our work shows that it is necessary to take into account the physicochemical properties of the radical, fine-tuned by the microenvironment, as well as those of the preceding [Fe(IV)=O Por(*+)] intermediate to engineer a catalytically competent Trp site for a given substrate. Manipulation of the microenvironment of the Trp-171 site in LiP allowed the detection by EPR spectroscopy of the Trp-171(*), for which direct evidence has been missing so far. Our work also highlights the role of Trp residues as tunable redox-active cofactors for enzyme catalysis in the context of peroxidases with a unique reactivity toward recalcitrant substrates that require oxidation potentials not realized at the heme site.

Tryptophan depletion under conditions that imitate insulin resistance enhances fatty acid oxidation and induces endothelial dysfunction through reactive oxygen species-dependent and independent pathways.

PubMed

Eleftheriadis, Theodoros; Pissas, Georgios; Sounidaki, Maria; Antoniadi, Georgia; Rountas, Christos; Liakopoulos, Vassilios; Stefanidis, Loannis

2017-04-01

In atherosclerosis-associated pathologic entities characterized by malnutrition and inflammation, L-tryptophan (TRP) levels are low. Insulin resistance is an independent cardiovascular risk factor and induces endothelial dysfunction by increasing fatty acid oxidation. It is also associated with inflammation and low TRP levels. Low TRP levels have been related to worse cardiovascular outcome. This study evaluated the effect of TRP depletion on endothelial dysfunction under conditions that imitate insulin resistance. Fatty acid oxidation, harmful pathways due to increased fatty acid oxidation, and endothelial dysfunction were assessed in primary human aortic endothelial cells cultured under normal glucose, low insulin conditions in the presence or absence of TRP. TRP depletion activated general control non-derepressible 2 kinase and inhibited aryl hydrocarbon receptor. It increased fatty acid oxidation by increasing expression and activity of carnitine palmitoyltransferase 1. Elevated fatty acid oxidation increased the formation of reactive oxygen species (ROS) triggering the polyol and hexosamine pathways, and enhancing protein kinase C activity and methylglyoxal production. TRP absence inhibited nitric oxide synthase activity in a ROS-dependent way, whereas it increased the expression of ICAM-1 and VCAM-1 in a ROS independent and possibly p53-dependent manner. Thus, TRP depletion, an amino acid whose low levels have been related to worse cardiovascular outcome and to inflammatory atherosclerosis-associated pathologic entities, under conditions that imitate insulin resistance enhances fatty acid oxidation and induces endothelial dysfunction through ROS-dependent and independent pathways. These findings may offer new insights at the molecular mechanisms involved in accelerated atherosclerosis that frequently accompanies malnutrition and inflammation.

Ethyl acetate extract from Panax ginseng C.A. Meyer and its main constituents inhibit α-melanocyte-stimulating hormone-induced melanogenesis by suppressing oxidative stress in B16 mouse melanoma cells.

PubMed

Jiang, Rui; Xu, Xiao-Hao; Wang, Ke; Yang, Xin-Zhao; Bi, Ying-Fei; Yan, Yao; Liu, Jian-Zeng; Chen, Xue-Nan; Wang, Zhen-Zhong; Guo, Xiao-Li; Zhao, Da-Qing; Sun, Li-Wei

2017-08-17

Hyperpigmentation disease involves darkening of the skin color due to melanin overproduction. Panax ginseng C.A. Meyer is a well-known traditional Chinese medicine and has a long history of use as a skin lightener to inhibit melanin formation in China, Korea and some other Asian countries. However, the constituents and the molecular mechanisms by which they affect melanogenesis are not fully clear. The purpose of this study was to identify the active ingredient in Panax ginseng C.A. Meyer extract that inhibits mushroom tyrosinase activity and to investigate the antioxidative capacity and molecular mechanisms of the effective extract on melanogenesis in B16 mouse melanoma cells. Aqueous extracts of Panax ginseng C.A. Meyer were successively fractionated with an equal volume of chloroform, ethyl acetate, and n-butyl alcohol to determine the effects by examining the activity of mushroom tyrosinase. The effective fraction was analyzed using HPLC and LC-MS. The antioxidative capacity and the inhibitory effects on melanin content, cell intracellular tyrosinase activity, and melanogenesis protein levels were determined in α-melanocyte-stimulating hormone (α-MSH)-treated B16 mouse melanoma cells. The ethyl acetate extract from Panax ginseng C.A. Meyer (PG-2) had the highest inhibiting effect on mushroom tyrosinase, mainly contained phenolic acids, including protocatechuic acid, vanillic acid, p-coumaric acid, salicylic acid, and caffeic acid, and exhibited apparent antioxidant activity in vitro. PG-2 and its main constituents significantly decreased melanin content, suppressed cellular tyrosinase activity, and reduced expression of tyrosinase protein to inhibit B16 cells melanogenesis induced by α-MSH, and no cytotoxic effects were observed. They also inhibited cellular reactive oxygen species (ROS) generation, increased superoxide dismutase (SOD) activity and glutathione (GSH) level in α-MSH-treated B16 cells effectively. And those activities of its main constituents could reach more than 80% of PG-2. The ROS scavengers N-acetyl-L-cysteine (NAC) had a similar inhibitory effect on melanogenesis. These results suggest that ethyl acetate extract from Panax ginseng C.A. Meyer has the highest effect on inhibiting melanogenesis, and that its main components are polyphenolic compounds, which may inhibit melanogenesis by suppressing oxidative stress. This work provides new insight into the active constituents and molecular mechanisms underlying skin-lightening effect of Panax ginseng C.A. Meyer. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Inhibition of tyrosinase activity and melanine pigmentation by 2-hydroxytyrosol

PubMed Central

Uchida, Ryuji; Ishikawa, Seiko; Tomoda, Hiroshi

2014-01-01

2-Hydroxytyrosol (2-HT), originally reported as a synthetic compound, was isolated for the first time as a fungal metabolite. 2-HT was found to inhibit mushroom tyrosinase with an IC50 value of 13.0 µmol/L. Furthermore, 2-HT dose-dependently inhibited tyrosinase activity (IC50, 32.5 µmol/L) in the cell-free extract of B16 melanoma cells and α-melanocyte stimulating hormone (α-MSH)-stimulated melanin formation in intact B16 melanoma cells. PMID:26579376

Inhibition of tyrosinase activity and melanine pigmentation by 2-hydroxytyrosol.

PubMed

Uchida, Ryuji; Ishikawa, Seiko; Tomoda, Hiroshi

2014-04-01

2-Hydroxytyrosol (2-HT), originally reported as a synthetic compound, was isolated for the first time as a fungal metabolite. 2-HT was found to inhibit mushroom tyrosinase with an IC50 value of 13.0 µmol/L. Furthermore, 2-HT dose-dependently inhibited tyrosinase activity (IC50, 32.5 µmol/L) in the cell-free extract of B16 melanoma cells and α-melanocyte stimulating hormone (α-MSH)-stimulated melanin formation in intact B16 melanoma cells.

Synthesis, molecular docking studies of coumarinyl-pyrazolinyl substituted thiazoles as non-competitive inhibitors of mushroom tyrosinase.

PubMed

Saeed, Aamer; Mahesar, Parvez Ali; Channar, Pervaiz Ali; Abbas, Qamar; Larik, Fayaz Ali; Hassan, Mubashir; Raza, Hussain; Seo, Sung-Yum

2017-10-01

A series of coumarinyl-pyrazolinyl substituted thiazoles derivatives were synthesized and their inhibitory effects on the DPPH and mushroom tyrosinase were evaluated. The results showed that all of the synthesized compounds exhibited significant mushroom tyrosinase inhibitory activities. In particular, 3-(5-(4-(benzyloxy)-3-methoxyphenyl)-1-(4-(4-bromophenyl)thiazol-2-yl)-4,5-dihydro-1H-pyrazol-3-yl)-2H-chromen-2-one (7j) exhibited the most potent tyrosinase inhibitory activity with IC 50 value 0.00458±0.00022μM compared with the IC 50 value of kojic acid is 16.84±0.052μM. The inhibition mechanism analyzed by Lineweaver-Burk plots revealed that the type of inhibition of compound 7j on tyrosinase was noncompetitive. The docking study against tyrosinase enzyme was also performed to determine the binding affinity of the compounds. The compound 7a showed the highest binding affinity (-10.20kcal/mol) with active binding site of tyrosinase. The initial structure activity relationships (SARs) analysis suggested that further development of such compounds might be of interest. The statistics of our results endorses that compound 7j may serve asa structural template for the design and development of novel tyrosinase inhibitors. Copyright © 2017 Elsevier Inc. All rights reserved.

A Better Understanding of Protein Structure and Function by the Synthesis and Incorporation of Selenium- and Tellurium Containing Tryptophan Analogs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Helmey, Sherif Samir; Rice, Ambrose Eugene; Hatch, Duane Michael

Unnatural heavy metal-containing amino acid analogs have shown to be very important in the analysis of protein structure, using methods such as X-ray crystallography, mass spectroscopy, and NMR spectroscopy. Synthesis and incorporation of selenium-containing methionine analogs has already been shown in the literature however with some drawbacks due to toxicity to host organisms. Thus synthesis of heavy metal tryptophan analogs should prove to be more effective since the amino acid tryptophan is naturally less abundant in many proteins. For example, bioincorporation of β-seleno[3,2-b]pyrrolyl-L-alanine ([4,5]SeTrp) and β-selenolo[2,3-b]pyrrolyl-L-alanine ([6,7]SeTrp) has been shown in the following proteins without structural or catalytic perturbations: humanmore » annexin V, barstar, and dihydrofolate reductase. The reported synthesis of these Se-containing analogs is currently not efficient for commercial purposes. Thus a more efficient, concise, high-yield synthesis of selenotryptophan, as well as the corresponding, tellurotryptophan, will be necessary for wide spread use of these unnatural amino acid analogs. This research will highlight our progress towards a synthetic route of both [6,7]SeTrp and [6,7]TeTrp, which ultimately will be used to study the effect on the catalytic activity of Lignin Peroxidase (LiP).« less

Synthesis, kinetic mechanism and docking studies of vanillin derivatives as inhibitors of mushroom tyrosinase.

PubMed

Ashraf, Zaman; Rafiq, Muhammad; Seo, Sung-Yum; Babar, Mustafeez Mujtaba; Zaidi, Najam-us-Sahar Sadaf

2015-09-01

The purpose of the present study was to discover the extent of contribution to antityrosinase activity by adding hydroxy substituted benzoic acid, cinnamic acid and piperazine residues to vanillin. The study showed the transformation of vanillin into esters as shown in (4a-4d), (6a-6b), and (8a-8b). In addition, the relationship between structures of these esters and their mushroom tyrosinase inhibitory activity was explored. The kinetics of inhibition on mushroom tyrosinase by these esters was also investigated. It was found that hydroxyl substituted benzoic acid derivatives were weak inhibitors; however hydroxy or chloro substituted cinnamic acid and piperazine substituted derivatives were able to induce significant tyrosinase inhibition. The mushroom tyrosinase (PDBID 2ZWE) was docked with synthesized vanillin derivatives and their calculated binding energies were compared with experimental IC50 values which provided positive correlation. The most potent derivative 2-(4-formyl-2-methoxyphenoxy)-2-oxoethyl (2E)-3-(4-hydroxyphenyl)prop-2-enoate (6a) possesses hydroxy substituted cinnamic acid scaffold having IC50 value 16.13 μM with binding energy of -7.2 kcal/mol. The tyrosinase inhibitory activity of (6a) is comparable with standard kojic acid. Kinetic analysis indicated that compound 6a was mixed-type tyrosinase inhibitor with inhibition constant values Ki (13 μM) and Ki' (53 μM) and formed reversible enzyme inhibitor complex. The active vanillin analog (6a) was devoid of toxic effects as shown in cytotoxic studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

Design, synthesis, kinetic mechanism and molecular docking studies of novel 1-pentanoyl-3-arylthioureas as inhibitors of mushroom tyrosinase and free radical scavengers.

PubMed

Larik, Fayaz Ali; Saeed, Aamer; Channar, Pervaiz Ali; Muqadar, Urooj; Abbas, Qamar; Hassan, Mubashir; Seo, Sung-Yum; Bolte, Michael

2017-12-01

A series of novel 1-pentanoyl-3-arylthioureas was designed as new mushroom tyrosinase inhibitors and free radical scavengers. The title compounds were obtained in excellent yield and characterized by FTIR, 1 H NMR, 13 C NMR and X-ray crystallography in case of compound (4a). The inhibitory effects on mushroom tyrosinase and DPPH were evaluated and it was observed that 1-Pentanoyl-3-(4-methoxyphenyl) thiourea (4f) showed tyrosinase inhibitory activity (IC 50 1.568 ± 0.01 mM) comparable to Kojic acid (IC 50 16.051 ± 1.27 mM). Interestingly compound 4f exhibited higher antioxidant potential compared to other derivatives. The docking studies of synthesized 1-Pentanoyl-3-arylthioureas analogues were also carried out against tyrosinase protein (PDBID 2ZMX) to compare the binding affinities with IC 50 values. The predicted binding affinities are in good agreement with the IC 50 values as compound (4f) showed highest binding affinity (-7.50 kcal/mol) compared to others derivatives. The kinetic mechanism analyzed by Line-weavere Burk plots exhibited that compound (4f) inhibit the enzyme inhibits the tyrosinase non-competitively to form an enzyme inhibitor complex. The inhibition constants Ki calculated from Dixon plots for compound (4f) is 1.10 μM. It was also found from kinetic analysis that derivative 4f irreversible enzyme inhibitor complex. It is proposed on the basis of our investigation that title compound (4f) may serve as lead structure for the design of more potent tyrosinase inhibitors. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Fluorescence enhancement of fluorescent unnatural streptavidin by binding of a biotin analogue with spacer tail and its application to biotin sensing.

PubMed

Zhu, Xianwei; Shinohara, Hiroaki

2014-01-01

We designed a novel molecular biosensing system for the detection of biotin, an important vitamin by the combination of fluorescent unnatural streptavidin with a commercialized biotin-(AC5)2-hydrazide. A fluorescent unnatural amino acid, BODIPY-FL-aminophenylalanine (BFLAF), was position-specifically incorporated into Trp120 of streptavidin by four-base codon method. Fluorescence of the Trp120BFLAF mutant streptavidin was enhanced by the addition of biotin-(AC5)2-hydrazide with the concentration dependent, whereas fluorescence enhancement was not observed at all by the addition of natural biotin. It was considered that the spacer tail of biotin-(AC5)2-hydrazide may disturb the fluorescence quenching of the Trp120BFLAF by Trp79 and Trp108 of the neighbor subunit. Therefore, biotin sensing was carried out by the competitive binding reaction of biotin-(AC5)2-hydrazide and natural biotin to the fluorescent mutant streptavidin. The fluorescence intensity decreased by increasing free biotin concentration. The result suggested that molecular biosensor for small ligand could be successfully designed by the pair of fluorescent mutant binding protein and ligand analogue.

TRP Channels

NASA Astrophysics Data System (ADS)

Voets, Thomas; Owsianik, Grzegorz; Nilius, Bernd

The TRP superfamily represents a highly diverse group of cation-permeable ion channels related to the product of the Drosophila trp (transient receptor potential) gene. The cloning and characterization of members of this cation channel family has experienced a remarkable growth during the last decade, uncovering a wealth of information concerning the role of TRP channels in a variety of cell types, tissues, and species. Initially, TRP channels were mainly considered as phospholipase C (PLC)-dependent and/or store-operated Ca2+-permeable cation channels. More recent research has highlighted the sensitivity of TRP channels to a broad array of chemical and physical stimuli, allowing them to function as dedicated biological sensors involved in processes ranging from vision to taste, tactile sensation, and hearing. Moreover, the tailored selectivity of certain TRP channels enables them to play key roles in the cellular uptake and/or transepithelial transport of Ca2+, Mg2+, and trace metal ions. In this chapter we give a brief overview of the TRP channel superfamily followed by a survey of current knowledge concerning their structure and activation mechanisms.

Communication Between the Calcium and cAMP Pathways Regulate the Expression of the TSH Receptor: TRPC2 in the Center of Action

PubMed Central

Löf, Christoffer; Sukumaran, Pramod; Viitanen, Tero; Vainio, Minna; Kemppainen, Kati; Pulli, Ilari; Näsman, Johnny; Kukkonen, Jyrki P.

2012-01-01

Transient receptor potential (TRP) cation channels are widely expressed and function in many physiologically important processes. Perturbations in the expression or mutations of the channels have implications for diseases. Many thyroid disorders, as excessive growth or disturbed thyroid hormone production, can be a result of dysregulated TSH signaling. In the present study, we found that of TRP canonicals (TRPCs), only TRPC2 was expressed in Fischer rat thyroid low-serum 5% cells (FRTL-5 cells). To investigate the physiological importance of the channel, we developed stable TRPC2 knockdown cells using short hairpin RNA (shTRPC2 cells). In these cells, the ATP-evoked entry of calcium was significantly decreased. This led to increased cAMP production, because inhibitory signals from calcium to adenylate cyclase 5/6 were decreased. Enhanced cAMP signaling projected to Ras-related protein 1-MAPK kinase 1 (MAPK/ERK kinase 1) pathway leading to phosphorylation of ERK1/2. The activated ERK1/2 pathway increased the expression of the TSH receptor. In contrast, secretion of thyroglobulin was decreased in shTRPC2 cells, due to improper folding and glycosylation of the protein. We show here a novel role for TRPC2 in regulating thyroid cell function. PMID:23015753

Urinary profiling of tryptophan and its related metabolites in patients with metabolic syndrome by liquid chromatography-electrospray ionization/mass spectrometry.

PubMed

Oh, Ji Sun; Seo, Hong Seong; Kim, Kyoung Heon; Pyo, Heesoo; Chung, Bong Chul; Lee, Jeongae

2017-09-01

Tryptophan (Trp) is an essential amino acid that plays an important role in protein synthesis and is a precursor of various substances related to diverse biological functions. An imbalance in Trp metabolites is associated with inflammatory diseases. The accurate and precise measurement of these compounds in biological specimens would provide meaningful information for understanding the biochemical states of various metabolic syndrome-related diseases, such as hyperlipidemia, hypertension, diabetes, and obesity. In this study, we developed a rapid, accurate, and sensitive liquid chromatography-tandem mass spectrometry-based method for the simultaneous targeted analysis of Trp and its related metabolites of the kynurenine (Kyn), serotonin, and tryptamine pathways in urine. The application of the developed method was tested using urine samples after protein precipitation. The detection limits of Trp and its metabolites were in the range of 0.01 to 0.1 μg/mL. The method was successfully validated and applied to urine samples from controls and patients with metabolic syndrome. Our results revealed high concentrations of Kyn, kynurenic acid, xanthurenic acid, and quinolinic acid as well as a high Kyn-to-Trp ratio (KTR) in patients with metabolic syndromes. The levels of urine Kyn and KTR were significantly increased in patients under 60 years old. The profiling of urinary Trp metabolites could be a useful indicator for age-related diseases including metabolic syndrome. ᅟ.

Phylogenetic analysis of eIF4E-family members

PubMed Central

Joshi, Bhavesh; Lee, Kibwe; Maeder, Dennis L; Jagus, Rosemary

2005-01-01

Background Translation initiation in eukaryotes involves the recruitment of mRNA to the ribosome which is controlled by the translation factor eIF4E. eIF4E binds to the 5'-m7Gppp cap-structure of mRNA. Three dimensional structures of eIF4Es bound to cap-analogues resemble 'cupped-hands' in which the cap-structure is sandwiched between two conserved Trp residues (Trp-56 and Trp-102 of H. sapiens eIF4E). A third conserved Trp residue (Trp-166 of H. sapiens eIF4E) recognizes the 7-methyl moiety of the cap-structure. Assessment of GenBank NR and dbEST databases reveals that many organisms encode a number of proteins with homology to eIF4E. Little is understood about the relationships of these structurally related proteins to each other. Results By combining sequence data deposited in the Genbank databases, we have identified sequences encoding 411 eIF4E-family members from 230 species. These sequences have been deposited into an internet-accessible database designed for sequence comparisons of eIF4E-family members. Most members can be grouped into one of three classes. Class I members carry Trp residues equivalent to Trp-43 and Trp-56 of H. sapiens eIF4E and appear to be present in all eukaryotes. Class II members, possess Trp→Tyr/Phe/Leu and Trp→Tyr/Phe substitutions relative to Trp-43 and Trp-56 of H. sapiens eIF4E, and can be identified in Metazoa, Viridiplantae, and Fungi. Class III members possess a Trp residue equivalent to Trp-43 of H. sapiens eIF4E but carry a Trp→Cys/Tyr substitution relative to Trp-56 of H. sapiens eIF4E, and can be identified in Coelomata and Cnidaria. Some eIF4E-family members from Protista show extension or compaction relative to prototypical eIF4E-family members. Conclusion The expansion of sequenced cDNAs and genomic DNAs from all eukaryotic kingdoms has revealed a variety of proteins related in structure to eIF4E. Evolutionarily it seems that a single early eIF4E gene has undergone multiple gene duplications generating multiple structural classes, such that it is no longer possible to predict function from the primary amino acid sequence of an eIF4E-family member. The variety of eIF4E-family members provides a source of alternatives on the eIF4E structural theme that will benefit structure/function analyses and therapeutic drug design. PMID:16191198

On the efficient bio-incorporation of 5-hydroxy-tryptophan in recombinant proteins expressed in Escherichia coli with T7 RNA polymerase-based vectors.

PubMed

Oliveira-Souza, Wellington P; Bronze, Fellipe; Broos, Jaap; Marcondes, Marcelo F M; Oliveira, Vitor

2017-10-21

Biosynthetic incorporation of non-canonic amino acids is an attractive strategy to introduce new properties in recombinant proteins. Trp analogs can be incorporated in recombinant proteins replacing regular Trp during protein translation into a Trp-auxotrophic cell host. This straightforward method however, is limited to few analogs recognized and accepted by the cellular protein production machinery. 5-hydroxy-tryptophan (5OH-Trp) can be bio-incorporated using E. coli as expression host however; we have experienced very low incorporation yields - amount of protein containing regular Trp/amount of protein containing the Trp analog - during expressions of 5OH-Trp labeled proteins. Furthermore, this low incorporation yield were verified especially when the widely-used vectors based on the T7 RNA polymerase were used. Testing different 5OH-Trp incorporation protocols we verified that in these T7-based systems, the production of the T7 RNA polymerase is driven by the same elements - lac promoter/IPTG - as the target protein. Consequently, the bio-incorporation of the 5OH-Trp residues also occurs in this crucial enzyme, but, the produced T7 RNA polymerase labeled with 5OH-Trp is inactive or much less active. In the present work, we describe an efficient method to overcome this mentioned problem and bio-incorporate 5OH-Trp in proteins expressed in E. coli., using vectors based on the T7 RNA polymerase-T7 promoter. The two-step induction protocol here described showed incorporation efficiencies of 5OH-Trp higher than 90%. Copyright © 2017 Elsevier Inc. All rights reserved.

Discovery of novel dual inhibitors against Mdm2 and Mdmx proteins by in silico approaches and binding assay.

PubMed

Golestanian, Sahand; Sharifi, Amirhossein; Popowicz, Grzegorz M; Azizian, Homa; Foroumadi, Alireza; Szwagierczak, Aleksandra; Holak, Tad A; Amanlou, Massoud

2016-01-15

The p53 protein, also called guardian of the genome, has a key role in cell cycle regulation. It is activated under stressful circumstances, such as DNA damage which results in permanent arrest or cell death. The protein is disabled in several types of human cancer due to over-expression of the two regulators, Mdm2 and Mdmx. As a result, inhibiting Mdm subtypes could reactivate p53 and bring about a promising therapeutic strategy in cancers. Here a structure-based pharmacophore search and docking simulation are presented in order to filter our in-house library which contains 1035 compounds to find novel scaffolds that inhibit Mdm2 and Mdmx concomitantly. Afterwards, fluorescence polarization binding assay was used to obtain inhibition constant of final compounds. Thirty two ligands were introduced to bioassay as a result of in-silico methods. Twelve of them inhibit both proteins with almost balanced Ki value ranging from 18 to 162μM for Mdm2 and 18 to 233μM for Mdmx. It was observed that all compounds fill Phe19 and Trp23 pockets of Mdm2/x binding sites and form a hydrogen bond with Trp23 pocket's neighbor amino acids in a manner similar to p53 protein. Additionally, it was concluded that Trp23 pocket of Mdmx has a bigger hydrophobic volume comparing with the one of Mdm2. Three structure-activity relationship patterns are supposed which one of them presents usefulness features and can be used in future studies. This study presents first qualitative SAR for dual inhibitors against Mdm2/x. Copyright © 2016 Elsevier Inc. All rights reserved.

Uterine Deletion of Trp53 Compromises Antioxidant Responses in the Mouse Decidua

PubMed Central

Burnum, Kristin E.; Hirota, Yasushi; Baker, Erin S.; Yoshie, Mikihiro; Ibrahim, Yehia M.; Monroe, Matthew E.; Anderson, Gordon A.; Smith, Richard D.; Daikoku, Takiko

2012-01-01

Preterm birth is a global health issue impacting millions of mothers and babies. However, the etiology of preterm birth is not clearly understood. Our recent finding that premature decidual senescence with terminal differentiation is a cause of preterm birth in mice with uterine Trp53 deletion, encoding p53 protein, led us to explore other potential factors that are related to preterm birth. Using proteomics approaches, here, we show that 183 candidate proteins show significant changes in deciduae with Trp53 deletion as compared with normal deciduae. Functional categorization of these proteins unveiled new pathways that are influenced by p53. In particular, down-regulation of a cluster of antioxidant enzymes in p53-deficient deciduae suggests that increased oxidative stress could be one cause of preterm birth in mice harboring uterine deletion of Trp53. PMID:22759378

Tryptophan and Non-Tryptophan Fluorescence of the Eye Lens Proteins Provides Diagnostics of Cataract at the Molecular Level

PubMed Central

Gakamsky, Anna; Duncan, Rory R.; Howarth, Nicola M.; Dhillon, Baljean; Buttenschön, Kim K.; Daly, Daniel J.; Gakamsky, Dmitry

2017-01-01

The chemical nature of the non-tryptophan (non-Trp) fluorescence of porcine and human eye lens proteins was identified by Mass Spectrometry (MS) and Fluorescence Steady-State and Lifetime spectroscopy as post-translational modifications (PTM) of Trp and Arg amino acid residues. Fluorescence intensity profiles measured along the optical axis of human eye lenses with age-related nuclear cataract showed increasing concentration of fluorescent PTM towards the lens centre in accord with the increased optical density in the lens nucleolus. Significant differences between fluorescence lifetimes of “free” Trp derivatives hydroxytryptophan (OH-Trp), N-formylkynurenine (NFK), kynurenine (Kyn), hydroxykynurenine (OH-Kyn) and their residues were observed. Notably, the lifetime constants of these residues in a model peptide were considerably greater than those of their “free” counterparts. Fluorescence of Trp, its derivatives and argpyrimidine (ArgP) can be excited at the red edge of the Trp absorption band which allows normalisation of the emission spectra of these PTMs to the fluorescence intensity of Trp, to determine semi-quantitatively their concentration. We show that the cumulative fraction of OH-Trp, NFK and ArgP emission dominates the total fluorescence spectrum in both emulsified post-surgical human cataract protein samples, as well as in whole lenses and that this correlates strongly with cataract grade and age. PMID:28071717

Reactivity of dinuclear copper(II) complexes towards melanoma cells: Correlation with its stability, tyrosinase mimicking and nuclease activity.

PubMed

Nunes, Cléia Justino; Borges, Beatriz Essenfelder; Nakao, Lia Sumie; Peyroux, Eugénie; Hardré, Renaud; Faure, Bruno; Réglier, Marius; Giorgi, Michel; Prieto, Marcela Bach; Oliveira, Carla Columbano; Da Costa Ferreira, Ana M

2015-08-01

In this work, the influence of two new dinuclear copper(II) complexes in the viability of melanoma cells (B16F10 and TM1MNG3) was investigated, with the aim of verifying possible correlations between their cytotoxicity and their structure. One of the complexes had a polydentate dinucleating amine-imine ligand (complex 2), and the other a tridentate imine and a diamine-bridging ligand (complex 4). The analogous mononuclear copper(II) species (complexes 1 and 3, respectively) were also prepared for comparative studies. Crystal structure determination of complex 2 indicated a square-based pyramidal geometry around each copper, coordinated to three N atoms from the ligand and the remaining sites being occupied by either solvent molecules or counter-ions. Complex 4 has a tetragonal geometry. Interactions of these complexes with human albumin protein (HSA) allowed an estimation of their relative stabilities. Complementary studies of their reactivity towards DNA indicated that all of them are able of causing significant oxidative damage, with single and double strand cleavages, in the presence of hydrogen peroxide. However, nuclease activity of the dinuclear species was very similar and much higher than that of the corresponding mononuclear compounds. Although complex 2, with a more flexible structure, exhibits a much higher tyrosinase activity than complex 4, having a more rigid environment around the metal ion, both complexes showed comparable cytotoxicity towards melanoma cells. Corresponding mononuclear complexes showed to be remarkably less reactive as tyrosinase mimics as well as cytotoxic agents. Moreover, the dinuclear complexes showed higher cytotoxicity towards more melanogenic cells. The obtained results indicated that the structure of these species is decisive for its activity towards the malignant tumor cells tested. Copyright © 2015 Elsevier Inc. All rights reserved.

Heteromerization and colocalization of TrpV1 and TrpV2 in mammalian cell lines and rat dorsal root ganglia.

PubMed

Rutter, A Richard; Ma, Qing-Ping; Leveridge, Mathew; Bonnert, Timothy P

2005-11-07

Coassociation of the vanilloid transient receptor potential (Trp) ion channels, TrpV1 and TrpV2, was investigated by immunoprecipitation and immunofluorescence in transfected mammalian cell lines, rat dorsal root ganglia and spinal cord. TrpV1/TrpV2 heteromeric complexes were coimmunoprecipitated from human embryonic kidney cells and F-11 dorsal root ganglion hybridoma cells following their transient coexpression. Immunofluorescent labelling of transfected F-11 cells revealed colocalization of TrpV1 and TrpV2 at the cell surface. Immunoprecipitation from rat dorsal root ganglion lysates identified a minor population of receptor complexes composed of TrpV1/TrpV2 heteromers, consistent with a small proportion of cells double-labelled with TrpV1 and TrpV2 antibodies in rat dorsal root ganglion sections. TrpV1/TrpV2 receptor complexes may represent a functionally distinct ion channel complex that may increase the diversity observed within the Trp ion channel family.

The AlternativeTranslational Profile That Underlies the Immune-Evasive State of Persistence in Chlamydiaceae Exploits Differential Tryptophan Contents of the Protein Repertoire

PubMed Central

Lo, Chien-Chi; Bonner, Carol A.

2012-01-01

Summary: One form of immune evasion is a developmental state called “persistence” whereby chlamydial pathogens respond to the host-mediated withdrawal of l-tryptophan (Trp). A sophisticated survival mode of reversible quiescence is implemented. A mechanism has evolved which suppresses gene products necessary for rapid pathogen proliferation but allows expression of gene products that underlie the morphological and developmental characteristics of persistence. This switch from one translational profile to an alternative translational profile of newly synthesized proteins is proposed to be accomplished by maximizing the Trp content of some proteins needed for rapid proliferation (e.g., ADP/ATP translocase, hexose-phosphate transporter, phosphoenolpyruvate [PEP] carboxykinase, the Trp transporter, the Pmp protein superfamily for cell adhesion and antigenic variation, and components of the cell division pathway) while minimizing the Trp content of other proteins supporting the state of persistence. The Trp starvation mechanism is best understood in the human-Chlamydia trachomatis relationship, but the similarity of up-Trp and down-Trp proteomic profiles in all of the pathogenic Chlamydiaceae suggests that Trp availability is an underlying cue relied upon by this family of pathogens to trigger developmental transitions. The biochemically expensive pathogen strategy of selectively increased Trp usage to guide the translational profile can be leveraged significantly with minimal overall Trp usage by (i) regional concentration of Trp residue placements, (ii) amplified Trp content of a single protein that is required for expression or maturation of multiple proteins with low Trp content, and (iii) Achilles'-heel vulnerabilities of complex pathways to high Trp content of one or a few enzymes. PMID:22688818

Singlet oxygen generation during the oxidation of L-tyrosine and L-dopa with mushroom tyrosinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miyaji, Akimitsu; Kohno, Masahiro; Inoue, Yoshihiro

2016-03-18

The generation of singlet oxygen during the oxidation of tyrosine and L-dopa using mushroom tyrosinase in a phosphate buffer (pH 7.4), the model of melanin synthesis in melanocytes, was examined. The reaction was performed in the presence of 2,2,6,6-tetramethyl-4-piperidone (4-oxo-TEMP), an acceptor of singlet oxygen and the electron spin resonance (ESR) of the spin adduct, 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMPO), was measured. An increase in the ESR signal attributable to 4-oxo-TEMPO was observed during the oxidation of tyrosine and L-dopa with tyrosinase, indicating the generation of singlet oxygen. The results suggest that {sup 1}O{sub 2} generation via tyrosinase-catalyzed melanin synthesis occurs in melanocyte.more » - Highlights: • Generation of singlet oxygen was observed during tyrosinase-catalyzed tyrosine oxidation. • The singlet oxygen generated when tyrosine was converted into dopachrome. • The amount of singlet oxygen is not sufficient for cell toxicity. • It decreased when the hydroxyl radicals and/or superoxide anions were trapped.« less

Singlet oxygen generation during the oxidation of L-tyrosine and L-dopa with mushroom tyrosinase.

PubMed

Miyaji, Akimitsu; Kohno, Masahiro; Inoue, Yoshihiro; Baba, Toshihide

2016-03-18

The generation of singlet oxygen during the oxidation of tyrosine and L-dopa using mushroom tyrosinase in a phosphate buffer (pH 7.4), the model of melanin synthesis in melanocytes, was examined. The reaction was performed in the presence of 2,2,6,6-tetramethyl-4-piperidone (4-oxo-TEMP), an acceptor of singlet oxygen and the electron spin resonance (ESR) of the spin adduct, 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMPO), was measured. An increase in the ESR signal attributable to 4-oxo-TEMPO was observed during the oxidation of tyrosine and L-dopa with tyrosinase, indicating the generation of singlet oxygen. The results suggest that (1)O2 generation via tyrosinase-catalyzed melanin synthesis occurs in melanocyte. Copyright © 2016 Elsevier Inc. All rights reserved.

Transient Receptor Potential Channels in the Vasculature

PubMed Central

Earley, Scott; Brayden, Joseph E.

2015-01-01

The mammalian genome encodes 28 distinct members of the transient receptor potential (TRP) superfamily of cation channels, which exhibit varying degrees of selectivity for different ionic species. Multiple TRP channels are present in all cells and are involved in diverse aspects of cellular function, including sensory perception and signal transduction. Notably, TRP channels are involved in regulating vascular function and pathophysiology, the focus of this review. TRP channels in vascular smooth muscle cells participate in regulating contractility and proliferation, whereas endothelial TRP channel activity is an important contributor to endothelium-dependent vasodilation, vascular wall permeability, and angiogenesis. TRP channels are also present in perivascular sensory neurons and astrocytic endfeet proximal to cerebral arterioles, where they participate in the regulation of vascular tone. Almost all of these functions are mediated by changes in global intracellular Ca2+ levels or subcellular Ca2+ signaling events. In addition to directly mediating Ca2+ entry, TRP channels influence intracellular Ca2+ dynamics through membrane depolarization associated with the influx of cations or through receptor- or store-operated mechanisms. Dysregulation of TRP channels is associated with vascular-related pathologies, including hypertension, neointimal injury, ischemia-reperfusion injury, pulmonary edema, and neurogenic inflammation. In this review, we briefly consider general aspects of TRP channel biology and provide an in-depth discussion of the functions of TRP channels in vascular smooth muscle cells, endothelial cells, and perivascular cells under normal and pathophysiological conditions. PMID:25834234

Endocannabinoids Stimulate Human Melanogenesis via Type-1 Cannabinoid Receptor*

PubMed Central

Pucci, Mariangela; Pasquariello, Nicoletta; Battista, Natalia; Di Tommaso, Monia; Rapino, Cinzia; Fezza, Filomena; Zuccolo, Michela; Jourdain, Roland; Finazzi Agrò, Alessandro; Breton, Lionel; Maccarrone, Mauro

2012-01-01

We show that a fully functional endocannabinoid system is present in primary human melanocytes (normal human epidermal melanocyte cells), including anandamide (AEA), 2-arachidonoylglycerol, the respective target receptors (CB1, CB2, and TRPV1), and their metabolic enzymes. We also show that at higher concentrations AEA induces normal human epidermal melanocyte apoptosis (∼3-fold over controls at 5 μm) through a TRPV1-mediated pathway that increases DNA fragmentation and p53 expression. However, at lower concentrations, AEA and other CB1-binding endocannabinoids dose-dependently stimulate melanin synthesis and enhance tyrosinase gene expression and activity (∼3- and ∼2-fold over controls at 1 μm). This CB1-dependent activity was fully abolished by the selective CB1 antagonist SR141716 or by RNA interference of the receptor. CB1 signaling engaged p38 and p42/44 mitogen-activated protein kinases, which in turn activated the cyclic AMP response element-binding protein and the microphthalmia-associated transcription factor. Silencing of tyrosinase or microphthalmia-associated transcription factor further demonstrated the involvement of these proteins in AEA-induced melanogenesis. In addition, CB1 activation did not engage the key regulator of skin pigmentation, cyclic AMP, showing a major difference compared with the regulation of melanogenesis by α-melanocyte-stimulating hormone through melanocortin 1 receptor. PMID:22431736

Regulation of Transient Receptor Potential channels by the phospholipase C pathway

PubMed Central

Rohacs, Tibor

2013-01-01

Transient Receptor Potential (TRP) channels were discovered while analyzing visual mutants in drosophila. The protein encoded by the transient receptor potential (trp) gene is a Ca2+ permeable cation channel activated downstream of the phospholipase C (PLC) pathway. While searching for homologues in other organisms, a surprisingly large number of mammalian TRP channels were cloned. The regulation of TRP channels is quite diverse, but many of them are either activated downstream of the PLC pathway, or modulated by it. This review will summarize the current knowledge on regulation of TRP channels by the PLC pathway, with special focus on TRPC-s, which can be considered as effectors of the PLC pathway, and the heat and capsaicin sensitive TRPV1, which is modulated by the PLC pathway in a complex manner. PMID:23916247

Acute Dietary Tryptophan Manipulation Differentially Alters Social Behavior, Brain Serotonin and Plasma Corticosterone in Three Inbred Mouse Strains

PubMed Central

Zhang, Wynne Q.; Smolik, Corey M.; Barba-Escobedo, Priscilla A.; Gamez, Monica; Sanchez, Jesus J.; Javors, Martin A.; Daws, Lynette C.; Gould, Georgianna G.

2014-01-01

Clinical evidence indicates brain serotonin (5-HT) stores and neurotransmission may be inadequate in subpopulations of individuals with autism, and this may contribute to characteristically impaired social behaviors. Findings that depletion of the 5-HT precursor tryptophan (TRP) worsens autism symptoms support this hypothesis. Yet dietetic studies show and parents report that many children with autism consume less TRP than peers. To measure the impact of dietary TRP content on social behavior, we administered either diets devoid of TRP, with standard TRP (0.2 gm%), or with 1% added TRP (1.2 gm%) overnight to three mouse strains. Of these, BTBRT+Itpr3tf/J and 129S1/SvImJ consistently exhibit low preference for social interaction relative to C57BL/6. We found that TRP depletion reduced C57BL/6 and 129S social interaction preference, while TRP enhancement improved BTBR sociability (p < 0.05; N= 8–10). Subsequent marble burying was similar regardless of grouping. After behavior tests, brain TRP levels and plasma corticosterone were higher in TRP enhanced C57BL/6 and BTBR, while 5-HT levels were reduced in all strains by TRP depletion (p <0.05; N= 4 −10). Relative hyperactivity of BTBR and hypoactivity of 129S, evident in self-grooming and chamber entries during sociability tests, were uninfluenced by dietary TRP. Our findings demonstrate mouse sociability and brain 5-HT turnover are reduced by acute TRP depletion, and can be enhanced by TRP supplementation. This outcome warrants further basic and/or clinical studies employing biomarker combinations such as TRP metabolism and 5-HT regulated hormones to characterize the conditions wherein TRP supplementation can best ameliorate sociability deficits. PMID:25445490

In vitro and cellular activities of the selected fruits residues for skin aging treatment.

PubMed

Lourith, Nattaya; Kanlayavattanakul, Mayuree; Chaikul, Puxvadee; Chansriniyom, Chaisak; Bunwatcharaphansakun, Pichaporn

2017-05-01

Peel extracts of litchi and rambutan, and that of tamarind seed coat were investigated in relation to their utility in skin-aging treatments. Standardized extracts of tamarind were significantly (p < 0.05) more efficient at O2 •- scavenging (IC50 = 27.44 ± 0.09) than those of litchi and rambutan (IC50 = 29.57 ± 0.30 and 39.49 ± 0.52 μg/ml, respectively) and the quercetin standard (IC50 = 31.88 ± 0.15 μg/ml). Litchi extract proved significantly (p < 0.05) more effective for elastase and collagenase inhibition (88.29 ± 0.25% and 79.46 ± 0.92%, respectively) than tamarind (35.43 ± 0.68% and 57.69 ± 5.97%) or rambutan (31.08 ± 0.38% and 53.99 ± 6.18%). All extracts were safe to human skin fibroblasts and inhibit MMP-2, with litchi extract showing significantly (p < 0.01) enhanced inhibition over the standard, vitamin C (23.75 ± 2.74% and 10.42 ± 5.91% at 0.05 mg/ml, respectively). Extracts suppress melanin production in B16F10 melanoma cells through inhibition of tyrosinase and TRP-2, with litchi extract being the most potent, even more so than kojic acid (standard). These results highlight the potential for adding value to agro-industrial waste, as the basis for the sustainable production of innovative, safe, anti-aging cosmetic products.

Biosensor based on tyrosinase immobilized on a single-walled carbon nanotube-modified glassy carbon electrode for detection of epinephrine

PubMed Central

Apetrei, Irina Mirela; Apetrei, Constantin

2013-01-01

A biosensor comprising tyrosinase immobilized on a single-walled carbon nanotube-modified glassy carbon electrode has been developed. The sensitive element, ie, tyrosinase, was immobilized using a drop-and-dry method followed by cross-linking. Tyrosinase maintained high bioactivity on this nanomaterial, catalyzing the oxidation of epinephrine to epinephrine-quinone, which was electrochemically reduced (−0.07 V versus Ag/AgCl) on the biosensor surface. Under optimum conditions, the biosensor showed a linear response in the range of 10–110 μM. The limit of detection was calculated to be 2.54 μM with a correlation coefficient of 0.977. The repeatability, expressed as the relative standard deviation for five consecutive determinations of 10−5 M epinephrine solution was 3.4%. A good correlation was obtained between results obtained by the biosensor and those obtained by ultraviolet spectrophotometric methods. PMID:24348034

Activity, Stability, and Structure of Native and Modified by Woodward Reagent K Mushroom Tyrosinase

NASA Astrophysics Data System (ADS)

Emami, S.; Piri, H.; Gheibi, N.

2018-01-01

Mushroom tyrosinase (MT) was considered a good model for studying the inhibition, activation, and mutation of tyrosinase as the key enzyme of melanogenesis. In the present study, the activity, structure, reduction, and stability of native and modified enzymes were investigated after the modification of MT carboxylic residues by the Woodward reagent K (WRK). The relative activity of the sole enzyme was reduced from 100 to 77.9, 53.8, 39.4, and 26.4% after its modification by 2.5, 5, 25, and 50 ratios of [WRK]/[MT], respectively. The Tm values were calculated from thermal denaturation curves at 61.2, 60.1, 58.3, 53.9, and 45.5oC for the sole and modified enzymes. The reduction of the Δ {G}_{{H}_2O} values for the modified enzyme in chemical denaturation indicated instability. A structural study by CD and intrinsic fluorescence technique revealed the fluctuation of the secondary and tertiary structures of MT.

A novel bioactive chalcone of Morus australis inhibits tyrosinase activity and melanin biosynthesis in B16 melanoma cells.

PubMed

Takahashi, Makoto; Takara, Kensaku; Toyozato, Tomonao; Wada, Koji

2012-01-01

The methanol extract of Morus australis (shimaguwa) acts as a whitening agent due to the inhibition of tyrosinase activity. In order to explore the mechanism(s) of the whitening action, constituents of the 95% methanol extract from the dried stems of shimaguwa were isolated and their skin-whitening capacity was examined. Bioassay-guided fractionation of the methanol soluble extract of shimaguwa led to the isolation of 2, 4, 2', 4'-hydroxycalcone (chalcone 1) and three analogues of chalcone 1 with 3'-substituted resorcinol moieties (chalcones 2-4). Chalcone derivative 4 proved to be a novel compound and was fully characterized. Chalcones 1-4 were evaluated for inhibition activity on mushroom tyrosinase using L-tyrosine as the substrate. The parent chalcone 1 was a highly effective inhibitor of tyrosinase activity (IC₅₀ = 0.21 μM) compared to arbutin (IC₅₀ = 164 μM). Compared to chalcone 1, chalcones 2 and 3, which possess 3'-substituted isoprenyl or bulky 2-benzoylbiphenyl, showed significantly decreased tyrosinase activity, while chalcone 4, possessing 3'-substituted 2-hydroxy-1-pentene group, showed slightly increased activity.The effects of chalcones 1-4 on melanin synthesis, without affecting cell growth, were assayed in melanin-producing B16 murine melanoma cells. Chalcone 3 significantly reduced cell viability before reaching the IC₅₀ value for melanin synthesis. In contrast, the inhibitory effects of chalcones 1, 2 and 4 were more than 100-fold greater than that of arbutin, with little or no cytotoxicity. More significantly, chalcone 2, which exhibited less tyrosinase inhibitory activity compared to the parent chalcone 1, showed the highest inhibition of melanin synthesis in B16 cells among the chalcones tested. Accordingly, chalcones 1 and 2, and the novel chalcone 4 might be the active components responsible for the whitening ability of shimaguwa. Moreover, whitening ability was not exclusively due to tyrosinase inhibition.

A novel plasminogen activator from Agkistrodon blomhoffii Ussurensis venom (ABUSV-PA): Purification and characterization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Shuqing; Sun Mingzhong; Greenaway, Frederick T.

2006-10-06

A plasminogen activator with arginine ester hydrolysis activity (ABUSV-PA) has been identified and purified to homogeneity from Chinese Agkistrodon blomhoffii Ussurensis snake venom. ABUSV-PA, a monomeric protein with molecular mass of 27815.2 Da, was purified 180-fold with 0.02% recovery for protein and 3.6% recovery for esterase activity. ABUSV-PA reacts optimally with its substrate N {sub {alpha}}-tosyl-L-arginine-methyl ester (TAME) at {approx}pH 7.5 and at 51 {sup o}C. Measurement from inductively coupled plasma-atomic emission spectroscopy (ICP-AES) reveals that ABUSV-PA is a Zn{sup 2+}-containing protein with a stoichiometry of 1:1 [Zn{sup 2+}]:[ABUSV-PA]. Analyses of esterase hydrolysis and UV absorption and CD spectra indicatemore » that Zn{sup 2+} plays an important role in maintaining the structural integrity rather than the esterase activity of ABUSV-PA. Divalent metal ions, including Ca{sup 2+}, Mg{sup 2+}, Cu{sup 2+}, Ni{sup 2+}, Mn{sup 2+}, and Co{sup 2+}, increase the TAME hydrolysis activity of ABUSV-PA. A red-shift of the emission wavelengths of the synchronous fluorescence of ABUSV-PA, compared to those of free Tyr and Trp, indicates a conformation where the Tyr and Trp residues are in exposed hydrophilic environments. The presence of zinc increases the hydrophobicity of the conformational environments surrounding the Trp residues of ABUSV-PA and affects the secondary structure of ABUSV-PA, as proved by UV absorption and CD spectroscopy.« less

Expression and distribution of transient receptor potential (TRP) channels in bladder epithelium.

PubMed

Yu, Weiqun; Hill, Warren G; Apodaca, Gerard; Zeidel, Mark L

2011-01-01

The urothelium is proposed to be a sensory tissue that responds to mechanical stress by undergoing dynamic membrane trafficking and neurotransmitter release; however, the molecular basis of this function is poorly understood. Transient receptor potential (TRP) channels are ideal candidates to fulfill such a role as they can sense changes in temperature, osmolarity, and mechanical stimuli, and several are reported to be expressed in the bladder epithelium. However, their complete expression profile is unknown and their cellular localization is largely undefined. We analyzed expression of all 33 TRP family members in mouse bladder and urothelium by RT-PCR and found 22 specifically expressed in the urothelium. Of the latter, 10 were chosen for closer investigation based on their known mechanosensory or membrane trafficking functions in other cell types. Western blots confirmed urothelial expression of TRPC1, TRPC4, TRPV1, TRPV2, TRPV4, TRPM4, TRPM7, TRPML1, and polycystins 1 and 2 (PKD1 and PKD2) proteins. We further defined the cellular and subcellular localization of all 10 TRP channels. TRPV2 and TRPM4 were prominently localized to the umbrella cell apical membrane, while TRPC4 and TRPV4 were identified on their abluminal surfaces. TRPC1, TRPM7, and TRPML1 were localized to the cytoplasm, while PKD1 and PKD2 were expressed on the apical and basolateral membranes of umbrella cells as well as in the cytoplasm. The cellular location of TRPV1 in the bladder has been debated, but colocalization with neuronal marker calcitonin gene-related peptide indicated clearly that it is present on afferent neurons that extend into the urothelium, but may not be expressed by the urothelium itself. These findings are consistent with the hypothesis that the urothelium acts as a sentinel and by expressing multiple TRP channels it is likely it can detect and presumably respond to a diversity of external stimuli and suggest that it plays an important role in urothelial signal transduction.

Physiological effects of formulation containing tannase-converted green tea extract on skin care: physical stability, collagenase, elastase, and tyrosinase activities.

PubMed

Hong, Yang-Hee; Jung, Eun Young; Noh, Dong Ouk; Suh, Hyung Joo

2014-03-01

Green tea contains numerous polyphenols, which have health-promoting effects. The purpose of this study was to evaluate the effect of tannase-converted green tea extract (TGE) formulation on the physical stability and activities of skin-related enzymes. Physical stability was evaluated by measuring the pH, precipitation, and colors at 25 ± 2 °C/ambient humidity and at 40 ± 2 °C/70% ± 5% relative humidity for 4 months. Activities of collagenase, elastase, and tyrosinase as skin-related enzymes were assessed on TGE formulation. The concentrations of epigallocatechin-3-gallate and epicatechin-3-gallate in green tea extract were greatly decreased to the extent of negligible level when treated with tannase. The formulation containing 5% tannase-converted green tea extract showed relatively stable pH, precipitation, and color features for 16 weeks. When TGE was added to the formulation, there was a significant increase in the inhibition of elastase and tyrosinase activities ( p  < 0.05) compared with the formulation containing 5% normal green tea extract. The TGE could be used in cosmetics as skin antiwrinkling or depigmenting agent.

Inhibition of melanin production by a combination of Siberian larch and pomegranate fruit extracts.

PubMed

Diwakar, Ganesh; Rana, Jatinder; Scholten, Jeffrey D

2012-09-01

In an effort to find botanicals containing polyphenolic compounds with the capacity to inhibit melanin biosynthesis, we identified a novel combination of Siberian larch (Larix sibirica) extract, standardized to 80% taxifolin, and pomegranate fruit (Punica granatum) extract, containing 20% punicalagins, that demonstrates a synergistic reduction of melanin biosynthesis in Melan-a cells. The combination of Siberian larch and pomegranate extracts (1:1) produced a 2-fold reduction in melanin content compared to Siberian larch or pomegranate extracts alone with no corresponding effect on cell viability. Siberian larch and pomegranate fruit extracts inhibited expression of melanocyte specific genes, tyrosinase (Tyr), microphthalmia transcription factor (Mitf), and melanosome structural proteins (Pmel17 and Mart1) but did not inhibit tyrosinase enzyme activity. These results suggest that the mechanism of inhibition of melanin biosynthesis by Siberian larch and pomegranate extracts, alone and in combination, is through downregulation of melanocyte specific genes and not due to inhibition of tyrosinase enzyme activity. Copyright © 2012 Elsevier B.V. All rights reserved.

Structure and Function of the Sterol Carrier Protein-2 N-Terminal Presequence†

PubMed Central

Martin, Gregory G.; Hostetler, Heather A.; McIntosh, Avery L.; Tichy, Shane E.; Williams, Brad J.; Russell, David H.; Berg, Jeremy M.; Spencer, Thomas A.; Ball, Judith; Kier, Ann B.; Schroeder, Friedhelm

2008-01-01

Although sterol carrier protein-2 (SCP-2) is encoded as a precursor protein (proSCP-2), little is known regarding the structure and function of the 20-amino acid N-terminal presequence. As shown herein, the presequence contains significant secondary structure and alters SCP-2: (i) secondary structure (CD), (ii) tertiary structure (aqueous exposure of Trp shown by UV absorbance, fluorescence, fluorescence quenching), (iii) ligand binding site [Trp response to ligands, peptide cross-linked by photoactivatable free cholesterol (FCBP)], (iv) selectivity for interaction with anionic phospholipid-rich membranes, (v) interaction with a peroxisomal import protein [FRET studies of Pex5p(C) binding], the N-terminal presequence increased SCP-2’s affinity for Pex5p(C) by 10-fold, and (vi) intracellular targeting in living and fixed cells (confocal microscopy). Nearly 5-fold more SCP-2 than proSCP-2 colocalized with plasma membrane lipid rafts/caveolae (AF488-CTB), 2.8-fold more SCP-2 than proSCP-2 colocalized with a mitochondrial marker (Mitotracker), but nearly 2-fold less SCP-2 than proSCP-2 colocalized with peroxisomes (AF488-antibody to PMP70). These data indicate the importance of the N-terminal presequence in regulating SCP-2 structure, cholesterol localization within the ligand binding site, membrane association, and, potentially, intracellular targeting. PMID:18465878

Data for a proteomic analysis of p53-independent induction of apoptosis by bortezomib

PubMed Central

Yerlikaya, Azmi; Okur, Emrah; Tarık Baykal, Ahmet; Acılan, Ceyda; Boyacı, İhsan; Ulukaya, Engin

2014-01-01

This data article contains data related to the research article entitled, “A proteomic analysis of p53-independent induction of apoptosis by bortezomib in 4T1 breast cancer cell line” by Yerlikaya et al. [1]. The research article presented 2-DE and nLC-MS/MS based proteomic analysis of proteasome inhibitor bortezomib-induced changes in the expression of cellular proteins. The report showed that GRP78 and TCEB2 were over-expressed in response to treatment with bortezomib for 24 h. In addition, the report demonstrated that Hsp70, the 26S proteasome non-ATPase regulatory subunit 14 and sequestosome 1 were increased at least 2 fold in p53-deficient 4T1 cells. The data here show for the first time the increased expressions of Card10, Dffb, Traf3 and Trp53bp2 in response to inhibition of the 26S proteasome. The information presented here also shows that both Traf1 and Xiap (a member of IAPs) are also downregulated simultaneously upon proteasomal inhibition. The increases in the level of Card10 and Trp53bp2 proteins were verified by Western blot analysis in response to varying concentrations of bortezomib for 24 h. PMID:26217687

Magnesium-adenosine diphosphate binding sites in wild-type creatine kinase and in mutants: role of aromatic residues probed by Raman and infrared spectroscopies.

PubMed

Hagemann, H; Marcillat, O; Buchet, R; Vial, C

2000-08-08

Two distinct methods were used to investigate the role of Trp residues during Mg-ADP binding to cytosolic creatine kinase (CK) from rabbit muscle: (1) Raman spectroscopy, which is very sensitive to the environment of aromatic side-chain residues, and (2) reaction-induced infrared difference spectroscopy (RIDS) and photolabile substrate (ADP[Et(PhNO(2))]), combined with site-directed mutagenesis on the four Trp residues of CK. Our Raman results indicated that the environment of Trp and of Tyr were not affected during Mg-ADP binding to CK. Analysis of RIDS of wild-type CK, inactive W227Y, and active W210,217,272Y mutants suggested that Trp227 was not involved in the stacking interactions. Results are consistent with Trp227 being essential to prevent water molecules from entering in the active site [as suggested by Gross, M., Furter-Graves, E. M., Wallimann, T., Eppenberger, H. M., and Furter, R. (1994) Protein Sci. 3, 1058-1068] and that another Trp could in addition help to steer the nucleotide in the binding site, although it is not essential for the activity of CK. Raman and infrared spectra indicated that Mg-ADP binding does not involve large secondary structure changes. Only 3-4 residues absorbing in the amide I region are directly implicated in the Mg-ADP binding (corresponding to secondary structure changes less than 1%), suggesting that movement of protein domains due to Mg-nucleotide binding do not promote large secondary structure changes.

Structural correlates of carrier protein recognition in tetanus toxoid-conjugated bacterial polysaccharide vaccines.

PubMed

Lockyer, Kay; Gao, Fang; Derrick, Jeremy P; Bolgiano, Barbara

2015-03-10

An analysis of structure-antibody recognition relationships in nine licenced polysaccharide-tetanus toxoid (TT) conjugate vaccines was performed. The panel of conjugates used included vaccine components to protect against disease caused by Haemophilus influenzae type b, Neisseria meningitidis groups A, C, W and Y and Streptococcus pneumoniae serotype 18C. Conformation and structural analysis included size exclusion chromatography with multi-angle light scattering to determine size, and intrinsic fluorescence spectroscopy and fluorescence quenching to evaluate the protein folding and exposure of Trp residues. A capture ELISA measured the recognition of TT epitopes in the conjugates, using four rat monoclonal antibodies: 2 localised to the HC domain, and 2 of which were holotoxoid conformation-dependent. The conjugates had a wide range of average molecular masses ranging from 1.8×10(6) g/mol to larger than 20×10(6) g/mol. The panel of conjugates were found to be well folded, and did not have spectral features typical of aggregated TT. A partial correlation was found between molecular mass and epitope recognition. Recognition of the epitopes either on the HC domain or the whole toxoid was not necessarily hampered by the size of the molecule. Correlation was also found between the accessibility of Trp side chains and polysaccharide loading, suggesting also that a higher level of conjugated PS does not necessarily interfere with toxoid accessibility. There were different levels of carrier protein Trp side-chain and epitope accessibility that were localised to the HC domain; these were related to the saccharide type, despite the conjugates being independently manufactured. These findings extend our understanding of the molecular basis for carrier protein recognition in TT conjugate vaccines. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

Structural correlates of carrier protein recognition in tetanus toxoid-conjugated bacterial polysaccharide vaccines

PubMed Central

Lockyer, Kay; Gao, Fang; Derrick, Jeremy P.; Bolgiano, Barbara

2015-01-01

An analysis of structure-antibody recognition relationships in nine licenced polysaccharide-tetanus toxoid (TT) conjugate vaccines was performed. The panel of conjugates used included vaccine components to protect against disease caused by Haemophilus influenzae type b, Neisseria meningitidis groups A, C, W and Y and Streptococcus pneumoniae serotype 18C. Conformation and structural analysis included size exclusion chromatography with multi-angle light scattering to determine size, and intrinsic fluorescence spectroscopy and fluorescence quenching to evaluate the protein folding and exposure of Trp residues. A capture ELISA measured the recognition of TT epitopes in the conjugates, using four rat monoclonal antibodies: 2 localised to the HC domain, and 2 of which were holotoxoid conformation-dependent. The conjugates had a wide range of average molecular masses ranging from 1.8 × 106 g/mol to larger than 20 × 106 g/mol. The panel of conjugates were found to be well folded, and did not have spectral features typical of aggregated TT. A partial correlation was found between molecular mass and epitope recognition. Recognition of the epitopes either on the HC domain or the whole toxoid was not necessarily hampered by the size of the molecule. Correlation was also found between the accessibility of Trp side chains and polysaccharide loading, suggesting also that a higher level of conjugated PS does not necessarily interfere with toxoid accessibility. There were different levels of carrier protein Trp side-chain and epitope accessibility that were localised to the HC domain; these were related to the saccharide type, despite the conjugates being independently manufactured. These findings extend our understanding of the molecular basis for carrier protein recognition in TT conjugate vaccines. PMID:25640334

Quantitative structure activity relationship studies of mushroom tyrosinase inhibitors

NASA Astrophysics Data System (ADS)

Xue, Chao-Bin; Luo, Wan-Chun; Ding, Qi; Liu, Shou-Zhu; Gao, Xing-Xiang

2008-05-01

Here, we report our results from quantitative structure-activity relationship studies on tyrosinase inhibitors. Interactions between benzoic acid derivatives and tyrosinase active sites were also studied using a molecular docking method. These studies indicated that one possible mechanism for the interaction between benzoic acid derivatives and the tyrosinase active site is the formation of a hydrogen-bond between the hydroxyl (aOH) and carbonyl oxygen atoms of Tyr98, which stabilized the position of Tyr98 and prevented Tyr98 from participating in the interaction between tyrosinase and ORF378. Tyrosinase, also known as phenoloxidase, is a key enzyme in animals, plants and insects that is responsible for catalyzing the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones. In the present study, the bioactivities of 48 derivatives of benzaldehyde, benzoic acid, and cinnamic acid compounds were used to construct three-dimensional quantitative structure-activity relationship (3D-QSAR) models using comparative molecular field (CoMFA) and comparative molecular similarity indices (CoMSIA) analyses. After superimposition using common substructure-based alignments, robust and predictive 3D-QSAR models were obtained from CoMFA ( q 2 = 0.855, r 2 = 0.978) and CoMSIA ( q 2 = 0.841, r 2 = 0.946), with 6 optimum components. Chemical descriptors, including electronic (Hammett σ), hydrophobic (π), and steric (MR) parameters, hydrogen bond acceptor (H-acc), and indicator variable ( I), were used to construct a 2D-QSAR model. The results of this QSAR indicated that π, MR, and H-acc account for 34.9, 31.6, and 26.7% of the calculated biological variance, respectively. The molecular interactions between ligand and target were studied using a flexible docking method (FlexX). The best scored candidates were docked flexibly, and the interaction between the benzoic acid derivatives and the tyrosinase active site was elucidated in detail. We believe that the QSAR models built here provide important information necessary for the design of novel tyrosinase inhibitors.

Preparation of tyrosinase inhibitors and antibrowning agents using green technology.

PubMed

Dong, Xue; Zhang, Yinan; He, Jia-Liang; Zhang, Shuang; Zeng, Mao-Mao; Chen, Jie; Zheng, Zong-Ping

2016-04-15

Chalcones and their derivatives have attracted great interests in recent years for their comprehensive biological activities. In this study, 2,4,2',4'-tetrahydroxychalcone and its two derivatives, 1,3,5-tris-(2,4-dihydroxy-phenyl)pentane-1,5-dione (new compound) and 7,2',4'-trihydroxyflavanone, were synthesized through one-pot green procedure catalyzed by boric acid in polyethylene glycol 400. Their structures were identified by ESI-MS and NMR spectral. Tyrosinase inhibitory activity and antibrowning test results showed that compounds 1-3 exhibited strong tyrosinase inhibitory activities and significant antibrowning effects on the fresh-cut lotus root slices at room temperature in 48 h. Among them, 0.01% 1,3,5-tris-(2,4-dihydroxy-phenyl)pentane-1,5-dione combined with 0.5% VC showed the best antibrowning ability. In brief, this study offers a protocol for one-pot green synthesis of high efficiency tyrosinase inhibitors which may be suitable as antibrowning agents for fresh-cut vegetables. More important, this study developed a new type of 1,5-dione derivative which may serve as new lead structures for novel tyrosinase inhibitors discovery. Copyright © 2015 Elsevier Ltd. All rights reserved.

Antioxidant activity and inhibitory effects of 2-hydroxy-3-methylcyclopent-2-enone isolated from ribose-histidine Maillard reaction products on aldose reductase and tyrosinase.

PubMed

Hwang, Seung Hwan; Wang, Zhiqiang; Suh, Hong-Won; Lim, Soon Sung

2018-03-01

This study aimed to better understand the functional properties of ribose and 20 amino acid Maillard reaction products (MRPs). The ABTS + radical scavenging ability of the ribose-20 amino acid MRPs was evaluated. Among the MRPs, ribose-histidine MRPs (RH-MRPs) showed the highest inhibitory activities on the ABTS + radical scavenging ability, aldose reductase (AR), and tyrosinase compared to other MRPs. Functional compounds with antioxidant and AR inhibitory activities have been recognized as an important strategy in the prevention and treatment of diabetic complications, and the search for tyrosinase inhibitors is important for the treatment of hyperpigmentation, development of skin-whitening agents, and use as preservatives in the food industry. On this basis, we sought to isolate and identify compounds with inhibitory activities against AR and tyrosinase. RH-MRPs were heated at 120 °C for 2 h and fractionated using four solvents: methylene chloride (MC), ethyl acetate, n-butanol, and water. The highest inhibitions were found in the MC fraction. The two compounds from this fraction were purified by silica gel column and preparative thin layer chromatography, and identified as 2-hydroxy-3-methylcyclopent-2-enone and furan-3-carboxylic acid. AR inhibition, tyrosinase inhibition, and ABTS + scavenging (IC 50 ) of 2-hydroxy-3-methylcyclopent-2-enone were 4.47, 721.91 and 9.81 μg mL -1 , respectively. In this study, inhibitory effects of 2-hydroxy-3-methylcyclopent-2-enone isolated from RH-MRP were demonstrated on AR, tyrosinase, and its antioxidant activity for the first time. RH-MRP and its constituents can be developed as beneficial functional food sources and cosmetic materials and should be investigated further as potential functional food sources.

Wnt/β-catenin signaling inhibitor ICG-001 enhances pigmentation of cultured melanoma cells.

PubMed

Kim, Kyung-Il; Jeong, Do-Sun; Jung, Eui Chang; Lee, Jeung-Hoon; Kim, Chang Deok; Yoon, Tae-Jin

2016-11-01

Wnt/β-catenin signaling is important in development and differentiation of melanocytes. The object of this study was to evaluate the effects of several Wnt/β-catenin signaling inhibitors on pigmentation using melanoma cells. Melanoma cells were treated with Wnt/β-catenin signaling inhibitors, and then melanin content and tyrosinase activity were checked. Although some inhibitors showed slight inhibition of pigmentation, we failed to observe potential inhibitory effect of those chemicals on pigmentation of HM3KO melanoma cells. Rather, one of powerful Wnt/β-catenin signaling inhibitors, ICG-001, increased the pigmentation of HM3KO melanoma cells. Pigmentation-enhancing effect of ICG-001 was reproducible in other melanoma cell line MNT-1. Consistent with these results. ICG-001 increased the expression of pigmentation-related genes, such as MITF, tyrosinase and TRP1. When ICG-001 was treated, the phosphorylation of CREB was significantly increased. In addition, ICG-001 treatment led to quick increase of intracellular cAMP level, suggesting that ICG-001 activated PKA signaling. The blockage of PKA signaling with pharmaceutical inhibitor H89 inhibited the ICG-001-induced pigmentation significantly. These results suggest that PKA signaling is pivotal in pigmentation process itself, while the importance of Wnt/β-catenin signaling should be emphasized in the context of development and differentiation. Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Distinct modes of perimembrane TRP channel turnover revealed by TIR-FRAP.

PubMed

Ghosh, Debapriya; Segal, Andrei; Voets, Thomas

2014-11-19

Transient Receptor Potential (TRP) channels form a broadly expressed and functionally diverse family of cation channels involved in various (patho)physiological processes. Whereas the mechanisms that control opening of TRP channels have been extensively studied, little is known about the transport processes of TRP channels to and within the plasma membrane. Here we used Total Internal Reflection--Fluorescence Recovery after Photobleaching (TIR-FRAP) to selectively visualize and bleach the fluorescently labeled TRP channels TRPV2 and TRPM4 in close proximity of the glass-plasma membrane interface, allowing detailed analysis of their perimembrane dynamics. We show that recovery of TRPM4 occurs via 200-nm diameter transport vesicles, and demonstrate the full fusion of such vesicles with the plasma membrane. In contrast, TRPV2 recovery proceeded mainly via lateral diffusion from non-bleached areas of the plasma membrane. Analysis of the two-dimensional channel diffusion kinetics yielded 2D diffusion coefficients ranging between 0.1 and 0.3 μm(2)/s, suggesting that these TRP channels move relatively unrestricted within the plasma membrane. These data demonstrate distinct modes of TRP channel turnover at the plasma membrane and illustrate the usefulness of TIR-FRAP to monitor these processes with high resolution.

Molecular dynamics studies of a DNA-binding protein: 2. An evaluation of implicit and explicit solvent models for the molecular dynamics simulation of the Escherichia coli trp repressor.

PubMed Central

Guenot, J.; Kollman, P. A.

1992-01-01

Although aqueous simulations with periodic boundary conditions more accurately describe protein dynamics than in vacuo simulations, these are computationally intensive for most proteins. Trp repressor dynamic simulations with a small water shell surrounding the starting model yield protein trajectories that are markedly improved over gas phase, yet computationally efficient. Explicit water in molecular dynamics simulations maintains surface exposure of protein hydrophilic atoms and burial of hydrophobic atoms by opposing the otherwise asymmetric protein-protein forces. This properly orients protein surface side chains, reduces protein fluctuations, and lowers the overall root mean square deviation from the crystal structure. For simulations with crystallographic waters only, a linear or sigmoidal distance-dependent dielectric yields a much better trajectory than does a constant dielectric model. As more water is added to the starting model, the differences between using distance-dependent and constant dielectric models becomes smaller, although the linear distance-dependent dielectric yields an average structure closer to the crystal structure than does a constant dielectric model. Multiplicative constants greater than one, for the linear distance-dependent dielectric simulations, produced trajectories that are progressively worse in describing trp repressor dynamics. Simulations of bovine pancreatic trypsin were used to ensure that the trp repressor results were not protein dependent and to explore the effect of the nonbonded cutoff on the distance-dependent and constant dielectric simulation models. The nonbonded cutoff markedly affected the constant but not distance-dependent dielectric bovine pancreatic trypsin inhibitor simulations. As with trp repressor, the distance-dependent dielectric model with a shell of water surrounding the protein produced a trajectory in better agreement with the crystal structure than a constant dielectric model, and the physical properties of the trajectory average structure, both with and without a nonbonded cutoff, were comparable. PMID:1304396

Cavity-Creating Mutations in Pseudomonas aeruginosa Azurin: Effects on Protein Dynamics and Stability

PubMed Central

Gabellieri, Edi; Balestreri, Ettore; Galli, Alvaro; Cioni, Patrizia

2008-01-01

Changes in flexibility and structural stability of Pseudomonas aeruginosa azurin in response to cavity-creating mutations were probed by the phosphorescence emission of Trp-48, which was deeply buried in the compact hydrophobic core of the macromolecule, and by measurements of guanidinum hydrochloride unfolding, respectively. Replacement of the bulky side chains Phe-110, Phe-29, and Tyr-108 with the smaller Ala introduced cavities at different distances from the hydrophobic core. The phosphorescence lifetime (τ0) of Trp-48, buried inside the protein core, and the acrylamide quenching rate constant (kq) were used to monitor local and global flexibility changes induced by the introduction of the cavity. The results of this work demonstrate the following: 1), the effect on core flexibility of the insertion of cavities is not correlated readily to the distance of the cavity from the core; 2), the protein global flexibility results are related to the cavity distance from the packed core of the macromolecule; and 3), the increase in protein flexibility does not correspond necessarily to a comparable destabilizing effect of some mutations. PMID:18424505

Raman scattering and red fluorescence in the photochemical transformation of dry tryptophan particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lai, Chih Wei; Schwab, Mark; Hill, Steven C.

Tryptophan is a fluorescent amino acid common in proteins. Its absorption is largest for wavelengths λ ≲ 290 nm and its fluorescence emissions peak around 300–350 nm, depending upon the local environment. Here we report the observation of red fluorescence near 600 nm emerging from 488-nm continuous-wave (CW) laser photoexcitation of dry tryptophan (Trp) particles. With an excitation intensity below 0.5 kW/cm 2, dry Trp particles yield distinctive Raman scattering peaks in the presence of relatively weak and spectrally broad emissions with λ ~500–700 nm, allowing estimation of particle temperature at low excitation intensities. When the photoexcitation intensity is increasedmore » to 1 kW/cm 2 or more for a few minutes, fluorescence intensity dramatically increases by more than two orders of magnitude. The fluorescence continues to increase in intensity and gradually shift to the red when photoexcitation intensity and the duration of exposure are increased. The resulting products absorb at visible wavelengths and generate red fluorescence with λ ~ 650–800 nm with 633-nm CW laser excitation. In conclusion, we attribute the emergence of orange and red fluorescence in the Trp products to a photochemical transformation that is instigated by weak optical transitions to triplet states in Trp with 488-nm excitation and which may be expedited by a photothermal effect.« less

Raman scattering and red fluorescence in the photochemical transformation of dry tryptophan particles

DOE PAGES

Lai, Chih Wei; Schwab, Mark; Hill, Steven C.; ...

2016-05-19

Tryptophan is a fluorescent amino acid common in proteins. Its absorption is largest for wavelengths λ ≲ 290 nm and its fluorescence emissions peak around 300–350 nm, depending upon the local environment. Here we report the observation of red fluorescence near 600 nm emerging from 488-nm continuous-wave (CW) laser photoexcitation of dry tryptophan (Trp) particles. With an excitation intensity below 0.5 kW/cm 2, dry Trp particles yield distinctive Raman scattering peaks in the presence of relatively weak and spectrally broad emissions with λ ~500–700 nm, allowing estimation of particle temperature at low excitation intensities. When the photoexcitation intensity is increasedmore » to 1 kW/cm 2 or more for a few minutes, fluorescence intensity dramatically increases by more than two orders of magnitude. The fluorescence continues to increase in intensity and gradually shift to the red when photoexcitation intensity and the duration of exposure are increased. The resulting products absorb at visible wavelengths and generate red fluorescence with λ ~ 650–800 nm with 633-nm CW laser excitation. In conclusion, we attribute the emergence of orange and red fluorescence in the Trp products to a photochemical transformation that is instigated by weak optical transitions to triplet states in Trp with 488-nm excitation and which may be expedited by a photothermal effect.« less

Raman scattering and red fluorescence in the photochemical transformation of dry tryptophan particles.

PubMed

Lai, Chih Wei; Schwab, Mark; Hill, Steven C; Santarpia, Joshua; Pan, Yong-Le

2016-05-30

Tryptophan is a fluorescent amino acid common in proteins. Its absorption is largest for wavelengths λ ≲ 290 nm and its fluorescence emissions peak around 300-350 nm, depending upon the local environment. Here we report the observation of red fluorescence near 600 nm emerging from 488-nm continuous-wave (CW) laser photoexcitation of dry tryptophan (Trp) particles. With an excitation intensity below 0.5 kW/cm², dry Trp particles yield distinctive Raman scattering peaks in the presence of relatively weak and spectrally broad emissions with λ ∼500-700 nm, allowing estimation of particle temperature at low excitation intensities. When the photoexcitation intensity is increased to 1 kW/cm² or more for a few minutes, fluorescence intensity dramatically increases by more than two orders of magnitude. The fluorescence continues to increase in intensity and gradually shift to the red when photoexcitation intensity and the duration of exposure are increased. The resulting products absorb at visible wavelengths and generate red fluorescence with λ ∼ 650-800 nm with 633-nm CW laser excitation. We attribute the emergence of orange and red fluorescence in the Trp products to a photochemical transformation that is instigated by weak optical transitions to triplet states in Trp with 488-nm excitation and which may be expedited by a photothermal effect.

The Metarhizium anisopliae trp1 gene: cloning and regulatory analysis.

PubMed

Staats, Charley Christian; Silva, Marcia Suzana Nunes; Pinto, Paulo Marcos; Vainstein, Marilene Henning; Schrank, Augusto

2004-07-01

The trp1 gene from the entomopathogenic fungus Metarhizium anisopliae, cloned by heterologous hybridization with the plasmid carrying the trpC gene from Aspergillus nidulans, was sequence characterized. The predicted translation product has the conserved catalytic domains of glutamine amidotransferase (G domain), indoleglycerolphosphate synthase (C domain), and phosphoribosyl anthranilate isomerase (F domain) organized as NH2-G-C-F-COOH. The ORF is interrupted by a single intron of 60 nt that is position conserved in relation to trp genes from Ascomycetes and length conserved in relation to Basidiomycetes species. RT-PCR analysis suggests constitutive expression of trp1 gene in M. anisopliae.

Agonist properties of a stable hexapeptide analog of neurotensin, N alpha MeArg-Lys-Pro-Trp-tLeu-Leu (NT1).

PubMed

Akunne, H C; Demattos, S B; Whetzel, S Z; Wustrow, D J; Davis, D M; Wise, L D; Cody, W L; Pugsley, T A; Heffner, T G

1995-04-18

The major signal transduction pathway for neurotensin (NT) receptors is the G-protein-dependent stimulation of phospholipase C, leading to the mobilization of intracellular free Ca2+ ([Ca2+]i) and the stimulation of cyclic GMP. We investigated the functional actions of an analog of NT(8-13), N alpha MeArg-Lys-Pro-Trp-tLeu-Leu (NT1), and other NT related analogs by quantitative measurement of the cytosolic free Ca2+ concentration in HT-29 (human colonic adenocarcinoma) cells using the Ca(2+)-sensitive dye fura-2/AM and by effects on cyclic GMP levels in rat cerebellar slices. The NT receptor binding affinities for these analogs to HT-29 cell membranes and newborn (10-day-old) mouse brain membranes were also investigated. Data obtained from HT-29 cell and mouse brain membrane preparations showed saturable single high-affinity sites and binding densities (Bmax) of 130.2 and 87.5 fmol/mg protein, respectively. The respective KD values were 0.47 and 0.39 nM, and the Hill coefficients were 0.99 and 0.92. The low-affinity levocabastine-sensitive site was not present (K1 > 10,000) in either membrane preparation. Although the correlation of binding between HT-29 cell membranes and mouse brain membranes was quite significant (r = 0.92), some of the reference agents had lower binding affinities in the HT-29 cell membranes. The metabolically stable compound NT1 plus other NT analogs and related peptides [NT, NT(8-13), xenopsin, neuromedin N, NT(9-13), kinetensin and (D-Trp11)-NT] increased intracellular Ca2+ levels in HT-29 cells, indicating NT receptor agonist properties. The effect of NT1 in mobilizing [Ca2+]i blocked by SR 48692, a non-peptide NT antagonist. Receptor binding affinities of NT analogs to HT-29 cell membranes were positively correlated with potencies for mobilizing intracellular calcium in the same cells. In addition, NT1 increased cyclic GMP levels in rat cerebellar slices, confirming the latter findings of its NT agonist action. These results substantiate the in vitro NT agonist properties of the hexapeptide NT analog NT1.

A stationary-phase protein of Escherichia coli that affects the mode of association between the trp repressor protein and operator-bearing DNA.

PubMed

Yang, W; Ni, L; Somerville, R L

1993-06-15

Highly purified preparations of trp repressor (TrpR) protein derived from Escherichia coli strains that were engineered to overexpress this material were found to contain another protein, of 21 kDa. The second protein, designated WrbA [for tryptophan (W) repressor-binding protein] remained associated with its namesake through several sequential protein fractionation steps. The N-terminal amino acid sequence of the WrbA protein guided the design of two degenerate oligonucleotides that were used as probes in the cloning of the wrbA gene (198 codons). The WrbA protein, in purified form, was found by several criteria to enhance the formation and/or stability of noncovalent complexes between TrpR holorepressor and its primary operator targets. The formation of an operator-holorepressor-WrbA ternary complex was demonstrated by gel mobility-shift analysis. The WrbA protein alone does not interact with the trp operator. During the stationary phase, cells deficient in the WrbA protein were less efficient than wild type in their ability to repress the trp promoter. It is proposed that the WrbA protein functions as an accessory element in blocking TrpR-specific transcriptional processes that might be physiologically disadvantageous in the stationary phase of the bacterial life cycle.

The nature of the apolar phase influences the structure of the protein emulsifier in oil-in-water emulsions stabilized by bovine serum albumin. A front-surface fluorescence study.

PubMed

Rampon, Vincent; Brossard, Chantal; Mouhous-Riou, Nadine; Bousseau, Benoît; Llamas, Geneviève; Genot, Claude

2004-05-20

Proteins are widely used as emulsifiers in food emulsions. Model emulsions, designed to study emulsifying properties of proteins and their conformation at the interfaces often contain a hydrocarbon as apolar phase instead of natural triglycerides as found in food products. Yet, some results indicate that the protein conformation at the interface depends on the nature of the apolar phase. Front-surface fluorescence spectroscopy was used to evidence differences in the structure of bovine serum albumin (BSA) adsorbed at the interface of emulsions prepared with different apolar phases: an hydrocarbon (n-dodecane), a synthetic medium-chain triglyceride (miglyol) and a natural vegetable oil (sunflower oil). Emulsions had similar size distributions of oil droplets. Front-surface fluorescence emission spectra of tryptophanyl residues of the protein (Trp) in emulsions, creams and serums varied as a function of the nature of hydrophobic phase. In emulsions and creams, wavelength of the maximum fluorescence intensities was blue-shifted as compared to the BSA solution. The shift was larger in creams than in emulsions and in samples containing dodecane than with the other apolar phases. Fourth derivative spectra of emulsions and creams exhibited two peaks assigned, respectively, to Trp located in hydrophilic and hydrophobic environments. The peaks were slightly red-shifted in the presence of sunflower oil as compared to miglyol and dodecane and the relative intensity of the "hydrophobic peak" was higher in dodecane. The effects were greater in creams than in emulsions. Fluorescence intensity of Trp was the highest in the serums of emulsions prepared with dodecane as compared to serums issued from sunflower oil and miglyol emulsions. Thus, proportion of adsorbed protein was lower in dodecane emulsions than with the other apolar phases. These results evidence that the mean environment of Trp was more hydrophobic in emulsions and creams than in solutions due to a displacement of some of the Trp of the protein to a more hydrophobic environment. Dodecane had the greatest impact on Trp environment (more hydrophobic) followed by miglyol and then by sunflower oil. This is likely due to differences in the conformation of the protein at the hydrocarbon-water interface as compared to the triacylglycerol-water ones. In addition, sunflower oil provoked a large decrease of Trp fluorescence intensity in emulsions and creams as compared to miglyol or dodecane. This could be due to contaminant quenchers in the oil or to interactions of the unsaturated fatty chains with the protein inducing quenching of the Trp. These observations should be related to the physical properties of the apolar phase and its molecular organization and interactions with the protein at the interface.

Induction of Melanogenesis by Rapamycin in Human MNT-1 Melanoma Cells

PubMed Central

Hah, Young-Sool; Cho, Hee Young; Lim, Tae-Yeon; Park, Dong Hwa; Kim, Hwa Mi; Yoon, Jimi; Kim, Jin Gu; Kim, Chi Yeon

2012-01-01

Background Melanogenesis is one of the characteristic parameters of differentiation in melanocytes and melanoma cells. Specific inhibitors of phosphatidylinositol 3-kinase (PI3K), such as wortmannin and LY294002, stimulate melanin production in mouse and in human melanoma cells, suggesting that PI3K and mammalian target of rapamycin (mTOR) might be involved in the regulation of melanogenesis. Objective The involvement of the mTOR pathway in regulating melanogenesis was examined using human MNT-1 melanoma cells, and the effects of the potent inhibitor of mTOR, rapamycin, in the presence or absence of α-melanocyte-stimulating hormone (α-MSH) were evaluated. Methods In cells treated with rapamycin, cell viability, melanin content, and tyrosinase (TYR) activity were measured and compared with untreated controls. Protein levels of TYR, tyrosinase-related protein (TYRP)-1, TYRP-2, and microphthalmia-associated transcription factor (MITF) were also analyzed by Western blot. Results In rapamycin-treated cells, the melanin content increased concomitantly with an elevation in TYR activity, which plays a major role in melanogenesis. There was also an up-regulation of TYR, TYRP-1, and MITF proteins. Combined treatment with rapamycin or wortmannin and α-MSH increased melanogenesis more strongly than α-MSH alone. Conclusion Rapamycin-induced melanin formation may be mediated through the up-regulation of TYR protein and activity. Furthermore, rapamycin and wortmannin, inhibitors of mTOR and PI3K, respectively, have co-stimulatory effects with α-MSH in enhancing melanogenesis in melanocyte cells. PMID:22577264

Induction of melanogenesis by rapamycin in human MNT-1 melanoma cells.

PubMed

Hah, Young-Sool; Cho, Hee Young; Lim, Tae-Yeon; Park, Dong Hwa; Kim, Hwa Mi; Yoon, Jimi; Kim, Jin Gu; Kim, Chi Yeon; Yoon, Tae-Jin

2012-05-01

Melanogenesis is one of the characteristic parameters of differentiation in melanocytes and melanoma cells. Specific inhibitors of phosphatidylinositol 3-kinase (PI3K), such as wortmannin and LY294002, stimulate melanin production in mouse and in human melanoma cells, suggesting that PI3K and mammalian target of rapamycin (mTOR) might be involved in the regulation of melanogenesis. The involvement of the mTOR pathway in regulating melanogenesis was examined using human MNT-1 melanoma cells, and the effects of the potent inhibitor of mTOR, rapamycin, in the presence or absence of α-melanocyte-stimulating hormone (α-MSH) were evaluated. In cells treated with rapamycin, cell viability, melanin content, and tyrosinase (TYR) activity were measured and compared with untreated controls. Protein levels of TYR, tyrosinase-related protein (TYRP)-1, TYRP-2, and microphthalmia-associated transcription factor (MITF) were also analyzed by Western blot. In rapamycin-treated cells, the melanin content increased concomitantly with an elevation in TYR activity, which plays a major role in melanogenesis. There was also an up-regulation of TYR, TYRP-1, and MITF proteins. Combined treatment with rapamycin or wortmannin and α-MSH increased melanogenesis more strongly than α-MSH alone. Rapamycin-induced melanin formation may be mediated through the up-regulation of TYR protein and activity. Furthermore, rapamycin and wortmannin, inhibitors of mTOR and PI3K, respectively, have co-stimulatory effects with α-MSH in enhancing melanogenesis in melanocyte cells.

Electrode Potentials of l-Tryptophan, l-Tyrosine, 3-Nitro-l-tyrosine, 2,3-Difluoro-l-tyrosine, and 2,3,5-Trifluoro-l-tyrosine.

PubMed

Mahmoudi, Leila; Kissner, Reinhard; Nauser, Thomas; Koppenol, Willem H

2016-05-24

Electrode potentials for aromatic amino acid radical/amino acid couples were deduced from cyclic voltammograms and pulse radiolysis experiments. The amino acids investigated were l-tryptophan, l-tyrosine, N-acetyl-l-tyrosine methyl ester, N-acetyl-3-nitro-l-tyrosine ethyl ester, N-acetyl-2,3-difluoro-l-tyrosine methyl ester, and N-acetyl-2,3,5-trifluoro-l-tyrosine methyl ester. Conditional potentials were determined at pH 7.4 for all compounds listed; furthermore, Pourbaix diagrams for l-tryptophan, l-tyrosine, and N-acetyl-3-nitro-l-tyrosine ethyl ester were obtained. Electron transfer accompanied by proton transfer is reversible, as confirmed by detailed analysis of the current waves, and because the slopes of the Pourbaix diagrams obey Nernst's law. E°'(Trp(•),H(+)/TrpH) and E°'(TyrO(•),H(+)/TyrOH) at pH 7 are 0.99 ± 0.01 and 0.97 ± 0.01 V, respectively. Pulse radiolysis studies of two dipeptides that contain both amino acids indicate a difference in E°' of approximately 0.06 V. Thus, in small peptides, we recommend values of 1.00 and 0.96 V for E°'(Trp(•),H(+)/TrpH) and E°'(TyrO(•),H(+)/TyrOH), respectively. The electrode potential of N-acetyl-3-nitro-l-tyrosine ethyl ester is higher, while because of mesomeric stabilization of the radical, those of N-acetyl-2,3-difluoro-l-tyrosine methyl ester and N-acetyl-2,3,5-trifluoro-l-tyrosine methyl ester are lower than that of tyrosine. Given that the electrode potentials at pH 7 of E°'(Trp(•),H(+)/TrpH) and E°'(TyrO(•),H(+)/TyrOH) are nearly equal, they would be, in principle, interchangeable. Proton-coupled electron transfer pathways in proteins that use TrpH and TyrOH are thus nearly thermoneutral.

Canonical Transient Receptor Potential (TRPC) 1 Acts as a Negative Regulator for Vanilloid TRPV6-mediated Ca2+ Influx*

PubMed Central

Schindl, Rainer; Fritsch, Reinhard; Jardin, Isaac; Frischauf, Irene; Kahr, Heike; Muik, Martin; Riedl, Maria Christine; Groschner, Klaus; Romanin, Christoph

2012-01-01

TRP proteins mostly assemble to homomeric channels but can also heteromerize, preferentially within their subfamilies. The TRPC1 protein is the most versatile member and forms various TRPC channel combinations but also unique channels with the distantly related TRPP2 and TRPV4. We show here a novel cross-family interaction between TRPC1 and TRPV6, a Ca2+ selective member of the vanilloid TRP subfamily. TRPV6 exhibited substantial co-localization and in vivo interaction with TRPC1 in HEK293 cells, however, no interaction was observed with TRPC3, TRPC4, or TRPC5. Ca2+ and Na+ currents of TRPV6-overexpressing HEK293 cells are significantly reduced by co-expression of TRPC1, correlating with a dramatically suppressed plasma membrane targeting of TRPV6. In line with their intracellular retention, remaining currents of TRPC1 and TRPV6 co-expression resemble in current-voltage relationship that of TRPV6. Studying the N-terminal ankyrin like repeat domain, structurally similar in the two proteins, we have found that these cytosolic segments were sufficient to mediate a direct heteromeric interaction. Moreover, the inhibitory role of TRPC1 on TRPV6 influx was also maintained by expression of only its N-terminal ankyrin-like repeat domain. Our experiments provide evidence for a functional interaction of TRPC1 with TRPV6 that negatively regulates Ca2+ influx in HEK293 cells. PMID:22932896

The Standardized Extract of Juniperus communis Alleviates Hyperpigmentation in Vivo HRM-2 Hairless Mice and in Vitro Murine B16 Melanoma Cells.

PubMed

Jegal, Jonghwan; Chung, Ki Wung; Chung, Hae Young; Jeong, Eun Ju; Yang, Min Hye

2017-01-01

In European folk medicine, the fruits of Juniperus communis are used in the treatment of skin-related disorders such as skin infection, itching, and psoriasis. Previously, we reported that the EtOAc fraction of J. communis (EAJC) contained tyrosinase inhibition properties in vitro non-cellular experiment. The aim of this study was to evaluate anti-melanogenic effect of standardized EAJC on a hyperpigmentation animal model. Therapeutic effects of EAJC toward skin hyperpigmentation were confirmed by both in vivo experiment and in vitro cell-based assay. Skin depigmenting effect was detected by topical treatment of EAJC for 11 d to HRM-2 melanin-possessing hairless mice. Histologic findings including significantly decreased melanin depositions could be observed in dorsal skin samples of EAJC-treated group. In addition, the EAJC (50 µg/mL) attenuated melanin production through down-regulation of tyrosinase activity and protein expression in B16 murine melanoma cells. According to the phytochemical analysis, EAJC was found to contain hypolaetin-7-O-β-D-xylopyranoside and isoscutellarein-7-O-β-D-xylopyranoside as main components. Hypolaetin-7-O-β-D-xylopyranoside was responsible for the skin-lightening effect of EAJC by reducing the number of melanocytes in dorsal skins of HRM-2 mice. The present study provided direct experimental evidence for skin-lightening effect of EAJC in UV-irradiated hairless mouse model. Therapeutic attempts with the J. communis might be useful in the management of skin pigmentation-related diseases.

Tumor-derived heat shock protein 70 peptide complexes are cross-presented by human dendritic cells.

PubMed

Noessner, Elfriede; Gastpar, Robert; Milani, Valeria; Brandl, Anna; Hutzler, Peter J S; Kuppner, Maria C; Roos, Miriam; Kremmer, Elisabeth; Asea, Alexzander; Calderwood, Stuart K; Issels, Rolf D

2002-11-15

Our study demonstrates that tumor-derived heat shock protein (HSP)70 chaperones a tyrosinase peptide and mediates its transfer to human immature dendritic cells (DCs) by receptor-dependent uptake. Human tumor-derived HSP70 peptide complexes (HSP70-PC) thus have the immunogenic potential to instruct DCs to cross-present endogenously expressed, nonmutated, and tumor antigenic peptides that are shared among tumors of the melanocytic lineage for T cell recognition. T cell stimulation by HSP70-instructed DCs is dependent on the Ag bound to HSP70 in that only DCs incubated with HSP70-PC purified from tyrosinase-positive (HSP70-PC/tyr(+)) but not from tyrosinase-negative (HSP70-PC/tyr(-)) melanoma cells resulted in the specific activation of the HLA-A*0201-restricted tyrosinase peptide-specific cytotoxic T cell clone. HSP70-PC-mediated T cell stimulation is very efficient, delivering the tyrosinase peptide at concentrations as low as 30 ng/ml of HSP70-PC for T cell recognition. Receptor-dependent binding of HSP70-PC and active cell metabolism are prerequisites for MHC class I-restricted cross-presentation and T cell stimulation. T cell stimulation does not require external DC maturation signals (e.g., exogenously added TNF-alpha), suggesting that signaling DC maturation is an intrinsic property of the HSP70-PC itself and related to receptor-mediated binding. The cross-presentation of a shared human tumor Ag together with the exquisite efficacy are important new aspects for HSP70-based immunotherapy in clinical anti-cancer vaccination strategies, and suggest a potential extension of HSP70-based vaccination protocols from a patient-individual treatment modality to its use in an allogeneic setting.

Glycine inhibits melanogenesis in vitro and causes hypopigmentation in vivo.

PubMed

Ishikawa, Masago; Kawase, Ichiro; Ishii, Fumio

2007-11-01

The simplest amino acid, glycine, is important in protein composition and plays a significant role in numerous physiological events in mammals. Despite the inhibitory effect of glycine on spontaneous melanogenesis in B16F0 melanoma cells, the details of the underlying mechanisms remain unknown. The present study was conducted to investigate the further effects and the mechanisms of inhibitory effect of glycine on melanogenesis using B16F0 melanoma cells and hair follicle melanogenesis in C57BL/6J mice. Treatment with glycine (1-16 mM) for 72 h inhibited alpha-melanocyte stimulating hormone (alpha-MSH)-induced melanogenesis in a concentration-dependent manner without any effects on cell proliferation in B16F0 melanoma cells. Treatment with kojic acid (2.5 mM) for 72 h also inhibited alpha-MSH-induced melanogenesis in B16F0 melanoma cells. The highest dose of glycine inhibited the alpha-MSH-induced increment of tyrosinase protein levels in B16F0 melanoma cells. In hair follicle melanogenesis in C57BL/6J mice, treatment with glycine (1250 or 2500 mg/kg, i.p.) for 5 d prevented the decrement of L* and C* values and inhibited the increment of tyrosinase protein levels and melanin content within the skin. Treatment with hydroquinone (100 mg/kg, i.p.) for 5 d had a similar hypopigmenting effect to that of high dose glycine. These results suggest that glycine has an inhibitory effect on melanogenesis that is mediated by down-regulation of tyrosinase protein levels, leading to a hypopigmenting effect in C57BL/6J mice.

Tyrosinase inhibitory constituents from a polyphenol enriched fraction of rose oil distillation wastewater.

PubMed

Solimine, Jessica; Garo, Eliane; Wedler, Jonas; Rusanov, Krasimir; Fertig, Orlando; Hamburger, Matthias; Atanassov, Ivan; Butterweck, Veronika

2016-01-01

During the water steam distillation process of rose flowers, the non-volatile phenolic compounds remain in the waste. We recently developed a strategy to separate rose oil distillation water (RODW) into a polyphenol depleted water fraction and a polyphenol enriched fraction (RF20-SP207). Bioassay-guided investigation of RF20-SP207 led to the isolation of quercetin, kaempferol and ellagic acid. Their structures were elucidated by spectroscopic analysis as well as by comparison with literature data. Tyrosinase inhibition studies were performed with RF20-SP207, fractions I-IV, and the isolated compounds of the most active fraction. RF20-SP207 strongly inhibited the enzyme with an IC50 of 0.41 μg/mL. From the tested fractions only fraction IV (IC50=5.81 μg/mL) exhibited strong anti-tyrosinase activities. Quercetin, kaempferol and ellagic acid were identified in fraction IV and inhibited mushroom tyrosinase with IC50 values of 4.2 μM, 5.5 μM and 5.2 μM, respectively, which is approximately 10 times more potent than that of the positive control kojic acid (56.1μM). The inhibition kinetics, analyzed by Lineweaver-Burk plots, indicated that RF20-SP207 and fraction IV are uncompetitive inhibitors of tyrosinase when l-tyrosine is used as a substrate. A mixed inhibition was determined for ellagic acid, and a competitive inhibition for quercetin and kaempferol. In conclusion, the recovered polyphenol fraction RF20-SP207 from RODW was found to be a potent tyrosinase inhibitor. This value-added product could be used as an active ingredient in cosmetic products related to hyperpigmentation. Copyright © 2015 Elsevier B.V. All rights reserved.

Microwave-assisted synthesis and tyrosinase inhibitory activity of chalcone derivatives.

PubMed

Liu, Jinbing; Chen, Changhong; Wu, Fengyan; Zhao, Liangzhong

2013-07-01

A series of chalcones and their derivatives were synthesized, and their inhibitory effects on the diphenolase activity of mushroom tyrosinase were evaluated. The results showed that some of the synthesized compounds exhibited significant inhibitory activity, and four compounds exhibited more potent tyrosinase inhibitory activity than the reference standard inhibitor kojic acid (5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one). Specifically, 1-(-1-(4-methoxyphen- yl)-3-phenylallylidene)thiosemicarbazide (18) exhibited the most potent tyrosinase inhibitory activity with IC₅₀ value of 0.274 μM. The inhibition mechanism analysis of 1-(-1-(2,4-dihydroxyphenyl)-3-phenylallylidene) thiosemicarbazide (16) and 1-(-1-(4-methoxyphenyl)-3-phenylallylidene) thiosemicarbazide (18) demonstrated that the inhibitory effects of the two compounds on the tyrosinase were irreversible. Preliminary structure activity relationships' analysis suggested that further development of such compounds might be of interest. © 2013 John Wiley & Sons A/S.

Tryptophan-to-Tryptophan Energy Transfer in UV-B photoreceptor UVR8

NASA Astrophysics Data System (ADS)

Li, Xiankun; Zhong, Dongping

UVR8 (UV RESISTANCE LOCUS 8) protein is a UV-B photoreceptor in high plants. UVR8 is a homodimer that dissociates into monomers upon UV-B irradiation (280 nm to 315 nm), which triggers various protective mechanisms against UV damages. Uniquely, UVR8 does not contain any external chromophores and utilizes the UV-absorbing natural amino acid tryptophan (Trp) to perceive UV-B. Each UVR8 monomer has 14 tryptophan residues. However, only 2 epicenter Trp (W285 W233) are critical to the light induced dimer-to-monomer transformation. Here, we revealed, using site-directed mutagenesis and spectroscopy, a striking energy flow network, in which other tryptophan chromophores serve as antenna to transfer excitation energy to epicenter Trp, greatly enhancing UVR8 light-harvesting efficiency. Furthermore, Trp-to-Trp energy transfer rates were measured and agree well with theoretical values.

In Silico Screening and Molecular Dynamics Simulation of Disease-Associated nsSNP in TYRP1 Gene and Its Structural Consequences in OCA3

PubMed Central

Kamaraj, Balu

2013-01-01

Oculocutaneous albinism type III (OCA3), caused by mutations of TYRP1 gene, is an autosomal recessive disorder characterized by reduced biosynthesis of melanin pigment in the hair, skin, and eyes. The TYRP1 gene encodes a protein called tyrosinase-related protein-1 (Tyrp1). Tyrp1 is involved in maintaining the stability of tyrosinase protein and modulating its catalytic activity in eumelanin synthesis. Tyrp1 is also involved in maintenance of melanosome structure and affects melanocyte proliferation and cell death. In this work we implemented computational analysis to filter the most probable mutation that might be associated with OCA3. We found R326H and R356Q as most deleterious and disease associated by using PolyPhen 2.0, SIFT, PANTHER, I-mutant 3.0, PhD-SNP, SNP&GO, Pmut, and Mutpred tools. To understand the atomic arrangement in 3D space, the native and mutant (R326H and R356Q) structures were modelled. Finally the structural analyses of native and mutant Tyrp1 proteins were investigated using molecular dynamics simulation (MDS) approach. MDS results showed more flexibility in native Tyrp1 structure. Due to mutation in Tyrp1 protein, it became more rigid and might disturb the structural conformation and catalytic function of the structure and might also play a significant role in inducing OCA3. The results obtained from this study would facilitate wet-lab researches to develop a potent drug therapies against OCA3. PMID:23862152

Chirality detection of amino acid enantiomers by organic electrochemical transistor.

PubMed

Zhang, Lijun; Wang, Guiheng; Xiong, Can; Zheng, Lei; He, Jianbo; Ding, Yunsheng; Lu, Hongbo; Zhang, Guobing; Cho, Kilwon; Qiu, Longzhen

2018-05-15

Chiral recognition of α-amino acids is attracting increasing interest due to the importance of α-amino acids in protein metabolism as well as in food products and pharmaceuticals. Organic electrochemical transistors (OECTs) with gate electrodes modified with molecularly imprinted polymer (MIP) films were fabricated and successfully used as highly selective and sensitive chiral recognition biosensors for d/l-tryptophan (d/l-Trp) and d/l-tyrosine (d/l-Tyr). The MIP films, which can specifically recognize and has an electrocatalytic effect on the oxidation of Trp and Tyr, together with the amplification function of an OECT, provide a highly sensitive and selective OECT biosensor. The sensor showed a linear response range for l-Trp and L-Tyr from 300 nM to 10 μM with a sensitivity of 3.19 and 3.64 μA/μM, respectivity. And the detection limit for L-Trp and L-Tyr is of 2 nM and 30 nM (S/N > 3). The selectivity factors of L-Trp, D-Trp, L-Tyr and D-Tyr to their enantiomers are 11.6, 3.5, 14.5 and 2.6, respectively. This method can pave the way for widespread applications of OECT-based sensors in chiral material identification. Copyright © 2018 Elsevier B.V. All rights reserved.

Effects of the immobilization supports on the catalytic properties of immobilized mushroom tyrosinase: a comparative study using several substrates.

PubMed

Marín-Zamora, María Elisa; Rojas-Melgarejo, Francisco; García-Cánovas, Francisco; García-Ruiz, Pedro Antonio

2007-09-30

Mushroom tyrosinase was immobilized from an extract onto glass beads covered with one of the following compounds: the crosslinked totally cinnamoylated derivatives of glycerine, D-sorbitol, D-manitol, 1,2-O-isopropylidene-alpha-D-glucofuranose, D-glucuronic acid, D-gulonic acid, sucrose, D-glucosone, D-arabinose, D-fructose, D-glucose, ethyl-D-glucopyranoside, inuline, dextrine, dextrane or starch, or the partially cinnamoylated derivative 3,5,6-tricinnamoyl-D-glucofuranose which was obtained by the acid hydrolysis of 1,2-O-isopropylidene-alpha-d-glucofuranose. The enzyme was immobilized by direct adsorption onto the support and the quantity of tyrosinase immobilized was found to increase with the hydrophobicity of the supports. The kinetic constants of immobilized tyrosinase acting on the substrates, 4-tert-butylcatechol, dopamine and DL-dopa, were studied. When immobilized tyrosinase acted on 4-tert-butylcatechol, the values of K(m)(app) were lower than these obtained for tyrosinase in solution while, when dopamine and DL-dopa were used, the K(m)(app) were higher. The order of the substrates as regards their ionizable groups, DL-dopa (two ionizable groups)>dopamine (one ionizable group)>4-tert-butylcatechol (no ionizable group) coincided with the order of the K(m)(app) values shown by tyrosinase immobilized on the hydrophobic supports, and was the inverse of that observed for tyrosinase in solution. The K(m)(app) values of immobilized tyrosinase were in all cases higher than those of soluble tyrosinase and depended on the nature of the support and the hydrophobicity of the substrate, meaning that it is possible to design supports with different degrees of selectivity towards a mixture of enzyme substrates in the reaction medium.

A stationary-phase protein of Escherichia coli that affects the mode of association between the trp repressor protein and operator-bearing DNA.

PubMed Central

Yang, W; Ni, L; Somerville, R L

1993-01-01

Highly purified preparations of trp repressor (TrpR) protein derived from Escherichia coli strains that were engineered to overexpress this material were found to contain another protein, of 21 kDa. The second protein, designated WrbA [for tryptophan (W) repressor-binding protein] remained associated with its namesake through several sequential protein fractionation steps. The N-terminal amino acid sequence of the WrbA protein guided the design of two degenerate oligonucleotides that were used as probes in the cloning of the wrbA gene (198 codons). The WrbA protein, in purified form, was found by several criteria to enhance the formation and/or stability of noncovalent complexes between TrpR holorepressor and its primary operator targets. The formation of an operator-holorepressor-WrbA ternary complex was demonstrated by gel mobility-shift analysis. The WrbA protein alone does not interact with the trp operator. During the stationary phase, cells deficient in the WrbA protein were less efficient than wild type in their ability to repress the trp promoter. It is proposed that the WrbA protein functions as an accessory element in blocking TrpR-specific transcriptional processes that might be physiologically disadvantageous in the stationary phase of the bacterial life cycle. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 PMID:8516330

Mutations in Arabidopsis thaliana genes involved in the tryptophan biosynthesis pathway affect root waving on tilted agar surfaces

NASA Technical Reports Server (NTRS)

Rutherford, R.; Gallois, P.; Masson, P. H.

1998-01-01

Arabidopsis thaliana roots grow in a wavy pattern upon a slanted surface. A novel mutation in the anthranilate synthase alpha 1 (ASA1) gene, named trp5-2wvc1, and mutations in the tryptophan synthase alpha and beta 1 genes (trp3-1 and trp2-1, respectively) confer a compressed root wave phenotype on tilted agar surfaces. When trp5-2wvc1 seedlings are grown on media supplemented with anthranilate metabolites, their roots wave like wild type. Genetic and pharmacological experiments argue that the compressed root wave phenotypes of trp5-2wvc1, trp2-1 and trp3-1 seedlings are not due to reduced IAA biosynthetic potential, but rather to a deficiency in L-tryptophan (L-Trp), or in a L-Trp derivative. Although the roots of 7-day-old seedlings possess higher concentrations of free L-Trp than the shoot as a whole, trp5-2wvc1 mutants show no detectable alteration in L-Trp levels in either tissue type, suggesting that a very localized shortage of L-Trp, or of a L-Trp-derived compound, is responsible for the observed phenotype.

New insights into highly potent tyrosinase inhibitors based on 3-heteroarylcoumarins: Anti-melanogenesis and antioxidant activities, and computational molecular modeling studies.

PubMed

Pintus, Francesca; Matos, Maria J; Vilar, Santiago; Hripcsak, George; Varela, Carla; Uriarte, Eugenio; Santana, Lourdes; Borges, Fernanda; Medda, Rosaria; Di Petrillo, Amalia; Era, Benedetta; Fais, Antonella

2017-03-01

Melanogenesis is a physiological pathway for the formation of melanin. Tyrosinase catalyzes the first step of this process and down-regulation of its activity is responsible for the inhibition of melanogenesis. The search for molecules capable of controlling hyperpigmentation is a trend topic in health and cosmetics. A series of heteroarylcoumarins have been synthesized and evaluated. Compounds 4 and 8 exhibited higher tyrosinase inhibitory activities (IC 50 =0.15 and 0.38μM, respectively), than the reference compound, kojic acid (IC 50 =17.9μM). Compound 4 acts as competitive, while compound 8 as uncompetitive inhibitor of mushroom tyrosinase. Furthermore, compounds 2 and 8 inhibited tyrosinase activity and melanin production in B16F10 cells. In addition, compounds 2-4 and 8 proved to have an interesting antioxidant profile in both ABTS and DPPH radicals scavenging assays. Docking experiments were carried out in order to study the interactions between these heteroarylcoumarins and mushroom tyrosinase. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tyrosinase, a new innate humoral immune parameter in large yellow croaker ( Pseudosciaena crocea R)

NASA Astrophysics Data System (ADS)

Wang, Shuhong; Wang, Yilei; Zhang, Ziping; Xie, Fangjing; Lin, Peng; Tai, Zhengang

2009-09-01

We evaluated the immune response to infection with a pathogen in large yellow croaker ( Pseudosciaena crocea Richardson). The fish were given an intraperitoneal (i.p.) injection of Vibrio parahaemolyticus or sterile sea water (control). We collected blood sera from the fish 0.17, 1, 2, 4, 8, 12, or 16 d after injection (dpi). We measured tyrosinase activity and the concentrations of lysozyme, NOS, and antibodies. Serum tyrosinase activity was significantly higher at 0.17 and 4 dpi than in the control group, and peaked at 8 dpi. Lysozyme activity was significantly higher at 2 and 12 dpi than in the control group, but lower at 16 dpi. There is no statistical difference in the level of nitric oxides synthase (NOS) activity or antibodies between the control and injection groups. This is the first report of the tyrosinase activity in the serum of large yellow croaker. Our results indicate that tyrosinase plays an important role in the immediate immune defense against V. parahaemolyticus in large yellow croaker. Tyrosinase is a candidate parameter for investigation of fish innate immune defense.

Overexpression of EAR1 and SSH4 that encode PPxY proteins in the multivesicular body provides stability to tryptophan permease Tat2, allowing yeast cells to grow under high hydrostatic pressure

NASA Astrophysics Data System (ADS)

Hiraki, Toshiki; Usui, Keiko; Abe, Fumiyoshi

2010-12-01

Tryptophan uptake in yeast Saccharomyces cerevisiae is susceptible to high hydrostatic pressure and it limits the growth of tryptophan auxotrophic (Trp-) strains under pressures of 15-25 MPa. The susceptibility of tryptophan uptake is accounted for by the pressure-induced degradation of tryptophan permease Tat2 occurring in a Rsp5 ubiquitin ligase-dependent manner. Ear1 and Ssh4 are multivesicular body proteins that physically interact with Rsp5. We found that overexpression of either of the EAR1 or SSH4 genes enabled the Trp- cells to grow at 15-25 MPa. EAR1 and SSH4 appeared to provide stability to the Tat2 protein when overexpressed. The result suggests that Ear1 and Ssh4 negatively regulate Rsp5 on ubiquitination of Tat2. Currently, high hydrostatic pressure is widely used in bioscience and biotechnology for structurally perturbing macromolecules such as proteins and lipids or in food processing and sterilizing microbes. We suggest that hydrostatic pressure is an operative experimental parameter to screen yeast genes specifically for regulation of Tat2 through the function of Rsp5 ubiquitin ligase.

Characterization of a New Flavone and Tyrosinase Inhibition Constituents from the Twigs of Morus alba L.

PubMed

Zhang, Long; Tao, Guanjun; Chen, Jie; Zheng, Zong-Ping

2016-09-02

The twigs of Morus alba L. were found to show strong tyrosinase inhibition activity, and the responsible active components in the extract were further investigated in this study. A flavone, named morusone (1), and sixteen known compounds 2-17 were isolated from M. alba twigs and their structures were identified by interpretation of the corresponding ESI-MS and NMR spectral data. In the tyrosinase inhibitory test, the compounds steppogenin (IC50 0.98 ± 0.01 µM), 2,4,2',4'-tetrahydroxychalcone (IC50 0.07 ± 0.02 µM), morachalcone A (IC50 0.08 ± 0.02 µM), oxyresveratrol (IC50 0.10 ± 0.01 µM), and moracin M (8.00 ± 0.22 µM) exhibited significant tyrosinase inhibition activities, much stronger than that of the positive control kojic acid. These results suggest that M. alba twig extract should served as a good source of natural tyrosinase inhibitors for use in foods as antibrowning agents or in cosmetics as skin-whitening agents.

Cellular and molecular defects in a patient with Hermansky-Pudlak syndrome type 5.

PubMed

Stephen, Joshi; Yokoyama, Tadafumi; Tolman, Nathanial J; O'Brien, Kevin J; Nicoli, Elena-Raluca; Brooks, Brian P; Huryn, Laryssa; Titus, Steven A; Adams, David R; Chen, Dong; Gahl, William A; Gochuico, Bernadette R; Malicdan, May Christine V

2017-01-01

Hermansky-Pudlak syndrome (HPS) is a heterogeneous group of genetic disorders typically manifesting with tyrosinase-positive oculocutaneous albinism, bleeding diathesis, and pulmonary fibrosis, in some subtypes. Most HPS subtypes are associated with defects in Biogenesis of Lysosome-related Organelle Complexes (BLOCs), which are groups of proteins that function together in the formation and/or trafficking of lysosomal-related endosomal compartments. BLOC-2, for example, consists of the proteins HPS3, HPS5, and HPS6. Here we present an HPS patient with defective BLOC-2 due to a novel intronic mutation in HPS5 that activates a cryptic acceptor splice site. This mutation leads to the insertion of nine nucleotides in-frame and results in a reduced amount of HPS5 at the transcript and protein level. In studies using skin fibroblasts derived from the proband and two other individuals with HPS-5, we found a perinuclear distribution of acidified organelles in patient cells compared to controls. Our results suggest the role of HPS5 in the endo-lysosomal dynamics of skin fibroblasts.

Anti-Pigmentation Effects of Eight Phellinus linteus-Fermented Traditional Crude Herbal Extracts on Brown Guinea Pigs of Ultraviolet B-Induced Hyperpigmentation.

PubMed

Ahn, Hee-Young; Choo, Young-Moo; Cho, Young-Su

2018-03-28

We have previously found that mycelia culture broth of eight kinds of traditional herbal extracts fermented with Phellinus linteus (previously named as 8-HsPLCB) not only inhibited melanin and tyrosinase activity, but also reduced the contents of melanogenesis-related proteins, including tyrosinase and microphthalmia-associated transcription factor, in 3-isobutyl-1-methylxanthine-stimulated B16F0 melanoma cells. For a further study, the effect of 8-HsPLCB against skin pigmentation in brown guinea pigs with ultraviolet B (UVB)-induced hyperpigmentation was investigated. 8-HsPLCB (3%) and arbutin (2%) as positive controls were applied topically twice daily for 4 weeks to the hyperpigmented areas. 8-HsPLCB showed skin-lightening effect as effective as arbutin, one of the most widely used in whitening cosmetics. Melanin index values as the degree of pigmentation showed a significant reduction week by week post 8-HsPLCB treatment and then substantially reduced by 4 weeks. The degree of depigmentation after 4 weeks of topical application with 8-HsPLCB was 32.2% as compared with before treatment (0 week). Moreover, using Fontana-Masson staining and hematoxylin-eosin staining, 8-HsPLCB reduced melanin pigmentation in the basal layer of the epidermis and epidermal thickness changes exposed to the UV-B irradiation as compared with non-treatment and vehicle treatment. The intensity of the skin-lightening effect of 8-HsPLCB was similar to arbutin. These results suggest that the skin-lightening effect of 8-HsPLCB might be resulted from inhibition of melanin synthesis by tyrosinase in melanocytes. To conclude, 8-HsPLCB treatment showed reduction of the melanin pigment and histological changes induced by UV irradiation in brown guinea pigs.

Bond-based 2D TOMOCOMD-CARDD approach for drug discovery: aiding decision-making in 'in silico' selection of new lead tyrosinase inhibitors.

PubMed

Marrero-Ponce, Yovani; Khan, Mahmud Tareq Hassan; Casañola-Martín, Gerardo M; Ather, Arjumand; Sultankhodzhaev, Mukhlis N; García-Domenech, Ramón; Torrens, Francisco; Rotondo, Richard

2007-04-01

In this paper, we present a new set of bond-level TOMOCOMD-CARDD molecular descriptors (MDs), the bond-based bilinear indices, based on a bilinear map similar to those defined in linear algebra. These novel MDs are used here in Quantitative Structure-Activity Relationship (QSAR) studies of tyrosinase inhibitors, for finding functions that discriminate between the tyrosinase inhibitor compounds and inactive ones. In total 14 models were obtained and the best two discriminant functions (Eqs. 32 and 33) shown globally good classification of 91.00% and 90.17%, respectively, in the training set. The test set had accuracies of 93.33% and 88.89% for the models 32 and 33, correspondingly. A simulated virtual screening was also carried out to prove the quality of the determined models. In a final step, the fitted models were used in the biosilico identification of new synthesized tetraketones, where a good agreement could be observed between the theoretical and experimental results. Four compounds of the novel bioactive chemicals discovered as tyrosinase inhibitors: TK10 (IC(50) = 2.09 microM), TK11 (IC(50) = 2.61 microM), TK21 (IC(50) = 2.06 microM), TK23 (IC(50) = 3.19 microM), showed more potent activity than L-mimose (IC(50) = 3.68 microM). Besides, for this study a heterogeneous database of tyrosinase inhibitors was collected, and could be a useful tool for the scientist in the domain of tyrosinase enzyme researches. The current report could help to shed some clues in the identification of new chemicals that inhibits enzyme tyrosinase, for entering in the pipeline of drug discovery development.

Computational analysis of histidine mutations on the structural stability of human tyrosinases leading to albinism insurgence.

PubMed

Hassan, Mubashir; Abbas, Qamar; Raza, Hussain; Moustafa, Ahmed A; Seo, Sung-Yum

2017-07-25

Misfolding and structural alteration in proteins lead to serious malfunctions and cause various diseases in humans. Mutations at the active binding site in tyrosinase impair structural stability and cause lethal albinism by abolishing copper binding. To evaluate the histidine mutational effect, all mutated structures were built using homology modelling. The protein sequence was retrieved from the UniProt database, and 3D models of original and mutated human tyrosinase sequences were predicted by changing the residual positions within the target sequence separately. Structural and mutational analyses were performed to interpret the significance of mutated residues (N 180 , R 202 , Q 202 , R 211 , Y 363 , R 367 , Y 367 and D 390 ) at the active binding site of tyrosinases. CSpritz analysis depicted that 23.25% residues actively participate in the instability of tyrosinase. The accuracy of predicted models was confirmed through online servers ProSA-web, ERRAT score and VERIFY 3D values. The theoretical pI and GRAVY generated results also showed the accuracy of the predicted models. The CCA negative correlation results depicted that the replacement of mutated residues at His within the active binding site disturbs the structural stability of tyrosinases. The predicted CCA scores of Tyr 367 (-0.079) and Q/R 202 (0.032) revealed that both mutations have more potential to disturb the structural stability. MD simulation analyses of all predicted models justified that Gln 202 , Arg 202 , Tyr 367 and D 390 replacement made the protein structures more susceptible to destabilization. Mutational results showed that the replacement of His with Q/R 202 and Y/R 363 has a lethal effect and may cause melanin associated diseases such as OCA1. Taken together, our computational analysis depicts that the mutated residues such as Q/R 202 and Y/R 363 actively participate in instability and misfolding of tyrosinases, which may govern OCA1 through disturbing the melanin biosynthetic pathway.

[Phylogenetic analysis of tyrosinase gene family in the Pacific oyster (Crassostrea gigas Thunberg)].

PubMed

Yu, Xue; Yu, Hong; Kong, Lingfeng; Li, Qi

2014-02-01

The deduced amino acid sequence characteristics, classification and phylogeny of tyrosinase gene family in the Pacific oyster (Crassostrea gigas Thunberg) were analyzed using bioinformatics methods. The results showed that gene duplication was the major cause of tyrosinase gene expansion in the Pacific oyster. The tyrosinase gene family in the Pacific oyster can be further classified into three types: secreted form (Type A), cytosolic form (Type B) and membrane-bound form (Type C). Based on the topology of the phylogenetic tree of the Pacific oyster tyrosinases, among Type A isoforms, tyr18 seemed divergent from other Type A tyrosinases early, while tyr2 and tyr9 appeared divergent early in Type B. In Type C tyrosinses, tyr8 was divergent early. The cluster of the Pacific oyster tyrosinasesis determined by their classifications and positions in the scaffolds. Further analysis suggested that Type A tyrosinases of C. gigas clustered with those from cephalopods and then with nematodes and cnidarians. Type B tyrosinases were generally clustered with the same type of tyrosinases from molluscas and nematodes, and then with those from platyhelminths, cnidarians and chordates. Type A tyrosinases in the Pacific oyster and the Pearl oyster expanded independently and were divergent from membrane-bound form of tyrosinases in chordata, platyhelminthes and annelida. These observations suggested that Type C tyrosinases in the bivalve had a distinct evolution direction.

Structure-activity relationship of cyclic peptide penta-c[Asp-His(6)-DPhe(7)-Arg(8)-Trp(9)-Lys]-NH(2) at the human melanocortin-1 and -4 receptors: His(6) substitution.

PubMed

Cheung, Adrian Wai-Hing; Danho, Waleed; Swistok, Joseph; Qi, Lida; Kurylko, Grazyna; Rowan, Karen; Yeon, Mitch; Franco, Lucia; Chu, Xin-Jie; Chen, Li; Yagaloff, Keith

2003-04-07

A series of MT-II related cyclic peptides, based on potent but non-selective hMC4R agonist (Penta-c[Asp-His(6)-DPhe(7)-Arg(8)-Trp(9)-Lys]-NH(2)) was prepared in which His(6) residue was systematically substituted. Two of the most interesting peptides identified in this study are Penta-c[Asp-5-ClAtc-DPhe-Arg-Trp-Lys]-NH(2) and Penta-c[Asp-5-ClAtc-DPhe-Cit-Trp-Lys]-NH(2) which are potent hMC4R agonists and are either inactive or weak partial agonists (not tested for their antagonist activities) in hMC1R, hMC3R and hMC5R agonist assays.

The Transient Receptor Potential (TRP) Channel Family in Colletotrichum graminicola: A Molecular and Physiological Analysis.

PubMed

Lange, Mario; Weihmann, Fabian; Schliebner, Ivo; Horbach, Ralf; Deising, Holger B; Wirsel, Stefan G R; Peiter, Edgar

2016-01-01

Calcium (Ca2+) is a universal second messenger in all higher organisms and centrally involved in the launch of responses to environmental stimuli. Ca2+ signals in the cytosol are initiated by the activation of Ca2+ channels in the plasma membrane and/or in endomembranes. Yeast (Saccharomyces cerevisiae) contains a Ca2+-permeable channel of the TRP family, TRPY1, which is localized in the vacuolar membrane and contributes to cytosolic free Ca2+ ([Ca2+]cyt) elevations, for example in response to osmotic upshock. A TRPY1 homologue in the rice blast fungus is known to be important for growth and pathogenicity. To determine the role of the TRP channel family in the maize pathogen Colletotrichum graminicola, proteins homologous to TRPY1 were searched. This identified not one, but four genes in the C. graminicola genome, which had putative orthologs in other fungi, and which we named CgTRPF1 through 4. The topology of the CgTRPF proteins resembled that of TRPY1, albeit with a variable number of transmembrane (TM) domains additional to the six-TM-domain core and a diverse arrangement of putatively Ca2+-binding acidic motifs. All CgTRPF genes were expressed in axenic culture and throughout the infection of maize. Like TRPY1, all TRPF proteins of C. graminicola were localized intracellularly, albeit three of them were found not in large vacuoles, but co-localized in vesicular structures. Deletion strains for the CgTRPF genes were not altered in processes thought to involve Ca2+ release from internal stores, i.e. spore germination, the utilization of complex carbon sources, and the generation of tip-focussed [Ca2+]cyt spikes. Heterologous expression of CgTRPF1 through 4 in a tryp1Δ yeast mutant revealed that none of the channels mediated the release of Ca2+ in response to osmotic upshock. Accordingly, aequorin-based [Ca2+]cyt measurements of C. graminicola showed that in this fungus, osmotic upshock-triggered [Ca2+]cyt elevations were generated entirely by influx of Ca2+ from the extracellular space. Cgtrpf mutants did not show pathogenicity defects in leaf infection assays. In summary, our study reveals major differences between different fungi in the contribution of TRP channels to Ca2+-mediated signal transduction.

The Transient Receptor Potential (TRP) Channel Family in Colletotrichum graminicola: A Molecular and Physiological Analysis

PubMed Central

Lange, Mario; Weihmann, Fabian; Schliebner, Ivo; Horbach, Ralf; Deising, Holger B.; Wirsel, Stefan G. R.

2016-01-01

Calcium (Ca2+) is a universal second messenger in all higher organisms and centrally involved in the launch of responses to environmental stimuli. Ca2+ signals in the cytosol are initiated by the activation of Ca2+ channels in the plasma membrane and/or in endomembranes. Yeast (Saccharomyces cerevisiae) contains a Ca2+-permeable channel of the TRP family, TRPY1, which is localized in the vacuolar membrane and contributes to cytosolic free Ca2+ ([Ca2+]cyt) elevations, for example in response to osmotic upshock. A TRPY1 homologue in the rice blast fungus is known to be important for growth and pathogenicity. To determine the role of the TRP channel family in the maize pathogen Colletotrichum graminicola, proteins homologous to TRPY1 were searched. This identified not one, but four genes in the C. graminicola genome, which had putative orthologs in other fungi, and which we named CgTRPF1 through 4. The topology of the CgTRPF proteins resembled that of TRPY1, albeit with a variable number of transmembrane (TM) domains additional to the six-TM-domain core and a diverse arrangement of putatively Ca2+-binding acidic motifs. All CgTRPF genes were expressed in axenic culture and throughout the infection of maize. Like TRPY1, all TRPF proteins of C. graminicola were localized intracellularly, albeit three of them were found not in large vacuoles, but co-localized in vesicular structures. Deletion strains for the CgTRPF genes were not altered in processes thought to involve Ca2+ release from internal stores, i.e. spore germination, the utilization of complex carbon sources, and the generation of tip-focussed [Ca2+]cyt spikes. Heterologous expression of CgTRPF1 through 4 in a tryp1Δ yeast mutant revealed that none of the channels mediated the release of Ca2+ in response to osmotic upshock. Accordingly, aequorin-based [Ca2+]cyt measurements of C. graminicola showed that in this fungus, osmotic upshock-triggered [Ca2+]cyt elevations were generated entirely by influx of Ca2+ from the extracellular space. Cgtrpf mutants did not show pathogenicity defects in leaf infection assays. In summary, our study reveals major differences between different fungi in the contribution of TRP channels to Ca2+-mediated signal transduction. PMID:27359114

Pharmacological Modulation of Diacylglycerol-Sensitive TRPC3/6/7 Channels

PubMed Central

Harteneck, Christian; Gollasch, Maik

2011-01-01

Members of the classic type of transient receptor potential channels (TRPC) represent important molecules involved in hormonal signal transduction. TRPC3/6/7 channels are of particular interest as they are components of phospholipase C driven signalling pathways. Upon receptor-activation, G-protein-mediated stimulation of phospholipase C results in breakdown of phosphatidylinositides leading to increased intracellular diacylglycerol and inositol-trisphosphate levels. Diacylglycerol activates protein kinase C, but more interestingly diacylglycerol directly activates TRPC2/3/6/7 channels. Molecular cloning, expression and characterization of TRP channels enabled reassignment of traditional inhibitors of receptor-dependent calcium entry such as SKF-96365 and 2-APB as blockers of TRPC3/6/7 and several members of non-classic TRP channels. Furthermore, several enzyme inhibitors have also been identified as TRP channel blockers, such as ACA, a phospholipase A2 inhibitor, and W-7, a calmodulin antagonist. Finally, the naturally occurring secondary plant compound hyperforin has been identified as TRPC6-selective drug, providing an exciting proof of concept that it is possible to generate TRPC-selective channel modulators. The description of Pyr3 as the first TRPC3-selective inhibitor shows that not only nature but also man is able to generate TRP-selective modulators. The review sheds lights on the current knowledge and historical development of pharmacological modulators of TRPC3/6/7. Our analysis indicates that Pyr3 and hyperforin provide promising core structures for the development of new, selective and more potent modulators of TRPC3/6/7 activity. PMID:20932261

Impact of GNB3-C825T, ADRB3-Trp64Arg, UCP2-3′UTR 45 bp del/ins, and PPARγ-Pro12Ala Polymorphisms on Bofutsushosan Response in Obese Subjects: A Randomized, Double-Blind, Placebo-Controlled Trial

PubMed Central

Park, Junghyun; Bose, Shambhunath; Hong, Sun-Woo; Lee, Dong-Ki; Yoo, Jae-Wook; Lim, Chi-Yeon; Lee, Myeongjong

2014-01-01

Abstract Obesity is known to be influenced by a number of genes, including the β3 subunit of G protein (GNB3), β3-adrenergic receptor (ADRB3), uncoupling protein 2 (UCP2), and peroxisome proliferator activated receptor gamma (PPARγ). The single nucleotide polymorphisms (SNPs) of the above genes, such as GNB3-C825T, ADRB3-Trp64Arg, UCP2-3′UTR 45 bp del/ins, and PPARγ-Pro12Ala, are associated with obesity and body mass index. The present study evaluates the impact of Bofutsushosan, a traditional Eastern Asian herbal medicine with known anti-obesity properties, on obese subjects according to the presence of the above-mentioned SNPs. Upon randomization, the volunteers were allocated to receive Bofutsushosan (n=55) or placebo (n=56) treatments for 8 weeks. Following the treatment schedule, significant reductions in total cholesterol and significant improvement in the Korean version of obesity-related quality of life scale were seen in the Bofutsushosan-treated group, but not in placebo. Bofutsushosan exerted significant anti-obesity effects on a number of parameters in the carriers of the GNB3-825T allele, but only on waist circumference in the GNB3-C/C homozygote. Significant anti-obesity impact of Bofutsushosan was also seen on a number of obesity-indices in both ADRB3-Arg64 carriers and ADRB3-Trp64 homozygotes, as well as in UCP2-D/D carriers, but not in UCP2-D/I+I/I variants. The effect of Bofutsushosan was more pronounced in PPARγ-Pro/Pro genotype compared to PPARγ-Pro/Ala variants. Thus, the results revealed differential responses of the subjects to the anti-obesity effects of Bofutsushosan treatment according to the polymorphism of the vital obesity-related genes. Our study provides new insight into individualized clinical applications of Bofutsushosan for obesity. PMID:24827746

The Mechanism of Melanocytes-Specific Cytotoxicity Induced by Phenol Compounds Having a Prooxidant Effect, relating to the Appearance of Leukoderma

PubMed Central

Ito, Shinobu; Kanazawa, Hideko; Masaki, Hitoshi

2015-01-01

Specific phenol compounds including rhododendrol (RD), a skin-brightening ingredient in cosmetics, are reported to induce leukoderma, inducing a social problem, and the elucidation of mechanism of leukoderma is strongly demanded. This study investigated the relationship among the cytotoxicities of six phenol compounds on B16F10 melanoma cells and HaCaT keratinocytes and generated reactive oxygen species (ROS). As a result, the cytotoxicity of RD on B16F10 cells was higher than that on HaCaT cells, and RD significantly increased intracellular ROS and hydrogen peroxide (H2O2) levels in B16F10 cells. Furthermore, although raspberry ketone (RK), RD derivative, also increased intracellular ROS in B16F10 cells, increase in ROS was suppressed by disodium dihydrogen ethylenediaminetetraacetate dehydrate (EDTA). The amounts of increased ROS with RK in HaCaT cells without melanocyte were further increased by tyrosinase. Therefore, tyrosinase, a metalloprotein having copper, was speculated to be one of causative agents allowing phenol compounds to work as a prooxidant. Hydroxyl radical was generated by adding a mixture of tyrosinase and H2O2 to RD, and the amount of the radical was further increased by UVB, indicating that RD cytotoxicity was caused by intracellularly increased ROS, which possibly related to phenol induced prooxidants. PMID:25861631

Photodynamic therapy for melanoma: efficacy and immunologic effects

NASA Astrophysics Data System (ADS)

Avci, Pinar; Gupta, Gaurav K.; Kawakubo, Masayoshi; Hamblin, Michael R.

2014-02-01

Malignant melanoma is one of the fastest growing cancers and if it cannot be completely surgically removed the prognosis is bleak. Melanomas are known to be particularly resistant to both chemotherapy and radiotherapy. Various types of immunotherapy have however been investigated with mixed reports of success. Photodynamic therapy (PDT) has also been tested against melanoma, again with mixed effects as the melanin pigment is thought to act as both an optical shield and as an antioxidant. We have been investigating PDT against malignant melanoma in mouse models. We have compared B16F10 melanoma syngenic to C57BL/6 mice and S91 Cloudman melanoma syngenic to DBA2 mice. We have tested the hypothesis that S91 will respond better than B16 because of higher expression of immunocritical molecules such as MHC-1, tyrosinase, tyrosinase related protein-2 gp100, and intercellular adhesion molecule-1. Some of these molecules can act as tumor rejection antigens that can be recognized by antigen-specific cytotoxic CD8 T cells that have been stimulated by PDT. Moreover it is possible that DBA2 mice are intrinsically better able to mount an anti-tumor immune response than C57BL/6 mice. We are also studying intratumoral injection of photosensitzers such as benzoporphyrin monoacid ring A and comparing this route with the more usual route of intravenous administration.

Disturbed tryptophan metabolism in cardiovascular disease.

PubMed

Mangge, H; Stelzer, I; Reininghaus, E Z; Weghuber, D; Postolache, T T; Fuchs, D

2014-06-01

Atherosclerosis (AS), a major pathologic consequence of obesity, is the main etiological factor of cardiovascular disease (CVD), which is the most common cause of death in the western world. A systemic chronic low grade immune- mediated inflammation (scLGI) is substantially implicated in AS and its consequences. In particular, proinflammatory cytokines play a major role, with Th1-type cytokine interferon-γ (IFN-γ) being a key mediator. Among various other molecular and cellular effects, IFN-γ activates the enzyme indoleamine 2,3-dioxygenase (IDO) in monocyte-derived macrophages, dendritic, and other cells, which, in turn, decreases serum levels of the essential amino acid tryptophan (TRP). Thus, people with CVD often have increased serum kynurenine to tryptophan ratios (KYN/TRP), a result of an increased TRP breakdown. Importantly, increased KYN/TRP is associated with a higher likelihood of fatal cardiovascular events. A scLGI with increased production of the proinflammatory adipokine leptin, in combination with IFN-γ and interleukin-6 (IL-6), represents another central link between obesity, AS, and CVD. Leptin has also been shown to contribute to Th1-type immunity shifting, with abdominal fat being thus a direct contributor to KYN/TRP ratio. However, TRP is not only an important source for protein production but also for the generation of one of the most important neurotransmitters, 5-hydroxytryptamine (serotonin), by the tetrahydrobiopterin-dependent TRP 5-hydroxylase. In prolonged states of scLGI, availability of free serum TRP is strongly diminished, affecting serotonin synthesis, particularly in the brain. Additionally, accumulation of neurotoxic KYN metabolites such as quinolinic acid produced by microglia, can contribute to the development of depression via NMDA glutamatergic stimulation. Depression had been reported to be associated with CVD endpoints, but it most likely represents only a secondary loop connecting excess adipose tissue, scLGI and cardiovascular morbidity and mortality. Accelerated catabolism of TRP is further involved in the pathogenesis of the anemia of scLGI. The pro-inflammatory cytokine IFN-γ suppresses growth and differentiation of erythroid progenitor cells, and the depletion of TRP limits protein synthesis and thus hemoglobin production, and, through reduction in oxygen supply, may contribute to ischemic vascular disease. In this review we discuss the impact of TRP breakdown and the related complex mechanisms on the prognosis and individual course of CVD. Measurement of TRP, KYN concentrations, and calculation of the KYN/TRYP ratio will contribute to a better understanding of the interplay between inflammation, metabolic syndrome, mood disturbance, and anemia, all previously described as significant predictors of an unfavorable outcome in patients with CVD. The review leads to a novel framework for successful therapeutic modification of several cardinal pathophysiological processes leading to adverse cardiovascular outcome.

Significance of two distinct types of tryptophan synthase beta chain in Bacteria, Archaea and higher plants.

PubMed

Xie, Gary; Forst, Christian; Bonner, Carol; Jensen, Roy A

2002-01-01

Tryptophan synthase consists of two subunits, alpha and beta. Two distinct subgroups of beta chain exist. The major group (TrpEb_1) includes the well-studied beta chain of Salmonella typhimurium. The minor group of beta chain (TrpEb_2) is most frequently found in the Archaea. Most of the amino-acid residues important for catalysis are highly conserved between both TrpE subfamilies. Conserved amino-acid residues of TrpEb_1 that make allosteric contact with the TrpEa subunit (the alpha chain) are absent in TrpEb_2. Representatives of Archaea, Bacteria and higher plants all exist that possess both TrpEb_1 and TrpEb_2. In those prokaryotes where two trpEb genes coexist, one is usually trpEb_1 and is adjacent to trpEa, whereas the second is trpEb_2 and is usually unlinked with other tryptophan-pathway genes. TrpEb_1 is nearly always partnered with TrpEa in the tryptophan synthase reaction. However, by default at least six lineages of the Archaea are likely to use TrpEb_2 as the functional beta chain, as TrpEb_1 is absent. The six lineages show a distinctive divergence within the overall TrpEa phylogenetic tree, consistent with the lack of selection for amino-acid residues in TrpEa that are otherwise conserved for interfacing with TrpEb_1. We suggest that the standalone function of TrpEb_2 might be to catalyze the serine deaminase reaction, an established catalytic capability of tryptophan synthase beta chains. A coincident finding of interest is that the Archaea seem to use the citramalate pathway, rather than threonine deaminase (IlvA), to initiate the pathway of isoleucine biosynthesis.

Identification of geranic acid, a tyrosinase inhibitor in lemongrass (Cymbopogon citratus).

PubMed

Masuda, Toshiya; Odaka, Yuka; Ogawa, Natsuko; Nakamoto, Katsuo; Kuninaga, Hideki

2008-01-23

Lemongrass is a popular Asian herb having a lemon-like flavor. Very recently, potent tyrosinase inhibitory activity has been found in lemongrass in addition to various biological activities reported in the literature. The aim of the present study is to identify the active compounds in the lemongrass. An assay-guided purification revealed that one of the active substances was geranic acid. Geranic acid has two stereoisomers, which are responsible for the trans and cis geometry on the conjugated double bond. Both isomers are present in the active ethyl acetate-soluble extract of the lemongrass, and their IC50 values were calculated to be 0.14 and 2.3 mM, respectively. The structure requirement of geranic acid for the potent tyrosinase inhibitory activity was investigated using geranic acid-related compounds.

The X-ray Crystal Structure of the Phage Tail Terminator Protein Reveals the Biologically Relevant Hexameric Rang Structure and Demonstrates a Conserved mechanism of Tail Termination among Divrse Long Tailed Phages

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pell, L.; Liu, A; Edmonds, L

The tail terminator protein (TrP) plays an essential role in phage tail assembly by capping the rapidly polymerizing tail once it has reached its requisite length and serving as the interaction surface for phage heads. Here, we present the 2.7-A crystal structure of a hexameric ring of gpU, the TrP of phage ?. Using sequence alignment analysis and site-directed mutagenesis, we have shown that this multimeric structure is biologically relevant and we have delineated its functional surfaces. Comparison of the hexameric crystal structure with the solution structure of gpU that we previously solved using NMR spectroscopy shows large structural changesmore » occurring upon multimerization and suggests a mechanism that allows gpU to remain monomeric at high concentrations on its own, yet polymerize readily upon contact with an assembled tail tube. The gpU hexamer displays several flexible loops that play key roles in head and tail binding, implying a role for disorder-to-order transitions in controlling assembly as has been observed with other ? morphogenetic proteins. Finally, we have found that the hexameric structure of gpU is very similar to the structure of a putative TrP from a contractile phage tail even though it displays no detectable sequence similarity. This finding coupled with further bioinformatic investigations has led us to conclude that the TrPs of non-contractile-tailed phages, such as ?, are evolutionarily related to those of contractile-tailed phages, such as P2 and Mu, and that all long-tailed phages may utilize a conserved mechanism for tail termination.« less

Homotropic Cooperativity from the Activation Pathway of the Allosteric Ligand-Responsive Regulatory Protein TRAP†

PubMed Central

Kleckner, Ian R.; McElroy, Craig A.; Kuzmic, Petr; Gollnick, Paul; Foster, Mark P.

2014-01-01

The trp RNA-binding Attenuation Protein (TRAP) assembles into an 11-fold symmetric ring that regulates transcription and translation of trp-mRNA in bacilli via heterotropic allosteric activation by the amino acid tryptophan (Trp). Whereas nuclear magnetic resonance studies have revealed that Trp-induced activation coincides with both μs-ms rigidification and local structural changes in TRAP, the pathway of binding of the 11 Trp ligands to the TRAP ring remains unclear. Moreover, because each of eleven bound Trp molecules is completely surrounded by protein, its release requires flexibility of Trp-bound (holo) TRAP. Here, we used stopped-flow fluorescence to study the kinetics of Trp binding by Bacillus stearothermophilus TRAP over a range of temperatures and we observed well-separated kinetic steps. These data were analyzed using non-linear least-squares fitting of several two- and three-step models. We found that a model with two binding steps best describes the data, although the structural equivalence of the binding sites in TRAP implies a fundamental change in the time-dependent structure of the TRAP rings upon Trp binding. Application of the two binding step model reveals that Trp binding is much slower than the diffusion limit, suggesting a gating mechanism that depends on the dynamics of apo TRAP. These data also reveal that Trp dissociation from the second binding mode is much slower than after the first Trp binding mode, revealing insight into the mechanism for positive homotropic allostery, or cooperativity. Temperature dependent analyses reveal that both binding modes imbue increases in bondedness and order toward a more compressed active state. These results provide insight into mechanisms of cooperative TRAP activation, and underscore the importance of protein dynamics for ligand binding, ligand release, protein activation, and allostery. PMID:24224873

Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein.

PubMed

Andersen, O M; Petersen, H H; Jacobsen, C; Moestrup, S K; Etzerodt, M; Andreasen, P A; Thøgersen, H C

2001-07-01

The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.

Modifying effects of carboxyl group on the interaction of recombinant S100A8/A9 complex with tyrosinase.

PubMed

NematiNiko, Fatemeh; Chegini, Koorosh Goodarzvand; Asghari, Hamideh; Amini, Abbas; Gheibi, Nematollah

2017-03-01

Tyrosinase is a determinant enzyme for modulating melanin production as its abnormal activity can result in an increased amount of melanin. Reduction of tyrosinase activity has been targeted for preventing and healing hyperpigmentation of skin, such as melanoma and age related spots. The aim of this systematic study is to investigate whether recombinant S100A8/A9 and its modified form reduce the activity of mushroom tyrosinase (MT) through changing its structure. Recombinant His-Tagged S100A8 and S100A9 are expressed in Escherichia coli BL21 (DE3) and modified using Woodward's reagent K which is a carboxyl group modifier. The structures of S100A8/A9 and its modified form are studied using fluorescence and circular dichroism spectroscopy, and the activity of MT is measured using UV-visible spectrophotometry in the presence of its substrate, L-3,4-dihydroxyphenylalanine (L-DOPA). The results show a lower stability of the modified protein when compared with its unmodified form. The interaction of S100A8/A9 with MT changes the structure and successfully reduces the activity of mushroom tyrosinase. Recombinant S100A8/A9 complex decreases MT activity which can control malignant melanoma, the most dangerous type of skin cancer. Copyright © 2016 Elsevier B.V. All rights reserved.

The unfolded protein response in melanocytes: activation in response to chemical stressors of the endoplasmic reticulum and tyrosinase misfolding.

PubMed

Manga, Prashiela; Bis, Sabina; Knoll, Kristen; Perez, Beremis; Orlow, Seth J

2010-10-01

Accumulation of proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), comprising three signaling pathways initiated by Ire1, Perk and Atf6 respectively. Unfolded protein response activation was compared in chemically stressed murine wildtype melanocytes and mutant melanocytes that retain tyrosinase in the ER. Thapsigargin, an ER stressor, activated all pathways in wildtype melanocytes, triggering Caspase 12-mediated apoptosis at toxic doses. Albino melanocytes expressing mutant tyrosinase showed evidence of ER stress with increased Ire1 expression, but the downstream effector, Xbp1, was not activated even following thapsigargin treatment. Attenuation of Ire1 signaling was recapitulated in wildtype melanocytes treated with thapsigargin for 8 days, with diminished Xbp1 activation observed after 4 days. Atf6 was also activated in albino melanocytes, with no response to thapsigargin, while the Perk pathway was not activated and thapsigargin treatment elicited robust expression of the downstream effector CCAAT-enhancer-binding protein homologous protein. Thus, melanocytes adapt to ER stress by attenuating two UPR pathways.

Tyrosinase inhibitory components from Aloe vera and their antiviral activity.

PubMed

Kim, Jang Hoon; Yoon, Ju-Yeon; Yang, Seo Young; Choi, Seung-Kook; Kwon, Sun Jung; Cho, In Sook; Jeong, Min Hee; Ho Kim, Young; Choi, Gug Seoun

2017-12-01

A new compound, 9-dihydroxyl-2'-O-(Z)-cinnamoyl-7-methoxy-aloesin (1), and eight known compounds (2-9) were isolated from Aloe vera. Their structures were elucidated using 1D/2D nuclear magnetic resonance and mass spectra. Compound 9 exhibited reversible competitive inhibitory activity against the enzyme tyrosinase, with an IC 50 value of 9.8 ± 0.9 µM. A molecular simulation revealed that compound 9 interacts via hydrogen bonding with residues His244, Thr261, and Val283 of tyrosinase. Additionally, compounds 3 and 7 were shown by half-leaf assays to exhibit inhibitory activity towards Pepper mild mottle virus.

Homologous desensitization of HEL cell thrombin receptors. Distinguishable roles for proteolysis and phosphorylation.

PubMed

Brass, L F

1992-03-25

Loss of sensitivity to thrombin following an initial response is characteristic of a number of cell types, including platelets. It has recently been proposed that thrombin receptors resemble other G protein-coupled receptors, but that activation involves a novel mechanism in which thrombin cleaves the receptor, exposing a new N terminus that serves as the ligand for the receptor. Based upon this model, we have examined the mechanism of thrombin receptor desensitization by comparing the effects of thrombin with those of a peptide corresponding to the N-terminal sequence of the receptor following proteolysis by thrombin: SFLLRNPNDKYEPF or TRP42/55. Like thrombin, TRP42/55 stimulated pertussis toxin-sensitive inositol 1,4,5-trisphosphate formation, raised cytosolic Ca2+, and inhibited cAMP formation in the megakaryoblastic HEL cell line. Exposure to either thrombin or TRP42/55 desensitized the cells to both, but not to a third agonist, neuropeptide Y. The rate of recovery after desensitization depended upon the order of agonist addition. Resensitization of the cell to thrombin following a brief exposure to thrombin required up to 24 h and could be inhibited with cycloheximide. Resensitization to TRP42/55 after exposure to thrombin, or to thrombin after exposure to TRP42/55, on the other hand, was detectable within 30 min and could be inhibited by serine/threonine phosphatase inhibitors, but not by cycloheximide. Loss of responsiveness to thrombin and TRP42/55 was also observed following addition of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). However, while the protein kinase inhibitor staurosporine completely prevented the desensitization caused by TPA, it had only a limited effect on the desensitization caused by TRP42/55. These results demonstrate that the G protein-mediated effects of thrombin can be reproduced by a receptor-derived peptide and suggest that desensitization occurs by at least two mechanisms. The first, which is seen with thrombin, but not TRP42/55, involves proteolysis and requires protein synthesis for recovery. The second, which occurs with TRP42/55 and TPA, as well as with thrombin, involves phosphorylation, possibly of the receptor itself. Although protien kinase C is activated by thrombin and is presumably responsible for the desensitization caused by TPA, it does not appear to play a major role in receptor desensitization caused by thrombin and TRP42/55. This suggests that other kinases, such as those which inactivate adrenergic receptors and rhodopsin, are involved in the down-regulation of thrombin receptor function.

TRPM2 activation by cyclic ADP-ribose at body temperature is involved in insulin secretion

PubMed Central

Togashi, Kazuya; Hara, Yuji; Tominaga, Tomoko; Higashi, Tomohiro; Konishi, Yasunobu; Mori, Yasuo; Tominaga, Makoto

2006-01-01

There are eight thermosensitive TRP (transient receptor potential) channels in mammals, and there might be other TRP channels sensitive to temperature stimuli. Here, we demonstrate that TRPM2 can be activated by exposure to warm temperatures (>35°C) apparently via direct heat-evoked channel gating. β-NAD+- or ADP-ribose-evoked TRPM2 activity is robustly potentiated at elevated temperatures. We also show that, even though cyclic ADP-ribose (cADPR) does not activate TRPM2 at 25°C, co-application of heat and intracellular cADPR dramatically potentiates TRPM2 activity. Heat and cADPR evoke similar responses in rat insulinoma RIN-5F cells, which express TRPM2 endogenously. In pancreatic islets, TRPM2 is coexpressed with insulin, and mild heating of these cells evokes increases in both cytosolic Ca2+ and insulin release, which is KATP channel-independent and protein kinase A-mediated. Heat-evoked responses in both RIN-5F cells and pancreatic islets are significantly diminished by treatment with TRPM2-specific siRNA. These results identify TRPM2 as a potential molecular target for cADPR, and suggest that TRPM2 regulates Ca2+ entry into pancreatic β-cells at body temperature depending on the production of cADPR-related molecules, thereby regulating insulin secretion. PMID:16601673

Inhibition of tyrosinase by 4H-chromene analogs: Synthesis, kinetic studies, and computational analysis.

PubMed

Brasil, Edikarlos M; Canavieira, Luciana M; Cardoso, Érica T C; Silva, Edilene O; Lameira, Jerônimo; Nascimento, José L M; Eifler-Lima, Vera L; Macchi, Barbarella M; Sriram, Dharmarajan; Bernhardt, Paul V; Silva, José Rogério Araújo; Williams, Craig M; Alves, Cláudio N

2017-11-01

Inhibition of mushroom tyrosinase was observed with synthetic dihydropyrano[3,2-b]chromenediones. Among them, DHPC04 displayed the most potent tyrosinase inhibitory activity with a K i value of 4 μm, comparable to the reference standard inhibitor kojic acid. A kinetic study suggested that these synthetic heterocyclic compounds behave as competitive inhibitors for the L-DOPA binding site of the enzyme. Furthermore, molecular modeling provided important insight into the mechanism of binding interactions with the tyrosinase copper active site. © 2017 John Wiley & Sons A/S.

Preparation of insect-cuticle-like biomimetic materials.

PubMed

Miessner, M; Peter, M G; Vincent, J F

2001-01-01

A model system of tanning of a protein matrix within a fibrous structure, such as most commonly found in insect cuticle, was developed, using the cellulose of paper in place of chitin. The paper was impregnated with a tripeptide, DOPA-Gly-Gly, or a protein (BSA) plus catechol and treated with tyrosinase to oxidize the catechol. The resulting material was waterproof and had very high wet strength. If the material was wetted and dried repeatedly its water retention decreased by a factor of at least 2.

Tyrosinase-catalyzed oxidation of fluorophenols.

PubMed

Battaini, Giuseppe; Monzani, Enrico; Casella, Luigi; Lonardi, Emanuela; Tepper, Armand W J W; Canters, Gerard W; Bubacco, Luigi

2002-11-22

The activity of the type 3 copper enzyme tyrosinase toward 2-, 3-, and 4-fluorophenol was studied by kinetic methods and (1)H and (19)F NMR spectroscopy. Whereas 3- and 4-fluorophenol react with tyrosinase to give products that undergo a rapid polymerization process, 2-fluorophenol is not reactive and actually acts as a competitive inhibitor in the enzymatic oxidation of 3,4-dihydroxyphenylalanine (L-dopa). The tyrosinase-mediated polymerization of 3- and 4-fluorophenols has been studied in detail. It proceeds through a phenolic coupling pathway in which the common reactive fluoroquinone, produced stereospecifically by tyrosinase, eliminates an inorganic fluorine ion. The enzymatic reaction studied as a function of substrate concentration shows a prominent lag that is completely depleted in the presence of L-dopa. The kinetic parameters of the reactions can be correlated to the electronic and steric effects of the fluorine substituent position. Whereas the fluorine electron withdrawing effect appears to control the binding of the substrates (K(m) for 3- and 4-fluorophenols and K(I) for 2-fluorophenol), the k(cat) parameters do not follow the expected trend, indicating that in the transition state some additional steric effect rules the reactivity.

Biochemical Characterization of Ferulic Acid and Caffeic Acid Which Effectively Inhibit Melanin Synthesis via Different Mechanisms in B16 Melanoma Cells.

PubMed

Maruyama, Hiroko; Kawakami, Fumitaka; Lwin, Thet-Thet; Imai, Motoki; Shamsa, Fazel

2018-01-01

In this study, we examined the inhibitory effects of ferulic acid and caffeic acid on melanin production using a murine B16 melanoma cell line. The mechanisms by which the two acids inhibit melanin production were investigated by evaluating their effects on the activity of tyrosinase, which is involved is the first step of melanin biosynthesis. Ferulic acid showed no toxicity against the melanoma cells at any dose, whereas caffeic acid exerted cellular toxicity at concentrations higher than 0.35 mM. Both ferulic and caffeic acids effectively inhibited melanin production in the B16 melanoma cells. Ferulic acid reduced tyrosinase activity by directly binding to the enzyme, whereas no binding was observed between caffeic acid and tyrosinase. Both ferulic acid and caffeic acid inhibited casein kinase 2 (CK2)-induced phosphorylation of tyrosinase in a dose-dependent manner in vitro. Ferulic acid was found to be a more effective inhibitor of melanin production than caffeic acid; this difference in the inhibitory efficacy between the two substances could be attributable to the difference in their tyrosine-binding activity. Our analysis revealed that both substances also inhibited the CK2-mediated phosphorylation of tyrosinase.

TRP and rhodopsin transport depends on dual XPORT ER chaperones encoded by an operon

PubMed Central

Chen, Zijing; Chen, Hsiang-Chin; Montell, Craig

2015-01-01

Summary TRP channels and G protein-coupled receptors (GPCR) play critical roles in sensory reception. However, the identities of the chaperones that assist GPCRs in translocating from the endoplasmic reticulum (ER) are limited, and TRP ER chaperones are virtually unknown. The one exception for TRPs is Drosophila XPORT. Here, we show that the xport locus is bicistronic, and encodes unrelated transmembrane proteins, which enable the signaling proteins that initiate and culminate phototransduction, rhodopsin 1 (Rh1) and TRP, to traffic to the plasma membrane. XPORT-A and XPORT-B are ER proteins, and loss of either has a profound impact on TRP and Rh1 targeting to the light-sensing compartment of photoreceptor cells. XPORT-B complexed in vivo with the Drosophila homolog of the mammalian HSP70 protein, GRP78/BiP, which in turn associated with Rh1. Our work highlights a coordinated network of chaperones required for the biosynthesis of the TRP channel and rhodopsin in Drosophila photoreceptor cells. PMID:26456832

Mechanism of the melanogenesis stimulation activity of (-)-cubebin in murine B16 melanoma cells.

PubMed

Hirata, Noriko; Naruto, Shunsuke; Ohguchi, Kenji; Akao, Yukihiro; Nozawa, Yoshinori; Iinuma, Munekazu; Matsuda, Hideaki

2007-07-15

(-)-Cubebin showed a melanogenesis stimulation activity in a concentration-dependent manner in murine B16 melanoma cells without any significant effects on cell proliferation. Tyrosinase activity was increased at 24-72 h after addition of cubebin to B16 cells, and then intracellular melanin amount was increased at 48-96 h after the treatment. The expression levels of tyrosinase were time-dependently enhanced after the treatment with cubebin. At the same time, the expression levels of tyrosinase mRNA were also increased after addition of cubebin. Furthermore Western blot analysis revealed that cubebin elevated the level of phosphorylation of p38 mitogen-activated protein kinase (MAPK). SB203580, a selective inhibitor of p38 MAPK, completely blocked cubebin-induced expression of tyrosinase mRNA in B16 cells. These results suggested that cubebin increased melanogenesis in B16 cells through the enhancement of tyrosinase expression mediated by activation of p38 MAPK.

π-Cation interactions as the origin of the weak absorption at 532 nm observed in tryptophan-containing polypeptides.

PubMed

Roveri, O A; Braslavsky, S E

2012-06-01

We have previously reported that bovine serum albumin (BSA) and other proteins that do not contain prosthetic groups exhibited a weak light absorption in the visible, only detectable by pulsed laser-induced optoacoustic spectroscopy (LIOAS). Human serum albumin (HSA) exhibited signals 25% higher than those observed with BSA. Signals comparable to those obtained with BSA were observed with poly(L-Trp, L-Lys), poly(L-Trp, L-Arg) or poly(L-Trp, L-Orn) at pH 7.0. No signals were obtained when tryptophan was replaced by other amino acids or when free tryptophan or the tripeptide Lys-Trp-Lys was assayed (pH 7.0). Tryptophan in HCl 5 N produced LIOAS signals similar to those produced by tryptophan-containing copolymers. Moreover, the absorption peak could be observed in a UV-VIS spectrophotometer. Therefore, the LIOAS signals obtained with BSA, HSA, and tryptophan-containing random copolymers may be attributed to a new transition of the indole moiety of their tryptophan residues when "protonated". Tryptophan residues of proteins are known to participate in π-cation interactions, which are important in protein stability and function. As a matter of fact, HSA and BSA contain an internal tryptophan in close proximity to lysine and arginine residues and therefore suitable for π-cation interactions. The strength of this type of interaction strongly depends on distances and relative orientations of both amino acid residues. Accordingly, these interactions should be highly sensitive to conformational changes. Based on preliminary results that have shown that LIOAS signal at 532 nm depended on the aggregation state of BSA and/or on the oxidation state of its Cys-34, we postulate that the LIOAS signal observed with proteins and tryptophan-containing polypeptides are related to Trp-Lys or Trp-Arg interactions and that the intensity of the signal depends on the strength of such interactions.

The TRP channel superfamily: insights into how structure, protein-lipid interactions and localization influence function.

PubMed

Reaves, B J; Wolstenholme, A J

2007-02-01

TRP (transient receptor potential) cationic channels are key molecules that are involved in a variety of diverse biological processes ranging from fertility to osmosensation and nociception. Increasing our knowledge of these channels will help us to understand a range of physiological and pathogenic processes, as well as highlighting potential therapeutic drug targets. The founding members of the TRP family, Drosophila TRP and TRPL (TRP-like) proteins, were identified within the last two decades and there has been a subsequent explosion in the number and type of TRP channel described. Although information is accumulating as to the function of some of the TRP channels, the activation and inactivation mechanisms, structure, and interacting proteins of many, if not most, are awaiting elucidation. The Cell and Molecular Biology of TRP Channels Meeting held at the University of Bath included speakers working on a number of the different subfamilies of TRP channels and provided a basis for highlighting both similarities and differences between these groups. As the TRP channels mediate diverse functions, this meeting also brought together an audience with wide-ranging research interests, including biochemistry, cell biology, physiology and neuroscience, and inspired lively discussion on the issues reviewed herein.

Mutagenic activities of heterocyclic amines in Chinese hamster lung cells in culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Terada, M.; Nagao, M.; Nakayasu, M.

1986-01-01

A mutation assay system with Chinese hamster lung cells (CHL) using diphtheria toxin resistance as a selective marker has been established. The mutagenic activities of heterocyclic amines, originally isolated from pyrolyzates of amino acids and proteins, broiled fish and fried beef were assayed in cultured CHL cells in the absence and presence of a metabolic activation system, with diphtheria toxin resistance as a marker. All the heterocyclic amines tested except 3-amino-1,4-dimethyl-5H-pyrido (4,3-b)indole (Trp-P-1) required the presence of a metabolic activation system for mutagenicity on CHL cells. 3-Amino-1-methyl-5H-pyrido(4,3-b)indole (Trp-P-2) was the most mutagenic among the heterocyclic amines tested. Other compounds weremore » also mutagenic in the following order of decreasing potency: Trp-P-1, 2-amino-3,4-dimethylimidazo(4,5-f)quinoline (MeIQ), 2-amino-3-methylimidazo(4,5-f)quinoline (IQ), 2-amino-9H-pyrido(2,3-b)indole (A..cap alpha..C), 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline (MeIQx), 2-amino-6-methyldipyrido(1,2-a:3',2'-d)imidazole (Glu-P-1) and 2-aminodipyrido(1,2--a:3',2'-d)imidazole (Glu-P-2).« less

Effective L-Tyrosine Hydroxylation by Native and Immobilized Tyrosinase

PubMed Central

Lewańczuk, Marcin; Koźlecki, Tomasz; Liesiene, Jolanta; Bryjak, Jolanta

2016-01-01

Hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA) by immobilized tyrosinase in the presence of ascorbic acid (AH2), which reduces DOPA-quinone to L-DOPA, is characterized by low reaction yields that are mainly caused by the suicide inactivation of tyrosinase by L-DOPA and AH2. The main aim of this work was to compare processes with native and immobilized tyrosinase to identify the conditions that limit suicide inactivation and produce substrate conversions to L-DOPA of above 50% using HPLC analysis. It was shown that immobilized tyrosinase does not suffer from partitioning and diffusion effects, allowing a direct comparison of the reactions performed with both forms of the enzyme. In typical processes, additional aeration was applied and boron ions to produce the L-DOPA and AH2 complex and hydroxylamine to close the cycle of enzyme active center transformations. It was shown that the commonly used pH 9 buffer increased enzyme stability, with concomitant reduced reactivity of 76%, and that under these conditions, the maximal substrate conversion was approximately 25 (native) to 30% (immobilized enzyme). To increase reaction yield, the pH of the reaction mixture was reduced to 8 and 7, producing L-DOPA yields of approximately 95% (native enzyme) and 70% (immobilized). A three-fold increase in the bound enzyme load achieved 95% conversion in two successive runs, but in the third one, tyrosinase lost its activity due to strong suicide inactivation caused by L-DOPA processing. In this case, the cost of the immobilized enzyme preparation is not overcome by its reuse over time, and native tyrosinase may be more economically feasible for a single use in L-DOPA production. The practical importance of the obtained results is that highly efficient hydroxylation of monophenols by tyrosinase can be obtained by selecting the proper reaction pH and is a compromise between complexation and enzyme reactivity. PMID:27711193

Products of cholecystokinin (CCK)-octapeptide proteolysis interact with central CCK receptors.

PubMed

Steardo, L; Knight, M; Tamminga, C A; Chase, T N

1985-03-15

Peptidases present in central nervous system (CNS) synaptic membranes, hydrolyze the neuroactive peptide cholecystokinin-octapeptide (CCK-8; Asp-Tyr-SO3H-Met-Gly-Trp-Met-Asp-Phe-NH2). In order to determine the pathway of degradation, synthetic CCK-8 was incubated at 37 degrees C with purified synaptic membranes; at various intervals reaction samples were removed from the reaction mixture and analysed by high-performance liquid chromatography to identify and quantify the peptide fragments. The results indicate an initial endopeptidase cleavage at the Met-Gly bond producing CCK-5 (Gly-Trp-Met-Asp-Phe-NH2). The carboxyl-terminal pentapeptide is further proteolysed to CCK-4 (Trp-Met-Asp-Phe-NH2) by a puromycin-sensitive aminopeptidase and to CCK-3 (Met-Asp-Phe-NH2) and Gly-Trp by an endopeptidase action. CCK-3 and CCK-2 appear to be relatively stable end-products. Moreover, these proteolytic fragments are shown to bind to the CCK receptor in brain with varying potencies.

MALDI Imaging Analysis of Neuropeptides in Africanized Honeybee (Apis mellifera) Brain: Effect of Aggressiveness.

PubMed

Pratavieira, Marcel; Menegasso, Anally Ribeiro da Silva; Esteves, Franciele Grego; Sato, Kenny Umino; Malaspina, Osmar; Palma, Mario Sérgio

2018-05-18

The aggressiveness in honeybees seems to be regulated by multiple genes, under the influence of different factors, such as polyethism of workers, environmental factors, and response to alarm pheromones, creating a series of behavioral responses. It is suspected that neuropeptides seem to be involved with the regulation of the aggressive behavior. The role of allatostatin and tachykinin-related neuropeptides in honeybee brain during the aggressive behavior is unknown; thus, worker honeybees were stimulated to attack and to sting leather targets hanged in front of the colonies. The aggressive individuals were collected and immediately frozen in liquid nitrogen; the heads were removed, and sliced at sagittal plan. The brain slices were submitted to MALDI-Spectral-Imaging analysis, and the results of the present study reported the processing of the precursors proteins into mature forms of the neuropeptides AmAST A (59-76) (AYTYVSEYKRLPVYNFGL-NH2), AmAST A (69-76) (LPVYNFGL-NH2), AmTRP (88 - 96) (APMGFQGMR-NH2), and AmTRP (254 - 262) (ARMGFHGMR-NH2), which apparently acted in different neuropils of honeybee brain, during the aggressive behavior, possibly playing the neuromodulation of different aspects of this complex behavior. These results were biologically validated performing aggressiveness-related behavioral assays, using young honeybee workers that received 1 ng of AmAST A (69-76) or AmTRP (88 - 96) via hemocele. The young workers that were not expected to be aggressive individuals, presented a complete series of the aggressive behaviors, in presence of the neuropeptides, corroborating the hypothesis that correlates the presence of mature AmASTs A and AmTRPs in honeybee brain with the aggressiveness of this insect.

Structural Insight into the Assembly of TRPV Channels

PubMed Central

Huynh, Kevin W.; Cohen, Matthew R.; Chakrapani, Sudha; Holdaway, Heather A.; Stewart, Phoebe L.; Moiseenkova-Bell, Vera Y.

2017-01-01

SUMMARY Transient receptor potential (TRP) proteins are a large family of polymodal nonselective cation channels. The TRP vanilloid (TRPV) subfamily consists of six homologous members with diverse functions. TRPV1–TRPV4 are nonselective cation channels proposed to play a role in nociception, while TRPV5 and TRPV6 are involved in epithelial Ca2+ homeostasis. Here we present the cryo-electron microscopy (cryo-EM) structure of functional, full-length TRPV2 at 13.6 Å resolution. The map reveals that the TRPV2 cytoplasmic domain displays a 4-fold petal-like shape in which high-resolution N-terminal ankyrin repeat domain (ARD) structures can be unambiguously fitted. Fitting of the available ARD structures for other TRPV subfamily members into the TRPV2 EM map suggests that TRPV subfamily members have highly homologous structural topologies. These results allowed us to postulate a structural explanation for the functional diversity among TRPV channels and their differential regulation by proteins and ligands. PMID:24373766

Anti-Melanogenic Activity of Gagunin D, a Highly Oxygenated Diterpenoid from the Marine Sponge Phorbas sp., via Modulating Tyrosinase Expression and Degradation

PubMed Central

Lee, Ho Yeon; Jang, Eun Jeong; Bae, Song Yi; Jeon, Ju-eun; Park, Hyen Joo; Shin, Jongheon; Lee, Sang Kook

2016-01-01

Tyrosinase is the rate-limiting enzyme critical for melanin synthesis and controls pigmentation in the skin. The inhibition of tyrosinase is currently the most common approach for the development of skin-whitening cosmetics. Gagunin D (GD), a highly oxygenated diterpenoid isolated from the marine sponge Phorbas sp., has exhibited cytotoxicity toward human leukemia cells. However, the effect of GD on normal cells and the molecular mechanisms remain to be elucidated. In the present study, we identified for the first time the anti-melanogenic activity of GD and its precise underlying mechanisms in mouse melan-a cells. GD significantly inhibited melanin synthesis in the melan-a cells and a reconstructed human skin model. Further analysis revealed that GD suppressed the expression of tyrosinase and increased the rate of tyrosinase degradation. GD also inhibited tyrosinase enzymatic activity. In addition, GD effectively suppressed the expression of proteins associated with melanosome transfer. These findings suggest that GD is a potential candidate for cosmetic formulations due to its multi-functional properties. PMID:27869664

Antimelanogenic effect of c-phycocyanin through modulation of tyrosinase expression by upregulation of ERK and downregulation of p38 MAPK signaling pathways

PubMed Central

2011-01-01

Background Pigmentation is one of the essential defense mechanisms against oxidative stress or UV irradiation; however, abnormal hyperpigmentation in human skin may pose a serious aesthetic problem. C-phycocyanin (Cpc) is a phycobiliprotein from spirulina and functions as an antioxidant and a light harvesting protein. Though it is known that spirulina has been used to reduce hyperpigmentation, little literature addresses the antimelanogenic mechanism of Cpc. Herein, we investigated the rationale for the Cpc-induced inhibitory mechanism on melanin synthesis in B16F10 melanoma cells. Methods Cpc-induced inhibitory effects on melanin synthesis and tyrosinase expression were evaluated. The activity of MAPK pathways-associated molecules such as MAPK/ERK and p38 MAPK, were also examined to explore Cpc-induced antimelanogenic mechanisms. Additionally, the intracellular localization of Cpc was investigated by confocal microscopic analysis to observe the migration of Cpc. Results Cpc significantly (P < 0.05) reduced both tyrosinase activity and melanin production in a dose-dependent manner. This phycobiliprotein elevated the abundance of intracellular cAMP leading to the promotion of downstream ERK1/2 phosphorylation and the subsequent MITF (the transcription factor of tyrosinase) degradation. Further, Cpc also suppressed the activation of p38 causing the consequent disturbed activation of CREB (the transcription factor of MITF). As a result, Cpc negatively regulated tyrosinase gene expression resulting in the suppression of melanin synthesis. Moreover, the entry of Cpc into B16F10 cells was revealed by confocal immunofluorescence localization and immunoblot analysis. Conclusions Cpc exerted dual antimelanogenic mechanisms by upregulation of MAPK/ERK-dependent degradation of MITF and downregulation of p38 MAPK-regulated CREB activation to modulate melanin formation. Cpc may have potential applications in biomedicine, food, and cosmetic industries. PMID:21988805

Role of microRNA508-3p in melanogenesis by targeting microphthalmia transcription factor in melanocytes of alpaca.

PubMed

Zhang, J; Liu, Y; Zhu, Z; Yang, S; Ji, K; Hu, S; Liu, X; Yao, J; Fan, R; Dong, C

2017-02-01

It has been demonstrated that microRNAs (miRNAs) play important roles in the control of melanogenesis and hair color in mammals. By comparing miRNA expression profiles between brown and white alpaca skin, we previously identified miR508-3p as a differentially expressed miRNA suggesting its potential role in melanogenesis and hair color formation. The present study was conducted to determine the role of miR508-3p in melanogenesis in alpaca melanocytes. In situ hybridization showed that miR508-3p is abundantly present in the cytoplasma of alpaca melanocytes. miR508-3p was predicted to target the gene encoding microphthalmia transcription factor (MITF) and a luciferase reporter assay indicated that miR508-3p regulates MITF expression by directly targeting its 3'UTR. Overexpression of miR508-3p in alpaca melanocytes down-regulated MITF expression both at the messenger RNA and protein level and resulted in decreased expression of key melanogenic genes including tyrosinase and tyrosinase-related protein 2. Overexpression of miR508-3p in melanocytes also resulted in decreased melanin production including total alkali-soluble melanogenesis, eumelanogenesis and pheomelanogenesis. Results support a functional role of miR508-3p in regulating melanogenesis in alpaca melanocytes by directly targeting MITF.

Promotion and computation of inhibitory effect on tyrosinase activity of herbal cream by incorporating indigenous medicinal plants.

PubMed

Sahu, Ram Kumar; Roy, Amit; Dwivedi, Jaya; Jha, Arvind Kumar

2014-01-01

Herbal cream imparts a chief role in regulating melanin production of skin. The phytoconstituents present in herbal cream impact biological functions of skin and contribute nutrients required for the healthy skin. In the present study, it was envisaged to prepare three batches of herbal cream (HC1, HC2 and HC3) containing ethanol extracts of Emblica officinalis (fruits), Daucus carota (root), Mangifera indica (leaves), Mentha arvensis (leaves), Terminalia arjuna (bark) and Cucumis sativus (fruits) and investigated the prepared cream for inhibitory effect on tyrosinase activity. The herbal cream was formulated by incorporating different ratio of extracts, by using cream base. Each formulation HC1, HC2 and HC3 were segregated into three different formulations (HC1.1, HC1.2, HC1.3, HC2.1, HC2.2, HC2.3, HC3.1, HC3.2 and HC3.3) by incorporating increasing ratio of extract in formulation. The HC3.2 cream produces highest tyrosinase inhibitory effect 65.23 +/- 0.07%, while the HC2.1 exhibited minimum tyrosinase inhibitory effect 26.19 +/- 0.08% compared to other prepared cream. Comparison of the inhibitory activity of the formulations demonstrated that the rank order was HC3.2 > HC3.3 > HC1.2 > HC1.3 > HC3.1 > HC1.1 > HC2.3 > HC2.2 > HC2.1. It has been observed from the result that the formulations of antityrosinase activity were not concentrate dependent. This finding suggests that decrease in antityrosinase activity of HC1 and HC3 might be considering that the incompatibility of the higher extract content with the base of cream. The HC3 produce the maximum inhibitory effects on tyrosinase activity might be due to higher level of polyphenol and flavonoids present in extracts.

Ehrlichia chaffeensis Tandem Repeat Proteins and Ank200 are Type 1 Secretion System Substrates Related to the Repeats-in-Toxin Exoprotein Family

PubMed Central

Wakeel, Abdul; den Dulk-Ras, Amke; Hooykaas, Paul J. J.; McBride, Jere W.

2011-01-01

Ehrlichia chaffeensis has type 1 and 4 secretion systems (T1SS and T4SS), but the substrates have not been identified. Potential substrates include secreted tandem repeat protein (TRP) 47, TRP120, and TRP32, and the ankyrin repeat protein, Ank200, that are involved in molecular host–pathogen interactions including DNA binding and a network of protein–protein interactions with host targets associated with signaling, transcriptional regulation, vesicle trafficking, and apoptosis. In this study we report that E. chaffeensis TRP47, TRP32, TRP120, and Ank200 were not secreted in the Agrobacterium tumefaciens Cre recombinase reporter assay routinely used to identify T4SS substrates. In contrast, all TRPs and the Ank200 proteins were secreted by the Escherichia coli complemented with the hemolysin secretion system (T1SS), and secretion was reduced in a T1SS mutant (ΔTolC), demonstrating that these proteins are T1SS substrates. Moreover, T1SS secretion signals were identified in the C-terminal domains of the TRPs and Ank200, and a detailed bioinformatic analysis of E. chaffeensis TRPs and Ank200 revealed features consistent with those described in the repeats-in-toxins (RTX) family of exoproteins, including glycine- and aspartate-rich tandem repeats, homology with ATP-transporters, a non-cleavable C-terminal T1SS signal, acidic pIs, and functions consistent with other T1SS substrates. Using a heterologous E. coli T1SS, this investigation has identified the first Ehrlichia T1SS substrates supporting the conclusion that the T1SS and corresponding substrates are involved in molecular host–pathogen interactions that contribute to Ehrlichia pathobiology. Further investigation of the relationship between Ehrlichia TRPs, Ank200, and the RTX exoprotein family may lead to a greater understanding of the importance of T1SS substrates and specific functions of T1SS in the pathobiology of obligately intracellular bacteria. PMID:22919588

Association of XRCC1 Trp194 allele with risk of breast cancer, and Ki67 protein status in breast tumor tissues

PubMed Central

Jalali, Chiya; Ghaderi, Bayazid; Amini, Sabrieh; Abdi, Mohammad; Roshani, Daem

2016-01-01

Objectives: To evaluate the role of this polymorphism as a risk factor for breast cancer in Kurdish patients and to investigate the possible association between Arg194Trp x-ray repair cross-complementing group 1 (XRCC1) gene polymorphisms with clinical and histopathological outcomes of patients with breast cancer. Methods: A total of 100 breast cancer patients and 200 cancer-free controls in Kurdish population of Kurdistan state admitted to Tohid Hospital, Sanandaj, Kurdistan, Iran between January 2012 and May 2015 were enrolled in this cross-sectional study. Tissue expression of estrogen receptor (ER), progesteron receptor (PR), human epidermal growth factor receptor 2 (Her2/neu), and Ki67 were evaluated by immunohistochemistry (IHC). The Arg194Trp genotypes were determined by polymerase chain reaction- restriction fragment length polymorphism method. Results: Our data showed that the risk for breast cancer increased significantly among the Trp variant of XRCC1. Statistically significant association was found between codon 194 polymorphisms and tissue expression of Ki67. Conclusion: The Trp allele of codon 194 XRCC1 is a potential risk factor for breast cancer in Kurdish ethnicity. Furthermore, effect of this polymorphism on clinical and histological features of breast cancer was significant. PMID:27279507

Host-Guest Recognition-Assisted Electrochemical Release: Its Reusable Sensing Application Based on DNA Cross Configuration-Fueled Target Cycling and Strand Displacement Reaction Amplification.

PubMed

Chang, Yuanyuan; Zhuo, Ying; Chai, Yaqin; Yuan, Ruo

2017-08-15

In this work, an elegantly designed host-guest recognition-assisted electrochemical release was established and applied in a reusable electrochemical biosensor for the detection of microRNA-182-5p (miRNA-182-5p), a prostate cancer biomarker in prostate cancer, based on the DNA cross configuration-fueled target cycling and strand displacement reaction (SDR) amplification. With such a design, the single target miRNA input could be converted to large numbers of single-stranded DNA (S1-Trp and S2-Trp) output, which could be trapped by cucurbit[8]uril methyl viologen (CB-8-MV 2+ ) based on the host-guest recognition, significantly enhancing the sensitivity for miRNA detection. Moreover, the nucleic acids products obtained from the process of cycling amplification could be utilized sufficiently, avoiding the waste and saving the experiment cost. Impressively, by resetting a settled voltage, the proposed biosensor could release S1-Trp and S2-Trp from the electrode surface, attributing that the guest ion methyl viologen (MV 2+ ) was reduced to MV +· under this settled voltage and formed a more-stable CB-8-MV +· -MV +· complex. Once O 2 was introduced in this system, MV +· could be oxidized to MV 2+ , generating the complex of CB-8-MV 2+ for capturing S1-Trp and S2-Trp again in only 5 min. As a result, the simple and fast regeneration of biosensor for target detection was realized on the base of electrochemical redox-driven assembly and release, overcoming the challenges of time-consuming, burdensome operations and expensive experimental cost in traditional reusable biosensors and updating the construction method for a reusable bisensor. Furthermore, the biosensor could be reused for more than 10 times with a regeneration rate of 93.20%-102.24%. After all, the conception of this work provides a novel thought for the construction of effective reusable biosensor to detect miRNA and other biomarkers and has great potential application in the area requiring the release of nucleic acids or proteins.

Side-chain conformation of the M2 transmembrane peptide proton channel of influenza a virus from 19F solid-state NMR.

PubMed

Luo, Wenbin; Mani, Rajeswari; Hong, Mei

2007-09-13

The M2 transmembrane peptide (M2TMP) of the influenza A virus forms a tetrameric helical bundle that acts as a proton-selective channel important in the viral life cycle. The side-chain conformation of the peptide is largely unknown and is important for elucidating the proton-conducting mechanism and the channel stability. Using a 19F spin diffusion NMR technique called CODEX, we have measured the oligomeric states and interhelical side chain-side chain 19F-19F distances at several residues using singly fluorinated M2TMP bound to DMPC bilayers. 19F CODEX data at a key residue of the proton channel, Trp41, confirm the tetrameric state of the peptide and yield a nearest-neighbor interhelical distance of approximately 11 A under both neutral and acidic pH. Since the helix orientation is precisely known from previous 15N NMR experiments and the backbone channel diameter has a narrow allowed range, this 19F distance constrains the Trp41 side-chain conformation to t90 (chi1 approximately 180 degrees , chi2 approximately 90 degrees ). This Trp41 rotamer, combined with a previously measured 15N-13C distance between His37 and Trp411, suggests that the His37 rotamer is t-160. The implication of the proposed (His37, Trp41) rotamers to the gating mechanism of the M2 proton channel is discussed. Binding of the antiviral drug amantadine to the peptide does not affect the F-F distance at Trp41. Interhelical 19F-19F distances are also measured at residues 27 and 38, each mutated to 4-19F-Phe. For V27F-M2TMP, the 19F-19F distances suggest a mixture of dimers and tetramers, whereas the L38F-M2TMP data indicate two tetramers of different sizes, suggesting side chain conformational heterogeneity at this lipid-facing residue. This work shows that 19F spin diffusion NMR is a valuable tool for determining long-range intermolecular distances that shed light on the mechanism of action and conformational heterogeneity of membrane protein oligomers.

Comparative Depigmentation Effects of Resveratrol and Its Two Methyl Analogues in α-Melanocyte Stimulating Hormone-Triggered B16/F10 Murine Melanoma Cells

PubMed Central

Yoon, Hoon-Seok; Hyun, Chang-Gu; Lee, Nam-Ho; Park, Sung-Soo; Shin, Dong-Bum

2016-01-01

Previous research showed that resveratrol (trans-3,4′,5-trihydroxystilbene) and pinostilbene (trans-3-methoxy-4′,5-dihydroxystilbene) were able to inhibit tyrosinase directly; however, anti-melanogenic effects of pterostilbene (trans-3,5-dimethoxy-4′-hydroxystilbene) and resveratrol trimethyl ether (RTE) have not been compared. To investigate the hypopigmentation effects of pterostilbene and RTE, melanin contents and intracellular tyrosinase activity were determined by western blot analysis. Firstly, pterostilbene showed the inhibitory effects on α-melanocyte stimulating hormone (MSH)-induced melanin synthesis stronger than RTE, resveratrol, and arbutin. Pterostilbene inhibited melanin biosynthesis in a dose-dependent manner in α-MSH-stimulated B16/F10 murine melanoma cells. Specifically, melanin content and intracellular tyrosinase activity were inhibited by 63% and 58%, respectively, in response to treatment with 10 μM of pterostilbene. The results of western blot analysis indicated that pterostilbene induced downregulation of tyrosinase protein expression and suppression of α-MSH-stimulated melan-A protein expression stronger than RTE or resveratrol. Based on these results, our study suggests that pterostilbene can induce hypopigmentation effects more effectively than resveratrol and RTE, and it functions via downregulation of protein expression associated with hyperpigmentation in α-MSH-triggered B16/F10 murine melanoma cells. PMID:27390733

Response of GWALP Transmembrane Peptides to Changes in the Tryptophan Anchor Positions†

PubMed Central

Vostrikov, Vitaly V.; Koeppe, Roger E.

2011-01-01

While the interfacial partitioning of charged or aromatic anchor residues may determine the preferred orientations of transmembrane peptide helices, the dependence of helix orientation on anchor residue position is not well understood. When anchor residue locations are changed systematically, some adaptations of the peptide-lipid interactions may be required to compensate the altered interfacial interactions. Recently we have developed a novel transmembrane peptide, termed GW5,19ALP23 (acetyl-GGALW5LALALALALALALW19LAGA-ethanolamide), which proves to be a well behaved sequence for an orderly investigation of protein-lipid interactions. Its roughly symmetric nature allows for shifting the anchoring Trp residues by one Leu-Ala pair inward (GW7,17ALP23) or outward (GW3,21ALP23), thus providing fine adjustments of the formal distance between the tryptophan residues. With no other obvious anchoring features present, we postulate that the inter-Trp distance may be crucial for aspects of the peptide-lipid interaction. Importantly, the amino acid composition is identical for each of the resulting related GWALP23 sequences, and the radial separation between the pairs of Trp residues on each side of the transmembrane α-helix remains similar. Here we address the adaptation of the aforementioned peptides to the varying Trp locations by means of solid-state 2H NMR experiments in varying lipid bilayer membrane environments. All of the GWx,yALP23 sequence isomers adopt transmembrane orientations in DOPC, DMPC and DLPC environments, even when the Trp residues are quite closely spaced, in GW7,17ALP23. Furthermore, the dynamics for each peptide isomer are less extensive than for peptides possessing additional interfacial Trp residues. The helical secondary structure is maintained more strongly within the Trp-flanked core region than outside of the Trp boundaries. Deuterium labeled tryptophan indole rings in the GWx,yALP23 peptides provide additional insights into the behavior of the Trp side chains. A Trp side chain near the C-terminus adopts a different orientation and undergoes somewhat faster dynamics than a corresponding Trp side chain located an equivalent distance from the N-terminus. In contrast, as the inter-Trp distance changes, the variations among the average orientations of the Trp indole rings at either terminus are systematic yet fairly small. We conclude that subtle adjustments to the peptide tilt, and to the N- and C-terminal Trp side-chain torsion angles, permit the GWx,yALP23 peptides to maintain preferred transmembrane orientations while adapting to lipid bilayers of differing hydrophobic thickness. PMID:21800919

Altered sexual and social behaviors in trp2 mutant mice

PubMed Central

Leypold, Bradley G.; Yu, C. Ron; Leinders-Zufall, Trese; Kim, Michelle M.; Zufall, Frank; Axel, Richard

2002-01-01

We have used gene targeting to generate mice with a homozygous deficiency in trp2, a cation channel expressed in the vomeronasal organ (VNO). Trp2 mutant animals reveal a striking reduction in the electrophysiological response to pheromones in the VNO, suggesting that trp2 plays a central role in mediating the pheromone response. These mutants therefore afford the opportunity to examine the role of the VNO in the generation of innate sexual and social behaviors in mice. Trp2 mutant males and nursing females are docile and fail to initiate aggressive attacks on intruder males. Male–female sexual behavior appears normal, but trp2 mutant males also vigorously mount other males. These results suggest that the cation channel trp2 is required in the VNO to detect male-specific pheromones that elicit aggressive behaviors and dictate the choice of sexual partners. PMID:11972034

Design and synthesis of chalcone derivatives as potent tyrosinase inhibitors and their structural activity relationship

NASA Astrophysics Data System (ADS)

Akhtar, Muhammad Nadeem; Sakeh, Nurshafika M.; Zareen, Seema; Gul, Sana; Lo, Kong Mun; Ul-Haq, Zaheer; Shah, Syed Adnan Ali; Ahmad, Syahida

2015-04-01

Browning of fruits and vegetables is a serious issue in the food industry, as it damages the organoleptic properties of the final products. Overproduction of melanin causes aesthetic problems such as melisma, freckles and lentigo. In this study, a series of chalcones (1-10) have been synthesized and examined for their tryrosinase inhibitory activity. The results showed that flavokawain B (1), flavokawain A (2) and compound 3 were found to be potential tyrosinase inhibitors, indicating IC50 14.20-14.38 μM values. This demonstrates that 4-substituted phenolic compound especially at ring A exhibited significant tyrosinase inhibition. Additionally, molecular docking results showed a strong binding affinity for compounds 1-3 through chelation between copper metal and ligands. The detailed molecular docking and SARs studies correlate well with the tyrosinase inhibition studies in vitro. The structures of these compounds were elucidated by the 1D and 2D NMR spectroscopy, mass spectrometry and single X-ray crystallographic techniques. These findings could lead to design and discover of new tyrosinase inhibitors to control the melanine overproduction and overcome the economic loss of food industry.

Chemical components and tyrosinase inhibitors from the twigs of Artocarpus heterophyllus.

PubMed

Zheng, Zong-Ping; Chen, Sibao; Wang, Shiyun; Wang, Xia-Chang; Cheng, Ka-Wing; Wu, Jia-Jun; Yang, Dajiang; Wang, Mingfu

2009-08-12

An HPLC method was developed and validated to compare the chemical profiles and tyrosinase inhibitors in the woods, twigs, roots, and leaves of Artocarpus heterophyllus . Five active tyrosinase inhibitors including dihydromorin, steppogenin, norartocarpetin, artocarpanone, and artocarpesin were used as marker compounds in this HPLC method. It was discovered that the chemical profiles of A. heterophyllus twigs and woods are quite different. Systematic chromatographic methods were further applied to purify the chemicals in the twigs of A. heterophyllus. Four new phenolic compounds, including one isoprenylated 2-arylbenzofuran derivative, artoheterophyllin A (1), and three isoprenylated flavonoids, artoheterophyllin B (2), artoheterophyllin C (3), and artoheterophyllin D (4), together with 16 known compounds, were isolated from the ethanol extract of the twigs of A. heterophyllus. The structures of compounds 1-4 were elucidated by spectroscopic analysis. However, the four new compounds did not show significant inhibitory activities against mushroom tyrosinase compared to kojic acid. It was found that similar compounds, such as norartocarpetin and artocarpesin in the twigs and woods of A. heterophyllus, contributed to their tyrosinase inhibitory activity.

Potent microbial and tyrosinase inhibitors from stem bark of Bauhinia rufescens (Fabaceae).

PubMed

Muhammad, Aminu; Sirat, Hasnah Mohd

2013-10-01

The stem bark extracts of Bauhinia rufescens Lam. (Fabaceae) yielded 6-methoxy-7-methyl-8-hydroxydibenz[b,f]oxepin, alpha-amyrin acetate, beta-sitosterol 3-O-beta-D-xylopyranoside, 4-(2'-Hydroxyphenethyl)-5-methoxy-2-methylphenol, menisdaurin and sequoyitol. Their structures were determined using spectroscopic methods and comparisons with the literature data. For the antimicrobial assay Gram-positive and Gram-negative bacterial and fungal strains were tested, while the tyrosinase inhibition assay utilized L-DOPA as a substrate for the tyrosinase enzyme. 6-Methoxy-7-methyl-8-hydroxydibenz[b,f]oxepin, a-amyrin acetate, beta-sitosterol 3-O-D-xylopyranoside, menisdaurin and sequoyitol showed weak to moderate activities with minimum inhibition concentration (MIC) values in the range of 112.5-900 microg/mL against all bacterial strains, while the MIC values for the fungal strains were in the range of 28.1-450 microg/mL. In the tyrosinase inhibition assay, a-amyrin acetate was found to be moderately active against tyrosinase with an inhibition of 62% at 0.1 mg/mL. This activity was lower than that of the positive control, kojic acid (85%).

Modeling tyrosinase activity. Effect of ligand topology on aromatic ring hydroxylation: an overview.

PubMed

De, Anindita; Mandal, Sukanta; Mukherjee, Rabindranath

2008-01-01

Synthetic modeling of tyrosinase (o-phenol ring hydroxylation) has emerged as a novel class of successful biomimetic studies. It is a well-established fact that the reaction of dioxygen with copper(I) complexes of m-xylyl-based ligands generate putative copper-oxygen intermediate species such as side-on peroxo {CuII2(mu-O2)}2+ [in some cases bis-oxo {CuIII2(mu-O)2}2+ in equilibrium with isomeric side-on peroxo], due to oxygen activation. Electrophilic attack of such species brings about monooxygenase activity by incorporating one of the oxygens to m-xylyl ring of the ligand and the other oxygen is reduced to hydroxide ion. The goal of this review is to provide a concise overview of the present day knowledge in this field of research to emphasize the important role the designed ligands play in eliciting the desired tyrosinase-like chemistry.

Tyrosine- and tryptophan-coated gold nanoparticles inhibit amyloid aggregation of insulin.

PubMed

Dubey, Kriti; Anand, Bibin G; Badhwar, Rahul; Bagler, Ganesh; Navya, P N; Daima, Hemant Kumar; Kar, Karunakar

2015-12-01

Here, we have strategically synthesized stable gold (AuNPs(Tyr), AuNPs(Trp)) and silver (AgNPs(Tyr)) nanoparticles which are surface functionalized with either tyrosine or tryptophan residues and have examined their potential to inhibit amyloid aggregation of insulin. Inhibition of both spontaneous and seed-induced aggregation of insulin was observed in the presence of AuNPs(Tyr), AgNPs(Tyr), and AuNPs(Trp) nanoparticles. These nanoparticles also triggered the disassembly of insulin amyloid fibrils. Surface functionalization of amino acids appears to be important for the inhibition effect since isolated tryptophan and tyrosine molecules did not prevent insulin aggregation. Bioinformatics analysis predicts involvement of tyrosine in H-bonding interactions mediated by its C=O, -NH2, and aromatic moiety. These results offer significant opportunities for developing nanoparticle-based therapeutics against diseases related to protein aggregation.

Human tyrosinase produced in insect cells: a landmark for the screening of new drugs addressing its activity.

PubMed

Fogal, Stefano; Carotti, Marcello; Giaretta, Laura; Lanciai, Federico; Nogara, Leonardo; Bubacco, Luigi; Bergantino, Elisabetta

2015-01-01

Human tyrosinase is the first enzyme of the multistep process of melanogenesis. It catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine and the following oxidation of o-diphenol to the corresponding quinone, L-dopaquinone. In spite of its biomedical relevance, its reactivity is far from being fully understood, mostly because of the lack of a suitable expression system. Indeed, until now, studies on substrates and inhibitors of tyrosinases have been performed in vitro almost exclusively using mushroom or bacterial enzymes. We report on the production of a recombinant human tyrosinase in insect cells (Sf9 line). Engineering the protein, improving cell culture conditions, and setting a suitable purification protocol optimized product yield. The obtained active enzyme was truthfully characterized with a number of substrate and inhibitor molecules. These results were compared to those gained from a parallel analysis of the bacterial (Streptomyces antibioticus) enzyme and those acquired from the literature for mushroom tyrosinase, showing that the reactivity of the human enzyme appears unique and pointing out the great bias introduced when using non-human tyrosinases to measure the inhibitory efficacy of new molecules. The described enzyme is therefore an indispensable paradigm in testing pharmaceutical or cosmetic agents addressing tyrosinase activity.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brandao, T.; Robinson, H; Johnson, S

Catalysis by the Yersinia protein-tyrosine phosphatase YopH is significantly impaired by the mutation of the conserved Trp354 residue to Phe. Though not a catalytic residue, this Trp is a hinge residue in a conserved flexible loop (the WPD-loop) that must close during catalysis. To learn why this seemingly conservative mutation reduces catalysis by 2 orders of magnitude, we have solved high-resolution crystal structures for the W354F YopH in the absence and in the presence of tungstate and vanadate. Oxyanion binding to the P-loop in W354F is analogous to that observed in the native enzyme. However, the WPD-loop in the presencemore » of oxyanions assumes a half-closed conformation, in contrast to the fully closed state observed in structures of the native enzyme. This observation provides an explanation for the impaired general acid catalysis observed in kinetic experiments with Trp mutants. A 1.4 Angstroms structure of the W354F mutant obtained in the presence of vanadate reveals an unusual divanadate species with a cyclic [VO]2 core, which has precedent in small molecules but has not been previously reported in a protein crystal structure.« less

Critical Roles of Two Hydrophobic Residues within Human Glucose Transporter 9 (hSLC2A9) in Substrate Selectivity and Urate Transport*

PubMed Central

Long, Wentong; Panwar, Pankaj; Witkowska, Kate; Wong, Kenneth; O'Neill, Debbie; Chen, Xing-Zhen; Lemieux, M. Joanne; Cheeseman, Chris I.

2015-01-01

High blood urate levels (hyperuricemia) have been found to be a significant risk factor for cardiovascular diseases and inflammatory arthritis, such as hypertension and gout. Human glucose transporter 9 (hSLC2A9) is an essential protein that mainly regulates urate/hexose homeostasis in human kidney and liver. hSLC2A9 is a high affinity-low capacity hexose transporter and a high capacity urate transporter. Our previous studies identified a single hydrophobic residue in trans-membrane domain 7 of class II glucose transporters as a determinant of fructose transport. A mutation of isoleucine 335 to valine (I355V) in hSLC2A9 can reduce fructose transport while not affecting glucose fluxes. This current study demonstrates that the I335V mutant transports urate similarly to the wild type hSLC2A9; however, Ile-335 is necessary for urate/fructose trans-acceleration exchange to occur. Furthermore, Trp-110 is a critical site for urate transport. Two structural models of the class II glucose transporters, hSLC2A9 and hSLC2A5, based on the crystal structure of hSLC2A1 (GLUT1), reveal that Ile-335 (or the homologous Ile-296 in hSLC2A5) is a key component for protein conformational changes when the protein translocates substrates. The hSLC2A9 model also predicted that Trp-110 is a crucial site that could directly interact with urate during transport. Together, these studies confirm that hSLC2A9 transports both urate and fructose, but it interacts with them in different ways. Therefore, this study advances our understanding of how hSLC2A9 mediates urate and fructose transport, providing further information for developing pharmacological agents to treat hyperuricemia and related diseases, such as gout, hypertension, and diabetes. PMID:25922070

Activation mechanism of melB tyrosinase from Aspergillus oryzae by acidic treatment.

PubMed

Fujieda, Nobutaka; Murata, Michiaki; Yabuta, Shintaro; Ikeda, Takuya; Shimokawa, Chizu; Nakamura, Yukihiro; Hata, Yoji; Itoh, Shinobu

2013-01-01

The pro form of recombinant tyrosinase from Aspergillus oryzae (melB) shows no catalytic activity, but acid treatment (around pH 3.5) of protyrosinase activates it to induce tyrosinase activity. Circular dichroism spectra, gel filtration analysis, and colorimetric assay have indicated that acid treatment around pH 3.5 induced the disruption of the conformation of the C-terminal domain covering the enzyme active site. These structural changes induced by the acid treatment may open the entrance to the enzyme active site for substrate incorporation. To compare the mechanism of hydroxylation by the acid-treated tyrosinase with that by trypsin-treated tyrosinase, a detailed steady-state kinetic analysis of the phenolase activity was performed by monitoring the O(2)-consumption rate using a Clark-type oxygen electrode. The results clearly show that the phenolase activity (phenol hydroxylation) of the activated tyrosinase involves an electrophilic aromatic substitution mechanism as in the case of mushroom tyrosinase (Yamazaki and Itoh in J. Am. Chem. Soc. 125:13034-13035, 2003) and activated hemocyanin with urea (Morioka et al. in J. Am. Chem. Soc. 128:6788-6789, 2006).

High l-Trp affinity of indoleamine 2,3-dioxygenase 1 is attributed to two residues located in the distal heme pocket.

PubMed

Yuasa, Hajime J

2016-10-01

Indoleamine 2, 3-dioxygenase (IDO) catalyzes the oxidative cleavage of the pyrrole ring of l-Trp to generate N-formyl-kynurenine. Two IDO genes, IDO1 and IDO2, are found in vertebrates. Mammalian IDO1s are high-affinity, l-Trp-degrading enzymes, whereas IDO2s generally have a relatively low affinity. It has been suggested that the distal-Ser (corresponding to Ser167 of human IDO1) was crucial for improvement in the affinity for l-Trp but this idea was insufficient to explain the high affinity shown by mammalian IDO1. In this study, the amino acid sequences of vertebrate ancestral IDO1 and ancestral IDO2 were inferred, and bacterially expressed ancestral IDOs were characterized. Although the amino acid sequences of the enzymes shared high identity (86%) with each other, they showed distinct enzymatic properties. In analyses of a series of ancestral IDO1/IDO2 chimeric enzymes and their variants, the distal-Tyr (corresponding to Tyr126 of human IDO1) was detected as another and was probably the most crucial residue for high l-Trp affinity. The two amino acid substitutions (distal-Ser to Thr and distal-Tyr to His) drastically decreased the l-Trp affinity and catalytic efficiency of IDO1s. Conversely, two substitutions (distal-Thr to Ser and distal-His to Tyr) were sufficient to bestow IDO1-like high affinity on ancestral and chicken IDO2. © 2016 Federation of European Biochemical Societies.

Structural insight into the active site of mushroom tyrosinase using phenylbenzoic acid derivatives.

PubMed

Oyama, Takahiro; Yoshimori, Atsushi; Takahashi, Satoshi; Yamamoto, Tetsuya; Sato, Akira; Kamiya, Takanori; Abe, Hideaki; Abe, Takehiko; Tanuma, Sei-Ichi

2017-07-01

So far, many inhibitors of tyrosinase have been discovered for cosmetic and clinical agents. However, the molecular mechanisms underlying the inhibition in the active site of tyrosinase have not been well understood. To explore this problem, we examined here the inhibitory effects of 4'-hydroxylation and methoxylation of phenylbenzoic acid (PBA) isomers, which have a unique scaffold to inhibit mushroom tyrosinase. The inhibitory effect of 3-PBA, which has the most potent inhibitory activity among the isomers, was slightly decreased by 4'-hydroxylation and further decreased by 4'-methoxylation against mushroom tyrosinase. Surprisingly, 4'-hydroxylation but not methoxylation of 2-PBA appeared inhibitory activity. On the other hand, both 4'-hydroxylation and methoxylation of 4-PBA increased the inhibitory activity against mushroom tyrosinase. In silico docking analyses using the crystallographic structure of mushroom tyrosinase indicated that the carboxylic acid or 4'-hydroxyl group of PBA derivatives could chelate with cupric ions in the active site of mushroom tyrosinase, and that the interactions of Asn260 and Phe264 in the active site with the adequate-angled biphenyl group are involved in the inhibitory activities of the modified PBAs, by parallel and T-shaped π-π interactions, respectively. Furthermore, Arg268 could fix the angle of the aromatic ring of Phe264, and Val248 is supposed to interact with the inhibitors as a hydrophobic manner. These results may enhance the structural insight into mushroom tyrosinase for the creation of novel tyrosinase inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Kushenol A and 8-prenylkaempferol, tyrosinase inhibitors, derived from Sophora flavescens.

PubMed

Kim, Jang Hoon; Cho, In Sook; So, Yang Kang; Kim, Hyeong-Hwan; Kim, Young Ho

2018-12-01

Tyrosinase is known for an enzyme that plays a key role in producing the initial precursor of melanin biosynthesis. Inhibition of the catalytic reaction of this enzyme led to some advantage such as skin-whitening and anti-insect agents. To find a natural compound with inhibitory activity towards tyrosinase, the five flavonoids of kushenol A (1), 8-prenylkaempferol (2), kushenol C (3), formononetin (4) and 8-prenylnaringenin (5) were isolated by column chromatography from a 95% methanol extract of Sophora flavescens. The ability of these flavonoids to block the conversion of L-tyrosine to L-DOPA by tyrosinase was tested in vitro. Compounds 1 and 2 exhibited potent inhibitory activity, with IC50 values less than 10 µM. Furthermore, enzyme kinetics and molecular docking analysis revealed the formation of a binary encounter complex between compounds 1-4 and the enzyme. Also, all of the isolated compounds (1-5) were confirmed to possess antioxidant activity.

Chemometric profile, antioxidant and tyrosinase inhibitory activity of Camel's foot creeper leaves (Bauhinia vahlii).

PubMed

Panda, Pritipadma; Dash, Priyanka; Ghosh, Goutam

2018-03-01

The present study is the first effort to a comprehensive evaluation of antityrosinase activity and chemometric analysis of Bauhinia vahlii. The experimental results revealed that the methanol extract of Bauhinia vahlii (BVM) possesses higher polyphenolic compounds and total antioxidant activity than those reported elsewhere for other more conventionally and geographically different varieties. The BVM contain saturated fatty acids such as hexadecanoic acid (10.15%), octadecanoic acid (1.97%), oleic acid (0.61%) and cis-vaccenic acid (2.43%) along with vitamin E (12.71%), α-amyrin (9.84%), methyl salicylate (2.39%) and β-sitosterol (17.35%), which were mainly responsible for antioxidant as well as tyrosinase inhibitory activity. Tyrosinase inhibitory activity of this extract was comparable to that of Kojic acid. These findings suggested that the B. vahlii leaves could be exploited as potential source of natural antioxidant and tyrosinase inhibitory agent, as well.

Integration of Next Generation Sequencing and EPR Analysis to Uncover Molecular Mechanism Underlying Shell Color Variation in Scallops

PubMed Central

Sun, Xiujun; Liu, Zhihong; Zhou, Liqing; Wu, Biao; Dong, Yinghui; Yang, Aiguo

2016-01-01

The Yesso scallop Patinopecten yessoensis displays polymorphism in shell colors, which is of great interest for the scallop industry. To identify genes involved in the shell coloration, in the present study, we investigate the transcriptome differences by Illumina digital gene expression (DGE) analysis in two extreme color phenotypes, Red and White. Illumina sequencing yields a total of 62,715,364 clean sequence reads, and more than 85% reads are mapped into our previously sequenced transcriptome. There are 25 significantly differentially expressed genes between Red and White scallops. EPR (Electron paramagnetic resonance) analysis has identified EPR spectra of pheomelanin and eumelanin in the red shells, but not in the white shells. Compared to the Red scallops, the White scallops have relatively higher mRNA expression in tyrosinase genes, but lower expression in other melanogensis-associated genes. Meantime, the relatively lower tyrosinase protein and decreased tyrosinase activity in White scallops are suggested to be associated with the lack of melanin in the white shells. Our findings highlight the functional roles of melanogensis-associated genes in the melanization process of scallop shells, and shed new lights on the transcriptional and post-transcriptional mechanisms in the regulation of tyrosinase activity during the process of melanin synthesis. The present results will assist our molecular understanding of melanin synthesis underlying shell color polymorphism in scallops, as well as other bivalves, and also help the color-based breeding in shellfish aquaculture. PMID:27563719

RUTBC1 Functions as a GTPase-activating Protein for Rab32/38 and Regulates Melanogenic Enzyme Trafficking in Melanocytes*

PubMed Central

Marubashi, Soujiro; Shimada, Hikaru; Fukuda, Mitsunori; Ohbayashi, Norihiko

2016-01-01

Two cell type-specific Rab proteins, Rab32 and Rab38 (Rab32/38), have been proposed as regulating the trafficking of melanogenic enzymes, including tyrosinase and tyrosinase-related protein 1 (Tyrp1), to melanosomes in melanocytes. Like other GTPases, Rab32/38 function as switch molecules that cycle between a GDP-bound inactive form and a GTP-bound active form; the cycle is thought to be regulated by an activating enzyme, guanine nucleotide exchange factor (GEF), and an inactivating enzyme, GTPase-activating protein (GAP), which stimulates the GTPase activity of Rab32/38. Although BLOC-3 has already been identified as a Rab32/38-specific GEF that regulates the trafficking of tyrosinase and Tyrp1, no physiological GAP for Rab32/38 in melanocytes has ever been identified, and it has remained unclear whether Rab32/38 is involved in the trafficking of dopachrome tautomerase, another melanogenic enzyme, in mouse melanocytes. In this study we investigated RUTBC1, which was originally characterized as a Rab9-binding protein and GAP for Rab32 and Rab33B in vitro, and the results demonstrated that RUTBC1 functions as a physiological GAP for Rab32/38 in the trafficking of all three melanogenic enzymes in mouse melanocytes. The results of this study also demonstrated the involvement of Rab9A in the regulation of the RUTBC1 localization and in the trafficking of all three melanogenic enzymes. We discovered that either excess activation or inactivation of Rab32/38 achieved by manipulating RUTBC1 inhibits the trafficking of all three melanogenic enzymes. These results collectively indicate that proper spatiotemporal regulation of Rab32/38 is essential for the trafficking of all three melanogenic enzymes in mouse melanocytes. PMID:26620560

Biological assessment of neonicotinoids imidacloprid and its major metabolites for potentially human health using globular proteins as a model.

PubMed

Ding, Fei; Peng, Wei

2015-06-01

The assessment of biological activities of imidacloprid and its two major metabolites, namely 6-chloronicotinic acid and 2-imidazolidone for nontarget organism, by employing essentially functional biomacromolecules, albumin and hemoglobin as a potentially model with the use of circular dichroism (CD), fluorescence, extrinsic 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence as well as molecular modeling is the theme of this work. By dint of CD spectra and synchronous fluorescence, it was clear that the orderly weak interactions between amino acid residues within globular proteins were disturbed by imidacloprid, and this event led to marginally alterations or self-regulations of protein conformation so as to lodge imidacloprid more tightly. Both steady state and time-resolved fluorescence suggested that the fluorescence of Trp residues in proteins was quenched after the presence of imidacloprid, corresponding to noncovalent protein-imidacloprid complexes formation and, the reaction belongs to moderate association (K=1.888/1.614×10(4)M(-1) for albumin/hemoglobin-imidacloprid, respectively), hydrogen bonds and π stacking performed a vital role in stabilizing the complexes, as derived from thermodynamic analysis and molecular modeling. With the aid of hydrophobic ANS experiments, subdomain IIA and α1β2 interface of albumin and hemoglobin, respectively, were found to be preserved high-affinity for imidacloprid. These results ties in with the subsequently molecular modeling laying imidacloprid in the Sudlow's site I and close to Trp-213 residue on albumin, while settling down B/Trp-37 residue nearby in hemoglobin, and these conclusions further confirmed by site-directed mutagenesis and molecular dynamics simulation. But, at the same time, several crucial noncovalent bonds came from other amino acid residues, e.g. Arg-194 and Arg-198 (albumin) and B/Arg-40, B/Asp-99 and B/Asn-102 (hemoglobin) cannot be ignored completely. Based on the comparative studies of binding modes between imidacloprid and its two primary metabolites with globular proteins, it is evident to us that the noncovalent interactions of 6-chloronicotinic acid and 2-imidazolidone with biopolymers are not always to be decreased obviously as a result of the relatively small molecular structures of these metabolites, compared with parent compound imidacloprid. Conversely, this could probably strengthen the weak interactions existed in the macromolecules-metabolites conjugation, or rather, the metabolites such as 6-chloronicotinic acid and 2-imidazolidone contributed drastically to the overall toxicity of imidacloprid. Copyright © 2015 Elsevier B.V. All rights reserved.

Detection of Misdistribution of Tyrosinase from Melanosomes to Lysosomes and Its Upregulation under Psoralen/Ultraviolet A with a Melanosome-Targeting Tyrosinase Fluorescent Probe.

PubMed

Zhou, Jin; Shi, Wen; Li, Lihong; Gong, Qiuyu; Wu, Xiaofeng; Li, Xiaohua; Ma, Huimin

2016-04-19

Tyrosinase is regarded as an important biomarker of melanoma cancer, and its metabolism is closely related to some severe skin diseases such as vitiligo. Since tyrosinase is mainly located in the melanosomes of melanocytes, a probe that can specifically detect and image tysosinase in melanosomes would be in urgent demand to study the behavior of the enzyme in cells, but unfortunately, no melanosome-targeting tyrosinase fluorescent probe has been reported so far to the best of our knowledge. In this work, we have developed such a new probe, Mela-TYR, which bears morpholine as a melanosome-targeting group and 4-aminophenol as a tyrosinase reaction group. The probe exhibits not only a highly sensitive and selective off-on response to tyrosinase via oxidization cleavage, but also an accurate targeting ability toward the acidic organelles of melanosomes and lyososomes, which is validated by colocalization experiments with mCherry-tagged melanosomes as well as DND-99 (a commercial dye). The probe has been used to image the relative contents of tyrosinase in different cells. Notably, because of the tyrosinase deficiency in normal lysosomes, the probe only fluoresces in melanosomes in principle although it can accumulate in other acidic organelles like lysosomes. By virtue of this property, the misdistribution of tyrosinase from melanosomes to lysosomes in murine melanoma B16 cells under the stimulation of inulavosin is imaged in real time for the first time. Moreover, the upregulation of melanosomal tyrosinase in live B16 cells under the stimulation of psoralen/ultraviolet A is detected with our probe, and this upregulation is further verified by standard colorimetric assay. The probe provides a simple, visual method to study the metabolism of tyrosinase in cells and shows great potential in clinical diagnosis and treatments of tyrosinase-associated diseases.

Coumestrol Down-Regulates Melanin Production in Melan-a Murine Melanocytes through Degradation of Tyrosinase.

PubMed

Hwang, Jeong Ah; Park, Nok Hyun; Na, Yong Joo; Lee, Hae Kwang; Lee, John Hwan; Kim, Yong Jin; Lee, Chang Seok

2017-01-01

Pigmentation reflects skin darkening caused by melanin production, but excessive melanin synthesis may cause problems, such as melasma, solar lentigo, dark spots, and freckles. Considerable effort has been devoted to alleviating these undesired symptoms through the development of safe and effective depigmenting agents. Coumestrol, a plant-derived natural isoflavone with an estrogen-like structure and actions, is known to have anti-aging ability, but its potential depigmenting efficacy has not been evaluated. In the present study, we investigated the effects of coumestrol on melanin synthesis in normal melan-a murine melanocytes. Coumestrol significantly reduced melanin synthesis in a concentration-dependent manner up to a concentration of 25 µM without causing cytotoxicity. It also brightened tissue in an artificial skin model (MelanoDerm) that incorporates both human keratinocytes and melanocytes. Interestingly, although coumestrol did not inhibit tyrosinase activity or transcript level in melan-a cells, it clearly decreased the expression level of tyrosinase protein at a concentration of 25 µM. This coumestrol-induced reduction in tyrosinase protein levels was prevented by pretreatment with the proteasome inhibitor MG-132 or the lysosomal proteolysis inhibitor chloroquine. Collectively, our findings indicate that coumestrol exerts an inhibitory effect on melanin synthesis in melan-a cells, at least in part, through degradation of tyrosinase. These findings suggest that coumestrol is a good candidate for use in depigmentary reagents from a cosmetic and clinical perspective.

TRP channel–associated factors are a novel protein family that regulates TRPM8 trafficking and activity

PubMed Central

Lemonnier, Loic; Shapovalov, George; Gordienko, Dmitri; Poux, Céline; Bernardini, Michela; Bokhobza, Alexandre; Bidaux, Gabriel; Degerny, Cindy; Verreman, Kathye; Guarmit, Basma; Benahmed, Mohamed; de Launoit, Yvan; Bindels, Rene J.M.; Pla, Alessandra Fiorio; Prevarskaya, Natalia

2015-01-01

TRPM8 is a cold sensor that is highly expressed in the prostate as well as in other non-temperature-sensing organs, and is regulated by downstream receptor–activated signaling pathways. However, little is known about the intracellular proteins necessary for channel function. Here, we identify two previously unknown proteins, which we have named “TRP channel–associated factors” (TCAFs), as new TRPM8 partner proteins, and we demonstrate that they are necessary for channel function. TCAF1 and TCAF2 both bind to the TRPM8 channel and promote its trafficking to the cell surface. However, they exert opposing effects on TRPM8 gating properties. Functional interaction of TCAF1/TRPM8 also leads to a reduction in both the speed and directionality of migration of prostate cancer cells, which is consistent with an observed loss of expression of TCAF1 in metastatic human specimens, whereas TCAF2 promotes migration. The identification of TCAFs introduces a novel mechanism for modulation of TRPM8 channel activity. PMID:25559186

Structure of the full-length TRPV2 channel by cryo-EM

NASA Astrophysics Data System (ADS)

Huynh, Kevin W.; Cohen, Matthew R.; Jiang, Jiansen; Samanta, Amrita; Lodowski, David T.; Zhou, Z. Hong; Moiseenkova-Bell, Vera Y.

2016-03-01

Transient receptor potential (TRP) proteins form a superfamily Ca2+-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a `minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2-6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ~5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels.

Structure of the full-length TRPV2 channel by cryo-EM

PubMed Central

Huynh, Kevin W.; Cohen, Matthew R.; Jiang, Jiansen; Samanta, Amrita; Lodowski, David T.; Zhou, Z. Hong; Moiseenkova-Bell, Vera Y.

2016-01-01

Transient receptor potential (TRP) proteins form a superfamily Ca2+-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a ‘minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2–6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ∼5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels. PMID:27021073

Structure of the full-length TRPV2 channel by cryo-EM.

PubMed

Huynh, Kevin W; Cohen, Matthew R; Jiang, Jiansen; Samanta, Amrita; Lodowski, David T; Zhou, Z Hong; Moiseenkova-Bell, Vera Y

2016-03-29

Transient receptor potential (TRP) proteins form a superfamily Ca(2+)-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a 'minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2-6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ∼5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels.

Identification of putative agouti-related protein(87-132)-melanocortin-4 receptor interactions by homology molecular modeling and validation using chimeric peptide ligands.

PubMed

Wilczynski, Andrzej; Wang, Xiang S; Joseph, Christine G; Xiang, Zhimin; Bauzo, Rayna M; Scott, Joseph W; Sorensen, Nicholas B; Shaw, Amanda M; Millard, William J; Richards, Nigel G; Haskell-Luevano, Carrie

2004-04-22

Agouti-related protein (AGRP) is one of only two naturally known antagonists of G-protein-coupled receptors (GPCRs) identified to date. Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis. Alpha-melanocyte stimulating hormone (alpha-MSH) is one of the known endogenous agonists for these melanocortin receptors. Insight into putative interactions between the antagonist AGRP amino acids with the melanocortin-4 receptor (MC4R) may be important for the design of unique ligands for the treatment of obesity related diseases and is currently lacking in the literature. A three-dimensional homology molecular model of the mouse MC4 receptor complex with the hAGRP(87-132) ligand docked into the receptor has been developed to identify putative antagonist ligand-receptor interactions. Key putative AGRP-MC4R interactions include the Arg111 of hAGRP(87-132) interacting in a negatively charged pocket located in a cavity formed by transmembrane spanning (TM) helices 1, 2, 3, and 7, capped by the acidic first extracellular loop (EL1) and specifically with the conserved melanocortin receptor residues mMC4R Glu92 (TM2), mMC4R Asp114 (TM3), and mMC4R Asp118 (TM3). Additionally, Phe112 and Phe113 of hAGRP(87-132) putatively interact with an aromatic hydrophobic pocket formed by the mMC4 receptor residues Phe176 (TM4), Phe193 (TM5), Phe253 (TM6), and Phe254 (TM6). To validate the AGRP-mMC4R model complex presented herein from a ligand perspective, we generated nine chimeric peptide ligands based on a modified antagonist template of the hAGRP(109-118) (Tyr-c[Asp-Arg-Phe-Phe-Asn-Ala-Phe-Dpr]-Tyr-NH(2)). In these chimeric ligands, the antagonist AGRP Arg-Phe-Phe residues were replaced by the melanocortin agonist His/D-Phe-Arg-Trp amino acids. These peptides resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3-5Rs). The most notable results include the identification of a novel subnanomolar melanocortin peptide template Tyr-c[Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) that is equipotent to alpha-MSH at the mMC1, mMC3, and mMC5 receptors but is 30-fold more potent than alpha-MSH at the mMC4R. Additionally, these studies identified a new and novel >200-fold MC4R versus MC3R selective peptide Tyr-c[Asp-D-Phe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) template. Furthermore, when the His-DPhe-Arg-Trp sequence is used to replace the hAGRP Arg-Phe-Phe residues in the "mini"-AGRP (hAGRP87-120, C105A) template, a potent nanomolar agonist resulted at the mMC1R and MC3-5Rs.

A fluorescence-based method for direct measurement of submicrosecond intramolecular contact formation in biopolymers: an exploratory study with polypeptides.

PubMed

Hudgins, Robert R; Huang, Fang; Gramlich, Gabriela; Nau, Werner M

2002-01-30

A fluorescent amino acid derivative (Fmoc-DBO) has been synthesized, which contains 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) as a small, hydrophilic fluorophore with an extremely long fluorescence lifetime (325 ns in H2O and 505 ns in D2O under air). Polypeptides containing both the DBO residue and an efficient fluorescence quencher allow the measurement of rate constants for intramolecular end-to-end contact formation. Bimolecular quenching experiments indicated that Trp, Cys, Met, and Tyr are efficient quenchers of DBO (k(q) = 20, 5.1, 4.5, and 3.6 x 10(8) M(-1) x s(-1) in D2O), while the other amino acids are inefficient. The quenching by Trp, which was selected as an intrinsic quencher, is presumed to involve exciplex-induced deactivation. Flexible, structureless polypeptides, Trp-(Gly-Ser)n-DBO-NH2, were prepared by standard solid-phase synthesis, and the rates of contact formation were measured through the intramolecular fluorescence quenching of DBO by Trp with time-correlated single-photon counting, laser flash photolysis, and steady-state fluorometry. Rate constants of 4.1, 6.8, 4.9, 3.1, 2.0, and 1.1 x 10(7) s(-1) for n = 0, 1, 2, 4, 6, and 10 were obtained. Noteworthy was the relatively slow quenching for the shortest peptide (n = 0). The kinetic data are in agreement with recent transient absorption studies of triplet probes for related peptides, but the rate constants are significantly larger. In contrast to the flexible structureless Gly-Ser polypeptides, the polyproline Trp-Pro4-DBO-NH2 showed insignificant fluorescence quenching, suggesting that a high polypeptide flexibility and the possibility of probe-quencher contact is essential to induce quenching. Advantages of the new fluorescence-based method for measuring contact formation rates in biopolymers include high accuracy, fast time range (100 ps-1 micros), and the possibility to perform measurements in water under air.

Reorientational Dynamics of Enzymes Adsorbed on Quartz: A Temperature-Dependent Time-Resolved TIRF Anisotropy Study

PubMed Central

Czeslik, C.; Royer, C.; Hazlett, T.; Mantulin, W.

2003-01-01

The preservation of enzyme activity and protein binding capacity upon protein adsorption at solid interfaces is important for biotechnological and medical applications. Because these properties are partly related to the protein flexibility and mobility, we have studied the internal dynamics and the whole-body reorientational rates of two enzymes, staphylococcal nuclease (SNase) and hen egg white lysozyme, over the temperature range of 20–80°C when the proteins are adsorbed at the silica/water interface and, for comparison, when they are dissolved in buffer. The data were obtained using a combination of two experimental techniques, total internal reflection fluorescence spectroscopy and time-resolved fluorescence anisotropy measurements in the frequency domain, with the protein Trp residues as intrinsic fluorescence probes. It has been found that the internal dynamics and the whole-body rotation of SNase and lysozyme are markedly reduced upon adsorption over large temperature ranges. At elevated temperatures, both protein molecules appear completely immobilized and the fractional amplitudes for the whole-body rotation, which are related to the order parameter for the local rotational freedom of the Trp residues, remain constant and do not approach zero. This behavior indicates that the angular range of the Trp reorientation within the adsorbed proteins is largely restricted even at high temperatures, in contrast to that of the dissolved proteins. The results of this study thus provide a deeper understanding of protein activity at solid surfaces. PMID:12668461

Immobilization of tyrosinase in carboxylic and carbonyl group-modified MWNT electrode and its application for sensing phenolics in red wines.

PubMed

Kim, Kyo-Il; Lee, Jae-Chan; Robards, Kevin; Choi, Seong-Ho

2010-06-01

Tyrosinase-immobilized biosensor was fabricated based on PAAc-g-MWNT and PMAn-g-MWNT, respectively. The poly(acrylic acid)-grafted multi-wall carbon nanotubes, PAAc-g-MWNT, and poly(maleic anhydride)-grafted multi-wall carbon nanotube, PMAn-g-MWNT, were prepared by radiation-induced graft polymerization of acrylic acid (AAc) and maleic anhydride (MAn) on the surface of MWNT. The biosensor was prepared on ITO glass electrode by coating of chitosan solution with tyrosinase-immobilized PAAc-g-MWNT and PMAn-g-MWNT, respectively. The sensing ranges of the tyrosinase-immobilized biosensor based on PAAc-g-MWNT and PMAn were in the range of 0.2-0.9 mM concentration and in the range of 0.1-0.5 mM for phenol in phosphate buffer solution, respectively. Optimal pH and temperature conditions for sensing various phenolic compounds with tyrosinase-immobilized biosensor were determined. Total phenolic content for three commercial red wines on tyrosinase-immobilized biosensor were also determined.

RACK1 promotes radiation resistance in esophageal cancer via regulating AKT pathway and Bcl-2 expression.

PubMed

Liu, Bowen; Wang, Cong; Chen, Pengxiang; Wang, Lu; Cheng, Yufeng

2017-09-23

RACK1 is a seven Trp-Asp 40 repeat protein, which interacts with a wide range of kinases and proteins. RACK1 plays an important role in the proliferation and progression of various cancers. The aim of this study is to detect the role of RACK1 in the radioresistance in esophageal cancer. The results indicated that downregulation of RACK1 reduced the colony formation ability, proliferation ability and resistance of cells to radiation effection through regulating the radiation-related proteins including pAKT, Bcl-2 and Bim; whereas upregulation of RACK1 promoted the ability and radioresistance of ESCC cells. Our findings suggest that RACK1 promotes proliferation and radioresistance in ESCC cells by activating the AKT pathway, upregulating Bcl-2 expression and downregulating protein levels of Bim. Our study fills in gaps in the field of RACK1 and radiation resistance and may provide new possibilities for improving strategies of radiotherapy in esophageal cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Involvement of phenoloxidase in browning during grinding of Tenebrio molitor larvae.

PubMed

Janssen, Renske H; Lakemond, Catriona M M; Fogliano, Vincenzo; Renzone, Giovanni; Scaloni, Andrea; Vincken, Jean-Paul

2017-01-01

Insects are investigated as alternative protein source to meet the increasing demand for proteins in the future. Enzymatic browning occurring during grinding of insect and subsequent extraction of proteins can influence the proteins' properties, but it is unclear which enzymes are responsible for this phenomenon. This study was performed on larvae of three commonly used insect species, namely Tenebrio molitor, Alphitobius diaperinus and Hermetia illucens. Oxygen consumption measurements on protein extracts showed activity on L-tyrosine, L-3,4-di-hydroxy-phenylalanine (L-DOPA) and L-dopamine, indicating phenoloxidase as a key player in browning. Furthermore, no reaction on 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) was observed, ruling out an important contribution of laccase to browning. The browning reaction was most prominent at pH 6 for T. molitor and A. diaperinus, and 7 for H. illucens. As the enzyme activity of H. illucens was the lowest with the darkest color formation, this was likely caused by another factor. The activity of phenoloxidase was confirmed for T. molitor and A. diaperinus by activity measurements after fractionation by anion-exchange chromatography. Color measurements showed the presence of activity on both L-DOPA and L-tyrosine in the same fractions. Both substrates were converted into dopachrome after incubation with enzyme-enriched fractions. No DOPA-decarboxylase, tyrosine hydroxylase and peroxidase activities were observed. By using native PAGE with L-DOPA as staining-solution, active T. molitor protein bands were resolved and characterized, identifying a tyrosinase/phenoloxidase as the active enzyme species. All together, these data confirmed that tyrosinase is an important enzyme in causing enzymatic browning in T. molitor and likely in A. diaperinus.

Decreased Anxiety-Like Behavior and Gαq/11-Dependent Responses in the Amygdala of Mice Lacking TRPC4 Channels

PubMed Central

Riccio, Antonio; Li, Yan; Tsvetkov, Evgeny; Gapon, Svetlana; Yao, Gui Lan; Smith, Kiersten S.; Engin, Elif; Rudolph, Uwe; Bolshakov, Vadim Y.

2014-01-01

Transient receptor potential (TRP) channels are abundant in the brain where they regulate transmission of sensory signals. The expression patterns of different TRPC subunits (TRPC1, 4, and 5) are consistent with their potential role in fear-related behaviors. Accordingly, we found recently that mutant mice lacking a specific TRP channel subunit, TRPC5, exhibited decreased innate fear responses. Both TRPC5 and another member of the same subfamily, TRPC4, form heteromeric complexes with the TRPC1 subunit (TRPC1/5 and TRPC1/4, respectively). As TRP channels with specific subunit compositions may have different functional properties, we hypothesized that fear-related behaviors could be differentially controlled by TRPCs with distinct subunit arrangements. In this study, we focused on the analysis of mutant mice lacking the TRPC4 subunit, which, as we confirmed in experiments on control mice, is expressed in brain areas implicated in the control of fear and anxiety. In behavioral experiments, we found that constitutive ablation of TRPC4 was associated with diminished anxiety levels (innate fear). Furthermore, knockdown of TRPC4 protein in the lateral amygdala via lentiviral-mediated gene delivery of RNAi mimicked the behavioral phenotype of constitutive TRPC4-null (TRPC4−/−) mouse. Recordings in brain slices demonstrated that these behavioral modifications could stem from the lack of TRPC4 potentiation in neurons in the lateral nucleus of the amygdala through two Gαq/11 protein-coupled signaling pathways, activated via Group I metabotropic glutamate receptors and cholecystokinin 2 receptors, respectively. Thus, TRPC4 and the structurally and functionally related subunit, TRPC5, may both contribute to the mechanisms underlying regulation of innate fear responses. PMID:24599464

Screening of tyrosinase inhibitors by capillary electrophoresis with immobilized enzyme microreactor and molecular docking.

PubMed

Cheng, Mengxia; Chen, Zilin

2017-02-01

A new method for screening tyrosinase inhibitors from traditional Chinese medicines (TCMs) was successfully developed by capillary electrophoresis with reliable online immobilized enzyme microreactor (IMER). In addition, molecular docking study has been used for supporting inhibition interaction between enzyme and inhibitors. The IMER of tyrosinase was constructed at the outlet of the capillary by using glutaraldehyde as cross-linker. The parameters including enzyme reaction, separation of the substrate and product, and the performance of immobilized tyrosinase were investigated systematically. Because of using short-end injection procedure, the product and substrate were effectively separated within 2 min. The immobilized tyrosinase could remain 80% active for 30 days at 4°C. The Michaelis-Menten constant of tyrosinase was determined as 1.78 mM. Kojic acid, a known tyrosinase inhibitor, was used as a model compound for the validation of the inhibitors screening method. The half-maximal inhibitory concentration of kojic acid was 5.55 μM. The method was successfully applied for screening tyrosinase inhibitors from 15 compounds of TCM. Four compounds including quercetin, kaempferol, bavachinin, and bakuchiol were found having inhibitory potentials. The results obtained in this work were supported by molecular docking study. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Spectroscopic and theoretical investigation of conformational changes of proteins by synthesized pyrimidine derivative and its sensitivity towards FRET application

NASA Astrophysics Data System (ADS)

Ghosh, Swadesh; Singharoy, Dipti; Bhattacharya, Subhash Chandra

2018-04-01

Interest in synthesizing and characterizing (IR, NMR and HRMS spectroscopic methods) a pyrimidine based Schiff-base ligand, 2-(2-(Anthracen-9-ylmethylene) hydrazinyl)-4,6-dimethyl pyrimidine (ANHP) has been developed for its application to ascertain the conformational change of protein and sensitivity towards fluorescence resonance energy transfer (FRET) process. Location of ANHP in bovine serum albumin (BSA) and human serum albumin (HSA) proteins environment has been determined using different spectroscopic techniques. Weakly fluorescent ANHP have shown greater protein induced fluorescence enhancement (PIFE) in case of HSA than BSA, though in both cases energy transfer efficiency are almost same but difference in binding constant values encourages us to find the location of ANHP within the complex protein environment. From the FRET parameter and α-helicity change, it has been found that ANHP bound with Trp-214 of HSA and surface Trp-134 of BSA. Conformational changes of proteins have been observed more for HSA than BSA in presence of ANHP, which has confirmed the location of ANHP in both the protein environments. Coupled with experimental studies, molecular docking analysis has also been done to explain the locations and distance dependent FRET process of ANHP in both proteins.

An evaluation of extracts of five traditional medicinal plants from Iran on the inhibition of mushroom tyrosinase activity and scavenging of free radicals.

PubMed

Khazaeli, P; Goldoozian, R; Sharififar, F

2009-10-01

This study aimed to evaluate the free radical scavenging and inhibition properties of five medicinal plants, including Quercus infectoria Olive., Terminalia chebula Retz., Lavendula stoechas L., Mentha longifolia L., Rheum palmatum L., toward the activity of mushroom tyrosinase using L-tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA) as the substrate.The methanol extracts of Q. infectoria and T. chebula showed strong radical scavenging effect in 2,2'-dipheny L-1-picrylhydrazyl (DPPH) assay(IC50 = 15.3 and 82.2 microg mL)1 respectively).These plants also showed inhibitory effects against the activity of mushroom tyrosinase in hydroxylation of L-tyrosine (85.9% and 82.2% inhibition,respectively). These two plants also inhibited the oxidation of l-DOPA similar to kojic acid as positive control (IC50 = 102.8 and 192.6 microg mL)1 respectively). In general Q. infectoria and T. chebula significantly inhibited tyrosinase activity and DPPH radical. Both activities were concentration dependant but not in linear manner. It is needed to study the cytotoxicity of these plant extracts in pigment cell culture before further evaluation and moving to in vivo conditions.

Aspergillus niger PA2: a novel strain for extracellular biotransformation of L-tyrosine into L-DOPA.

PubMed

Agarwal, Pragati; Pareek, Nidhi; Dubey, Swati; Singh, Jyoti; Singh, R P

2016-05-01

L-DOPA (3,4-dihydroxyphenyl-L-alanine), an amino acid derivative is the most widely used drug of choice for the treatment of Parkinson's disease and other neurologic injuries. The present study deals with the elevated biochemical transformation of L-tyrosine to L-DOPA by Aspergillus niger PA2, a potent tyrosinase producer, isolated from decomposed food wastes. This appears to be the first report on A. niger as a notable extracellular tyrosinase producer. The extracellular tyrosinase activity produced remarkably higher levels of L-DOPA, i.e. 2.44 mg mL(-1) when the media was supplemented with 5 mg mL(-1) L-tyrosine. The optimum pH for tyrosinase production was 6.0, with the maximal L-DOPA production at the same pH. The product thus produced was analyzed by thin-layer chromatography, UV spectroscopy, high-performance liquid chromatography and Fourier transform infrared spectroscopy, that had denoted this to be L-DOPA. Kinetic parameters viz. Y p/s, Q s and Q p had further indicated the notable levels of production. Thus, Aspergillus niger PA2 could be a promising resource and may be further exploited for large-scale production of L-DOPA.

Thermally activated TRP channels: molecular sensors for temperature detection.

PubMed

Castillo, Karen; Diaz-Franulic, Ignacio; Canan, Jonathan; Gonzalez-Nilo, Fernando; Latorre, Ramon

2018-01-24

Temperature sensing is one of the oldest capabilities of living organisms, and is essential for sustaining life, because failure to avoid extreme noxious temperatures can result in tissue damage or death. A subset of members of the transient receptor potential (TRP) ion channel family is finely tuned to detect temperatures ranging from extreme cold to noxious heat, giving rise to thermoTRP channels. Structural and functional experiments have shown that thermoTRP channels are allosteric proteins, containing different domains that sense changes in temperature, among other stimuli, triggering pore opening. Although temperature-dependence is well characterized in thermoTRP channels, the molecular nature of temperature-sensing elements remains unknown. Importantly, thermoTRP channels are involved in pain sensation, related to pathological conditions. Here, we provide an overview of thermoTRP channel activation. We also discuss the structural and functional evidence supporting the existence of an intrinsic temperature sensor in this class of channels, and we explore the basic thermodynamic principles for channel activation. Finally, we give a view of their role in painful pathophysiological conditions.

Cinnamomum cassia Essential Oil Inhibits α-MSH-Induced Melanin Production and Oxidative Stress in Murine B16 Melanoma Cells

PubMed Central

Chou, Su-Tze; Chang, Wen-Lun; Chang, Chen-Tien; Hsu, Shih-Lan; Lin, Yu-Che; Shih, Ying

2013-01-01

Essential oils extracted from aromatic plants exhibit important biological activities and have become increasingly important for the development of aromatherapy for complementary and alternative medicine. The essential oil extracted from Cinnamomum cassia Presl (CC-EO) has various functional properties; however, little information is available regarding its anti-tyrosinase and anti-melanogenic activities. In this study, 16 compounds in the CC-EO have been identified; the major components of this oil are cis-2-methoxycinnamic acid (43.06%) and cinnamaldehyde (42.37%). CC-EO and cinnamaldehyde exhibited anti-tyrosinase activities; however, cis-2-methoxycinnamic acid did not demonstrate tyrosinase inhibitory activity. In murine B16 melanoma cells stimulated with α-melanocyte-stimulating hormone (α-MSH), CC-EO and cinnamaldehyde not only reduced the melanin content and tyrosinase activity of the cells but also down-regulated tyrosinase expression without exhibiting cytotoxicity. Moreover, CC-EO and cinnamaldehyde decreased thiobarbituric acid-reactive substance (TBARS) levels and restored glutathione (GSH) and catalase activity in the α-MSH-stimulated B16 cells. These results demonstrate that CC-EO and its major component, cinnamaldehyde, possess potent anti-tyrosinase and anti-melanogenic activities that are coupled with antioxidant properties. Therefore, CC-EO may be a good source of skin-whitening agents and may have potential as an antioxidant in the future development of complementary and alternative medicine-based aromatherapy. PMID:24051402

A Lys-Trp cation-π interaction mediates the dimerization and function of the chloride intracellular channel protein 1 transmembrane domain.

PubMed

Peter, Bradley; Polyansky, Anton A; Fanucchi, Sylvia; Dirr, Heini W

2014-01-14

Chloride intracellular channel protein 1 (CLIC1) is a dual-state protein that can exist either as a soluble monomer or in an integral membrane form. The oligomerization of the transmembrane domain (TMD) remains speculative despite it being implicated in pore formation. The extent to which electrostatic and van der Waals interactions drive folding and association of the dimorphic TMD is unknown and is complicated by the requirement of interactions favorable in both aqueous and membrane environments. Here we report a putative Lys37-Trp35 cation-π interaction and show that it stabilizes the dimeric form of the CLIC1 TMD in membranes. A synthetic 30-mer peptide comprising a K37M TMD mutant was examined in 2,2,2-trifluoroethanol, sodium dodecyl sulfate micelles, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes using far-ultraviolet (UV) circular dichroism, fluorescence, and UV absorbance spectroscopy. Our data suggest that Lys37 is not implicated in the folding, stability, or membrane insertion of the TMD peptide. However, removal of this residue impairs the formation of dimers and higher-order oligomers. This is accompanied by a 30-fold loss of chloride influx activity, suggesting that dimerization modulates the rate of chloride conductance. We propose that, within membranes, individual TMD helices associate via a Lys37-mediated cation-π interaction to form active dimers. The latter findings are also supported by results of modeling a putative TMD dimer conformation in which Lys37 and Trp35 form cation-π pairs at the dimer interface. Dimeric helix bundles may then associate to form fully active ion channels. Thus, within a membrane-like environment, aromatic interactions involving a polar lysine side chain provide a thermodynamic driving force for helix-helix association.

Control of Transmembrane Helix Dynamics by Interfacial Tryptophan Residues.

PubMed

McKay, Matthew J; Martfeld, Ashley N; De Angelis, Anna A; Opella, Stanley J; Greathouse, Denise V; Koeppe, Roger E

2018-06-05

Transmembrane protein domains often contain interfacial aromatic residues, which may play a role in the insertion and stability of membrane helices. Residues such as Trp or Tyr, therefore, are often found situated at the lipid-water interface. We have examined the extent to which the precise radial locations of interfacial Trp residues may influence peptide helix orientation and dynamics. To address these questions, we have modified the GW 5,19 ALP23 (acetyl-GGALW 5 (LA) 6 LW 19 LAGA-[ethanol]amide) model peptide framework to relocate the Trp residues. Peptide orientation and dynamics were analyzed by means of solid-state nuclear magnetic resonance (NMR) spectroscopy to monitor specific 2 H- and 15 N-labeled residues. GW 5,19 ALP23 adopts a defined, tilted orientation within lipid bilayer membranes with minimal evidence of motional averaging of NMR observables, such as 2 H quadrupolar or 15 N- 1 H dipolar splittings. Here, we examine how peptide dynamics are impacted by relocating the interfacial Trp (W) residues on both ends and opposing faces of the helix, for example by a 100° rotation on the helical wheel for positions 4 and 20. In contrast to GW 5,19 ALP23, the modified GW 4,20 ALP23 helix experiences more extensive motional averaging of the NMR observables in several lipid bilayers of different thickness. Individual and combined Gaussian analyses of the 2 H and 15 N NMR signals confirm that the extent of dynamic averaging, particularly rotational "slippage" about the helix axis, is strongly coupled to the radial distribution of the interfacial Trp residues as well as the bilayer thickness. Additional 2 H labels on alanines A3 and A21 reveal partial fraying of the helix ends. Even within the context of partial unwinding, the locations of particular Trp residues around the helix axis are prominent factors for determining transmembrane helix orientation and dynamics within the lipid membrane environment. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Tyrosinase autoactivation and the problem of the lag period.

PubMed

Naish-Byfield, S; Riley, P A

1998-06-01

Evidence is presented for the binding of the quinone oxidation product of the monohydric phenol substrate, 4-hydroxyanisole, to mushroom tyrosinase. Column chromatography and SDS-PAGE separation showed labelling of the enzyme when incubated with 14C ring-labelled 4-hydroxyanisole. It is proposed that covalent binding to the enzyme and other proteins is through reaction of accessible nucleophilic groups, including thiols and amino groups, with the anisylquinone. This reductive addition enables the indirect generation of the catecholic substrate, which acts as an electron donor for the bicupric active site of met-tyrosinase and explains the lag kinetics of tyrosinase oxidation of non-cyclizing substrates. The effects of diluting the enzyme or the addition of amino acids on the lag period was consistent with a mechanism involving indirect generation of the dihydric phenol, which acts as the met-enzyme-recruiting substrate.

Crystal structure of Agaricus bisporus mushroom tyrosinase: identity of the tetramer subunits and interaction with tropolone.

PubMed

Ismaya, Wangsa T; Rozeboom, Henriëtte J; Weijn, Amrah; Mes, Jurriaan J; Fusetti, Fabrizia; Wichers, Harry J; Dijkstra, Bauke W

2011-06-21

Tyrosinase catalyzes the conversion of phenolic compounds into their quinone derivatives, which are precursors for the formation of melanin, a ubiquitous pigment in living organisms. Because of its importance for browning reactions in the food industry, the tyrosinase from the mushroom Agaricus bisporus has been investigated in depth. In previous studies the tyrosinase enzyme complex was shown to be a H(2)L(2) tetramer, but no clues were obtained of the identities of the subunits, their mode of association, and the 3D structure of the complex. Here we unravel this tetramer at the molecular level. Its 2.3 Å resolution crystal structure is the first structure of the full fungal tyrosinase complex. The complex comprises two H subunits of ∼392 residues and two L subunits of ∼150 residues. The H subunit originates from the ppo3 gene and has a fold similar to other tyrosinases, but it is ∼100 residues larger. The L subunit appeared to be the product of orf239342 and has a lectin-like fold. The H subunit contains a binuclear copper-binding site in the deoxy-state, in which three histidine residues coordinate each copper ion. The side chains of these histidines have their orientation fixed by hydrogen bonds or, in the case of His85, by a thioether bridge with the side chain of Cys83. The specific tyrosinase inhibitor tropolone forms a pre-Michaelis complex with the enzyme. It binds near the binuclear copper site without directly coordinating the copper ions. The function of the ORF239342 subunits is not known. Carbohydrate binding sites identified in other lectins are not conserved in ORF239342, and the subunits are over 25 Å away from the active site, making a role in activity unlikely. The structures explain how calcium ions stabilize the tetrameric state of the enzyme.

Structure-activity relationships of the melanocortin tetrapeptide Ac-His-DPhe-Arg-Trp-NH2 at the mouse melanocortin receptors. Part 3: modifications at the Arg position.

PubMed

Holder, Jerry Ryan; Xiang, Zhimin; Bauzo, Rayna M; Haskell-Luevano, Carrie

2003-01-01

The melanocortin pathway is involved in the regulation of several physiological functions including skin pigmentation, steroidogenesis, obesity, energy homeostasis, and exocrine gland function. This melanocortin pathway consists of five known G-protein coupled receptors, endogenous agonists derived from the proopiomelanocortin (POMC) gene transcript, the endogenous antagonists Agouti and the Agouti-related protein (AGRP) and signals through the intracellular cAMP signal transduction pathway. The melanocortin-3 receptor (MC3R) and melanocortin-4 receptor (MC4R) located in the brain are implicated as participating in the metabolic and food intake aspects of energy homeostasis and are stimulated by melanocortin agonists such as alpha-melanocyte stimulation hormone (alpha-MSH). All the endogenous (POMC-derived) melanocortin agonists contain the putative message sequence "His-Phe-Arg-Trp." Herein, we report 12 tetrapeptides, based upon the template Ac-His(6)-DPhe(7)-Arg(8)-Trp(9)-NH(2) (alpha-MSH numbering) that have been modified at the Arg(8) position by neutral, basic, or acidic amino acid side chains. These peptides have been pharmacologically characterized for agonist activity at the mouse melanocortin receptors MC1R, MC3R, MC4R, and MC5R. The most notable results of this study include the observation that removal of the guanidinyl side chain moiety results in decreased melanocortin receptor potency, but that this Arg(8) side chain is not critical for melanocortin receptor agonist activity. Additionally, incorporation of the homoArg(8) residue results in 56-fold MC4R versus MC3R selectivity, and the Orn(8) residue results in 123-fold MC4R versus MC5R and 63-fold MC5R versus MC3R selectivity. Copyright 2002 Elsevier Science Inc.

Virus-cell fusion inhibitory activity of novel analogue peptides based on the HP (2-20) derived from N-terminus of Helicobacter pylori Ribosomal Protein L1.

PubMed

Woo, Eun-Rhan; Lee, Dong Gun; Chang, Young-Su; Park, Yoonkyung; Hahm, Kyung-Soo

2002-12-01

HP (2-20) (AKKVFKRLEKLFSKIQNDK) is the antibacterial sequence derived from N-terminus of Helicobacter pylori Ribosomal Protein L1 (RPL1). It has a broad-spectrum microbicidal activity in vitro that is thought to be related to the membrane-disruptive properties of the peptide. Based on the putative membrane-targeted mode of action, we postulated that HP (2-20) might be possessed virus-cell fusion inhibitory activity. To develop the novel virus-cell fusion inhibitory peptides, several analogues with amino acid substitution were designed to increase or decrease only net hydrophobic region. In particular, substitution of Gln and Asp for hydrophobic amino acid, Trp at position 17 and 19 of HP (2-20) (Anal 3) caused a dramatic increase in virus-cell fusion inhibitory activity without hemolytic effect.

Identification of three signaling molecules required for calcineurin-dependent monopolar growth induced by the DNA replication checkpoint in fission yeast.

PubMed

Kume, Kazunori; Hashimoto, Tomoyo; Suzuki, Masashi; Mizunuma, Masaki; Toda, Takashi; Hirata, Dai

2017-09-30

Cell polarity is coordinately regulated with the cell cycle. Growth polarity of the fission yeast Schizosaccharomyces pombe transits from monopolar to bipolar during G2 phase, termed NETO (new end take off). Upon perturbation of DNA replication, the checkpoint kinase Cds1/CHK2 induces NETO delay through activation of Ca 2+ /calmodulin-dependent protein phosphatase calcineurin (CN). CN in turn regulates its downstream targets including the microtubule (MT) plus-end tracking CLIP170 homologue Tip1 and the Casein kinase 1γ Cki3. However, whether and which Ca 2+ signaling molecules are involved in the NETO delay remains elusive. Here we show that 3 genes (trp1322, vcx1 and SPAC6c3.06c encoding TRP channel, antiporter and P-type ATPase, respectively) play vital roles in the NETO delay. Upon perturbation of DNA replication, these 3 genes are required for not only the NETO delay but also for the maintenance of cell viability. Trp1322 and Vcx1 act downstream of Cds1 and upstream of CN for the NETO delay, whereas SPAC6c3.06c acts downstream of CN. Consistently, Trp1322 and Vcx1, but not SPAC6c3.06c, are essential for activation of CN. Interestingly, we have found that elevated extracellular Ca 2+ per se induces a NETO delay, which depends on CN and its downstream target genes. These findings imply that Ca 2+ -CN signaling plays a central role in cell polarity control by checkpoint activation. Copyright © 2017 Elsevier Inc. All rights reserved.

Coarse kMC-based replica exchange algorithms for the accelerated simulation of protein folding in explicit solvent.

PubMed

Peter, Emanuel K; Shea, Joan-Emma; Pivkin, Igor V

2016-05-14

In this paper, we present a coarse replica exchange molecular dynamics (REMD) approach, based on kinetic Monte Carlo (kMC). The new development significantly can reduce the amount of replicas and the computational cost needed to enhance sampling in protein simulations. We introduce 2 different methods which primarily differ in the exchange scheme between the parallel ensembles. We apply this approach on folding of 2 different β-stranded peptides: the C-terminal β-hairpin fragment of GB1 and TrpZip4. Additionally, we use the new simulation technique to study the folding of TrpCage, a small fast folding α-helical peptide. Subsequently, we apply the new methodology on conformation changes in signaling of the light-oxygen voltage (LOV) sensitive domain from Avena sativa (AsLOV2). Our results agree well with data reported in the literature. In simulations of dialanine, we compare the statistical sampling of the 2 techniques with conventional REMD and analyze their performance. The new techniques can reduce the computational cost of REMD significantly and can be used in enhanced sampling simulations of biomolecules.

Synthesis of chiral pyrazolo[4,3-e][1,2,4]triazine sulfonamides with tyrosinase and urease inhibitory activity.

PubMed

Mojzych, Mariusz; Tarasiuk, Paweł; Kotwica-Mojzych, Katarzyna; Rafiq, Muhammad; Seo, Sung-Yum; Nicewicz, Michał; Fornal, Emilia

2017-12-01

A new series of sulfonamide derivatives of pyrazolo[4,3-e][1,2,4]triazine with chiral amino group has been synthesized and characterized. The compounds were tested for their tyrosinase and urease inhibitory activity. Evaluation of prepared derivatives demonstrated that compounds (8b) and (8j) are most potent mushroom tyrosinase inhibitors whereas all of the obtained compounds showed higher urease inhibitory activity than the standard thiourea. The compounds (8a), (8f) and (8i) exhibited excellent enzyme inhibitory activity with IC 50 0.037, 0.044 and 0.042 μM, respectively, while IC 50 of thiourea is 20.9 μM.

Effects of intraduodenal infusion of L-tryptophan on ad libitum eating, antropyloroduodenal motility, glycemia, insulinemia, and gut peptide secretion in healthy men.

PubMed

Steinert, Robert E; Luscombe-Marsh, Natalie D; Little, Tanya J; Standfield, Scott; Otto, Bärbel; Horowitz, Michael; Feinle-Bisset, Christine

2014-09-01

Changes in gut motor and hormonal function contribute to the eating-inhibitory and glucose-lowering effects of protein. The effect of amino acids, the digestive products of protein, on gastrointestinal function, eating, and glycemia has not been investigated comprehensively. We tested the hypothesis that L-tryptophan (L-Trp) stimulates gastrointestinal motor and hormonal functions, inhibits eating, and modulates glycemia. Design, Settings, Participants, and Intervention: Ten healthy, normal-weight men were studied in randomized, double-blind fashion, each receiving a 90-minute intraduodenal infusion of L-Trp at 0.075 (total 6.75 kcal) or 0.15 (total 13.5 kcal) kcal/min or saline (control). Antropyloroduodenal motility, plasma ghrelin, cholecystokinin, glucagon-like peptide-1, peptide tyrosine tyrosine, insulin, glucagon, blood glucose, and appetite perceptions were measured. Food intake was quantified from a buffet meal after the infusion. Intraduodenal L-Trp suppressed antral pressures (P < .05) and stimulated pyloric pressures (P < .01) and markedly increased cholecystokinin and glucagon (both P < .001). Glucagon-like peptide-1 and peptide tyrosine tyrosine increased modestly (both P < .001), but there was no effect on total ghrelin. Insulin increased slightly (P < .05) without affecting blood glucose. Plasma L-Trp increased substantially (P < .001). All effects were dose-related and associated with increased fullness and substantially decreased energy intake (P < .001). There was a strong inverse correlation between energy intake and plasma L-Trp (r = -0.70; P < .001). Low caloric intraduodenal loads of L-Trp affect gut motor and hormonal function and markedly reduce energy intake. A strong inverse correlation between energy intake and plasma L-Trp suggests that, beyond gut mechanisms, direct effects of circulating L-Trp mediate its eating-inhibitory effect.

Glu-Trp-ONa or its acylated analogue (R-Glu-Trp-ONa) administration enhances the wound healing in the model of chronic skin wounds in rabbits.

PubMed

Shevtsov, Maxim A; Smagina, Larisa V; Kudriavtceva, Tatiana A; Petlenko, Sergey V; Voronkina, Irina V

2015-01-01

The management of chronic skin wounds represents a major therapeutic challenge. The synthesized dipeptide (Glu-Trp-ONa) and its acylated analogue (R-Glu-Trp-ONa) were assessed in the model of nonhealing dermal wounds in rabbits in relation to their healing properties in wound closure. Following wound modeling, the rabbits received a course of intraperitoneal injections of Glu-Trp-ONa or R-Glu-Trp-ONa. Phosphate-buffered saline and Solcoseryl® were applied as negative and positive control agents, respectively. An injection of Glu-Trp-ONa and R-Glu-Trp-ONa decreased the period of wound healing in animals in comparison to the control and Solcoseryl-treated groups. Acylation of Glu-Trp-ONa proved to be beneficial as related to the healing properties of the dipeptide. Subsequent zymography analyses showed that the applied peptides decreased the proteolytic activity of matrix metalloproteinases MMP-9, MMP-8, and MMP-2 in the early inflammatory phase and reversely increased the activity of MMP-9, MMP-8, and MMP-1 in the remodeling phase. Histological analyses of the wound sections (hematoxylin-eosin, Mallory's staining) confirmed the enhanced formation of granulation tissue and re-epithelialization in the experimental groups. By administering the peptides, wound closures increased significantly through the modulation of the MMPs' activity, indicating their role in wound healing.

Inhibitory effects of Morinda citrifolia extract and its constituents on melanogenesis in murine B16 melanoma cells.

PubMed

Masuda, Megumi; Itoh, Kimihisa; Murata, Kazuya; Naruto, Shunsuke; Uwaya, Akemi; Isami, Fumiyuki; Matsuda, Hideaki

2012-01-01

The objective of this study was to examine the effects of Morinda citrifolia (noni) extract and its constituents on α-melanocyte stimulating hormone (α-MSH)-stimulated melanogenesis in cultured murine B16 melanoma cells (B16 cells). A 50% ethanolic extract of noni seeds (MCS-ext) showed significant inhibition of melanogenesis with no effect on cell proliferation. MCS-ext was more active than noni leaf and fruit flesh extracts. Activity guided fractionation of MCS-ext led to the isolation of two lignans, 3,3'-bisdemethylpinoresinol (1) and americanin A (2), as active constituents. To elucidate the mechanism of melanogenesis inhibition by the lignans, α-MSH-stimulated B16 cells were treated with 1 (5 μM) and 2 (200 μM). Time-dependent increases of intracellular melanin content and tyrosinase activity, during 24 to 72 h, were inhibited significantly by treatment with the lignans. The activity of 1 was greater than that of 2. Western blot analysis suggested that the lignans inhibited melanogenesis by down regulation of the levels of phosphorylation of p38 mitogen-activated protein kinase, resulting in suppression of tyrosinase expression.

Development of a robust, sensitive and selective liquid chromatography-tandem mass spectrometry assay for the quantification of the novel macrocyclic peptide kappa opioid receptor antagonist [D-Trp]CJ-15,208 in plasma and application to an initial pharmacokinetic study.

PubMed

Khaliq, Tanvir; Williams, Todd D; Senadheera, Sanjeewa N; Aldrich, Jane V

2016-08-15

Selective kappa opioid receptor (KOR) antagonists may have therapeutic potential as treatments for substance abuse and mood disorders. Since [D-Trp]CJ-15,208 (cyclo[Phe-d-Pro-Phe-d-Trp]) is a novel potent KOR antagonist in vivo, it is imperative to evaluate its pharmacokinetic properties to assist the development of analogs as potential therapeutic agents, necessitating the development and validation of a quantitative method for determining its plasma levels. A method for quantifying [D-Trp]CJ-15,208 was developed employing high performance liquid chromatography-tandem mass spectrometry in mouse plasma. Sample preparation was accomplished through a simple one-step protein precipitation method with acetonitrile, and [D-Trp]CJ-15,208 analyzed following HPLC separation on a Hypersil BDS C8 column. Multiple reaction monitoring (MRM), based on the transitions m/z 578.1→217.1 and 245.0, was specific for [D-Trp]CJ-15,208, and MRM based on the transition m/z 566.2→232.9 was specific for the internal standard without interference from endogenous substances in blank mouse plasma. The assay was linear over the concentration range 0.5-500ng/mL with a mean r(2)=0.9987. The mean inter-day accuracy and precision for all calibration standards were 93-118% and 8.9%, respectively. The absolute recoveries were 85±6% and 81±9% for [D-Trp]CJ-15,208 and the internal standard, respectively. The analytical method had excellent sensitivity with a lower limit of quantification of 0.5ng/mL using a sample volume of 20μL. The method was successfully applied to an initial pharmacokinetic study of [D-Trp]CJ-15,208 following intravenous administration to mice. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of a robust, sensitive and selective liquid chromatography-tandem mass spectrometry assay for the quantification of the novel macrocyclic peptide kappa opioid receptor antagonist [D-Trp]CJ-15,208 in plasma and application to an initial pharmacokinetic study

PubMed Central

Khaliq, Tanvir; Williams, Todd D.; Senadheera, Sanjeewa N.; Aldrich, Jane V.

2016-01-01

Selective kappa opioid receptor (KOR) antagonists may have therapeutic potential as treatments for substance abuse and mood disorders. Since [D-Trp]CJ-15,208 (cyclo[Phe-D-Pro-Phe-D-Trp]) is a novel potent KOR antagonist in vivo, it is imperative to evaluate its pharmacokinetic properties to assist the development of analogs as potential therapeutic agents, necessitating the development and validation of a quantitative method for determining its plasma levels. A method for quantifying [D-Trp]CJ-15,208 was developed employing high performance liquid chromatography-tandem mass spectrometry in mouse plasma. Sample preparation was accomplished through a simple one-step protein precipitation method with acetonitrile, and [D-Trp]CJ-15,208 analyzed following HPLC separation on a Hypersil BDS C8 column. Multiple reaction monitoring (MRM), based on the transitions m/z 578.1 → 217.1 and 245.0, was specific for [D-Trp]CJ-15,208, and MRM based on the transition m/z 566.2 → 232.9 was specific for the internal standard without interference from endogenous substances in blank mouse plasma. The assay was linear over the concentration range 0.5–500 ng/mL with a mean r2 = 0.9987. The mean inter-day accuracy and precision for all calibration standards was 93–118% and 8.9%, respectively. The absolute recoveries were 85±6% and 81±9% for [D-Trp]CJ-15,208 and the internal standard, respectively. The analytical method had excellent sensitivity with a lower limit of quantification of 0.5 ng/mL using a sample volume of 20 μL. The method was successfully applied to an initial pharmacokinetic study of [D-Trp]CJ-15,208 following intravenous administration to mice. PMID:27318293

Structure-activity relationship of linear peptide Bu-His6-DPhe7-Arg8-Trp9-Gly10-NH2 at the human melanocortin-1 and -4 receptors: DPhe7 and Trp9 substitution.

PubMed

Danho, Waleed; Swistok, Joseph; Cheung, Adrian Wai-Hing; Kurylko, Grazyna; Franco, Lucia; Chu, Xin-Jie; Chen, Li; Yagaloff, Keith

2003-02-24

A series of pentapeptides, based on hMC4R pentapeptide agonist (Bu-His(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2)), was prepared in which either DPhe(7) or Trp(9) residue was systematically substituted. A number of interesting DPhe surrogates (D-Thi, D-3-CF(3)Phe, D-2-Nal and D-3,4-diClPhe) as well as Trp surrogates (2-Nal and Bta) were identified in this study.

Inhibitory Effects of (2'R)-2',3'-dihydro-2'-(1-hydroxy-1-methylethyl)-2,6'-bibenzofuran-6,4'-diol on Mushroom Tyrosinase and Melanogenesis in B16-F10 Melanoma Cells.

PubMed

Zhu, Jing-Jie; Yan, Gui-Rui; Xu, Zhi-Jian; Hu, Xiao; Wang, Gai-Hong; Wang, Ting; Zhu, Wei-Liang; Hou, Ai-Jun; Wang, He-Yao

2015-07-01

(2'R)-2',3'-Dihydro-2'-(1-hydroxy-1-methylethyl)-2,6'-bibenzofuran-6,4'-diol (DHMB) is a natural compound extracted from Morus notabilis. It was found that DHMB acts as a competitive inhibitor against mushroom tyrosinase with a Ki value of 14.77 μM. Docking results further indicated that it could form strong interactions with one copper ion with a distance of 2.7 Å, suggesting the mechanism of inhibition might be due to chelating copper ions in the active site. Furthermore, melanin production in B16-F10 murine melanoma cells was significantly inhibited by DHMB in a concentration-dependent manner without cytotoxicity. The results of western blotting also showed that DHMB decreased 3-isobuty-1-methxlzanthine-induced mature tyrosinase expression. Taken together, these findings indicated that DHMB may be a new promising pigmentation-altering agent for agriculture, cosmetic, and therapeutic applications. Copyright © 2015 John Wiley & Sons, Ltd.

Phytol suppresses melanogenesis through proteasomal degradation of MITF via the ROS-ERK signaling pathway.

PubMed

Ko, Gyeong-A; Cho, Somi Kim

2018-04-25

Phytol (3,7,11,15-tetramethyl-2-hexadecen-1-ol) is an acyclic monounsaturated diterpene alcohol generated from chlorophyll metabolism that exerts anti-inflammatory, antithrombotic, antimicrobial, and antitumor effects. However, the effect of phytol on melanogenesis and the underlying molecular mechanisms of its inhibition remain unknown. Here, we found that phytol suppressed α-melanocyte-stimulating hormone-induced melanogenesis in B16F10 murine melanoma cells without any toxic effects. Phytol significantly attenuated melanin production by reducing the expression of tyrosinase and tyrosinase related protein 1. Treatment with phytol inhibited the expression of microphthalmia-associated transcription factor (MITF) by phosphorylating extracellular signal-regulated protein kinase (ERK). The ERK inhibitor PD98059 restored MITF expression and prevented the anti-melanogenic effect of phytol. We found that the ERK inhibitor coincidently abrogated MITF ubiquitination and degradation, suggesting that the ERK pathway is involved in phytol-induced ubiquitination of MITF. Furthermore, our data show that reactive oxygen species (ROS) production was increased in cells treated with phytol. Consistently, a ROS scavenger inhibited ERK phosphorylation and restored MITF degradation. Accordingly, the intermediary role of ROS was confirmed in phytol-induced MITF degradation. Taken together, these results demonstrate that phytol stimulates ROS production and modulates ERK-mediated proteasomal degradation of MITF in B16F10 murine melanoma cells. These findings suggest that phytol may have potential to be utilized as a whitening agent in cosmetics and as a therapy for skin hyperpigmentation. Copyright © 2018. Published by Elsevier B.V.

Molecular basis of the specific subcellular localization of the C2-like domain of 5-lipoxygenase.

PubMed

Kulkarni, Shilpa; Das, Sudipto; Funk, Colin D; Murray, Diana; Cho, Wonhwa

2002-04-12

The activation of 5-lipoxygenase (5-LO) involves its calcium-dependent translocation to the nuclear envelope, where it catalyzes the two-step transformation of arachidonic acid into leukotriene A(4), leading to the synthesis of various leukotrienes. To understand the mechanism by which 5-LO is specifically targeted to the nuclear envelope, we studied the membrane binding properties of the amino-terminal domain of 5-LO, which has been proposed to have a C2 domain-like structure. The model building, electrostatic potential calculation, and in vitro membrane binding studies of the isolated C2-like domain of 5-LO and selected mutants show that this Ca(2+)-dependent domain selectively binds zwitterionic phosphatidylcholine, which is conferred by tryptophan residues (Trp(13), Trp(75), and Trp(102)) located in the putative Ca(2+)-binding loops. The spatiotemporal dynamics of the enhanced green fluorescence protein-tagged C2-like domain of 5-LO and mutants in living cells also show that the phosphatidylcholine selectivity of the C2-like domain accounts for the specific targeting of 5-LO to the nuclear envelope. Together, these results show that the C2-like domain of 5-LO is a genuine Ca(2+)-dependent membrane-targeting domain and that the subcellular localization of the domain is governed in large part by its membrane binding properties.

The effect of sampling methods on the apparent constituents of ink from the squid Sepioteuthis australis.

PubMed

Madaras, F; Gerber, J P; Peddie, F; Kokkinn, M J

2010-11-01

Results of experiments conducted on ink recovered from the squid Sepioteuthis australis indicate that there is no epinephrine or protein naturally present in the ink as it would be ejected in vivo. Protein content was effectively zero when ink was syringed from the duct end of the ink sac of freshly killed animals. By contrast, there were proteins in samples collected from dead specimens where ink was collected by a stripping method. From these samples, a single large molecular weight protein was identified as having tyrosinase activity. Digestion of syringed ink did not yield signs of melanin-bound proteins. Analysis of supernatants after centrifugation of squid ink consistently revealed the presence of DOPA, dopamine, and taurine, whereas epinephrine and nor-epinephrine were recorded from what was believed to be contaminated ink. Histological investigations of the ink sac revealed a compartmentalised glandular structure distal to the duct end. Closer observation of the glandular tissue showed that compartments increased in size as they matured and moved further into the lumen. It was concluded that the presence of epinephrine and tyrosinase (or a related protein) in the ink of S. australis could be attributed to rupturing of basal glandular compartments or contamination from other sources during the extraction process.

Involvement of phenoloxidase in browning during grinding of Tenebrio molitor larvae

PubMed Central

Lakemond, Catriona M. M.; Fogliano, Vincenzo; Renzone, Giovanni; Scaloni, Andrea; Vincken, Jean-Paul

2017-01-01

Insects are investigated as alternative protein source to meet the increasing demand for proteins in the future. Enzymatic browning occurring during grinding of insect and subsequent extraction of proteins can influence the proteins’ properties, but it is unclear which enzymes are responsible for this phenomenon. This study was performed on larvae of three commonly used insect species, namely Tenebrio molitor, Alphitobius diaperinus and Hermetia illucens. Oxygen consumption measurements on protein extracts showed activity on L-tyrosine, L-3,4-di-hydroxy-phenylalanine (L-DOPA) and L-dopamine, indicating phenoloxidase as a key player in browning. Furthermore, no reaction on 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) was observed, ruling out an important contribution of laccase to browning. The browning reaction was most prominent at pH 6 for T. molitor and A. diaperinus, and 7 for H. illucens. As the enzyme activity of H. illucens was the lowest with the darkest color formation, this was likely caused by another factor. The activity of phenoloxidase was confirmed for T. molitor and A. diaperinus by activity measurements after fractionation by anion-exchange chromatography. Color measurements showed the presence of activity on both L-DOPA and L-tyrosine in the same fractions. Both substrates were converted into dopachrome after incubation with enzyme-enriched fractions. No DOPA-decarboxylase, tyrosine hydroxylase and peroxidase activities were observed. By using native PAGE with L-DOPA as staining-solution, active T. molitor protein bands were resolved and characterized, identifying a tyrosinase/phenoloxidase as the active enzyme species. All together, these data confirmed that tyrosinase is an important enzyme in causing enzymatic browning in T. molitor and likely in A. diaperinus. PMID:29244828

The N-terminal strand modulates immunoglobulin light chain fibrillogenesis.

PubMed

del Pozo-Yauner, Luis; Wall, Jonathan S; González Andrade, Martín; Sánchez-López, Rosana; Rodríguez-Ambriz, Sandra L; Pérez Carreón, Julio I; Ochoa-Leyva, Adrián; Fernández-Velasco, D Alejandro

2014-01-10

It has been suggested that the N-terminal strand of the light chain variable domain (V(L)) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stability and kinetic of fibrillogenesis of the V(L) protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein. Copyright © 2013 Elsevier Inc. All rights reserved.

Generation Mechanism of Radical Species by Tyrosine-Tyrosinase Reaction

PubMed Central

Tada, Mika; Kohno, Masahiro; Kasai, Shigenobu; Niwano, Yoshimi

2010-01-01

Alleviated melanin formation in the skin through inhibition of tyrosine-tyrosinase reaction is one of the major targets of cosmetics for whitening ability. Since melanin has a pivotal role for photoprotection, there are pros and cons of inhibition of melanin formation. This study applying electron spin resonance (ESR)-spin trapping method revealed that •H and •OH are generated through tyrosine-tyrosinase reaction. When deuterium water was used instead of H2O, the signal of 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-H (a spin adduct of DMPO and •H) greatly decreased, whilst DMPO-OH (a spin adduct of DMPO and •OH) did not. Thus, it is suggested that •H was derived from H2O, and •OH through oxidative catalytic process of tyrosine to dopaquinone. Our study suggests that tyrosinase inhibitors might contribute to alleviate the oxidative damage of the skin by inhibiting •OH generation via the enzyme reaction. PMID:20838572

Tyrosinase-Mediated Construction of a Silk Fibroin/Elastin Nanofiber Bioscaffold.

PubMed

Hong, Yanqing; Zhu, Xueke; Wang, Ping; Fu, Haitian; Deng, Chao; Cui, Li; Wang, Qiang; Fan, Xuerong

2016-04-01

Elastin has characteristics of elasticity, biological activity, and mechanical stability. In the present work, tyrosinase-mediated construction of a bioscaffold with silk fibroin and elastin was carried out, aiming at developing a novel medical biomaterial. The efficiency of enzymatic oxidation of silk fibroin and the covalent reaction between fibroin and elastin were examined by spectrophotometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and size exclusion chromatography (SEC). The properties of composite air-dried and nanofiber scaffolds were investigated. The results reveal that elastin was successfully bonded to silk fibroins, resulting in an increase in molecular weight of fibroin proteins. ATR-FTIR spectra indicated that tyrosinase treatment impacted the conformational structure of fibroin-based membrane. The thermal behaviors and mechanical properties of the tyrosinase-treated scaffolds were also improved compared with the untreated group. NIH/3T3 cells exhibited optimum densities when grown on the nanofiber scaffold, implying that the nanofiber scaffold has enhanced biocompatibility compared to the air-dried scaffold. A biological nanofiber scaffold constructed from tyrosinase-treated fibroin and elastin could potentially be utilized in biomedical applications.

Application of amphipols for structure-functional analysis of TRP channels.

PubMed

Huynh, Kevin W; Cohen, Matthew R; Moiseenkova-Bell, Vera Y

2014-10-01

Amphipathic polymers (amphipols), such as A8-35 and SApol, are a new tool for stabilizing integral membrane proteins in detergent-free conditions for structural and functional studies. Transient receptor potential (TRP) ion channels function as tetrameric protein complexes in a diverse range of cellular processes including sensory transduction. Mammalian TRP channels share ~20 % sequence similarity and are categorized into six subfamilies: TRPC (canonical), TRPV (vanilloid), TRPA (ankyrin), TRPM (melastatin), TRPP (polycystin), and TRPML (mucolipin). Due to the inherent difficulties in purifying eukaryotic membrane proteins, structural studies of TRP channels have been limited. Recently, A8-35 was essential in resolving the molecular architecture of the nociceptor TRPA1 and led to the determination of a high-resolution structure of the thermosensitive TRPV1 channel by cryo-EM. Newly developed maltose-neopentyl glycol (MNG) detergents have also proven to be useful in stabilizing TRP channels for structural analysis. In this review, we will discuss the impacts of amphipols and MNG detergents on structural studies of TRP channels by cryo-EM. We will compare how A8-35 and MNG detergents interact with the hydrophobic transmembrane domains of TRP channels. In addition, we will discuss what these cryo-EM studies reveal on the importance of screening different types of surfactants toward determining high-resolution structures of TRP channels.

Application of amphipols for structure-functional analysis of TRP channels

PubMed Central

Huynh, Kevin W.; Cohen, Matthew R.; Moiseenkova-Bell, Vera Y.

2014-01-01

Amphipathic polymers (amphipols), such as A8-35 and SApol, are a new tool for stabilizing integral membrane proteins in detergent-free conditions for structural and functional studies. Transient receptor potential (TRP) ion channels function as tetrameric protein complexes in a diverse range of cellular processes including sensory transduction. Mammalian TRP channels share ~20% sequence similarity and are categorized into six subfamilies: TRPC (canonical), TRPV (vanilloid), TRPA (ankyrin), TRPM (melastatin), TRPP (polycystin), and TRPML (mucolipin). Due to the inherent difficulties in purifying eukaryotic membrane proteins, structural studies of TRP channels have been limited. Recently, A8-35 was essential in resolving the molecular architecture of the nociceptor TRPA1 and led to the determination of a high resolution structure of the thermosensitive TRPV1 channel by cryo-EM. Newly developed maltose-neopentyl glycol (MNG) detergents have also proven useful in stabilizing TRP channels for structural analysis. In this review, we will discuss the impact of amphipols and MNG detergents on structural studies of TRP channels by cryo-EM. We will compare how A8-35 and MNG detergents interact with the hydrophobic transmembrane (TM) domains of TRP channels. In addition, we will discuss what these cryo-EM studies reveal on the importance of screening different types of surfactants towards determining high resolution structures of TRP channels. PMID:24894720

A novel o-aminophenol oxidase responsible for formation of the phenoxazinone chromophore of grixazone.

PubMed

Suzuki, Hirokazu; Furusho, Yasuhide; Higashi, Tatsuichiro; Ohnishi, Yasuo; Horinouchi, Sueharu

2006-01-13

Grixazone contains a phenoxazinone chromophore and is a secondary metabolite produced by Streptomyces griseus. In the grixazone biosynthesis gene cluster, griF (encoding a tyrosinase homolog) and griE (encoding a protein similar to copper chaperons for tyrosinases) are encoded. An expression study of GriE and GriF in Escherichia coli showed that GriE activated GriF by transferring copper ions to GriF, as has been observed for a Streptomyces melanogenesis system in which the MelC1 copper chaperon transfers copper ions to MelC2 tyrosinase. In contrast with tyrosinases, GriF showed no monophenolase activity, although it oxidized various o-aminophenols as preferable substrates rather than catechol-type substrates. Deletion of the griEF locus on the chromosome resulted in accumulation of 3-amino-4-hydroxybenzaldehyde (3,4-AHBAL) and its acetylated compound, 3-acetylamino-4-hydroxybenzaldehyde. GriF oxidized 3,4-AHBAL to yield an o-quinone imine derivative, which was then non-enzymatically coupled with another molecule of the o-quinone imine to form a phenoxazinone. The coexistence of N-acetylcysteine in the in vitro oxidation of 3,4-AH-BAL by GriF resulted in the formation of grixazone A, suggesting that the -SH group of N-acetylcysteine is conjugated to the o-quinone imine formed from 3,4-AHBAL and that the conjugate is presumably coupled with another molecule of the o-quinone imine. GriF is thus a novel o-aminophenol oxidase that is responsible for the formation of the phenoxazinone chromophore in the grixazone biosynthetic pathway.

Assessment of Cuscuta chinensis seeds׳ effect on melanogenesis: comparison of water and ethanol fractions in vitro and in vivo.

PubMed

Wang, Tian-Jing; An, Jing; Chen, Xiao-Hui; Deng, Qiu-Di; Yang, Liu

2014-05-28

Cuscuta chinensis seeds have traditionally been used to treat freckles and melasma in Asia, although recent reports have revealed that Semen cuscutae is a promoter of melanogenesis. The present study aims to investigate the mechanism of this opposite effect of Semen cuscutae on melanogenesis. In accordance with traditional usage, the water fraction and the ethanol fraction from Semen cuscutae (WFSC/EFSC) were extracted to determine the herbal effects by examining the activity of mushroom tyrosinase, cellular melanin contents, tyrosinase activity assay, quantitative-reverse transcription polymerase chain reaction (qRT-PCR), and Western blot analysis for tyrosinase in B16F10 mouse melanoma cells. The melanocyte phenotypes of zebrafish larvae were observed while the in vivo melanin contents and tyrosinase activity were determined. The activity of mushroom tyrosinase assay shown that WFSC was an uncompetitive inhibitor of mushroom tyrosinase, while EFSC indicated dose-dependent activation of the mushroom tyrosinase activity. The WFSC markedly inhibited 3-isobutyl-1-methylxanthine (IBMX)-stimulated melanin synthesis and tyrosinase activity in vitro. Howeveran accelerant role in melanin synthesis and tyosinase activity. Neither fraction had any effect on the IBMX-induced expression of tyrosinase protein or mRNA. The WFSC strongly inhibited melanin synthesis and cellular tyrosinase activity in vivo. Furthermore, with the function of WFSC at a higher concentration, a punctate melanocyte pattern appeared that was similar to the pattern induced by arbutin or Mequinol (MQ). The EFSC had no effect on the melanocytes of zebrafish larvae. It was discovered that WFSC did not show a stable inhibitory effect until it was extracted 1 month later. These results suggest that the opposite effects of Cuscuta chinensis seeds were caused by the extraction methods and that time has an important role on the effect of WFSC. Both WFSC and EFSC significantly influence melanogenesis by regulating enzymatic activity of tyrosinase. In addition, the data indicate that wildtype (WT) zebrafish may be an ideal model for testing inhibitors of melanogenesis from clinically active herbs. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Effect of Zingiber officinale Supplementation on Obesity Management with Respect to the Uncoupling Protein 1 -3826A>G and ß3-adrenergic Receptor Trp64Arg Polymorphism.

PubMed

Ebrahimzadeh Attari, Vahideh; Asghari Jafarabadi, Mohammad; Zemestani, Maryam; Ostadrahimi, Alireza

2015-07-01

The present study aimed to investigate the effect of ginger (Zingiber officinale) supplementation on some obesity-associated parameters, with nutrigenetics approach. Accordingly, 80 eligible obese women (aged 18-45 years) were randomly assigned to receive either ginger (2-g ginger rhizomes powder as two 1-g tablets per day) or placebo supplements (corn starch with the same amount) for 12 weeks. Subjects were tested for changes in body weight, body mass index, waist and hip circumferences, body composition, appetite score, and dietary intake. Moreover, participants were genotyped for the -3826A>G and Trp64Arg polymorphisms of uncoupling protein 1 and ß3-adrenergic receptor genes, respectively. Over 12 weeks, ginger supplementation resulted in a slight but statistically significant decrease in all anthropometric measurements and total appetite score as compared with placebo group, which were more pronounced in subjects with the AA genotype for uncoupling protein 1 and Trp64Trp genotype for ß3-adrenergic receptor gene. However, there was no significant difference in changes of body composition and total energy and macronutrients intake between groups. In conclusion, our findings suggest that ginger consumption has potential in managing obesity, accompanying with an intervention-genotype interaction effect. However, further clinical trials need to explore ginger's efficacy as an anti-obesity agent in the form of powder, extract, or its active components. Copyright © 2015 John Wiley & Sons, Ltd.

Anti-melanoma vaccines engineered to simultaneously modulate cytokine priming and silence PD-L1 characterized using ex vivo myeloid-derived suppressor cells as a readout of therapeutic efficacy.

PubMed

Liechtenstein, Therese; Perez-Janices, Noemi; Blanco-Luquin, Idoia; Goyvaerts, Cleo; Schwarze, Julia; Dufait, Ines; Lanna, Alessio; Ridder, Mark De; Guerrero-Setas, David; Breckpot, Karine; Escors, David

Efficacious antitumor vaccines strongly stimulate cancer-specific effector T cells and counteract the activity of tumor-infiltrating immunosuppressive cells. We hypothesised that combining cytokine expression with silencing programmed cell death ligand 1 (PD-L1) could potentiate anticancer immune responses of lentivector vaccines. Thus, we engineered a collection of lentivectors that simultaneously co-expressed an antigen, a PD-L1-silencing shRNA, and various T cell-polarising cytokines, including interferon γ (IFNγ), transforming growth factor β (TGFβ) or interleukins (IL12, IL15, IL23, IL17A, IL6, IL10, IL4). In a syngeneic B16F0 melanoma model and using tyrosinase related protein 1 (TRP1) as a vaccine antigen, we found that simultaneous delivery of IL12 and a PD-L1-silencing shRNA was the only combination that exhibited therapeutically relevant anti-melanoma activities. Mechanistically, we found that delivery of the PD-L1 silencing construct boosted T cell numbers, inhibited in vivo tumor growth and strongly cooperated with IL12 cytokine priming and antitumor activities. Finally, we tested the capacities of our vaccines to counteract tumor-infiltrating myeloid-derived suppressor cell (MDSC) activities ex vivo . Interestingly, the lentivector co-expressing IL12 and the PD-L1 silencing shRNA was the only one that counteracted MDSC suppressive activities, potentially underlying the observed anti-melanoma therapeutic benefit. We conclude that (1) evaluation of vaccines in healthy mice has no significant predictive value for the selection of anticancer treatments; (2) B16 cells expressing xenoantigens as a tumor model are of limited value; and (3) vaccines which inhibit the suppressive effect of MDSC on T cells in our ex vivo assay show promising and relevant antitumor activities.

Anti-melanoma vaccines engineered to simultaneously modulate cytokine priming and silence PD-L1 characterized using ex vivo myeloid-derived suppressor cells as a readout of therapeutic efficacy

PubMed Central

Liechtenstein, Therese; Perez-Janices, Noemi; Blanco-Luquin, Idoia; Goyvaerts, Cleo; Schwarze, Julia; Dufait, Ines; Lanna, Alessio; Ridder, Mark De; Guerrero-Setas, David; Breckpot, Karine; Escors, David

2014-01-01

Efficacious antitumor vaccines strongly stimulate cancer-specific effector T cells and counteract the activity of tumor-infiltrating immunosuppressive cells. We hypothesised that combining cytokine expression with silencing programmed cell death ligand 1 (PD-L1) could potentiate anticancer immune responses of lentivector vaccines. Thus, we engineered a collection of lentivectors that simultaneously co-expressed an antigen, a PD-L1-silencing shRNA, and various T cell-polarising cytokines, including interferon γ (IFNγ), transforming growth factor β (TGFβ) or interleukins (IL12, IL15, IL23, IL17A, IL6, IL10, IL4). In a syngeneic B16F0 melanoma model and using tyrosinase related protein 1 (TRP1) as a vaccine antigen, we found that simultaneous delivery of IL12 and a PD-L1-silencing shRNA was the only combination that exhibited therapeutically relevant anti-melanoma activities. Mechanistically, we found that delivery of the PD-L1 silencing construct boosted T cell numbers, inhibited in vivo tumor growth and strongly cooperated with IL12 cytokine priming and antitumor activities. Finally, we tested the capacities of our vaccines to counteract tumor-infiltrating myeloid-derived suppressor cell (MDSC) activities ex vivo. Interestingly, the lentivector co-expressing IL12 and the PD-L1 silencing shRNA was the only one that counteracted MDSC suppressive activities, potentially underlying the observed anti-melanoma therapeutic benefit. We conclude that (1) evaluation of vaccines in healthy mice has no significant predictive value for the selection of anticancer treatments; (2) B16 cells expressing xenoantigens as a tumor model are of limited value; and (3) vaccines which inhibit the suppressive effect of MDSC on T cells in our ex vivo assay show promising and relevant antitumor activities. PMID:25954597

A Macrocyclic Agouti-Related Protein/[Nle4,DPhe7]α-Melanocyte Stimulating Hormone Chimeric Scaffold Produces Subnanomolar Melanocortin Receptor Ligands.

PubMed

Ericson, Mark D; Freeman, Katie T; Schnell, Sathya M; Haskell-Luevano, Carrie

2017-01-26

The melanocortin system consists of five receptor subtypes, endogenous agonists, and naturally occurring antagonists. These receptors and ligands have been implicated in numerous biological pathways including processes linked to obesity and food intake. Herein, a truncation structure-activity relationship study of chimeric agouti-related protein (AGRP)/[Nle4,DPhe7]α-melanocyte stimulating hormone (NDP-MSH) ligands is reported. The tetrapeptide His-DPhe-Arg-Trp or tripeptide DPhe-Arg-Trp replaced the Arg-Phe-Phe sequence in the AGRP active loop derivative c[Pro-Arg-Phe-Phe-Xxx-Ala-Phe-DPro], where Xxx was the native Asn of AGRP or a diaminopropionic (Dap) acid residue previously shown to increase antagonist potency at the mMC4R. The Phe, Ala, and Dap/Asn residues were successively removed to generate a 14-member library that was assayed for agonist activity at the mouse MC1R, MC3R, MC4R, and MC5R. Two compounds possessed nanomolar agonist potency at the mMC4R, c[Pro-His-DPhe-Arg-Trp-Asn-Ala-Phe-DPro] and c[Pro-His-DPhe-Arg-Trp-Dap-Ala-DPro], and may be further developed to generate novel melanocortin probes and ligands for understanding and treating obesity.

Effect of osmolytes on the thermal stability of proteins: replica exchange simulations of Trp-cage in urea and betaine solutions.

PubMed

Adamczak, Beata; Kogut, Mateusz; Czub, Jacek

2018-04-25

Although osmolytes are known to modulate the folding equilibrium, the molecular mechanism of their effect on thermal denaturation of proteins is still poorly understood. Here, we simulated the thermal denaturation of a small model protein (Trp-cage) in the presence of denaturing (urea) and stabilizing (betaine) osmolytes, using the all-atom replica exchange molecular dynamics simulations. We found that urea destabilizes Trp-cage by enthalpically-driven association with the protein, acting synergistically with temperature to induce unfolding. In contrast, betaine is sterically excluded from the protein surface thereby exerting entropic depletion forces that contribute to the stabilization of the native state. In fact, we find that while at low temperatures betaine slightly increases the folding free energy of Trp-cage by promoting another near-native conformation, it protects the protein against temperature-induced denaturation. This, in turn, can be attributed to enhanced exclusion of betaine at higher temperatures that arises from less attractive interactions with the protein surface.

Mutagens in cooked foods - metabolism and genetic toxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Felton, J.S.; Bjeldanes, L.F.; Hatch, F.T.

1984-02-17

Recently developed in our laboratories is an efficient extraction procedure incorporating XAD resin adsorption which yields from 200/sup 0/C grilled ground beef an extract containing 230 Salmonella TA1538 revertants per g fresh weight of original ground beef. These mutagenic components are specific for frameshift-sensitive Salmonella strains and have an absolute requirement for metabolic activation. Normal-phase HPLC separation of methanol-extractable metabolites generated from reaction of 2-amino-3-methylimidazo (4,5-f)quinoline (IQ), a mutagenic component of broiled food with rat liver microsomes resulted in one direct-acting mutagenic peak and a second more polar peak still requiring metabolic activation. Two potent thermally-produced bacterial mutagens, 3-amino-1-methyl-5H-pyrido (4,3-b)more » indole (Trp-P-2) and IQ, were examined in mammalian cells. In excision repair-deficient CHO cells, Trp-P-2 exposure caused cytotoxicity, mutagenicity, sister chromatid exchange, and chromosomal aberrations at concentrations more than 30-fold lower than those for IQ. In normal repair-proficient CHO cells Trp-P-2 was one-half as active and IQ was inactive. Relative to Trp-P-2, IQ is much more potent in the Salmonella bacterial system than in mammalian CHO cells.« less

An Exquisitely Specific PDZ/Target Recognition Revealed by the Structure of INAD PDZ3 in Complex with TRP Channel Tail.

PubMed

Ye, Fei; Liu, Wei; Shang, Yuan; Zhang, Mingjie

2016-03-01

The vast majority of PDZ domains are known to bind to a few C-terminal tail residues of target proteins with modest binding affinities and specificities. Such promiscuous PDZ/target interactions are not compatible with highly specific physiological functions of PDZ domain proteins and their targets. Here, we report an unexpected PDZ/target binding occurring between the scaffold protein inactivation no afterpotential D (INAD) and transient receptor potential (TRP) channel in Drosophila photoreceptors. The C-terminal 15 residues of TRP are required for the specific interaction with INAD PDZ3. The INAD PDZ3/TRP peptide complex structure reveals that only the extreme C-terminal Leu of TRP binds to the canonical αB/βB groove of INAD PDZ3. The rest of the TRP peptide, by forming a β hairpin structure, binds to a surface away from the αB/βB groove of PDZ3 and contributes to the majority of the binding energy. Thus, the INAD PDZ3/TRP channel interaction is exquisitely specific and represents a new mode of PDZ/target recognitions. Copyright © 2016 Elsevier Ltd. All rights reserved.

The N-terminal strand modulates immunoglobulin light chain fibrillogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pozo-Yauner, Luis del, E-mail: ldelpozo@inmegen.gob.mx; Wall, Jonathan S.; González Andrade, Martín

2014-01-10

Highlights: •We evaluated the impact of mutations in the N-terminal strand of 6aJL2 protein. •Mutations destabilized the protein in a position-dependent manner. •Destabilizing mutations accelerated the fibrillogenesis by shortening the lag time. •The effect on the kinetic of fibril elongation by seeding was of different nature. •The N-terminal strand is buried in the fibrillar state of 6aJL2 protein. -- Abstract: It has been suggested that the N-terminal strand of the light chain variable domain (V{sub L}) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stabilitymore » and kinetic of fibrillogenesis of the V{sub L} protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein.« less

Tyrosinase Inhibitory Activities of Carissa opaca Stapf ex Haines Roots Extracts and Their Phytochemical Analysis

PubMed Central

Malik, Wajeeha; Ahmed, Dildar; Izhar, Sania

2017-01-01

Objective: Carissa opaca is a medicinal plant with rich folkloric applications. The present research was conducted to explore the tyrosinase inhibitory potential of aqueous decoction (AD) and methanolic extract (ME) of roots of C. opaca and its fractions in various solvents and their phytochemical analysis. Materials and Methods: AD of the dried powdered roots of C. opaca was prepared by boiling in water. ME was prepared by cold maceration. Its fractions were obtained in solvents of increasing polarity, i.e., hexane, chloroform, ethyl acetate, n-butanol, and water. The biomass left after extraction with methanol was boiled in water to get its decoction Biomass aqueous decoction (BAD). Tyrosinase inhibitory activities of the samples were studied according to a reported method. Chemical compounds in the samples were identified by gas chromatography-mass spectrometry (GC-MS). Results: The AD, BAD, and ME and its fractions displayed remarkable tyrosinase inhibitory activity. The IC50 of AD was 23.33 μg/mL as compared to 15.80 μg/mL of the standard arbutin and that of BAD was 21.24 μg/mL. The IC50 of ME was 34.76 μg/mL while that of hexane, chloroform, ethyl acetate, n-butanolic, and aqueous fractions was 21.0, 44.73, 43.40, 27.66, and 25.06 μg/mL, respectively. The hexane fraction was thus most potent followed by aqueous fraction. By phytochemical analysis, campesterol, stigmasterol, gamma-sitosterol, alpha-amyrin, 9,19-cyclolanostan-3-ol, 24-methylene-,(3 β)-, lupeol, lup-20(29)-en-3-one, lup-20(29)-en-3-ol, acetate,(3 β)-, 2(1H) naphthalenone, 3,5,6,7,8,8a-hexahydro-4,8a-dimethyl-6-(1-methylethenyl)-, and 2,3,3-trimethyl-2-(3-methylbuta-1,3-dienyl)-6-methylenecyclohexanone were identified in the extracts by GC-MS. Other compounds included fatty acids and their esters. Some of these compounds are being first time reported here from this plant. Conclusions: The roots extracts exhibited considerable tyrosinase inhibitory activities, alluding to a possible application of the plant in cosmetic as whitening agent subject to further pharmacological studies. SUMMARY The present study aimed to explore the tyrosinase inhibitory potential of aqueous decoction and methanolic extract of roots of Carissa opaca and its fractions in various solvents and their phytochemical constituents. GCMS analysis was conducted to identify the phytochemicals. The extracts and fractions of C. opaca roots showed remarkable anti-tyrosinase activities alluding to their possible application to treat disorders related to overproduction of melanin. Abbreviations used: AD: Aqueous decoction; ME: Methanolic extract; BAD: Biomass aqueous decoction; GC-MS: Gas chromatography-mass spectrometry. PMID:29142412

Loss of Oca2 disrupts the unfolded protein response and increases resistance to endoplasmic reticulum stress in melanocytes

PubMed Central

Cheng, Tsing; Orlow, Seth J.; Manga, Prashiela

2013-01-01

Summary Accumulation of proteins in the endoplasmic reticulum (ER) typically induces stress and initiates the unfolded protein response (UPR) to facilitate recovery. If homeostasis is not restored, apoptosis is induced. However, adaptation to chronic UPR activation can increase resistance to subsequent acute ER stress. We therefore investigated adaptive mechanisms in Oculocutaneous albinism type 2 (Oca2)-null melanocytes where UPR signaling is arrested despite continued tyrosinase accumulation leading to resistance to the chemical ER stressor thapsigargin. Although thapsigargin triggers UPR activation, instead of Perk-mediated phosphorylation of eIF2α, in Oca2-null melanocytes, eIF2α was rapidly dephosphorylated upon treatment. Dephosphorylation was mediated by the Gadd34-PP1α phosphatase complex. Gadd34-complex inhibition blocked eIF2α dephosphorylation and significantly increased Oca2-null melanocyte sensitivity to thapsigargin. Thus, Oca2-null melanocytes adapt to acute ER stress by disruption of proapoptotic Perk signaling, which promotes cell survival. This is the first study to demonstrate rapid eIF2α dephosphorylation as an adaptive mechanism to ER stress. PMID:23962237

α-Glucosidase and tyrosinase inhibitory effects of an abietane type diterpenoid taxoquinone from Metasequoia glyptostroboides.

PubMed

Bajpai, Vivek K; Park, Yong-Ha; Na, MinKyun; Kang, Sun Chul

2015-03-26

Nowadays plant derived natural compounds have gained huge amount of research attention especially in food and medicine industries due to their multitude of biological and therapeutic properties as alternative medicines. In this study, a diterpenoid compound taxoquinone, isolated from Metasequoia glyptostroboides was evaluated for its α-glucosidase and tyrosinase inhibitory efficacy in terms of its potent anti-diabetic and depigmentation potential, respectively. As a result, taxoquinone at the concentration range of 100-3,000 μg/mL and 200-1,000 μg/mL showed potent efficacy on inhibiting α-glucosidase and tyrosinase enzymes by 9.24-51.32% and 11.14-52.32%, respectively. The findings of this study clearly evident potent therapeutic efficacy of an abietane diterpenoid taxoquinone isolated from M. glyptostroboides with a possibility for using it as a novel candidate in food and medicine industry as a natural alternative medicine to prevent diabetes mellitus type-2 related disorders and as a depigmentation agent.

The perturbation of tryptophan fluorescence by phenylalanine to alanine mutations identifies the hydrophobic core in a subset of bacterial Ig-like domains.

PubMed

Raman, Rajeev; Ptak, Christopher P; Hsieh, Ching-Lin; Oswald, Robert E; Chang, Yung-Fu; Sharma, Yogendra

2013-07-09

Many host-parasite interactions are mediated via surface-exposed proteins containing bacterial immunoglobulin-like (Big) domains. Here, we utilize the spectral properties of a conserved Trp to provide evidence that, along with a Phe, these residues are positioned within the hydrophobic core of a subset of Big_2 domains. The mutation of the Phe to Ala decreases Big_2 domain stability and impairs the ability of LigBCen2 to bind to the host protein, fibronectin.

TRPM4 Is a Novel Component of the Adhesome Required for Focal Adhesion Disassembly, Migration and Contractility

PubMed Central

Cáceres, Mónica; Ortiz, Liliana; Recabarren, Tatiana; Romero, Anibal; Colombo, Alicia; Leiva-Salcedo, Elías; Varela, Diego; Rivas, José; Silva, Ian; Morales, Diego; Campusano, Camilo; Almarza, Oscar; Simon, Felipe; Toledo, Hector; Park, Kang-Sik; Trimmer, James S.; Cerda, Oscar

2015-01-01

Cellular migration and contractility are fundamental processes that are regulated by a variety of concerted mechanisms such as cytoskeleton rearrangements, focal adhesion turnover, and Ca2+ oscillations. TRPM4 is a Ca2+-activated non-selective cationic channel (Ca2+-NSCC) that conducts monovalent but not divalent cations. Here, we used a mass spectrometry-based proteomics approach to identify putative TRPM4-associated proteins. Interestingly, the largest group of these proteins has actin cytoskeleton-related functions, and among these nine are specifically annotated as focal adhesion-related proteins. Consistent with these results, we found that TRPM4 localizes to focal adhesions in cells from different cellular lineages. We show that suppression of TRPM4 in MEFs impacts turnover of focal adhesions, serum-induced Ca2+ influx, focal adhesion kinase (FAK) and Rac activities, and results in reduced cellular spreading, migration and contractile behavior. Finally, we demonstrate that the inhibition of TRPM4 activity alters cellular contractility in vivo, affecting cutaneous wound healing. Together, these findings provide the first evidence, to our knowledge, for a TRP channel specifically localized to focal adhesions, where it performs a central role in modulating cellular migration and contractility. PMID:26110647

Finding New Enzymes from Bacterial Physiology: A Successful Approach Illustrated by the Detection of Novel Oxidases in Marinomonas mediterranea

PubMed Central

Sanchez-Amat, Antonio; Solano, Francisco; Lucas-Elío, Patricia

2010-01-01

The identification and study of marine microorganisms with unique physiological traits can be a very powerful tool discovering novel enzymes of possible biotechnological interest. This approach can complement the enormous amount of data concerning gene diversity in marine environments offered by metagenomic analysis, and can help to place the activities associated with those sequences in the context of microbial cellular metabolism and physiology. Accordingly, the detection and isolation of microorganisms that may be a good source of enzymes is of great importance. Marinomonas mediterranea, for example, has proven to be one such useful microorganism. This Gram-negative marine bacterium was first selected because of the unusually high amounts of melanins synthesized in media containing the amino acid l-tyrosine. The study of its molecular biology has allowed the cloning of several genes encoding oxidases of biotechnological interest, particularly in white and red biotechnology. Characterization of the operon encoding the tyrosinase responsible for melanin synthesis revealed that a second gene in that operon encodes a protein, PpoB2, which is involved in copper transfer to tyrosinase. This finding made PpoB2 the first protein in the COG5486 group to which a physiological role has been assigned. Another enzyme of interest described in M. mediterranea is a multicopper oxidase encoding a membrane-associated enzyme that shows oxidative activity on a wide range of substrates typical of both laccases and tyrosinases. Finally, an enzyme very specific for l-lysine, which oxidises this amino acid in epsilon position and that has received a new EC number (1.4.3.20), has also been described for M. mediterranea. Overall, the studies carried out on this bacterium illustrate the power of exploring the physiology of selected microorganisms to discover novel enzymes of biotechnological relevance. PMID:20411113

Violacein and related tryptophan metabolites produced by Chromobacterium violaceum: biosynthetic mechanism and pathway for construction of violacein core.

PubMed

Hoshino, Tsutomu

2011-09-01

Violacein is a natural violet pigment produced by several gram-negative bacteria, including Chromobacterium violaceum, Janthinobacterium lividum, and Pseudoalteromonas tunicata D2, among others. This pigment has potential medical applications as antibacterial, anti-trypanocidal, anti-ulcerogenic, and anticancer drugs. The structure of violacein consists of three units: a 5-hydroxyindole, an oxindole, and a 2-pyrrolidone. The biosynthetic origins of hydrogen, nitrogen, and carbon in the pyrrolidone nucleus were established by feeding experiments using various stable isotopically labeled tryptophans (Trps). Pro-S hydrogen of CH(2) at the 3-position of Trp is retained during biosynthesis. The nitrogen atom is exclusively from the α-amino group, and the skeletal carbon atoms originate from the side chains of the two Trp molecules. All three oxygen atoms in the violacein core are derived from molecular oxygen. The most interesting biosynthetic mechanism is the 1,2-shift of the indole nucleus on the left side of the violacein scaffold. The alternative Trp molecule is directly incorporated into the right side of the violacein core. This indole shift has been observed only in violacein biosynthesis, despite the large number of natural products having been isolated. There were remarkable advances in biosynthetic studies in 2006-2008. During the 3 years, most of the intermediates and the complete pathway were established. Two independent processes are involved: the enzymatic process catalyzed by the five proteins VioABCDE or the alternative nonenzymatic oxidative decarboxylation reactions. The X-ray crystallographic structure of VioE that mediates the indole rearrangement reaction was recently identified, and the mechanism of the indole shift is discussed here.

Very Short Peptides with Stable Folds

PubMed Central

Eidenschink, Lisa; Kier, Brandon L.; Huggins, Kelly N. L.; Andersen, Niels H.

2008-01-01

By combining a favorable turn sequence with a turn flanking Trp/Trp interaction and a C-terminal H-bonding interaction between a backbone amide and an i - 2 Trp ring, a particularly stable (ΔGU > 7 kJ/mol) truncated hairpin, Ac-WI-(D-Pro-D-Asn)-KWTG-NH2, results. In this construct and others with a W-(4-residue turn)-W motif in severely truncated hairpins, the C-terminal Trp is the edge residue in a well-defined face-to-edge (FtE) aryl/aryl interaction. Longer hairpins and those with six-residue turns retain the reversed “edge-to-face” Trp/Trp geometry first observed for the trpzip peptides. Mutational studies suggest that the W-(4-residue turn)-W interaction provides at least 3 kJ/mol of stabilization in excess of that due to the greater β-propensity of Trp. The β-propensity of Trp is context dependent; but, for the systems studied, always greater than that of Thr (by 0.4 - 4.7 kJ/mol). At non-H-bonded positions remote from the turn, two alternative edge-to-face geometries are observed and there is no evidence of additional stabilization due to the Trp/Trp interaction. The NMR structuring shift diagnostics of edge-to-face Trp/Trp, Trp/Lys π-cation, and Trp/Gly-HN interactions have been defined. The latter can give rise to > 3 ppm upfield shifts for the Gly-HN in -WXnG- units both in turns (n = 2) and at the C-termini (n = 1) of hairpins. Terminal YTG units result in somewhat smaller shifts (extrapolated to 2 ppm for 100% folding). In peptides with both the EtF and FtE W/W interaction geometries, Trp to Tyr mutations indicate that Trp is the preferred “face” residue in aryl/aryl pairings, presumably due to its greater π basicity. PMID:18831035

Oxidation of monohydric phenol substrates by tyrosinase. An oximetric study.

PubMed

Naish-Byfield, S; Riley, P A

1992-11-15

The purity of commercially available mushroom tyrosinase was investigated by non-denaturing PAGE. Most of the protein in the preparation migrated as a single band under these conditions. This band contained both tyrosinase and dopa oxidase activity. No other activity of either classification was found in the preparation. Oxygen consumption by tyrosinase during oxidation of the monohydric phenol substrates tyrosine and 4-hydroxyanisole (4HA) was monitored by oximetry in order to determine the stoichiometry of the reactions. For complete oxidation, the molar ratio of oxygen: 4HA was 1:1. Under identical conditions, oxidation of tyrosine required 1.5 mol of oxygen/mol of tyrosine. The additional oxygen uptake during tyrosine oxidation is due to the internal cyclization of dopaquinone to form cyclodopa, which undergoes a redox reaction with dopaquinone to form dopachrome and dopa, which is then oxidized by the enzyme, leading to an additional 0.5 mol of oxygen/mol of original substrate. Oxygen consumption for complete oxidation of 200 nmol of 4HA was constant over a range of concentrations of tyrosinase of 33-330 units/ml of substrate. The maximum rate of reaction was directly proportional to the concentration of tyrosinase, whereas the length of the lag phase decreased non-linearly with increasing tyrosinase concentration. Activation of the enzyme by exposure to citrate was not seen, nor was the lag phase abolished by exposure of the enzyme to low pH. Michaelis-Menten analysis of tyrosinase in which the lag phase is abolished by pre-exposure of the enzyme to a low concentration of dithiothreitol gave Km values for tyrosine and 4HA of 153 and 20 microM respectively.

ULK1 regulates melanin levels in MNT-1 cells independently of mTORC1.

PubMed

Kalie, Eyal; Razi, Minoo; Tooze, Sharon A

2013-01-01

Melanosomes are lysosome-related organelles that serve as specialized sites of melanin synthesis and storage in melanocytes. The progression of melanosomes through the different stages of their formation requires trafficking of specific proteins and membrane constituents in a sequential manner, which is likely to deploy ubiquitous cellular machinery along with melanocyte-specific proteins. Recent evidence revealed a connection between melanogenesis and the autophagy machinery, suggesting a novel role for members of the latter in melanocytes. Here we focused on ULK1, a key autophagy protein which is negatively regulated by mTORC1, to assess its potential role in melanogenesis in MNT-1 cells. We found that ULK1 depletion causes an increase in melanin levels, suggesting an inhibitory function for this protein in melanogenesis. Furthermore, this increase was accompanied by increased transcription of MITF (microphthalmia-associated transcription factor) and tyrosinase and by elevated protein levels of tyrosinase, the rate-limiting factor in melanin biogenesis. We also provide evidence to show that ULK1 function in this context is independent of the canonical ULK1 autophagy partners, ATG13 and FIP200. Furthermore we show that regulation of melanogenesis by ULK1 is independent of mTORC1 inhibition. Our data thus provide intriguing insights regarding the involvement of the key regulatory autophagy machinery in melanogenesis.

Anti-Oxidant, Anti-Aging, and Anti-Melanogenic Properties of the Essential Oils from Two Varieties of Alpinia zerumbet.

PubMed

Tu, Pham Thi Be; Tawata, Shinkichi

2015-09-14

Here, we investigated the anti-oxidant and anti-aging effects of essential oils (EOs) from the leaves of Alpinia zerumbet (tairin and shima) in vitro and anti-melanogenic effects in B16F10 melanoma cells. The anti-oxidant activities were performed with 2,2-diphenyl-1-picrylhydrazyl (DPPH); 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS); nitric oxide; singlet oxygen; hydroxyl radical scavenging; and xanthine oxidase. The inhibitory activities against collagenase, elastase, hyaluronidase, and tyrosinase were employed for anti-aging. The anti-melanogenic was assessed in B16F10 melanoma cells by melanin synthesis and intracellular tyrosinase inhibitory activity. The volatile chemical composition of the essential oil was analyzed with gas chromatography-mass spectrometry (GC/MS). The EO was a complex mixture mainly consisting of monoterpenes and sesquiterpenes. The results revealed that tairin and shima EOs showed strong anti-oxidant activities against DPPH and nitric oxide, hydroxyl radical scavenging activity, and xanthine oxidase inhibition. Compared to shima EO; tairin EO exhibited strong anti-aging activity by inhibiting collagenase, tyrosinase, hyaluronidase, and elastase (IC50 = 11 ± 0.1; 25 ± 1.2; 83 ± 1.6; and 213 ± 2 μg/mL, respectively). Both EOs inhibited intracellular tyrosinase activity; thus, reducing melanin synthesis. These results suggest that tairin EO has better anti-oxidant/anti-aging activity than shima EO, but both are equally anti-melanogenic.

Betaxanthins as Substrates for Tyrosinase. An Approach to the Role of Tyrosinase in the Biosynthetic Pathway of Betalains1

PubMed Central

Gandía-Herrero, Fernando; Escribano, Josefa; García-Carmona, Francisco

2005-01-01

Tyrosinase or polyphenol oxidase (EC 1.14.18.1) is the key enzyme in melanin biosynthesis and in the enzymatic browning of fruits and vegetables. The role of tyrosinase in the secondary metabolism of plants still remains unclear, but its implication in betalain biosynthesis has been proposed. Betalains are an important class of water-soluble pigments, characteristic of plants belonging to the order Caryophyllales. In this article, the betaxanthins, tyrosine-betaxanthin (portulacaxanthin II) and dopaxanthin, are reported to be physiological substrates for tyrosinase. The direct activity of tyrosinase on selected betaxanthins is characterized in depth, and conversion of tyrosine-betaxanthin to dopaxanthin and its further oxidation to a series of compounds are described. Identity of the reaction products was studied by high-performance liquid chromatography and electrospray ionization-mass spectrometry. Masses determined for the reaction products were the same in all cases, 389 m/z ([M + H]+) and equal to that determined for betanidin. Data indicate that dopaxanthin-quinone is obtained and evolves to more stable species by intramolecular cyclization. Kinetic parameters for tyrosinase acting on dopaxanthin were evaluated, showing a high affinity for this substrate (Km = 84.3 μm). The biosynthetic scheme of betalains is reviewed and a branch is proposed based on the description of physiological substrates for tyrosinase. Lampranthus productus, Glottiphylum oligocarpum, and Glottiphylum pigmaeum are described as sources of stereopure (2S/S)-dopaxanthin. PMID:15805475

Mutual synergy between catalase and peroxidase activities of the bifunctional enzyme KatG is facilitated by electron hole-hopping within the enzyme.

PubMed

Njuma, Olive J; Davis, Ian; Ndontsa, Elizabeth N; Krewall, Jessica R; Liu, Aimin; Goodwin, Douglas C

2017-11-10

KatG is a bifunctional, heme-dependent enzyme in the front-line defense of numerous bacterial and fungal pathogens against H 2 O 2 -induced oxidative damage from host immune responses. Contrary to the expectation that catalase and peroxidase activities should be mutually antagonistic, peroxidatic electron donors (PxEDs) enhance KatG catalase activity. Here, we establish the mechanism of synergistic cooperation between these activities. We show that at low pH values KatG can fully convert H 2 O 2 to O 2 and H 2 O only if a PxED is present in the reaction mixture. Stopped-flow spectroscopy results indicated rapid initial rates of H 2 O 2 disproportionation slowing concomitantly with the accumulation of ferryl-like heme states. These states very slowly returned to resting ( i.e. ferric) enzyme, indicating that they represented catalase-inactive intermediates. We also show that an active-site tryptophan, Trp-321, participates in off-pathway electron transfer. A W321F variant in which the proximal tryptophan was replaced with a non-oxidizable phenylalanine exhibited higher catalase activity and less accumulation of off-pathway heme intermediates. Finally, rapid freeze-quench EPR experiments indicated that both WT and W321F KatG produce the same methionine-tyrosine-tryptophan (MYW) cofactor radical intermediate at the earliest reaction time points and that Trp-321 is the preferred site of off-catalase protein oxidation in the native enzyme. Of note, PxEDs did not affect the formation of the MYW cofactor radical but could reduce non-productive protein-based radical species that accumulate during reaction with H 2 O 2 Our results suggest that catalase-inactive intermediates accumulate because of off-mechanism oxidation, primarily of Trp-321, and PxEDs stimulate KatG catalase activity by preventing the accumulation of inactive intermediates. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Evidence for a single class of somatostatin receptors in ground squirrel cerebral cortex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krantic, S.; Petrovic, V.M.; Quirion, R.

1989-01-01

In the present study we characterized high-affinity somatostatin (SRIF) binding sites (Kd = 2.06 +/- 0.32 nM and Bmax = 295 +/- 28 fmol/mg protein) in cerebral cortex membrane preparations of European ground squirrel using /sup 125/I-(Tyr0-D-Trp8)-SRIF14 as a radioligand. The inhibition of radioligand specific binding by SRIF14, as well as by its agonists (SRIF28, Tyr0-D-Trp8-SRIF14, SMS 201 995) was complete and monophasic, thus revealing a single population of somatostatinergic binding sites. Radioautographic analysis of /sup 125/I-(Tyr0-D-Trp8)-SRIF14 labeled brain sections confirmed the results of our biochemical study. The homogeneity of SRIF binding sites in the ground squirrel neocortex was notmore » dependent on the animal's life-cycle phase.« less

Structure-activity relationship of linear peptide Bu-His-DPhe-Arg-Trp-Gly-NH(2) at the human melanocortin-1 and -4 receptors: histidine substitution.

PubMed

Cheung, Adrian Wai-Hing; Danho, Waleed; Swistok, Joseph; Qi, Lida; Kurylko, Grazyna; Rowan, Karen; Yeon, Mitch; Franco, Lucia; Chu, Xin-Jie; Chen, Li; Yagaloff, Keith

2003-01-06

Systematic substitution of His(6) residue using non-selective hMC4R pentapeptide agonist (Bu-His(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2)) as the template led to the identification of Bu-Atc(6)(2-aminotetraline-2-carboxylic acid)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2) which showed moderate selectivity towards hMC4R over hMC1R. Further SAR studies resulted in the discovery of Penta-5-BrAtc(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2) and Penta-5-Me(2)NAtc(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2) which are potent hMC4R agonists and are inactive in hMC1R, hMC3R and hMC5R agonist assays.

Regulation of Drosophila transient receptor potential-like (TrpL) channels by phospholipase C-dependent mechanisms.

PubMed

Estacion, M; Sinkins, W G; Schilling, W P

2001-01-01

Patch clamp and fura-2 fluorescence were employed to characterize receptor-mediated activation of recombinant Drosophila TrpL channels expressed in Sf9 insect cells. TrpL was activated by receptor stimulation and by exogenous application of diacylglycerol (DAG) or poly-unsaturated fatty acids (PUFAs). Activation of TrpL was blocked more than 70% by U73122, suggesting that the effect of these agents was dependent upon phospholipase C (PLC). In fura-2 assays, extracellular application of bacterial phosphatidylinositol (PI)-PLC or phosphatidylcholine (PC)-PLC caused a transient increase in TrpL channel activity, the magnitude of which was significantly less than that observed following receptor stimulation. TrpL channels were also activated in excised inside-out patches by cytoplasmic application of mammalian PLC-b2, bacterial PI-PLC and PC-PLC, but not by phospholipase D (PLD). The phospholipases had little or no effect when examined in either whole-cell or cell-attached configurations.TrpL activity was inhibited by addition of phosphatidylinositol-4,5-bisphosphate (PIP2) to excised inside-out membrane patches exhibiting spontaneous channel activity or to patches pre-activated by treatment with PLC. The effect was reversible, specific for PIP2, and was not observed with phosphatidylethanolamine (PE), PI, PC or phosphatidylserine (PS). However, antibodies against PIP2 consistently failed to activate TrpL in inside-out patches. It is concluded that both the hydrolysis of PIP2 and the generation of DAG are required to rapidly activate TrpL following receptor stimulation, or that some other PLC-dependent mechanism plays a crucial role in the activation process.

Characterization of inhibitory effects of the potential therapeutic inhibitors, benzoic acid and pyridine derivatives, on the monophenolase and diphenolase activities of tyrosinase.

PubMed

Gheibi, Nematollah; Taherkhani, Negar; Ahmadi, Abolfazl; Haghbeen, Kamahldin; Ilghari, Dariush

2015-02-01

Involvement of tyrosinase in the synthesis of melanin and cell signaling pathway has made it an attractive target in the search for therapeutic inhibitors for treatment of different skin hyperpigmentation disorders and melanoma cancers. In the present study, we conducted a comprehensive kinetic analysis to understand the mechanisms of inhibition imposed by 2-amino benzoic acid, 4-amino benzoic acid, nicotinic acid, and picolinic acid on the monophenolase and diphenolase activities of the mushroom tyrosinase, and then MTT assay was exploited to evaluate their toxicity on the melanoma cells. Kinetic analysis revealed that nicotinic acid and picolinic acid competitively restricted the monophenolase activity with inhibition constants (Ki) of 1.21 mM and 1.97 mM and the diphenolase activity with Kis of 2.4 mM and 2.93 mM, respectively. 2-aminobenzoic acid and 4-aminobenzoic acid inhibited the monophenolase activity in a non-competitive fashion with Kis of 5.15 µM and 3.8 µM and the diphenolase activity with Kis of 4.72 µM and 20 µM, respectively. Our cell-based data revealed that only the pyridine derivatives imposed cytotoxicity in melanoma cells. Importantly, the concentrations of the inhibitors leading to 50% decrease in the cell density (IC50) were comparable to those causing 50% drop in the enzyme activity, implying that the observed cytotoxicity is highly likely due to the tyrosinase inhibition. Moreover, our cell-based data exhibited that the pyridine derivatives acted as anti-proliferative agents, perhaps inducing cytotoxicity in the melanoma cells through inhibition of the tyrosinase activities.

Characterization of inhibitory effects of the potential therapeutic inhibitors, benzoic acid and pyridine derivatives, on the monophenolase and diphenolase activities of tyrosinase

PubMed Central

Gheibi, Nematollah; Taherkhani, Negar; Ahmadi, Abolfazl; Haghbeen, Kamahldin; Ilghari, Dariush

2015-01-01

Objective(s): Involvement of tyrosinase in the synthesis of melanin and cell signaling pathway has made it an attractive target in the search for therapeutic inhibitors for treatment of different skin hyperpigmentation disorders and melanoma cancers. Materials and Methods: In the present study, we conducted a comprehensive kinetic analysis to understand the mechanisms of inhibition imposed by 2-amino benzoic acid, 4-amino benzoic acid, nicotinic acid, and picolinic acid on the monophenolase and diphenolase activities of the mushroom tyrosinase, and then MTT assay was exploited to evaluate their toxicity on the melanoma cells. Results: Kinetic analysis revealed that nicotinic acid and picolinic acid competitively restricted the monophenolase activity with inhibition constants (Ki) of 1.21 mM and 1.97 mM and the diphenolase activity with Kis of 2.4 mM and 2.93 mM, respectively. 2-aminobenzoic acid and 4-aminobenzoic acid inhibited the monophenolase activity in a non-competitive fashion with Kis of 5.15 µM and 3.8 µM and the diphenolase activity with Kis of 4.72 µM and 20 µM, respectively. Conclusion: Our cell-based data revealed that only the pyridine derivatives imposed cytotoxicity in melanoma cells. Importantly, the concentrations of the inhibitors leading to 50% decrease in the cell density (IC50) were comparable to those causing 50% drop in the enzyme activity, implying that the observed cytotoxicity is highly likely due to the tyrosinase inhibition. Moreover, our cell-based data exhibited that the pyridine derivatives acted as anti-proliferative agents, perhaps inducing cytotoxicity in the melanoma cells through inhibition of the tyrosinase activities. PMID:25810885

Cleavage Specificity of Mycobacterium tuberculosis ClpP1P2 Protease and Identification of Novel Peptide Substrates and Boronate Inhibitors with Anti-bacterial Activity*

PubMed Central

Akopian, Tatos; Kandror, Olga; Tsu, Christopher; Lai, Jack H.; Wu, Wengen; Liu, Yuxin; Zhao, Peng; Park, Annie; Wolf, Lisa; Dick, Lawrence R.; Rubin, Eric J.; Bachovchin, William; Goldberg, Alfred L.

2015-01-01

The ClpP1P2 protease complex is essential for viability in Mycobacteria tuberculosis and is an attractive drug target. Using a fluorogenic tripeptide library (Ac-X3X2X1-aminomethylcoumarin) and by determining specificity constants (kcat/Km), we show that ClpP1P2 prefers Met ≫ Leu > Phe > Ala in the X1 position, basic residues or Trp in the X2 position, and Pro ≫ Ala > Trp in the X3 position. We identified peptide substrates that are hydrolyzed up to 1000 times faster than the standard ClpP substrate. These positional preferences were consistent with cleavage sites in the protein GFPssrA by ClpXP1P2. Studies of ClpP1P2 with inactive ClpP1 or ClpP2 indicated that ClpP1 was responsible for nearly all the peptidase activity, whereas both ClpP1 and ClpP2 contributed to protein degradation. Substrate-based peptide boronates were synthesized that inhibit ClpP1P2 peptidase activity in the submicromolar range. Some of them inhibited the growth of Mtb cells in the low micromolar range indicating that cleavage specificity of Mtb ClpP1P2 can be used to design novel anti-bacterial agents. PMID:25759383

Human biallelic MFN2 mutations induce mitochondrial dysfunction, upper body adipose hyperplasia, and suppression of leptin expression.

PubMed

Rocha, Nuno; Bulger, David A; Frontini, Andrea; Titheradge, Hannah; Gribsholt, Sigrid Bjerge; Knox, Rachel; Page, Matthew; Harris, Julie; Payne, Felicity; Adams, Claire; Sleigh, Alison; Crawford, John; Gjesing, Anette Prior; Bork-Jensen, Jette; Pedersen, Oluf; Barroso, Inês; Hansen, Torben; Cox, Helen; Reilly, Mary; Rossor, Alex; Brown, Rebecca J; Taylor, Simeon I; McHale, Duncan; Armstrong, Martin; Oral, Elif A; Saudek, Vladimir; O'Rahilly, Stephen; Maher, Eamonn R; Richelsen, Bjørn; Savage, David B; Semple, Robert K

2017-04-19

MFN2 encodes mitofusin 2, a membrane-bound mediator of mitochondrial membrane fusion and inter-organelle communication. MFN2 mutations cause axonal neuropathy, with associated lipodystrophy only occasionally noted, however homozygosity for the p.Arg707Trp mutation was recently associated with upper body adipose overgrowth. We describe similar massive adipose overgrowth with suppressed leptin expression in four further patients with biallelic MFN2 mutations and at least one p.Arg707Trp allele. Overgrown tissue was composed of normal-sized, UCP1-negative unilocular adipocytes, with mitochondrial network fragmentation, disorganised cristae, and increased autophagosomes. There was strong transcriptional evidence of mitochondrial stress signalling, increased protein synthesis, and suppression of signatures of cell death in affected tissue, whereas mitochondrial morphology and gene expression were normal in skin fibroblasts. These findings suggest that specific MFN2 mutations cause tissue-selective mitochondrial dysfunction with increased adipocyte proliferation and survival, confirm a novel form of excess adiposity with paradoxical suppression of leptin expression, and suggest potential targeted therapies.

Polyphenoloxidases immobilized in organic gels: Properties and applications in the detoxification of aromatic compounds

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crecchio, C.; Ruggiero, P.; Pizzigallo, M.D.R.

1995-12-20

Gelatine gels originate from water in oil microemulsions in which the ternary system consists of isooctane/sulfosuccinic acid bis [2-ethyl hexyl] ester/water; the solubilization of gelatin in the water pool of these microemulsions transforms them into viscous gels in which it is possible to cosolubilize various reactive molecules. These gels were used to immobilize two phenoloxidases, a laccase from Trametes versicolor and a tyrosinase from mushroom. The best balance between gel retention and catalytic activity was reached at a gelatine concentration of 2.5% (w/v) in the case of tyrosinase, while laccase immobilization was independent of gelatine concentration. Both enzymes kept themore » same optimum pH as the corresponding soluble controls, while a partial loss of activity was observed when they were immobilized. Immobilized enzymes showed an increased stability when incubated for several days at 4 C with a very low release from the gels in the incubation solutions. The immobilization of tyrosinase and of laccase enhanced stability to thermal inactivation. Furthermore, gel-entrapped tyrosinase was almost completely preserved from proteolysis: more than 80% of the activity was maintained, while only 25% of the soluble control activity was detected after the same proteolytic treatments. A column packed with gel-immobilized tyrosinase was used to demonstrate that enzymes immobilized with this technique may be reused several times in the same reaction without loosing their efficiency. Finally, gel-entrapped tyrosinase and laccase were capable of removing naturally occurring and xenobiotic aromatic compounds from aqueous suspensions with different degrees of efficiency.« less

Targeted Recombinant Progeny: a design for ultra-high resolution mapping of Quantitative Trait Loci in crosses between inbred or pure lines.

PubMed

Heifetz, Eliyahu M; Soller, Morris

2015-07-07

High-resolution mapping of the loci (QTN) responsible for genetic variation in quantitative traits is essential for positional cloning of candidate genes, and for effective marker assisted selection. The confidence interval (QTL) flanking the point estimate of QTN-location is proportional to the number of individuals in the mapping population carrying chromosomes recombinant in the given interval. Consequently, many designs for high resolution QTN mapping are based on increasing the proportion of recombinants in the mapping population. The "Targeted Recombinant Progeny" (TRP) design is a new design for high resolution mapping of a target QTN in crosses between pure, or inbred lines. It is a three-generation procedure generating a large number of recombinant individuals within a QTL previously shown to contain a QTN. This is achieved by having individuals that carry chromosomes recombinant across the target QTL interval as parents of a large mapping population; most of whom will therefore carry recombinant chromosomes targeted to the given QTL. The TRP design is particularly useful for high resolution mapping of QTN that differentiate inbred or pure lines, and hence are not amenable to high resolution mapping by genome-wide association tests. In the absence of residual polygenic variation, population sizes required for achieving given mapping resolution by the TRP-F2 design relative to a standard F2 design ranged from 0.289 for a QTN with standardized allele substitution effect = 0.2, mapped to an initial QTL of 0.2 Morgan to 0.041 for equivalent QTN mapped to an initial QTL of 0.02 M. In the presence of residual polygenic variation, the relative effectiveness of the TRP design ranges from 1.068 to 0.151 for the same initial QTL intervals and QTN effect. Thus even in the presence of polygenic variation, the TRP can still provide major savings. Simulation showed that mapping by TRP should be based on 30-50 markers spanning the initial interval; and on at least 50 or more G2 families representing this number of recombination points,. The TRP design can be an effective procedure for achieving high and ultra-high mapping resolution of a target QTN previously mapped to a known confidence interval (QTL).

Assessment of local friction in protein folding dynamics using a helix cross-linker.

PubMed

Markiewicz, Beatrice N; Jo, Hyunil; Culik, Robert M; DeGrado, William F; Gai, Feng

2013-11-27

Internal friction arising from local steric hindrance and/or the excluded volume effect plays an important role in controlling not only the dynamics of protein folding but also conformational transitions occurring within the native state potential well. However, experimental assessment of such local friction is difficult because it does not manifest itself as an independent experimental observable. Herein, we demonstrate, using the miniprotein trp-cage as a testbed, that it is possible to selectively increase the local mass density in a protein and hence the magnitude of local friction, thus making its effect directly measurable via folding kinetic studies. Specifically, we show that when a helix cross-linker, m-xylene, is placed near the most congested region of the trp-cage it leads to a significant decrease in both the folding rate (by a factor of 3.8) and unfolding rate (by a factor of 2.5 at 35 °C) but has little effect on protein stability. Thus, these results, in conjunction with those obtained with another cross-linked trp-cage and two uncross-linked variants, demonstrate the feasibility of using a nonperturbing cross-linker to help quantify the effect of internal friction. In addition, we estimate that a m-xylene cross-linker could lead to an increase in the roughness of the folding energy landscape by as much as 0.4-1.0k(B)T.

RUTBC1 Functions as a GTPase-activating Protein for Rab32/38 and Regulates Melanogenic Enzyme Trafficking in Melanocytes.

PubMed

Marubashi, Soujiro; Shimada, Hikaru; Fukuda, Mitsunori; Ohbayashi, Norihiko

2016-01-15

Two cell type-specific Rab proteins, Rab32 and Rab38 (Rab32/38), have been proposed as regulating the trafficking of melanogenic enzymes, including tyrosinase and tyrosinase-related protein 1 (Tyrp1), to melanosomes in melanocytes. Like other GTPases, Rab32/38 function as switch molecules that cycle between a GDP-bound inactive form and a GTP-bound active form; the cycle is thought to be regulated by an activating enzyme, guanine nucleotide exchange factor (GEF), and an inactivating enzyme, GTPase-activating protein (GAP), which stimulates the GTPase activity of Rab32/38. Although BLOC-3 has already been identified as a Rab32/38-specific GEF that regulates the trafficking of tyrosinase and Tyrp1, no physiological GAP for Rab32/38 in melanocytes has ever been identified, and it has remained unclear whether Rab32/38 is involved in the trafficking of dopachrome tautomerase, another melanogenic enzyme, in mouse melanocytes. In this study we investigated RUTBC1, which was originally characterized as a Rab9-binding protein and GAP for Rab32 and Rab33B in vitro, and the results demonstrated that RUTBC1 functions as a physiological GAP for Rab32/38 in the trafficking of all three melanogenic enzymes in mouse melanocytes. The results of this study also demonstrated the involvement of Rab9A in the regulation of the RUTBC1 localization and in the trafficking of all three melanogenic enzymes. We discovered that either excess activation or inactivation of Rab32/38 achieved by manipulating RUTBC1 inhibits the trafficking of all three melanogenic enzymes. These results collectively indicate that proper spatiotemporal regulation of Rab32/38 is essential for the trafficking of all three melanogenic enzymes in mouse melanocytes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The effect of oxaloacetic acid on tyrosinase activity and structure: Integration of inhibition kinetics with docking simulation.

PubMed

Gou, Lin; Lee, Jinhyuk; Hao, Hao; Park, Yong-Doo; Zhan, Yi; Lü, Zhi-Rong

2017-08-01

Oxaloacetic acid (OA) is naturally found in organisms and well known as an intermediate of citric acid cycle producing ATP. We evaluated the effects of OA on tyrosinase activity and structure via integrating methods of enzyme kinetics and computational simulations. OA was found to be a reversible inhibitor of tyrosinase and its induced mechanism was the parabolic non-competitive inhibition type (IC 50 =17.5±0.5mM and K i =6.03±1.36mM). Kinetic measurements by real-time interval assay showed that OA induced multi-phasic inactivation process composing with fast (k 1 ) and slow (k 2 ) phases. Spectrofluorimetry studies showed that OA mainly induced regional changes in the active site of tyrosinase accompanying with hydrophobic disruption at high dose. The computational docking simulations further revealed that OA could interact with several residues near the tyrosinase active site pocket such as HIS61, HIS259, HIS263, and VAL283. Our study provides insight into the mechanism by which energy producing intermediate such as OA inhibit tyrosinase and OA is a potential natural anti-pigmentation agent. Copyright © 2017 Elsevier B.V. All rights reserved.

Standardized ileal digestible tryptophan-to-lysine ratios in growing pigs fed corn-based and non-corn-based diets.

PubMed

Quant, A D; Lindemann, M D; Kerr, B J; Payne, R L; Cromwell, G L

2012-04-01

Two 21-d experiments were conducted to determine the optimum standardized ileal digestible (SID) Trp:Lys in growing pigs fed corn-based diets compared with non-corn-based diets. The primary response variables in both experiments were ADG and plasma urea N (PUN) concentrations with the optimum SID Trp:Lys determined using broken-line analysis. Experiment 1 evaluated the optimum SID Trp:Lys in growing pigs fed corn-based diets consisting primarily of corn with minor inclusion of Canadian field peas and corn gluten meal to keep the SID Trp:Lys low. This experiment used 120 crossbred pigs (initial BW: 25.73 ± 2.46 kg) that were blocked by sex and initial BW and allotted to 5 SID Trp:Lys with 5 pens each for the first 4 treatments and 4 pens for the last treatment and 5 pigs/pen. Diets were formulated by the addition of supplemental Trp to create various SID Trp:Lys (12.77, 14.07, 15.50, 16.91, and 17.94%) with a constant SID Lys of 0.66%, which was determined to be 83% of the Lys requirement for pigs at this location. As the SID Trp:Lys increased from 12.77 to 17.94%, ADG increased (0.562, 0.648, 0.788, 0.787, and 0.815 kg/d) linearly (P < 0.001) and quadratically (P = 0.009), resulting in an optimum SID Trp:Lys of 15.73% (P < 0.001). Plasma urea N decreased (10.43, 9.30, 8.21, 8.55, and 9.25 mg/dL) linearly (P = 0.069) and quadratically (P = 0.015), resulting in an optimum SID Trp:Lys of 15.83% (P = 0.007). Experiment 2 evaluated the optimum SID Trp:Lys in growing pigs fed non-corn-based diets consisting primarily of barley and Canadian field peas, with smaller proportions of corn and wheat. Experiment 2 used 120 crossbred pigs (initial BW: 28.49 ± 2.92 kg) that were allotted to 5 increasing SID Trp:Lys (13.05, 14.32, 15.59, 16.85, and 18.11%; 0.66% SID Lys) in the same manner as Exp. 1. As SID Trp:Lys increased in Exp. 2, ADG increased linearly (P = 0.007) with the optimum SID Trp:Lys of 15.99% (P = 0.048). Plasma urea N concentrations decreased linearly (P = 0.056) and quadratically (P = 0.067) as SID Trp:Lys increased, resulting in an optimum SID Trp:Lys of 15.29% (P = 0.009). Averaging the break point values for ADG and PUN obtained from broken-line analysis for Exp. 1 and 2 produced optimum SID Trp:Lys of 15.78 and 15.64%, respectively. Based on the results from these 2 experiments, it seems that the optimum SID Trp:Lys is virtually unaffected by the dietary feedstuffs used as long as the diets are formulated on an SID AA basis.

The Waardenburg Syndrome Type 4 Gene, SOX10, Is a Novel Tumor-associated Antigen Identified in a Patient with a Dramatic Response to Immunotherapy

PubMed Central

Khong, Hung T.; Rosenberg, Steven A.

2008-01-01

In this study, we have identified, for the first time, the presence of de novo cellular immune reactivity against the transcription factor SOX10, using tumor-infiltrating lymphocytes obtained from a patient who experienced a dramatic clinical response to immunotherapy. SOX10 acts as a critical transactivator of tyrosinase-related protein-2 during melanoblast development and a potent transactivator of micropthalmia-associated transcription factor, which is considered to be a master gene that controls the development and postnatal survival of melanocytes. Mutations in SOX10 result in Waardenburg syndrome type 4. The overlapping epitopes AWISKPPGV and SAWISKPPGV, designated SOX10: 332–340 and SOX10: 331–340, respectively, were recognized by tumor-infiltrating lymphocyte clone M37 in an HLA-A2-restricted fashion. PMID:12036907

Astaxanthin and withaferin A block paracrine cytokine interactions between UVB-exposed human keratinocytes and human melanocytes via the attenuation of endothelin-1 secretion and its downstream intracellular signaling.

PubMed

Niwano, Takao; Terazawa, Shuko; Nakajima, Hiroaki; Wakabayashi, Yuki; Imokawa, Genji

2015-06-01

Paracrine interactions between keratinocytes and melanocytes via cytokines play an essential role in regulating pigmentation in epidermal hyperpigmentary disorders. There is an urgent need for a human epidermal model in which melanogenic paracrine interactions between UVB-exposed keratinocytes and melanocytes can be precisely evaluated because human epidermal equivalents consisting of multilayered keratinocytes and melanocytes have significant limitations in this respect. To resolve this challenge, we established a co-culture system with cell inserts using human keratinocytes and human melanocytes that serves as an appropriate new model for UVB-induced hyperpigmentation. Using that new model, we examined the blocking effects of two natural chemicals, astaxanthin and withaferin A, on paracrine cytokine interactions between UVB-exposed keratinocytes and melanocytes and characterized their mechanisms of action. RT-PCR analysis showed that co-culture of human keratinocytes that had been exposed to UVB significantly stimulated human melanocytes to increase their expression of genes encoding microphthalmia-associated transcription factor, tyrosinase and tyrosinase-related protein 1. The catalytic activity of tyrosinase was also increased. ELISA assays revealed that UVB significantly increased the secretion of interleukin-1α, interleukin-6/8, granulocyte macrophage stimulatory factor and endothelin-1 but not α-melanocyte stimulating hormone. The addition of an endothelin-1 neutralizing antibody significantly abrogated the increase of tyrosinase activity. Post-irradiation treatment with astaxanthin or withaferin A significantly abolished the up-regulation of tyrosinase activity induced by UVB. Treatment with astaxanthin or withaferin A significantly reduced the increased levels of interleukin-1α, interleukin-6/8, granulocyte macrophage stimulatory factor and endothelin-1. Withaferin A but not astaxanthin also significantly abrogated the endothelin-1-stimulated activity of tyrosinase in melanocytes. Western blot analysis of intracellular signaling factors revealed that withaferin A but not astaxanthin significantly abolished the endothelin-1-stimulated phosphorylation of Raf-1, MEK, ERK, MITF and CREB in human melanocytes. These results demonstrate that this co-culture system is an appropriate model to characterize melanogenic paracrine interactions and that astaxanthin and withaferin A serve as potent inhibitors of those interactions. Their effects are caused not only by down-regulating the increased secretion of an intrinsic melanogenic cytokine, endothelin-1, by UVB-exposed human keratinocytes, but also by interrupting the endothelin-1-triggered downstream intracellular signaling between protein kinase C and Raf-1 in human melanocytes (only for withaferin A). Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular dissection of the interaction between the SH3 domain and the SH2-Kinase Linker region in PTK6.

PubMed

Kim, Han Ie; Jung, Jinwon; Lee, Eun-Saem; Kim, Yong-Chul; Lee, Weontae; Lee, Seung-Taek

2007-11-03

PTK6 (also known as Brk) is an intracellular tyrosine kinase that contains SH3, SH2, and tyrosine kinase catalytic (Kinase) domains. The SH3 domain of PTK6 interacts with the N-terminal half of the linker (Linker) region between the SH2 and Kinase domains. Site-directed mutagenesis and surface plasmon resonance studies showed that a tryptophan residue (Trp44) in the SH3 domain and proline residues in the Linker region, in the order of Pro177, Pro175, and Pro179, contribute to the interaction. The three-dimensional modeled structure of the SH3-Linker complex was in agreement with the biochemical data. Disruption of the intramolecular interaction between the SH3 domain and the Linker region by mutation of Trp44, Pro175, Pro177, and Pro179 markedly increased the catalytic activity of PTK6 in HEK 293 cells. These results demonstrate that Trp44 in the SH3 domain and Pro177, Pro175, and Pro179 in the N-terminal half of the Linker region play important roles in the SH3-Linker interaction to maintain the protein in an inactive conformation along with the phosphorylated Tyr447-SH2 interaction.

The Variant p.(Arg183Trp) in SPTLC2 Causes Late-Onset Hereditary Sensory Neuropathy.

PubMed

Suriyanarayanan, Saranya; Auranen, Mari; Toppila, Jussi; Paetau, Anders; Shcherbii, Maria; Palin, Eino; Wei, Yu; Lohioja, Tarja; Schlotter-Weigel, Beate; Schön, Ulrike; Abicht, Angela; Rautenstrauss, Bernd; Tyynismaa, Henna; Walter, Maggie C; Hornemann, Thorsten; Ylikallio, Emil

2016-03-01

Hereditary sensory and autonomic neuropathy 1 (HSAN1) is an autosomal dominant disorder that can be caused by variants in SPTLC1 or SPTLC2, encoding subunits of serine palmitoyl-CoA transferase. Disease variants alter the enzyme's substrate specificity and lead to accumulation of neurotoxic 1-deoxysphingolipids. We describe two families with autosomal dominant HSAN1C caused by a new variant in SPTLC2, c.547C>T, p.(Arg183Trp). The variant changed a conserved amino acid and was not found in public variant databases. All patients had a relatively mild progressive distal sensory impairment, with onset after age 50. Small fibers were affected early, leading to abnormalities on quantitative sensory testing. Sural biopsy revealed a severe chronic axonal neuropathy with subtotal loss of myelinated axons, relatively preserved number of non-myelinated fibers and no signs for regeneration. Skin biopsy with PGP9.5 labeling showed lack of intraepidermal nerve endings early in the disease. Motor manifestations developed later in the disease course, but there was no evidence of autonomic involvement. Patients had elevated serum 1-deoxysphingolipids, and the variant protein produced elevated amounts of 1-deoxysphingolipids in vitro, which proved the pathogenicity of the variant. Our results expand the genetic spectrum of HSAN1C and provide further detail about the clinical characteristics. Sequencing of SPTLC2 should be considered in all patients presenting with mild late-onset sensory-predominant small or large fiber neuropathy.

Tryptophan metabolism, growth responses, and postprandial insulin metabolism in weaned piglets according to the dietary provision of niacin (vitamin B) and tryptophan.

PubMed

Matte, J Jacques; Corrent, Etienne; Simongiovanni, Aude; Le Floc'h, Nathalie

2016-05-01

The present experiment aimed to determine if Trp metabolism and growth responses to dietary Trp are modulated by dietary niacin (B) in weanling piglets. Piglets weaned at 3 wk of age were distributed 1 wk later (7.6 kg of BW, SEM = 0.1) in 52 pens of 2 animals each. Pens were assigned to factorial dietary treatments with 2 additions of B, 15 mg/kg (LB3) vs. 45 mg/kg (HB3) and 2 additions of Trp, 0 mg/kg (-Trp) vs. 1 mg/kg (+Trp) for Trp to Lys ratios of 0.16 vs. 0.24, respectively. Growth performance was recorded every week from 4 to 10 wk of age. Fasting blood samples were taken at 4, 6, 8, and 10 wk of age. From 4 to 10 wk of age, ADFI tended to be greater ( = 0.10) in HB3 than in LB3 (1,031 vs. 1,003 g, SEM = 7), and this was reflected ( = 0.06) by ADG (642 vs. 623 g, SEM = 7). No treatment effect was observed on plasma Trp or kynurenine (Kyn), an intermediate metabolite of Trp catabolism. The response of plasma nicotinamide (Nam), a product of Trp catabolism and an indicator of B status, to dietary B differed according to treatments (interaction Trp × B, < 0.01) with values of 1.4, 3.3, 4.1, and 5.3 μM (SEM = 0.1) in LB3-Trp, HB3-Trp, LB3+Trp, and HB3+Trp, respectively. At 11 wk of age, postprandial blood samples were collected from 6 piglets per treatment for measurements of Trp and insulin metabolism. Postprandial plasma Trp (96.4 vs. 72.2 μ, SEM = 3.4) and Kyn (1.7 vs. 1.3 μ, SEM = 0.1) were greater ( < 0.01) in +Trp vs. -Trp. Postprandial plasma Nam was greater ( < 0.01) in +Trp vs. -Trp (3.4 vs. 1.9 µ, SEM = 0.3) and in HB3 vs. LB3 piglets (3.4 vs. 1.9 µ, SEM = 0.3). Postprandial peaks and areas under curves of C-peptide and glucose were not affected by treatments. However, for insulin, the postprandial peak was lower in +Trp vs. -Trp piglets in the LB3 group (interaction Trp × B, < 0.05); values were 1.3, 1.0, 0.7, and 1.0 n (SEM = 0.1) in LB3-Trp, HB3-Trp, LB3+Trp, and HB3+Trp, respectively. The peak value of the molar ratio insulin:C-peptide was lower ( < 0.02) in +Trp vs. -Trp piglets (0.56 vs. 0.73, SEM = 0.05). The responses observed on growth performance and plasma Nam suggest that the LB3 level was suboptimal. According also to plasma Nam, it appears that supplemental dietary B can attenuate Trp oxidation toward niacin metabolites. Postprandial profiles of insulin and C-peptide indicate that Trp action is exerted on insulin clearance rather than on insulin secretion in piglets, without apparent consequences on glucose utilization.

[Association of beta 3-adrenergic receptor gene with obesity in patients with type 2 diabetes mellitus].

PubMed

Chen, Y; Xu, Y; Zhou, L

2001-09-01

To investigate the association between the mutation of beta 3-adrenergoc receptor gene and obesity in patients with type 2 diabetes. Body mass, waist-hip ratio, blood pressure and blood lipids were measured in 154 type 2 diabetic patients. Polymerase chain reaction and the restriction fragment length polymorphism analysis were used to determine the wild, heterozygous and homozygous forms of beta 3-adrenergoc receptor gene. The frequency of the Trp64Arg mutation was 42.5% and the frequency of Arg64 allele was 22.6%. The mutation frequency of the genetic types was significantly different between the obese and non-obese type 2 diabetes mellitus patients. The body mass, systolic blood pressure, diastolic blood pressure, HDL-cholesterol were significantly different, when those with Trp64Arg heterozygous were compared with those with Trp64 homozygous. The genetic mutation of beta 3-adrenegoc receptor in patients with type 2 diabetes is probably related to obesity.

Tyrosinase-Based Biosensors for Selective Dopamine Detection

PubMed Central

Florescu, Monica; David, Melinda

2017-01-01

A novel tyrosinase-based biosensor was developed for the detection of dopamine (DA). For increased selectivity, gold electrodes were previously modified with cobalt (II)-porphyrin (CoP) film with electrocatalytic activity, to act both as an electrochemical mediator and an enzyme support, upon which the enzyme tyrosinase (Tyr) was cross-linked. Differential pulse voltammetry was used for electrochemical detection and the reduction current of dopamine-quinone was measured as a function of dopamine concentration. Our experiments demonstrated that the presence of CoP improves the selectivity of the electrode towards dopamine in the presence of ascorbic acid (AA), with a linear trend of concentration dependence in the range of 2–30 µM. By optimizing the conditioning parameters, a separation of 130 mV between the peak potentials for ascorbic acid AA and DA was obtained, allowing the selective detection of DA. The biosensor had a sensitivity of 1.22 ± 0.02 µA·cm−2·µM−1 and a detection limit of 0.43 µM. Biosensor performances were tested in the presence of dopamine medication, with satisfactory results in terms of recovery (96%), and relative standard deviation values below 5%. These results confirmed the applicability of the biosensors in real samples such as human urine and blood serum. PMID:28590453

Receptors for luteinizing hormone-releasing hormone (LHRH) in Dunning R3327 prostate cancers and rat anterior pituitaries after treatment with a sustained delivery system of LHRH antagonist SB-75.

PubMed

Srkalovic, G; Bokser, L; Radulovic, S; Korkut, E; Schally, A V

1990-12-01

Membrane receptors for LHRH were evaluated in Dunning R3327 prostate cancers and rat anterior pituitaries. The receptors were characterized both in untreated animals and after in vivo treatment with microcapsules of the agonist D-Trp6-LHRH and a sustained delivery system releasing different doses (23.8, 47.6, 71.4 micrograms/day) of LHRH antagonist [Ac-D-Nal(2)1-D-Phe(4Cl)2-D-Pal(3)3,D-Cit6, D-Ala10]-LHRH (SB-75). The therapy, which lasted 8 weeks, strongly inhibited tumor growth. A group of normal Sprague-Dawley male rats was also treated for 6 weeks with microcapsules of SB-75 releasing 25 micrograms/day. In the Dunning tumors from the control group, ligand [125I, D-Trp6]-LHRH was bound to two classes of binding sites [dissociation constant, class a (Kda) = 1.01 +/- 0.30 x 10(-9) M; Kdb = 1.71 +/- 0.41 x 10(-6) M; maximal binding capacity of receptors, class a (Bmaxa) = 48.66 +/- 22.13 fmol/mg of protein; Bmaxb = 92.10 +/- 29.40 pmol/mg of protein] in both kinetic and equilibrium studies. Treatment with D-Trp6-LHRH produced down-regulation of membrane receptors for LHRH in Dunning tumors. Microcapsules of SB-75 resulted in dose-dependent up-regulation of binding sites for LHRH in Dunning tumors. Analysis of the binding data showed that interaction of labeled D-Trp6-LHRH with binding sites in anterior pituitaries was consistent with the presence of a single class of noncooperative receptors (Kd = 43.75 x 10(-9) M; Bmax = 5.25 pmol/mg membrane proteins). Prolonged treatment with microcapsules of D-Trp6-LHRH reduced both Bmax and Kd. Lower doses of SB-75 (23.8 and 47.6 micrograms/day) produced up-regulation, whereas the highest dose (71.4 micrograms/day) resulted in down-regulation of binding sites for LHRH in rat pituitaries. In normal Sprague-Dawley rats, treatment with microcapsules of SB-75 (25 micrograms/day) for 6 weeks produced a slight increase in the number of available binding sites (Bmax = 2.35 +/- 0.82 pmol/mg membrane protein) and a moderate decrease in affinity (Kd = 35.10 +/- 15.19 x 10(-9) M) of pituitary membrane receptors for LHRH. The findings provide additional support for the view that LHRH analogs exert direct effects on tumor cells. Our findings indicate that prolonged treatment with high doses of modern LHRH antagonists produces down-regulation of pituitary receptors. Our work in tumors also implies that some differences may exist between LHRH receptors, even in the same tissue, leading to the concept of subclassification of LHRH receptors.

Design of specific peptide inhibitors of phospholipase A2: structure of a complex formed between Russell's viper phospholipase A2 and a designed peptide Leu-Ala-Ile-Tyr-Ser (LAIYS).

PubMed

Chandra, Vikas; Jasti, Jayasankar; Kaur, Punit; Dey, Sharmistha; Srinivasan, A; Betzel, Ch; Singh, T P

2002-10-01

Phospholipase A(2) (EC 3.1.1.4) is a key enzyme of the cascade mechanism involved in the production of proinflammatory compounds known as eicosanoids. The binding of phospholipase A(2) to membrane surfaces and the hydrolysis of phospholipids are thought to involve the formation of a hydrophobic channel into which a single substrate molecule diffuses before cleavage. In order to regulate the production of proinflammatory compounds, a specific peptide inhibitor of PLA(2), Leu-Ala-Ile-Tyr-Ser, has been designed. Phospholipase A(2) from Daboia russelli pulchella (DPLA(2)) and peptide Leu-Ala-Ile-Tyr-Ser (LAIYS) have been co-crystallized. The structure of the complex has been determined and refined to 2.0 A resolution. The structure contains two crystallographically independent molecules of DPLA(2), with one molecule of peptide specifically bound to one of them. The overall conformations of the two molecules are essentially similar except in three regions; namely, the calcium-binding loop including Trp31 (residues 25-34), the beta-wing consisting of two antiparallel beta-strands (residues 74-85) and the C-terminal region (residues 119-133). Of these, the most striking difference pertains to the orientation of Trp31 in the two molecules. The conformation of Trp31 in molecule A was suitable to allow the binding of peptide LAIYS, while that in molecule B prevented the entry of the ligand into the hydrophobic channel. The structure of the complex clearly showed that the OH group of Tyr of the inhibitor formed hydrogen bonds with both His48 N(delta1) and Asp49 O(delta1), while O(gamma)H of Ser was involved in a hydrogen bond with Trp31. Other peptide backbone atoms interact with protein through water molecules, while Leu, Ala and Ile form strong hydrophobic interactions with the residues of the hydrophobic channel.

Pure Δ9-tetrahydrocannabivarin and a Cannabis sativa extract with high content in Δ9-tetrahydrocannabivarin inhibit nitrite production in murine peritoneal macrophages.

PubMed

Romano, Barbara; Pagano, Ester; Orlando, Pierangelo; Capasso, Raffaele; Cascio, Maria Grazia; Pertwee, Roger; Marzo, Vincenzo Di; Izzo, Angelo A; Borrelli, Francesca

2016-11-01

Historical and scientific evidence suggests that Cannabis use has immunomodulatory and anti-inflammatory effects. We have here investigated the effect of the non-psychotropic phytocannabinoid Δ 9 -tetrahydrocannabivarin (THCV) and of a Cannabis sativa extract with high (64.8%) content in THCV (THCV-BDS) on nitric oxide (NO) production, and on cannabinoid and transient receptor potential (TRP) channel expression in lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages. THCV-BDS and THCV exhibited similar affinity in radioligand binding assays for CB 1 and CB 2 receptors, and inhibited, via CB 2 but not CB 1 cannabinoid receptors, nitrite production evoked by LPS in peritoneal macrophages. THCV down-regulated the over-expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and interleukin 1β (IL-1β) proteins induced by LPS. Furthermore, THCV counteracted LPS-induced up-regulation of CB 1 receptors, without affecting the changes in CB 2 , TRPV2 or TRPV4 mRNA expression caused by LPS. Other TRP channels, namely, TRPA1, TRPV1, TRPV3 and TRPM8 were poorly expressed or undetectable in both unstimulated and LPS-challenged macrophages. It is concluded that THCV - via CB 2 receptor activation - inhibits nitrite production in macrophages. The effect of this phytocannabinoid was associated with a down-regulation of CB 1 , but not CB 2 or TRP channel mRNA expression. Copyright © 2016 Elsevier Ltd. All rights reserved.

Association of β1 and β3 adrenergic receptors gene polymorphisms with insulin resistance and high lipid profiles related to type 2 diabetes and metabolic syndrome.

PubMed

Burguete-Garcia, Ana I; Martinez-Nava, Gabriela A; Valladares-Salgado, Adan; Bermudez Morales, V H; Estrada-Velasco, Barbara; Wacher, Niels; Peralta-Romero, Jesus; Garcia-Mena, Jaime; Parra, Esteban; Cruz, Miguel

2014-06-01

Among the diverse genes associated to type 2 diabetes (T2D), the β-adrenergic receptors are an excellent candidate to study in Mexican population. The objective of this work was to analyze the association of polymorphisms in ADRB1 (rs1801253) (Arg389Gly) and ADRB3 (Trp64Arg) genes with T2D and metabolic syndrome (MS). We studied 445 MS patients, 502 with T2D and 552 healthy controls. Anthropometric features and complete biochemical profile were evaluated, and Arg389Gly and Trp64Arg SNPs were determined by TaqMan assays. Data analysis was adjusted by African, Caucasian and Amerindian ancestral percentage. The variant Arg389Gly of ADRB1 was statistically associated with an increase of LDL levels (P < 0.008), and the variant ADRB3 Trp64Arg was associated to larger HOMA-IR (P < 0.018) and with an increase of insulin levels (P < 0.001). A multiple logistic regression analysis was made in three grouping models: For ADRB3 in the codominant model Trp/Arg genotype, there was an OR of 1.53 (1.09-2.13, P < 0.003) which was increased up to OR 2.99 (1.44-6.22, P < 0.003) for the Arg/Arg genotype. Similar risk association was found under the dominant model Trp/Arg-Arg/Arg genotype with OR 1.67 (1.21-2.30; P < 0.002). In the recessive model (Arg/Arg genotype), there was also a high association OR 2.56 (1.24-5.26, P < 0.01). The ADRB3 Trp64Arg variant is a susceptibility gene polymorphism for T2D and the ADRB1 Gly389Arg for lipid metabolism disruption. These results show that these variants are potential biomarkers for predicting metabolic alterations and evolution in diabetic and metabolic syndrome patients. Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.

A prospective study of XRCC1 haplotypes and their interaction with plasma carotenoids on breast cancer risk

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mohrenweiser, H W; Han, J; Hankinson, S E

2004-01-15

The XRCC1 protein is involved in the base excision repair pathway through interactions with other proteins. Polymorphisms in the XRCC1 gene may lead to variation in repair proficiency and confer inherited predisposition to cancer. We prospectively assessed the associations between polymorphisms and haplotypes in XRCC1 and breast cancer risk in a nested case-control study within the Nurses' Health Study (incident cases, n 1004; controls, n 1385). We further investigated gene-environment interactions between the XRCC1 variations and plasma carotenoids on breast cancer risk. We genotyped four haplotype-tagging single nucleotide polymorphisms Arg {sup 194}Trp, C26602T, Arg{sup 399}Gln, and Gln{sup 632}Gln in themore » XRCC1 gene. Five common haplotypes accounted for 99% of the chromosomes in the present study population of mostly Caucasian women. We observed a marginally significant reduction in the risk of breast cancer among {sup 194}Trp carriers. As compared with no-carriers, women with at least one {sup 194}Trp allele had a multivariate odds ratio of 0.79 (95% of the confidence interval, 0.60 -1.04). The inferred haplotype harboring the {sup 194}Trp allele was more common in controls than in cases (6.6 versus 5.3%, P 0.07). We observed that the Arg {sup 194}Trp modified the inverse associations of plasma -carotene level (P, ordinal test for interaction 0.02) and plasma -carotene level (P, ordinal test for interaction 0.003) with breast cancer risk. No suggestion of an interaction was observed between the Arg {sup 194}Trp and cigarette smoking. Our results suggest an inverse association between XRCC1 {sup 194}Trp allele and breast cancer risk. The findings of the effect modification of the Arg {sup 194}Trp on the relations of plasma -and -carotene levels with breast cancer risk suggest a potential protective effect of carotenoids in breast carcinogenesis by preventing oxidative DNA damage.« less

Development of tyrosinase-based reporter genes for preclinical photoacoustic imaging of mesenchymal stem cells

NASA Astrophysics Data System (ADS)

Märk, Julia; Ruschke, Karen; Dortay, Hakan; Schreiber, Isabelle; Sass, Andrea; Qazi, Taimoor; Pumberger, Matthias; Laufer, Jan

2014-03-01

The capability to image stem cells in vivo in small animal models over extended periods of time is important to furthering our understanding of the processes involved in tissue regeneration. Photoacoustic imaging is suited to this application as it can provide high resolution (tens of microns) absorption-based images of superficial tissues (cm depths). However, stem cells are rare, highly migratory, and can divide into more specialised cells. Genetic labelling strategies are therefore advantageous for their visualisation. In this study, methods for the transfection and viral transduction of mesenchymal stem cells with reporter genes for the co-expression of tyrosinase and a fluorescent protein (mCherry). Initial photoacoustic imaging experiments of tyrosinase expressing cells in small animal models of tissue regeneration were also conducted. Lentiviral transduction methods were shown to result in stable expression of tyrosinase and mCherry in mesenchymal stem cells. The results suggest that photoacoustic imaging using reporter genes is suitable for the study of stem cell driven tissue regeneration in small animals.

Membrane-tethered peptides patterned after the TRP domain (TRPducins) selectively inhibit TRPV1 channel activity.

PubMed

Valente, Pierluigi; Fernández-Carvajal, Asia; Camprubí-Robles, María; Gomis, Ana; Quirce, Susana; Viana, Félix; Fernández-Ballester, Gregorio; González-Ros, José M; Belmonte, Carlos; Planells-Cases, Rosa; Ferrer-Montiel, Antonio

2011-05-01

The transient receptor potential vanilloid 1 (TRPV1) channel is a thermosensory receptor implicated in diverse physiological and pathological processes. The TRP domain, a highly conserved region in the C terminus adjacent to the internal channel gate, is critical for subunit tetramerization and channel gating. Here, we show that cell-penetrating, membrane-anchored peptides patterned after this protein domain are moderate and selective TRPV1 antagonists both in vitro and in vivo, blocking receptor activity in intact rat primary sensory neurons and their peripheral axons with mean decline time of 30 min. The most potent lipopeptide, TRP-p5, blocked all modes of TRPV1 gating with micromolar efficacy (IC(50)<10 μM), without significantly affecting other thermoTRP channels. In contrast, its retrosequence or the corresponding sequences of other TRPV channels did not alter TRPV1 channel activity (IC(50)>100 μM). TRP-p5 did not affect the capsaicin sensitivity of the vanilloid receptor. Our data suggest that TRP-p5 interferes with protein-protein interactions at the level of the TRP domain that are essential for the "conformational" change that leads to gate opening. Therefore, these palmitoylated peptides, which we termed TRPducins, are noncompetitive, voltage-independent, sequence-specific TRPV1 blockers. Our findings indicate that TRPducin-like peptides may embody a novel molecular strategy that can be exploited to generate a selective pharmacological arsenal for the TRP superfamily of ion channels.

Low plasma tryptophan is associated with olfactory function in healthy elderly community dwellers in Japan.

PubMed

Adachi, Yusuke; Shimodaira, Yoshiki; Nakamura, Hidehiro; Imaizumi, Akira; Mori, Maiko; Kageyama, Yoko; Noguchi, Yasushi; Seki, Asuka; Okabe, Yuki; Miyake, Yuko; Ono, Kaori; Kumagai, Shu

2017-10-16

Decreased circulating tryptophan (Trp) levels are frequently observed in elderly patients with neurodegenerative disease including Alzheimer's disease. Trp may serve as a potential biomarker for monitoring disease risk in elderly people. We aimed to investigate the association between low plasma Trp levels and olfactory function, which is known to predict age-related diseases including dementia in elderly people. A total of 144 healthy elderly Japanese community (≥ 65 years old) dwellers from the Health, Aging and Nutritional Improvement study (HANI study) were the subjects of our analysis. Low Trp levels were classified using the lower limit values of the reference interval according to a previous report. Olfactory function was assessed using a card-type test called Open Essence, which includes 12 odour items that are familiar to Japanese people. The elderly subjects with low circulating Trp levels were compared to a control group with normal plasma Trp levels. We conducted the analyses using 144 people aged 65 years or older (mean age 73.7 ± 5.5 years; 36.1% men). The subjects showed normal serum albumin levels (4.4 ± 0.2 g/dL) and no daily living disabilities. Low plasma Trp levels (low Trp group) were found in 11.1% of the study population. The low Trp group showed a significantly lower correct-answer rate for the items india ink, perfume, curry and sweaty smelling socks than control group (P < 0.05). There was also a significant association between low Trp levels and low olfactory ability, after adjusting for age and sex. Lower plasma Trp levels were associated with a decrease in olfactory function in functionally competent older individuals. Because olfactory dysfunction predicts age-related diseases, low plasma Trp levels may represent a clinical sign of disease risk in elderly people.

Microphthalmia-associated transcription factor as the molecular target of cadmium toxicity in human melanocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chantarawong, Wipa; Inter Departmental Multidisciplinary Graduate Program in Bioscience, Faculty of Science, Kasetsart University, Bangkok; Takeda, Kazuhisa

Highlights: • In human melanocytes, cadmium decreases the expression of MITF-M and tyrosinase and their mRNAs. • In human melanoma cells, cadmium decreases the expression of MITF-M protein and tyrosinase mRNA. • Expression of MITF-H is less sensitive to cadmium toxicity in melanocyte-linage cells. • Cadmium does not decrease the expression of MITF-H in retinal pigment epithelial cells. • MITF-M is the molecular target of cadmium toxicity in melanocytes. - Abstract: Dietary intake of cadmium is inevitable, causing age-related increase in cadmium accumulation in many organs, including hair, choroid and retinal pigment epithelium (RPE). Cadmium has been implicated in themore » pathogenesis of hearing loss and macular degeneration. The functions of cochlea and retina are maintained by melanocytes and RPE, respectively, and the differentiation of these pigment cells is regulated by microphthalmia-associated transcription factor (MITF). In the present study, we explored the potential toxicity of cadmium in the cochlea and retina by using cultured human melanocytes and human RPE cell lines. MITF consists of multiple isoforms, including melanocyte-specific MITF-M and widely expressed MITF-H. Levels of MITF-M protein and its mRNA in human epidermal melanocytes and HMV-II melanoma cells were decreased significantly by cadmium. In parallel with the MITF reduction, mRNA levels of tyrosinase, the key enzyme of melanin biosynthesis that is regulated by MITF-M, were also decreased. In RPE cells, however, the levels of total MITF protein, constituting mainly MITF-H, were not decreased by cadmium. We thus identify MITF-M as the molecular target of cadmium toxicity in melanocytes, thereby accounting for the increased risk of disability from melanocyte malfunction, such as hearing and vision loss among people with elevated cadmium exposure.« less

Nucks1 synergizes with Trp53 to promote radiation lymphomagenesis in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yue, Yangbo; Leung, Stanley G.; Liu, Yueyong

NUCKS1 is a 27 kD vertebrate-specific protein, with a role in the DNA damage response. Here, we show that after 4 Gy total-body X-irradiation, Trp53+/- Nucks1+/- mice more rapidly developed tumors, particularly thymic lymphoma (TL), than Trp53+/- mice. TLs in both cohorts showed loss of heterozygosity (LOH) of the Trp53+ allele in essentially all cases. In contrast, LOH of the Nucks1+ allele was rare. Nucks1 expression correlated well with Nucks1 gene dosage in normal thymi, but was increased in the majority of TLs from Trp53+/- Nucks1+/- mice, suggesting that elevated Nucks1 message may be associated with progression towards malignancy inmore » vivo. Trp53+/- Nucks1+/- mice frequently succumbed to CD4- CD8- TLs harboring translocations involving Igh but not Tcra/d, indicating TLs in Trp53+/- Nucks1+/- mice mostly originated prior to the double positive stage and at earlier lineage than TLs in Trp53+/- mice. Monoclonal rearrangements at Tcrb were more prevalent in TLs from Trp53+/- Nucks1+/- mice, as was infiltration of primary TL cells to distant organs (liver, kidney and spleen). We propose that, in the context of Trp53 deficiency, wild type levels of Nucks1 are required to suppress radiation-induced TL, likely through the role of the NUCKS1 protein in the DNA damage response.« less

Nucks1 synergizes with Trp53 to promote radiation lymphomagenesis in mice

DOE PAGES

Yue, Yangbo; Leung, Stanley G.; Liu, Yueyong; ...

2016-08-16

NUCKS1 is a 27 kD vertebrate-specific protein, with a role in the DNA damage response. Here, we show that after 4 Gy total-body X-irradiation, Trp53+/- Nucks1+/- mice more rapidly developed tumors, particularly thymic lymphoma (TL), than Trp53+/- mice. TLs in both cohorts showed loss of heterozygosity (LOH) of the Trp53+ allele in essentially all cases. In contrast, LOH of the Nucks1+ allele was rare. Nucks1 expression correlated well with Nucks1 gene dosage in normal thymi, but was increased in the majority of TLs from Trp53+/- Nucks1+/- mice, suggesting that elevated Nucks1 message may be associated with progression towards malignancy inmore » vivo. Trp53+/- Nucks1+/- mice frequently succumbed to CD4- CD8- TLs harboring translocations involving Igh but not Tcra/d, indicating TLs in Trp53+/- Nucks1+/- mice mostly originated prior to the double positive stage and at earlier lineage than TLs in Trp53+/- mice. Monoclonal rearrangements at Tcrb were more prevalent in TLs from Trp53+/- Nucks1+/- mice, as was infiltration of primary TL cells to distant organs (liver, kidney and spleen). We propose that, in the context of Trp53 deficiency, wild type levels of Nucks1 are required to suppress radiation-induced TL, likely through the role of the NUCKS1 protein in the DNA damage response.« less

Carvacrol derivatives as mushroom tyrosinase inhibitors; synthesis, kinetics mechanism and molecular docking studies.

PubMed

Ashraf, Zaman; Rafiq, Muhammad; Nadeem, Humaira; Hassan, Mubashir; Afzal, Samina; Waseem, Muhammad; Afzal, Khurram; Latip, Jalifah

2017-01-01

The present work describesthe development of highly potent mushroom tyrosinase inhibitor better than the standard kojic acid. Carvacrol derivatives 4a-f and 6a-d having substituted benzoic acid and cinnamic acidresidues were synthesized with the aim to possess potent tyrosinase inhibitory activity.The structures of the synthesized compounds were ascertained by their spectroscopic data (FTIR, 1HNMR, 13CNMR and Mass Spectroscopy).Mushroom tyrosinase inhibitory activity of synthesized compounds was determined and it was found that one of the derivative 6c possess higher activity (IC50 0.0167μM) than standard kojic acid (IC50 16.69μM). The derivatives 4c and 6b also showed good tyrosinase inhibitory activity with (IC50 16.69μM) and (IC50 16.69μM) respectively.Lineweaver-Burk and Dixon plots were used for the determination of kinetic mechanism of the compounds 4c and 6b and 6c. The kinetic analysis revealed that compounds 4c and 6b showed mixed-type inhibition while 6c is a non-competitive inhibitor having Ki values19 μM, 10 μM, and 0.05 μMrespectively. The enzyme inhibitory kinetics further showed thatcompounds 6b and 6c formed irreversible enzyme inhibitor complex while 4c bind reversibly with mushroom tyrosinase.The docking studies showed that compound 6c have maximum binding affinity against mushroom tyrosinase (PDBID: 2Y9X) with binding energy value (-7.90 kcal/mol) as compared to others.The 2-hydroxy group in compound 6c interacts with amino acid HIS85 which is present in active binding site. The wet lab results are in good agreement with the dry lab findings.Based upon our investigation we may propose that the compound 6c is promising candidate for the development of safe cosmetic agent.

Common mechanisms regulating dark noise and quantum bump amplification in Drosophila photoreceptors

PubMed Central

Chu, Brian; Liu, Che-Hsiung; Sengupta, Sukanya; Gupta, Amit; Raghu, Padinjat

2013-01-01

Absolute visual thresholds are limited by “dark noise,” which in Drosophila photoreceptors is dominated by brief (∼10 ms), small (∼2 pA) inward current events, occurring at ∼2/s, believed to reflect spontaneous G protein activations. These dark events were increased in rate and amplitude by a point mutation in myosin III (NINAC), which disrupts its interaction with the scaffolding protein, INAD. This phenotype mimics that previously described in null mutants of ninaC (no inactivation no afterpotential; encoding myosin III) and an associated protein, retinophilin (rtp). Dark noise was similarly increased in heterozygote mutants of diacylglycerol kinase (rdgA/+). Dark noise in ninaC, rtp, and rdgA/+ mutants was greatly suppressed by mutations of the Gq α-subunit (Gαq) and the major light-sensitive channel (trp) but not rhodopsin. ninaC, rtp, and rdgA/+ mutations also all facilitated residual light responses in Gαq and PLC hypomorphs. Raising cytosolic Ca2+ in the submicromolar range increased dark noise, facilitated activation of transient receptor potential (TRP) channels by exogenous agonist, and again facilitated light responses in Gαq hypomorphs. Our results indicate that RTP, NINAC, INAD, and diacylglycerol kinase, together with a Ca2+-dependent threshold, share common roles in suppressing dark noise and regulating quantum bump generation; consequently, most spontaneous G protein activations fail to generate dark events under normal conditions. By contrast, quantum bump generation is reliable but delayed until sufficient G proteins and PLC are activated to overcome threshold, thereby ensuring generation of full-size bumps with high quantum efficiency. PMID:23365183

COSTS OF CHILDHOOD ASTHMA DUE TO TRAFFIC-RELATED POLLUTION IN TWO CALIFORNIA COMMUNITIES

PubMed Central

Brandt, Sylvia J.; Perez, Laura; Künzli, Nino; Lurmann, Fred; McConnell, Rob

2015-01-01

Recent research suggests the burden of childhood asthma attributable to air pollution has been underestimated in traditional risk assessments, and there are no estimates of these associated costs. We estimated the yearly childhood asthma-related costs attributable to air pollution for Riverside and Long Beach, California, including: 1) the indirect and direct costs of health care utilization due to asthma exacerbations linked to traffic-related pollution (TRP); and 2) the costs of health care for asthma cases attributable to local TRP exposure. We estimated these costs using estimates from peer-reviewed literature and the authors' analysis of surveys (Medical Expenditure Panel Survey, California Health Interview Survey, National Household Travel Survey, and Health Care Utilization Project). A lower-bound estimate of the asthma burden attributable to air pollution was $18 million yearly. Asthma cases attributable to TRP exposure accounted for almost half of this cost. The cost of bronchitic episodes was a major proportion of both the annual cost of asthma cases attributable to TRP and of pollution-linked exacerbations. Traditional risk assessment methods underestimate both the burden of disease and cost of asthma associated with air pollution, and these costs are borne disproportionately by communities with higher than average TRP. PMID:22267764

Transient Receptor Potential Channels as Targets for Phytochemicals

PubMed Central

2015-01-01

To date, 28 mammalian transient receptor potential (TRP) channels have been cloned and characterized. They are grouped into six subfamilies on the basis of their amino acid sequence homology: TRP Ankyrin (TRPA), TRP Canonical (TRPC), TRP Melastatin (TRPM), TRP Mucolipin (TRPML), TRP Polycystin (TRPP), and TRP Vanilloid (TRPV). Most of the TRP channels are nonselective cation channels expressed on the cell membrane and exhibit variable permeability ratios for Ca2+ versus Na+. They mediate sensory functions (such as vision, nociception, taste transduction, temperature sensation, and pheromone signaling) and homeostatic functions (such as divalent cation flux, hormone release, and osmoregulation). Significant progress has been made in our understanding of the specific roles of these TRP channels and their activation mechanisms. In this Review, the emphasis will be on the activation of TRP channels by phytochemicals that are claimed to exert health benefits. Recent findings complement the anecdotal evidence that some of these phytochemicals have specific receptors and the activation of which is responsible for the physiological effects. Now, the targets for these phytochemicals are being unveiled; a specific hypothesis can be proposed and tested experimentally to infer a scientific validity of the claims of the health benefits. The broader and pressing issues that have to be addressed are related to the quantities of the active ingredients in a given preparation, their bioavailability, metabolism, adverse effects, excretion, and systemic versus local effects. PMID:24926802

Inhibitory effect of corn silk on skin pigmentation.

PubMed

Choi, Sang Yoon; Lee, Yeonmi; Kim, Sung Soo; Ju, Hyun Min; Baek, Ji Hwoon; Park, Chul-Soo; Lee, Dong-Hyuk

2014-03-03

In this study, the inhibitory effect of corn silk on melanin production was evaluated. This study was performed to investigate the inhibitory effect of corn silk on melanin production in Melan-A cells by measuring melanin production and protein expression. The corn silk extract applied on Melan-A cells at a concentration of 100 ppm decreased melanin production by 37.2% without cytotoxicity. This was a better result than arbutin, a positive whitening agent, which exhibited a 26.8% melanin production inhibitory effect at the same concentration. The corn silk extract did not suppress tyrosinase activity but greatly reduced the expression of tyrosinase in Melan-A cells. In addition, corn silk extract was applied to the human face with hyperpigmentation, and skin color was measured to examine the degree of skin pigment reduction. The application of corn silk extract on faces with hyperpigmentation significantly reduced skin pigmentation without abnormal reactions. Based on the results above, corn silk has good prospects for use as a material for suppressing skin pigmentation.

Investigation of the Anti-Melanogenic and Antioxidant Characteristics of Eucalyptus camaldulensis Flower Essential Oil and Determination of Its Chemical Composition.

PubMed

Huang, Huey-Chun; Ho, Ya-Chi; Lim, Jia-Min; Chang, Tzu-Yun; Ho, Chen-Lung; Chang, Tsong-Min

2015-05-07

The effects of essential oil from Eucalyptus camaldulensis flowers oil on melanogenesis and the oil's antioxidant characteristics were investigated. Assays of mushroom and cellular tyrosinase activities and melanin content of mouse melanoma cells were performed spectrophotometrically, and the expression of melanogenesis-related proteins was determined by Western blotting. The possible signaling pathways involved in essential oil-mediated depigmentation were also investigated using specific protein kinase inhibitors. The results revealed that E. camaldulensis flower essential oil effectively suppresses intracellular tyrosinase activity and decreases melanin amount in B16F10 mouse melanoma cells. The essential oil also exhibits antioxidant properties and effectively decreases intracellular reactive oxygen species (ROS) levels. The volatile chemical composition of the essential oil was analyzed with gas chromatography-mass spectrometry (GC/MS). The chemical constituents in the essential oil are predominately oxygenated monoterpenes (34.9%), followed by oxygenated sesquiterpenes (31.8%), monoterpene hydrocarbons (29.0%) and sesquiterpene hydrocarbons (4.3%). Our results indicated that E. camaldulensis flower essential oil inhibits melanogenesis through its antioxidant properties and by down-regulating both mitogen-activated protein kinases (MAPK) and protein kinase A (PKA) signaling pathways. The present study indicates that the essential oil has the potential to be developed into a skin care product.

Investigation of the Anti-Melanogenic and Antioxidant Characteristics of Eucalyptus camaldulensis Flower Essential Oil and Determination of Its Chemical Composition

PubMed Central

Huang, Huey-Chun; Ho, Ya-Chi; Lim, Jia-Min; Chang, Tzu-Yun; Ho, Chen-Lung; Chang, Tsong-Min

2015-01-01

The effects of essential oil from Eucalyptus camaldulensis flowers oil on melanogenesis and the oil’s antioxidant characteristics were investigated. Assays of mushroom and cellular tyrosinase activities and melanin content of mouse melanoma cells were performed spectrophotometrically, and the expression of melanogenesis-related proteins was determined by Western blotting. The possible signaling pathways involved in essential oil-mediated depigmentation were also investigated using specific protein kinase inhibitors. The results revealed that E. camaldulensis flower essential oil effectively suppresses intracellular tyrosinase activity and decreases melanin amount in B16F10 mouse melanoma cells. The essential oil also exhibits antioxidant properties and effectively decreases intracellular reactive oxygen species (ROS) levels. The volatile chemical composition of the essential oil was analyzed with gas chromatography–mass spectrometry (GC/MS). The chemical constituents in the essential oil are predominately oxygenated monoterpenes (34.9%), followed by oxygenated sesquiterpenes (31.8%), monoterpene hydrocarbons (29.0%) and sesquiterpene hydrocarbons (4.3%). Our results indicated that E. camaldulensis flower essential oil inhibits melanogenesis through its antioxidant properties and by down-regulating both mitogen-activated protein kinases (MAPK) and protein kinase A (PKA) signaling pathways. The present study indicates that the essential oil has the potential to be developed into a skin care product. PMID:25961954

Anti-melanogenic activity of schaftoside in Rhizoma Arisaematis by increasing autophagy in B16F1 cells.

PubMed

Kim, Pan Soo; Shin, Ji Hyun; Jo, Doo Sin; Shin, Dong Woon; Choi, Dong-Hwa; Kim, Woo Jung; Park, Kyuhee; Kim, Jin Kyu; Joo, Chul Gue; Lee, Jong Suk; Choi, Yongmun; Shin, Yong Won; Shin, Joong Jin; Jeon, Hong Bae; Seo, Jin-Ho; Cho, Dong-Hyung

2018-06-08

Skin pigmentation involves multiple processes, including melanin synthesis, transport, and melanosome release. Melanin content determines skin color and protects against UV radiation-induced damage. Autophagy is a cooperative process between autophagosomes and lysosomes that degrades cellular components and organelles. In the present study, B16F1 cells were treated with Rhizoma Arisaematis extract (RA) and assessed for pigmentation and autophagy regulation. RA treatment suppressed the α-MSH-stimulated increase of melanogenesis and down-regulated the expression of tyrosinase and TRP1 proteins in B16F1 cells. In addition, autophagy was activated in RA-treated cells. Inhibition of autophagy reduced the anti-melanogenic activity of RA in α-MSH-treated B16F1 cells. We identified schaftoside as an effector molecule by LC-MS analysis of RA. Consistently, treatment of schaftoside showed anti-melanogenic effect and induced autophagy activation in B16F1 cells. Inhibition of autophagy by 3 MA treatment reduced the anti-melanogenic effect of the schaftoside and recovered expression level of melanogenesis regulators in α-MSH-treated B16F1 cells. Taken together, our results suggest that schaftoside from RA inhibits skin pigmentation through modulation of autophagy. Copyright © 2018. Published by Elsevier Inc.

Thrombin-receptor agonist peptides, in contrast to thrombin itself, are not full agonists for activation and signal transduction in human platelets in the absence of platelet-derived secondary mediators.

PubMed Central

Lau, L F; Pumiglia, K; Côté, Y P; Feinstein, M B

1994-01-01

Synthetic thrombin receptor peptides (TRPs), comprising the first 6-14 amino acids of the new N-terminus tethered ligand of the thrombin receptor that is generated by thrombin's proteolytic activity, were reported to activate platelets equally with thrombin itself and are considered to be full agonists [Vu et al. (1991) Cell 64, 1057-1068]. Using aspirin plus ADP-scavengers or the ADP-receptor antagonist adenosine 5'-[alpha-thio]triphosphate to prevent the secondary effects of the potent agonists that are normally released from stimulated platelets (i.e. ADP and thromboxane A2), we assessed the direct actions of thrombin and TRPs (i.e. TRP42-47 and TRP42-55). Compared with thrombin, under these conditions, TRPs: (1) failed to aggregate platelets completely; (2) produced less activation of glycoprotein (GP)IIb-IIIa; (3) did not cause association of GPIIb and pp60c-src with the cytoskeleton; and (4) caused less alpha-granule secretion, phosphorylation of cytoplasmic phospholipase A2, arachidonic acid release and phosphatidyl inositol (PtdOH) production. Furthermore, TRPs induced transient increases in protein phosphorylation mediated by protein kinase C and protein tyrosine phosphorylation, whereas these same responses to thrombin were greater and more sustained. Hirudin added after thrombin accelerated protein dephosphorylation, thereby mimicking the rate of spontaneous dephosphorylation seen after stimulation by TRPs. Platelets totally desensitized to very high concentrations of TRPs, by prior exposure to maximally effective concentrations of the peptides, remained responsive to alpha- and gamma-thrombins. Thrombin-stimulated PtdOH production in permeabilized platelets desensitized to TRPs was abolished by guanosine 5'-[beta-thio]diphosphate (GDP[beta S]), as in normal platelets. These results are discussed in terms of the allosteric Ternary Complex Model for G-protein linked receptors [Samama et al. (1993) J. Biol. Chem. 268, 4625-4636]. We conclude that: (1) TRPs are partial agonists for the thrombin receptor and produce incomplete receptor desensitization in keeping with their lower intrinsic activity; (2) thrombin's effects in platelets, even in TRP-desensitized platelets, are entirely mediated through the recently cloned G-protein linked receptor, and (3) thrombin's ability to produce sustained signals, compared with TRPs, may require the continued progressive proteolytic activation of naive thrombin receptors. Images Figure 3 PMID:7526841

Mutations of the tyrosinase gene in patients with oculocutaneous albinism from various ethnic groups in Israel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gershoni-Baruch, R.; Rosenmann, A.; Droetto, S.

1994-04-01

The authors have analyzed the tyrosinase (TYR) gene in 38 unrelated patients with oculocutaneous albinism (OCA), derived from several different ethnic groups of the diverse population of Israel. They detected TYR gene mutations in 23 of the 34 patients with apparent type I (i.e., tyrosinase-deficient) OCA and in none of the patients with other clinical forms of albinism. Among Moroccan Jews with type IA (i.e., tyrosinase-negative) OCA, they detected a highly predominant mutant allele containing a missense substitution, Gly47Asp (G47D). This mutation occurs on the same haplotype as in patients from the Canary Islands and Puerto Rico, suggesting that themore » G47D mutation in these ethnically distinct populations may stem from a common origin. 28 refs., 1 fig., 2 tabs.« less

The Phosphorylation State of the Drosophila TRP Channel Modulates the Frequency Response to Oscillating Light In Vivo

PubMed Central

Rhodes-Mordov, Elisheva; Katz, Ben; Oberegelsbacher, Claudia; Yasin, Bushra; Tzadok, Hanan; Huber, Armin

2017-01-01

Drosophila photoreceptors respond to oscillating light of high frequency (∼100 Hz), while the detected maximal frequency is modulated by the light rearing conditions, thus enabling high sensitivity to light and high temporal resolution. However, the molecular basis for this adaptive process is unclear. Here, we report that dephosphorylation of the light-activated transient receptor potential (TRP) ion channel at S936 is a fast, graded, light-dependent, and Ca2+-dependent process that is partially modulated by the rhodopsin phosphatase retinal degeneration C (RDGC). Electroretinogram measurements of the frequency response to oscillating lights in vivo revealed that dark-reared flies expressing wild-type TRP exhibited a detection limit of oscillating light at relatively low frequencies, which was shifted to higher frequencies upon light adaptation. Strikingly, preventing phosphorylation of the S936-TRP site by alanine substitution in transgenic Drosophila (trpS936A) abolished the difference in frequency response between dark-adapted and light-adapted flies, resulting in high-frequency response also in dark-adapted flies. In contrast, inserting a phosphomimetic mutation by substituting the S936-TRP site to aspartic acid (trpS936D) set the frequency response of light-adapted flies to low frequencies typical of dark-adapted flies. Light-adapted rdgC mutant flies showed relatively high S936-TRP phosphorylation levels and light–dark phosphorylation dynamics. These findings suggest that RDGC is one but not the only phosphatase involved in pS936-TRP dephosphorylation. Together, this study indicates that TRP channel dephosphorylation is a regulatory process that affects the detection limit of oscillating light according to the light rearing condition, thus adjusting dynamic processing of visual information under varying light conditions. SIGNIFICANCE STATEMENT Drosophila photoreceptors exhibit high temporal resolution as manifested in frequency response to oscillating light of high frequency (≤∼100 Hz). Light rearing conditions modulate the maximal frequency detected by photoreceptors, thus enabling them to maintain high sensitivity to light and high temporal resolution. However, the precise mechanisms for this process are not fully understood. Here, we show by combination of biochemistry and in vivo electrophysiology that transient receptor potential (TRP) channel dephosphorylation at a specific site is a fast, light-activated and Ca2+-dependent regulatory process. TRP dephosphorylation affects the detection limit of oscillating light according to the adaptation state of the photoreceptor cells by shifting the detection limit to higher frequencies upon light adaptation. This novel mechanism thus adjusts dynamic processing of visual information under varying light conditions. PMID:28314815

Two Novel Tyrosinase Inhibitory Sesquiterpenes Induced by CuCl2 from a Marine-Derived Fungus Pestalotiopsis sp. Z233

PubMed Central

Wu, Bin; Wu, Xiaodan; Sun, Min; Li, Minhui

2013-01-01

Two new sesquiterpenes, 1β,5α,6α,14-tetraacetoxy-9α-benzoyloxy-7βH-eudesman-2β,11-diol (1) and 4α,5α-diacetoxy-9α-benzoyloxy-7βH-eudesman-1β,2β,11,14-tetraol (2), were produced as stress metabolites in the cultured mycelia of Pestalotiopsis sp. Z233 isolated from the algae Sargassum horneri in response to abiotic stress elicitation by CuCl2. Their structures were established by spectroscopic means. New compounds 1 and 2 showed tyrosinase inhibitory activities with IC50 value of 14.8 µM and 22.3 µM. PMID:23917067

Two novel tyrosinase inhibitory sesquiterpenes induced by CuCl2 from a marine-derived fungus Pestalotiopsis sp. Z233.

PubMed

Wu, Bin; Wu, Xiaodan; Sun, Min; Li, Minhui

2013-08-02

Two new sesquiterpenes, 1β,5α,6α,14-tetraacetoxy-9α-benzoyloxy-7β H-eudesman-2β,11-diol (1) and 4α,5α-diacetoxy-9α-benzoyloxy-7βH-eudesman-1β,2β,11, 14-tetraol (2), were produced as stress metabolites in the cultured mycelia of Pestalotiopsis sp. Z233 isolated from the algae Sargassum horneri in response to abiotic stress elicitation by CuCl2. Their structures were established by spectroscopic means. New compounds 1 and 2 showed tyrosinase inhibitory activities with IC50 value of 14.8 µM and 22.3 µM.

Induction of cell death by tospoviral protein NSs and the motif critical for cell death does not control RNA silencing suppression activity.

PubMed

Singh, Ajeet; Permar, Vipin; Jain, R K; Goswami, Suneha; Kumar, Ranjeet Ranjan; Canto, Tomas; Palukaitis, Peter; Praveen, Shelly

2017-08-01

Groundnut bud necrosis virus induces necrotic symptoms in different hosts. Previous studies showed reactive oxygen species-mediated programmed cell death (PCD) resulted in necrotic symptoms. Transgenic expression of viral protein NSs mimics viral symptoms. Here, we showed a role for NSs in influencing oxidative burst in the cell, by analyzing H 2 O 2 accumulation, activities of antioxidant enzymes and expression levels of vacuolar processing enzymes, H 2 O 2 -responsive microRNA 319a.2 plus its possible target metacaspase-8. The role of NSs in PCD, was shown using two NSs mutants: one in the Trp/GH3 motif (a homologue of pro-apototic domain) (NSs S189R ) and the other in a non-Trp/GH3 motif (NSs L172R ). Tobacco rattle virus (TRV) expressing NSs S189R enhanced the PCD response, but not TRV-NSs L172R , while RNA silencing suppression activity was lost in TRV-NSs L172R , but not in TRV-NSs S189R . Therefore, we propose dual roles of NSs in RNA silencing suppression and induction of cell death, controlled by different motifs. Copyright © 2017 Elsevier Inc. All rights reserved.

Parametric models to compute tryptophan fluorescence wavelengths from classical protein simulations.

PubMed

Lopez, Alvaro J; Martínez, Leandro

2018-02-26

Fluorescence spectroscopy is an important method to study protein conformational dynamics and solvation structures. Tryptophan (Trp) residues are the most important and practical intrinsic probes for protein fluorescence due to the variability of their fluorescence wavelengths: Trp residues emit in wavelengths ranging from 308 to 360 nm depending on the local molecular environment. Fluorescence involves electronic transitions, thus its computational modeling is a challenging task. We show that it is possible to predict the wavelength of emission of a Trp residue from classical molecular dynamics simulations by computing the solvent-accessible surface area or the electrostatic interaction between the indole group and the rest of the system. Linear parametric models are obtained to predict the maximum emission wavelengths with standard errors of the order 5 nm. In a set of 19 proteins with emission wavelengths ranging from 308 to 352 nm, the best model predicts the maximum wavelength of emission with a standard error of 4.89 nm and a quadratic Pearson correlation coefficient of 0.81. These models can be used for the interpretation of fluorescence spectra of proteins with multiple Trp residues, or for which local Trp environmental variability exists and can be probed by classical molecular dynamics simulations. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

Homozygosity Mapping and Whole Exome Sequencing to Detect SLC45A2 and G6PC3 Mutations in a Single Patient with Oculocutaneous Albinism and Neutropenia

PubMed Central

Cullinane, Andrew R.; Vilboux, Thierry; O’Brien, Kevin; Curry, James A.; Maynard, Dawn M.; Carlson-Donohoe, Hannah; Ciccone, Carla; Markello, Thomas C.; Gunay-Aygun, Meral; Huizing, Marjan; Gahl, William A.

2011-01-01

We evaluated a 32 year-old woman whose oculocutaneous albinism, bleeding diathesis, neutropenia, and history of recurrent infections prompted consideration of the diagnosis of Hermansky-Pudlak syndrome type 2 (HPS-2). This was ruled out due to the presence of platelet delta granules and absence of AP3B1 mutations. Since parental consanguinity suggested an autosomal recessive mode of inheritance, we employed homozygosity mapping, followed by whole exome sequencing, to identify two candidate disease-causing genes, SLC45A2 and G6PC3. Conventional di-deoxy sequencing confirmed pathogenic mutations in SLC45A2, associated with oculocutaneous albinism type 4 (OCA-4), and G6PC3, associated with neutropenia. The substantial reduction of SLC45A2 protein in the patient’s melanocytes caused the mis-localization of tyrosinase from melanosomes to the plasma membrane and also led to the incorporation of tyrosinase into exosomes and secretion into the culture medium, explaining the hypopigmentation in OCA-4. Our patient’s G6PC3 mRNA expression level was also reduced, leading to increased apoptosis of her fibroblasts under ER stress. This report describes the first North American patient with OCA-4, the first culture of human OCA-4 melanocytes, and the use of homozygosity mapping followed by whole exome sequencing to identify disease-causing mutations in multiple genes in a single affected individual. PMID:21677667

Variation in Hsp70-1A expression contributes to skin color diversity

PubMed Central

Murase, Daiki; Hachiya, Akira; Fullenkamp, Rachel; Beck, Anita; Moriwaki, Shigeru; Hase, Tadashi; Takema, Yoshinori; Manga, Prashiela

2017-01-01

The wide range in human skin color results from varying levels of the pigment melanin. Genetic mechanisms underlying coloration differences have been explored, but identified genes do not account for all variation seen in the skin color spectrum. Post-transcriptional and post-translational regulation of factors that determine skin color, including melanin synthesis in epidermal melanocytes, melanosome transfer to keratinocytes and melanosome degradation, is also critical for pigmentation. We therefore investigated proteins that are differentially expressed in melanocytes derived from either White or African American (AA) skin. Two dimensional gel electrophoresis (2-DGE) and mass spectrometry demonstrated that Heat Shock Protein 70-1A (Hsp70-1A) protein levels were significantly higher in AA melanocytes compared to White melanocytes. Hsp70-1A expression significantly correlated with levels of tyrosinase, the rate-limiting melanogenic enzyme, consistent with a proposed role for Hsp70-family members in tyrosinase post-translational modification. Additionally, pharmacologic inhibition and siRNA-mediated down-regulation of Hsp70-1A correlated with pigmentation changes in cultured melanocytes, modified human skin substitutes and ex vivo skin. Furthermore, Hsp70-1A inhibition led to increased autophagy-mediated melanosome degradation in keratinocytes. Our data thus reveal that epidermal Hsp70-1A contributes to the diversity of skin color by regulating the amount of melanin synthesized in melanocytes and modulating autophagic melanosome degradation in keratinocytes. PMID:27094592

Loss of Oca2 disrupts the unfolded protein response and increases resistance to endoplasmic reticulum stress in melanocytes.

PubMed

Cheng, Tsing; Orlow, Seth J; Manga, Prashiela

2013-11-01

Accumulation of proteins in the endoplasmic reticulum (ER) typically induces stress and initiates the unfolded protein response (UPR) to facilitate recovery. If homeostasis is not restored, apoptosis is induced. However, adaptation to chronic UPR activation can increase resistance to subsequent acute ER stress. We therefore investigated adaptive mechanisms in Oculocutaneous albinism type 2 (Oca2)-null melanocytes where UPR signaling is arrested despite continued tyrosinase accumulation leading to resistance to the chemical ER stressor thapsigargin. Although thapsigargin triggers UPR activation, instead of Perk-mediated phosphorylation of eIF2α, in Oca2-null melanocytes, eIF2α was rapidly dephosphorylated upon treatment. Dephosphorylation was mediated by the Gadd34-PP1α phosphatase complex. Gadd34-complex inhibition blocked eIF2α dephosphorylation and significantly increased Oca2-null melanocyte sensitivity to thapsigargin. Thus, Oca2-null melanocytes adapt to acute ER stress by disruption of pro-apoptotic Perk signaling, which promotes cell survival. This is the first study to demonstrate rapid eIF2α dephosphorylation as an adaptive mechanism to ER stress. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The melanin synthesis inhibition and radical scavenging activities of compounds isolated from the aerial part of Lespedeza cyrtobotrya.

PubMed

Lee, Mi Yeon; Kim, Jin Hee; Choi, Jung Nam; Kim, Jiyoung; Hwang, Geum Sook; Lee, Choonghwan

2010-06-01

The EtOAc fraction of Lespedeza cyrtobotrya showed mushroom tyrosinase inhibitory and radical scavenging activity. Four active compounds were isolated based on LH-20 chromatography and HPLC, and the structures were elucidated on the basis of their LC-MS and NMR spectral data, as 2-(2,4-Dihydroxyphenyl)-6-hydroxybenzofuran (1), eriodictyol-7-O-glucopyranoside (2), haginin A (3), and dalbergioidin (4), respectively. 2-(2,4-Dihydroxyphenyl)-6-hydroxybenzofuran (1) showed mushroom tyrosinase inhibitory activity with an IC50 value of 5.2 micronM and acted as a competitive inhibitor. Furthermore, 37.3 micronM of compound 1 reduced 50 % of the melanin content on a human melanoma (MNT-1) cells. The radical scavenging activity of 2-(2,4-dihydroxyphenyl)-6-hydroxybenzofuran (1), eriodictyol-7-O-glucopyranoside (2), haginin A (3), and dalbergioidin (4) was shown with IC50 values of 11.0, 24.5, 9.0 and 36.5 micronM in an ABTS system and with IC50 values of 42.7, 36.0, 37.7 and 61.7 micronM in a DPPH system, respectively. The mushroom tyrosinase inhibitory activity of EtOAc fraction of Lespedeza cyrtobotrya was contributed by compound 1, 3 and 4, and radical scavenging activity of it was contributed by compound 1-4.

Hair-Cell Mechanotransduction Persists in TRP Channel Knockout Mice

PubMed Central

Niksch, Paul D.; Webber, Roxanna M.; Garcia-Gonzalez, Miguel; Watnick, Terry; Zhou, Jing; Vollrath, Melissa A.; Corey, David P.

2016-01-01

Members of the TRP superfamily of ion channels mediate mechanosensation in some organisms, and have been suggested as candidates for the mechanotransduction channel in vertebrate hair cells. Some TRP channels can be ruled out based on lack of an inner ear phenotype in knockout animals or pore properties not similar to the hair-cell channel. Such studies have excluded Trpv4, Trpa1, Trpml3, Trpm1, Trpm3, Trpc1, Trpc3, Trpc5, and Trpc6. However, others remain reasonable candidates. We used data from an RNA-seq analysis of gene expression in hair cells as well as data on TRP channel conductance to narrow the candidate group. We then characterized mice lacking functional Trpm2, Pkd2, Pkd2l1, Pkd2l2 and Pkd1l3, using scanning electron microscopy, auditory brainstem response, permeant dye accumulation, and single-cell electrophysiology. In all of these TRP-deficient mice, and in double and triple knockouts, mechanotransduction persisted. Together with published studies, these results argue against the participation of any of the 33 mouse TRP channels in hair cell transduction. PMID:27196058

A role for tyrosinase-related protein 1 in 4-tert-butylphenol-induced toxicity in melanocytes: Implications for vitiligo.

PubMed

Manga, Prashiela; Sheyn, David; Yang, Fan; Sarangarajan, Rangaprasad; Boissy, Raymond E

2006-11-01

Vitiligo presents with depigmented cutaneous lesions following localized melanocyte death. Multiple factors contribute to cell death, including genetically determined susceptibility to trauma, and environmental factors, such as exposure to 4-tert-butylphenol (4-TBP). We demonstrate that 4-TBP induces oxidative stress that is more readily overcome by melanocytes from normally pigmented individuals than from two individuals with vitiligo. The antioxidant catalase selectively and significantly reduced death of melanocytes derived from two individuals with vitiligo, indicating a role for oxidative stress in vitiligo pathogenesis. In normal melanocytes, oxidative stress results in reduced expression of microphthalmia-associated transcription factor (MITF). Melanocyte-stimulating hormone-induced expression of MITF protein caused increased sensitivity to 4-TBP, whereas sensitivity of melanomas correlated with MITF expression. MITF stimulates melanin synthesis by up-regulating expression of melanogenic enzymes such as tyrosinase-related protein-1 (Tyrp1). Although melanin content per se did not affect sensitivity to 4-TBP, expression of Tyrp1 significantly increased sensitivity. Melanocytes and melanomas that express functional Tyrp1 were significantly more sensitive to 4-TBP than Tyrp1-null cells. Thus, normal melanocytes respond to 4-TBP by reducing expression of MITF and Tyrp1. We hypothesize that melanocytes in vitiligo demonstrate reduced ability to withstand oxidative stress due, partly, to a disruption in MITF regulation of Tyrp1.

ULK1 Regulates Melanin Levels in MNT-1 Cells Independently of mTORC1

PubMed Central

Tooze, Sharon A.

2013-01-01

Melanosomes are lysosome-related organelles that serve as specialized sites of melanin synthesis and storage in melanocytes. The progression of melanosomes through the different stages of their formation requires trafficking of specific proteins and membrane constituents in a sequential manner, which is likely to deploy ubiquitous cellular machinery along with melanocyte-specific proteins. Recent evidence revealed a connection between melanogenesis and the autophagy machinery, suggesting a novel role for members of the latter in melanocytes. Here we focused on ULK1, a key autophagy protein which is negatively regulated by mTORC1, to assess its potential role in melanogenesis in MNT-1 cells. We found that ULK1 depletion causes an increase in melanin levels, suggesting an inhibitory function for this protein in melanogenesis. Furthermore, this increase was accompanied by increased transcription of MITF (microphthalmia-associated transcription factor) and tyrosinase and by elevated protein levels of tyrosinase, the rate-limiting factor in melanin biogenesis. We also provide evidence to show that ULK1 function in this context is independent of the canonical ULK1 autophagy partners, ATG13 and FIP200. Furthermore we show that regulation of melanogenesis by ULK1 is independent of mTORC1 inhibition. Our data thus provide intriguing insights regarding the involvement of the key regulatory autophagy machinery in melanogenesis. PMID:24066173

Dietary tryptophan depletion in humans using a simplified two amino acid formula - a pilot study.

PubMed

Linden, Maike; Helmbold, Katrin; Kempf, Janina; Sippas, Shabnam; Filss, Christian; Langen, Karl-Josef; Eisert, Albrecht; Zepf, Florian Daniel

2016-01-01

Acute tryptophan depletion (ATD) is a well-established dietary method in translational brain research used to briefly lower central nervous serotonin (5-hydroxytryptamine (5-HT)) synthesis. A simplified two amino acid ATD formula (ATD PHE/LEU ) was developed while reducing the overall amount of amino acids (AAs), with the objective of administration especially in children and adolescents in future studies. This study investigated tryptophan (TRP) influx rates across the blood-brain barrier (BBB) after dietary ATD PHE/LEU administration relative to the ATD Moja-De protocol that has been established for use in children and adolescents. Seventy-two healthy adults (50% females) were randomized into four groups and administered ATD Moja-De, its TRP-balanced control condition (BAL), ATD PHE/LEU , or its respective control mixture (BAL PHE/LEU ) in a counterbalanced, double-blind, between-subjects design. Blood samples were collected at baseline and at hourly intervals for 6 h after AA intake. Questionnaires about mood, taste, and challenge tolerance were completed at fixed time points. Both challenge mixtures significantly reduced central nervous TRP influx as calculated by Michaelis-Menten kinetics relative to baseline and the respective control conditions with only mild and comparable side effects. A greater decline in TRP influx over the BBB after ATD PHE/LEU administration when compared with ATD Moja-De was detected without group effects for taste, challenge tolerance, and mood. There was unintended initial short increase in plasma TRP concentrations observed after ATD PHE/LEU intake, and a possible redistribution between free and protein-bound TRP triggered by protein synthesis stimulated by the ingested AAs may account for this finding. Moreover, a decline in TRP influx after BAL PHE/LEU administration over a 6-h period was observed, and the large amount of PHE in the BAL PHE/LEU mixture may be a possible explanation for this particular phenomenon, which could have led to an unexpected increase in displacement of TRP at the BBB in this control condition. This pilot study provides preliminary evidence for the possibility of lowering TRP influx as calculated by Michaelis-Menten kinetics into the brain by using a simplified ATD protocol in humans. The simplified composition of only two AAs, the lower overall AA amount, and the appropriate tolerance are characteristics of the newly developed ATD PHE/LEU protocol. Future studies focusing on the effects of the ATD PHE/LEU protocol and its respective control condition on CSF 5-HIAA concentrations, as well as neurochemical studies in rodents, are needed to further validate this newly developed AA mixture before definite conclusions about its usability in ATD-related research in humans, its specificity, and additional effects can be made.

Molecular docking studies of (1E,3E,5E)-1,6-Bis(substituted phenyl)hexa-1,3,5-triene and 1,4-Bis(substituted trans-styryl)benzene analogs as novel tyrosinase inhibitors.

PubMed

Ha, Young Mi; Lee, Hye Jin; Park, Daeui; Jeong, Hyoung Oh; Park, Ji Young; Park, Yun Jung; Lee, Kyung Jin; Lee, Ji Yeon; Moon, Hyung Ryong; Chung, Hae Young

2013-01-01

We simulated the docking of the tertiary structure of mushroom tyrosinase with our compounds. From the structure-tyrosinase inhibitory activity relationship, it is notable that compounds 4, 8 and 11 showed similar or better activity rates than kojic acid which was used as a positive control. Compounds 17, 21, and 23 among benzene analogs that possess the same substituent showed significantly lower tyrosinase inhibitory effects. Therefore, we have confirmed that among the compounds showing better tyrosinase inhibitory effects than kojic acid, the compounds with triene analogs have better tyrosinase inhibitory effect than the compounds with benzene analogs. Docking simulation suggested the mechanism of compounds by several key residues which had possible hydrogen bonding interactions. The pharmacophore model underlined the features of active compounds, 4,4'-((1E,3E,5E)-hexa-1,3,5-triene-1,6-diyl)diphenol, 5,5'-((1E,3E,5E)-hexa-1,3,5-triene-1,6-diyl)bis(2-methoxy-phenol), and 5,5'-((1E,3E,5E)-hexa-1,3,5-triene-1,6-diyl)dibenzene-1,3-diol among triene derivatives which had several hydrogen bond groups on both terminal rings. The soundness of the docking results and the agreement with the pharmacophores suggest that it can be conveniently exploited to design inhibitors with an improved affinity for tyrosinase.

Distance Mapping in Proteins Using Fluorescence Spectroscopy: The Tryptophan-Induced Quenching (TrIQ) Method

PubMed Central

Mansoor, Steven E.; DeWitt, Mark A.; Farrens, David L.

2014-01-01

Studying the interplay between protein structure and function remains a daunting task. Especially lacking are methods for measuring structural changes in real time. Here we report our most recent improvements to a method that can be used to address such questions. This method, which we now call Tryptophan induced quenching (TrIQ), provides a straightforward, sensitive and inexpensive way to address questions of conformational dynamics and short-range protein interactions. Importantly, TrIQ only occurs over relatively short distances (~5 to 15 Å), making it complementary to traditional fluorescence resonance energy transfer (FRET) methods that occur over distances too large for precise studies of protein structure. As implied in the name, TrIQ measures the efficient quenching induced in some fluorophores by tryptophan (Trp). We present here our analysis of the TrIQ effect for five different fluorophores that span a range of sizes and spectral properties. Each probe was attached to four different cysteine residues on T4 lysozyme and the extent of TrIQ caused by a nearby Trp was measured. Our results show that for smaller probes, TrIQ is distance dependent. Moreover, we also demonstrate how TrIQ data can be analyzed to determine the fraction of fluorophores involved in a static, non-fluorescent complex with Trp. Based on this analysis, our study shows that each fluorophore has a different TrIQ profile, or "sphere of quenching", which correlates with its size, rotational flexibility, and the length of attachment linker. This TrIQ-based "sphere of quenching" is unique to every Trp-probe pair and reflects the distance within which one can expect to see the TrIQ effect. It provides a straightforward, readily accessible approach for mapping distances within proteins and monitoring conformational changes using fluorescence spectroscopy. PMID:20886836

"TRP inflammation" relationship in cardiovascular system.

PubMed

Numata, Tomohiro; Takahashi, Kiriko; Inoue, Ryuji

2016-05-01

Despite considerable advances in the research and treatment, the precise relationship between inflammation and cardiovascular (CV) disease remains incompletely understood. Therefore, understanding the immunoinflammatory processes underlying the initiation, progression, and exacerbation of many cardiovascular diseases is of prime importance. The innate immune system has an ancient origin and is well conserved across species. Its activation occurs in response to pathogens or tissue injury. Recent studies suggest that altered ionic balance, and production of noxious gaseous mediators link to immune and inflammatory responses with altered ion channel expression and function. Among plausible candidates for this are transient receptor potential (TRP) channels that function as polymodal sensors and scaffolding proteins involved in many physiological and pathological processes. In this review, we will first focus on the relevance of TRP channel to both exogenous and endogenous factors related to innate immune response and transcription factors related to sustained inflammatory status. The emerging role of inflammasome to regulate innate immunity and its possible connection to TRP channels will also be discussed. Secondly, we will discuss about the linkage of TRP channels to inflammatory CV diseases, from a viewpoint of inflammation in a general sense which is not restricted to the innate immunity. These knowledge may serve to provide new insights into the pathogenesis of various inflammatory CV diseases and their novel therapeutic strategies.

Inhibitions by hydrogen-occluding silica microcluster to melanogenesis in human pigment cells and tyrosinase reaction.

PubMed

Kato, Shinya; Saitoh, Yasukazu; Miwa, Nobuhiko

2013-01-01

We investigated the anti-melanogenetic efficacy of hydrogen-occluding silica microcluster (H2-Silica), which is a silsesquioxane-based compound with hydrogen interstitially embedded in a matrix of caged silica, against melanogenesis in HMV-II human melanoma cells and L-DOPA-tyrosinase reaction [EC1.14.18.1]. HMV-II cells were subjected to oxidative stress by ultraviolet ray-A (UVA) exposure of 3-times of 0.65 J/cm2 summed up to 1.95 J/cm2. After UVA irradiation, HMV-II cells were stimulated to produce melanin by 2.72-fold more abundantly than unirradiated control. When HMV-II cells were treated with H2-Silica of 20 ppm or kojic acid of 28.4 ppm before and after UVA-irradiation, the amount of melanin was repressed to 12.2% or 14.5% as compared to that of UVA-irradiated control, respectively. That is, H2-Silica exhibited a comparable efficacy to the whitening agent kojic acid. The H2-Silica could prevent melanogenesis in HMV-II cells by low-level doses at 1-10 ppm, and cell viability and apoptosis event did not change even by high-level doses at 100-1000 ppm. On the contrary, kojic acid was cytotoxic at the concentration of 14-28 ppm or more. By microscopic observation, H2-Silica suppressed such properties indicative of melanin-rich cells as cellular hypertrophy, cell process formation, and melanogenesis around the outside of nuclei. The enzymatic assay using L-DOPA and mushroom tyrosinase demonstrated that H2-Silica restrained UVA-mediated melanin formation owing to down-regulation of tyrosinase activity, which could be attributed to scavenging of free radicals and inhibition of L-DOPA-to-dopachrome oxidation by hydrogen released from H2-Silica. Thus H2-Silica has a potential to prevent melanin production against UVA and serves as a skin-lightening ingredient for supplements or cosmetics.

Design and discovery of mushroom tyrosinase inhibitors and their therapeutic applications.

PubMed

Mendes, Eduarda; Perry, Maria de Jesus; Francisco, Ana Paula

2014-05-01

Tyrosinase inhibitors could have a huge importance in medicine, cosmetics and agriculture. Although many tyrosinase inhibitors are available, they have demonstrated only mild efficacy and safety concerns. This has led to the discovery of novel tyrosinase inhibitors that are more safe, potent and efficacious. The authors provide an overview of the recent scientific accounts describing the design of new molecules. These compounds belong to different chemical families. The review emphasizes the rationale behind the discovery, the study of structure-activity relationships, the study of the mechanism and kinetic of inhibition and the cellular effect of the inhibitors. The article is based on the literature published from 2007 onward related with the development of synthetic tyrosinase inhibitors. Although a great number of tyrosinase inhibitors have been published in the literature, none, as of yet, have reached the potency and safety requirements needed to enter clinical trials. The emergence of new in vitro and in vivo tests will finally allow the arrival of new compounds that are more potent and safe.

In vitro assessment of the structure-activity relationship of tyrosinase-dependent cytotoxicity of a series of substituted phenols.

PubMed

Naish-Byfield, S; Cooksey, C J; Latter, A M; Johnson, C I; Riley, P A

1991-01-01

The rate of oxidation by purified mushroom tyrosinase of 30 compounds was measured by oximetry, and the tyrosinase-dependent cytotoxicity of each estimated in an in vitro assay using exposure of non-melanogenic cells to the agents in the presence and absence of tyrosinase. Cytotoxicity was estimated by immediate inhibition of DNA synthesis; 4-hydroxyanisole was used as the reference material. Compounds that were not oxidized by tyrosinase were found to be non-toxic but there was no direct relationship between the rate of oxidation and the relative cytotoxicity of those materials that acted as substrates for the enzyme. Thioethers were found to be more cytotoxic than the corresponding phenoxyethers. This was partly due to their greater rate of oxidation by tyrosinase and, in the case of propylthiophenol, the consequence of higher effective toxicity of the lipophilic species. The optimum chain length for the side chain of the oxyethers was three saturated carbon atoms and the toxicity appeared to be influenced by the lipophilicity of the compounds, possibly reflecting the relative lipid solubility of the putative toxic ortho-quinones generated from them. The maximum tyrosinase-dependent toxicity observed was in the range 5-6 times the relative toxicity of 4-hydroxyanisole. Sulphinyl and sulphonyl derivatives were inactive. In addition to oxyethers and thioethers, esters and glycosides of oxyethers were also examined and were found to be toxic in the presence of tyrosinase when hydrolysed. The succinates were found to be oxidized and toxic in our test system, suggesting that they rapidly underwent spontaneous hydrolysis. Oximetry data suggest that slight spontaneous hydrolysis of the other compounds occurs but they were not toxic in our assay. Ring-methylated phenoxyethers were oxidized relatively slowly and were non-toxic. Fluorine-substituted phenoxyethers were oxidized slightly more rapidly and exhibited clear toxicity in our system. Sesamol was oxidized to a black pigment but was non-toxic in our assay. A water-soluble vitamin E derivative was not oxidized and was non-toxic. Allyl hydroquinone was not oxidized but exhibited significant direct toxicity.

Generation of hydroxyl radicals and singlet oxygen during oxidation of rhododendrol and rhododendrol-catechol.

PubMed

Miyaji, Akimitsu; Gabe, Yu; Kohno, Masahiro; Baba, Toshihide

2017-03-01

The generation of hydroxyl radicals and singlet oxygen during the oxidation of 4-(4-hydroxyphenyl)-2-butanol (rhododendrol) and 4-(3,4-dihydroxyphenyl)-2-butanol (rhododendrol-catechol) with mushroom tyrosinase in a phosphate buffer (pH 7.4) was examined as the model for the reactive oxygen species generation via the two rhododendrol compounds in melanocytes. The reaction was performed in the presence of 5,5-dimethyl-1-pyrroline- N -oxide (DMPO) spin trap reagents for hydroxyl radical or 2,2,6,6-tetramethyl-4-piperidone (4-oxo-TEMP), an acceptor of singlet oxygen, and their electron spin resonances were measured. An increase in the electron spin resonances signal attributable to the adduct of DMPO reacting with the hydroxyl radical and that of 4-oxo-TEMP reacting with singlet oxygen was observed during the tyrosinase-catalyzed oxidation of rhododendrol and rhododendrol-catechol, indicating the generation of hydroxyl radical and singlet oxygen. Moreover, hydroxyl radical generation was also observed in the autoxidation of rhododendrol-catechol. We show that generation of intermediates during tyrosinase-catalyzed oxidation of rhododendrol enhances oxidative stress in melanocytes.

Distinct Protein Sorting and Localization to Premelanosomes, Melanosomes, and Lysosomes in Pigmented Melanocytic Cells✪

PubMed Central

Raposo, Graça; Tenza, Danielle; Murphy, Diane M.; Berson, Joanne F.; Marks, Michael S.

2001-01-01

Melanosomes and premelanosomes are lysosome-related organelles with a unique structure and cohort of resident proteins. We have positioned these organelles relative to endosomes and lysosomes in pigmented melanoma cells and melanocytes. Melanosome resident proteins Pmel17 and TRP1 localized to separate vesicular structures that were distinct from those enriched in lysosomal proteins. In immunogold-labeled ultrathin cryosections, Pmel17 was most enriched along the intralumenal striations of premelanosomes. Increased pigmentation was accompanied by a decrease in Pmel17 and by an increase in TRP1 in the limiting membrane. Both proteins were largely excluded from lysosomal compartments enriched in LAMP1 and cathepsin D. By kinetic analysis of fluid phase uptake and immunogold labeling, premelanosomal proteins segregated from endocytic markers within an unusual endosomal compartment. This compartment contained Pmel17, was accessed by BSA–gold after 15 min, was acidic, and displayed a cytoplasmic planar coat that contained clathrin. Our results indicate that premelanosomes and melanosomes represent a distinct lineage of organelles, separable from conventional endosomes and lysosomes within pigmented cells. Furthermore, they implicate an unusual clathrin-coated endosomal compartment as a site from which proteins destined for premelanosomes and lysosomes are sorted. PMID:11266471

Nucks1 synergizes with Trp53 to promote radiation lymphomagenesis in mice

PubMed Central

Yue, Yangbo; Leung, Stanley G.; Liu, Yueyong; Huang, Yurong; Grundt, Kirsten; Østvold, Anne-Carine; Jen, Kuang-Yu; Schild, David; Mao, Jian-Hua; Wiese, Claudia

2016-01-01

NUCKS1 is a 27 kD vertebrate-specific protein, with a role in the DNA damage response. Here, we show that after 4 Gy total-body X-irradiation, Trp53+/− Nucks1+/− mice more rapidly developed tumors, particularly thymic lymphoma (TL), than Trp53+/− mice. TLs in both cohorts showed loss of heterozygosity (LOH) of the Trp53+ allele in essentially all cases. In contrast, LOH of the Nucks1+ allele was rare. Nucks1 expression correlated well with Nucks1 gene dosage in normal thymi, but was increased in the majority of TLs from Trp53+/− Nucks1+/− mice, suggesting that elevated Nucks1 message may be associated with progression towards malignancy in vivo. Trp53+/− Nucks1+/− mice frequently succumbed to CD4- CD8- TLs harboring translocations involving Igh but not Tcra/d, indicating TLs in Trp53+/− Nucks1+/− mice mostly originated prior to the double positive stage and at earlier lineage than TLs in Trp53+/- mice. Monoclonal rearrangements at Tcrb were more prevalent in TLs from Trp53+/− Nucks1+/− mice, as was infiltration of primary TL cells to distant organs (liver, kidney and spleen). We propose that, in the context of Trp53 deficiency, wild type levels of Nucks1 are required to suppress radiation-induced TL, likely through the role of the NUCKS1 protein in the DNA damage response. PMID:27542204

Fluidization of Membrane Lipids Enhances the Tolerance of Saccharomyces cerevisiae to Freezing and Salt Stress▿

PubMed Central

Rodríguez-Vargas, Sonia; Sánchez-García, Alicia; Martínez-Rivas, Jose Manuel; Prieto, Jose Antonio; Randez-Gil, Francisca

2007-01-01

Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Δ9 position. We expressed two sunflower (Helianthus annuus) oleate Δ12 desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Δ9,12, the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15°C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp+ or Trp− strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30°C or 15°C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains. PMID:17071783

Microbial Tyrosinases: Promising Enzymes for Pharmaceutical, Food Bioprocessing, and Environmental Industry

PubMed Central

Zaidi, Kamal Uddin; Ali, Ayesha S.; Ali, Sharique A.; Naaz, Ishrat

2014-01-01

Tyrosinase is a natural enzyme and is often purified to only a low degree and it is involved in a variety of functions which mainly catalyse the o-hydroxylation of monophenols into their corresponding o-diphenols and the oxidation of o-diphenols to o-quinones using molecular oxygen, which then polymerizes to form brown or black pigments. The synthesis of o-diphenols is a potentially valuable catalytic ability and thus tyrosinase has attracted a lot of attention with respect to industrial applications. In environmental technology it is used for the detoxification of phenol-containing wastewaters and contaminated soils, as biosensors for phenol monitoring, and for the production of L-DOPA in pharmaceutical industries, and is also used in cosmetic and food industries as important catalytic enzyme. Melanin pigment synthesized by tyrosinase has found applications for protection against radiation cation exchangers, drug carriers, antioxidants, antiviral agents, or immunogen. The recombinant V. spinosum tryosinase protein can be used to produce tailor-made melanin and other polyphenolic materials using various phenols and catechols as starting materials. This review compiles the recent data on biochemical and molecular properties of microbial tyrosinases, underlining their importance in the industrial use of these enzymes. After that, their most promising applications in pharmaceutical, food processing, and environmental fields are presented. PMID:24895537

Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes

PubMed Central

Varma, Sandeep R.; Sivaprakasam, Thiyagarajan O.; Mishra, Abheepsa; Kumar, L. M. Sharath; Prakash, N. S.; Prabhu, Sunil; Ramakrishnan, Shyam

2016-01-01

Human skin is body’s vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated in vitro. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC50 values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 μg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H2O2) induced RBC haemolysis (IC50 64.95 μg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H2O2-induced damage; inhibited H2O2 induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells in vitro and can be used as a potential ingredient in skin care formulations. PMID:26731545

Effect of different tryptophan sources on amino acids availability to the brain and mood in healthy volunteers.

PubMed

Markus, C Rob; Firk, Christine; Gerhardt, Cindy; Kloek, Joris; Smolders, Gertjan F

2008-11-01

Reduced brain serotonin function is acknowledged as a vulnerability factor for affective disturbances. Since the production of serotonin is limited by the availability of its plasma dietary amino acid precursor tryptophan (TRP), the beneficial effects of tryptophan-rich alpha-lactalbumin whey protein (ALAC) have recently been studied. The effects of ALAC remain rather modest, and alternative protein sources of tryptophan may be more effective. We tested whether hydrolyzed protein (HPROT) has greater effects on the plasma TRP/large neutral amino acids (LNAA) ratio and mood than intact ALAC protein in healthy volunteers. In a double-blind, randomized cross-over study, plasma amino acids and mood were repeatedly measured in 18 healthy subjects before and after intake of ALAC and HPROT as well as after placebo protein, pure tryptophan, and a tryptophan-containing synthetic peptide. Except for the placebo protein, all interventions contained 0.8 g TRP. Significantly faster and greater increases in plasma TRP/LNAA were found after HPROT than after ALAC. In addition, the effects of HPROT on plasma TRP/LNAA were comparable with the effects of the tryptophan-containing synthetic peptide and even exceeded the effect of pure tryptophan. Sixty minutes after intake, mood was improved only following intake of HPROT and pure tryptophan, whereas longer-lasting mood effects were only found after intake of HPROT. The use of a tryptophan-rich hydrolyzed protein source may be more adequate to increase brain tryptophan and 5-HT function compared with intact alpha-lactalbumin protein or pure tryptophan.

A PTPN11 allele encoding a catalytically impaired SHP2 protein in a patient with a Noonan syndrome phenotype.

PubMed

Edwards, Jonathan J; Martinelli, Simone; Pannone, Luca; Lo, Ivan Fai-Man; Shi, Lisong; Edelmann, Lisa; Tartaglia, Marco; Luk, Ho-Ming; Gelb, Bruce D

2014-09-01

The RASopathies are a relatively common group of phenotypically similar and genetically related autosomal dominant genetic syndromes caused by missense mutations affecting genes participating in the RAS/mitogen-activated protein kinase (MAPK) pathway that include Noonan syndrome (NS) and Noonan syndrome with multiple lentigines (NSML, formerly LEOPARD syndrome). NS and NSML can be difficult to differentiate during infancy, but the presence of multiple lentigines, café au lait spots, and specific cardiac defects facilitate the diagnosis. Furthermore, individual PTPN11 missense mutations are highly specific to each syndrome and engender opposite biochemical alterations on the function of SHP-2, the protein product of that gene. Here, we report on a 5-year-old male with two de novo PTPN11 mutations in cis, c.1471C>T (p.Pro491Ser), and c.1492C>T (p.Arg498Trp), which are associated with NS and NSML, respectively. This boy's phenotype is intermediate between NS and NSML with facial dysmorphism, short stature, mild global developmental delay, pulmonic stenosis, and deafness but absence of café au lait spots or lentigines. The double-mutant SHP-2 was found to be catalytically impaired. This raises the question of whether clinical differences between NS and NSML can be ascribed solely to the relative SHP-2 catalytic activity. © 2014 Wiley Periodicals, Inc.

Surplus dietary tryptophan reduces plasma cortisol and noradrenaline concentrations and enhances recovery after social stress in pigs.

PubMed

Koopmans, Sietse Jan; Ruis, Marko; Dekker, Ruud; van Diepen, Hans; Korte, Mechiel; Mroz, Zdzislaw

2005-07-21

Social stress occurs in intensive pig farming due to aggressive behavior. This stress may be reduced at elevated dietary levels of tryptophan (TRP). In this study, we compared the effects of high (13.2%) vs. normal (3.4%) dietary TRP to large neutral amino acid (LNAA) ratios on behavior and stress hormones in catheterized pigs ( approximately 50 kg BW), which were exposed to social stress by placing them twice into the territory of a dominant pig ( approximately 60 kg) for 15 min. Pre-stress plasma TRP concentrations were 156+/-15 vs. 53+/-6 micromol/l (p<0.01) in pigs on the high vs. normal TRP diets, respectively. Pre-stress plasma cortisol and noradrenaline concentrations were twofold (p<0.01) and 1.4-fold (p<0.05) lower but plasma adrenaline concentration was similar in pigs on the high vs. normal TRP diets, respectively. During the social confrontations, pigs on the high vs. normal TRP diets show a tendency towards reduced active avoidance behavior (3.2+/-1.1 vs. 6.7+/-1.2 min, p<0.1) but their physical activity (8.5+/-0.6 vs. 10.2+/-0.8 min) and aggressive attitude towards the dominant pig (11+/-3 vs. 7+/-2 times biting) were similar. Immediate (+5 min) post-stress plasma cortisol, noradrenaline and adrenaline responses were similar among dietary groups. After the social confrontations, the post-stress plasma cortisol, noradrenaline and adrenaline concentrations and/or curves (from +5 min to 2 h) were lower/steeper (p<0.05) in pigs on the high vs. normal TRP diets. In summary, surplus TRP in diets for pigs (1) does not significantly affect behavior when exposed to social stress, (2) reduces basal plasma cortisol and noradrenaline concentrations, (3) does not affect the immediate hormonal response to stress, and (4) reduces the long-term hormonal response to stress. In general, pigs receiving high dietary TRP were found to be less affected by stress.

Influence of heme-thiolate in shaping the catalytic properties of a bacterial nitric-oxide synthase.

PubMed

Hannibal, Luciana; Somasundaram, Ramasamy; Tejero, Jesús; Wilson, Adjele; Stuehr, Dennis J

2011-11-11

Nitric-oxide synthases (NOS) are heme-thiolate enzymes that generate nitric oxide (NO) from L-arginine. Mammalian and bacterial NOSs contain a conserved tryptophan (Trp) that hydrogen bonds with the heme-thiolate ligand. We mutated Trp(66) to His and Phe (W66H, W66F) in B. subtilis NOS to investigate how heme-thiolate electronic properties control enzyme catalysis. The mutations had opposite effects on heme midpoint potential (-302, -361, and -427 mV for W66H, wild-type (WT), and W66F, respectively). These changes were associated with rank order (W66H < WT < W66F) changes in the rates of oxygen activation and product formation in Arg hydroxylation and N-hydroxyarginine (NOHA) oxidation single turnover reactions, and in the O(2) reactivity of the ferrous heme-NO product complex. However, enzyme ferrous heme-O(2) autoxidation showed an opposite rank order. Tetrahydrofolate supported NO synthesis by WT and the mutant NOS. All three proteins showed similar extents of product formation (L-Arg → NOHA or NOHA → citrulline) in single turnover studies, but the W66F mutant showed a 2.5 times lower activity when the reactions were supported by flavoproteins and NADPH. We conclude that Trp(66) controls several catalytic parameters by tuning the electron density of the heme-thiolate bond. A greater electron density (as in W66F) improves oxygen activation and reactivity toward substrate, but decreases heme-dioxy stability and lowers the driving force for heme reduction. In the WT enzyme the Trp(66) residue balances these opposing effects for optimal catalysis.

New antagonists of LHRH. II. Inhibition and potentiation of LHRH by closely related analogues.

PubMed

Bajusz, S; Csernus, V J; Janaky, T; Bokser, L; Fekete, M; Schally, A V

1988-12-01

Modifications of the previously described LHRH antagonists, [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Trp3, D-Cit6, D-Ala10]LHRH and the corresponding D-Hci6 analogue, have been made to alter the hydrophobicity of the N-terminal acetyl-tripeptide portion. Substitution of D-Trp3 with the less hydrophobic D-Pal(3) had only marginal effects on the antagonistic activities and receptor binding potencies of the D-Cit/D-Hci6 analogues, but it appeared to further improve the toxicity lowering effect of D-Cit/D-Hci6 substitution. Antagonists containing D-Pal(3)3 and D-Cit/D-Hci6 residues, i.e. [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10]LHRH (SB-75) and [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Hci6, D-Ala10]LHRH (SB-88), were completely free of the toxic effects, such as cyanosis and respiratory depression leading to death, which have been observed in rats with the D-Trp3, D-Arg6 antagonist and related antagonists. Replacement of the N-acetyl group with the hydrophilic carbamoyl group caused a slight decrease in antagonistic activities, particularly in vitro. Introduction of urethane type acyl group such as methoxycarbonyl (Moc) or t-butoxycarbonyl (Boc) led to analogues that showed LHRH-potentiating effect. The increase in potency induced by these analogues, e.g. [Moc-D-Nal(2)1, D-Phe(4Cl)2, D-Trp3, D-Cit6, D-Ala10]LHRH and [Boc-D-Phe1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10]LHRH, was 170-260% and persisted for more than 2 h when studied in a superfused rat pituitary system.

The Potency of White Rice (Oryza sativa), Black Rice (Oryza sativa L. indica), and Red Rice (Oryza nivara) as Antioxidant and Tyrosinase Inhibitor

NASA Astrophysics Data System (ADS)

Batubara, I.; Maharni, M.; Sadiah, S.

2017-04-01

Rice is known to have many beneficial biological activities and is often used as “bedak dingin”, a face powder. The content of vitamins, minerals, fiber, and several types of antioxidants, such as ferulic acid, phytic acid, tocopherol, and oryzanols [1-2] are predicted to be potential as a tyrosinase inhibitor. The purpose of this study is to determine the potency of extracts from there types of rice, namely white, red, and black rice as an antioxidant and tyrosinase inhibitor. The rice was extracted with three different solvents, n-hexane, ethyl acetate, and methanol. The results showed that the highest antioxidant activity using 1,1-diphenyl-2-picrylhydrazyl method was found in the methanol extract of black rice (IC50 290 μg/mL). Meanwhile, ethyl acetate extract of white rice has the highest antioxidant activity withphosphomolybdic acid method (41 mmol α-tocopherol equivalents/g sample). Thus, methanol extract of black rice and ethyl acetate extract of white rice are potential as an antioxidant. For tyrosinase inhibitor, n-hexane extract of red rice (IC50 3156 μg/mL) was the most active extract. The active component for radical scavenging is polar compound and for antioxidant by phosphomolybdate method is less polar compounds in black rice methanol extract based on TLC bioautogram. In conclusion, the black rice is the most potent in antioxidant while red rice is for tyrosinase inhibition.

Codon Usage Patterns of Tyrosinase Genes in Clonorchis sinensis.

PubMed

Bae, Young-An

2017-04-01

Codon usage bias (CUB) is a unique property of genomes and has contributed to the better understanding of the molecular features and the evolution processes of particular gene. In this study, genetic indices associated with CUB, including relative synonymous codon usage and effective numbers of codons, as well as the nucleotide composition, were investigated in the Clonorchis sinensis tyrosinase genes and their platyhelminth orthologs, which play an important role in the eggshell formation. The relative synonymous codon usage patterns substantially differed among tyrosinase genes examined. In a neutrality analysis, the correlation between GC 12 and GC 3 was statistically significant, and the regression line had a relatively gradual slope (0.218). NC-plot, i.e., GC 3 vs effective number of codons (ENC), showed that most of the tyrosinase genes were below the expected curve. The codon adaptation index (CAI) values of the platyhelminth tyrosinases had a narrow distribution between 0.685/0.714 and 0.797/0.837, and were negatively correlated with their ENC. Taken together, these results suggested that CUB in the tyrosinase genes seemed to be basically governed by selection pressures rather than mutational bias, although the latter factor provided an additional force in shaping CUB of the C. sinensis and Opisthorchis viverrini genes. It was also apparent that the equilibrium point between selection pressure and mutational bias is much more inclined to selection pressure in highly expressed C. sinensis genes, than in poorly expressed genes.

Structure-function analysis of amino acid 74 of human RAMP1 and RAMP3 and its role in peptide interactions with adrenomedullin and calcitonin gene-related peptide receptors.

PubMed

Qi, Tao; Ly, Kien; Poyner, David R; Christopoulos, George; Sexton, Patrick M; Hay, Debbie L

2011-05-01

The receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are complexes of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMP). The CGRP receptor is a CLR/RAMP1 pairing whereas CLR/RAMP2 and CLR/RAMP3 constitute two subtypes of AM receptor: AM(1) and AM(2), respectively. Previous studies identified Glu74 in RAMP3 to be important for AM binding and potency. To further understand the importance of this residue and its equivalent in RAMP1 (Trp74) we substituted the native amino acids with several others. In RAMP3, these were Trp, Phe, Tyr, Ala, Ser, Thr, Arg and Asn; in RAMP1, Glu, Phe, Tyr, Ala and Asn substitutions were made. The mutant RAMPs were co-expressed with CLR in Cos7 cells; receptor function in response to AM, AM(2)/intermedin and CGRP was measured in a cAMP assay and cell surface expression was determined by ELISA. Phe reduced AM potency in RAMP3 but had no effect in RAMP1. In contrast, Tyr had no effect in RAMP3 but enhanced AM potency in RAMP1. Most other substitutions had a small effect on AM potency in both receptors whereas there was little impact on CGRP or AM(2) potency. Overall, these data suggest that the geometry and charge of the residue at position 74 contribute to how AM interacts with the AM(2) and CGRP receptors and confirms the role of this position in dictating differential AM pharmacology at the AM(2) and CGRP receptors. Copyright © 2011. Published by Elsevier Inc.

Combining molecular docking and QSAR studies for modeling the anti-tyrosinase activity of aromatic heterocycle thiosemicarbazone analogues

NASA Astrophysics Data System (ADS)

Dong, Huanhuan; Liu, Jing; Liu, Xiaoru; Yu, Yanying; Cao, Shuwen

2018-01-01

A collection of thirty-six aromatic heterocycle thiosemicarbazone analogues presented a broad span of anti-tyrosinase activities were designed and obtained. A robust and reliable two-dimensional quantitative structure-activity relationship model, as evidenced by the high q2 and r2 values (0.848 and 0.893, respectively), was gained based on the analogues to predict the quantitative chemical-biological relationship and the new modifier direction. Inhibitory activities of the compounds were found to greatly depend on molecular shape and orbital energy. Substituents brought out large ovality and high highest-occupied molecular orbital energy values helped to improve the activity of these analogues. The molecular docking results provided visual evidence for QSAR analysis and inhibition mechanism. Based on these, two novel tyrosinase inhibitors O04 and O05 with predicted IC50 of 0.5384 and 0.8752 nM were designed and suggested for further research.

Biometal binding-site mimicry with modular, hetero-bifunctionally modified architecture encompassing a Trp/His motif: insights into spatiotemporal noncovalent interactions from a comparative spectroscopic study.

PubMed

Yang, Chi Ming

2011-03-28

Metal-site Trp/His interactions are crucial to diverse metalloprotein functions. This paper presents a study using metal-motif mimicry to capture and dissect the static and transient components of physicochemical properties underlying the Trp/His aromatic side-chain noncovalent interactions across the first- and second-coordination spheres of biometal ions. Modular biomimetic constructs, EDTA-(L-Trp, L-His) or EWH and DTPA-(L-Trp, L-His) or DWH, featuring a function-significant Trp/His pair, enabled extracting the putative hydrophobic/hydrophilic aromatic interactions surrounding metal centers. Fluorescence, circular dichroism (CD) spectroscopic titrations and ESI mass spectrometry demonstrated that both the constructs stoichiometrically bind to Ca(2+), Co(2+), Cu(2+), Ni(2+), Mn(2+), Zn(2+), Cd(2+), and Fe(2+), and such binding was strongly coupled to stereospecific side-chain structure reorientations of the Trp indole and His imidazole rings. A mechanistic dichotomy corresponding to the participation of the indole unit in the binding event was revealed by a scaffold-platform correlation of steady-state fluorescence-response landscape, illuminating that secondary-coordination-sphere ligand cation-π interactions were immediately followed by subsequent transient physicochemical processes including through-space energy transfer, charge transfer and/or electron transfer, depending on the type of metals. The fluorescence quenching of Trp side chain by 3d metal ions can be ascribed to through-space d-π interactions. While the fluorescence titration was capable of illuminating a two-component energetic model, clean isosbestic/isodichroic points in the CD titration spectra indicated that the metallo-constructs, such as Cu(2+)-EWH complex, fold thermodynamically by means of a two-state equilibrium. Further, the metal-ion dependence of Trp conformational variation in the modular architecture of metal-bound scaffolds was evidenced unambiguously by the CD spectra and supported by MMFF calculations; both were capable of distinguishing between the coordination geometry and the preference for metal binding mode. The study thus helps understand how aromatic rings around metal-sites have unique capabilities through the control of the spatiotemporal distribution of noncovalent interaction elements to achieve diverse chemical functionality.

Ser298 of MITF, a mutation site in Waardenburg syndrome type 2, is a phosphorylation site with functional significance.

PubMed

Takeda, K; Takemoto, C; Kobayashi, I; Watanabe, A; Nobukuni, Y; Fisher, D E; Tachibana, M

2000-01-01

MITF (microphthalmia-associated transcription factor) is a basic-helix-loop-helix-leucine zipper (bHLHZip) factor which regulates expression of tyrosinase and other melanocytic genes via a CATGTG promoter sequence, and is involved in melanocyte differentiation. Mutations of MITF in mice or humans with Waardenburg syndrome type 2 (WS2) often severely disrupt the bHLHZip domain, suggesting the importance of this structure. Here, we show that Ser298, which locates downstream of the bHLHZip and was previously found to be mutated in individuals with WS2, plays an important role in MITF function. Glycogen synthase kinase 3 (GSK3) was found to phosphorylate Ser298 in vitro, thereby enhancing the binding of MITF to the tyrosinase promoter. The same serine was found to be phosphorylated in vivo, and expression of dominant-negative GSK3beta selectively suppressed the ability of MITF to transactivate the tyrosinase promoter. Moreover, mutation of Ser298, as found in a WS2 family, disabled phos-phorylation of MITF by GSK3beta and impaired MITF function. These findings suggest that the Ser298 is important for MITF function and is phosphorylated probably by GSK3beta.

Odontoblasts as sensory receptors: transient receptor potential channels, pannexin-1, and ionotropic ATP receptors mediate intercellular odontoblast-neuron signal transduction.

PubMed

Shibukawa, Yoshiyuki; Sato, Masaki; Kimura, Maki; Sobhan, Ubaidus; Shimada, Miyuki; Nishiyama, Akihiro; Kawaguchi, Aya; Soya, Manabu; Kuroda, Hidetaka; Katakura, Akira; Ichinohe, Tatsuya; Tazaki, Masakazu

2015-04-01

Various stimuli induce pain when applied to the surface of exposed dentin. However, the mechanisms underlying dentinal pain remain unclear. We investigated intercellular signal transduction between odontoblasts and trigeminal ganglion (TG) neurons following direct mechanical stimulation of odontoblasts. Mechanical stimulation of single odontoblasts increased the intracellular free calcium concentration ([Ca(2+)]i) by activating the mechanosensitive-transient receptor potential (TRP) channels TRPV1, TRPV2, TRPV4, and TRPA1, but not TRPM8 channels. In cocultures of odontoblasts and TG neurons, increases in [Ca(2+)]i were observed not only in mechanically stimulated odontoblasts, but also in neighboring odontoblasts and TG neurons. These increases in [Ca(2+)]i were abolished in the absence of extracellular Ca(2+) and in the presence of mechanosensitive TRP channel antagonists. A pannexin-1 (ATP-permeable channel) inhibitor and ATP-degrading enzyme abolished the increases in [Ca(2+)]i in neighboring odontoblasts and TG neurons, but not in the stimulated odontoblasts. G-protein-coupled P2Y nucleotide receptor antagonists also inhibited the increases in [Ca(2+)]i. An ionotropic ATP (P2X3) receptor antagonist inhibited the increase in [Ca(2+)]i in neighboring TG neurons, but not in stimulated or neighboring odontoblasts. During mechanical stimulation of single odontoblasts, a connexin-43 blocker did not have any effects on the [Ca(2+)]i responses observed in any of the cells. These results indicate that ATP, released from mechanically stimulated odontoblasts via pannexin-1 in response to TRP channel activation, transmits a signal to P2X3 receptors on TG neurons. We suggest that odontoblasts are sensory receptor cells and that ATP released from odontoblasts functions as a neurotransmitter in the sensory transduction sequence for dentinal pain.

Bioinformatic Analysis Reveals Archaeal tRNATyr and tRNATrp Identities in Bacteria

PubMed Central

Mukai, Takahito; Reynolds, Noah M.; Crnković, Ana; Söll, Dieter

2017-01-01

The tRNA identity elements for some amino acids are distinct between the bacterial and archaeal domains. Searching in recent genomic and metagenomic sequence data, we found some candidate phyla radiation (CPR) bacteria with archaeal tRNA identity for Tyr-tRNA and Trp-tRNA synthesis. These bacteria possess genes for tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase (TrpRS) predicted to be derived from DPANN superphylum archaea, while the cognate tRNATyr and tRNATrp genes reveal bacterial or archaeal origins. We identified a trace of domain fusion and swapping in the archaeal-type TyrRS gene of a bacterial lineage, suggesting that CPR bacteria may have used this mechanism to create diverse proteins. Archaeal-type TrpRS of bacteria and a few TrpRS species of DPANN archaea represent a new phylogenetic clade (named TrpRS-A). The TrpRS-A open reading frames (ORFs) are always associated with another ORF (named ORF1) encoding an unknown protein without global sequence identity to any known protein. However, our protein structure prediction identified a putative HIGH-motif and KMSKS-motif as well as many α-helices that are characteristic of class I aminoacyl-tRNA synthetase (aaRS) homologs. These results provide another example of the diversity of molecular components that implement the genetic code and provide a clue to the early evolution of life and the genetic code. PMID:28230768

Analytic gradient for second order Møller-Plesset perturbation theory with the polarizable continuum model based on the fragment molecular orbital method.

PubMed

Nagata, Takeshi; Fedorov, Dmitri G; Li, Hui; Kitaura, Kazuo

2012-05-28

A new energy expression is proposed for the fragment molecular orbital method interfaced with the polarizable continuum model (FMO/PCM). The solvation free energy is shown to be more accurate on a set of representative polypeptides with neutral and charged residues, in comparison to the original formulation at the same level of the many-body expansion of the electrostatic potential determining the apparent surface charges. The analytic first derivative of the energy with respect to nuclear coordinates is formulated at the second-order Møller-Plesset (MP2) perturbation theory level combined with PCM, for which we derived coupled perturbed Hartree-Fock equations. The accuracy of the analytic gradient is demonstrated on test calculations in comparison to numeric gradient. Geometry optimization of the small Trp-cage protein (PDB: 1L2Y) is performed with FMO/PCM/6-31(+)G(d) at the MP2 and restricted Hartree-Fock with empirical dispersion (RHF/D). The root mean square deviations between the FMO optimized and NMR experimental structure are found to be 0.414 and 0.426 Å for RHF/D and MP2, respectively. The details of the hydrogen bond network in the Trp-cage protein are revealed.

Analytic gradient for second order Møller-Plesset perturbation theory with the polarizable continuum model based on the fragment molecular orbital method

NASA Astrophysics Data System (ADS)

Nagata, Takeshi; Fedorov, Dmitri G.; Li, Hui; Kitaura, Kazuo

2012-05-01

A new energy expression is proposed for the fragment molecular orbital method interfaced with the polarizable continuum model (FMO/PCM). The solvation free energy is shown to be more accurate on a set of representative polypeptides with neutral and charged residues, in comparison to the original formulation at the same level of the many-body expansion of the electrostatic potential determining the apparent surface charges. The analytic first derivative of the energy with respect to nuclear coordinates is formulated at the second-order Møller-Plesset (MP2) perturbation theory level combined with PCM, for which we derived coupled perturbed Hartree-Fock equations. The accuracy of the analytic gradient is demonstrated on test calculations in comparison to numeric gradient. Geometry optimization of the small Trp-cage protein (PDB: 1L2Y) is performed with FMO/PCM/6-31(+)G(d) at the MP2 and restricted Hartree-Fock with empirical dispersion (RHF/D). The root mean square deviations between the FMO optimized and NMR experimental structure are found to be 0.414 and 0.426 Å for RHF/D and MP2, respectively. The details of the hydrogen bond network in the Trp-cage protein are revealed.

Dynamic Folding Pathway Models of the Trp-Cage Protein

PubMed Central

Kim, Seung-Yeon

2013-01-01

Using action-derived molecular dynamics (ADMD), we study the dynamic folding pathway models of the Trp-cage protein by providing its sequential conformational changes from its initial disordered structure to the final native structure at atomic details. We find that the numbers of native contacts and native hydrogen bonds are highly correlated, implying that the native structure of Trp-cage is achieved through the concurrent formations of native contacts and native hydrogen bonds. In early stage, an unfolded state appears with partially formed native contacts (~40%) and native hydrogen bonds (~30%). Afterward, the folding is initiated by the contact of the side chain of Tyr3 with that of Trp6, together with the formation of the N-terminal α-helix. Then, the C-terminal polyproline structure docks onto the Trp6 and Tyr3 rings, resulting in the formations of the hydrophobic core of Trp-cage and its near-native state. Finally, the slow adjustment processes of the near-native states into the native structure are dominant in later stage. The ADMD results are in agreement with those of the experimental folding studies on Trp-cage and consistent with most of other computational studies. PMID:23865078

Distribution of stable free radicals among amino acids of isolated soy proteins.

PubMed

Lei, Qingxin; Liebold, Christopher M; Boatright, William L; Shah Jahan, M

2010-09-01

Application of deuterium sulfide to powdered isolated soy proteins (ISP) was used to quench stable free radicals and produce a single deuterium label on amino acids where free radicals reside. The deuterium labels rendered increases of isotope ratio for the specific ions of radical-bearing amino acids. Isotope ratio measurements were achieved by gas chromatography/mass spectrometry (GC/MS) analyses after the amino acids were released by acidic hydrolysis and converted to volatile derivatives with propyl chloroformate. The isotope enrichment data showed the stable free radicals were located on Ala, Gly, Leu, Ile, Asx (Asp+Asn), Glx (Glu+Gln), and Trp but not on Val, Pro, Met, Phe, Lys, and His. Due to the low abundance of Ser, Thr, and Cys derivatives and the impossibility to accurately measure their isotope ratios, the radical bearing status for these amino acids remained undetermined even though their derivatives were positively identified from ISP hydrolysates. The relative isotope enrichment for radical-bearing amino acids Ala, Gly, Leu, Ile, Asx (Asp+Asn), Glx (Glu+Gln), and Trp were 8.67%, 2.96%, 2.90%, 3.94%, 6.03%, 3.91%, and 21.48%, respectively. Isotope ratio increase for Tyr was also observed but further investigation revealed such increase was mainly from nonspecific deuterium-hydrogen exchange not free radical quenching. The results obtained from the present study provide important information for a better understanding of the mechanisms of free radical formation and stabilization in "dry" ISP.

Costs of childhood asthma due to traffic-related pollution in two California communities.

PubMed

Brandt, Sylvia J; Perez, Laura; Künzli, Nino; Lurmann, Fred; McConnell, Rob

2012-08-01

Recent research suggests the burden of childhood asthma that is attributable to air pollution has been underestimated in traditional risk assessments, and there are no estimates of these associated costs. We aimed to estimate the yearly childhood asthma-related costs attributable to air pollution for Riverside and Long Beach, CA, USA, including: 1) the indirect and direct costs of healthcare utilisation due to asthma exacerbations linked with traffic-related pollution (TRP); and 2) the costs of health care for asthma cases attributable to local TRP exposure. We calculated costs using estimates from peer-reviewed literature and the authors' analysis of surveys (Medical Expenditure Panel Survey, California Health Interview Survey, National Household Travel Survey, and Health Care Utilization Project). A lower-bound estimate of the asthma burden attributable to air pollution was US$18 million yearly. Asthma cases attributable to TRP exposure accounted for almost half of this cost. The cost of bronchitic episodes was a major proportion of both the annual cost of asthma cases attributable to TRP and of pollution-linked exacerbations. Traditional risk assessment methods underestimate both the burden of disease and cost of asthma associated with air pollution, and these costs are borne disproportionately by communities with higher than average TRP.

Activation of a promyelocytic leukemia-tumor protein 53 axis underlies acute promyelocytic leukemia cure.

PubMed

Ablain, Julien; Rice, Kim; Soilihi, Hassane; de Reynies, Aurélien; Minucci, Saverio; de Thé, Hugues

2014-02-01

Acute promyelocytic leukemia (APL) is driven by the promyelocytic leukemia (PML)-retinoic acid receptor-α (PML-RARA) fusion protein, which interferes with nuclear receptor signaling and PML nuclear body (NB) assembly. APL is the only malignancy definitively cured by targeted therapies: retinoic acid (RA) and/or arsenic trioxide, which both trigger PML-RARA degradation through nonoverlapping pathways. Yet, the cellular and molecular determinants of treatment efficacy remain disputed. We demonstrate that a functional Pml-transformation-related protein 53 (Trp53) axis is required to eradicate leukemia-initiating cells in a mouse model of APL. Upon RA-induced PML-RARA degradation, normal Pml elicits NB reformation and induces a Trp53 response exhibiting features of senescence but not apoptosis, ultimately abrogating APL-initiating activity. Apart from triggering PML-RARA degradation, arsenic trioxide also targets normal PML to enhance NB reformation, which may explain its clinical potency, alone or with RA. This Pml-Trp53 checkpoint initiated by therapy-triggered NB restoration is specific for PML-RARA-driven APL, but not the RA-resistant promyelocytic leukemia zinc finger (PLZF)-RARA variant. Yet, as NB biogenesis is druggable, it could be therapeutically exploited in non-APL malignancies.

Small Peptides Derived from Penetratin as Antibacterial Agents.

PubMed

Parravicini, Oscar; Somlai, Csaba; Andujar, Sebastián A; Garro, Adriana D; Lima, Beatriz; Tapia, Alejandro; Feresin, Gabriela; Perczel, Andras; Tóth, Gabor; Cascales, Javier López; Rodríguez, Ana M; Enriz, Ricardo D

2016-04-01

The synthesis, in vitro evaluation and conformational study of several small-size peptides acting as antibacterial agents are reported. Among the compounds evaluated, the peptides Arg-Gln-Ile-Lys-Ile-Trp-Arg-Arg-Met-Lys-Trp-Lys-Lys-NH2 , Arg-Gln-Ile-Lys-Ile-Arg-Arg-Met-Lys-Trp-Arg-NH2 , and Arg-Gln-Ile-Trp-Trp-Trp-Trp-Gln-Arg-NH2 exhibited significant antibacterial activity. These were found to be very active antibacterial compounds, considering their small molecular size. In order to better understand the antibacterial activity obtained for these peptides, an exhaustive conformational analysis was performed, using both theoretical calculations and experimental measurements. Molecular dynamics simulations using two different media (water and trifluoroethanol/water) were employed. The results of these theoretical calculations were corroborated by experimental circular dichroism measurements. A brief discussion on the possible mechanism of action of these peptides at molecular level is also presented. Some of the peptides reported here constitute very interesting structures to be used as starting compounds for the design of new small-size peptides possessing antibacterial activity. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bioinorganic Chemical Modeling of Dioxygen-Activating Copper Proteins.

ERIC Educational Resources Information Center

Karlin, Kenneth D.; Gultneh, Yilma

1985-01-01

Discusses studies done in modeling the copper centers in the proteins hemocyanin (a dioxygen carrier), tyrosinase, and dopamine beta-hydroxylase. Copper proteins, model approach in copper bioinorganic chemistry, characterization of reversible oxygen carriers and dioxygen-metal complexes, a copper mono-oxygenase model reaction, and other topics are…

The SANT2 domain of the murine tumor cell DnaJ-like protein 1 human homologue interacts with alpha1-antichymotrypsin and kinetically interferes with its serpin inhibitory activity.

PubMed

Kroczynska, Barbara; Evangelista, Christina M; Samant, Shalaka S; Elguindi, Ebrahim C; Blond, Sylvie Y

2004-03-19

The murine tumor cell DnaJ-like protein 1 or MTJ1/ERdj1 is a membrane J-domain protein enriched in microsomal and nuclear fractions. We previously showed that its lumenal J-domain stimulates the ATPase activity of the molecular chaperone BiP/GRP78 (Chevalier, M., Rhee, H., Elguindi, E. C., and Blond, S. Y. (2000) J. Biol. Chem. 275, 19620-19627). MTJ1/ERdj1 also contains a large carboxyl-terminal cytosolic extension composed of two tryptophan-mediated repeats or SANT domains for which the function(s) is unknown. Here we describe the cloning of the human homologue HTJ1 and its interaction with alpha(1)-antichymotrypsin (ACT), a member of the serine proteinase inhibitor (serpin) family. The interaction was initially identified in a two-hybrid screening and further confirmed in vitro by dot blots, native electrophoresis, and fluorescence studies. The second SANT domain of HTJ1 (SANT2) was found to be sufficient for binding to ACT, both in yeast and in vitro. Single tryptophan-alanine substitutions at two strictly conserved residues significantly (Trp-497) or totally (Trp-520) abolished the interaction with ACT. SANT2 binds to human ACT with an intrinsic affinity equal to 0.5 nm. Preincubation of ACT with nearly stoichiometric concentrations of SANT2 wild-type but not SANT2: W520A results in an apparent loss of ACT inhibitory activity toward chymotrypsin. Kinetic analysis indicates that the formation of the covalent inhibitory complex ACT-chymotrypsin is significantly delayed in the presence of SANT2 with no change on the catalytic efficiency of the enzyme. This work demonstrates for the first time that the SANT2 domain of MTJ1/HTJ1/ERdj1 mediates stable and high affinity protein-protein interactions.

Hair Dyes Resorcinol and Lawsone Reduce Production of Melanin in Melanoma Cells by Tyrosinase Activity Inhibition and Decreasing Tyrosinase and Microphthalmia-Associated Transcription Factor (MITF) Expression

PubMed Central

Lee, Shu-Mei; Chen, Yi-Shyan; Lin, Chih-Chien; Chen, Kuan-Hung

2015-01-01

Hair coloring products are one of the most important cosmetics for modern people; there are three major types of hair dyes, including the temporary, semi-permanent and permanent hair dyes. The selected hair dyes (such as ammonium persulfate, sodium persulfate, resorcinol and lawsone) are the important components for hair coloring products. Therefore, we analyzed the effects of these compounds on melanogenesis in B16-F10 melanoma cells. The results proved that hair dyes resorcinol and lawsone can reduce the production of melanin. The results also confirmed that resorcinol and lawsone inhibit mushroom and cellular tyrosinase activities in vitro. Resorcinol and lawsone can also downregulate the protein levels of tyrosinase and microphthalmia-associated transcription factor (MITF) in B16-F10 cells. Thus, we suggest that frequent use of hair dyes may have the risk of reducing natural melanin production in hair follicles. Moreover, resorcinol and lawsone may also be used as hypopigmenting agents to food, agricultural and cosmetic industry in the future. PMID:25584612

The Relationships among Tryptophan, Kynurenine, Indoleamine 2,3-Dioxygenase, Depression, and Neuropsychological Performance.

PubMed

Hestad, Knut A; Engedal, Knut; Whist, Jon E; Farup, Per G

2017-01-01

It has been suggested that the metabolic enzyme indoleamine 2,3-dioxygenase (IDO) is a biological mediator of inflammation related to the psychopathology of depression, with a Kynurenine (KYN) increase in the Tryptophan (TRP) metabolic pathway, resulting in reduced Serotonin. In this study, we examined KYN, TRP, and the ratio of KYN to TRP concentrations × 10 3 (KT Ratio) in serum and cerebrospinal fluid (CSF) in (a) a group of depressed patients and (b) a control group of patients referred to a neurologic outpatient clinic for whom no specific diagnosis could be established. The KT Ratio is considered an index that represents IDO. The participants were examined with the Beck Depression Inventory II (BDI-II), the Montgomery Aasberg Depression Rating Scale (MADRS), and a neuropsychological test battery. We found no significant differences between the two study groups with respect to TRP, KYN, or KT Ratio in serum or CSF. Differences in neuropsychological performance between the two patient groups could be seen in the following tests: Animal Fluency, Digit Symbol, the DKEFS Color-Interference Test (Naming Part), Trail Making Test A and B, and the Grooved Pegboard Non-dominant Hand. KYN in serum correlated highly with KYN in CSF. KYN in serum correlated significantly with both age and gender. When analyzing males and females separately, we found that women had a lower level of TRP in both serum (Mann-Whitney U -test: TRP in Serum; p = 0.001) and CSF (Mann-Whitney U -test: TRP in CSF; p = 0.003). Women had a lower level of KYN in serum ( p = 0.029) than men did. Age was positively associated with KYN. KYN in CSF correlated only with age, however; there were no gender differences. No significant relationship was seen between BDI-II and MADRS on the one hand, and KYN and TRP on the other. KYN in CSF as the KT Ratio in both serum and CSF was associated with neuropsychological performance. Thus, we suggest that KYN and KT Ratio are related more strongly to neuropsychological performance than to affective symptoms in depression.

GPR139, an Orphan Receptor Highly Enriched in the Habenula and Septum, Is Activated by the Essential Amino Acids L-Tryptophan and L-Phenylalanine.

PubMed

Liu, Changlu; Bonaventure, Pascal; Lee, Grace; Nepomuceno, Diane; Kuei, Chester; Wu, Jiejun; Li, Qingqin; Joseph, Victory; Sutton, Steven W; Eckert, William; Yao, Xiang; Yieh, Lynn; Dvorak, Curt; Carruthers, Nicholas; Coate, Heather; Yun, Sujin; Dugovic, Christine; Harrington, Anthony; Lovenberg, Timothy W

2015-11-01

GPR139 is an orphan G-protein-coupled receptor expressed in the central nervous system. To identify its physiologic ligand, we measured GPR139 receptor activity from recombinant cells after treatment with amino acids, orphan ligands, serum, and tissue extracts. GPR139 activity was measured using guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding, calcium mobilization, and extracellular signal-regulated kinases phosphorylation assays. Amino acids L-tryptophan (L-Trp) and L-phenylalanine (L-Phe) activated GPR139, with EC50 values in the 30- to 300-μM range, consistent with the physiologic concentrations of L-Trp and L-Phe in tissues. Chromatography of rat brain, rat serum, and human serum extracts revealed two peaks of GPR139 activity, which corresponded to the elution peaks of L-Trp and L-Phe. With the purpose of identifying novel tools to study GPR139 function, a high-throughput screening campaign led to the identification of a selective small-molecule agonist [JNJ-63533054, (S)-3-chloro-N-(2-oxo-2-((1-phenylethyl)amino)ethyl) benzamide]. The tritium-labeled JNJ-63533054 bound to cell membranes expressing GPR139 and could be specifically displaced by L-Trp and L-Phe. Sequence alignment revealed that GPR139 is highly conserved across species, and RNA sequencing studies of rat and human tissues indicated its exclusive expression in the brain and pituitary gland. Immunohistochemical analysis showed specific expression of the receptor in circumventricular regions of the habenula and septum in mice. Together, these findings suggest that L-Trp and L-Phe are candidate physiologic ligands for GPR139, and we hypothesize that this receptor may act as a sensor to detect dynamic changes of L-Trp and L-Phe in the brain. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

In vitro quantitative study of the degradation of endomorphins.

PubMed

Tömböly, Csaba; Péter, Antal; Tóth, Géza

2002-09-01

The catabolism of the endomorphins was investigated in detail. The endomorphins were degraded relatively slowly in the rat brain homogenate (t1/2(endomorphin-1)=4.94 min; t1/2(endomorphin-2)=3.81 min). The inhibition of metalloproteases and aminopeptidases stabilised the endomorphins to the greatest extent. The digestion of endomorphins tritiated specifically on Tyr(1), Pro(2) or Phe(3) established also that only the aminopeptidase pathways were essential for inactivation of the endomorphins, and that the tetrapeptides were degraded by cleavage of the Pro(2)-Trp(3) or Pro(2)-Phe(3) bond. The end-products of the catabolism were amino acids; the fragments Tyr-Pro-OH and Pro-Trp-Phe-NH2 were present as intermediates. Metabolites produced by brain carboxypeptidases were not detected.

Spectroscopic and molecular docking studies on the charge transfer complex of bovine serum albumin with quinone in aqueous medium and its influence on the ligand binding property of the protein.

PubMed

Satheshkumar, Angupillai; Elango, Kuppanagounder P

2014-09-15

The spectral techniques such as UV-Vis, (1)H NMR and fluorescence and electrochemical experiments have been employed to investigate the interaction between 2-methoxy-3,5,6-trichloro-1,4-benzoquinone (MQ; a water soluble quinone) and bovine serum albumin (BSA) in aqueous medium. The fluorescence of BSA was quenched by MQ via formation of a 1:1 BSA-MQ charge transfer adduct with a formation constant of 3.3×10(8) L mol(-1). Based on the Forster's theory the binding distance between them is calculated as 2.65 nm indicating high probability of binding. For the first time, influence of quinone on the binding property of various types of ligands such as aspirin, ascorbic acid, nicotinimide and sodium stearate has also been investigated. The results indicated that the strong and spontaneous binding existing between BSA and MQ, decreased the intensity of binding of these ligands with BSA. Since Tryptophan (Trp) is the basic residue present in BSA, a comparison between binding property of Trp-MQ adduct with that of BSA-MQ with these ligands has also been attempted. 1H NMR titration study indicated that the Trp forms a charge transfer complex with MQ, which reduces the interaction of Trp with the ligands. Molecular docking study supported the fact that the quinone interacts with the Trp212 unit of the BSA and the free energy change of binding (ΔG) for the BSA-MQ complex was found to be -46 kJ mol(-1), which is comparable to our experimental free energy of binding (-49 kJ mol(-1)) obtained from fluorescence study. Copyright © 2014 Elsevier B.V. All rights reserved.

Spectroscopic and molecular docking studies on the charge transfer complex of bovine serum albumin with quinone in aqueous medium and its influence on the ligand binding property of the protein

NASA Astrophysics Data System (ADS)

Satheshkumar, Angupillai; Elango, Kuppanagounder P.

2014-09-01

The spectral techniques such as UV-Vis, 1H NMR and fluorescence and electrochemical experiments have been employed to investigate the interaction between 2-methoxy-3,5,6-trichloro-1,4-benzoquinone (MQ; a water soluble quinone) and bovine serum albumin (BSA) in aqueous medium. The fluorescence of BSA was quenched by MQ via formation of a 1:1 BSA-MQ charge transfer adduct with a formation constant of 3.3 × 108 L mol-1. Based on the Forster’s theory the binding distance between them is calculated as 2.65 nm indicating high probability of binding. For the first time, influence of quinone on the binding property of various types of ligands such as aspirin, ascorbic acid, nicotinimide and sodium stearate has also been investigated. The results indicated that the strong and spontaneous binding existing between BSA and MQ, decreased the intensity of binding of these ligands with BSA. Since Tryptophan (Trp) is the basic residue present in BSA, a comparison between binding property of Trp-MQ adduct with that of BSA-MQ with these ligands has also been attempted. 1H NMR titration study indicated that the Trp forms a charge transfer complex with MQ, which reduces the interaction of Trp with the ligands. Molecular docking study supported the fact that the quinone interacts with the Trp212 unit of the BSA and the free energy change of binding (ΔG) for the BSA-MQ complex was found to be -46 kJ mol-1, which is comparable to our experimental free energy of binding (-49 kJ mol-1) obtained from fluorescence study.

Truncation studies of alpha-melanotropin peptides identify tripeptide analogues exhibiting prolonged agonist bioactivity.

PubMed

Haskell-Luevano, C; Sawyer, T K; Hendrata, S; North, C; Panahinia, L; Stum, M; Staples, D J; Castrucci, A M; Hadley, M F; Hruby, V J

1996-01-01

Truncation studies of alpha-melanotropin peptides identify tripeptide analogues exhibiting prolonged agonist bioactivity: PEPTIDES 17(6) 995-1002, 1996.-Systematic analysis of fragment derivatives of the superpotent alpha-MSH analogue. Ac-Ser.Tyr-Ser-Nle4-Glu- His-DPhe7-Arg-Trp-Gly-Lys-Pro-Val-NH2(NDP-MSH), led to the discovery of tripeptide agonists possessing prolonged bioactivity in the frog skin assay. Of particular significance to this discovery was Ac-DPhe-Arg-DTrp-NH2, which was the most potent tripeptide in this series exhibiting sustained melanotropic activity. Different pharmacophore models appear to exist that are dependent on the substructure and stereochemistry of the MSH(6-9) "active site." The tripeptides Ac-DPhe-Arg-Trp-NH2, Ac-DPhe-Arg-DTrp-NH2, and Ac-DPhe-DArg-Trp-NH2 stereo-chemical combinations require only Phe7-Xaa8-Trp9, whereas Ac-DPhe-DArg-DTrp-NH2, Ac-Phe-Arg-DTrp-NH2, and Ac-Phe-Arg-Trp-NH2 additionally require His4 for minimal biological activity. Ac-DPhe-Arg-DTrp-NH2 represents a novel prototype lead for the development of MSH-based peptidomimetic agonists.

The Structure of the Flavoprotein Tryptophan-2-Monooxygenase, a Key Enzyme in the Formation of Galls in Plants†

PubMed Central

Gaweska, Helena M.; Taylor, Alexander B.; Hart, P. John; Fitzpatrick, Paul F.

2013-01-01

The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to yield indole-3-acetamide. This is the initial step in the biosynthesis of the plant growth hormone indole-acetic-acid by bacterial pathogens that cause crown gall and related diseases. The structure of the enzyme from Pseudomonas savastanoi has been determined by X-ray diffraction methods to a resolution of 1.95 Å. The overall structure of the protein shows that it has the same fold as the monoamine oxidase family of flavoproteins, with the greatest similarities to the L-amino acid oxidases. The location of bound indole-3-acetamide in the active site enables identification of residues responsible for substrate binding and specificity. Two residues in the enzyme are conserved in all members of the monoamine oxidase family, Lys365 and Trp466. The K365M mutation decreases the kcat and kcat/KTrp values by 60,000 and 2 million-fold, respectively. The deuterium kinetic isotope effect increases to 3.2, consistent with carbon-hydrogen bond cleavage becoming rate-limiting in the mutant enzyme. The W466F mutation decreases the kcat value less than 2-fold and the kcat/KTrp value only 5-fold, while the W466M mutation results in enzyme lacking flavin and detectable activity. This is consistent with a role for Trp466 in maintaining the structure of the flavin binding site in the more conserved FAD domain. PMID:23521653

Type 2 diabetes mellitus: association study of five candidate genes in an Indian population of Guadeloupe, genetic contribution of FABP2 polymorphism.

PubMed

Boullu-Sanchis, S; Leprêtre, F; Hedelin, G; Donnet, J P; Schaffer, P; Froguel, P; Pinget, M

1999-06-01

We studied by PCR-RFLP 6 polymorphisms in these 5 candidate genes: Ala54Thr in the fatty acid binding protein 2 gene (FABP2), A to G substitution in the uncoupling protein type 1 gene (UCP1), Asp905Tyr in the protein phosphatase type 1 gene (PP1G), Trp64Arg in the human beta 3 adrenergic receptor gene (beta 3AR) and 2 RFLP sites of the vitamin D receptor (VDR) gene (VDRTaq1 and VDRApa1). This study was conducted among 89 cases and 100 controls matched according to age, gender and absence of first degree family link (11 triplets with 2 controls for 1 case and 78 pairs with 1 control for 1 case). Cases and controls were taken among a sample of 429 individuals selected for the study of the prevalence of diabetes in this ethnic group from Guadeloupe. By conditional logistic regression analysis, there was a significant relation (p = 0.02) between the Ala54Thr FABP2 polymorphism and Type 2 DM. Multivariate analysis discriminate the FABP2 polymorphism (p = 0.10), a triglyceridemia over 2 g/l (p < 10(-3)) and high blood pressure (p = 10(-2)) as variables associated with Type 2 DM in this population. These findings suggest that FABP2 does not represent a major gene for Type 2 DM in this migrant Indian population living in Guadeloupe, but seems to be related to the metabolic insulin resistance syndrome.

Homology modeling and docking studies of human Bcl-2L10 protein.

PubMed

Bhargavi, K; Kalyan Chaitanya, P; Ramasree, D; Vasavi, M; Murthy, D K; Uma, V

2010-12-01

Cancer, an unrestrained proliferation of cells, is one of the lead cause of death. Nearly 12.5 million people are diagnosed with cancer worldwide, 7.5 million people die of which 2.5 million cases are from India. Major cause for cancer is restriction of programmed cell death (apoptosis). Multiple signaling pathways regulate apoptosis. Bcl-2 (B - Cell Lymphomas-2) family proteins play a vital role as central regulators of apoptosis. Bcl-2L10, a novel anti-apoptotic protein, blocks apoptosis by mitochondrial dependent mechanism. The present study evaluates the 3D structure of Bcl-2L10 protein using homology modeling and aims to understand plausible functional and binding interactions between Bcl-2L10 with BH3 domain of BAX using protein - protein docking. The docking studies show binding of BH3 domain at Lys 110, Trp-111, Pro-115, Glu-119 and Asp-127 in the groove of BH 1, 2 and 3 domains of Bcl-2L10. Heterodimerization of anti-apoptotic Bcl-2 and BH3 domain of pro-apoptotic Bcl-2 proteins instigates apoptosis. Profound understanding of Bcl-2 pathway may prove useful in identification of future therapeutic targets for cancer.

The alpha-adducin Gly460Trp polymorphism and the antihypertensive effects of exercise among men with high blood pressure.

PubMed

Pescatello, Linda S; Blanchard, Bruce E; Tsongalis, Gregory J; Maresh, Carl M; O'Connell, Ann; Thompson, Paul D

2007-09-01

The alpha-adducin Gly460Trp polymorphism alters renal sodium transport and is associated with hypertension. Despite the immediate sodium- and volume-depleting effects of aerobic exercise, the influence of the alpha-adducin Gly460Trp polymorphism on PEH (postexercise hypotension) has not been studied. In the present study we examined the effects of the alpha-adducin Gly460Trp polymorphism on PEH among 48 men (42.6+/-1.6 years; mean+/-S.E.M.) with high BP (blood pressure; 144.0+/-1.7/84.7+/-1.1 mmHg). Subjects completed three experiments: non-exercise control and two cycle exercise sessions at 40% (light exercise) and 60% (moderate exercise) of maximal oxygen consumption. Subjects left the laboratory wearing an ambulatory BP monitor. PCR and restriction enzyme digestion determined the genotypes. No subjects had the Trp460Trp genotype due to the low frequency of 5% in the population. Repeated measure ANCOVA tested whether BP differed over time between experimental conditions and genotypes (Gly460Gly, n=36; Gly460Trp, n=12). Among Gly460Gly genotypes, SBP (systolic BP) was reduced by 5.2+/-1.4 mmHg after moderate exercise compared with non-exercise controls over 9 h (P<0.01). Among Gly460Trp genotypes, SBP was lowered by 7.8+/-2.3 mmHg; after light exercise compared with non-exercise controls over 9 h (P<0.05). The SBP reductions after light exercise (0.6+/-1.3 compared with 7.8+/-2.3 mmHg; P<0.05) but not moderate exercise (5.2+/-1.4 compared with 3.8+/-2.4 mmHg; P> or =0.05) differed between the Gly460Gly and Gly460Trp genotypes respectively. Men with Gly460Gly had a reduced SBP after moderate exercise, whereas men with Gly460Trp had a reduced SBP after light exercise. However, only the SBP reductions after light exercise differed between genotypes. Our findings indicate that the alpha-adducin Gly460Trp genotype may be useful in identifying men who have a reduced BP after lower intensity aerobic exercise.

[Mechanism for synergistic effect of IRF4 and MITF on tyrosinase promoter].

PubMed

Song, Jian; Liu, Xueming; Li, Jiada; Liu, Huadie; Peng, Zhen; Chen, Hongsheng; Mei, Lingyun; He, Chufeng; Feng, Yong

2018-05-28

To investigate the mechanism for the synergistic effect of interferon regulatory factor 4 (IRF4) and microphthalmia-associated transcription factor (MITF) on tyrosinase (TYR) promoter.
 Methods: The synergistic transcriptional effect, subcellular localization, and protein-protein interaction for IRF4 and MITF were observed by luciferase assay, immunofluorescence, GST-pull down, and co-immunoprecipitation, respectively.
 Results: IRF4 and MITF proteins were co-expressed in the cell nucleus. IRF4 augmented the transcriptional function of MITF (but not the mutant MITF) to activate the expression of the TYR promoter, but with no effect on other MITF-specific target promoters. IRF4 alone did not affect TYR promoter significantly. No direct interaction between the two proteins was noted.
 Conclusion: IRF4 and MITF exert a specifically synergistic effect on activation of TYR promoter through IRF4-mediated upregulation of transcriptional function of MITF. This synergistic effect is mainly regulated by MITF; DNA might be involved in the interaction between the two proteins.

Design and characterization of an engineered gp41 protein from human immunodeficiency virus-1 as a tool for drug discovery

NASA Astrophysics Data System (ADS)

Stewart, Kent D.; Steffy, Kevin; Harris, Kevin; Harlan, John E.; Stoll, Vincent S.; Huth, Jeffrey R.; Walter, Karl A.; Gramling-Evans, Emily; Mendoza, Renaldo R.; Severin, Jean M.; Richardson, Paul L.; Barrett, Leo W.; Matayoshi, Edmund D.; Swift, Kerry M.; Betz, Stephen F.; Muchmore, Steve W.; Kempf, Dale J.; Molla, Akhter

2007-01-01

Two new proteins of approximately 70 amino acids in length, corresponding to an unnaturally-linked N- and C-helix of the ectodomain of the gp41 protein from the human immunodeficiency virus (HIV) type 1, were designed and characterized. A designed tripeptide links the C-terminus of the C-helix with the N-terminus of the N-helix in a circular permutation so that the C-helix precedes the N-helix in sequence. In addition to the artificial peptide linkage, the C-helix is truncated at its N-terminus to expose a region of the N-helix known as the "Trp-Trp-Ile" binding pocket. Sedimentation, crystallographic, and nuclear magnetic resonance studies confirmed that the protein had the desired trimeric structure with an unoccupied binding site. Spectroscopic and centrifugation studies demonstrated that the engineered protein had ligand binding characteristics similar to previously reported constructs. Unlike previous constructs which expose additional, shallow, non-conserved, and undesired binding pockets, only the single deep and conserved Trp-Trp-Ile pocket is exposed in the proteins of this study. This engineered version of gp41 protein will be potentially useful in research programs aimed at discovery of new drugs for therapy of HIV-infection in humans.

Evolutionary differences in chromosomal locations of four early genes of the tryptophan pathway in fluorescent pseudomonads: DNA sequences and characterization of Pseudomonas putida trpE and trpGDC.

PubMed

Essar, D W; Eberly, L; Crawford, I P

1990-02-01

Pseudomonas putida possesses seven structural genes for enzymes of the tryptophan pathway. All but one, trpG, which encodes the small (beta) subunit of anthranilate synthase, have been mapped on the circular chromosome. This report describes the cloning and sequencing of P. putida trpE, trpG, trpD, and trpC. In P. putida and Pseudomonas aeruginosa, DNA sequence analysis as well as growth and enzyme assays of insertionally inactivated strains indicated that trpG is the first gene in a three-gene operon that also contains trpD and trpC. In P. putida, trpE is 2.2 kilobases upstream from the trpGDC cluster, whereas in P. aeruginosa, they are separated by at least 25 kilobases (T. Shinomiya, S. Shiga, and M. Kageyama, Mol. Gen. Genet., 189:382-389, 1983). The DNA sequence in P. putida shows an open reading frame on the opposite strand between trpE and trpGDC; this putative gene was not characterized. Evidence is also presented for sequence similarities in the 5' untranslated regions of trpE and trpGDC in both pseudomonads; the function of these regions is unknown, but it is possible that they play some role in regulation of these genes, since all the genes respond to repression by tryptophan. The sequences of the anthranilate synthase genes in the fluorescent pseudomonads resemble those of p-aminobenzoate synthase genes of the enteric bacteria more closely than the anthranilate synthase genes of those organisms; however, no requirement for p-aminobenzoate was found in the Pseudomonas mutants created in this study.

Galvanic zinc-copper microparticles inhibit melanogenesis via multiple pigmentary pathways.

PubMed

Won, Yen-Kim; Lin, Connie B; Seiberg, Miri; Chen, Nannan; Hu, Yaping; Rossetti, Dianne; Saliou, Claude; Loy, Chong-Jin

2014-01-01

The endogenous electrical field of human skin plays an important role in many skin functions. However, the biological effects and mechanism of action of externally applied electrical stimulation on skin remain unclear. Recent study showed that galvanic zinc-copper microparticles produce electrical stimulation and reduce inflammatory and immune responses in intact skin, suggesting the important role of electrical stimulation in non-wounded skin. The objective of this study is to investigate the biological effect of galvanic zinc-copper microparticles on skin pigmentation. Our findings showed that galvanic zinc-copper microparticles inhibited melanogenesis in a human melanoma cell line (MNT-1), human keratinocytes and melanoma cells co-cultures, and in pigmented epidermal equivalents. Treatment of galvanic zinc-copper microparticles inhibited melanogenesis by reducing the promoter transactivation of tyrosinase and tyrosinase-related protein-1 in human melanoma cells. In a co-culture Transwell system of keratinocytes and melanoma cells, galvanic zinc-copper microparticles reduced melanin production via downregulation of endothelin-1 secretion from keratinocytes and reduced tyrosinase gene expression in melanoma cells. In addition, exposure of pigmented epidermal equivalents to galvanic zinc-copper microparticles resulted in reduced melanin deposition. In conclusion, our data demonstrated for the first time that galvanic zinc-copper microparticles reduced melanogenesis in melanoma cells and melanin deposition in pigmented epidermal equivalents by affecting multiple pigmentary pathways.

The glycoprotein TRP36 of Ehrlichia sp. UFMG-EV and related cattle pathogen Ehrlichia sp. UFMT-BV evolved from a highly variable clade of E. canis under adaptive diversifying selection.

PubMed

Cabezas-Cruz, Alejandro; Valdés, James J; de la Fuente, José

2014-12-10

A new species of Ehrlichia, phylogenetically distant from E. ruminantium, was found in 2010 infecting cattle in Canada. In 2012 and 2013, we reported the in vitro propagation, molecular and ultrastructural characterization of Ehrlichia sp. UFMG-EV (E. mineirensis), a new species of Ehrlichia isolated from the haemolymph of Brazilian Rhipicephalus (Boophilus) microplus ticks. A new organism, named Ehrlichia sp. UFMT-BV, closely related to Ehrlichia sp. UFMG-EV, was recently described in Brazil and after experimental infection it was shown to be pathogenic for cattle. This new emerging clade of cattle Ehrlichia pathogens is closely related to E. canis. The major immunogenic Tandem Repeat Protein (TRP36; also known as gp36) is extensively used to characterize the genetic diversity of E. canis. Homologs of TRP36 were found in both Ehrlichia sp. UFMG-EV and Ehrlichia sp. UFMT-BV. Herein, we characterized the evolution of this new Ehrlichia clade using TRP36 sequences. Our working hypothesis is that Ehrlichia sp. UFMG-EV and related microorganisms evolved from a highly variable E. canis clade. In support of our hypothesis we found that Ehrlichia sp. UFMG-EV and Ehrlichia sp. UFMT-BV TRP36 evolved from a highly divergent and variable clade within E. canis and this clade evolved under episodic diversifying selection with a high proportion of sites under positive selection. Our results suggest that Ehrlichia sp. UFMG-EV and Ehrlichia sp. UFMT-BV evolved from a variable clade within E. canis.

Inhibitory effects of constituents of Morinda citrifolia seeds on elastase and tyrosinase.

PubMed

Masuda, Megumi; Murata, Kazuya; Fukuhama, Akiko; Naruto, Shunsuke; Fujita, Tadashi; Uwaya, Akemi; Isami, Fumiyuki; Matsuda, Hideaki

2009-07-01

A 50% ethanolic extract (MCS-ext) from seeds of Morinda citrifolia ("noni" seeds) showed more potent in vitro inhibition of elastase and tyrosinase, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity than extracts of M. citrifolia leaves or flesh. Activity-guided fractionation of MCS-ext using in vitro assays led to the isolation of ursolic acid as an active constituent of elastase inhibitory activity. 3,3'-Bisdemethylpinoresinol, americanin A, and quercetin were isolated as active constituents having both tyrosinase inhibitory and radical scavenging activities. Americanin A and quercetin also showed superoxide dismutase (SOD)-like activity. These active compounds were isolated from noni seeds for the first time.

Direct Binding between Pre-S1 and TRP-like Domains in TRPP Channels Mediates Gating and Functional Regulation by PIP2.

PubMed

Zheng, Wang; Cai, Ruiqi; Hofmann, Laura; Nesin, Vasyl; Hu, Qiaolin; Long, Wentong; Fatehi, Mohammad; Liu, Xiong; Hussein, Shaimaa; Kong, Tim; Li, Jingru; Light, Peter E; Tang, Jingfeng; Flockerzi, Veit; Tsiokas, Leonidas; Chen, Xing-Zhen

2018-02-06

Transient receptor potential (TRP) channels are regulated by diverse stimuli comprising thermal, chemical, and mechanical modalities. They are also commonly regulated by phosphatidylinositol-4,5-bisphosphate (PIP2), with underlying mechanisms largely unknown. We here revealed an intramolecular interaction of the TRPP3 N and C termini (N-C) that is functionally essential. The interaction was mediated by aromatic Trp81 in pre-S1 domain and cationic Lys568 in TRP-like domain. Structure-function analyses revealed similar N-C interaction in TRPP2 as well as TRPM8/-V1/-C4 via highly conserved tryptophan and lysine/arginine residues. PIP2 bound to cationic residues in TRPP3, including K568, thereby disrupting the N-C interaction and negatively regulating TRPP3. PIP2 had similar negative effects on TRPP2. Interestingly, we found that PIP2 facilitates the N-C interaction in TRPM8/-V1, resulting in channel potentiation. The intramolecular N-C interaction might represent a shared mechanism underlying the gating and PIP2 regulation of TRP channels. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Acetylcholinesterase complexed with bivalent ligands related to huperzine a: experimental evidence for species-dependent protein-ligand complementarity.

PubMed

Wong, Dawn M; Greenblatt, Harry M; Dvir, Hay; Carlier, Paul R; Han, Yi-Fan; Pang, Yuan-Ping; Silman, Israel; Sussman, Joel L

2003-01-15

Acetylcholinesterase (AChE) inhibitors improve the cognitive abilities of Alzheimer patients. (-)-Huperzine A [(-)-HupA], an alkaloid isolated from the club moss, Huperzia serrata, is one such inhibitor, but the search for more potent and selective drugs continues. Recently, alkylene-linked dimers of 5-amino-5,6,7,8-tetrahydroquinolinone (hupyridone, 1a), a fragment of HupA, were shown to serve as more potent inhibitors of AChE than (-)-HupA and monomeric 1a. We soaked two such dimers, (S,S)-(-)-bis(10)-hupyridone [(S,S)-(-)-2a] and (S,S)-(-)-bis(12)-hupyridone [(S,S)-(-)-2b] containing, respectively, 10 and 12 methylenes in the spacer, into trigonal TcAChE crystals, and solved the X-ray structures of the resulting complexes using the difference Fourier technique, both to 2.15 A resolution. The structures revealed one HupA-like 1a unit bound to the "anionic" subsite of the active-site, near the bottom of the active-site gorge, adjacent to Trp84, as seen for the TcAChE/(-)-HupA complex, and the second 1a unit near Trp279 in the "peripheral" anionic site at the top of the gorge, both bivalent molecules thus spanning the active-site gorge. The results confirm that the increased affinity of the dimeric HupA analogues for AChE is conferred by binding to the two "anionic" sites of the enzyme. Inhibition data show that (-)-2a binds to TcAChE approximately 6-7- and > 170-fold more tightly than (-)-2b and (-)-HupA, respectively. In contrast, previous data for rat AChE show that (-)-2b binds approximately 3- and approximately 2-fold more tightly than (-)-2a and (-)-HupA, respectively. Structural comparison of TcAChE with rat AChE, as represented by the closely related mouse AChE structure (1maa.pdb), reveals a narrower gorge for rat AChE, a perpendicular alignment of the Tyr337 ring to the gorge axis, and its conformational rigidity, as a result of hydrogen bonding between its hydroxyl group and that of Tyr341, relative to TcAChE Phe330. These structural differences in the active-site gorge explain the switch in inhibitory potency of (-)-2a and 2b and the larger dimer/(-)-HupA potency ratios observed for TcAChE relative to rat AChE. The results offer new insights into factors affecting protein-ligand complementarity within the gorge and should assist the further development of improved AChE inhibitors.