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Sample records for intracellular serpin regulates

  1. Ostrinia furnacalis serpin-3 regulates melanization cascade by inhibiting a prophenoloxidase-activating protease.

    PubMed

    Chu, Yuan; Zhou, Fan; Liu, Yang; Hong, Fang; Wang, Guirong; An, Chunju

    2015-06-01

    Serine protease cascade-mediated prophenolxidase activation is a prominent innate immune response in insect defense against the invading pathogens. Serpins regulate this reaction to avoid excessive activation. However, the function of serpins in most insect species, especially in some non-model agriculture insect pests, is largely unknown. We here cloned a full-length cDNA for a serpin, named as serpin-3, from Asian corn borer, Ostrinia furnacalis (Guenée). The open reading frame of serpin-3 encodes 462-amino acid residue protein with a 19-residue signal peptide. It contains a reactive center loop strikingly similar to the proteolytic activation site in prophenoloxidase. Sequence comparison indicates that O. furnacalis serpin-3 is an apparent ortholog of Manduca sexta serpin-3, a defined negative regulator of melanization reaction. Serpin-3 mRNA and protein levels significantly increase after a bacterial or fungal injection. Recombinant serpin-3 efficiently blocks prophenoloxidase activation in larval plasma in a concentration-dependent manner. It forms SDS-stable complexes with serine protease 13 (SP13), and prevents SP13 from cleaving prophenoloxidase. Injection of recombinant serpin-3 into larvae results in decreased fungi-induced melanin synthesis and reduced the expression of attacin, cecropin, gloverin, and peptidoglycan recognition protein-1 genes in the fat body. Altogether, serpin-3 plays important roles in the regulation of prophenoloxidase activation and antimicrobial peptide production in O. furnacalis larvae. PMID:25818483

  2. EGFR Signaling Regulates Maspin/SerpinB5 Phosphorylation and Nuclear Localization in Mammary Epithelial Cells

    PubMed Central

    Reina, Jeffrey; Morais Freitas, Vanessa

    2016-01-01

    Maspin (SerpinB5) is a non-inhibitory serpin (serine protease inhibitor) with very diverse biological activities including regulation of cell adhesion, migration, death, control of gene expression and oxidative stress response. Initially described as a tumor and metastasis suppressor, clinical data brought controversies to the field, as some studies reported no correlation between SerpinB5 expression and prognosis value. These data underscore the importance of understanding SerpinB5 function in a normal physiological context and the molecular mechanism involved. Several SerpinB5 phosphoforms have been detected in different cell lines, but the signaling pathways involved and the biological significance of this post-translational modification in vivo remains to be explored. In this study we investigated SerpinB5 expression, subcellular localization and phosphorylation in different stages of the mouse mammary gland development and the signaling pathway involved. Here we show that SerpinB5 is first detected in late pregnancy, reaches its highest levels in lactation and remains at constant levels during post-lactational regression (involution). Using high resolution isoelectric focusing followed but immunoblot, we found at least 8 different phosphoforms of SerpinB5 during lactation, which decreases steadily at the onset of involution. In order to investigate the signaling pathway involved in SerpinB5 phosphorylation, we took advantage of the non-transformed MCF-10A model system, as we have previously observed SerpinB5 phosphorylation in these cells. We detected basal levels of SerpinB5 phosphorylation in serum- and growth factor-starved cells, which is due to amphiregulin autocrine activity on MCF-10A cells. EGF and TGF alpha, two other EGFR ligands, promote important SerpinB5 phosphorylation. Interestingly, EGF treatment is followed by SerpinB5 nuclear accumulation. Altogether, these data indicate that SerpinB5 expression and phosphorylation are developmentally

  3. Inhibitory serpins. New insights into their folding, polymerization, regulation and clearance.

    PubMed

    Gettins, Peter G W; Olson, Steven T

    2016-08-01

    Serpins are a widely distributed family of high molecular mass protein proteinase inhibitors that can inhibit both serine and cysteine proteinases by a remarkable mechanism-based kinetic trapping of an acyl or thioacyl enzyme intermediate that involves massive conformational transformation. The trapping is based on distortion of the proteinase in the complex, with energy derived from the unique metastability of the active serpin. Serpins are the favoured inhibitors for regulation of proteinases in complex proteolytic cascades, such as are involved in blood coagulation, fibrinolysis and complement activation, by virtue of the ability to modulate their specificity and reactivity. Given their prominence as inhibitors, much work has been carried out to understand not only the mechanism of inhibition, but how it is fine-tuned, both spatially and temporally. The metastability of the active state raises the question of how serpins fold, whereas the misfolding of some serpin variants that leads to polymerization and pathologies of liver disease, emphysema and dementia makes it clinically important to understand how such polymerization might occur. Finally, since binding of serpins and their proteinase complexes, particularly plasminogen activator inhibitor-1 (PAI-1), to the clearance and signalling receptor LRP1 (low density lipoprotein receptor-related protein 1), may affect pathways linked to cell migration, angiogenesis, and tumour progression, it is important to understand the nature and specificity of binding. The current state of understanding of these areas is addressed here. PMID:27470592

  4. TEL2 suppresses metastasis by down-regulating SERPINE1 in nasopharyngeal carcinoma

    PubMed Central

    Zhang, Ru-Hua; Wang, Li; Li, Mei; Luo, Rongzhen; Qian, Chao-Nan; Shao, Jian-Yong; Zeng, Yi-Xin; Kang, Tiebang

    2015-01-01

    Metastasis is the major cause of treatment failure in patients with nasopharyngeal carcinoma (NPC). However, the molecular mechanisms of NPC metastasis are poorly understood. Here, using our customized gene microarray containing all of the known human transcription factors and the current markers for epithelial-mesenchymal transition, we report that TEL2 was down-regulated in highly metastatic NPC cells and the metastatic tissues in lymph node. Mechanistically, TEL2 inhibits the cell migration and invasion in vitro and metastasis in vivo by releasing its direct suppression on the SERPINE1 promoter in NPC. Consistently, an inverse correlation was observed between the protein levels of TEL2 and SERPINE1 using clinical NPC samples. Collectively, we have provided the first evidence that TEL2 plays a key role in NPC metastasis by directly down-regulating SERPINE1, and that this novel axis of TEL2 / SERPINE1 may be valuable to develop new strategies for treating NPC patients with metastasis. PMID:26335051

  5. Serpin-5 regulates prophenoloxidase activation and antimicrobial peptide pathways in the silkworm, Bombyx mori.

    PubMed

    Li, Junlan; Ma, Li; Lin, Zhe; Zou, Zhen; Lu, Zhiqiang

    2016-06-01

    The prophenoloxidase (PPO) activation pathway and Toll pathway are two critical insect immune responses against microbial infection. Activation of these pathways is mediated by an extracellular serine protease cascade, which is negatively regulated by serpins. In this study, we found that the mRNA abundance of silkworm serpin-5 (BmSpn-5) increased dramatically in the fat body after bacterial infection. The expression level of antimicrobial peptides (AMPs), gloverin-3, cecropin-D and -E decreased in the silkworm larvae injected with recombinant BmSpn-5 protein. Meanwhile, the inhibition of beads melanization, systemic melanization and PPO activation by BmSpn-5 was also observed. By means of immunoaffinity purification and analysis by mass spectrometry, we identified that the silkworm clip domain serine proteases BmHP6 and BmSP21 form a complex with BmSpn-5, which suggests that BmHP6 and SP21 are the cognate proteases of BmSpn-5 and are essential in the serine protease cascade that activates the Toll and PPO pathways. Our study provides a comprehensive characterization of BmSpn-5 and sheds light on the multiple pathways leading to PPO activation and their regulation by serpins. PMID:27084699

  6. SerpinE2 promotes multiple cell proliferation and drug resistance in osteosarcoma.

    PubMed

    Mao, Minzhi; Wang, Wanchun

    2016-07-01

    SerpinE2 is a member of the Serpins family, which could inhibit serine protease and promote tumor progression, particularly in tumor metastasis. However, at present, its role in the progression of osteosarcoma has not been determined. The present study analyzed the expression profiles of SerpinE2 in cancer tissues, including tissues from osteosarcoma of different stages. Higher expression of SerpinE2 was shown in osteosarcoma tissues, particularly in tissue from patients with metastasis and a tumor-node-metastasis stage II‑III. Following chemotherapy, the SerpinE2 expression levels were shown to be higher than those at diagnosis. Cell proliferation and colony formation were increased after transfection with SerpinE2 over‑expression vector. Additionally, drug resistance to bortezomib and doxorubicin treatment following SerpinE2 transfection was analyzed. MG‑63 and SAOS‑2 cells showed less sensitivity following transfection with SerpinE2. The cell cycle‑related genes, cyclin‑dependent kinase (CDK)4 and cyclin D1 were positively correlated with SerpinE2 expression in patient‑derived tissue and in osteosarcoma cells. Finally, the high expression of SerpinE2 contributes to poor survival rates in patients with osteosarcoma. In conclusion, high expression of SerpinE2 in osteosarcoma stimulates cell proliferation, promotes drug‑resistance, and results in poor survival by regulating CDK4 and cyclin D1. Thus, SerpinE2 could be a potential target for treatment of patients with osteosarcoma. PMID:27221371

  7. Down-regulation of SerpinB2 is associated with gefitinib resistance in non-small cell lung cancer and enhances invadopodia-like structure protrusions

    PubMed Central

    Bae, Song Yi; Park, Hyen Joo; Hong, Ji-Young; Lee, Hye-Jung; Lee, Sang Kook

    2016-01-01

    The failure of targeted therapy due to the resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, is considered a major problem in the treatment of non-small cell lung cancer (NSCLC) patients. SerpinB2, a component of the urokinase plasminogen activator (uPA) system, has been recognized as a biomarker for the progression and metastasis of lung cancer. Nevertheless, the relationship between SerpinB2 and EGFR-TKI resistance has not been elucidated. Here, we report that SerpinB2 is down-regulated in gefitinib-resistant (H292-Gef) cells compared to gefitinib-sensitive (H292) cells. The low SerpinB2 levels in H292-Gef cells were also associated with an enhancement in invasiveness and increase in the length of invadopodia-like structures in the cells. The effect on invasiveness and gefitinib sensitivity was confirmed by knockdown and overexpression of SerpinB2. In addition, the possibility to overcome the resistance through the up-regulation of SerpinB2 was supported by employing an antitumor agent yuanhuadine (YD). Treatment with YD effectively elevated SerpinB2 levels and suppressed invasive properties in H292-Gef cells. Collectively, these findings demonstrate the prospective role of SerpinB2 as a novel biomarker for acquired gefitinib resistance and a potential target for NSCLC treatment. PMID:27558531

  8. Characterization of cucurbita maxima phloem serpin-1 (CmPS-1). A developmentally regulated elastase inhibitor.

    PubMed

    Yoo, B C; Aoki, K; Xiang, Y; Campbell, L R; Hull, R J; Xoconostle-Cázares, B; Monzer, J; Lee, J Y; Ullman, D E; Lucas, W J

    2000-11-10

    We report on the molecular, biochemical, and functional characterization of Cucurbita maxima phloem serpin-1 (CmPS-1), a novel 42-kDa serine proteinase inhibitor that is developmentally regulated and has anti-elastase properties. CmPS-1 was purified to near homogeneity from C. maxima (pumpkin) phloem exudate and, based on microsequence analysis, the cDNA encoding CmPS-1 was cloned. The association rate constant (k(a)) of phloem-purified and recombinant His(6)-tagged CmPS-1 for elastase was 3.5 +/- 1.6 x 10(5) and 2.7 +/- 0.4 x 10(5) m(-)(1) s(-)(1), respectively. The fraction of complex-forming CmPS-1, X(inh), was estimated at 79%. CmPS-1 displayed no detectable inhibitory properties against chymotrypsin, trypsin, or thrombin. The elastase cleavage sites within the reactive center loop of CmPS-1 were determined to be Val(347)-Gly(348) and Val(350)-Ser(351) with a 3:2 molar ratio. In vivo feeding assays conducted with the piercing-sucking aphid, Myzus persicae, established a close correlation between the developmentally regulated increase in CmPS-1 within the phloem sap and the reduced ability of these insects to survive and reproduce on C. maxima. However, in vitro feeding experiments, using purified phloem CmPS-1, failed to demonstrate a direct effect on aphid survival. Likely roles of this novel phloem serpin in defense against insects/pathogens are discussed. PMID:10960478

  9. Cloning, expression and characterization of Ostrinia furnacalis serpin1, a regulator of the prophenoloxidase activation system.

    PubMed

    Zhang, Bing; Wu, Taoyan; Tang, Xiaowei; Zhang, Shiyang; Xu, Qiuwen; Zhao, Ya; Wang, Yingjuan; Feng, Congjing

    2016-02-01

    Serine protease inhibitors of the serpin superfamily are regulators of proteases involved in a variety of physiological processes including immune responses. In this study, we have isolated a full-length serpin cDNA from Ostrinia furnacalis. The 1188 bp open reading frame encodes a 395-residue protein with a theoretical molecular mass of 43.3 kDa and an isoelectric point of 4.92. Ofserpin1 contains a putative signal peptide followed by a conserved domain including a reactive center loop (RCL) with a hinge region (E(344) to S(353)) and a predicted P1-P1' cleavage site (Leu(360)-Ser(361)). Ofserpin1 mRNA and protein were detected in all the tested tissues, particularly in hemocytes and integument. The recombinant protein inhibited chymotrypsin and trypsin in a dose-dependent manner, and were significantly cleaved by the enzyme trypsin and chymotrypsin. Ofserpin1 impeded the prophenoloxidase activation cascade by 45.6% at 16.5 μg, and affected activity of prophenoloxidase activating protease. Levels of Ofserpin1 transcripts in the integument were higher than those in hemocytes, fat body and midgut. After an immune challenge with Staphylococcus aureus and Escherichia coli, the relative mRNA levels of Ofserpin1 decreased in 2-10h post-infection (hpi) in integument and hemocytes compared to the untreated control. Our results suggested that Ofserpin1 has serine protease inhibitory activity and is likely involved in the regulation of prophenoloxidase activation system in O. furnacalis. PMID:26589634

  10. Molecular mechanisms of antithrombin-heparin regulation of blood clotting proteinases. a paradigm for understanding proteinase regulation by serpin family protein proteinase inhibitors

    PubMed Central

    Olson, Steven T.; Richard, Benjamin; Izaguirre, Gonzalo; Schedin-Weiss, Sophia; Gettins, Peter G. W.

    2010-01-01

    Serpin family protein proteinase inhibitors regulate the activity of serine and cysteine proteinases by a novel conformational trapping mechanism that may itself be regulated by cofactors to provide a finely-tuned time and location-dependent control of proteinase activity. The serpin, antithrombin, together with its cofactors, heparin and heparan sulfate, perform a critical anticoagulant function by preventing the activation of blood clotting proteinases except when needed at the site of a vascular injury. Here, we review the detailed molecular understanding of this regulatory mechanism that has emerged from numerous X-ray crystal structures of antithrombin and its complexes with heparin and target proteinases together with mutagenesis and functional studies of heparin-antithrombin-proteinase interactions in solution. Like other serpins, antithrombin achieves specificity for its target blood clotting proteinases by presenting recognition determinants in an exposed reactive center loop as well as in exosites outside the loop. Antithrombin reactivity is repressed in the absence of its activator because of unfavorable interactions that diminish the favorable RCL and exosite interactions with proteinases. Binding of a specific heparin or heparan sulfate pentasaccharide to antithrombin induces allosteric activating changes that mitigate the unfavorable interactions and promote template bridging of the serpin and proteinase. Antithrombin has thus evolved a sophisticated means of regulating the activity of blood clotting proteinases in a time and location-dependent manner that exploits the multiple conformational states of the serpin and their differential stabilization by glycosaminoglycan cofactors. PMID:20685328

  11. Update of the human and mouse SERPIN gene superfamily

    PubMed Central

    2013-01-01

    The serpin family comprises a structurally similar, yet functionally diverse, set of proteins. Named originally for their function as serine proteinase inhibitors, many of its members are not inhibitors but rather chaperones, involved in storage, transport, and other roles. Serpins are found in genomes of all kingdoms, with 36 human protein-coding genes and five pseudogenes. The mouse has 60 Serpin functional genes, many of which are orthologous to human SERPIN genes and some of which have expanded into multiple paralogous genes. Serpins are found in tissues throughout the body; whereas most are extracellular, there is a class of intracellular serpins. Serpins appear to have roles in inflammation, immune function, tumorigenesis, blood clotting, dementia, and cancer metastasis. Further characterization of these proteins will likely reveal potential biomarkers and therapeutic targets for disease. PMID:24172014

  12. Update of the human and mouse SERPIN gene superfamily.

    PubMed

    Heit, Claire; Jackson, Brian C; McAndrews, Monica; Wright, Mathew W; Thompson, David C; Silverman, Gary A; Nebert, Daniel W; Vasiliou, Vasilis

    2013-01-01

    The serpin family comprises a structurally similar, yet functionally diverse, set of proteins. Named originally for their function as serine proteinase inhibitors, many of its members are not inhibitors but rather chaperones, involved in storage, transport, and other roles. Serpins are found in genomes of all kingdoms, with 36 human protein-coding genes and five pseudogenes. The mouse has 60 Serpin functional genes, many of which are orthologous to human SERPIN genes and some of which have expanded into multiple paralogous genes. Serpins are found in tissues throughout the body; whereas most are extracellular, there is a class of intracellular serpins. Serpins appear to have roles in inflammation, immune function, tumorigenesis, blood clotting, dementia, and cancer metastasis. Further characterization of these proteins will likely reveal potential biomarkers and therapeutic targets for disease. PMID:24172014

  13. Involvement of a Serpin serine protease inhibitor (OoSerpin) from mollusc Octopus ocellatus in antibacterial response.

    PubMed

    Wei, Xiumei; Xu, Jie; Yang, Jianmin; Liu, Xiangquan; Zhang, Ranran; Wang, Weijun; Yang, Jialong

    2015-01-01

    Serpin is an important member of serine protease inhibitors (SPIs), which is capable of regulating proteolytic events and involving in a variety of physiological processes. In present study, a Serpin homolog was identified from Octopus ocellatus (designated as OoSerpin). Full-length cDNA of OoSerpin was of 1735 bp, containing a 5' untranslated region of 214 bp, a 3' UTR of 282 bp, and an open reading frame of 1239 bp. The open reading frame encoded a polypeptide of 412 amino acids which has a predicted molecular weight of 46.5 kDa and an isoelectric point of 8.52. The OoSerpin protein shares 37% sequence identity with other Serpins from Mus musculus (NP_941373) and Ixodes scapularis (XP_002407493). The existence of a conserved SERPIN domain strongly suggested that OoSerpin was a member of the Serpin subfamily. Expression patterns of OoSerpin, both in tissues and towards bacterial stimulation, were then characterized. The mRNA of OoSerpin was constitutively expressed at different levels in all tested tissues of untreated O. ocellatus, including mantle (lowest), muscle, renal sac, gill, hemocyte, gonad, systemic heart, and hepatopancreas (highest). The transcriptional level of OoSerpin was significantly up-regulated (P<0.01) in O. ocellatus upon bacterial challenges with Vibrio anguillarum and Micrococcus luteus, indicating its involvement in the antibacterial immune response. Furthermore, rOoSerpin, the recombinant protein of OoSerpin, exhibited strong abilities to inhibit proteinase activities of trypsin and chymotrypsin as well as the growth of Escherichia coli. Our results demonstrate that OoSerpin is a potential antibacterial factor involved in the immune response of O. ocellatus against bacterial infection. PMID:25449372

  14. Production of serpins using yeast expression systems.

    PubMed

    Pemberton, Philip A; Bird, Phillip I

    2004-02-01

    Serpins occupy a unique niche in the field of biology. As more of them are discovered, the need to produce sufficient quantities of each to aid experimental and therapeutic research increases. Yeast expression systems are well suited for the production of recombinant serpins. The genetics of many yeast species is well understood and readily manipulated to induce the targeted over-production of many different serpins. In addition, protease-deficient strains of certain species are available and a few species carry out post-translational modifications resembling those of humans. Yeasts are easy to grow and multiply readily in simple culture media hence the cost of production is low, while the scale of production can be small or large. The disadvantages are the inability of most yeast(s) to perform complex post-translational modifications and a lower product yield of secreted protein compared to intracellular protein production. However, for the intracellular production of serpins, in particular the clade B serpins that do not have complex post-translational modifications, yeast expression systems should be among the first systems considered. PMID:14698631

  15. Differential expansion and evolution of the exon family encoding the Serpin-1 reactive centre loop has resulted in divergent serpin repertoires among the Lepidoptera.

    PubMed

    Hegedus, Dwayne D; Erlandson, Martin; Baldwin, Douglas; Hou, Xingwei; Chamankhah, Mahmood

    2008-07-15

    Serpins are a unique class of serine protease inhibitors that are becoming increasingly recognized as important regulators of insect defense mechanisms and developmental processes. Previously, we identified three Mamestra configurata serpins that were similar in structure to those encoded by the Manduca sexta Serpin-1 gene. To gain insight into the evolution and function of serpins in lepidopterans, we developed a bacterial artificial chromosome library and sequenced the entire M. configurata gene. The Serpin-1 gene was 28 kbp and had the capacity to encode nine serpin isoforms via alternate splicing of exons encoding variant reactive center loops onto a common scaffold. The relative abundance of each isoform was estimated by expressed sequence tag analysis and their expression patterns examined in various developmental stages and larval tissues. The organization of the M. configurata Serpin-1 gene was very similar to that of M. sexta Serpin-1; however, only the Ms Serpin-1Z (1 of 12) and the Mc Serpin-1a isoforms exhibited a high degree of similarity. Orthologs similar to this variant were also found in other lepidopterans, namely Bombyx mori and Plutella xylostella, suggesting that they are involved in a conserved biochemical process and likely represent the ancestral serpin variant. Expansion of the exon family encoding the Serpin-1 reactive centre loop region appears to be a product of recent duplication events that has given rise to different serpin repertoires in related insect taxa. PMID:18495381

  16. Cytoskeletal Network Morphology Regulates Intracellular Transport Dynamics.

    PubMed

    Ando, David; Korabel, Nickolay; Huang, Kerwyn Casey; Gopinathan, Ajay

    2015-10-20

    Intracellular transport is essential for maintaining proper cellular function in most eukaryotic cells, with perturbations in active transport resulting in several types of disease. Efficient delivery of critical cargos to specific locations is accomplished through a combination of passive diffusion and active transport by molecular motors that ballistically move along a network of cytoskeletal filaments. Although motor-based transport is known to be necessary to overcome cytoplasmic crowding and the limited range of diffusion within reasonable timescales, the topological features of the cytoskeletal network that regulate transport efficiency and robustness have not been established. Using a continuum diffusion model, we observed that the time required for cellular transport was minimized when the network was localized near the nucleus. In simulations that explicitly incorporated network spatial architectures, total filament mass was the primary driver of network transit times. However, filament traps that redirect cargo back to the nucleus caused large variations in network transport. Filament polarity was more important than filament orientation in reducing average transit times, and transport properties were optimized in networks with intermediate motor on and off rates. Our results provide important insights into the functional constraints on intracellular transport under which cells have evolved cytoskeletal structures, and have potential applications for enhancing reactions in biomimetic systems through rational transport network design. PMID:26488648

  17. Histone Acetylation Regulates Intracellular pH

    PubMed Central

    McBrian, Matthew A.; Behbahan, Iman Saramipoor; Ferrari, Roberto; Su, Trent; Huang, Ta-Wei; Li, Kunwu; Hong, Candice S.; Christofk, Heather R.; Vogelauer, Maria; Seligson, David B.; Kurdistani, Siavash K.

    2014-01-01

    SUMMARY Differences in global levels of histone acetylation occur in normal and cancer cells, although the reason why cells regulate these levels has been unclear. Here we demonstrate a role for histone acetylation in regulating intracellular pH (pHi). As pHi decreases, histones are globally deacetylated by histone deacetylases (HDACs), and the released acetate anions are coexported with protons out of the cell by monocarboxylate transporters (MCTs), preventing further reductions in pHi. Conversely, global histone acetylation increases as pHi rises, such as when resting cells are induced to proliferate. Inhibition of HDACs or MCTs decreases acetate export and lowers pHi, particularly compromising pHi maintenance in acidic environments. Global deacetylation at low pH is reflected at a genomic level by decreased abundance and extensive redistribution of acetylation throughout the genome. Thus, acetylation of chromatin functions as a rheostat to regulate pHi with important implications for mechanism of action and therapeutic use of HDAC inhibitors. PMID:23201122

  18. Secretion of SerpinB2 from endothelial cells activated with inflammatory stimuli

    SciTech Connect

    Boncela, Joanna; Przygodzka, Patrycja; Wyroba, Elzbieta; Papiewska-Pajak, Izabela; Cierniewski, Czeslaw S.

    2013-05-01

    Due to the lack of an N-terminal signal peptide, SerpinB2 (plasminogen activator inhibitor type 2) accumulates in cells and only a small percentage of it is secreted. The extracellular concentration of SerpinB2 significantly increases during inflammation. In the present study we investigated the mechanism with which SerpinB2 can be secreted from endothelial cells activated with LPS. We evaluated the intracellular distribution of SerpinB2 by double immunogold labeling followed by a high resolution electron microscopy analysis. We found that SerpinB2 gathers in the vesicular structures and in the endothelial cell periphery. These vesicles stained positive for the trans-Golgi network marker TGN46, which is consistent with their formation by the endoplasmatic reticulum (ER) and Golgi-dependent pathways. SerpinB2 was delivered to the plasma membrane, apparently together with TGN46 in the same vesicles, which after fusion with the membranes released cargo. Secretion of SerpinB2 was partially inhibited by brefeldin A. The secreted SerpinB2 was predominantly in its nonglycosylated 43 kDa form as evaluated by Western immunoblotting. Our data suggest that increased expression of SerpinB2 by an inflammatory stimulus is sufficient to generate structures that resemble secretory vesicles. These vesicles may represent the mechanism by which high local concentrations of SerpinB2 are released at inflammation sites from endothelial cells. - Highlights: ► LPS stimulates generation of secretory vesicles containing SerpinB2. ► SerpinB2 concentrates in TGN46 positive vesicles close to the plasma membrane. ► Brefeldin A inhibits secretion of SerpinB2. ► The secreted SerpinB2 was predominantly in its nonglycosylated 43 kDa.

  19. Secretion of SerpinB2 from endothelial cells activated with inflammatory stimuli.

    PubMed

    Boncela, Joanna; Przygodzka, Patrycja; Wyroba, Elzbieta; Papiewska-Pajak, Izabela; Cierniewski, Czeslaw S

    2013-05-01

    Due to the lack of an N-terminal signal peptide, SerpinB2 (plasminogen activator inhibitor type 2) accumulates in cells and only a small percentage of it is secreted. The extracellular concentration of SerpinB2 significantly increases during inflammation. In the present study we investigated the mechanism with which SerpinB2 can be secreted from endothelial cells activated with LPS. We evaluated the intracellular distribution of SerpinB2 by double immunogold labeling followed by a high resolution electron microscopy analysis. We found that SerpinB2 gathers in the vesicular structures and in the endothelial cell periphery. These vesicles stained positive for the trans-Golgi network marker TGN46, which is consistent with their formation by the endoplasmatic reticulum (ER) and Golgi-dependent pathways. SerpinB2 was delivered to the plasma membrane, apparently together with TGN46 in the same vesicles, which after fusion with the membranes released cargo. Secretion of SerpinB2 was partially inhibited by brefeldin A. The secreted SerpinB2 was predominantly in its nonglycosylated 43kDa form as evaluated by Western immunoblotting. Our data suggest that increased expression of SerpinB2 by an inflammatory stimulus is sufficient to generate structures that resemble secretory vesicles. These vesicles may represent the mechanism by which high local concentrations of SerpinB2 are released at inflammation sites from endothelial cells. PMID:23474086

  20. Snail and serpinA1 promote tumor progression and predict prognosis in colorectal cancer

    PubMed Central

    Choi, Jin Hwa; Lee, Ja Rang; Kim, Hye Kyung; Jo, Hong-jae; Kim, Hyun Sung; Oh, Nahmgun; Song, Geun Am; Park, Do Youn

    2015-01-01

    The role of Snail and serpin peptidase inhibitor clade A member 1 (serpinA1) in tumorigenesis has been previously identified. However, the exact role and mechanism of these proteins in progression of colorectal cancer (CRC) are controversial. In this study, we investigated the role of Snail and serpinA1 in colorectal cancer (CRC) and examined the mechanisms through which these proteins mediate CRC progression. Immunohistochemical analysis of 528 samples from patients with CRC showed that elevated expression of Snail or serpinA1 was correlated with advanced stage, lymph node metastasis, and poor prognosis. Moreover, we detected a correlation between Snail and serpinA1 expression. Functional studies performed using the CRC cell lines DLD-1 and SW-480 showed that overexpression of Snail or serpinA1 significantly increased CRC cell invasion and migration. Conversely, knockdown of Snail or serpinA1 expression suppressed CRC cell invasion and migration. ChIP analysis revealed that Snail regulated serpinA1 by binding to its promoter. In addition, fibronectin mediated Snail and serpinA1 signaling was involved in CRC cell invasion and migration. Taken together, our data showed that Snail and serpinA1 promoted CRC progression through fibronectin. These findings suggested that Snail and serpinA1 were novel prognostic biomarkers and candidate therapeutic targets in CRC. PMID:26015410

  1. SerpinB2 (PAI-2) Modulates Proteostasis via Binding Misfolded Proteins and Promotion of Cytoprotective Inclusion Formation

    PubMed Central

    Farrawell, Natalie; Shearer, Robert F.; Constantinescu, Patrick; Hatters, Danny M.; Schroder, Wayne A.; Suhrbier, Andreas; Wilson, Mark R.; Saunders, Darren N.; Ranson, Marie

    2015-01-01

    SerpinB2 (PAI-2), a member of the clade B family of serine protease inhibitors, is one of the most upregulated proteins following cellular stress. Originally described as an inhibitor of urokinase plasminogen activator, its predominant cytoplasmic localisation suggests an intracellular function. SerpinB2 has been reported to display cytoprotective properties in neurons and to interact with intracellular proteins including components of the ubiquitin-proteasome system (UPS). In the current study we explored the potential role of SerpinB2 as a modulator of proteotoxic stress. Initially, we transiently transfected wild-type SerpinB2 and SerpinB2-/- murine embryonic fibroblasts (MEFs) with Huntingtin exon1-polyglutamine (fused C-terminally to mCherry). Inclusion body formation as result of Huntingtin aggregation was evident in the SerpinB2 expressing cells but significantly impaired in the SerpinB2-/- cells, the latter concomitant with loss in cell viability. Importantly, recovery of the wild-type phenotype and cell viability was rescued by retroviral transduction of SerpinB2 expression. SerpinB2 modestly attenuated Huntingtin and amyloid beta fibril formation in vitro and was able to bind preferentially to misfolded proteins. Given the modest chaperone-like activity of SerpinB2 we tested the ability of SerpinB2 to modulate UPS and autophagy activity using a GFP reporter system and autophagy reporter, respectively. Activity of the UPS was reduced and autophagy was dysregulated in SerpinB2-/- compared to wild-type MEFs. Moreover, we observed a non-covalent interaction between ubiquitin and SerpinB2 in cells using GFP-pulldown assays and bimolecular fluorescence complementation. We conclude that SerpinB2 plays an important role in proteostasis as its loss leads to a proteotoxic phenotype associated with an inability to compartmentalize aggregating proteins and a reduced capacity of the UPS. PMID:26083412

  2. The meteoric rise of regulated intracellular proteolysis.

    PubMed

    Mayer, R J

    2000-11-01

    It is often the case in biology that research into breaking things down lags behind research into synthesizing them, and this is certainly true for intracellular proteolysis. Now that we recognize that intracellular proteolysis, triggered by attaching multiple copies of a small protein called ubiquitin to target proteins, is fundamental to life, it is hard to believe that 20 years ago this field was little more than a backwater of biochemistry studied by a handful of laboratories. Among the few were Avram Hershko, Aaron Ciechanover and Alexander Varshavsky, who were recently awarded the Albert Lasker award for basic medical research for discovering the importance of protein degradation in cellular physiology. This Timeline traces how they and their collaborators triggered the rapid movement of ubiquitin-mediated proteolysis to centre stage. PMID:11253367

  3. Host metabolism regulates intracellular growth of Trypanosoma cruzi.

    PubMed

    Caradonna, Kacey L; Engel, Juan C; Jacobi, David; Lee, Chih-Hao; Burleigh, Barbara A

    2013-01-16

    Metabolic coupling of intracellular pathogens with host cells is essential for successful colonization of the host. Establishment of intracellular infection by the protozoan Trypanosoma cruzi leads to the development of human Chagas' disease, yet the functional contributions of the host cell toward the infection process remain poorly characterized. Here, a genome-scale functional screen identified interconnected metabolic networks centered around host energy production, nucleotide metabolism, pteridine biosynthesis, and fatty acid oxidation as key processes that fuel intracellular T. cruzi growth. Additionally, the host kinase Akt, which plays essential roles in various cellular processes, was critical for parasite replication. Targeted perturbations in these host metabolic pathways or Akt-dependent signaling pathways modulated the parasite's replicative capacity, highlighting the adaptability of this intracellular pathogen to changing conditions in the host. These findings identify key cellular process regulating intracellular T. cruzi growth and illuminate the potential to leverage host pathways to limit T. cruzi infection. PMID:23332160

  4. Host metabolism regulates intracellular growth of Trypanosoma cruzi

    PubMed Central

    Caradonna, Kacey L.; Engel, Juan C.; Jacobi, David; Lee, Chih-Hao; Burleigh, Barbara A.

    2012-01-01

    SUMMARY Metabolic coupling of intracellular pathogens with host cells is essential for successful colonization of the host. Establishment of intracellular infection by the protozoan Trypanosoma cruzi leads to the development of human Chagas disease, yet the functional contributions of the host cell toward the infection process remain poorly characterized. Here, a genome-scale functional screen identified interconnected metabolic networks centered around host energy production, nucleotide metabolism, pteridine biosynthesis, and fatty acid oxidation as key processes that fuel intracellular T. cruzi growth. Additionally, the host kinase Akt, which plays essential roles in various cellular processes, was critical for parasite replication. Targeted perturbations in these host metabolic pathways or Akt-dependent signaling pathways modulated the parasite’s replicative capacity, highlighting the adaptability of this intracellular pathogen to changing conditions in the host. These findings identify key cellular process regulating intracellular T. cruzi growth and illuminate the potential to leverage host pathways to limit T. cruzi infection. PMID:23332160

  5. An RNA Aptamer Inhibits a Mutation-Induced Inactivating Misfolding of a Serpin.

    PubMed

    Madsen, Jeppe B; Andersen, Lisbeth M; Dupont, Daniel M; Trelle, Morten B; Johansen, Jesper S; Jensen, Jan K; Jørgensen, Thomas J D; Andreasen, Peter A

    2016-06-23

    Most serpins are fast and specific inhibitors of extracellular serine proteases controlling biological processes such as blood coagulation, fibrinolysis, tissue remodeling, and inflammation. The inhibitory activity of serpins is based on a conserved metastable structure and their conversion to a more stable state during reaction with the target protease. However, the metastable state also makes serpins vulnerable to mutations, resulting in disease caused by inactive and misfolded monomeric or polymeric forms ("serpinopathy"). Misfolding can occur either intracellularly (type-I serpinopathies) or extracellularly (type-II serpinopathies). We have isolated a 2'-fluoropyrimidine-modified RNA aptamer, which inhibits a mutation-induced inactivating misfolding of the serpin α1-antichymotrypsin. It is the first agent able to stabilize a type-II mutation of a serpin without interfering with the inhibitory mechanism, thereby presenting a solution for the long-standing challenge of preventing pathogenic misfolding without compromising the inhibitory function. PMID:27265748

  6. Serpins in rice: protein sequence analysis, phylogeny and gene expression during development

    PubMed Central

    2012-01-01

    point to a range of target proteases with different proteolytic specificities. Large differences in basal expression levels of the eight selected rice serpin genes during development further suggest a range of functions in regulation and in plant defence for the corresponding proteins. PMID:22947050

  7. Intracellular calcium ions as regulators of renal tubular sodium transport.

    PubMed

    Windhager, E; Frindt, G; Yang, J M; Lee, C O

    1986-09-15

    This review addresses the putative role of intracellular calcium ions in the regulation of sodium transport by renal tubules. Cytoplasmic calcium-ion activities in proximal tubules of Necturus are less than 10(-7) M and can be increased by lowering the electrochemical potential gradient for sodium ions across the peritubular cell membrane, or by addition of quinidine or ionomycin to peritubular fluid. Whereas lowering of the peritubular Na concentration increases cytosolic [Ca++] and [H+], ionomycin, a calcium ionophore, raises intracellular [Ca++] without decreasing pHi. The intracellular calcium-ion level is maintained by transport processes in the plasma membrane and membranes of intracellular organelles, as well as by calcium-binding proteins. Calcium ions inhibit net transport of sodium by reducing the rate of sodium entry across the luminal cell membrane. In the collecting tubule this inhibition is caused, at least in part, by an indirect reduction in the activity of the amiloride-sensitive sodium channel. PMID:2430134

  8. SerpinB2 is critical to Th2 immunity against enteric nematode infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    SerpinB2, a member of the serine protease inhibitor family, is expressed by macrophages and up-regulated significantly by inflammation. Recent studies implicated a role for SerpinB2 in the control of Th1 and Th2 immune responses, but the mechanisms of these effects are unknown. In the current study...

  9. SerpinB3 and Yap Interplay Increases Myc Oncogenic Activity

    PubMed Central

    Turato, Cristian; Cannito, Stefania; Simonato, Davide; Villano, Gianmarco; Morello, Elisabetta; Terrin, Liliana; Quarta, Santina; Biasiolo, Alessandra; Ruvoletto, Mariagrazia; Martini, Andrea; Fasolato, Silvano; Zanus, Giacomo; Cillo, Umberto; Gatta, Angelo; Parola, Maurizio; Pontisso, Patrizia

    2015-01-01

    SerpinB3 has been recently described as an early marker of liver carcinogenesis, but the potential mechanistic role of this serpin in tumor development is still poorly understood. Overexpression of Myc often correlates with more aggressive tumour forms, supporting its involvement in carcinogenesis. Yes-associated protein (Yap), the main effector of the Hippo pathway, is a central regulator of proliferation and it has been found up-regulated in hepatocellular carcinomas. The study has been designed to investigate and characterize the interplay and functional modulation of Myc by SerpinB3 in liver cancer. Results from this study indicate that Myc was up-regulated by SerpinB3 through calpain and Hippo-dependent molecular mechanisms in transgenic mice and hepatoma cells overexpressing human SerpinB3, and also in human hepatocellular carcinomas. Human recombinant SerpinB3 was capable to inhibit the activity of Calpain in vitro, likely reducing its ability to cleave Myc in its non oncogenic Myc-nick cytoplasmic form. SerpinB3 indirectly increased the transcription of Myc through the induction of Yap pathway. These findings provide for the first time evidence that SerpinB3 can improve the production of Myc through direct and indirect mechanisms that include the inhibition of generation of its cytoplasmic form and the activation of Yap pathway. PMID:26634820

  10. A proprotein convertase-inhibiting serpin with an endoplasmic reticulum targeting signal from Branchiostoma lanceolatum, a close relative of vertebrates

    PubMed Central

    Bentele, Caterina; Krüger, Olaf; Tödtmann, Ulf; Oley, Mareke; Ragg, Hermann

    2006-01-01

    Lancelets are considered to take a key position in the evolution of lineages leading to vertebrates. Herein, a serpin from the lancelet Branchiostoma lanceolatum, Bl-Spn1, was identified that inhibits the PCs (proprotein convertases) PC1/3 and furin. The inhibitor forms SDS-stable complexes with either of its targets. Analysis of the inhibitor/furin reaction products by mass spectroscopy assigns the enzyme's cleavage position C-terminally to Met-Met-Lys-Arg↓ in the reactive site loop of Spn1, in concordance with the classical recognition/cleavage site of the principal vertebrate PCs. The inhibitor is equipped with a canonical ER (endoplasmic reticulum) retrieval signal, Lys-Asp-Glu-Leu (KDEL), marking the inhibitor as a guardian of the cellular secretory routes. Deletion of the ER retrieval signal results in the export of the inhibitor into the medium of transfected COS-7 cells, consistent with the assigned intracellular location. These results identify Bl-Spn1 as the first serpin that may inhibit PC1/3-like subtilases at their natural sites of action. Phylogenetic comparisons support a concept implying a general role for ER-residing serpins in the surveillance of subtilase-like enzymes along the constitutive and regulated secretory pathways of metazoans including a role in the defence of intruders that turn PCs to their propagation. PMID:16445382

  11. Serpine2, a potential novel target for combating melanoma metastasis

    PubMed Central

    Wu, Qi Wei

    2016-01-01

    Early stages of melanoma can be treated by surgical resection of tumor, but there is still no effective treatment once it is progressed to metastatic phases. Although growing family of both metastasis promoting and metastasis suppressor genes have been reported, the molecular mechanisms governing melanoma metastatic cascade are still not completely understood. Therefore, defining the molecules that govern melanoma metastasis may aid the development of more effective therapeutic strategies for combating cancer. In the present study, we found that Serpin Peptidase Inhibitor 2, Serpine2 was involved in the metastasis of melanoma cells. The requirement of Serpine2 in the migration of melanoma cells was confirmed by gene silencing and over-expression in vitro. Moreover, down-regulation of Serpine2 expression strikingly inhibited melanoma cellular metastasis in vivo. Finally, we found that Serpine2 promotes melanoma metastasis through the glycogen synthesis kinase 3β, GSK-3β signaling pathway. To conclude, our findings suggested a novel mechanism underlying the metastasis of melanoma cells which might serve as a new intervention target for the treatment of melanoma. PMID:27347308

  12. Preferential intracellular pH regulation: hypotheses and perspectives.

    PubMed

    Shartau, Ryan B; Baker, Daniel W; Crossley, Dane A; Brauner, Colin J

    2016-08-01

    The regulation of vertebrate acid-base balance during acute episodes of elevated internal PCO2  is typically characterized by extracellular pH (pHe) regulation. Changes in pHe are associated with qualitatively similar changes in intracellular tissue pH (pHi) as the two are typically coupled, referred to as 'coupled pH regulation'. However, not all vertebrates rely on coupled pH regulation; instead, some preferentially regulate pHi against severe and maintained reductions in pHe Preferential pHi regulation has been identified in several adult fish species and an aquatic amphibian, but never in adult amniotes. Recently, common snapping turtles were observed to preferentially regulate pHi during development; the pattern of acid-base regulation in these species shifts from preferential pHi regulation in embryos to coupled pH regulation in adults. In this Commentary, we discuss the hypothesis that preferential pHi regulation may be a general strategy employed by vertebrate embryos in order to maintain acid-base homeostasis during severe acute acid-base disturbances. In adult vertebrates, the retention or loss of preferential pHi regulation may depend on selection pressures associated with the environment inhabited and/or the severity of acid-base regulatory challenges to which they are exposed. We also consider the idea that the retention of preferential pHi regulation into adulthood may have been a key event in vertebrate evolution, with implications for the invasion of freshwater habitats, the evolution of air breathing and the transition of vertebrates from water to land. PMID:27489212

  13. Increasing transcriptome response of serpins during the ontogenetic stages in the salmon louse Caligus rogercresseyi (Copepoda: Caligidae).

    PubMed

    Maldonado-Aguayo, W; Gallardo-Escárate, C

    2014-06-01

    Serine protease inhibitors, or serpins, target serine proteases, and are important regulators of intra- and extracellular proteolysis. For parasite survival, parasite-derived protease inhibitors have been suggested to play essential roles in evading the host's immune system and protecting against exogenous host proteases. The aim of this work was to identify serpins via high throughput transcriptome sequencing and elucidate their potential functions during the lifecycle of the salmon louse Caligus rogercresseyi. Eleven putative, partial serpin sequences in the C. rogercresseyi transcriptome were identified and denoted as Cr-serpins 1 to 11. Comparative analysis of the deduced serpin-like amino acid sequences revealed a highly conserved reactive center loop region. Interestingly, P1 residues suggest putative functions involved with the trypsin/subtilisin, elastase, or subtilisin inhibitors, which evidenced increasing gene expression profiles from the copepodid to adult stage in C. rogercresseyi. Concerning this, Cr-serpin 10 was mainly expressed in the copepodid stage, while Cr-serpins 3, 4, 5, and 11 were mostly expressed in chalimus and adult stages. These results suggest that serpins could be involved in evading the immune response of the host fish. The identification of these serpins furthers the understanding of the immune system in this important ectoparasite species. PMID:24798872

  14. Characterisation of serpin polymers in vitro and in vivo.

    PubMed

    Belorgey, Didier; Irving, James A; Ekeowa, Ugo I; Freeke, Joanna; Roussel, Benoit D; Miranda, Elena; Pérez, Juan; Robinson, Carol V; Marciniak, Stefan J; Crowther, Damian C; Michel, Claire H; Lomas, David A

    2011-03-01

    Neuroserpin is a member of the serine protease inhibitor or serpin superfamily of proteins. It is secreted by neurones and plays an important role in the regulation of tissue plasminogen activator at the synapse. Point mutations in the neuroserpin gene cause the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. This is one of a group of disorders caused by mutations in the serpins that are collectively known as the serpinopathies. Others include α(1)-antitrypsin deficiency and deficiency of C1 inhibitor, antithrombin and α(1)-antichymotrypsin. The serpinopathies are characterised by delays in protein folding and the retention of ordered polymers of the mutant serpin within the cell of synthesis. The clinical phenotype results from either a toxic gain of function from the inclusions or a loss of function, as there is insufficient protease inhibitor to regulate important proteolytic cascades. We describe here the methods required to characterise the polymerisation of neuroserpin and draw parallels with the polymerisation of α(1)-antitrypsin. It is important to recognise that the conditions in which experiments are performed will have a major effect on the findings. For example, incubation of monomeric serpins with guanidine or urea will produce polymers that are not found in vivo. The characterisation of the pathological polymers requires heating of the folded protein or alternatively the assessment of ordered polymers from cell and animal models of disease or from the tissues of humans who carry the mutation. PMID:21115126

  15. KRIT1 Regulates the Homeostasis of Intracellular Reactive Oxygen Species

    PubMed Central

    Goitre, Luca; Balzac, Fiorella; Degani, Simona; Degan, Paolo; Marchi, Saverio; Pinton, Paolo; Retta, Saverio Francesco

    2010-01-01

    KRIT1 is a gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhage. Comprehensive analysis of the KRIT1 gene in CCM patients has suggested that KRIT1 functions need to be severely impaired for pathogenesis. However, the molecular and cellular functions of KRIT1 as well as CCM pathogenesis mechanisms are still research challenges. We found that KRIT1 plays an important role in molecular mechanisms involved in the maintenance of the intracellular Reactive Oxygen Species (ROS) homeostasis to prevent oxidative cellular damage. In particular, we demonstrate that KRIT1 loss/down-regulation is associated with a significant increase in intracellular ROS levels. Conversely, ROS levels in KRIT1−/− cells are significantly and dose-dependently reduced after restoration of KRIT1 expression. Moreover, we show that the modulation of intracellular ROS levels by KRIT1 loss/restoration is strictly correlated with the modulation of the expression of the antioxidant protein SOD2 as well as of the transcriptional factor FoxO1, a master regulator of cell responses to oxidative stress and a modulator of SOD2 levels. Furthermore, we show that the KRIT1-dependent maintenance of low ROS levels facilitates the downregulation of cyclin D1 expression required for cell transition from proliferative growth to quiescence. Finally, we demonstrate that the enhanced ROS levels in KRIT1−/− cells are associated with an increased cell susceptibility to oxidative DNA damage and a marked induction of the DNA damage sensor and repair gene Gadd45α, as well as with a decline of mitochondrial energy metabolism. Taken together, our results point to a new model where KRIT1 limits the accumulation of intracellular oxidants and prevents oxidative stress-mediated cellular dysfunction and DNA damage by enhancing the

  16. Reduced serpinB9-mediated caspase-1 inhibition can contribute to autoinflammatory disease

    PubMed Central

    van der Burgh, Robert; Meeldijk, Jan; Jongeneel, Lieneke; Frenkel, Joost; Bovenschen, Niels

    2016-01-01

    Patients who suffer from autoinflammatory disease (AID) exhibit seemingly uncontrolled release of interleukin (IL)-1β. The presence of this inflammatory cytokine triggers immune activation in absence of pathogens and foreign material. The mechanisms that contribute to ‘sterile inflammation’ episodes in AID patients are not fully understood, although for some AIDs underlying genetic causes have been identified. We show that the serine protease inhibitor B9 (serpinB9) regulates IL-1β release in human monocytes. SerpinB9 function is more commonly known for its role in control of granzyme B. SerpinB9 however also serves to restrain IL-1β maturation through caspase-1 inhibition. We here describe an autoinflammatory disease-associated serpinB9 (c.985G>T, A329S) variant, which we discovered in a patient with unknown AID. Using patient cells and serpinB9 overexpressing monocytic cells, we show the A329S variant of serpinB9 exhibits unobstructed granzyme B inhibition, but compromised caspase-1 inhibition. SerpinB9 gene variants might contribute to AID development. PMID:26992230

  17. Role of intracellular calcium in cellular volume regulation

    SciTech Connect

    Wong, S.M.; Chase, H.S. Jr.

    1986-06-01

    We investigated the role of intracellular calcium in epithelial cell volume regulation using cells isolated from the toad urinary bladder. A suspension of cells was prepared by treatment of the bladder with collagenase followed by ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid. The cells retained their ion-transporting capabilities: ouabain (1 mM) and amiloride (10 microM) inhibited cellular uptake of /sup 86/Rb and /sup 22/Na, respectively. Using a Coulter counter to measure cellular volume, we found that we could swell cells either by reducing the extracellular osmolality or by adding the permeant solute urea (45 mM) isosmotically. Under both conditions, cells first swelled and then returned to their base-line volume, in spite of the continued presence of the stimulus to swell. Volume regulation was inhibited when cells were swelled at low extracellular (Ca) (100 nM) and was retarded in cells preloaded with the calcium buffer quin 2. Swelling increased the intracellular free calcium concentration ((Ca)i), as measured by quin 2 fluorescence: (Ca)i increased 35 +/- 9 nM (n = 6) after hypotonic swelling and 42 +/- 3 nM (n = 3) after urea swelling. Reducing extracellular (Ca) to less than 100 nM prevented the swelling-induced increase in (Ca)i, suggesting that the source of the increase in (Ca)i was extracellular. This result was confirmed in measurements of cellular uptake of 45Ca: the rate of uptake was significantly higher in swollen cells compared with control (1.1 +/- 0.2 vs. 0.4 +/- 0.1 fmol . cell-1 X 5 min-1). Our experiments provide the first demonstration that cellular swelling increases (Ca)i. This increase is likely to play a critical role in cellular volume regulation.

  18. SerpinB2 Deficiency Results in a Stratum Corneum Defect and Increased Sensitivity to Topically Applied Inflammatory Agents.

    PubMed

    Schroder, Wayne A; Anraku, Itaru; Le, Thuy T; Hirata, Thiago D C; Nakaya, Helder I; Major, Lee; Ellis, Jonathan J; Suhrbier, Andreas

    2016-06-01

    SerpinB2 (plasminogen activator inhibitor type 2) is constitutively expressed at high levels by differentiating keratinocytes in mice and humans; however, the physiological function of keratinocyte SerpinB2 remains unclear. Herein, we show that SerpinB2(-/-) mice are more susceptible to contact dermatitis after topical application of dinitrofluorobenzene, and show enhanced inflammatory lesions after topical applications of phorbol ester. Untreated SerpinB2(-/-) mice showed no overt changes in epithelial structure, and we were unable to find evidence for a role for keratinocyte SerpinB2 in regulating immunity, apoptosis, IL-1β production, proteasomal activity, or wound healing. Instead, the phenotype was associated with impaired skin barrier function and a defective stratum corneum, with SerpinB2(-/-) mice showing increased transepidermal water loss, increased overt loss of stratum corneum in inflammatory lesions, and impaired stratum corneum thickening after phorbol ester treatment. Immunoblotting suggested that SerpinB2 (cross-linked into the cornified envelope) is present in the stratum corneum and retains the ability to form covalent inhibitory complexes with urokinase. Data suggest that the function of keratinocyte SerpinB2 is protection of the stratum corneum from proteolysis via inhibition of urokinase, thereby maintaining the integrity and barrier function of the stratum corneum, particularly during times of skin inflammation. Implications for studies involving genetically modified mice treated with topical agents and human dermatological conditions, such as contact dermatitis, are discussed. PMID:27109612

  19. Ornithine decarboxylase antizyme inhibitor 2 regulates intracellular vesicle trafficking

    SciTech Connect

    Kanerva, Kristiina; Maekitie, Laura T.; Baeck, Nils; Andersson, Leif C.

    2010-07-01

    Antizyme inhibitor 1 (AZIN1) and 2 (AZIN2) are proteins that activate ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. Both AZINs release ODC from its inactive complex with antizyme (AZ), leading to formation of the catalytically active ODC. The ubiquitously expressed AZIN1 is involved in cell proliferation and transformation whereas the role of the recently found AZIN2 in cellular functions is unknown. Here we report the intracellular localization of AZIN2 and present novel evidence indicating that it acts as a regulator of vesicle trafficking. We used immunostaining to demonstrate that both endogenous and FLAG-tagged AZIN2 localize to post-Golgi vesicles of the secretory pathway. Immuno-electron microscopy revealed that the vesicles associate mainly with the trans-Golgi network (TGN). RNAi-mediated knockdown of AZIN2 or depletion of cellular polyamines caused selective fragmentation of the TGN and retarded the exocytotic release of vesicular stomatitis virus glycoprotein. Exogenous addition of polyamines normalized the morphological changes and reversed the inhibition of protein secretion. Our findings demonstrate that AZIN2 regulates the transport of secretory vesicles by locally activating ODC and polyamine biosynthesis.

  20. Stochastic focusing: Fluctuation-enhanced sensitivity of intracellular regulation

    PubMed Central

    Paulsson, Johan; Berg, Otto G.; Ehrenberg, Måns

    2000-01-01

    Many regulatory molecules are present in low copy numbers per cell so that significant random fluctuations emerge spontaneously. Because cell viability depends on precise regulation of key events, such signal noise has been thought to impose a threat that cells must carefully eliminate. However, the precision of control is also greatly affected by the regulatory mechanisms' capacity for sensitivity amplification. Here we show that even if signal noise reduces the capacity for sensitivity amplification of threshold mechanisms, the effect on realistic regulatory kinetics can be the opposite: stochastic focusing (SF). SF particularly exploits tails of probability distributions and can be formulated as conventional multistep sensitivity amplification where signal noise provides the degrees of freedom. When signal fluctuations are sufficiently rapid, effects of time correlations in signal-dependent rates are negligible and SF works just like conventional sensitivity amplification. This means that, quite counterintuitively, signal noise can reduce the uncertainty in regulated processes. SF is exemplified by standard hyperbolic inhibition, and all probability distributions for signal noise are first derived from underlying chemical master equations. The negative binomial is suggested as a paradigmatic distribution for intracellular kinetics, applicable to stochastic gene expression as well as simple systems with Michaelis–Menten degradation or positive feedback. SF resembles stochastic resonance in that noise facilitates signal detection in nonlinear systems, but stochastic resonance is related to how noise in threshold systems allows for detection of subthreshold signals and SF describes how fluctuations can make a gradual response mechanism work more like a threshold mechanism. PMID:10852944

  1. IQGAP1: A Regulator of Intracellular Spacetime Relativity

    PubMed Central

    Malarkannan, Subramaniam; Awasthi, Aradhana; Kamalakannan, Rajasekaran; Kumar, Pawan; Schuldt, Kristina M; Bartoszek, Allison; Manoharan, Niranjan; Goldner, Nicholas K; Umhoefer, Colleen M; Thakar, Monica S

    2012-01-01

    Activating and inhibiting receptors of lymphocytes collect valuable information about their mikròs kósmos. This information is essential to initiate or to turn off complex signaling pathways. Irrespective of these advances, our knowledge on how these intracellular activation cascades are coordinated in a spatiotemporal manner is far from complete. Amongst multiple explanations, the scaffolding proteins have emerged as a critical piece of this evolutionary tangram. Amongst many, IQGAP1 is one of the essential scaffolding proteins that coordinate multiple signaling pathways. IQGAP1 possesses multiple protein interaction motifs to achieve its scaffolding functions. Using these domains, IQGAP1 has been shown to regulate a number of essential cellular events. This includes actin polymerization, tubulin multimerization, MTOC formation, calcium/calmodulin signaling, Pak/Raf/Mek1/2-mediated Erk1/2 activation, formation of maestrosome, E-cadherin and CD44-mediated signaling and GSK3/APC-mediated β-catenin activation. In this review we summarize the recent developments and exciting new findings of cellular functions of IQGAP1. PMID:22345702

  2. IQGAP1: a regulator of intracellular spacetime relativity.

    PubMed

    Malarkannan, Subramaniam; Awasthi, Aradhana; Rajasekaran, Kamalakannan; Kumar, Pawan; Schuldt, Kristina M; Bartoszek, Allison; Manoharan, Niranjan; Goldner, Nicholas K; Umhoefer, Colleen M; Thakar, Monica S

    2012-03-01

    Activating and inhibiting receptors of lymphocytes collect valuable information about their mikròs kósmos. This information is essential to initiate or to turn off complex signaling pathways. Irrespective of these advances, our knowledge on how these intracellular activation cascades are coordinated in a spatiotemporal manner is far from complete. Among multiple explanations, the scaffolding proteins have emerged as a critical piece of this evolutionary tangram. Among many, IQGAP1 is one of the essential scaffolding proteins that coordinate multiple signaling pathways. IQGAP1 possesses multiple protein interaction motifs to achieve its scaffolding functions. Using these domains, IQGAP1 has been shown to regulate a number of essential cellular events. This includes actin polymerization, tubulin multimerization, microtubule organizing center formation, calcium/calmodulin signaling, Pak/Raf/Mek1/2-mediated Erk1/2 activation, formation of maestrosome, E-cadherin, and CD44-mediated signaling and glycogen synthase kinase-3/adenomatous polyposis coli-mediated β-catenin activation. In this review, we summarize the recent developments and exciting new findings of cellular functions of IQGAP1. PMID:22345702

  3. Molecular gymnastics: serpin structure, folding and misfolding.

    PubMed

    Whisstock, James C; Bottomley, Stephen P

    2006-12-01

    The native state of serpins represents a long-lived intermediate or metastable structure on the serpin folding pathway. Upon interaction with a protease, the serpin trap is sprung and the molecule continues to fold into a more stable conformation. However, thermodynamic stability can also be achieved through alternative, unproductive folding pathways that result in the formation of inactive conformations. Our increasing understanding of the mechanism of protease inhibition and the dynamics of native serpin structures has begun to reveal how evolution has harnessed the actual process of protein folding (rather than the final folded outcome) to elegantly achieve function. The cost of using metastability for function, however, is an increased propensity for misfolding. PMID:17079131

  4. Regulating Intracellular Calcium in Plants: From Molecular Genetics to Physiology

    SciTech Connect

    Heven Sze

    2008-06-22

    To grow, develop, adapt, and reproduce, plants have evolved mechanisms to regulate the uptake, translocation and sorting of calcium ions into different cells and subcellular compartments. Yet how plants accomplish this remarkable feat is still poorly understood. The spatial and temporal changes in intracellular [Ca2+] during growth and during responses to hormonal and environmental stimuli indicate that Ca2+ influx and efflux transporters are diverse and tightly regulated in plants. The specific goals were to determine the biological roles of multiple Ca pumps (ECAs) in the model plant Arabidopsis thaliana. We had pioneered the use of K616 yeast strain to functionally express plant Ca pumps, and demonstrated two distinct types of Ca pumps in plants (Sze et al., 2000. Annu Rev Plant Biol. 51,433). ACA2 represented one type that was auto-inhibited by the N-terminal region and stimulated by calmodulin. ECA1 represented another type that was not sensitive to calmodulin and phylogenetically distinct from ACAs. The goal to determine the biological roles of multiple ECA-type Ca pumps in Arabidopsis has been accomplished. Although we demonstrated ECA1 was a Ca pump by functional expression in yeast, the in vivo roles of ECAs was unclear. A few highlights are described. ECA1 and/or ECA4 are Ca/Mn pumps localized to the ER and are highly expressed in all cell types. Using homozygous T-DNA insertional mutants of eca1, we demonstrated that the ER-bound ECA1 supports growth and confers tolerance of plants growing on medium low in Ca or containing toxic levels of Mn. This is the first genetic study to determine the in vivo function of a Ca pump in plants. A phylogenetically distinct ECA3 is also a Ca/Mn pump that is localized to endosome, such as post-Golgi compartments. Although it is expressed at lower levels than ECA1, eca3 mutants are impaired in Ca-dependent root growth and in pollen tube elongation. Increased secretion of wall proteins in mutants suggests that Ca and Mn

  5. Intracellular calcium levels can regulate Importin-dependent nuclear import

    SciTech Connect

    Kaur, Gurpreet; Ly-Huynh, Jennifer D.; Jans, David A.

    2014-07-18

    Highlights: • High intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import. • The effect of Ca{sup 2+} on nuclear import does not relate to changes in the nuclear pore. • High intracellular calcium can result in mislocalisation of Impβ1, Ran and RCC1. - Abstract: We previously showed that increased intracellular calcium can modulate Importin (Imp)β1-dependent nuclear import of SRY-related chromatin remodeling proteins. Here we extend this work to show for the first time that high intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import generally. The basis of this relates to the mislocalisation of the transport factors Impβ1 and Ran, which show significantly higher nuclear localization in contrast to various other factors, and RCC1, which shows altered subnuclear localisation. The results here establish for the first time that intracellular calcium modulates conventional nuclear import through direct effects on the nuclear transport machinery.

  6. Reactive Center Loop (RCL) Peptides Derived from Serpins Display Independent Coagulation and Immune Modulating Activities.

    PubMed

    Ambadapadi, Sriram; Munuswamy-Ramanujam, Ganesh; Zheng, Donghang; Sullivan, Colin; Dai, Erbin; Morshed, Sufi; McFadden, Baron; Feldman, Emily; Pinard, Melissa; McKenna, Robert; Tibbetts, Scott; Lucas, Alexandra

    2016-02-01

    Serpins regulate coagulation and inflammation, binding serine proteases in suicide-inhibitory complexes. Target proteases cleave the serpin reactive center loop scissile P1-P1' bond, resulting in serpin-protease suicide-inhibitory complexes. This inhibition requires a near full-length serpin sequence. Myxomavirus Serp-1 inhibits thrombolytic and thrombotic proteases, whereas mammalian neuroserpin (NSP) inhibits only thrombolytic proteases. Both serpins markedly reduce arterial inflammation and plaque in rodent models after single dose infusion. In contrast, Serp-1 but not NSP improves survival in a lethal murine gammaherpesvirus68 (MHV68) infection in interferon γ-receptor-deficient mice (IFNγR(-/-)). Serp-1 has also been successfully tested in a Phase 2a clinical trial. We postulated that proteolytic cleavage of the reactive center loop produces active peptide derivatives with expanded function. Eight peptides encompassing predicted protease cleavage sites for Serp-1 and NSP were synthesized and tested for inhibitory function in vitro and in vivo. In engrafted aorta, selected peptides containing Arg or Arg-Asn, not Arg-Met, with a 0 or +1 charge, significantly reduced plaque. Conversely, S-6 a hydrophobic peptide of NSP, lacking Arg or Arg-Asn with -4 charge, induced early thrombosis and mortality. S-1 and S-6 also significantly reduced CD11b(+) monocyte counts in mouse splenocytes. S-1 peptide had increased efficacy in plasminogen activator inhibitor-1 serpin-deficient transplants. Plaque reduction correlated with mononuclear cell activation. In a separate study, Serp-1 peptide S-7 improved survival in the MHV68 vasculitis model, whereas an inverse S-7 peptide was inactive. Reactive center peptides derived from Serp-1 and NSP with suitable charge and hydrophobicity have the potential to extend immunomodulatory functions of serpins. PMID:26620556

  7. Optogenetic oligomerization of Rab GTPases regulates intracellular membrane trafficking.

    PubMed

    Nguyen, Mai Khanh; Kim, Cha Yeon; Kim, Jin Man; Park, Byung Ouk; Lee, Sangkyu; Park, Hyerim; Heo, Won Do

    2016-06-01

    Intracellular membrane trafficking, which is involved in diverse cellular processes, is dynamic and difficult to study in a spatiotemporal manner. Here we report an optogenetic strategy, termed light-activated reversible inhibition by assembled trap of intracellular membranes (IM-LARIAT), that uses various Rab GTPases combined with blue-light-induced hetero-interaction between cryptochrome 2 and CIB1. In this system, illumination induces a rapid and reversible intracellular membrane aggregation that disrupts the dynamics and functions of the targeted membrane. We applied IM-LARIAT to specifically perturb several Rab-mediated trafficking processes, including receptor transport, protein sorting and secretion, and signaling initiated from endosomes. We finally used this tool to reveal different functions of local Rab5-mediated and Rab11-mediated membrane trafficking in growth cones and soma of young hippocampal neurons. Our results show that IM-LARIAT is a versatile tool that can be used to dissect spatiotemporal functions of intracellular membranes in diverse systems. PMID:27065232

  8. Functional characterization of hesp018, a baculovirus-encoded serpin gene.

    PubMed

    Ardisson-Araujo, Daniel M P; Rohrmann, George F; Ribeiro, Bergmann M; Clem, Rollie J

    2015-05-01

    The serpin family of serine proteinase inhibitors plays key roles in a variety of biochemical pathways. In insects, one of the important functions carried out by serpins is regulation of the phenoloxidase (PO) cascade - a pathway that produces melanin and other compounds that are important in insect humoral immunity. Recent sequencing of the baculovirus Hemileuca sp. nucleopolyhedrovirus (HespNPV) genome revealed the presence of a gene, hesp018, with homology to insect serpins. To our knowledge, hesp018 is the first viral serpin homologue to be characterized outside of the chordopoxviruses. The Hesp018 protein was found to be a functional serpin with inhibitory activity against a subset of serine proteinases. Hesp018 also inhibited PO activation when mixed with lepidopteran haemolymph. The Hesp018 protein was secreted when expressed in lepidopteran cells and a baculovirus expressing Hesp018 exhibited accelerated production of viral progeny during in vitro infection. Expression of Hesp018 also reduced caspase activity induced by baculovirus infection, but caused increased cathepsin activity. In infected insect larvae, expression of Hesp018 resulted in faster larval melanization, consistent with increased activity of viral cathepsin. Finally, expression of Hesp018 increased the virulence of a prototype baculovirus by fourfold in orally infected neonate Trichoplusia ni larvae. Based on our observations, we hypothesize that hesp018 may have been retained in HespNPV due to its ability to inhibit the activity of select host proteinases, possibly including proteinases involved in the PO response, during infection of host insects. PMID:25573886

  9. [Serpins in hyperplastic colon tissue].

    PubMed

    Kit, O I; Frantsiiants, E M; Kozlova, L S; Terpugov, A L

    2014-01-01

    The purpose of the study was to define α-2-macroglobulin (α-2M) and α-1-proteinase inhibitor (α-1PI) in tissues of malignant tumors and polyps of the lower parts of the colon. 28 patients had malignant tumors of the sigmoid colon or rectum (T3N0-1M0-2), 29 had polyps of the same location. Content of α-2M and α-1PI was studied in cytosols of the central, peripheral and conditionally healthy tissues (of resection line) of the mentioned hyperplasias by the ELISA method using standard test kits. Suppression of a-2M and increase of α-1PI (perifocal zone) were found in malignant tumor tissue, as well as α-1PI maintenance in tumorous focus. Increase of α-2M and decrease of α-1PI were detected in polyp tissue. Changes in physiological balance of serpins were assessed by α-1PI/α-2M ratio in comparison with the resection line. The risk of distortion of proliferation and differentiation processes increases in polyps in ineffective inhibition of proteolysis under the influence of released factors of malignancy. Endogenous or medicamentous restoration of balance of interaction of trypsin-like proteases and kallikrein with inhibitors will probably play the crucial role. PMID:25911925

  10. Bioinformatic analyses of male and female Amblyomma americanum tick expressed serine protease inhibitors (serpins)

    PubMed Central

    Porter, Lindsay; Radulovic, Zeljko; Kim, Tae; Braz, Gloria R. C.; Da Silva Vaz, Itabajara; Mulenga, Albert

    2014-01-01

    Serine protease inhibitors (serpins) are a diverse family of proteins that is conserved across taxa. The diversity of Amblyomma americanum serpins (AAS) is far more complex than previously thought as revealed by discovery of 57 and 33 AAS transcripts that are respectively expressed in male and female A. americanum ticks, with 30 found in both. While distinct reproductively, both male and female metastriate ticks, such as A. americanum, require a blood meal. Thus, 30 AAS sequences found in both male and female ticks could play important role(s) in regulating tick feeding and thus represent attractive candidates for anti-tick vaccine development. Of significant interest, 19 AAS sequences expressed in male and female ticks are also part of the 48 AAS sequences expressed in fed female tick salivary glands or midguts; two organs through which the tick interacts with host blood and immune response factors. Considered the most important domain for serpin function, the reactive center loop (RCL) is further characterized by a single ‘P1’ site amino acid residue, which is central to determining the protease regulated by the serpin. In this study, a diversity of 17 different P1 site amino acid residues were predicted, suggesting that A. americanum serpins potentially regulate a large number of proteolytic pathways. Our data also indicate that some serpins in this study could regulate target protease common to all tick species, in that more than 40% of AAS show 58–97% inter-species amino acid conservation. Of significance, 24% of AAS showed 62–100% inter-species conservation within the functional RCL domain, with 10 RCLs showing ≥90–100% conservation. In vertebrates, serpins with basic residues at the P1 site regulate key host defense pathways, which the tick must evade to feed successfully. Interestingly, we found that AAS sequences with basic or polar uncharged residues at the putative P1 site are more likely to be conserved across tick species. Another notable

  11. Vaspin inhibits kallikrein 7 by serpin mechanism.

    PubMed

    Heiker, John T; Klöting, Nora; Kovacs, Peter; Kuettner, E Bartholomeus; Sträter, Norbert; Schultz, Stephan; Kern, Matthias; Stumvoll, Michael; Blüher, Matthias; Beck-Sickinger, Annette G

    2013-07-01

    The molecular target of the adipokine vaspin (visceral adipose tissue-derived serpin; serpinA12) and its mode of action are unknown. Here, we provide the vaspin crystal structure and identify human kallikrein 7 (hK7) as a first protease target of vaspin inhibited by classical serpin mechanism with high specificity in vitro. We detect vaspin-hK7 complexes in human plasma and find co-expression of both proteins in murine pancreatic β-cells. We further demonstrate that hK7 cleaves human insulin in the A- and B-chain. Vaspin treatment of isolated pancreatic islets leads to increased insulin concentration in the media upon glucose stimulation without influencing insulin secretion. By application of vaspin and generated inactive mutants, we find the significantly improved glucose tolerance in C57BL/6NTac and db/db mice treated with recombinant vaspin fully dependent on the vaspin serpin activity and not related to vaspin-mediated changes in insulin sensitivity as determined by euglycemic-hyperinsulinemic clamp studies. Improved glucose metabolism could be mediated by increased insulin plasma concentrations 150 min after a glucose challenge in db/db mice, supporting the hypothesis that vaspin may inhibit insulin degradation by hK7 in the circulation. In conclusion, we demonstrate the inhibitory serpin nature and the first protease target of the adipose tissue-derived serpin vaspin, and our findings suggest hK7 inhibition by vaspin as an underlying physiological mechanism for its compensatory actions on obesity-induced insulin resistance. PMID:23370777

  12. Regulation of lung surfactant secretion by intracellular pH.

    PubMed

    Chander, A

    1989-12-01

    We investigated secretion of lung surfactant phosphatidylcholine (PC) using isolated perfused rat lung preparation after labeling the lung lipids in vitro with [methyl-3H]choline. The perfusion medium was Krebs-Ringer bicarbonate buffer (pH 7.4) containing 10 mM glucose and 3% fatty acid-poor bovine serum albumin. After ventilation of lungs with air containing 5% CO2 (control) for 1 h, 0.91% +/- 0.04 (mean +/- SE, n = 6) of total lung lipid radioactivity (greater than 95% in PC) was recovered in the cell-free lavage fluid. The secretion of PC was increased with terbutaline (50 microM), 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP, 100 microM), phorbol L2-myristate 13-acetate (30 ng/ml), and ATP (1 mM), in each case by approximately 150%. Secretion of PC was also increased by 160% if the lungs were ventilated with air containing 0% CO2. The low CO2-mediated PC secretion was time and concentration dependent. The dose-response curve for 0-10% CO2 was S-shaped. The low CO2-induced increase in PC secretion could be largely reversed with diffusible weak acids (25 mM, acetate or butyrate) in the perfusion medium. An increase (70%) in secretion was also induced with 10 mM NH4Cl, suggesting a role for intracellular alkalosis. These observations suggest that intracellular alkalosis stimulates lung surfactant secretion. Alkalosis-stimulated secretion of PC was additive with that with terbutaline (5 X 10(-7) to 5 X 10(-4) M) or 10(-4) M 8-BrcAMP, suggesting that alkalosis effect was not mediated through the beta-adrenergic pathway of surfactant secretion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2514603

  13. Rab proteins: The key regulators of intracellular vesicle transport

    SciTech Connect

    Bhuin, Tanmay; Roy, Jagat Kumar

    2014-10-15

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future.

  14. Regulation of intracellular heme trafficking revealed by subcellular reporters.

    PubMed

    Yuan, Xiaojing; Rietzschel, Nicole; Kwon, Hanna; Walter Nuno, Ana Beatriz; Hanna, David A; Phillips, John D; Raven, Emma L; Reddi, Amit R; Hamza, Iqbal

    2016-08-30

    Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous heme contribute to cellular labile heme and that extracellular heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile heme. Restoration of reporter activity by heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous heme trafficking. Expression of peroxidase reporters in Caenorhabditis elegans shows that environmental heme influences labile heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on heme trafficking in cellular and whole-animal models. PMID:27528661

  15. Role of heparin and non heparin binding serpins in coagulation and angiogenesis: A complex interplay.

    PubMed

    Bhakuni, Teena; Ali, Mohammad Farhan; Ahmad, Irshad; Bano, Shadabi; Ansari, Shoyab; Jairajpuri, Mohamad Aman

    2016-08-15

    Pro-coagulant, anti-coagulant and fibrinolytic pathways are responsible for maintaining hemostatic balance under physiological conditions. Any deviation from these pathways would result in hypercoagulability leading to life threatening diseases like myocardial infarction, stroke, portal vein thrombosis, deep vein thrombosis (DVT) and pulmonary embolism (PE). Angiogenesis is the process of sprouting of new blood vessels from pre-existing ones and plays a critical role in vascular repair, diabetic retinopathy, chronic inflammation and cancer progression. Serpins; a superfamily of protease inhibitors, play a key role in regulating both angiogenesis and coagulation. They are characterized by the presence of highly conserved secondary structure comprising of 3 β-sheets and 7-9 α-helices. Inhibitory role of serpins is modulated by binding to cofactors, specially heparin and heparan sulfate proteoglycans (HSPGs) present on cell surfaces and extracellular matrix. Heparin and HSPGs are the mainstay of anti-coagulant therapy and also have therapeutic potential as anti-angiogenic inhibitors. Many of the heparin binding serpins that regulate coagulation cascade are also potent inhibitors of angiogenesis. Understanding the molecular mechanism of the switch between their specific anti-coagulant and anti-angiogenic role during inflammation, stress and regular hemostasis is important. In this review, we have tried to integrate the role of different serpins, their interaction with cofactors and their interplay in regulating coagulation and angiogenesis. PMID:27372899

  16. Ectdomain shedding and regulated intracellular proteolysis in the central nervous system.

    PubMed

    Montes de Oca-B, Pavel

    2010-12-01

    The term Ectodomain Shedding (ES) refers to extracellular domain proteolytic release from cell membrane molecules. This proteolysis is mediated mainly by matrix metalloproteases (MMP) or disintegrin and metalloproteases (ADAM), although some other proteases may mediate it. Virtually, all functional categories of cell membrane molecules are subject of this kind of proteolysis, for this reason ES is involved in different cellular processes such as proliferation, apoptosis, migration, differentiation or pathologies such as inflammation, cancer and degeneration among others. ES releases membrane molecule's extracellular domain (or ectodomain) to the extracellular milieu where it can play different biological functions. ES of transmembrane molecules also generates membrane attached terminal fragments comprising transmembrane and intracellular domains that enable their additional processing by intracellular proteases known as Regulated Intracellular Proteolysis (RIP). This second proteolytic cleavage delivers molecule's intracellular domain (ICD) that carry out intracellular functions. RIP is mediated by the group of intracellular cleaving proteases (i-CLiPs) that include presenilin from the γ-secretase complex. In the CNS the best well known ES is that of the Amyloid Precursor Protein, although many other membrane molecules expressed by cells of the CNS are also subject to ES and RIP. In this review, these molecules are summarized, and some meaningful examples are highlighted and described. In addition, ES and RIP implications in the context of cell biology are discussed. Finally, some considerations that rise from the study of ES and RIP are formulated in view of the unexpected roles of intracellular fragments. PMID:20868353

  17. Sch9 regulates intracellular protein ubiquitination by controlling stress responses

    PubMed Central

    Qie, Beibei; Lyu, Zhou; Lyu, Lei; Liu, Jun; Gao, Xuejie; Liu, Yanyan; Duan, Wei; Zhang, Nianhui; Du, Linfang; Liu, Ke

    2015-01-01

    Protein ubiquitination and the subsequent degradation are important means by which aberrant proteins are removed from cells, a key requirement for long-term survival. In this study, we found that the overall level of ubiquitinated proteins dramatically decreased as yeast cell grew from log to stationary phase. Deletion of SCH9, a gene encoding a key protein kinase for longevity control, decreased the level of ubiquitinated proteins in log phase and this effect could be reversed by restoring Sch9 function. We demonstrate here that the decrease of ubiquitinated proteins in sch9Δ cells in log phase is not caused by changes in ubiquitin expression, proteasome activity, or autophagy, but by enhanced expression of stress response factors and a decreased level of oxidative stress. Our results revealed for the first time how Sch9 regulates the level of ubiquitinated proteins and provides new insight into how Sch9 controls longevity. PMID:26087116

  18. Sch9 regulates intracellular protein ubiquitination by controlling stress responses.

    PubMed

    Qie, Beibei; Lyu, Zhou; Lyu, Lei; Liu, Jun; Gao, Xuejie; Liu, Yanyan; Duan, Wei; Zhang, Nianhui; Du, Linfang; Liu, Ke

    2015-08-01

    Protein ubiquitination and the subsequent degradation are important means by which aberrant proteins are removed from cells, a key requirement for long-term survival. In this study, we found that the overall level of ubiquitinated proteins dramatically decreased as yeast cell grew from log to stationary phase. Deletion of SCH9, a gene encoding a key protein kinase for longevity control, decreased the level of ubiquitinated proteins in log phase and this effect could be reversed by restoring Sch9 function. We demonstrate here that the decrease of ubiquitinated proteins in sch9Δ cells in log phase is not caused by changes in ubiquitin expression, proteasome activity, or autophagy, but by enhanced expression of stress response factors and a decreased level of oxidative stress. Our results revealed for the first time how Sch9 regulates the level of ubiquitinated proteins and provides new insight into how Sch9 controls longevity. PMID:26087116

  19. Dissociation of SERPINE1 mRNA from the translational repressor proteins Ago2 and TIA-1 upon platelet activation.

    PubMed

    Corduan, Aurélie; Plé, Hélène; Laffont, Benoit; Wallon, Thérèse; Plante, Isabelle; Landry, Patricia; Provost, Patrick

    2015-05-01

    Platelets play an important role in haemostasis, as well as in thrombosis and coagulation processes. They harbour a wide variety of messenger RNAs (mRNAs), that can template de novo protein synthesis, and an abundant array of microRNAs, which are known to mediate mRNA translational repression through proteins of the Argonaute (Ago) family. The relationship between platelet microRNAs and proteins capable of mediating translational repression, however, remains unclear. Here, we report that half of platelet microRNAs is associated to mRNA-regulatory Ago2 protein complexes, in various proportions. Associated to these Ago2 complexes are platelet mRNAs known to support de novo protein synthesis. Reporter gene activity assays confirmed the capacity of the platelet microRNAs, found to be associated to Ago2 complexes, to regulate translation of these platelet mRNAs through their 3'UTR. Neither the microRNA repertoire nor the microRNA composition of Ago2 complexes of human platelets changed upon activation with thrombin. However, under conditions favoring de novo synthesis of Plasminogen Activator Inhibitor-1 (PAI-1) protein, we documented a rapid dissociation of the encoding platelet SERPINE1 mRNA from Ago2 protein complexes as well as from the translational repressor protein T-cell-restricted intracellular antigen-1 (TIA-1). These findings are consistent with a scenario by which lifting of the repressive effects of Ago2 and TIA-1 protein complexes, involving a rearrangement of proteinmRNA complexes rather than disassembly of Ago2microRNA complexes, would allow translation of SERPINE1 mRNA into PAI-1 in response to platelet activation. PMID:25673011

  20. Modification of bursting in a Helix neuron by drugs influencing intracellular regulation of calcium level.

    PubMed

    Salánki, J; Budai, D; Véró, M

    1983-01-01

    The effect of ruthenium red, caffein and EGTA (ethyleneglycol tetraacetic acid) influencing intracellular Ca2+ level as well as that of pH-lowering was investigated on identified RPal neuron of Helix pomatia characterized by bimodal pacemaker (bursting) activity. Drugs were applied both extracellularly and intracellularly. Intracellular injection was performed from micropipettes by pressure. It was found that intracellular injection of ruthenium red, caffein, EGTA and pH-lowering caused immediate short hyperpolarization and suspension of bursting. The effect of caffein and lowering of pH was biphasic, hyperpolarization was followed by an increase of spiking. Following EGTA injection the amplitudes of interburst hyperpolarizing waves decreased, and prolongation of spikes occurred. Extracellular application of ruthenium red caused slight depolarization, while caffein produced mainly effects that were similar to those of the intracellular injection. Adding EGTA into the bath resulted in cessation of bursting, and later on also spike generation was blocked. All these effects could be eliminated by washing. It is concluded that Ca-influx during spiking cannot be considered as a single factor in maintaining bursting activity, nevertheless, intracellular binding and liberation of Ca depending on the cell metabolism should also be taken into consideration as a possible mechanism of burst regulation. PMID:6198869

  1. Intracellular and extracellular ATP coordinately regulate the inverse correlation between osteoclast survival and bone resorption.

    PubMed

    Miyazaki, Tsuyoshi; Iwasawa, Mitsuyasu; Nakashima, Tomoki; Mori, Shuuichi; Shigemoto, Kazuhiro; Nakamura, Hiroaki; Katagiri, Hideki; Takayanagi, Hiroshi; Tanaka, Sakae

    2012-11-01

    Osteoclasts, highly differentiated bone-resorbing cells of hematopoietic origin, have two conflicting tendencies: a lower capacity to survive and a higher capacity to execute energy-consuming activities such as bone resorption. Here, we report that when compared with their precursors, mature mitochondria-rich osteoclasts have lower levels of intracellular ATP, which is associated with receptor activator of nuclear factor κ-B ligand (RANKL)-induced Bcl-x(L) down-regulation. Severe ATP depletion, caused by disrupting mitochondrial transcription factor A (Tfam) gene, leads to increased bone-resorbing activity despite accelerated apoptosis. Although AMP-activated protein kinase (AMPK) activation by ATP depletion is not involved in the regulation of osteoclast function, the release of ATP from intracellular stores negatively regulates bone-resorbing activity through an autocrine/paracrine feedback loop by altering cytoskeletal structures. Furthermore, osteoclasts derived from aged mice exhibit reduced mitochondrial DNA (mtDNA) and intracellular ATP levels with increased bone-resorbing activity, implicating the possible involvement of age-related mitochondrial dysfunction in osteoporosis. Thus, our study provides evidence for a mechanism underlying the control of cellular functions by reciprocal changes in intracellular and extracellular ATP, which regulate the negative correlation between osteoclast survival and bone resorption. PMID:22988253

  2. Intracellular pathways regulating ciliary beating of rat brain ependymal cells

    PubMed Central

    Nguyen, Thien; Chin, Wei-Chun; O’Brien, Jennifer A; Verdugo, Pedro; Berger, Albert J

    2001-01-01

    The mammalian brain ventricles are lined with ciliated ependymal cells. As yet little is known about the mechanisms by which neurotransmitters regulate cilia beat frequency (CBF). Application of 5-HT to ependymal cells in cultured rat brainstem slices caused CBF to increase. 5-HT had an EC50 of 30 μM and at 100 μM attained a near-maximal CBF increase of 52.7 ± 4.1 % (mean ± s.d.) (n= 8). Bathing slices in Ca2+-free solution markedly reduced the 5-HT-mediated increase in CBF. Fluorescence measurements revealed that 5-HT caused a marked transient elevation in cytosolic Ca2+ ([Ca2+]c) that then slowly decreased to a plateau level. Analysis showed that the [Ca2+]c transient was due to release of Ca2+ from inositol 1,4,5-trisphosphate (IP3)-sensitive stores; the plateau was probably due to extracellular Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels. Application of ATP caused a sustained decrease in CBF. ATP had an EC50 of about 50 μM and 100 μM ATP resulted in a maximal 57.5 ± 6.5 % (n= 12) decrease in CBF. The ATP-induced decrease in CBF was unaffected by lowering extracellular [Ca2+], and no changes in [Ca2+]c were observed. Exposure of ependymal cells to forskolin caused a decrease in CBF. Ciliated ependymal cells loaded with caged cAMP exhibited a 54.3 ± 7.5 % (n= 9) decrease in CBF following uncaging. These results suggest that ATP reduces CBF by a Ca2+-independent cAMP-mediated pathway. Application of 5-HT and adenosine-5′-O-3-thiotriphosphate (ATP-γ-S) to acutely isolated ciliated ependymal cells resulted in CBF responses similar to those of ependymal cells in cultured slices suggesting that these neurotransmitters act directly on these cells. The opposite response of ciliated ependymal cells to 5-HT and ATP provides a novel mechanism for their active involvement in central nervous system signalling. PMID:11179397

  3. Immune challenge induces N-terminal cleavage of the Drosophila serpin Necrotic

    PubMed Central

    Pelte, Nadège; Robertson, Andrew S.; Zou, Zhen; Belorgey, Didier; Dafforn, Timothy R.; Jiang, Haobo; Lomas, David; Reichhart, Jean-Marc; Gubb, David

    2007-01-01

    The Drosophila Necrotic protein is a serine proteinase inhibitor, which regulates the Toll-mediated innate immune response. Necrotic specifically inhibits an extracellular serine proteinase cascade leading to activation of the Toll ligand, Spätzle. Necrotic carries a polyglutamine extension amino-terminal to the core serpin structure. We show here that cleavage of this N-terminal extension occurs following immune challenge. This modification is blocked in PGRP-SAsemmelweiss mutants after Gram-positive bacterial challenge and in persephone mutants after fungal or Gram-positive bacterial challenge, indicating that activation of either of the Toll pathway upstream branches induces N-terminal cleavage of the serpin. The absolute requirement of persephone gene product for this cleavage indicates that Gram-positive bacteria activate a redundant set of proteinases upstream of Toll. Both full-length Necrotic and the core serpin are active inhibitors of a range of serine proteinases: the highest affinity being for cathepsin G and elastases. We found a 13-fold increase in the specificity of the core serpin over that of full-length Necrotic for one of the tested proteinases (porcine pancreatic elastase). This finding indicates that cleavage of the Necrotic amino-terminal extension might modulate Toll activation following the initial immune response. PMID:16360948

  4. A SerpinB1 regulatory mechanism is essential for restricting NETosis

    PubMed Central

    Farley, Kalamo; Stolley, J Michael; Zhao, Picheng; Cooley, Jessica; Remold-O’Donnell, Eileen

    2014-01-01

    NETosis (NET generation), a programmed death pathway initiated in mature neutrophils by pathogens and inflammatory mediators, can be a protective process that sequesters microbes and prevents spread of infection, but can also be a pathological process that causes inflammation and serious tissue injury. Little is known about the regulatory mechanism. Previously we demonstrated that serpinb1-deficient mice are highly susceptible to pulmonary bacterial and viral infections due to inflammation and tissue injury associated with increased neutrophilic death. Here we used in vitro and in vivo approaches to investigate whether SerpinB1 regulates NETosis. We found that serpinb1-deficient bone marrow and lung neutrophils are hyper-susceptible to NETosis induced by multiple mediators in both NADPH-dependent and independent manner, indicating a deeply rooted regulatory role in NETosis. This role is further supported by increased nuclear expansion (representing chromatin decondensation) of PMA-treated serpinb-1-deficient neutrophils compared to wild-type, by migration of SerpinB1 from the cytoplasm to the nucleus of human neutrophils coincident with, or before, early conversion of lobulated (segmented) nuclei to delobulated (spherical) morphology, and by finding that exogenous rSerpinB1 abrogates NET production. NETosis of serpinb1-deficient neutrophils is also increased in vivo during Pseudomonas aeruginosa lung infection. The findings identify a previously unrecognized regulatory mechanism involving SerpinB1 that restricts the production of NETs. PMID:23002442

  5. Stress-induced inhibition of nonsense mediated RNA decay regulates intracellular cystine transport and intracellular glutathione through regulation of the cystine/glutamate exchanger SLC7A11

    PubMed Central

    Martin, Leenus; Gardner, Lawrence B.

    2014-01-01

    SLC7A11 encodes a subunit of the xCT cystine/glutamate amino acid transport system and plays a critical role in the generation of glutathione and the protection of cells from oxidative stress. Expression of SLC7A11 promotes tumorigenesis and chemotherapy resistance, but while SLC7A11 has been previously noted to be upregulated in hypoxic cells its regulation has not been fully delineated. We have recently shown that nonsense mediated RNA decay (NMD) is inhibited by cellular stresses generated by the tumor microenvironment, including hypoxia, and augments tumorigenesis. Here we demonstrate that the inhibition of NMD by various cellular stresses leads to the stabilization and upregulation of SLC7A11 mRNA and protein. The inhibition of NMD and upregulation of SLC7A11 augments intracellular cystine transport, and increases intracellular levels of cysteine and glutathione. Accordinglyy, the inhibition of NMD protects cells against oxidative stress via SLC7A11 upregulation. Together our studies identify a mechanism for the dynamic regulation of SLC7A11, through the stress-inhibited regulation of NMD, and add to the growing evidence that the inhibition of NMD is an adaptive response. PMID:25399695

  6. Prevention of Serpin Misfolding by RNA Aptamers.

    PubMed

    Zhou, Xiaohua; Declerck, Paul J

    2016-06-23

    Owing to their structural flexibility, most serpins inhibit the cognate proteases in a fast and specific manner and also are susceptible to pathogenic misfolding. In this issue of Cell Chemical Biology, Madsen et al. (2016) report on the selection and characterization of an RNA aptamer that stabilizes α1-antichymotrypsin L55P mutant without interfering with the protease inhibitory activity. PMID:27341430

  7. Miro1 Regulates Activity-Driven Positioning of Mitochondria within Astrocytic Processes Apposed to Synapses to Regulate Intracellular Calcium Signaling.

    PubMed

    Stephen, Terri-Leigh; Higgs, Nathalie F; Sheehan, David F; Al Awabdh, Sana; López-Doménech, Guillermo; Arancibia-Carcamo, I Lorena; Kittler, Josef T

    2015-12-01

    It is fast emerging that maintaining mitochondrial function is important for regulating astrocyte function, although the specific mechanisms that govern astrocyte mitochondrial trafficking and positioning remain poorly understood. The mitochondrial Rho-GTPase 1 protein (Miro1) regulates mitochondrial trafficking and detachment from the microtubule transport network to control activity-dependent mitochondrial positioning in neurons. However, whether Miro proteins are important for regulating signaling-dependent mitochondrial dynamics in astrocytic processes remains unclear. Using live-cell confocal microscopy of rat organotypic hippocampal slices, we find that enhancing neuronal activity induces transient mitochondrial remodeling in astrocytes, with a concomitant, transient reduction in mitochondrial trafficking, mediated by elevations in intracellular Ca(2+). Stimulating neuronal activity also induced mitochondrial confinement within astrocytic processes in close proximity to synapses. Furthermore, we show that the Ca(2+)-sensing EF-hand domains of Miro1 are important for regulating mitochondrial trafficking in astrocytes and required for activity-driven mitochondrial confinement near synapses. Additionally, activity-dependent mitochondrial positioning by Miro1 reciprocally regulates the levels of intracellular Ca(2+) in astrocytic processes. Thus, the regulation of intracellular Ca(2+) signaling, dependent on Miro1-mediated mitochondrial positioning, could have important consequences for astrocyte Ca(2+) wave propagation, gliotransmission, and ultimately neuronal function. PMID:26631479

  8. Redox Regulation of Intracellular Zinc: Molecular Signaling in the Life and Death of Neurons

    PubMed Central

    Aizenman, Elias

    2011-01-01

    Abstract Zn2+ has emerged as a major regulator of neuronal physiology, as well as an important signaling agent in neural injury. The intracellular concentration of this metal is tightly regulated through the actions of Zn2+ transporters and the thiol-rich metal binding protein metallothionein, closely linking the redox status of the cell to cellular availability of Zn2+. Accordingly, oxidative and nitrosative stress during ischemic injury leads to an accumulation of neuronal free Zn2+ and the activation of several downstream cell death processes. While this Zn2+ rise is an established signaling event in neuronal cell death, recent evidence suggests that a transient, sublethal accumulation of free Zn2+ can also play a critical role in neuroprotective pathways activated during ischemic preconditioning. Thus, redox-sensitive proteins, like metallothioneins, may play a critical role in determining neuronal cell fate by regulating the localization and concentration of intracellular free Zn2+. Antioxid. Redox Signal. 15, 2249–2263. PMID:20849376

  9. The Spn4 gene of Drosophila encodes a potent furin-directed secretory pathway serpin

    PubMed Central

    Richer, Martin J.; Keays, Clairessa A.; Waterhouse, Jennifer; Minhas, Jessey; Hashimoto, Carl; Jean, François

    2004-01-01

    Proprotein convertases (PCs) are an important class of host-cell serine endoproteases implicated in many physiological and pathological processes. Owing to their expanding roles in the proteolytic events required for generating infectious microbial pathogens and for tumor growth and invasiveness, there is increasing interest in identifying endogenous PC inhibitors. Here we report the identification of Spn4A, a previously uncharacterized secretory pathway serine protease inhibitor (serpin) from Drosophila melanogaster that contains a consensus furin cleavage site, -ArgP4-Arg-Lys-ArgP1↓-, in its reactive site loop (RSL). Our biochemical and kinetics analysis revealed that recombinant Spn4A inhibits human furin (Ki, 13 pM; kass, 3.2 × 107 M–1·s–1) and Drosophila PC2 (Ki, 3.5 nM; kass, 9.2 × 104 M–1·s–1) by a slow-binding mechanism characteristic of serpin molecules and forms a kinetically trapped SDS-stable complex with each enzyme. For both PCs, the stoichiometry of inhibition by Spn4A is nearly 1, which is characteristic of known physiological serpin–protease interactions. Mass analysis of furin–Spn4A reaction products identified the actual reactive site center of Spn4A to be -ArgP4-Arg-Lys-ArgP1↓-. Moreover, we demonstrate that Spn4A's highly effective PC inhibition properties are critically dependent on the unusual length of its RSL, which is composed of 18 aa instead of the typical 17-residue RSL found in most other inhibitory serpins. The identification of Spn4A, the most potent and effective natural serpin of PCs identified to date, suggests that Spn4A could be a prototype of endogenous serpins involved in the precise regulation of PC-dependent proteolytic cleavage events in the secretory pathway of eukaryotic cells. PMID:15247425

  10. Intracellular pH regulation in isolated hepatopancreas cells from the Roman snail (Helix pomatia).

    PubMed

    Manzl, Claudia; Krumschnabel, Gerhard; Schwarzbaum, Pablo J; Chabicovsky, Monika; Dallinger, Reinhard

    2004-01-01

    The mechanisms of intracellular pH (pHi) regulation were studied in isolated hepatopancreas cells from the Roman snail, Helix pomatia. The relationship between intracellular and extracellular pH indicated that pHi is actively regulated in these cells. At least three pHi-regulatory ion transporters were found to be present in these cells and to be responsible for the maintenance of pHi: an amiloride-sensitive Na+/H+ exchanger, a 4-acetamido-4'-isothiocyanostilbene-2,2'disulfonic acid (SITS)-sensitive, presumably Na(+)-dependent, Cl-/HCO3-exchanger, and a bafilomycin-sensitive H(+)-pump. Inhibition of one of these transporters alone did not affect steady state pHi, whereas incubation with amiloride and SITS in combination resulted in a significant intracellular acidification. Following the induction of intracellular acidosis by addition of the weak acid Na+propionate, the Na+/H+ exchanger was immediately activated leading to a rapid recovery of pHi towards the baseline level. Both the SITS-sensitive mechanism and the H(+)-pump responded more slowly, but were of similar importance for pHi recovery. Measurement of pHi recovery from acidification in the three discernible types of hepatopancreas cells with a video fluorescence image system revealed slightly differing response patterns, the physiological significance of which remains to be determined. PMID:14695690

  11. Regulation of intracellular pH in cnidarians: response to acidosis in Anemonia viridis.

    PubMed

    Laurent, Julien; Venn, Alexander; Tambutté, Éric; Ganot, Philippe; Allemand, Denis; Tambutté, Sylvie

    2014-02-01

    The regulation of intracellular pH (pHi) is a fundamental aspect of cell physiology that has received little attention in studies of the phylum Cnidaria, which includes ecologically important sea anemones and reef-building corals. Like all organisms, cnidarians must maintain pH homeostasis to counterbalance reductions in pHi, which can arise because of changes in either intrinsic or extrinsic parameters. Corals and sea anemones face natural daily changes in internal fluids, where the extracellular pH can range from 8.9 during the day to 7.4 at night. Furthermore, cnidarians are likely to experience future CO₂-driven declines in seawater pH, a process known as ocean acidification. Here, we carried out the first mechanistic investigation to determine how cnidarian pHi regulation responds to decreases in extracellular and intracellular pH. Using the anemone Anemonia viridis, we employed confocal live cell imaging and a pH-sensitive dye to track the dynamics of pHi after intracellular acidosis induced by acute exposure to decreases in seawater pH and NH₄Cl prepulses. The investigation was conducted on cells that contained intracellular symbiotic algae (Symbiodinium sp.) and on symbiont-free endoderm cells. Experiments using inhibitors and Na⁺-free seawater indicate a potential role of Na⁺/H⁺ plasma membrane exchangers (NHEs) in mediating pHi recovery following intracellular acidosis in both cell types. We also measured the buffering capacity of cells, and obtained values between 20.8 and 43.8 mM per pH unit, which are comparable to those in other invertebrates. Our findings provide the first steps towards a better understanding of acid-base regulation in these basal metazoans, for which information on cell physiology is extremely limited. PMID:24256552

  12. Intracellular Na(+) inhibits volume-regulated anion channel in rat cortical astrocytes.

    PubMed

    Minieri, Laura; Pivonkova, Helena; Harantova, Lenka; Anderova, Miroslava; Ferroni, Stefano

    2015-02-01

    Accumulating evidence indicates that increased intracellular Na(+) concentration ([Na(+) ]i ) in astroglial cells is associated with the development of brain edema under ischemic conditions, but the underlying mechanisms are still elusive. Here, we report that in primary cultured rat cortical astrocytes, elevations of [Na(+) ]i reflecting those achieved during ischemia cause a marked decrease in hypotonicity-evoked current mediated by volume-regulated anion channel (VRAC). Pharmacological manipulations revealed that VRAC inhibition was not due to the reverse mode of the plasma membrane sodium/calcium exchanger. The negative modulation of VRAC was also observed in an astrocytic cell line lacking the predominant astrocyte water channel aquaporin 4, indicating that [Na(+) ]i effect was not mediated by the regulation of aquaporin 4 activity. The inward rectifier Cl(-) current, which is also expressed by cultured astrocytes, was not affected by [Na(+) ]i increase. VRAC depression by high [Na(+) ]i was confirmed in adult astrocytes, suggesting that it was not developmentally regulated. Altogether, these results provide the first evidence that intracellular Na(+) dynamics can modulate astrocytic membrane conductance that controls functional processes linked to cell volume regulation and add further support to the concept that limiting astrocyte intracellular Na(+) accumulation might be a favorable strategy to counteract the development of brain edema. PMID:25279950

  13. TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis

    PubMed Central

    Shambharkar, Prashant B.; Bittinger, Mark; Latario, Brian; Xiong, ZhaoHui; Bandyopadhyay, Somnath; Davis, Vanessa; Lin, Victor; Yang, Yi; Valdez, Reginald; Labow, Mark A.

    2015-01-01

    Intracellular calcium signaling is critical for initiating and sustaining diverse cellular functions including transcription, synaptic signaling, muscle contraction, apoptosis and fertilization. Trans-membrane 203 (TMEM203) was identified here in cDNA overexpression screens for proteins capable of modulating intracellular calcium levels using activation of a calcium/calcineurin regulated transcription factor as an indicator. Overexpression of TMEM203 resulted in a reduction of Endoplasmic Reticulum (ER) calcium stores and elevation in basal cytoplasmic calcium levels. TMEM203 protein was localized to the ER and found associated with a number of ER proteins which regulate ER calcium entry and efflux. Mouse Embryonic Fibroblasts (MEFs) derived from Tmem203 deficient mice had reduced ER calcium stores and altered calcium homeostasis. Tmem203 deficient mice were viable though male knockout mice were infertile and exhibited a severe block in spermiogenesis and spermiation. Expression profiling studies showed significant alternations in expression of calcium channels and pumps in testes and concurrently Tmem203 deficient spermatocytes demonstrated significantly altered calcium handling. Thus Tmem203 is an evolutionarily conserved regulator of cellular calcium homeostasis, is required for spermatogenesis and provides a causal link between intracellular calcium regulation and spermiogenesis. PMID:25996873

  14. Intracellular oxygen determined by respiration regulates localization of Ras and prenylated proteins

    PubMed Central

    Kim, A; Davis, R; Higuchi, M

    2015-01-01

    Reduction of mitochondrial DNA (mtDNA) content induces the reduction of oxidative phosphorylation and dependence on fermentative glycolysis, that is, the Warburg effect. In aggressive prostate cancer (PCa), the reduction of mtDNA reduces oxygen consumption, increases intracellular oxygen concentration, and induces constitutive activation of Ras. Many essential proteins for cell death, growth, differentiation, and development, such as Ras, require prenylation for subcellular localization and activation. Prenylation of a protein is defined as the attachment of isoprenoids to a cysteine residue at or near the C-terminus. 3-Hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) produces isoprenoids, and is posttranslationally regulated by oxygen. We investigated a critical role of intracellular oxygen in membrane localization of prenylated proteins. Localization of prenylated proteins (H-Ras, prelamin A/C, and Rab5a) was observed in poorly differentiated PCa (PC-3) and well-differentiated PCa (LNCaP) cells. PC-3 cells exhibited high intracellular oxygen concentration, and H-Ras, prelamin A/C, and Rab5a were localized to various membranes (Golgi and plasma membrane, nuclear membrane, and early endosomes, respectively). Remarkably, exogenous hypoxia (0.2% O2) in PC-3 cells induced intracellular hypoxia and changed the localization of the prenylated proteins. H-Ras and Rab5a were translocated to cytosol, and prelamin A/C was in the nucleus forming an abnormal nuclear envelope. The localization was reversed by mevalonate indicating the involvement of mevalonate pathway. In contrast, in LNCaP cells, exhibiting low intracellular oxygen concentration, H-Ras and Rab5a were localized in the cytosol, and prelamin A/C was inside the nucleus forming an inadequate nuclear envelope. Exogenous hyperoxia (40% O2) increased the intracellular oxygen concentration and induced Ras translocation from cytosol to the membrane. Prelamin A/C was translocated to the nuclear membrane and formed a

  15. Identification and characterization of a serpin from Emeria acervulina

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Serpins are serine protease inhibitors that are widely distributed in metazoans but have not been previously characterized in Eimeria. A serpin from Eimeria acervulina was cloned, expressed and partially characterized. Random screening of an E. acervulina sporozoite cDNA library identified a singl...

  16. Chemoattractant-Regulated Mobilization of a Novel Intracellular Compartment in Human Neutrophils

    NASA Astrophysics Data System (ADS)

    Borregaard, N.; Miller, L. J.; Springer, T. A.

    1987-09-01

    A novel mobilizable intracellular compartment was identified in human neutrophils by latent alkaline phosphatase activity. This compartment is mobilized to the plasma membrane much more readily than any identified granule subset and has kinetics of up-regulation in the membrane similar to those reported for a variety of receptor proteins. Triton X-100 permeabilization of both intact human neutrophils and subcellular fractions obtained by density-gradient centrifugation revelaed that 70 percent of the alkaline phosphatase is located in an intracellular compartment distinct from primary, secondary, and gelatinase granules and from the plasma membrane. This compartment fully translocates to the plasma membrane after stimulation with nanomolar concentrations of the chemotactic peptide N-formylmethionylleucylphenylalanine.

  17. Testosterone and interleukin-1β increase cardiac remodeling during coxsackievirus B3 myocarditis via serpin A 3n

    PubMed Central

    Coronado, Michael J.; Brandt, Jessica E.; Kim, Eunyong; Bucek, Adriana; Bedja, Djahida; Abston, Eric D.; Shin, Jaewook; Gabrielson, Kathleen L.; Mitzner, Wayne

    2012-01-01

    Myocarditis and dilated cardiomyopathy (DCM) are often caused by viral infections and occur more frequently in men than in women, but the reasons for the sex difference remain unclear. The aim of this study was to assess whether gene changes in the heart during coxsackievirus B3 (CVB3) myocarditis in male and female BALB/c mice predicted worse DCM in males. Although myocarditis (P = 4.2 × 10−5) and cardiac dilation (P = 0.008) were worse in males, there was no difference in viral replication in the heart. Fibrotic remodeling genes, such as tissue inhibitor of metalloproteinase (TIMP)-1 and serpin A 3n, were upregulated in males during myocarditis rather than during DCM. Using gonadectomy and testosterone replacement, we showed that testosterone increased cardiac TIMP-1 (P = 0.04), serpin A 3n (P = 0.007), and matrix metalloproteinase (MMP)-8 (P = 0.04) during myocarditis. Testosterone increased IL-1β levels in the heart (P = 0.02), a cytokine known to regulate cardiovascular remodeling, and IL-1β in turn increased cardiac serpin A 3n mRNA (P = 0.005). We found that 39 of 118 (33%) genes identified in acute DCM patients were significantly altered in the heart during CVB3 myocarditis in mice, including serpin A 3n (3.3-fold change, P = 0.0001). Recombinant serpin A 3n treatment induced cardiac fibrosis during CVB3 myocarditis (P = 0.0008) while decreasing MMP-3 (P = 0.04) and MMP-9 (P = 0.03) levels in the heart. Thus, serpin A 3n was identified as a gene associated with fibrotic cardiac remodeling and progression to DCM in male myocarditis patients and mice. PMID:22328081

  18. Galectin-3 regulates intracellular trafficking of epidermal growth factor receptor through Alix and promotes keratinocyte migration

    PubMed Central

    Liu, Wei; Hsu, Daniel K.; Chen, Huan-Yuan; Yang, Ri-Yao; Carraway, Kermit L.; Isseroff, Roslyn R.; Liu, Fu-Tong

    2012-01-01

    The epidermal growth factor receptor (EGFR)-mediated signaling pathways are important in a variety of cellular processes, including cell migration and wound re-epithelialization. Intracellular trafficking of EGFR is critical for maintaining EGFR surface expression. Galectin-3, a member of an animal lectin family, has been implicated in a number of physiological and pathological processes. Through studies of galectin-3-deficient mice and cells isolated from these mice, we demonstrated that absence of galectin-3 impairs keratinocyte migration and skin wound re-epithelialization. We have linked this pro-migratory function to a crucial role of cytosolic galectin-3 in controlling intracellular trafficking and cell surface expression of EGFR after EGF stimulation. Without galectin-3, the surface levels of EGFR are dramatically reduced and the receptor accumulates diffusely in the cytoplasm. This is associated with reduced rates of both endocytosis and recycling of the receptor. We have provided evidence that this novel function of galectin-3 may be mediated through interaction with its binding partner Alix, which is a protein component of the endosomal sorting complex required for transport (ESCRT) machinery. Our results suggest that galectin-3 is potentially a critical regulator of a number of important cellular responses through its intracellular control of trafficking of cell surface receptors. PMID:22785133

  19. Phosphate-Regulated Induction of Intracellular Ribonucleases in Cultured Tomato (Lycopersicon esculentum) Cells 1

    PubMed Central

    Löffler, Andreas; Abel, Steffen; Jost, Wolfgang; Beintema, Jaap J.; Glund, Konrad

    1992-01-01

    Four intracellular RNases were found to be induced in cultured tomato (Lycopersicon esculentum) cells upon phosphate starvation. Localization studies revealed three (RNases LV 1-3) in the vacuoles and one (RNase LX) outside these organelles. All of these RNases were purified to homogeneity and were shown to be type I RNases on the basis of type of splitting, substrate, and base specificity at the cleavage site, molecular weight, isoelectric point, and pH optimum. Moreover, RNase LV 3 was shown by fingerprinting of tryptic digests on reversed-phase high-performance liquid chromatography and sequencing the N terminus and two tryptic peptides to be structurally very similar to a recently characterized extracellular RNase LE which is also phosphate regulated (Nürnberger et al. [1990] Plant Physiol 92: 970-976; Jost et al. [1991] Eur J Biochem 198: 1-6). Expression of the four intracellular RNases is induced by depleting the cells of phosphate and repressed by adding phosphate. Our studies indicate that higher plants, in addition to secreting enzymes for scavanging phosphate under starvation conditions, also induce intracellularly emergency rescue systems. ImagesFigure 1Figure 2Figure 3Figure 4 PMID:16668816

  20. Apoplastic and intracellular plant sugars regulate developmental transitions in witches’ broom disease of cacao

    PubMed Central

    Barau, Joan; Grandis, Adriana; Carvalho, Vinicius Miessler de Andrade; Teixeira, Gleidson Silva; Zaparoli, Gustavo Henrique Alcalá; do Rio, Maria Carolina Scatolin; Rincones, Johana; Buckeridge, Marcos Silveira; Pereira, Gonçalo Amarante Guimarães

    2015-01-01

    Witches’ broom disease (WBD) of cacao differs from other typical hemibiotrophic plant diseases by its unusually long biotrophic phase. Plant carbon sources have been proposed to regulate WBD developmental transitions; however, nothing is known about their availability at the plant–fungus interface, the apoplastic fluid of cacao. Data are provided supporting a role for the dynamics of soluble carbon in the apoplastic fluid in prompting the end of the biotrophic phase of infection. Carbon depletion and the consequent fungal sensing of starvation were identified as key signalling factors at the apoplast. MpNEP2, a fungal effector of host necrosis, was found to be up-regulated in an autophagic-like response to carbon starvation in vitro. In addition, the in vivo artificial manipulation of carbon availability in the apoplastic fluid considerably modulated both its expression and plant necrosis rate. Strikingly, infected cacao tissues accumulated intracellular hexoses, and showed stunted photosynthesis and the up-regulation of senescence markers immediately prior to the transition to the necrotrophic phase. These opposite findings of carbon depletion and accumulation in different host cell compartments are discussed within the frame of WBD development. A model is suggested to explain phase transition as a synergic outcome of fungal-related factors released upon sensing of extracellular carbon starvation, and an early senescence of infected tissues probably triggered by intracellular sugar accumulation. PMID:25540440

  1. 3-Hydroxybutyrate dehydrogenase-2 and ferritin-H synergistically regulate intracellular iron.

    PubMed

    Liu, Zhuoming; Velpula, Kiran K; Devireddy, Lax

    2014-05-01

    Siderophores are best known as small iron-binding molecules that facilitate iron uptake in bacteria and fungi. In our previous study, we demonstrated that eukaryotes also produce siderophore-like molecules via a remarkably conserved biosynthetic pathway. A member of the short-chain dehydrogenase family of reductases, 3-hydroxybutyrate dehydrogenase-2, catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore. Physiologically, depletion of the mammalian siderophore by inhibiting expression of the 3-hydroxybutyrate dehydrogenase-2 gene (Bdh2) results in abnormal accumulation of intracellular iron, increased oxidative stress, and mitochondrial iron deficiency. Thus, the mammalian siderophore is an important regulator of cellular iron homeostasis. The cellular iron storage protein ferritin also regulates iron metabolism and protects cells from oxidative stress. Depletion of ferritin results in intracellular iron accumulation, predisposes to oxidative stress, and confers a growth advantage to cells. We therefore hypothesize that the siderophore and ferritin coregulate cellular iron metabolism/homeostasis in eukaryotes. We tested this prediction by depleting both the siderophore and ferritin. This resulted in a marked accumulation of cellular iron, and caused increased sensitivity to oxidants. Interestingly, cells lacking both the siderophore and ferritin proliferated at a higher rate than cells lacking either of these components alone. Taken together, our findings suggest that the siderophore and ferritin synergistically regulate cellular iron levels. PMID:24673886

  2. Intracellular Cl- as a signaling ion that potently regulates Na+/HCO3- transporters.

    PubMed

    Shcheynikov, Nikolay; Son, Aran; Hong, Jeong Hee; Yamazaki, Osamu; Ohana, Ehud; Kurtz, Ira; Shin, Dong Min; Muallem, Shmuel

    2015-01-20

    Cl(-) is a major anion in mammalian cells involved in transport processes that determines the intracellular activity of many ions and plasma membrane potential. Surprisingly, a role of intracellular Cl(-) (Cl(-) in) as a signaling ion has not been previously evaluated. Here we report that Cl(-) in functions as a regulator of cellular Na(+) and HCO3 (-) concentrations and transepithelial transport through modulating the activity of several electrogenic Na(+)-HCO3 (-) transporters. We describe the molecular mechanism(s) of this regulation by physiological Cl(-) in concentrations highlighting the role of GXXXP motifs in Cl(-) sensing. Regulation of the ubiquitous Na(+)-HCO3(-) co-transport (NBC)e1-B is mediated by two GXXXP-containing sites; regulation of NBCe2-C is dependent on a single GXXXP motif; and regulation of NBCe1-A depends on a cryptic GXXXP motif. In the basal state NBCe1-B is inhibited by high Cl(-) in interacting at a low affinity GXXXP-containing site. IP3 receptor binding protein released with IP3 (IRBIT) activation of NBCe1-B unmasks a second high affinity Cl(-) in interacting GXXXP-dependent site. By contrast, NBCe2-C, which does not interact with IRBIT, has a single high affinity N-terminal GXXP-containing Cl(-) in interacting site. NBCe1-A is unaffected by Cl(-) in between 5 and 140 mM. However, deletion of NBCe1-A residues 29-41 unmasks a cryptic GXXXP-containing site homologous with the NBCe1-B low affinity site that is involved in inhibition of NBCe1-A by Cl(-) in. These findings reveal a cellular Cl(-) in sensing mechanism that plays an important role in the regulation of Na(+) and HCO3 (-) transport, with critical implications for the role of Cl(-) in cellular ion homeostasis and epithelial fluid and electrolyte secretion. PMID:25561556

  3. C1 inhibitor serpin domain structure reveals the likely mechanism of heparin potentiation and conformational disease.

    PubMed

    Beinrohr, László; Harmat, Veronika; Dobó, József; Lörincz, Zsolt; Gál, Péter; Závodszky, Péter

    2007-07-20

    C1 inhibitor, a member of the serpin family, is a major down-regulator of inflammatory processes in blood. Genetic deficiency of C1 inhibitor results in hereditary angioedema, a dominantly inheritable, potentially lethal disease. Here we report the first crystal structure of the serpin domain of human C1 inhibitor, representing a previously unreported latent form, which explains functional consequences of several naturally occurring mutations, two of which are discussed in detail. The presented structure displays a novel conformation with a seven-stranded beta-sheet A. The unique conformation of the C-terminal six residues suggests its potential role as a barrier in the active-latent transition. On the basis of surface charge pattern, heparin affinity measurements, and docking of a heparin disaccharide, a heparin binding site is proposed in the contact area of the serpin-proteinase encounter complex. We show how polyanions change the activity of the C1 inhibitor by a novel "sandwich" mechanism, explaining earlier reaction kinetic and mutagenesis studies. These results may help to improve therapeutic C1 inhibitor preparations used in the treatment of hereditary angioedema, organ transplant rejection, and heart attack. PMID:17488724

  4. Regulation of Intracellular Structural Tension by Talin in the Axon Growth and Regeneration.

    PubMed

    Dingyu, Wang; Fanjie, Meng; Zhengzheng, Ding; Baosheng, Huang; Chao, Yang; Yi, Pan; Huiwen, Wu; Jun, Guo; Gang, Hu

    2016-09-01

    Intracellular tension is the most important characteristic of neuron polarization as well as the growth and regeneration of axons, which can be generated by motor proteins and conducted along the cytoskeleton. To better understand this process, we created Förster resonance energy transfer (FRET)-based tension probes that can be incorporated into microfilaments to provide a real-time measurement of forces in neuron cytoskeletons. We found that our probe could be used to assess the structural tension of neuron polarity. Nerve growth factor (NGF) upregulated structural forces, whereas the glial-scar inhibitors chondroitin sulfate proteoglycan (CSPG) and aggrecan weakened such forces. Notably, the tension across axons was distributed uniformly and remarkably stronger than that in the cell body in NGF-stimulated neurons. The mechanosensors talin/vinculin could antagonize the effect of glial-scar inhibitors via structural forces. However, E-cadherin was closely associated with glial-scar inhibitor-induced downregulation of structural forces. Talin/vinculin was involved in the negative regulation of E-cadherin transcription through the nuclear factor-kappa B pathway. Collectively, this study clarified the mechanism underlying intracellular tension in the growth and regeneration of axons which, conversely, can be regulated by talin and E-cadherin. PMID:26298665

  5. Hyperoside regulates the level of thymic stromal lymphopoietin through intracellular calcium signalling.

    PubMed

    Han, Na-Ra; Go, Ji-Hyun; Kim, Hyung-Min; Jeong, Hyun-Ja

    2014-07-01

    Hyperoside (HYP) is the principle active component of Crataegus pinnatifida. Thymic stromal lymphopoietin (TSLP) plays a vital role in the pathogenesis of allergic reactions. Here, we investigated how HYP regulates the levels of TSLP in a human mast cell line, HMC-1 cells. We analyzed the levels of TSLP by treatment with HYP in phorbol myristate acetate plus calcium ionophore A23187-stimulated HMC-1 cells with ELISA and a polymerase chain reaction analysis. We also analyzed the pathway that HYP regulates TSLP by measuring the level of fluorescent intracellular calcium and using a Western blot analysis. HYP decreased the level of intracellular calcium in stimulated HMC-1 cells. It also significantly decreased the production and mRNA expression of TSLP in stimulated HMC-1 cells. It significantly decreased the levels of receptor-interacting protein 2 and active caspase-1 in stimulated HMC-1 cells. HYP significantly decreased the translocation of NF-κB into the nucleus and degradation of IκBα in the cytoplasm in stimulated HMC-1 cells. Furthermore, it significantly decreased the production and mRNA expression of interleukin-1β and interleukin-6 in stimulated HMC-1 cells. Taken together, our findings establish HYP as a potential agent for the treatment of allergic reactions. PMID:24338918

  6. Regulation of intracellular Zn homeostasis in two intestinal epithelial cell models at various maturation time points.

    PubMed

    Gefeller, Eva-Maria; Bondzio, Angelika; Aschenbach, Jörg R; Martens, Holger; Einspanier, Ralf; Scharfen, Franziska; Zentek, Jürgen; Pieper, Robert; Lodemann, Ulrike

    2015-07-01

    After weaning, piglets are often fed diets supplemented with high concentrations of zinc (Zn) to decrease post-weaning diarrhea. The aim of this study was to elucidate the regulation of Zn homeostasis within intestinal epithelial cells during excessive Zn exposure. High Zn concentrations elevated the intracellular Zn level in IPEC-J2 and Caco-2 cells which was influenced by differentiation status and time of exposure. With increasing Zn concentrations, mRNA and protein levels of metallothionein (MT) and zinc transporter 1 (ZnT1) were upregulated, whereas zinc transporter 4 (ZIP4) expression was downregulated. Metal-regulatory transcription factor-1 (MTF1) mRNA expression was upregulated at high Zn concentrations in IPEC-J2 cells, which corresponded to higher intracellular Zn concentrations. Based on these results, we suggest that intestinal epithelial cells adapt the expression of these genes to the amount of extracellular Zn available in order to maintain Zn homeostasis. Cell line-dependent differences in the regulation of Zn homeostasis were detected. PMID:25757458

  7. Cellular folding pathway of a metastable serpin.

    PubMed

    Chandrasekhar, Kshama; Ke, Haiping; Wang, Ning; Goodwin, Theresa; Gierasch, Lila M; Gershenson, Anne; Hebert, Daniel N

    2016-06-01

    Although proteins generally fold to their thermodynamically most stable state, some metastable proteins populate higher free energy states. Conformational changes from metastable higher free energy states to lower free energy states with greater stability can then generate the work required to perform physiologically important functions. However, how metastable proteins fold to these higher free energy states in the cell and avoid more stable but inactive conformations is poorly understood. The serpin family of metastable protease inhibitors uses large conformational changes that are downhill in free energy to inhibit target proteases by pulling apart the protease active site. The serpin antithrombin III (ATIII) targets thrombin and other proteases involved in blood coagulation, and ATIII misfolding can thus lead to thrombosis and other diseases. ATIII has three disulfide bonds, two near the N terminus and one near the C terminus. Our studies of ATIII in-cell folding reveal a surprising, biased order of disulfide bond formation, with early formation of the C-terminal disulfide, before formation of the N-terminal disulfides, critical for folding to the active, metastable state. Early folding of the predominantly β-sheet ATIII domain in this two-domain protein constrains the reactive center loop (RCL), which contains the protease-binding site, ensuring that the RCL remains accessible. N-linked glycans and carbohydrate-binding molecular chaperones contribute to the efficient folding and secretion of functional ATIII. The inability of a number of disease-associated ATIII variants to navigate the folding reaction helps to explain their disease phenotypes. PMID:27222580

  8. Intracellular osteopontin stabilizes TRAF3 to positively regulate innate antiviral response

    PubMed Central

    Zhao, Kai; Zhang, Meng; Zhang, Lei; Wang, Peng; Song, Guanhua; Liu, Bingyu; Wu, Haifeng; Yin, Zhinan; Gao, Chengjiang

    2016-01-01

    Osteopontin (OPN) is a multifunctional protein involved in both innate immunity and adaptive immunity. However, the function of OPN, especially the intracellular form OPN (iOPN) on innate antiviral immune response remains elusive. Here, we demonstrated that iOPN is an essential positive regulator to protect the host from virus infection. OPN deficiency or knockdown significantly attenuated virus-induced IRF3 activation, IFN-β production and antiviral response. Consistently, OPN-deficient mice were more susceptible to VSV infection than WT mice. Mechanistically, iOPN was found to interact with tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) and inhibit Triad3A-mediated K48-linked polyubiquitination and degradation of TRAF3 through the C-terminal fragment of iOPN. Therefore, our findings delineated a new function for iOPN to act as a positive regulator in innate antiviral immunity through stabilization of TRAF3. PMID:27026194

  9. Potential Use of a Serpin from Arabidopsis for Pest Control

    PubMed Central

    Alvarez-Alfageme, Fernando; Maharramov, Jafar; Carrillo, Laura; Vandenabeele, Steven; Vercammen, Dominique; Van Breusegem, Frank; Smagghe, Guy

    2011-01-01

    Although genetically modified (GM) plants expressing toxins from Bacillus thuringiensis (Bt) protect agricultural crops against lepidopteran and coleopteran pests, field-evolved resistance to Bt toxins has been reported for populations of several lepidopteran species. Moreover, some important agricultural pests, like phloem-feeding insects, are not susceptible to Bt crops. Complementary pest control strategies are therefore necessary to assure that the benefits provided by those insect-resistant transgenic plants are not compromised and to target those pests that are not susceptible. Experimental GM plants producing plant protease inhibitors have been shown to confer resistance against a wide range of agricultural pests. In this study we assessed the potential of AtSerpin1, a serpin from Arabidopsis thaliana (L). Heynh., for pest control. In vitro assays were conducted with a wide range of pests that rely mainly on either serine or cysteine proteases for digestion and also with three non-target organisms occurring in agricultural crops. AtSerpin1 inhibited proteases from all pest and non-target species assayed. Subsequently, the cotton leafworm Spodoptera littoralis Boisduval and the pea aphid Acyrthosiphon pisum (Harris) were fed on artificial diets containing AtSerpin1, and S. littoralis was also fed on transgenic Arabidopsis plants overproducing AtSerpin1. AtSerpin1 supplied in the artificial diet or by transgenic plants reduced the growth of S. littoralis larvae by 65% and 38%, respectively, relative to controls. Nymphs of A. pisum exposed to diets containing AtSerpin1 suffered high mortality levels (LC50 = 637 µg ml−1). The results indicate that AtSerpin1 is a good candidate for exploitation in pest control. PMID:21655276

  10. [Roles of intracellular calcium and monomeric G-proteins in regulating exocytosis of human neutrophils].

    PubMed

    Zhu, Ying; Wang, Jun-Han; Wu, Jian-Min; Xu, Tao; Zhang, Chun-Guang

    2003-12-25

    Neutrophils play a major role in host defense against microbial infection. There are some clues indicate that neutrophils may also play a role in the pathophysiology of the airway obstruction in chronic asthma. We studied the roles of intracellular calcium and GTP gamma S in the regulation of neutrophils exocytosis using pipette perfusion and membrane capacitance measurement technique in whole cell patch clamp configuration. The results showed that the membrane capacitance increase induced by calcium revealed a biphasic process. The first phase occurred when the calcium level was between 0.2-14 micromol/L with a plateau amplitude of 1.23 pF and a calcium EC50 of 1.1 micromol/L. This phase might correspond to the release of the tertiary granules. The second phase occurred when the calcium concentration was between 20-70 micromol/L with a plateau increment of 6.36 pF, the calcium EC50 being about 33 micromol/L. This phase might represent the release of the primary and secondary granules. Intracellular calcium also simultaneously increased the exocytotic rate and the eventual extent in neutrophils. On the other hand, GTP gamma S can increase the exocytotic rate in a dose-dependent manner but had no effect on the eventual extent of membrane capacitance increment (>6 pF) if the cell was stimulated for a long period (>20 min). GTP gamma S (ranging from 20 to 100 micromol/L) induced the neutrophils to release all four types of the granules at very low intracellular calcium level. PMID:14695488

  11. Extracellular and intracellular regulation of oligodendrocyte development: roles of Sonic hedgehog and expression of E proteins.

    PubMed

    Sussman, Caroline R; Davies, Jeannette E; Miller, Robert H

    2002-10-01

    Recent advances in understanding oligodendrocyte development have revealed the importance of both extra- and intracellular molecules in regulating the induction, survival, and proliferation of early oligodendrocyte progenitors. The signaling molecule Sonic hedgehog (Shh) is critical for normal development of oligodendrocytes, although the precise influences of Shh on cells of the oligodendrocyte lineage are unclear. The present study shows that Shh increased the number of oligodendrocyte precursors in both pure cultures of oligodendrocyte precursors and mixed cultures from embryonic rat spinal cord. In pure precursor cultures Shh increased cell survival. In mixed cultures, Shh increased both the survival and proliferation of oligodendrocyte precursors in a concentration dependent manner. One intracellular consequence of exposure to Shh is the activation of transcription factors in oligodendrocyte lineage cells, which are critical for oligodendrocyte development, helix-loop-helix (HLH) transcription factors, Olig1 and 2. In many cases, HLH proteins such as Olig1 and Olig2 heterodimerize with other HLH proteins, such as members of the E subfamily, which are critical regulators of cell proliferation and differentiation. Immature (A2B5(+)) and more mature (O4(+)) rat oligodendrocyte precursors in dissociated cell culture expressed Olig1 as well as E proteins, HEB and E2A. Similarly, cells bearing the morphology of oligodendrocyte precursors expressed both Olig1 and HEB or E2A. We propose that E2A and/or HEB, possibly in combination with Olig1 and 2, are critical components of oligodendrogenesis and may regulate cell survival, proliferation, and fate decisions in the oligodendrocyte lineage. PMID:12237843

  12. APP intracellular domain acts as a transcriptional regulator of miR-663 suppressing neuronal differentiation

    PubMed Central

    Shu, R; Wong, W; Ma, Q H; Yang, Z Z; Zhu, H; Liu, F J; Wang, P; Ma, J; Yan, S; Polo, J M; Bernard, C C A; Stanton, L W; Dawe, G S; Xiao, Z C

    2015-01-01

    Amyloid precursor protein (APP) is best known for its involvement in the pathogenesis of Alzheimer's disease. We have previously demonstrated that APP intracellular domain (AICD) regulates neurogenesis; however, the mechanisms underlying AICD-mediated regulation of neuronal differentiation are not yet fully characterized. Using genome-wide chromatin immunoprecipitation approaches, we found that AICD is specifically recruited to the regulatory regions of several microRNA genes, and acts as a transcriptional regulator for miR-663, miR-3648 and miR-3687 in human neural stem cells. Functional assays show that AICD negatively modulates neuronal differentiation through miR-663, a primate-specific microRNA. Microarray data further demonstrate that miR-663 suppresses the expression of multiple genes implicated in neurogenesis, including FBXL18 and CDK6. Our results indicate that AICD has a novel role in suppression of neuronal differentiation via transcriptional regulation of miR-663 in human neural stem cells. PMID:25695604

  13. Viral Polymerase-Helicase Complexes Regulate Replication Fidelity To Overcome Intracellular Nucleotide Depletion

    PubMed Central

    Stapleford, Kenneth A.; Rozen-Gagnon, Kathryn; Das, Pratyush Kumar; Saul, Sirle; Poirier, Enzo Z.; Blanc, Hervé; Vidalain, Pierre-Olivier; Merits, Andres

    2015-01-01

    ABSTRACT To date, the majority of work on RNA virus replication fidelity has focused on the viral RNA polymerase, while the potential role of other viral replicase proteins in this process is poorly understood. Previous studies used resistance to broad-spectrum RNA mutagens, such as ribavirin, to identify polymerases with increased fidelity that avoid misincorporation of such base analogues. We identified a novel variant in the alphavirus viral helicase/protease, nonstructural protein 2 (nsP2) that operates in concert with the viral polymerase nsP4 to further alter replication complex fidelity, a functional linkage that was conserved among the alphavirus genus. Purified chikungunya virus nsP2 presented delayed helicase activity of the high-fidelity enzyme, and yet purified replication complexes manifested stronger RNA polymerization kinetics. Because mutagenic nucleoside analogs such as ribavirin also affect intracellular nucleotide pools, we addressed the link between nucleotide depletion and replication fidelity by using purine and pyrimidine biosynthesis inhibitors. High-fidelity viruses were more resistant to these conditions, and viral growth could be rescued by the addition of exogenous nucleosides, suggesting that mutagenesis by base analogues requires nucleotide pool depletion. This study describes a novel function for nsP2, highlighting the role of other components of the replication complex in regulating viral replication fidelity, and suggests that viruses can alter their replication complex fidelity to overcome intracellular nucleotide-depleting conditions. IMPORTANCE Previous studies using the RNA mutagen ribavirin to select for drug-resistant variants have highlighted the essential role of the viral RNA-dependent RNA polymerase in regulating replication fidelity. However, the role of other viral replicase components in replication fidelity has not been studied in detail. We identified here an RNA mutagen-resistant variant of the nsP2 helicase

  14. Vitamin E and Phosphoinositides Regulate the Intracellular Localization of the Hepatic α-Tocopherol Transfer Protein.

    PubMed

    Chung, Stacey; Ghelfi, Mikel; Atkinson, Jeffrey; Parker, Robert; Qian, Jinghui; Carlin, Cathleen; Manor, Danny

    2016-08-12

    α-Tocopherol (vitamin E) is an essential nutrient for all vertebrates. From the eight naturally occurring members of the vitamin E family, α-tocopherol is the most biologically active species and is selectively retained in tissues. The hepatic α-tocopherol transfer protein (TTP) preferentially selects dietary α-tocopherol and facilitates its transport through the hepatocyte and its secretion to the circulation. In doing so, TTP regulates body-wide levels of α-tocopherol. The mechanisms by which TTP facilitates α-tocopherol trafficking in hepatocytes are poorly understood. We found that the intracellular localization of TTP in hepatocytes is dynamic and responds to the presence of α-tocopherol. In the absence of the vitamin, TTP is localized to perinuclear vesicles that harbor CD71, transferrin, and Rab8, markers of the recycling endosomes. Upon treatment with α-tocopherol, TTP- and α-tocopherol-containing vesicles translocate to the plasma membrane, prior to secretion of the vitamin to the exterior of the cells. The change in TTP localization is specific to α-tocopherol and is time- and dose-dependent. The aberrant intracellular localization patterns of lipid binding-defective TTP mutants highlight the importance of protein-lipid interaction in the transport of α-tocopherol. These findings provide the basis for a proposed mechanistic model that describes TTP-facilitated trafficking of α-tocopherol through hepatocytes. PMID:27307040

  15. Role of Metal-Dependent Regulation of ESX-3 Secretion in Intracellular Survival of Mycobacterium tuberculosis.

    PubMed

    Tinaztepe, Emir; Wei, Jun-Rong; Raynowska, Jenelle; Portal-Celhay, Cynthia; Thompson, Victor; Philips, Jennifer A

    2016-08-01

    More people die every year from Mycobacterium tuberculosis infection than from infection by any other bacterial pathogen. Type VII secretion systems (T7SS) are used by both environmental and pathogenic mycobacteria to secrete proteins across their complex cell envelope. In the nonpathogen Mycobacterium smegmatis, the ESX-1 T7SS plays a role in conjugation, and the ESX-3 T7SS is involved in metal homeostasis. In M. tuberculosis, these secretion systems have taken on roles in virulence, and they also are targets of the host immune response. ESX-3 secretes a heterodimer composed of EsxG (TB9.8) and EsxH (TB10.4), which impairs phagosome maturation in macrophages and is essential for virulence in mice. Given the importance of EsxG and EsxH during infection, we examined their regulation. With M. tuberculosis, the secretion of EsxG and EsxH was regulated in response to iron and zinc, in accordance with the previously described transcriptional response of the esx-3 locus to these metals. While iron regulated the esx-3 expression in both M. tuberculosis and M. smegmatis, there is a significant difference in the dynamics of this regulation. In M. smegmatis, the esx-3 locus behaved like other iron-regulated genes such as mbtB In M. tuberculosis, both iron and zinc modestly repressed esx-3 expression. Diminished secretion of EsxG and EsxH in response to these metals altered the interaction of M. tuberculosis with macrophages, leading to impaired intracellular M. tuberculosis survival. Our findings detail the regulatory differences of esx-3 in M. tuberculosis and M. smegmatis and demonstrate the importance of metal-dependent regulation of ESX-3 for virulence in M. tuberculosis. PMID:27245412

  16. Spatial and Temporal Regulation of Receptor Tyrosine Kinase Activation and Intracellular Signal Transduction.

    PubMed

    Bergeron, John J M; Di Guglielmo, Gianni M; Dahan, Sophie; Dominguez, Michel; Posner, Barry I

    2016-06-01

    Epidermal growth factor (EGF) and insulin receptor tyrosine kinases (RTKs) exemplify how receptor location is coupled to signal transduction. Extracellular binding of ligands to these RTKs triggers their concentration into vesicles that bud off from the cell surface to generate intracellular signaling endosomes. On the exposed cytosolic surface of these endosomes, RTK autophosphorylation selects the downstream signaling proteins and lipids to effect growth factor and polypeptide hormone action. This selection is followed by the recruitment of protein tyrosine phosphatases that inactivate the RTKs and deliver them by membrane fusion and fission to late endosomes. Coincidentally, proteinases inside the endosome cleave the EGF and insulin ligands. Subsequent inward budding of the endosomal membrane generates multivesicular endosomes. Fusion with lysosomes then results in RTK degradation and downregulation. Through the spatial positioning of RTKs in target cells for EGF and insulin action, the temporal extent of signaling, attenuation, and downregulation is regulated. PMID:27023845

  17. Regulation of gamma T-cell antigen receptor expression by intracellular calcium in acute lymphoblastic leukemia cell line DND41.

    PubMed

    Peralta-Zaragoza, O; Martínez-Valdez, H; Madrid-Marina, V

    1996-01-01

    The calcium ionophore, ionomycin, promotes an increase of intracellular calcium and regulates mRNA expression of gamma/delta-TcR gene in human T lymphocytes. The mechanism of this regulation is not yet clear. Thus, the regulation by intracellular calcium requires elucidation. We studied the gamma-TcR gene expression in acute lymphoblastic leukemia cell line DND41 (CD4- CD8-) by Northern blot and flow cytometric analysis. The mRNA levels of gamma-TcR increased by ionomycin, anti-CD3, and with TPA. TPA had an antagonistic effect to both ionomycin and anti-CD3. Also, TPA inhibits the increased intracellular calcium promoted by ionomycin but not the increase promoted by anti-CD3 and ionomycin. Our results suggest that intracellular calcium induces mRNA and protein expression of gamma-TcR chain. This effect is antagonized by protein kinase C-activation. Thus, we conclude that the target cells of the differential regulation on gamma-TcR mRNA expression by intracellular calcium modulators are the CD4- CD8- cells, and this is due to cytosolic calcium mobilization. PMID:8854386

  18. α-Arrestins Aly1 and Aly2 Regulate Intracellular Trafficking in Response to Nutrient Signaling

    PubMed Central

    O'Donnell, Allyson F.; Apffel, Alex; Gardner, Richard G.

    2010-01-01

    Extracellular signals regulate trafficking events to reorganize proteins at the plasma membrane (PM); however, few effectors of this regulation have been identified. β-Arrestins relay signaling cues to the trafficking machinery by controlling agonist-stimulated endocytosis of G-protein–coupled receptors. In contrast, we show that yeast α-arrestins, Aly1 and Aly2, control intracellular sorting of Gap1, the general amino acid permease, in response to nutrients. These studies are the first to demonstrate association of α-arrestins with clathrin and clathrin adaptor proteins (AP) and show that Aly1 and Aly2 interact directly with the γ-subunit of AP-1, Apl4. Aly2-dependent trafficking of Gap1 requires AP-1, which mediates endosome-to-Golgi transport, and the nutrient-regulated kinase, Npr1, which phosphorylates Aly2. During nitrogen starvation, Npr1 phosphorylation of Aly2 may stimulate Gap1 incorporation into AP-1/clathrin-coated vesicles to promote Gap1 trafficking from endosomes to the trans-Golgi network. Ultimately, increased Aly1-/Aly2-mediated recycling of Gap1 from endosomes results in higher Gap1 levels within cells and at the PM by diverting Gap away from trafficking pathways that lead to vacuolar degradation. This work defines a new role for arrestins in membrane trafficking and offers insight into how α-arrestins coordinate signaling events with protein trafficking. PMID:20739461

  19. SorLA regulates the activity of lipoprotein lipase by intracellular trafficking.

    PubMed

    Klinger, Stine C; Glerup, Simon; Raarup, Merete K; Mari, Muriel C; Nyegaard, Mette; Koster, Gerbrand; Prabakaran, Thaneas; Nilsson, Stefan K; Kjaergaard, Maj M; Bakke, Oddmund; Nykjær, Anders; Olivecrona, Gunilla; Petersen, Claus Munck; Nielsen, Morten S

    2011-04-01

    Many different tissues and cell types exhibit regulated secretion of lipoprotein lipase (LPL). However, the sorting of LPL in the trans Golgi network has not, hitherto, been understood in detail. Here, we characterize the role of SorLA (officially known as SorLA-1 or sortilin-related receptor) in the intracellular trafficking of LPL. We found that LPL bound to SorLA under neutral and acidic conditions, and in cells this binding mainly occurred in vesicular structures. SorLA expression changed the subcellular distribution of LPL so it became more concentrated in endosomes. From the endosomes, LPL was further routed to the lysosomes, which resulted in a degradation of newly synthesized LPL. Consequently, an 80% reduction of LPL activity was observed in cells that expressed SorLA. By analogy, SorLA regulated the vesicle-like localization of LPL in primary neuronal cells. Thus, LPL binds to SorLA in the biosynthetic pathway and is subsequently transported to endosomes. As a result of this SorLA mediated-transport, newly synthesized LPL can be routed into specialized vesicles and eventually sent to degradation, and its activity thereby regulated. PMID:21385844

  20. β-PIX controls intracellular viscoelasticity to regulate lung cancer cell migration.

    PubMed

    Yu, Helen Wenshin; Chen, Yin-Quan; Huang, Chi-Ming; Liu, Ching-Yi; Chiou, Arthur; Wang, Yang-Kao; Tang, Ming-Jer; Kuo, Jean-Cheng

    2015-05-01

    Cancer metastasis occurs via a progress involving abnormal cell migration. Cell migration, a dynamic physical process, is controlled by the cytoskeletal system, which includes the dynamics of actin organization and cellular adhesive organelles, focal adhesions (FAs). However, it is not known whether the organization of actin cytoskeletal system has a regulatory role in the physiologically relevant aspects of cancer metastasis. In the present studies, it was found that lung adenocarcinoma cells isolated from the secondary lung cancer of the lymph nodes, H1299 cells, show specific dynamics in terms of the actin cytoskeleton and FAs. This results in a higher level of mobility and this is regulated by an immature FA component, β-PIX (PAK-interacting exchange factor-β). In H1299 cells, β-PIX's activity was found not to be down-regulated by sequestration onto stress fibres, as the cells did not bundle actin filaments into stress fibres. Thus, β-PIX mainly remained localized at FAs, which allowed maturation of nascent adhesions into focal complexes; this resulted in actin polymerization, increased actin network integrity, changes in the intracellular microrheology at the peripheral of the cell, and cell polarity, which in turn regulated cell migration. Perturbation of β-PIX caused an inhibition of cell migration, including migration velocity, accumulated distance and directional persistence. Our results demonstrate the importance of β-PIX to the regulation of high mobility of lung adenocarcinoma cell line H1299 and that this occurs via regulation of FA dynamics, changes in actin cytoskeleton organization and cell polarity. PMID:25683605

  1. SERPINE1, PAI-1 protein coding gene, methylation levels and epigenetic relationships with adiposity changes in obese subjects with metabolic syndrome features under dietary restriction

    PubMed Central

    Lopez-Legarrea, Patricia; Mansego, Maria Luisa; Zulet, Marian Angeles; Martinez, Jose Alfredo

    2013-01-01

    Plasminogen activator inhibitor 1 (PAI-1) has been associated with metabolic disorders, through different mechanisms, which could involve changes in DNA methylation. This work aimed to assess the potential relationships of the cytosine methylation levels within SERPINE1 gene transcriptional regulatory region, which codes for PAI-1, in peripheral white blood cells with anthropometrical, metabolic and inflammatory features. Forty-six obese subjects with metabolic syndrome features followed Control or Metabolic Syndrome Reduction in Navarra (RESMENA) energy-restricted (−30%E) diets for 8 weeks. SERPINE1 transcriptional regulatory region methylation at baseline was analyzed by a microarray technical. Both dietary strategies reduced anthropometric and biochemical parameters. The Control group significantly reduced plasma PAI-1 concentrations but not the RESMENA group. Participants from both nutritional interventions with higher SERPINE1 methylation levels at baseline showed significantly major reductions in body weight, total fat mass, android fat mass, total cholesterol and triglycerides, as compared with those with lower initial SERPINE1 methylation levels. In conclusion, the DNA methylation levels of SERPINE1 transcriptional regulatory region were associated with some metabolic and anthropometric changes in obese subjects with metabolic syndrome under energy restriction, suggesting a complex epigenetic network in the regulation of this recognized pro-inflammatory marker. (www.clinicaltrials.gov; NCT01087086) PMID:24249967

  2. Endoplasmic Reticulum-associated Degradation (ERAD) and Autophagy Cooperate to Degrade Polymerogenic Mutant Serpins*

    PubMed Central

    Kroeger, Heike; Miranda, Elena; MacLeod, Ian; Pérez, Juan; Crowther, Damian C.; Marciniak, Stefan J.; Lomas, David A.

    2009-01-01

    The serpinopathies are a family of diseases characterized by the accumulation of ordered polymers of mutant protein within the endoplasmic reticulum. They are a diverse group including α1-antitrypsin deficiency and the inherited dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. We have used transient transfection of COS7 cells and mouse embryonic fibroblasts, PC12 cell lines that conditionally express wild type and mutant neuroserpin and fly models of FENIB to assess the cellular handling of wild type and mutant serpins. By using a polymer-specific monoclonal antibody, we show that mutant neuroserpin forms polymers after a delay of at least 30 min and that polymers can be cleared in PC12 cell lines and from the brain in a fly model of FENIB. At steady state, the fractions of intracellular polymerogenic G392E mutant neuroserpin in the monomeric and polymeric states are comparable. Inhibition of the proteasome with MG132 reveals that both mutant neuroserpin and α1-antitrypsin are degraded predominantly by endoplasmic reticulum-associated degradation (ERAD). Pharmacological and genetic inhibitions demonstrate that autophagy is responsible for bulk turnover of wild type and mutant serpins, but can be stimulated by rapamycin to compensate for proteasome inhibition. The significance of these findings to the treatment of serpinopathies is discussed. PMID:19549782

  3. Serpin peptidase inhibitor clade A member 1 is a biomarker of poor prognosis in gastric cancer

    PubMed Central

    Kwon, C H; Park, H J; Lee, J R; Kim, H K; Jeon, T Y; Jo, H-J; Kim, D H; Kim, G H; Park, D Y

    2014-01-01

    Background: In a previous study, we reported that serpin peptidase inhibitor clade A member 1 (serpinA1) is upregulated in Snail-overexpressing gastric cancer. Although serpinA1 has been studied in several types of cancer, little is known about its roles and mechanisms of action. In this study, we examined the role of serpinA1 in the migration and invasion of gastric cancers and determined its underlying mechanism. Methods: Expression levels were assessed by western blot analyses and real-time PCR. Snail binding to serpinA1 promoter was analysed by chromatin immunoprecipitation (ChIP) assays. The roles of serpinA1 were studied using cell invasion and migration assays. In addition, the clinicopathologic and prognostic significance of serpinA1 expression were validated in 400 gastric cancer patients using immunohistochemical analysis. Results: Overexpression of Snail resulted in upregulation of serpinA1 in gastric cancer cell lines, AGS and MKN45, whereas knockdown of Snail inhibited serpinA1 expression. Chromatin immunoprecipitation analysis showed that overexpression of Snail increased Snail recruitment to the serpinA1 promoter. Overexpression of serpinA1 increased the migration and invasion of gastric cancer cells, whereas knockdown of serpinA1 decreased invasion and migration. Moreover, serpinA1 increased mRNA levels and release of metalloproteinase-8 in gastric cancer cells. Serpin peptidase inhibitor clade A member 1 was observed in the cytoplasm of tumour cells and the stroma by immunohistochemistry. Enhanced serpinA1 expression was significantly associated with increased tumour size, advanced T stage, perineural invasion, lymphovascular invasion, lymph node metastases, and shorter overall survival. Conclusions: Serpin peptidase inhibitor clade A member 1 induces the invasion and migration of gastric cancer cells and its expression is associated with the progression of gastric cancer. These results may provide a potential target to prevent invasion and

  4. Bile Acids Regulate Nuclear Receptor (Nur77) Expression and Intracellular Location to Control Proliferation and Apoptosis

    PubMed Central

    Hu, Ying; Chau, Thinh; Liu, Hui-xin; Liao, Degui; Keane, Ryan; Nie, Yuqiang; Yang, Hui; Wan, Yu-Jui Yvonne

    2014-01-01

    Bile acids (BAs) are endogenous agents capable of causing cancer throughout the gastrointestinal (GI) tract. To uncover the mechanism by which BAs exert carcinogenic effects, both human liver and colon cancer cells as well as mouse primary hepatocytes were treated with BAs and assayed for viability, genotoxic stress, and transcriptional response. BAs induced both Nur77 (NR4A1) and pro-inflammatory gene expression. The intracellular location of BA-induced Nur77 was time-dependent; short-term (1–3 h) exposure induced nuclear Nur77 whereas longer (1–2 days) exposure also increased cytosolic Nur77 expression and apoptosis. Inhibiting Nur77 nuclear export with leptomycin B decreased LCA-induced apoptosis. Extended (7 days) treatment with BA generated resistance to BA with increased nuclear Nur77, viability, and mobility. While, knockdown of Nur77 in BA-resistant cells increased cellular susceptibility to LCA-induced apoptosis. Moreover, in vivo mouse xenograft experiments demonstrated that BA-resistant cells form larger tumors with elevated Nur77 expression compared to parental controls. DNA-binding and gene expression assays identified multiple survival genes (CDK4, CCND2, MAP4K5, STAT5A, and RBBP8) and a pro-apoptosis gene (BID) as Nur77 targets. Consistently, BA-induced up-regulation of the aforementioned genes was abrogated by a lack of Nur77. Importantly, Nur77 was overexpressed in high percentage of human colon and liver cancer specimens and the intracellular location of Nur77 correlated with elevated serum total BA levels in colon cancer patients. These data show for the first time that BAs via Nur77 have a dual role in modulating cell survival and death. Implications: These findings establish a direct link between Nur77 and the carcinogenic effect of bile acids. PMID:25232032

  5. Design and engineering of intracellular-metabolite-sensing/regulation gene circuits in Saccharomyces cerevisiae.

    PubMed

    Wang, Meng; Li, Sijin; Zhao, Huimin

    2016-01-01

    The development of high-throughput phenotyping tools is lagging far behind the rapid advances of genotype generation methods. To bridge this gap, we report a new strategy for design, construction, and fine-tuning of intracellular-metabolite-sensing/regulation gene circuits by repurposing bacterial transcription factors and eukaryotic promoters. As proof of concept, we systematically investigated the design and engineering of bacterial repressor-based xylose-sensing/regulation gene circuits in Saccharomyces cerevisiae. We demonstrated that numerous properties, such as induction ratio and dose-response curve, can be fine-tuned at three different nodes, including repressor expression level, operator position, and operator sequence. By applying these gene circuits, we developed a cell sorting based, rapid and robust high-throughput screening method for xylose transporter engineering and obtained a sugar transporter HXT14 mutant with 6.5-fold improvement in xylose transportation capacity. This strategy should be generally applicable and highly useful for evolutionary engineering of proteins, pathways, and genomes in S. cerevisiae. PMID:26059511

  6. Intracellular RIG-I Signaling Regulates TLR4-Independent Endothelial Inflammatory Responses to Endotoxin.

    PubMed

    Moser, Jill; Heeringa, Peter; Jongman, Rianne M; Zwiers, Peter J; Niemarkt, Anita E; Yan, Rui; de Graaf, Inge A; Li, Ranran; Ravasz Regan, Erzsébet; Kümpers, Philipp; Aird, William C; van Nieuw Amerongen, Geerten P; Zijlstra, Jan G; Molema, Grietje; van Meurs, Matijs

    2016-06-01

    Sepsis is a systemic inflammatory response to infections associated with organ failure that is the most frequent cause of death in hospitalized patients. Exaggerated endothelial activation, altered blood flow, vascular leakage, and other disturbances synergistically contribute to sepsis-induced organ failure. The underlying signaling events associated with endothelial proinflammatory activation are not well understood, yet they likely consist of molecular pathways that act in an endothelium-specific manner. We found that LPS, a critical factor in the pathogenesis of sepsis, is internalized by endothelial cells, leading to intracellular signaling without the need for priming as found recently in immune cells. By identifying a novel role for retinoic acid-inducible gene-I (RIG-I) as a central regulator of endothelial activation functioning independent of TLR4, we provide evidence that the current paradigm of TLR4 solely being responsible for LPS-mediated endothelial responses is incomplete. RIG-I, as well as the adaptor protein mitochondrial antiviral signaling protein, regulates NF-κB-mediated induction of adhesion molecules and proinflammatory cytokine expression in response to LPS. Our findings provide essential new insights into the proinflammatory signaling pathways in endothelial cells and suggest that combined endothelial-specific inhibition of RIG-I and TLR4 will provide protection from aberrant endothelial responses associated with sepsis. PMID:27183587

  7. Intracellular Redox Compartmentation and ROS-Related Communication in Regulation and Signaling1[OPEN

    PubMed Central

    2016-01-01

    Recent years have witnessed enormous progress in understanding redox signaling related to reactive oxygen species (ROS) in plants. The consensus view is that such signaling is intrinsic to many developmental processes and responses to the environment. ROS-related redox signaling is tightly wedded to compartmentation. Because membranes function as barriers, highly redox-active powerhouses such as chloroplasts, peroxisomes, and mitochondria may elicit specific signaling responses. However, transporter functions allow membranes also to act as bridges between compartments, and so regulated capacity to transmit redox changes across membranes influences the outcome of triggers produced at different locations. As well as ROS and other oxidizing species, antioxidants are key players that determine the extent of ROS accumulation at different sites and that may themselves act as signal transmitters. Like ROS, antioxidants can be transported across membranes. In addition, the intracellular distribution of antioxidative enzymes may be modulated to regulate or facilitate redox signaling appropriate to the conditions. Finally, there is substantial plasticity in organellar shape, with extensions such as stromules, peroxules, and matrixules playing potentially crucial roles in organelle-organelle communication. We provide an overview of the advances in subcellular compartmentation, identifying the gaps in our knowledge and discussing future developments in the area. PMID:27208308

  8. A zinc-sensing receptor triggers the release of intracellular Ca2+ and regulates ion transport

    PubMed Central

    Hershfinkel, Michal; Moran, Arie; Grossman, Nili; Sekler, Israel

    2001-01-01

    Changes in extracellular zinc concentration participate in modulating fundamental cellular processes such as proliferation, secretion, and ion transport in a mechanism that is not well understood. Here, we show that a micromolar concentration of extracellular zinc triggers a massive release of calcium from thapsigargin-sensitive intracellular pools in the colonocytic cell line HT29. Calcium release was blocked by a phospholipase-C inhibitor, indicating that formation of inositol 1,4,5-triphosphate is required for zinc-dependent calcium release. Zinc influx was not observed, indicating that extracellular zinc triggered the release. The Cai2+ release was zinc specific and could not be triggered by other heavy metals. Furthermore, zinc failed to activate the Ca2+-sensing receptor heterologously expressed in HEK293 cells. The zinc-induced Cai2+ rise stimulated the activity of the Na+/H+ exchanger in HT29 cells. Our results indicate that a previously uncharacterized extracellular, G protein-coupled, Zn2+-sensing receptor is functional in colonocytes. Because Cai2+ rise is known to regulate key cellular and signal-transduction processes, the zinc-sensing receptor may provide the missing link between extracellular zinc concentration changes and the regulation of cellular processes. PMID:11573009

  9. Aluminum-dependent regulation of intracellular silicon in the aquatic invertebrate Lymnaea stagnalis

    PubMed Central

    Desouky, Mahmoud; Jugdaohsingh, Ravin; McCrohan, Catherine R.; White, Keith N.; Powell, Jonathan J.

    2002-01-01

    Silicon is essential for some plants, diatoms, and sponges but, in higher animals, its endogenous regulation has not been demonstrated. Silicate ions may be natural ligands for aluminum and here we show that, in the freshwater snail (Lymnaea stagnalis), intracellular silicon seems specifically up-regulated in response to sublethal aluminum exposure. X-ray microanalysis showed that exposure of snails to low levels of aluminum led to its accumulation in lysosomal granules, accompanied by marked up-regulation of silicon. Increased lysosomal levels of silicon were a specific response to aluminum because cadmium and zinc had no such effect. Furthermore, intra-lysosomal sulfur from metallothionein and other sulfur-containing ligands was increased after exposure to cadmium and zinc but not aluminum. To ensure that these findings indicated a specific in vivo response, and not ex vivo formation of hydroxy-aluminosilicates (HAS) from added aluminum (555 μg/liter) and water-borne silicon (43 μg/liter), two further studies were undertaken. In a ligand competition assay the lability of aluminum (527 μg/liter) was completely unaffected by the presence of silicon (46 μg/liter), suggesting the absence of HAS. In addition, exogenous silicon (6.5 mg/liter), added to the water column to promote formation of HAS, caused a decrease in lysosomal aluminum accumulation, showing that uptake of HAS would not explain the loading of aluminum into lysosomal granules. These findings, and arguments on the stability, lability, and kinetics of aluminum–silicate interactions, suggest that a silicon-specific mechanism exists for the in vivo detoxification of aluminum, which provides regulatory evidence of silicon in a multicellular organism. PMID:11891333

  10. Regulation of intracellular pH in cancer cell lines under normoxia and hypoxia.

    PubMed

    Hulikova, Alzbeta; Harris, Adrian L; Vaughan-Jones, Richard D; Swietach, Pawel

    2013-04-01

    Acid-extrusion by active transport is important in metabolically active cancer cells, where it removes excess intracellular acid and sets the intracellular resting pH. Hypoxia is a major trigger of adaptive responses in cancer, but its effect on acid-extrusion remains unclear. We studied pH-regulation under normoxia and hypoxia in eight cancer cell-lines (HCT116, RT112, MDA-MB-468, MCF10A, HT29, HT1080, MiaPaca2, HeLa) using the pH-sensitive fluorophore, cSNARF-1. Hypoxia responses were triggered by pre-incubation in low O(2) or with the 2-oxoglutarate-dependent dioxygenase inhibitor dimethyloxalylglycine (DMOG). By selective pharmacological inhibition or transport-substrate removal, acid-extrusion flux was dissected into components due to Na(+)/H(+) exchange (NHE) and Na(+)-dependent HCO(3)(-) transport. In half of the cell-lines (HCT116, RT112, MDA-MB-468, MCF10A), acid-extrusion on NHE was the dominant flux during an acid load, and in all of these, bar one (MDA-MB-468), NHE-flux was reduced following hypoxic incubation. Further studies in HCT116 cells showed that <4-h hypoxic incubation reduced NHE-flux reversibly with a time-constant of 1-2 h. This was not associated with a change in expression of NHE1, the principal NHE isoform. Following 48-h hypoxia, inhibition of NHE-flux persisted but became only slowly reversible and associated with reduced expression of the glycosylated form of NHE1. Acid-extrusion by Na(+)-dependent HCO(3)(-) transport was hypoxia-insensitive and comparable in all cell lines. This constitutive and stable element of pH-regulation was found to be important for setting and stabilizing resting pH at a mildly alkaline level (conducive for growth), irrespective of oxygenation status. In contrast, the more variable flux on NHE underlies cell-specific differences in their dynamic response to larger acid loads. PMID:22949268

  11. Intracellular transduction in the regulation of pheromone biosynthesis of the silkworm, Bombyx mori: suggested involvement of calmodulin and phosphoprotein phosphatase.

    PubMed

    Matsumoto, S; Ozawa, R; Nagamine, T; Kim, G H; Uchiumi, K; Shono, T; Mitsui, T

    1995-03-01

    We have tested the effects of chemicals on bombykol production in vitro in the silkworm, Bombyx mori, to probe the biochemical steps as well as underlying mechanisms regulated by PBAN. These results suggest the involvement of calmodulin and phosphoprotein phosphatase in the intracellular signal transduction of PBAN action. PMID:7766202

  12. Bicaudal-D1 regulates the intracellular sorting and signalling of neurotrophin receptors

    PubMed Central

    Terenzio, Marco; Golding, Matthew; Russell, Matthew R G; Wicher, Krzysztof B; Rosewell, Ian; Spencer-Dene, Bradley; Ish-Horowicz, David; Schiavo, Giampietro

    2014-01-01

    We have identified a new function for the dynein adaptor Bicaudal D homolog 1 (BICD1) by screening a siRNA library for genes affecting the dynamics of neurotrophin receptor-containing endosomes in motor neurons (MNs). Depleting BICD1 increased the intracellular accumulation of brain-derived neurotrophic factor (BDNF)-activated TrkB and p75 neurotrophin receptor (p75NTR) by disrupting the endosomal sorting, reducing lysosomal degradation and increasing the co-localisation of these neurotrophin receptors with retromer-associated sorting nexin 1. The resulting re-routing of active receptors increased their recycling to the plasma membrane and altered the repertoire of signalling-competent TrkB isoforms and p75NTR available for ligand binding on the neuronal surface. This resulted in attenuated, but more sustained, AKT activation in response to BDNF stimulation. These data, together with our observation that Bicd1 expression is restricted to the developing nervous system when neurotrophin receptor expression peaks, indicate that BICD1 regulates neurotrophin signalling by modulating the endosomal sorting of internalised ligand-activated receptors. PMID:24920579

  13. Regulation of the glutamine transporter SN1 by extracellular pH and intracellular sodium ions

    PubMed Central

    Bröer, Angelika; Albers, Alexandra; Setiawan, Iwan; Edwards, Robert H; Chaudhry, Farrukh A; Lang, Florian; Wagner, Carsten A; Bröer, Stefan

    2002-01-01

    The glutamine transporter SN1 has recently been identified as one of the major glutamine transporters in hepatocytes and brain astrocytes. It appears to be the molecular correlate of system N amino acid transport. Two different transport mechanisms have been proposed for this transporter. These are an electroneutral mechanism, in which glutamine uptake is coupled to an exchange of 1Na+ and 1H+, or an electrogenic mechanism coupled to the exchange of 2Na+ against 1H+. This study was performed to solve these discrepancies and to investigate the reversibility of the transporter. When SN1 was expressed in Xenopus laevis oocytes, glutamine uptake was accompanied by a cotransport of 2–3 Na+ ions as determined by 22Na+ fluxes. However, at the same time a rapid release of intracellular Na+ was observed indicating an active exchange of Na+ ions. The driving force of the proton electrochemical gradient was equivalent to that of the sodium electrochemical gradient. Acidification of the extracellular medium caused the transporter to run in reverse and to release glutamine. Determination of accumulation ratios at different driving forces were in agreement with an electroneutral 1Na+-glutamine cotransport-1H+ antiport. Inward currents that were observed during glutamine uptake were much smaller than expected for a stoichiometric cotransport of charges. A slippage mode in the transporter mechanism and pH-regulated endogenous oocyte cation channels are likely to contribute to the observed currents. PMID:11850497

  14. Regulation of Epidermal Growth Factor Receptor Signaling by Endocytosis and Intracellular Trafficking

    SciTech Connect

    Burke, Patrick; Schooler, Kevin; Wiley, H S.

    2001-06-01

    Ligand activation of the epidermal growth factor receptor (EGFR) leads to its rapid internalization and eventual delivery to lysosomes. This process is thought to be a mechanism to attenuate signaling, but signals could potentially be generated following endocytosis. To directly evaluate EGFR signaling during receptor trafficking, we developed a technique to rapidly and selectively isolate internalized EGFR and associated molecules using reversibly-biotinylated anti-EGFR antibodies. In addition, we developed antibodies specific to tyrosine-phosphorylated EGFR. Using a combination of fluorescence imaging and affinity precipitation approaches, we evaluated the state of EGFR activation and substrate association during trafficking in epithelial cells. We found that following internalization, EGFR remained active in the early endosomes. However, receptors were inactivated prior to degradation, apparently due to ligand removal from endosomes. Adapter molecules, such as Shc, were associated with EGFR both at the cell surface and within endosomes. Some molecules, such as Grb2, were primarily found associated with surface EGFR, while others, such as Eps8, were only found with intracellular receptors. During the inactivation phase, c-Cbl became EGFR-associated, consistent with its postulated role in receptor attenuation. We conclude that the association of the EGFR with different proteins is compartment-specific . In addition, ligand loss is the proximal cause of EGFR inactivation. Thus, regulated trafficking could potentially influence the pattern as well as the duration of signal transduction.

  15. Monitoring of Intracellular Tau Aggregation Regulated by OGA/OGT Inhibitors.

    PubMed

    Lim, Sungsu; Haque, Md Mamunul; Nam, Ghilsoo; Ryoo, Nayeon; Rhim, Hyewhon; Kim, Yun Kyung

    2015-01-01

    Abnormal phosphorylation of tau has been considered as a key pathogenic mechanism inducing tau aggregation in multiple neurodegenerative disorders, collectively called tauopathies. Recent evidence showed that tau phosphorylation sites are protected with O-linked β-N-acetylglucosamine (O-GlcNAc) in normal brain. In pathological condition, tau is de-glycosylated and becomes a substrate for kinases. Despite the importance of O-GlcNAcylation in tau pathology, O-GlcNAc transferase (OGT), and an enzyme catalyzing O-GlcNAc to tau, has not been carefully investigated in the context of tau aggregation. Here, we investigated intracellular tau aggregation regulated by BZX2, an inhibitor of OGT. Upon the inhibition of OGT, tau phosphorylation increased 2.0-fold at Ser199 and 1.5-fold at Ser396, resulting in increased tau aggregation. Moreover, the BZX2 induced tau aggregation was efficiently reduced by the treatment of Thiamet G, an inhibitor of O-GlcNAcase (OGA). Our results demonstrated the protective role of OGT in tau aggregation and also suggest the counter-regulatory mechanism of OGA and OGT in tau pathology. PMID:26343633

  16. Monitoring of Intracellular Tau Aggregation Regulated by OGA/OGT Inhibitors

    PubMed Central

    Lim, Sungsu; Haque, Md. Mamunul; Nam, Ghilsoo; Ryoo, Nayeon; Rhim, Hyewhon; Kim, Yun Kyung

    2015-01-01

    Abnormal phosphorylation of tau has been considered as a key pathogenic mechanism inducing tau aggregation in multiple neurodegenerative disorders, collectively called tauopathies. Recent evidence showed that tau phosphorylation sites are protected with O-linked β-N-acetylglucosamine (O-GlcNAc) in normal brain. In pathological condition, tau is de-glycosylated and becomes a substrate for kinases. Despite the importance of O-GlcNAcylation in tau pathology, O-GlcNAc transferase (OGT), and an enzyme catalyzing O-GlcNAc to tau, has not been carefully investigated in the context of tau aggregation. Here, we investigated intracellular tau aggregation regulated by BZX2, an inhibitor of OGT. Upon the inhibition of OGT, tau phosphorylation increased 2.0-fold at Ser199 and 1.5-fold at Ser396, resulting in increased tau aggregation. Moreover, the BZX2 induced tau aggregation was efficiently reduced by the treatment of Thiamet G, an inhibitor of O-GlcNAcase (OGA). Our results demonstrated the protective role of OGT in tau aggregation and also suggest the counter-regulatory mechanism of OGA and OGT in tau pathology. PMID:26343633

  17. Intracellular chloride channel protein CLIC1 regulates macrophage function through modulation of phagosomal acidification

    PubMed Central

    Jiang, Lele; Salao, Kanin; Li, Hui; Rybicka, Joanna M.; Yates, Robin M.; Luo, Xu Wei; Shi, Xin Xin; Kuffner, Tamara; Tsai, Vicky Wang-Wei; Husaini, Yasmin; Wu, Liyun; Brown, David A.; Grewal, Thomas; Brown, Louise J.; Curmi, Paul M. G.; Breit, Samuel N.

    2012-01-01

    Summary Intracellular chloride channel protein 1 (CLIC1) is a 241 amino acid protein of the glutathione S transferase fold family with redox- and pH-dependent membrane association and chloride ion channel activity. Whilst CLIC proteins are evolutionarily conserved in Metazoa, indicating an important role, little is known about their biology. CLIC1 was first cloned on the basis of increased expression in activated macrophages. We therefore examined its subcellular localisation in murine peritoneal macrophages by immunofluorescence confocal microscopy. In resting cells, CLIC1 is observed in punctate cytoplasmic structures that do not colocalise with markers for endosomes or secretory vesicles. However, when these macrophages phagocytose serum-opsonised zymosan, CLIC1 translocates onto the phagosomal membrane. Macrophages from CLIC1−/− mice display a defect in phagosome acidification as determined by imaging live cells phagocytosing zymosan tagged with the pH-sensitive fluorophore Oregon Green. This altered phagosomal acidification was not accompanied by a detectable impairment in phagosomal-lysosomal fusion. However, consistent with a defect in acidification, CLIC1−/− macrophages also displayed impaired phagosomal proteolytic capacity and reduced reactive oxygen species production. Further, CLIC1−/− mice were protected from development of serum transfer induced K/BxN arthritis. These data all point to an important role for CLIC1 in regulating macrophage function through its ion channel activity and suggest it is a suitable target for the development of anti-inflammatory drugs. PMID:22956539

  18. Strain-specific regulation of intracellular Wolbachia density in multiply infected insects.

    PubMed

    Mouton, L; Henri, H; Bouletreau, M; Vavre, F

    2003-12-01

    Vertically transmitted symbionts suffer a severe reduction in numbers when they pass through host generations, resulting in genetic homogeneity or even clonality of their populations. Wolbachia endosymbionts that induce cytoplasmic incompatibility in their hosts depart from this rule, because cytoplasmic incompatibility actively maintains multiple infection within hosts. Hosts and symbionts are thus probably under peculiar selective pressures that must shape the way intracellular bacterial populations are regulated. We studied the density and location of Wolbachia within adult Leptopilina heterotoma, a haplodiploid wasp that is parasitic on Drosophila and that is naturally infected with three Wolbachia strains, but for which we also obtained one simply infected and two doubly infected lines. Comparison of these four lines by quantitative polymerase chain reaction using a real-time detection system showed that total Wolbachia density varies according to the infection status of individuals, while the specific density of each Wolbachia strain remains constant regardless of the presence of other strains. This suggests that Wolbachia strains do not compete with one another within the same host individual, and that a strain-specific regulatory mechanism is operating. We discuss the regulatory mechanisms that are involved, and how this process might have evolved as a response to selective pressures acting on both partners. PMID:14629360

  19. Intracellular calcium regulation among subpopulations of rat dorsal root ganglion neurons

    PubMed Central

    Lu, Shao-Gang; Zhang, Xiulin; Gold, Michael S

    2006-01-01

    Primary afferent neurons are functionally heterogeneous. To determine whether this functional heterogeneity reflects, in part, heterogeneity in the regulation of the concentration of intracellular Ca2+ ([Ca2+]i), the magnitude and decay of evoked Ca2+ transients were assessed in subpopulations of dorsal root ganglion (DRG) neurons with voltage clamp and fura-2 ratiometric imaging. To determine whether differences in evoked Ca2+ transients among subpopulations of DRG neurons reflected differences in the contribution of Ca2+ regulatory mechanisms, pharmacological techniques were employed to assess the contribution of influx, efflux, release and uptake pathways. Subpopulations of DRG neurons were defined by cell body size, binding of the plant lectin IB4 and responsiveness to the algogenic compound capsaicin (CAP). Ca2+ transients were evoked with 30 mm K+ or voltage steps to 0 mV. There were marked differences between subpopulations of neurons with respect to both the magnitude and decay of the Ca2+ transient, with the largest and most slowly decaying Ca2+ transients in small-diameter, IB4-positive, CAP-responsive neurons. The smallest and most rapidly decaying transients were in large-diameter, IB4-negative and CAP-unresponsive DRG neurons. These differences were not due to a differential distribution of voltage-gated Ca2+ currents. However, these differences did appear to reflect a differential contribution of other influx, efflux, release and uptake mechanisms between subpopulations of neurons. These results suggest that electrical activity in subpopulations of DRG neurons will have a differential influence on Ca2+-regulated phenomena such as spike adaptation, transmitter release and gene transcription. Significantly more activity should be required in large-diameter non-nociceptive afferents than in small-diameter nociceptive afferents to have a comparable influence on these processes. PMID:16945973

  20. Endothelial mitochondria regulate the intracellular Ca2+ response to fluid shear stress.

    PubMed

    Scheitlin, Christopher G; Julian, Justin A; Shanmughapriya, Santhanam; Madesh, Muniswamy; Tsoukias, Nikolaos M; Alevriadou, B Rita

    2016-03-15

    Shear stress is known to stimulate an intracellular free calcium concentration ([Ca(2+)]i) response in vascular endothelial cells (ECs). [Ca(2+)]i is a key second messenger for signaling that leads to vasodilation and EC survival. Although it is accepted that the shear-induced [Ca(2+)]i response is, in part, due to Ca(2+) release from the endoplasmic reticulum (ER), the role of mitochondria (second largest Ca(2+) store) is unknown. We hypothesized that the mitochondria play a role in regulating [Ca(2+)]i in sheared ECs. Cultured ECs, loaded with a Ca(2+)-sensitive fluorophore, were exposed to physiological levels of shear stress. Shear stress elicited [Ca(2+)]i transients in a percentage of cells with a fraction of them displaying oscillations. Peak magnitudes, percentage of oscillating ECs, and oscillation frequencies depended on the shear level. [Ca(2+)]i transients/oscillations were present when experiments were conducted in Ca(2+)-free solution (plus lanthanum) but absent when ECs were treated with a phospholipase C inhibitor, suggesting that the ER inositol 1,4,5-trisphosphate receptor is responsible for the [Ca(2+)]i response. Either a mitochondrial uncoupler or an electron transport chain inhibitor, but not a mitochondrial ATP synthase inhibitor, prevented the occurrence of transients and especially inhibited the oscillations. Knockdown of the mitochondrial Ca(2+) uniporter also inhibited the shear-induced [Ca(2+)]i transients/oscillations compared with controls. Hence, EC mitochondria, through Ca(2+) uptake/release, regulate the temporal profile of shear-induced ER Ca(2+) release. [Ca(2+)]i oscillation frequencies detected were within the range for activation of mechanoresponsive kinases and transcription factors, suggesting that dysfunctional EC mitochondria may contribute to cardiovascular disease by deregulating the shear-induced [Ca(2+)]i response. PMID:26739489

  1. Intracellular signaling in the regulation of renal Na-K-ATPase. II. Role of eicosanoids.

    PubMed Central

    Satoh, T; Cohen, H T; Katz, A I

    1993-01-01

    We recently reported a novel intracellular mechanism of renal Na-K-ATPase regulation by agents that increase cell cAMP, which involves protein kinase A-phospholipase A2 and is mediated by one or more arachidonic acid metabolites (Satoh, T., H. T. Cohen, and A. I. Katz. 1992. J. Clin. Invest. 89:1496). The present studies were, therefore, designed to assess the role of eicosanoids in the modulation of Na-K-ATPase activity in the rat cortical collecting duct. The effect of various cAMP agonists (dopamine, fenoldopam, vasopressin, forskolin, and dibutyryl cAMP), which inhibited the pump to a similar extent (approximately 50%), was independent of altered Na entry as it was elicited in the presence of amiloride or nystatin, or when NaCl was replaced with choline Cl. This effect was completely blocked by SKF 525A or ethoxyresorufin, two inhibitors of the cytochrome P450-dependent monooxygenase pathway, or by pretreating the animals with CoCl2, which depletes cytochrome P450. Equimolar concentrations (10(-7) M) of the cyclooxygenase inhibitors indomethacin or meclofenamate caused only a partial inhibition of the cAMP agonists' effect on the pump, whereas nordihydroguaiaretic acid or A 63162, two inhibitors of the lipoxygenase pathway, were without effect. Furthermore, two products of this pathway, leukotriene B4 and leukotriene D4, had no effect on Na-K-ATPase activity, and ICI 198615, a leukotriene receptor antagonist, did not alter pump inhibition by cAMP agonists. Several P450 monoxygenase arachidonic acid metabolites (5,6-epoxyeicosatrienoic acid; 11,12-epoxyeicosatrienoic acid; 11,12-dihydroxyeicosatrienoic acid; and 12(R)-hydroxyeicosatetraenoic acid) as well as PGE2 inhibited the Na:K pump in dose-dependent manner, but the effect of PGE2 was blocked when Na availability was altered, whereas that of 12(R)-HETE remained unchanged. We conclude that the cytochrome P450-monooxygenase pathway of the arachidonic acid cascade plays a major role in the modulation of Na

  2. Candida albicans erythroascorbate peroxidase regulates intracellular methylglyoxal and reactive oxygen species independently of D-erythroascorbic acid.

    PubMed

    Kwak, Min-Kyu; Song, Sung-Hyun; Ku, MyungHee; Kang, Sa-Ouk

    2015-07-01

    Candida albicans D-erythroascorbate peroxidase (EAPX1), which can catalyze the oxidation of D-erythroascorbic acid (EASC) to water, was observed to be inducible in EAPX1-deficient and EAPX1-overexpressing cells via activity staining. EAPX1-deficient cells have remarkably increased intracellular reactive oxygen species and methylglyoxal independent of the intracellular EASC content. The increased methylglyoxal caused EAPX1-deficient cells to activate catalase-peroxidase and cytochrome c peroxidase, which led to defects in cell growth, viability, mitochondrial respiration, filamentation and virulence. These findings indicate that EAPX1 mediates cell differentiation and virulence by regulating intracellular methylglyoxal along with oxidative stresses, regardless of endogenous EASC biosynthesis or alternative oxidase expression. PMID:25957768

  3. Glucose-regulated protein 78 is an intracellular antiviral factor against hepatitis B virus.

    PubMed

    Ma, Yan; Yu, Jun; Chan, Henry L Y; Chen, Yang-chao; Wang, Hua; Chen, Ying; Chan, Chu-yan; Go, Minnie Y Y; Tsai, Sau-na; Ngai, Sai-ming; To, Ka-fai; Tong, Joanna H M; He, Qing-Yu; Sung, Joseph J Y; Kung, Hsiang-fu; Cheng, Christopher H K; He, Ming-liang

    2009-11-01

    Hepatitis B virus (HBV) infection is a global public health problem that plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. However, the pathogenesis of HBV infection and the mechanisms of host-virus interactions are still elusive. In this study, two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomics were applied to analyze the host response to HBV using an inducible HBV-producing cell line, HepAD38. Twenty-three proteins were identified as differentially expressed with glucose-regulated protein 78 (GRP78) as one of the most significantly up-regulated proteins induced by HBV replication. This induction was further confirmed in both HepAD38 and HepG2 cells transfected with HBV-producing plasmids by real time RT-PCR and Western blotting as well as in HBV-infected human liver biopsies by immunohistochemistry. Knockdown of GRP78 expression by RNA interference resulted in a significant increase of both intracellular and extracellular HBV virions in the transient HBV-producing HepG2 cells concomitant with enhanced levels of hepatitis B surface antigen and e antigen in the culture medium. Conversely overexpression of GRP78 in HepG2 cells led to HBV suppression concomitant with induction of the positive regulatory circuit of GRP78 and interferon-beta1 (IFN-beta1). In this connection, the IFN-beta1-mediated 2',5'-oligoadenylate synthetase and RNase L signaling pathway was noted to be activated in GRP78-overexpressing HepG2 cells. Moreover GRP78 was significantly down-regulated in the livers of chronic hepatitis B patients after effective anti-HBV treatment (p = 0.019) as compared with their counterpart pretreatment liver biopsies. In conclusion, the present study demonstrates for the first time that GRP78 functions as an endogenous anti-HBV factor via the IFN-beta1-2',5'-oligoadenylate synthetase-RNase L pathway in hepatocytes. Induction of hepatic GRP78 may provide a novel therapeutic

  4. Crystal structure of cleaved vaspin (serpinA12).

    PubMed

    Pippel, Jan; Kuettner, E Bartholomeus; Ulbricht, David; Daberger, Jan; Schultz, Stephan; Heiker, John T; Sträter, Norbert

    2016-01-01

    The adipokine vaspin (serpinA12) is mainly expressed in white adipose tissue and exhibits various beneficial effects on obesity-related processes. Kallikrein 7 is the only known target protease of vaspin and is inhibited by the classical serpin inhibitory mechanism involving a cleavage of the reactive center loop between P1 (M378) and P1' (E379). Here, we present the X-ray structure of vaspin, cleaved between M378 and E379. We provide a comprehensive analysis of differences between the uncleaved and cleaved forms in the shutter, breach, and hinge regions with relation to common molecular features underlying the serpin inhibitory mode. Furthermore, we point out differences towards other serpins and provide novel data underlining the remarkable stability of vaspin. We speculate that the previously reported FKGx1Wx2x3 motif in the breach region may play a decisive role in determining the reactive center loop configuration in the native vaspin state and might contribute to the high thermostability of vaspin. Thus, this structure may provide a basis for future mutational studies. PMID:26529565

  5. Intra-ChIP: studying gene regulation in an intracellular pathogen.

    PubMed

    Hanson, Brett R; Tan, Ming

    2016-08-01

    Intracellular bacteria that reside within a host cell use a variety of strategies to exploit this unique niche. While these organisms are technically challenging to study in the context of an infected host cell, recent advances have led to an improved understanding of how the intracellular environment impacts bacterial gene expression. We recently demonstrated that chromatin immunoprecipitation (ChIP) can be used to quantify transcription factor binding in the obligate intracellular pathogen Chlamydia trachomatis within infected cells. Furthermore, we showed it was possible to experimentally modulate transcription factor binding while simultaneously measuring changes in transcription. Here we discuss these findings as well as other recent work that has used ChIP to study intracellular pathogens within infected cells. We also discuss technical considerations associated with this approach and its possible future applications. PMID:26886234

  6. Effect of glucagon on intracellular pH regulation in isolated rat hepatocyte couplets.

    PubMed Central

    Alvaro, D; Della Guardia, P; Bini, A; Gigliozzi, A; Furfaro, S; La Rosa, T; Piat, C; Capocaccia, L

    1995-01-01

    To elucidate mechanisms of glucagon-induced bicarbonate-rich choleresis, we investigated the effect of glucagon on ion transport processes involved in the regulation of intracellular pH (pHi) in isolated rat hepatocyte couplets. It was found that glucagon (200 nM), without influencing resting pHi, significantly stimulates the Cl-/HCO3- exchange activity. The effect of glucagon was associated with a sevenfold increase in cAMP levels in rat hepatocytes. The activity of the Cl-/HCO3- exchanger was also stimulated by DBcAMP + forskolin. The effect of glucagon on the Cl-/HCO3- exchange was individually blocked by two specific and selective inhibitors of protein kinase A, Rp-cAMPs (10 microM) and H-89 (30 microM), the latter having no influence on the glucagon-induced cAMP accumulation in isolated rat hepatocytes. The Cl- channel blocker, NPPB (10 microM), showed no effect on either the basal or the glucagon-stimulated Cl-/HCO3 exchange. In contrast, the protein kinase C agonist, PMA (10 microM), completely blocked the glucagon stimulation of the Cl-/HCO3- exchange; however, this effect was achieved through a significant inhibition of the glucagon-stimulated cAMP accumulation in rat hepatocytes. Colchicine pretreatment inhibited the basal as well as the glucagon-stimulated Cl-/HCO3- exchange activity. The Na+/H+ exchanger was unaffected by glucagon either at basal pHi or at acid pHi values. In contrast, glucagon, at basal pHi, stimulated the Na(+)-HCO3- symport. The main findings of this study indicate that glucagon, through the cAMP-dependent protein kinase A pathway, stimulates the activity of the Cl-/HCO3- exchanger in isolated rat hepatocyte couplets, a mechanism which could account for the in vivo induced bicarbonate-rich choleresis. Images PMID:7635959

  7. Developmental expression of chicken antithrombin III is regulated by increased RNA abundance and intracellular processing.

    PubMed

    Amrani, D L; Rosenberg, J; Samad, F; Bergtrom, G; Banfield, D K

    1993-01-23

    We isolated and sequenced a 432 bp cDNA to cAT-III, that encoded 115 nucleotides of 5' untranslated sequence, a 17 amino acid long signal peptide and residues 1-88 of the mature protein, and used it to prepare a probe for measuring and correlating the developmental changes of steady-state cAT-III mRNA levels with known changes in antigen levels. Densitometric analysis of nuclease protection (n = 2), Northern blot (n = 4), and slot blots (n = 3) of total RNA from chick livers of 16-day-old embryos to 6-day-old chicks showed a 2.6 +/- 0.5-fold increase in steady-state cAT-III mRNA levels. Assay of functional mRNA levels by in vitro translation of poly(A)+ RNA and specific immunoprecipitation of 35S-Met-labelled cAT-III was comparable to RNA analysis (16-day-old embryos vs. 10-day-old hatchlings). We evaluated whether there were developmental differences in post-translational secretion which may also contribute to the regulation of the circulating level of this protein. Pulse-chase studies of freshly-isolated hepatocytes from 16-day-old embryos and 10-day-old hatchlings maintained in suspension demonstrated a approx. 5.0-5.5-fold increase in cAT-III levels at steady-state secretion. The above findings indicate that changes in circulating cAT-III levels during late embryonic development are primarily due to increased abundance of cAT-III mRNA. In addition, we postulate that post-translational intracellular processing may account for further differences in circulating protein levels. PMID:8424948

  8. Altered intracellular pH regulation in cells with high levels of P-glycoprotein expression.

    PubMed

    Young, Gregory; Reuss, Luis; Altenberg, Guillermo A

    2011-01-01

    P-glycoprotein is an ATP-binding-cassette transporter that pumps many structurally unrelated drugs out of cells through an ATP-dependent mechanism. As a result, multidrug-resistant cells that overexpress P-glycoprotein have reduced intracellular steady-state levels of a variety of chemotherapeutic agents. In addition, increased cytosolic pH has been a frequent finding in multidrug-resistant cells that express P-glycoprotein, and it has been proposed that this consequence of P-glycoprotein expression may contribute to the lower intracellular levels of chemotherapeutic agents. In these studies, we measured intracellular pH and the rate of acid extrusion in response to an acid load in two cells with very different levels of P-glycoprotein expression: V79 parental cells and LZ-8 multidrug resistant cells. Compared to the wild-type V79 cells, LZ-8 cells have a lower intracellular pH and a slower recovery of intracellular pH after an acid load. The data also show that LZ-8 cells have reduced ability to extrude acid, probably due to a decrease in Na(+)/H(+) exchanger activity. The alterations in intracellular pH and acid extrusion in LZ-8 cells are reversed by 24-h exposure to the multidrug-resistance modulator verapamil. The lower intracellular pH in LZ-8 indicates that intracellular alkalinization is not necessary for multidrug resistance. The reversal by verapamil of the decreased acid-extrusion suggests that P-glycoprotein can affect other membrane transport mechanism. PMID:22003434

  9. Modulation of iron metabolism by iron chelation regulates intracellular calcium and increases sensitivity to doxorubicin

    PubMed Central

    Yalcintepe, Leman; Halis, Emre

    2016-01-01

    Increased intracellular iron levels can both promote cell proliferation and death, as such; iron has a “two-sided effect” in the delicate balance of human health. Though the role of iron in the development of cancer remains unclear, investigations of iron chelators as anti-tumor agents have revealed promising results. Here, we investigated the influence of iron and desferrioxamine (DFO), the iron chelating agent on intracellular calcium in a human leukemia cell line, K562. Iron uptake is associated with increased reactive oxygen species (ROS) generation. Therefore, we showed that iron also caused dose-dependent ROS generation in K562 cells. The measurement of intracellular calcium was determined using Furo-2 with a fluorescence spectrophotometer. The iron delivery process to the cytoplasmic iron pool was examined by monitoring the fluorescence of cells loaded with calcein-acetoxymethyl. Our data showed that iron increased intracellular calcium, and this response was 8 times higher when cells were incubated with DFO. K562 cells with DFO caused a 3.5 times increase of intracellular calcium in the presence of doxorubicin (DOX). In conclusion, DFO induces intracellular calcium and increases their sensitivity to DOX, a chemotherapeutic agent. PMID:26773173

  10. High resolution structure of cleaved Serpin 42 Da from Drosophila melanogaster

    PubMed Central

    2014-01-01

    Background The Drosophila melanogaster Serpin 42 Da gene (previously Serpin 4) encodes a serine protease inhibitor that is capable of remarkable functional diversity through the alternative splicing of four different reactive centre loop exons. Eight protein isoforms of Serpin 42 Da have been identified to date, targeting the protease inhibitor to both different proteases and cellular locations. Biochemical and genetic studies suggest that Serpin 42 Da inhibits target proteases through the classical serpin ‘suicide’ inhibition mechanism, however the crystal structure of a representative Serpin 42 Da isoform remains to be determined. Results We report two high-resolution crystal structures of Serpin 42 Da representing the A/B isoforms in the cleaved conformation, belonging to two different space-groups and diffracting to 1.7 Å and 1.8 Å. Structural analysis reveals the archetypal serpin fold, with the major elements of secondary structure displaying significant homology to the vertebrate serpin, neuroserpin. Key residues known to have central roles in the serpin inhibitory mechanism are conserved in both the hinge and shutter regions of Serpin 42 Da. Furthermore, these structures identify important conserved interactions that appear to be of crucial importance in allowing the Serpin 42 Da fold to act as a versatile template for multiple reactive centre loops that have different sequences and protease specificities. Conclusions In combination with previous biochemical and genetic studies, these structures confirm for the first time that the Serpin 42 Da isoforms are typical inhibitory serpin family members with the conserved serpin fold and inhibitory mechanism. Additionally, these data reveal the remarkable structural plasticity of serpins, whereby the basic fold is harnessed as a template for inhibition of a large spectrum of proteases by reactive centre loop exon ‘switching’. This is the first structure of a Drosophila serpin reported to date

  11. CaMKII regulates intracellular Ca²⁺ dynamics in native endothelial cells.

    PubMed

    Toussaint, Fanny; Charbel, Chimène; Blanchette, Alexandre; Ledoux, Jonathan

    2015-09-01

    Localized endothelial Ca(2+) signalling, such as Ca(2+) pulsars, can modulate the contractile state of the underlying vascular smooth muscle cell through specific endothelial targets. In addition to K(Ca)3.1 as a target, Ca(2+) pulsars, an IP3R-dependent pulsatile Ca(2+) release from the endoplasmic reticulum (ER) could activate a frequency-sensitive Ca(2+)-dependent kinase such as CaMKII. In the absence of extracellular Ca(2+), acetylcholine increased endothelial CaMKII phosphorylation and activation, thereby suggesting CaMKII activation independently of Ca(2+) influx. Herein, a reciprocal relation where CaMKII controls endothelial Ca(2+) dynamics has been investigated in mesenteric arteries. Both CaMKIIα and β isoforms have been identified in endothelial cells and close proximity (<40 nm) suggests their association in heteromultimers. Intracellular Ca(2+) monitoring with high speed confocal microscopy then showed that inhibition of CaMKII with KN-93 significantly increased the population of Ca(2+) pulsars active sites (+89%), suggesting CaMKII as a major regulator of Ca(2+) pulsars in native endothelium. Mechanistic insights were then sought through the elucidation of the impact of CaMKII on ER Ca(2+) store. ER Ca(2+) emptying was accelerated by CaMKII inhibition and ER Ca(2+) content was assessed using ionomycin. Exposure to KN-93 strongly diminished ER Ca(2+) content (-61%) by relieving CaMKII-dependent inhibition of IP3 receptors (IP3R). Moreover, in situ proximity ligation assay suggested CaMKII-IP3R promiscuity, essential condition for a protein-protein interaction. Interestingly, segregation of IP3R within myoendothelial projection (MEP) appears to be isoform-specific. Hence, only IP3R type 1 and type 2 are detected within fenestrations of the internal elastic lamina, sites of MEP, whilst type 3 is absent from these structures. In summary, CaMKII seems to act as a Ca(2+)-sensitive switch of a negative feedback loop regulating endothelial Ca(2

  12. Dynamic changes in intracellular ROS levels regulate airway basal stem cell homeostasis through Nrf2-dependent Notch signaling

    PubMed Central

    Paul, MK; Bisht, B; Darmawan, DO; Chiou, R; Ha, VL; Wallace, WD; Chon, AC; Hegab, AE; Grogan, T; Elashoff, DA; Alva-Ornelas, JA; Gomperts, BN

    2014-01-01

    SUMMARY Airways are exposed to myriad environmental and damaging agents such as reactive oxygen species (ROS), which also have physiological roles as signaling molecules that regulate stem cell function. However, the functional significance of both steady and dynamically changing ROS levels in different stem cell populations, as well as downstream mechanisms that integrate ROS sensing into decisions regarding stem cell homeostasis, are unclear. Here, we show in mouse and human airway basal stem cells (ABSCs) that intracellular flux from low to moderate ROS levels is required for stem cell self-renewal and proliferation. Changing ROS levels activate Nrf2, which activates the Notch pathway to stimulate ABSC self-renewal as well an antioxidant program that scavenges intracellular ROS, returning overall ROS levels to a low state to maintain homeostatic balance. This redox-mediated regulation of lung stem cell function has significant implications for stem cell biology, repair of lung injuries, and diseases such as cancer. PMID:24953182

  13. FAD-linked Presenilin-1 V97L mutation impede tranport regulation and intracellular Ca2+ homeostasis under ER stress

    PubMed Central

    Shao, Yankun; Li, Miao; Wu, Miao; Shi, Kai; Fang, Boyan; Wang, Jie

    2015-01-01

    We report a PS1 gene mutation (Val 97Leu) in a Chinese familial Alzheimer’s disease (FAD) pedigree and a cell model of FAD built by transfecting PS1 v97L mutants into human neuroblastoma SH-SY5Y cells. To test our hypothesis that the PS1 v97L mutation is pathogenic, we investigated possible alterations in transport regulation and intracellular Ca2+ homeostasis in endoplasmic reticulum (ER). Grp78 is an ER-resident chaperone mediating the unfolded protein response (UPR) and is a key regulator of ER stress transducers. KDEL is a 4-amino-acid retention sequence made of Lys-Asp-Glu-Leu-COO. KDEL is a “resident” sequence as protein residence in ER is consistently associated with KDEL at the C-extremity. Our group used KDEL recognizing anti-Grp78 monoclonal antibody to detect the level of Grp78. We found increased KDEL level in all the transfected cells including cells transfected with PS1 V97L genes, wild-type and the mock. However cells with PS1 V97L mutation expressed a relatively lower KDEL compared with the wild-type and the mock, and a significantly lower Grp78 level compared with the wild-type, the mock and control. These results suggest that PS1 V97L mutation impedes intracellular transport regulation in ER. PS1 V97L mutation mediates increased ER Ca2+ content in human neuroblastoma SH-SY5Y cells. The increased intracellular Ca2+ release is due to depleted Ca2+ storing content of ER but not due to extracellular environment as capacitative Ca2+ entry (CCE) is invariant. PS1 V97L mutation interferes with intracellular Ca2+ homeostasis. Abnormal transport regulation and Ca2+ homeostasis attributed to PS1 V97L mutation may be associated with the pathology of Chinese familial FAD. PMID:26884997

  14. Regulation of biofilm formation and cellular buoyancy through modulating intracellular cyclic di-GMP levels in engineered cyanobacteria.

    PubMed

    Agostoni, Marco; Waters, Christopher M; Montgomery, Beronda L

    2016-02-01

    The second messenger cyclic dimeric (3'→5') GMP (cyclic di-GMP or c-di-GMP) has been implicated in the transition between motile and sessile lifestyles in bacteria. In this study, we demonstrate that biofilm formation, cellular aggregation or flocculation, and cellular buoyancy are under the control of c-di-GMP in Synechocystis sp. PCC 6803 (Synechocystis) and Fremyella diplosiphon. Synechocystis is a unicellular cyanobacterium and displays lower levels of c-di-GMP; F. diplosiphon is filamentous and displays higher intracellular c-di-GMP levels. We transformed Synechocystis and F. diplosiphon with a plasmid for constitutive expression of genes encoding diguanylate cylase (DGC) and phosphodiesterase (PDE) proteins from Vibrio cholerae or Escherichia coli, respectively. These engineered strains allowed us to modulate intracellular c-di-GMP levels. Biofilm formation and cellular deposition were induced in the DGC-expressing Synechocystis strain which exhibited high intracellular levels of c-di-GMP; whereas strains expressing PDE in Synechocystis and F. diplosiphon to drive low intracellular levels of c-di-GMP exhibited enhanced cellular buoyancy. In addition, the PDE-expressing F. diplosiphon strain showed elevated chlorophyll levels. These results imply roles for coordinating c-di-GMP homeostasis in regulating native cyanobacterial phenotypes. Engineering exogenous DGC or PDE proteins to regulate intracellular c-di-GMP levels represents an effective tool for uncovering cryptic phenotypes or modulating phenotypes in cyanobacteria for practical applications in biotechnology applicable in photobioreactors and in green biotechnologies, such as energy-efficient harvesting of cellular biomass or the treatment of metal-containing wastewaters. PMID:26192200

  15. Structural and Inhibitory Effects of Hinge Loop Mutagenesis in Serpin-2 from the Malaria Vector Anopheles gambiae*

    PubMed Central

    Zhang, Xin; Meekins, David A.; An, Chunju; Zolkiewski, Michal; Battaile, Kevin P.; Kanost, Michael R.; Lovell, Scott; Michel, Kristin

    2015-01-01

    Serpin-2 (SRPN2) is a key negative regulator of the melanization response in the malaria vector Anopheles gambiae. SRPN2 irreversibly inhibits clip domain serine proteinase 9 (CLIPB9), which functions in a serine proteinase cascade culminating in the activation of prophenoloxidase and melanization. Silencing of SRPN2 in A. gambiae results in spontaneous melanization and decreased life span and is therefore a promising target for vector control. The previously determined structure of SRPN2 revealed a partial insertion of the hinge region of the reactive center loop (RCL) into β sheet A. This partial hinge insertion participates in heparin-linked activation in other serpins, notably antithrombin III. SRPN2 does not contain a heparin binding site, and any possible mechanistic function of the hinge insertion was previously unknown. To investigate the function of the SRPN2 hinge insertion, we developed three SRPN2 variants in which the hinge regions are either constitutively expelled or inserted and analyzed their structure, thermostability, and inhibitory activity. We determined that constitutive hinge expulsion resulted in a 2.7-fold increase in the rate of CLIPB9Xa inhibition, which is significantly lower than previous observations of allosteric serpin activation. Furthermore, we determined that stable insertion of the hinge region did not appreciably decrease the accessibility of the RCL to CLIPB9. Together, these results indicate that the partial hinge insertion in SRPN2 does not participate in the allosteric activation observed in other serpins and instead represents a molecular trade-off between RCL accessibility and efficient formation of an inhibitory complex with the cognate proteinase. PMID:25525260

  16. Regulation of B Cell Differentiation by Intracellular Membrane-Associated Proteins and microRNAs: Role in the Antibody Response.

    PubMed

    Lou, Zheng; Casali, Paolo; Xu, Zhenming

    2015-01-01

    B cells are central to adaptive immunity and their functions in antibody responses are exquisitely regulated. As suggested by recent findings, B cell differentiation is mediated by intracellular membrane structures (including endosomes, lysosomes, and autophagosomes) and protein factors specifically associated with these membranes, including Rab7, Atg5, and Atg7. These factors participate in vesicle formation/trafficking, signal transduction and induction of gene expression to promote antigen presentation, class switch DNA recombination (CSR)/somatic hypermutation (SHM), and generation/maintenance of plasma cells and memory B cells. Their expression is induced in B cells activated to differentiate and further fine-tuned by immune-modulating microRNAs, which coordinates CSR/SHM, plasma cell differentiation, and memory B cell differentiation. These short non-coding RNAs would individually target multiple factors associated with the same intracellular membrane compartments and collaboratively target a single factor in addition to regulating AID and Blimp-1. These, together with regulation of microRNA biogenesis and activities by endosomes and autophagosomes, show that intracellular membranes and microRNAs, two broadly relevant cell constituents, play important roles in balancing gene expression to specify B cell differentiation processes for optimal antibody responses. PMID:26579118

  17. The Pseudomonas aeruginosa Chp Chemosensory System Regulates Intracellular cAMP Levels by Modulating Adenylate Cyclase Activity

    PubMed Central

    Fulcher, Nanette B.; Holliday, Phillip M.; Klem, Erich; Cann, Martin J.; Wolfgang, Matthew C.

    2010-01-01

    Summary Multiple virulence systems in the opportunistic pathogen Pseudomonas aeruginosa are regulated by the second messenger signaling molecule adenosine 3’, 5’-cyclic monophosphate (cAMP). Production of cAMP by the putative adenylate cyclase enzyme CyaB represents a critical control point for virulence gene regulation. To identify regulators of CyaB, we screened a transposon insertion library for mutants with reduced intracellular cAMP. The majority of insertions resulting in reduced cAMP mapped to the Chp gene cluster encoding a putative chemotaxis-like chemosensory system. Further genetic analysis of the Chp system revealed that it has both positive and negative effects on intracellular cAMP and that it regulates cAMP levels by modulating CyaB activity. The Chp system was previously implicated in the production and function of type IV pili (TFP). Given that cAMP and the cAMP-dependent transcriptional regulator Vfr control TFP biogenesis gene expression, we explored the relationship between cAMP, the Chp system and TFP regulation. We discovered that the Chp system controls TFP production through modulation of cAMP while control of TFP-dependent twitching motility is cAMP-independent. Overall, our data define a novel function for a chemotaxis-like system in controlling cAMP production and establish a regulatory link between the Chp system, TFP and other cAMP-dependent virulence systems. PMID:20345659

  18. The role of chloride-bicarbonate exchange in the regulation of intracellular chloride in guinea-pig vas deferens.

    PubMed Central

    Aickin, C C; Brading, A F

    1984-01-01

    The Cl-depleted smooth muscle cells of guinea-pig vas deferens rapidly restore their intracellular Cl activity ( aiCl ) to a level 5 times higher than that predicted by a passive distribution when Cl- ions are readmitted to the extracellular solution. Cl reaccumulation, measured using Cl-sensitive micro-electrodes and the uptake of 36Cl, was slowed about 3-fold by the nominal absence of CO2 and HCO3 and about 10-fold by the presence of the anion exchange inhibitor DIDS (4-4'-diisothiocyanostilbene-2,2'-disulphonic acid). However, the usual level of intracellular Cl was eventually attained and neither condition reduced intracellular Cl in normal tissues. The loss of intracellular Cl that occurs when Cl- ions are removed from the extracellular solution was slowed about 3-fold by the nominal absence of CO2 and HCO3 and was accelerated by their readdition. DIDS slowed the fall in aiCl about 10-fold and reduced 36Cl efflux. Intracellular pH (pHi), measured using pH-sensitive micro-electrodes, increased by a mean of 0.73 units when Cl was removed from the superfusing solution in the presence of CO2 and HCO3, and rapidly decreased when Cl was readmitted. These changes are equivalent to an intracellular accumulation and loss of HCO3- ions respectively. The net transmembrane movement of HCO3- ions which would have caused these changes in pHi was calculated using the measured intracellular buffering power ( Aickin , 1984). 25% fewer HCO3- ions than Cl- ions were accumulated and lost and the HCO3 movement was completed 2-3 times faster than the simultaneous Cl movement under the same conditions. The changes in pHi induced by alteration of extracellular Cl were reduced by the nominal absence of CO2 and HCO3 and abolished by the presence of DIDS. The acidification recorded on readmission of Cl in the nominal absence of CO2 and HCO3 was compatible with a metabolic production of about 0.1% CO2. We conclude that Cl-HCO3 exchange plays a major role in the regulation of

  19. MicroRNAs in the intracellular space, regulation of organelle specific pathways in health and disease.

    PubMed

    Nguyen, Thao T; Brenu, Ekua W; Staines, Don R; Marshall-Gradisnik, Sonya M

    2014-01-01

    MicroRNAs (miRNA) are small (~22 nucleotide] non-coding RNA molecules originally characterised as nonsense or junk DNA. Emerging research suggests that these molecules have diverse regulatory roles in an array of molecular, cellular and physiological processes. MiRNAs are versatile and highly stable molecules, therefore, they are able to exist as intracellular or extracellular miRNAs. The purpose of this paper is to review the function and role of miRNAs in the intracellular space with specific focus on the interactions between miRNAs and organelles such as the mitochondria and the rough endoplasmic reticulum. Understanding the role of miRNAs in the intracellular space may be vital in understanding the mechanism of certain diseases. PMID:25541912

  20. Regulation of intracellular calcium is closely linked to glucose metabolism in J774 macrophages.

    PubMed

    Darbha, S; Marchase, R B

    1996-10-01

    The effects of 2-deoxy-D-glucose (2dGlc) and glucose deprivation were investigated in the J774 murine macrophage-like cell line. 2dGlc addition or glucose deprivation for 4 min led to an inhibition in the transient increase in cytoplasmic free Ca2+ ([Ca2+]i) that otherwise occurs in response to three different agonists: IgG, ATP and platelet activating factor. This inhibition was preceded by a partial release of Ca2+ from intracellular, thapsigargin-sensitive stores. In contrast, the transition from 5 to 30 mM glucose caused a decrease in [Ca2+]i and a corresponding increase in thapsigargin-sensitive sequestered Ca2+. The effects of an alternate glycolytic inhibitor, NaF, and a mitochondrial inhibitor, rotenone, were also tested. These inhibitors caused neither a release of Ca2+ from intracellular stores nor an inhibition in any of the agonist responses. The capacitative influx of extracellular Ca2+ following depletion of intracellular stores was also found to be selectively inhibited by the prior addition of 2dGlc or with glucose deprivation. In addition, when an elevated plateau of [Ca2+]i was established by the irreversible depletion of intracellular Ca2+ stores, the addition of 2dGlc caused a decrease in the on-going capacitative entry of Ca2+. PMID:8939356

  1. The C-terminal tail of tetraspanin proteins regulates their intracellular distribution in the parasite Trichomonas vaginalis.

    PubMed

    Coceres, V M; Alonso, A M; Nievas, Y R; Midlej, V; Frontera, L; Benchimol, M; Johnson, P J; de Miguel, N

    2015-08-01

    The parasite Trichomonas vaginalis is the causative agent of trichomoniasis, a prevalent sexually transmitted infection. Here, we report the cellular analysis of T.vaginalis tetraspanin family (TvTSPs). This family of membrane proteins has been implicated in cell adhesion, migration and proliferation in vertebrates. We found that the expression of several members of the family is up-regulated upon contact with vaginal ectocervical cells. We demonstrate that most TvTSPs are localized on the surface and intracellular vesicles and that the C-terminal intracellular tails of surface TvTSPs are necessary for proper localization. Analyses of full-length TvTSP8 and a mutant that lacks the C-terminal tail indicates that surface-localized TvTSP8 is involved in parasite aggregation, suggesting a role for this protein in parasite : parasite interaction. PMID:25703821

  2. THE ROLE OF INTRACELLULAR SODIUM (Na+) IN THE REGULATION OF CALCIUM (Ca2+)-MEDIATED SIGNALING AND TOXICITY

    PubMed Central

    Yu, Xian-Min; Groveman, Bradley R; Fang, Xiao-Qian; Lin, Shuang-Xiu

    2010-01-01

    It is known that activated N-methyl-D-aspartate receptors (NMDARs) are a major route of excessive calcium ion (Ca2+) entry in central neurons, which may activate degradative processes and thereby cause cell death. Therefore, NMDARs are now recognized to play a key role in the development of many diseases associated with injuries to the central nervous system (CNS). However, it remains a mystery how NMDAR activity is recruited in the cellular processes leading to excitotoxicity and how NMDAR activity can be controlled at a physiological level. The sodium ion (Na+) is the major cation in extracellular space. With its entry into the cell, Na+ can act as a critical intracellular second messenger that regulates many cellular functions. Recent data have shown that intracellular Na+ can be an important signaling factor underlying the up-regulation of NMDARs. While Ca2+ influx during the activation of NMDARs down-regulates NMDAR activity, Na+ influx provides an essential positive feedback mechanism to overcome Ca2+-induced inhibition and thereby potentiate both NMDAR activity and inward Ca2+ flow. Extensive investigations have been conducted to clarify mechanisms underlying Ca2+-mediated signaling. This review focuses on the roles of Na+ in the regulation of Ca2+-mediated NMDAR signaling and toxicity. PMID:21243124

  3. Intracellular sodium activity and its regulation in guinea-pig atrial myocardium.

    PubMed Central

    Wang, G X; Schmied, R; Ebner, F; Korth, M

    1993-01-01

    1. Intracellular Na+ activity (aNai) and membrane resting potential were studied in quiescent guinea-pig atrial and papillary muscles by means of Na(+)-sensitive and conventional microelectrodes. The effects of the cardioactive steroid dihydroouabain (DHO) on aiNa, force of contraction and sarcolemmal Na+, K(+)-ATPase activity were also investigated. 2. In thirty atria and twenty-two papillary muscles, aNai amounted to 8.0 +/- 0.2 and 4.7 +/- 0.3 mM, respectively (mean +/- S.E.M.). When both tissues were from the same animal, with the same ion-sensitive microelectrode mean aNai values of 7.9 +/- 0.2 and 5.1 +/- 0.5 mM (P < 0.01) were obtained from eight atrial and eight papillary muscles, respectively. 3. Membrane resting potentials (Em) were significantly (P < 0.001) more negative in the papillary muscles (-83.5 +/- 0.7 mV; n = 8) than in the atrium (-78.1 +/- 0.5 mV; n = 8). Deviation of Em from EK (determined by K(+)-sensitive microelectrodes) was 3.0 +/- 0.2 mV in ventricular (P < 0.05) and 6.1 +/- 0.3 mV in atrial preparations (P < 0.05). 4. Inhibition of the Na+ pump by DHO increased aNai of the atrium within 10 min by 0.6 +/- 0.1 (n = 7), 1.3 +/- 0.1 (n = 5) and 3.2 +/- 0.2 mM (n = 5) at 5, 10 and 30 microM, respectively. In the papillary muscle, 10 microM DHO was without effect while aNai rose by 1.0 +/- 0.1 (n = 5) and 2.9 +/- 0.2 mM (n = 6) at 30 and 120 microM DHO. 5. Consistent with the aNai measurements, the potency of DHO to increase force of the isometric contraction was three times higher in atrium than in papillary muscle (stimulation frequency 0.2 Hz). 6. Hydrolytic activity of sarcolemmal Na+,K(+)-ATPase isolated from atria amounted to only one third of that detected in ventricles (0.07 +/- 0.01, n = 6, versus 0.2 +/- 0.01 mumol phosphate released min-1 (g tissue)-1, n = 5). The inhibitory potencies of DHO on sarcolemmal Na+,K(+)-ATPase preparations were found to be identical in the enzymes from either tissue. 7. It is concluded that a lower Na

  4. Ubiquitination of Innate Immune Regulator TRAF3 Orchestrates Expulsion of Intracellular Bacteria by Exocyst Complex.

    PubMed

    Miao, Yuxuan; Wu, Jianxuan; Abraham, Soman N

    2016-07-19

    Although the intracellular trafficking system is integral to most physiologic activities, its role in mediating immune responses to infection has remained elusive. Here, we report that infected bladder epithelial cells (BECs) mobilized the exocyst complex, a powerful exporter of subcellular vesicles, to rapidly expel intracellular bacteria back for clearance. Toll-like receptor (TLR) 4 signals emanating from bacteria-containing vesicles (BCVs) were found to trigger K33-linked polyubiquitination of TRAF3 at Lys168, which was then detected by RalGDS, a guanine nucleotide exchange factor (GEF) that precipitated the assembly of the exocyst complex. Although this distinct modification of TRAF3 served to connect innate immune signaling to the cellular trafficking apparatus, it crucially ensured temporal and spatial accuracy in determining which among the many subcellular vesicles was recognized and selected for expulsion in response to innate immune signaling. PMID:27438768

  5. Modulating intracellular acidification by regulating the incubation time of proton caged compounds.

    PubMed

    Carbone, Marilena; Sabbatella, Gianfranco; Antonaroli, Simonetta; Orlando, Viviana; Biagioni, Stefano; Nucara, Alessandro

    2016-09-01

    A proton caged compound, the 1-(2-nitrophenyl)- ethylhexadecyl sulfonate (HDNS), was dosed into HEK-293 at different incubation times. Samples were irradiated with filtered UV light for inducing photolysis of the HDNS and then probed by infrared spectroscopy. The intracellular acidification reaction can be followed by monitoring the consequent CO2 peak intensity variation. The total CO2 produced is similar for all the samples, hence it is only a function of the initial HDNS concentration. The way it is achieved, though, is different for the different incubation times and follows kinetics, which results in a combination of a linear CO2 increase and a steep CO2 increase followed by a decay. This is interpreted in terms of confinement of the HDNS into intracellular vesicles of variable average size and sensitive to UV light when they reach critical dimensions. PMID:27017356

  6. Serpine2/PN-1 Is Required for Proliferative Expansion of Pre-Neoplastic Lesions and Malignant Progression to Medulloblastoma

    PubMed Central

    Vaillant, Catherine; Kool, Marcel; Schwarzentruber-Schauerte, Alexandra; Méreau, Hélène; Cabuy, Erik; Lobrinus, Johannes A.; Pfister, Stefan; Zuniga, Aimée; Frank, Stephan; Zeller, Rolf

    2015-01-01

    Background Medulloblastomas are malignant childhood brain tumors that arise due to the aberrant activity of developmental pathways during postnatal cerebellar development and in adult humans. Transcriptome analysis has identified four major medulloblastoma subgroups. One of them, the Sonic hedgehog (SHH) subgroup, is caused by aberrant Hedgehog signal transduction due to mutations in the Patched1 (PTCH1) receptor or downstream effectors. Mice carrying a Patched-1 null allele (Ptch1∆/+) are a good model to study the alterations underlying medulloblastoma development as a consequence of aberrant Hedgehog pathway activity. Results Transcriptome analysis of human medulloblastomas shows that SERPINE2, also called Protease Nexin-1 (PN-1) is overexpressed in most medulloblastomas, in particular in the SHH and WNT subgroups. As siRNA-mediated lowering of SERPINE2/PN-1 in human medulloblastoma DAOY cells reduces cell proliferation, we analyzed its potential involvement in medulloblastoma development using the Ptch1∆/+ mouse model. In Ptch1∆/+ mice, medulloblastomas arise as a consequence of aberrant Hedgehog pathway activity. Genetic reduction of Serpine2/Pn-1 interferes with medulloblastoma development in Ptch1∆/+ mice, as ~60% of the pre-neoplastic lesions (PNLs) fail to develop into medulloblastomas and remain as small cerebellar nodules. In particular the transcription factor Atoh1, whose expression is essential for development of SHH subgroup medulloblastomas is lost. Comparative molecular analysis reveals the distinct nature of the PNLs in young Ptch1∆/+Pn-1Δ/+ mice. The remaining wild-type Ptch1 allele escapes transcriptional silencing in most cases and the aberrant Hedgehog pathway activity is normalized. Furthermore, cell proliferation and the expression of the cell-cycle regulators Mycn and Cdk6 are significantly reduced in PNLs of Ptch1∆/+Pn-1Δ/+ mice. Conclusions Our analysis provides genetic evidence that aberrant Serpine2/Pn-1 is required for

  7. Burkholderia pseudomallei Differentially Regulates Host Innate Immune Response Genes for Intracellular Survival in Lung Epithelial Cells

    PubMed Central

    Vellasamy, Kumutha Malar; Mariappan, Vanitha; Shankar, Esaki M.; Vadivelu, Jamuna

    2016-01-01

    Background Burkholderia pseudomallei, the causative agent of melioidosis poses a serious threat to humankind. B. pseudomallei secretes numerous virulence proteins that alter host cell functions to escape from intracellular immune sensors. However, the events underlying disease pathogenesis are poorly understood. Methods We determined the ability of B. pseudomallei to invade and survive intracellularly in A549 human lung epithelial cells, and also investigated the early transcriptional responses using an Illumina HumanHT-12 v4 microarray platform, after three hours of exposure to live B. pseudomallei (BCMS) and its secreted proteins (CCMS). Results We found that the ability of B. pseudomallei to invade and survive intracellularly correlated with increase of multiplicity of infection and duration of contact. Activation of host carbohydrate metabolism and apoptosis as well as suppression of amino acid metabolism and innate immune responses both by live bacteria and its secreted proteins were evident. These early events might be linked to initial activation of host genes directed towards bacterial dissemination from lungs to target organs (via proposed in vivo mechanisms) or to escape potential sensing by macrophages. Conclusion Understanding the early responses of A549 cells toward B. pseudomallei infection provide preliminary insights into the likely pathogenesis mechanisms underlying melioidosis, and could contribute to development of novel intervention strategies to combat B. pseudomallei infections. PMID:27367858

  8. Agouti regulation of intracellular calcium: Role in the insulin resistance of viable yellow mice

    SciTech Connect

    Zemel, M.B.; Kim, J.H.; Woychik, R.P.; Michaud, E.J.; Hadwell, S.H.; Patel, I.R.; Wilkison, W.O.

    1995-05-23

    Several dominant mutations at the agouti locus in the mouse cause a syndrome of marked obesity, hyperinsulinemia, and insulin resistance. Although it is known that the agouti gene is expressed in an ectopic manner in these mutants, the precise mechanism by which the agouti gene product mediates these effects is unclear. Since intracellular Ca{sup 2+} is believed to play a role in mediating insulin action and dysregulation of Ca{sup 2+} flux is observed in diabetic animals and humans, we examined the status of intracellular Ca{sup 2+} in mice carrying the dominant agouti allele, viable yellow (A{sup vy}). We show here that in mice carrying this mutation, the intracellular free calcium concentration ([Ca{sup 2+}]{sub i}) is elevated in skeletal muscle, and the degree of elevation is closely correlated with the degree to which the mutant traits are expressed in individual animals. Moreover, we demonstrate that the agouti gene product is capable of inducing increased [Ca{sup 2+}]{sub i} in cultured and freshly isolated skeletal muscle myocytes from wild-type mice. Based on these findings, we present a model in which we propose that the agouti polypeptide promotes insulin resistance in mutant animals through its ability to increase [Ca{sup 2+}]{sub i}. 36 refs., 3 figs., 2 tabs.

  9. Changes in intracellular copper concentration and copper-regulating gene expression after PC12 differentiation into neurons.

    PubMed

    Ogra, Yasumitsu; Tejima, Aya; Hatakeyama, Naohiro; Shiraiwa, Moeko; Wu, Siyuan; Ishikawa, Tsutomu; Yawata, Ayako; Anan, Yasumi; Suzuki, Noriyuki

    2016-01-01

    It is suspected that some neurodegenerative diseases are a result of the disturbance of copper (Cu) homeostasis, although it remains unclear whether the disturbance of Cu homeostasis has aberrant effects on neurons. Herein, we investigated Cu metabolism specifically in neurons in terms of changes in the intracellular Cu concentration and the expression of Cu-regulating genes, such as Cu transporters and metallothioneins (MTs), before and after the differentiation of rat pheochromocytoma cells (PC12 cells) into neurons. After the differentiation, Cu and Zn imaging with fluorescent probes revealed an increase in intracellular Cu concentration. The concentrations of other essential metals, which were determined by an inductively coupled plasma mass spectrometer, were not altered. The mRNA expression of the Cu influx transporter, Ctr1, was decreased after the differentiation, and the differentiated cells acquired tolerance to Cu and cisplatin, another substrate of Ctr1. In addition, the expression of MT-3, a brain-specific isoform, was increased, contrary to the decreased expression of MT-1 and MT-2. Taken together, the differentiation of PC12 cells into neurons induced MT-3 expression, thereby resulting in intracellular Cu accumulation. The decrease in Ctr1 expression was assumed to be a response aimed at abolishing the physiological accumulation of Cu after the differentiation. PMID:27623342

  10. Chloride Channels of Intracellular Membranes

    PubMed Central

    Edwards, John C.; Kahl, Christina R.

    2010-01-01

    Proteins implicated as intracellular chloride channels include the intracellular ClC proteins, the bestrophins, the cystic fibrosis transmembrane conductance regulator, the CLICs, and the recently described Golgi pH regulator. This paper examines current hypotheses regarding roles of intracellular chloride channels and reviews the evidence supporting a role in intracellular chloride transport for each of these proteins. PMID:20100480

  11. Protein-protein interactions involving voltage-gated sodium channels: Post-translational regulation, intracellular trafficking and functional expression.

    PubMed

    Shao, Dongmin; Okuse, Kenji; Djamgoz, Mustafa B A

    2009-07-01

    Voltage-gated sodium channels (VGSCs), classically known to play a central role in excitability and signalling in nerves and muscles, have also been found to be expressed in a range of 'non-excitable' cells, including lymphocytes, fibroblasts and endothelia. VGSC abnormalities are associated with various diseases including epilepsy, long-QT syndrome 3, Brugada syndrome, sudden infant death syndrome and, more recently, various human cancers. Given their pivotal role in a wide range of physiological and pathophysiological processes, regulation of functional VGSC expression has been the subject of intense study. An emerging theme is post-translational regulation and macro-molecular complexing by protein-protein interactions and intracellular trafficking, leading to changes in functional VGSC expression in plasma membrane. This partially involves endoplasmic reticulum associated degradation and ubiquitin-proteasome system. Several proteins have been shown to associate with VGSCs. Here, we review the interactions involving VGSCs and the following proteins: p11, ankyrin, syntrophin, beta-subunit of VGSC, papin, ERM and Nedd4 proteins. Protein kinases A and C, as well as Ca(2+)-calmodulin dependent kinase II that have also been shown to regulate intracellular trafficking of VGSCs by changing the balance of externalization vs. internalization, and an effort is made to separate these effects from the short-term phosphorylation of mature proteins in plasma membrane. Two further modulatory mechanisms are reciprocal interactions with the cytoskeleton and, late-stage, activity-dependent regulation. Thus, the review gives an updated account of the range of post-translational molecular mechanisms regulating functional VGSC expression. However, many details of VGSC subtype-specific regulation and pathophysiological aspects remain unknown and these are highlighted throughout for completeness. PMID:19401147

  12. Transmembrane protein OSTA-1 shapes sensory cilia morphology via regulation of intracellular membrane trafficking in C. elegans

    PubMed Central

    Olivier-Mason, Anique; Wojtyniak, Martin; Bowie, Rachel V.; Nechipurenko, Inna V.; Blacque, Oliver E.; Sengupta, Piali

    2013-01-01

    The structure and function of primary cilia are critically dependent on intracellular trafficking pathways that transport ciliary membrane and protein components. The mechanisms by which these trafficking pathways are regulated are not fully characterized. Here we identify the transmembrane protein OSTA-1 as a new regulator of the trafficking pathways that shape the morphology and protein composition of sensory cilia in C. elegans. osta-1 encodes an organic solute transporter alpha-like protein, mammalian homologs of which have been implicated in membrane trafficking and solute transport, although a role in regulating cilia structure has not previously been demonstrated. We show that mutations in osta-1 result in altered ciliary membrane volume, branch length and complexity, as well as defects in localization of a subset of ciliary transmembrane proteins in different sensory cilia types. OSTA-1 is associated with transport vesicles, localizes to a ciliary compartment shown to house trafficking proteins, and regulates both retrograde and anterograde flux of the endosome-associated RAB-5 small GTPase. Genetic epistasis experiments with sensory signaling, exocytic and endocytic proteins further implicate OSTA-1 as a crucial regulator of ciliary architecture via regulation of cilia-destined trafficking. Our findings suggest that regulation of transport pathways in a cell type-specific manner contributes to diversity in sensory cilia structure and might allow dynamic remodeling of ciliary architecture via multiple inputs. PMID:23482491

  13. Regulation of cAMP Intracellular Levels in Human Platelets Stimulated by 2-Arachidonoylglycerol.

    PubMed

    Signorello, Maria Grazia; Leoncini, Giuliana

    2016-05-01

    We demonstrated that in human platelets the endocannabinoid 2-arachidonoylglycerol (2-AG) decreased dose- and time-dependently cAMP intracellular levels. No effect on cAMP decrease induced by 2-AG was observed in the presence of the adenylate cyclase inhibitor SQ22536 as well in platelets pretreated with the thromboxane A2 receptor antagonist, SQ29548 or with aspirin, inhibitor of arachidonic acid metabolism through the cyclooxygenase pathway. An almost complete recovering of cAMP level was measured in platelets pretreated with the specific inhibitor of phosphodiesterase (PDE) 3A, milrinone. In platelets pretreated with LY294002 or MK2206, inhibitors of PI3K/AKT pathway, and with U73122, inhibitor of phospholipase C pathway, only a partial prevention was shown. cAMP intracellular level depends on synthesis by adenylate cyclase and hydrolysis by PDEs. In 2-AG-stimulated platelets adenylate cyclase activity seems to be unchanged. In contrast PDEs appear to be involved. In particular PDE3A was specifically activated, as milrinone reversed cAMP reduction by 2-AG. 2-AG enhanced PDE3A activity through its phosphorylation. The PI3K/AKT pathway and PKC participate to this PDE3A phosphorylation/activation mechanism as it was greatly inhibited by platelet pretreatment with LY294002, MK2206, U73122, or the PKC specific inhibitor GF109203X. Taken together these data suggest that 2-AG potentiates its power of platelet agonist reducing cAMP intracellular level. J. Cell. Biochem. 117: 1240-1249, 2016. © 2015 Wiley Periodicals, Inc. PMID:26460717

  14. Epigenetics: A New Model for Intracellular Parasite-Host Cell Regulation.

    PubMed

    Robert McMaster, W; Morrison, Charlotte J; Kobor, Michael S

    2016-07-01

    Intracellular protozoan parasites are an extremely important class of pathogens that cause a spectrum of diseases in human and animal hosts. There is a growing body of evidence suggesting that protozoan parasites, like other prokaryotic and viral pathogens, manipulate host cells via epigenetic modifications of the host genome that alter transcription and corresponding signaling pathways. In light of these data, we examine the role of epigenetics in downregulation of host macrophages by Leishmania that could potentially lead to a permanent state of inactivation, thus favoring pathogen survival and disease progression. PMID:27142564

  15. LacI(Ts)-regulated expression as an in situ intracellular biomolecular thermometer.

    PubMed

    McCabe, K M; Lacherndo, E J; Albino-Flores, I; Sheehan, E; Hernandez, M

    2011-05-01

    In response to needs for in situ thermometry, a temperature-sensitive vector was adapted to report changes in the intracellular heat content of Escherichia coli in near-real time. This model system utilized vectors expressing increasing quantities of β-galactosidase in response to stepwise temperature increases through a biologically relevant range (22 to 45°C). As judged by calibrated fluorometric and colorimetric reporters, both whole E. coli cells and lysates expressed significant repeatable changes in β-galactosidase activity that were sensitive to temperature changes of less than 1°C (35 to 45°C). This model system suggests that changes in cellular heat content can be detected independently of the medium in which cells are maintained, a feature of particular importance where the medium is heterogeneous or nonaqueous, or otherwise has a low heat transfer capacity. We report here that the intracellular temperature can be reliably obtained in near-real time using reliable fluorescent reporting systems from cellular scales, with a 20°C range of detection and at least 0.7°C sensitivity between 35 and 45°C. PMID:21378059

  16. Tumorigenic Poxviruses Up-Regulate Intracellular Superoxide To Inhibit Apoptosis and Promote Cell Proliferation

    PubMed Central

    Teoh, Melissa L. T.; Turner, Patricia V.; Evans, David H.

    2005-01-01

    Tumorigenic leporipoxviruses encode catalytically inactive homologs of cellular Cu-Zn superoxide dismutase (SOD1). The function of the orthologous myxoma virus M131R and Shope fibroma virus S131R gene products is uncertain, but they inhibit SOD1 activity by a process linked to binding its copper chaperone. Using a superoxide-sensitive dye (hydroethidine), we observed that virus infection increased intracellular superoxide levels in an M/S131R-dependent manner. To see whether this effect promotes infection, we deleted the Shope fibroma virus S131R gene and compared the clinical manifestations of wild-type and mutant virus infections in rabbits. S131RΔ virus produced significantly smaller fibroxanthosarcoma-like growths in vivo and, at a point where these growths were already receding, wild-type infections still showed extensive leukocyte infiltration, necrosis, and fibromatous cell proliferation. Coincidentally, whereas Jurkat cells are protected from mitochondria- and Fas-mediated apoptosis by wild-type myxoma virus in vitro, M131RΔ virus could not block Fas-initiated apoptosis as judged by DNA laddering, terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein nick end labeling, and caspase 3 cleavage assays. These data suggest that tumorigenic poxviruses can modulate intracellular redox status to their advantage to stimulate infected cell growth and inhibit programmed cell death. PMID:15827194

  17. Regulation of intracellular levels of calmodulin and tubulin in normal and transformed cells.

    PubMed Central

    Chafouleas, J G; Pardue, R L; Brinkley, B R; Dedman, J R; Means, A R

    1981-01-01

    Transformation of mammalian tissue culture cells by oncogenic viruses results in a 2-fold increase in the intracellular concentration of calmodulin quantitated by radioimmunoassay. The two pairs of companion cell lines used in this study were the Swiss mouse 3T3/simian virus 40-transformed 3T3 cells and the normal rat kidney (NRK)/Rous sarcoma virus-transformed NRK cells. The increased intracellular levels of calmodulin in the transformed cells are due to a greater increase in the rate of synthesis (3-fold) relative to the change in the rate of degradation (1.4-fold). On the other hand, no increases were observed in tubulin levels as quantitated by a colchicine-binding assay. The lack of change in tubulin concentration was accounted for by a 2-fold increase in the rate of degradation that is compensated by a similar increase in the rate of synthesis. The consequence of such changes in both transformed cell types is a 2-fold increase in the calmodulin-to-tubulin protein ratio relative to that in their nontransformed counterparts. PMID:6262788

  18. The Aβ-clearance protein transthyretin, like neprilysin, is epigenetically regulated by the amyloid precursor protein intracellular domain.

    PubMed

    Kerridge, Caroline; Belyaev, Nikolai D; Nalivaeva, Natalia N; Turner, Anthony J

    2014-08-01

    Proteolytic cleavage of the amyloid precursor protein (APP) by the successive actions of β- and γ-secretases generates several biologically active metabolites including the amyloid β-peptide (Aβ) and the APP intracellular domain (AICD). By analogy with the Notch signalling pathway, AICD has been proposed to play a role in transcriptional regulation. Among the cohort of genes regulated by AICD is the Aβ-degrading enzyme neprilysin (NEP). AICD binds to the NEP promoter causing transcriptional activation by competitive replacement with histone deacetylases (HDACs) leading to increased levels of NEP activity and hence increased Aβ clearance. We now show that the Aβ-clearance protein transthyretin (TTR) is also epigenetically up-regulated by AICD. Like NEP regulation, AICD derived specifically from the neuronal APP isoform, APP695 , binds directly to the TTR promoter displacing HDAC1 and HDAC3. Cell treatment with the tyrosine kinase inhibitor Gleevec (imatinib) or with the alkalizing agent NH4 Cl causes an accumulation of 'functional' AICD capable of up-regulating both TTR and NEP, leading to a reduction in total cellular Aβ levels. Pharmacological regulation of both NEP and TTR might represent a viable therapeutic target in Alzheimer's disease. PMID:24528201

  19. Opposite regulation of cocaine-induced intracellular signaling and gene expression by dopamine D1 and D3 receptors.

    PubMed

    Zhang, Jianhua; Xu, Ming

    2006-08-01

    Repeated exposure to cocaine induces persistent neuroadaptations that involve alterations in cellular signaling and gene expression mediated by dopamine (DA) receptors in the brain. Both dopamine D1 and D3 receptors mediate cocaine-induced behaviors and they are also coexpressed in the same neurons in the nucleus accumbens (NAc) and caudoputamen (CPu). We have investigated whether these two receptors coordinately regulate intracellular signaling and gene expression after acute and repeated cocaine administration. We found that extracellular signal-regulated kinase (ERK) activation and c-fos induction in the CPu following an acute cocaine administration is mediated by the D1 receptor and inhibited by the D3 receptor. ERK activation is necessary for acute cocaine-induced expression of fos family genes that include c-fos, fosB, and fra2. Furthermore, potential target genes of cAMP response element-binding (CREB) protein and/or AP-1 transcription complex, including dynorphin, neogenin, and synaptotagmin VII, are also oppositely regulated by D1 and D3 receptors after repeated cocaine injections. Lastly, such regulation requires proper ERK activation. These results suggest that D1 and D3 receptors oppositely regulate target gene expression by regulating ERK activation after cocaine administration. PMID:17105899

  20. Purinergic regulation of cation conductances and intracellular Ca2+ in cultured rat retinal pigment epithelial cells

    PubMed Central

    Ryan, Jennifer S; Baldridge, William H; Kelly, Melanie E M

    1999-01-01

    We used whole-cell patch clamp and fluorescent calcium imaging techniques to investigate the effects of adenosine 5′-triphosphate (ATP) on membrane currents and intracellular calcium concentration ([Ca2+]i)in rat retinal pigment epithelial (RPE) cells. In 62 % of RPE cells, application of 100 μM ATP elicited a fast inward current at negative membrane potentials. In 38 % of RPE cells recorded, a biphasic response to ATP was observed in which activation of the fast inward current was followed by activation of a delayed outward current. The ATP-activated inward current was a non-selective cation (NSC) current that showed inward rectification, reversed at −1.5 ± 1 mV and was permeable to monovalent cations. The NSC current was insensitive to the P2 purinoceptor antagonists, suramin or PPADS but was activated by the purinoceptor agonists UTP, ADP and 2MeSATP. The outward current activated by ATP reversed at −68 ± 3 mV (equilibrium potential for potassium (EK) = −84 mV) and was blocked by Ba2+ ions, consistent with the activation of a K+ conductance. The outward K+ conductance was also reduced by the maxi-KCa channel blocker iberiotoxin (IbTX; 10 nM), suggesting that ATP activated an outward Ca2+-activated K+ channel in rat RPE cells. The Ca2+-activated K+ current (IK(Ca)) was also activated by the purinoceptor agonists UTP, ADP and 2MeSATP. In fluo-3 or fluo-4 loaded RPE cells, ATP and the pyrimidine agonist UTP elevated [Ca2+]i. The increase in Ca2+ was not dependent on extracellular Ca2+ influx, but was sensitive to the Ca2+-ATPase inhibitor thapsigargin, confirming the involvement of intracellular Ca2+ stores release. These results suggest that rat RPE cells express both P2X purinoceptors that gate activation of a non-selective cation conductance and G protein-coupled P2Y purinoceptors that mediate Ca2+ release from intracellular stores and activation of a calcium-activated K+ current. PMID:10545141

  1. Intracellular calcium and its sodium-independent regulation in voltage-clamped snail neurones.

    PubMed Central

    Kennedy, H J; Thomas, R C

    1995-01-01

    1. We have used both Ca(2+)-sensitive microelectrodes and fura-2 to measure the intracellular free calcium ion concentration ([Ca2+]i or its negative log, pCai) of snail neurones voltage clamped to -50 or -60 mV. Using Ca(2+)-sensitive microelectrodes, [Ca2+]i was found to be approximately 174 nM and pCai, 6.76 +/- 0.09 (mean +/- S.E.M.; n = 11); using fura-2, [Ca2+]i was approximately 40 nM and pCai, 7.44 +/- 0.06 (mean +/- S.E.M., n = 10). 2. Depolarizations (1-20 s) caused an increase in [Ca2+]i which was abolished by removal of extracellular Ca2+, indicating that the rise in [Ca2+]i was due to Ca2+ influx through voltage-activated Ca2+ channels. 3. Caffeine (10-20 mM) caused an increase in [Ca2+]i in the presence or absence of extracellular Ca2+. The effects of caffeine on [Ca2+]i could be prevented by ryanodine. 4. Thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a small increase in resting [Ca2+]i and slowed the rate of recovery from Ca2+ loads following 20 s depolarizations. 5. Neither replacement of extracellular sodium with N-methyl-D-glucamine (NMDG), nor loading the cells with intracellular sodium, had any effect on resting [Ca2+]i or the rate of recovery of [Ca2+]i following depolarizations. 6. The mitochondrial uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (CCmP) caused a small gradual rise in resting [Ca2+]i. Removal of extracellular sodium during exposure to CCmP had no further effect on [Ca2+]i. 7. Intracellular orthovanadate caused an increase in resting [Ca2+]i and prevented the full recovery of [Ca2+]i following small Ca2+ loads, but removal of extracellular sodium did not cause a rise in [Ca2+]i. We conclude that there is no Na(+)-Ca2+ exchanger present in the cell body of these neurones and that [Ca2+]i is maintained by an ATP-dependent Ca2+ pump. Images Figure 1 PMID:7623274

  2. Purinergic regulation of cation conductances and intracellular Ca2+ in cultured rat retinal pigment epithelial cells.

    PubMed

    Ryan, J S; Baldridge, W H; Kelly, M E

    1999-11-01

    1. We used whole-cell patch clamp and fluorescent calcium imaging techniques to investigate the effects of adenosine 5'-triphosphate (ATP) on membrane currents and intracellular calcium concentration ([Ca2+]i)in rat retinal pigment epithelial (RPE) cells. In 62 % of RPE cells, application of 100 microM ATP elicited a fast inward current at negative membrane potentials. In 38 % of RPE cells recorded, a biphasic response to ATP was observed in which activation of the fast inward current was followed by activation of a delayed outward current. 2. The ATP-activated inward current was a non-selective cation (NSC) current that showed inward rectification, reversed at -1.5 +/- 1 mV and was permeable to monovalent cations. The NSC current was insensitive to the P2 purinoceptor antagonists, suramin or PPADS but was activated by the purinoceptor agonists UTP, ADP and 2MeSATP. 3. The outward current activated by ATP reversed at -68 +/- 3 mV (equilibrium potential for potassium (EK) = -84 mV) and was blocked by Ba2+ ions, consistent with the activation of a K+ conductance. The outward K+ conductance was also reduced by the maxi-KCa channel blocker iberiotoxin (IbTX; 10 nM), suggesting that ATP activated an outward Ca2+-activated K+ channel in rat RPE cells. The Ca2+-activated K+ current (IK(Ca)) was also activated by the purinoceptor agonists UTP, ADP and 2MeSATP. 4. In fluo-3 or fluo-4 loaded RPE cells, ATP and the pyrimidine agonist UTP elevated [Ca2+]i. The increase in Ca2+ was not dependent on extracellular Ca2+ influx, but was sensitive to the Ca2+-ATPase inhibitor thapsigargin, confirming the involvement of intracellular Ca2+ stores release. 5. These results suggest that rat RPE cells express both P2X purinoceptors that gate activation of a non-selective cation conductance and G protein-coupled P2Y purinoceptors that mediate Ca2+ release from intracellular stores and activation of a calcium-activated K+ current. PMID:10545141

  3. Jagged1 intracellular domain-mediated inhibition of Notch1 signalling regulates cardiac homeostasis in the postnatal heart

    PubMed Central

    Metrich, Mélanie; Bezdek Pomey, April; Berthonneche, Corinne; Sarre, Alexandre; Nemir, Mohamed; Pedrazzini, Thierry

    2015-01-01

    Aims Notch1 signalling in the heart is mainly activated via expression of Jagged1 on the surface of cardiomyocytes. Notch controls cardiomyocyte proliferation and differentiation in the developing heart and regulates cardiac remodelling in the stressed adult heart. Besides canonical Notch receptor activation in signal-receiving cells, Notch ligands can also activate Notch receptor-independent responses in signal-sending cells via release of their intracellular domain. We evaluated therefore the importance of Jagged1 (J1) intracellular domain (ICD)-mediated pathways in the postnatal heart. Methods and results In cardiomyocytes, Jagged1 releases J1ICD, which then translocates into the nucleus and down-regulates Notch transcriptional activity. To study the importance of J1ICD in cardiac homeostasis, we generated transgenic mice expressing a tamoxifen-inducible form of J1ICD, specifically in cardiomyocytes. Using this model, we demonstrate that J1ICD-mediated Notch inhibition diminishes proliferation in the neonatal cardiomyocyte population and promotes maturation. In the neonatal heart, a response via Wnt and Akt pathway activation is elicited as an attempt to compensate for the deficit in cardiomyocyte number resulting from J1ICD activation. In the stressed adult heart, J1ICD activation results in a dramatic reduction of the number of Notch signalling cardiomyocytes, blunts the hypertrophic response, and reduces the number of apoptotic cardiomyocytes. Consistently, this occurs concomitantly with a significant down-regulation of the phosphorylation of the Akt effectors ribosomal S6 protein (S6) and eukaryotic initiation factor 4E binding protein1 (4EBP1) controlling protein synthesis. Conclusions Altogether, these data demonstrate the importance of J1ICD in the modulation of physiological and pathological hypertrophy, and reveal the existence of a novel pathway regulating cardiac homeostasis. PMID:26249804

  4. Zinc is both an intracellular and extracellular regulator of KATP channel function.

    PubMed

    Prost, Anne-Lise; Bloc, Alain; Hussy, Nicolas; Derand, Renaud; Vivaudou, Michel

    2004-08-15

    Extracellular Zn(2+) has been identified as an activator of pancreatic K(ATP) channels. We further examined the action of Zn(2+) on recombinant K(ATP) channels formed with the inward rectifier K(+) channel subunit Kir6.2 associated with either the pancreatic/neuronal sulphonylurea receptor 1 (SUR1) subunit or the cardiac SUR2A subunit. Zn(2+), applied at either the extracellular or intracellular side of the membrane appeared as a potent, reversible activator of K(ATP) channels. External Zn(2+), at micromolar concentrations, activated SUR1/Kir6.2 but induced a small inhibition of SUR2A/Kir6.2 channels. Cytosolic Zn(2+) dose-dependently stimulated both SUR1/Kir6.2 and SUR2A/Kir6.2 channels, with half-maximal effects at 1.8 and 60 microm, respectively, but it did not affect the Kir6.2 subunit expressed alone. These observations point to an action of both external and internal Zn(2+) on the SUR subunit. Effects of internal Zn(2+) were not due to Zn(2+) leaking out, since they were unaffected by the presence of a Zn(2+) chelator on the external side. Similarly, internal chelators did not affect activation by external Zn(2+). Therefore, Zn(2+) is an endogenous K(ATP) channel opener being active on both sides of the membrane, with potentially distinct sites of action located on the SUR subunit. These findings uncover a novel regulatory pathway targeting K(ATP) channels, and suggest a new role for Zn(2+) as an intracellular signalling molecule. PMID:15218066

  5. Zinc is both an intracellular and extracellular regulator of KATP channel function

    PubMed Central

    Prost, Anne-Lise; Bloc, Alain; Hussy, Nicolas; Derand, Renaud; Vivaudou, Michel

    2004-01-01

    Extracellular Zn2+ has been identified as an activator of pancreatic KATP channels. We further examined the action of Zn2+ on recombinant KATP channels formed with the inward rectifier K+ channel subunit Kir6.2 associated with either the pancreatic/neuronal sulphonylurea receptor 1 (SUR1) subunit or the cardiac SUR2A subunit. Zn2+, applied at either the extracellular or intracellular side of the membrane appeared as a potent, reversible activator of KATP channels. External Zn2+, at micromolar concentrations, activated SUR1/Kir6.2 but induced a small inhibition of SUR2A/Kir6.2 channels. Cytosolic Zn2+ dose-dependently stimulated both SUR1/Kir6.2 and SUR2A/Kir6.2 channels, with half-maximal effects at 1.8 and 60 μm, respectively, but it did not affect the Kir6.2 subunit expressed alone. These observations point to an action of both external and internal Zn2+ on the SUR subunit. Effects of internal Zn2+ were not due to Zn2+ leaking out, since they were unaffected by the presence of a Zn2+ chelator on the external side. Similarly, internal chelators did not affect activation by external Zn2+. Therefore, Zn2+ is an endogenous KATP channel opener being active on both sides of the membrane, with potentially distinct sites of action located on the SUR subunit. These findings uncover a novel regulatory pathway targeting KATP channels, and suggest a new role for Zn2+ as an intracellular signalling molecule. PMID:15218066

  6. Positive Charges at the Intracellular Mouth of the Pore Regulate Anion Conduction in the CFTR Chloride Channel

    PubMed Central

    Aubin, Chantal N. St.; Linsdell, Paul

    2006-01-01

    Many different ion channel pores are thought to have charged amino acid residues clustered around their entrances. The so-called surface charges contributed by these residues can play important roles in attracting oppositely charged ions from the bulk solution on one side of the membrane, increasing effective local counterion concentration and favoring rapid ion movement through the channel. Here we use site-directed mutagenesis to identify arginine residues contributing important surface charges in the intracellular mouth of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel pore. While wild-type CFTR was associated with a linear current–voltage relationship with symmetrical solutions, strong outward rectification was observed after mutagenesis of two arginine residues (R303 and R352) located near the intracellular ends of the fifth and sixth transmembrane regions. Current rectification was dependent on the charge present at these positions, consistent with an electrostatic effect. Furthermore, mutagenesis-induced rectification was more pronounced at lower Cl− concentrations, suggesting that these mutants had a reduced ability to concentrate Cl− ions near the inner pore mouth. R303 and R352 mutants exhibited reduced single channel conductance, especially at negative membrane potentials, that was dependent on the charge of the amino acid residue present at these positions. However, the very low conductance of both R303E and R352E-CFTR could be greatly increased by elevating intracellular Cl− concentration. Modification of an introduced cysteine residue at position 303 by charged methanethiosulfonate reagents reproduced charge-dependent effects on current rectification. Mutagenesis of arginine residues in the second and tenth transmembrane regions also altered channel permeation properties, however these effects were not consistent with changes in channel surface charges. These results suggest that positively charged arginine

  7. Positive charges at the intracellular mouth of the pore regulate anion conduction in the CFTR chloride channel.

    PubMed

    Aubin, Chantal N St; Linsdell, Paul

    2006-11-01

    Many different ion channel pores are thought to have charged amino acid residues clustered around their entrances. The so-called surface charges contributed by these residues can play important roles in attracting oppositely charged ions from the bulk solution on one side of the membrane, increasing effective local counterion concentration and favoring rapid ion movement through the channel. Here we use site-directed mutagenesis to identify arginine residues contributing important surface charges in the intracellular mouth of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore. While wild-type CFTR was associated with a linear current-voltage relationship with symmetrical solutions, strong outward rectification was observed after mutagenesis of two arginine residues (R303 and R352) located near the intracellular ends of the fifth and sixth transmembrane regions. Current rectification was dependent on the charge present at these positions, consistent with an electrostatic effect. Furthermore, mutagenesis-induced rectification was more pronounced at lower Cl(-) concentrations, suggesting that these mutants had a reduced ability to concentrate Cl(-) ions near the inner pore mouth. R303 and R352 mutants exhibited reduced single channel conductance, especially at negative membrane potentials, that was dependent on the charge of the amino acid residue present at these positions. However, the very low conductance of both R303E and R352E-CFTR could be greatly increased by elevating intracellular Cl(-) concentration. Modification of an introduced cysteine residue at position 303 by charged methanethiosulfonate reagents reproduced charge-dependent effects on current rectification. Mutagenesis of arginine residues in the second and tenth transmembrane regions also altered channel permeation properties, however these effects were not consistent with changes in channel surface charges. These results suggest that positively charged arginine residues

  8. Ionic osmolytes and intracellular calcium regulate tissue production in chondrocytes cultured in a 3D charged hydrogel.

    PubMed

    Farnsworth, Nikki L; Mead, Benjamin E; Antunez, Lorena R; Palmer, Amy E; Bryant, Stephanie J

    2014-11-01

    The goal of this study was to investigate the role of fixed negative charges in regulating cartilage-like tissue production by chondrocytes under static and dynamic three-dimensional culture, and to determine whether intracellular calcium ([Ca(2+)]i) is involved in mediating this response. Initial experiments using the 3D neutral hydrogel were conducted in static isotonic culture with ionic and non-ionic osmolytes added to the culture medium. Tissue production by bovine chondrocytes with non-ionic osmolytes was 1.9-fold greater than with ionic osmolytes, suggesting that the ionic nature of the osmolyte is an important regulator of tissue production. To investigate fixed negative charges, a 3D culture system containing encapsulated chondrocytes was employed based on a synthetic and neutral hydrogel platform within which negatively charged chondroitin sulfate was incorporated in a controlled manner. Incorporation of negative charges did not affect the mechanical properties of the hydrogel; however, intracellular ion concentration was elevated from the culture medium (330 mOsm) and estimated to be similar to that in ~400 mOsm culture medium. With dynamic loading, GAG synthesis decreased by 26% in neutral hydrogels cultured in 400mOsm medium, and increased by 26% in charged gels cultured in 330 mOsm. Treatment of chondrocyte-seeded hydrogels with the Ca(2+) chelator BAPTA-AM decreased GAG synthesis by 32-46% and was similar among all conditions, suggesting multiple roles for Ca(2+) mediated tissue production including with ionic osmolytes. In conclusion, findings from this study suggest that a dynamic ionic environment regulates tissue synthesis and points to [Ca(2+)]i signaling as a potential mediator. PMID:25128592

  9. The space of enzyme regulation in HeLa cells can be inferred from its intracellular metabolome.

    PubMed

    Diener, Christian; Muñoz-Gonzalez, Felipe; Encarnación, Sergio; Resendis-Antonio, Osbaldo

    2016-01-01

    During the transition from a healthy state to a cancerous one, cells alter their metabolism to increase proliferation. The underlying metabolic alterations may be caused by a variety of different regulatory events on the transcriptional or post-transcriptional level whose identification contributes to the rational design of therapeutic targets. We present a mechanistic strategy capable of inferring enzymatic regulation from intracellular metabolome measurements that is independent of the actual mechanism of regulation. Here, enzyme activities are expressed by the space of all feasible kinetic constants (k-cone) such that the alteration between two phenotypes is given by their corresponding kinetic spaces. Deriving an expression for the transformation of the healthy to the cancer k-cone we identified putative regulated enzymes between the HeLa and HaCaT cell lines. We show that only a few enzymatic activities change between those two cell lines and that this regulation does not depend on gene transcription but is instead post-transcriptional. Here, we identify phosphofructokinase as the major driver of proliferation in HeLa cells and suggest an optional regulatory program, associated with oxidative stress, that affects the activity of the pentose phosphate pathway. PMID:27335086

  10. BMP regulates regional gene expression in the dorsal otocyst through canonical and non-canonical intracellular pathways.

    PubMed

    Ohta, Sho; Wang, Baolin; Mansour, Suzanne L; Schoenwolf, Gary C

    2016-06-15

    The inner ear consists of two otocyst-derived, structurally and functionally distinct components: the dorsal vestibular and ventral auditory compartments. BMP signaling is required to form the vestibular compartment, but how it complements other required signaling molecules and acts intracellularly is unknown. Using spatially and temporally controlled delivery of signaling pathway regulators to developing chick otocysts, we show that BMP signaling regulates the expression of Dlx5 and Hmx3, both of which encode transcription factors essential for vestibular formation. However, although BMP regulates Dlx5 through the canonical SMAD pathway, surprisingly, it regulates Hmx3 through a non-canonical pathway involving both an increase in cAMP-dependent protein kinase A activity and the GLI3R to GLI3A ratio. Thus, both canonical and non-canonical BMP signaling establish the precise spatiotemporal expression of Dlx5 and Hmx3 during dorsal vestibular development. The identification of the non-canonical pathway suggests an intersection point between BMP and SHH signaling, which is required for ventral auditory development. PMID:27151948

  11. The space of enzyme regulation in HeLa cells can be inferred from its intracellular metabolome

    PubMed Central

    Diener, Christian; Muñoz-Gonzalez, Felipe; Encarnación, Sergio; Resendis-Antonio, Osbaldo

    2016-01-01

    During the transition from a healthy state to a cancerous one, cells alter their metabolism to increase proliferation. The underlying metabolic alterations may be caused by a variety of different regulatory events on the transcriptional or post-transcriptional level whose identification contributes to the rational design of therapeutic targets. We present a mechanistic strategy capable of inferring enzymatic regulation from intracellular metabolome measurements that is independent of the actual mechanism of regulation. Here, enzyme activities are expressed by the space of all feasible kinetic constants (k-cone) such that the alteration between two phenotypes is given by their corresponding kinetic spaces. Deriving an expression for the transformation of the healthy to the cancer k-cone we identified putative regulated enzymes between the HeLa and HaCaT cell lines. We show that only a few enzymatic activities change between those two cell lines and that this regulation does not depend on gene transcription but is instead post-transcriptional. Here, we identify phosphofructokinase as the major driver of proliferation in HeLa cells and suggest an optional regulatory program, associated with oxidative stress, that affects the activity of the pentose phosphate pathway. PMID:27335086

  12. A model system using confocal fluorescence microscopy for examining real-time intracellular sodium ion regulation.

    PubMed

    Lee, Jacqueline A; Collings, David A; Glover, Chris N

    2016-08-15

    The gills of euryhaline fish are the ultimate ionoregulatory tissue, achieving ion homeostasis despite rapid and significant changes in external salinity. Cellular handling of sodium is not only critical for salt and water balance but is also directly linked to other essential functions such as acid-base homeostasis and nitrogen excretion. However, although measurement of intracellular sodium ([Na(+)]i) is important for an understanding of gill transport function, it is challenging and subject to methodological artifacts. Using gill filaments from a model euryhaline fish, inanga (Galaxias maculatus), the suitability of the fluorescent dye CoroNa Green as a probe for measuring [Na(+)]i in intact ionocytes was confirmed via confocal microscopy. Cell viability was verified, optimal dye loading parameters were determined, and the dye-ion dissociation constant was measured. Application of the technique to freshwater- and 100% seawater-acclimated inanga showed salinity-dependent changes in branchial [Na(+)]i, whereas no significant differences in branchial [Na(+)]i were determined in 50% seawater-acclimated fish. This technique facilitates the examination of real-time changes in gill [Na(+)]i in response to environmental factors and may offer significant insight into key homeostatic functions associated with the fish gill and the principles of sodium ion transport in other tissues and organisms. PMID:27235170

  13. Regulation of intracellular membrane trafficking and cell dynamics by syntaxin-6

    PubMed Central

    Jung, Jae-Joon; Inamdar, Shivangi M.; Tiwari, Ajit; Choudhury, Amit

    2012-01-01

    Intracellular membrane trafficking along endocytic and secretory transport pathways plays a critical role in diverse cellular functions including both developmental and pathological processes. Briefly, proteins and lipids destined for transport to distinct locations are collectively assembled into vesicles and delivered to their target site by vesicular fusion. SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins are required for these events, during which v-SNAREs (vesicle SNAREs) interact with t-SNAREs (target SNAREs) to allow transfer of cargo from donor vesicle to target membrane. Recently, the t-SNARE family member, syntaxin-6, has been shown to play an important role in the transport of proteins that are key to diverse cellular dynamic processes. In this paper, we briefly discuss the specific role of SNAREs in various mammalian cell types and comprehensively review the various roles of the Golgi- and endosome-localized t-SNARE, syntaxin-6, in membrane trafficking during physiological as well as pathological conditions. PMID:22489884

  14. PRG-1 Regulates Synaptic Plasticity via Intracellular PP2A/β1-Integrin Signaling.

    PubMed

    Liu, Xingfeng; Huai, Jisen; Endle, Heiko; Schlüter, Leslie; Fan, Wei; Li, Yunbo; Richers, Sebastian; Yurugi, Hajime; Rajalingam, Krishnaraj; Ji, Haichao; Cheng, Hong; Rister, Benjamin; Horta, Guilherme; Baumgart, Jan; Berger, Hendrik; Laube, Gregor; Schmitt, Ulrich; Schmeisser, Michael J; Boeckers, Tobias M; Tenzer, Stefan; Vlachos, Andreas; Deller, Thomas; Nitsch, Robert; Vogt, Johannes

    2016-08-01

    Alterations in dendritic spine numbers are linked to deficits in learning and memory. While we previously revealed that postsynaptic plasticity-related gene 1 (PRG-1) controls lysophosphatidic acid (LPA) signaling at glutamatergic synapses via presynaptic LPA receptors, we now show that PRG-1 also affects spine density and synaptic plasticity in a cell-autonomous fashion via protein phosphatase 2A (PP2A)/β1-integrin activation. PRG-1 deficiency reduces spine numbers and β1-integrin activation, alters long-term potentiation (LTP), and impairs spatial memory. The intracellular PRG-1 C terminus interacts in an LPA-dependent fashion with PP2A, thus modulating its phosphatase activity at the postsynaptic density. This results in recruitment of adhesome components src, paxillin, and talin to lipid rafts and ultimately in activation of β1-integrins. Consistent with these findings, activation of PP2A with FTY720 rescues defects in spine density and LTP of PRG-1-deficient animals. These results disclose a mechanism by which bioactive lipid signaling via PRG-1 could affect synaptic plasticity and memory formation. PMID:27453502

  15. Programmed Nanococktail for Intracellular Cascade Reaction Regulating Self-Synergistic Tumor Targeting Therapy.

    PubMed

    Chen, Wei-Hai; Luo, Guo-Feng; Qiu, Wen-Xiu; Lei, Qi; Hong, Sheng; Wang, Shi-Bo; Zheng, Di-Wei; Zhu, Cheng-Hui; Zeng, Xuan; Feng, Jun; Cheng, Si-Xue; Zhang, Xian-Zheng

    2016-02-10

    In this work, a ZnO based nanococktail with programmed functions is designed and synthesized for self-synergistic tumor targeting therapy. The nanococktail can actively target tumors via specific interaction of hyaluronic acid (HA) with CD44 receptors and respond to HAase-rich tumor microenvironment to induce intracellular cascade reaction for controlled therapy. The exposed cell-penetrating peptide (R8) potentiates the cellular uptake of therapeutic nanoparticles into targeted tumor cells. Then ZnO cocktail will readily degrade in acidic endo/lysosomes and induce the production of desired reactive oxygen species (ROS) in situ. The destructive ROS not only leads to serious cell damage but also triggers the on-demand drug release for precise chemotherapy, thus achieving enhanced antitumor efficiency synergistically. After tail vein injection of ZnO cocktail, a favorable tumor apoptosis rate (71.2 ± 8.2%) is detected, which is significantly superior to that of free drug, doxorubicin (12.9 ± 5.2%). Both in vitro and in vivo studies demonstrate that the tailor-made ZnO cocktail with favorable biocompatibility, promising tumor specificity, and self-synergistically therapeutic capacity opens new avenues for cancer therapy. PMID:26708101

  16. Expression and insulin-regulated distribution of caveolin in skeletal muscle. Caveolin does not colocalize with GLUT4 in intracellular membranes.

    PubMed

    Muñoz, P; Mora, S; Sevilla, L; Kaliman, P; Tomàs, E; Gumà, A; Testar, X; Palacín, M; Zorzano, A

    1996-04-01

    Caveolin is believed to play an important role in sorting processes, vesicular trafficking, transmembrane signaling, and molecular transport across membranes. In this study we have evaluated the expression and distribution of caveolin in skeletal muscle and its interaction with GLUT4 glucose carriers. Caveolin was expressed to substantial levels in muscle and its expression was regulated in muscle; aging and high fat diet enhanced caveolin expression in skeletal muscle and inversely, myogenesis down-regulated caveolin in L6E9 cells. Under fasting conditions, most of caveolin was found in intracellular membranes and the caveolin present in the cell surface was found in both sarcolemma and T-tubules. Insulin administration led to a redistribution of caveolin from intracellular high density membrane fractions to intracellular lighter density fractions and to the cell surface; this pattern of insulin-induced redistribution was different to what was shown by GLUT4. These results suggests that caveolin is a component of an insulin-regulated machinery of vesicular transport in muscle. Quantitative immunoisolation of GLUT4 vesicles obtained from different intracellular GLUT4 populations revealed the absence of caveolin which substantiates the lack of colocalization of intracellular GLUT4 and caveolin. This indicates that caveolin is not involved in intracellular GLUT4 trafficking in skeletal muscle. PMID:8626501

  17. Calcium-dependent regulation of Rab activation and vesicle fusion by an intracellular P2X ion channel.

    PubMed

    Parkinson, Katie; Baines, Abigail E; Keller, Thomas; Gruenheit, Nicole; Bragg, Laricia; North, R Alan; Thompson, Christopher R L

    2014-01-01

    Rab GTPases play key roles in the delivery, docking and fusion of intracellular vesicles. However, the mechanism by which spatial and temporal regulation of Rab GTPase activity is controlled is poorly understood. Here we describe a mechanism by which localized calcium release through a vesicular ion channel controls Rab GTPase activity. We show that activation of P2XA, an intracellular ion channel localized to the Dictyostelium discoideum contractile vacuole system, results in calcium efflux required for downregulation of Rab11a activity and efficient vacuole fusion. Vacuole fusion and Rab11a downregulation require the activity of CnrF, an EF-hand-containing Rab GAP found in a complex with Rab11a and P2XA. CnrF Rab GAP activity for Rab11a is enhanced by the presence of calcium and the EF-hand domain. These findings suggest that P2XA activation results in vacuolar calcium release, which triggers activation of CnrF Rab GAP activity and subsequent downregulation of Rab11a to allow vacuole fusion. PMID:24335649

  18. Polyamines regulate cell growth and cellular methylglyoxal in high-glucose medium independently of intracellular glutathione.

    PubMed

    Kwak, Min-Kyu; Lee, Mun-Hyoung; Park, Seong-Jun; Shin, Sang-Min; Liu, Rui; Kang, Sa-Ouk

    2016-03-01

    Polyamines can presumably inhibit protein glycation, when associated with the methylglyoxal inevitably produced during glycolysis. Herein, we hypothesized a nonenzymatic interaction between putrescine and methylglyoxal in putrescine-deficient or -overexpressing Dictyostelium cells in high-glucose medium, which can control methylglyoxal production. Putrescine was essentially required for growth rescue accompanying methylglyoxal detoxification when cells underwent growth defect and cell cycle G1-arrest when supplemented with high glucose. Furthermore, methylglyoxal regulation by putrescine seemed to be a parallel pathway independent of the changes in cellular glutathione content in high-glucose medium. Consequently, we suggest that Dictyostelium cells need polyamines for normal growth and cellular methylglyoxal regulation. PMID:26898161

  19. Intracellular pH regulation in isolated rat bile duct epithelial cells.

    PubMed Central

    Strazzabosco, M; Mennone, A; Boyer, J L

    1991-01-01

    To evaluate ion transport mechanisms in bile duct epithelium (BDE), BDE cells were isolated from bile duct-ligated rats. After short-term culture pHi was measured with a single cell microfluorimetric set-up using the fluorescent pHi indicator BCECF, and calibrated with nigericin in high K+ concentration buffer. Major contaminants were identified using vital markers. In HCO3(-)-free media, baseline pHi (7.03 +/- 0.12) decreased by 0.45 +/- 0.18 pH units after Na+ removal and by 0.12 +/- .04 after amiloride administration (1 mM). After acid loading (20 mM NH4Cl) pHi recovery was inhibited by both Na+ removal and amiloride (JH+ = 0.74 +/- 1.1, and JH+ = 2.28 +/- 0.8, respectively, vs. 5.47 +/- 1.97 and 5.97 +/- 1.76 mM/min, in controls, respectively). In HCO3- containing media baseline pHi was higher (7.16 +/- 0.1, n = 36, P less than 0.05) and was decreased by Na+ substitution but not by amiloride. Na+ removal inhibited pHi recovery after an intracellular acid load (0.27 +/- 0.26, vs. 7.7 +/- 4.1 mM/min, in controls), whereas amiloride reduced JH+ only by 27%. pH recovery was inhibited by DIDS (0.5-1 mM), but not by Cl- depletion. Finally, acute Cl- removal increased pHi by 0.18 pH units in the absence but not presence of DIDS. These data indicate that BDE cells possess mechanisms for Na+/H+ exchange, Na+:HCO3- symport and Cl-/HCO3 exchange. Therefore BDE may be capable of transepithelial H+/HCO3- transport. Images PMID:2022723

  20. By Regulating Mitochondrial Ca2+-Uptake UCP2 Modulates Intracellular Ca2+

    PubMed Central

    Gebing, Tina; Reda, Sara; Schwaiger, Astrid; Leitner, Johannes; Wolny, Martin; Eckardt, Lars; Hoppe, Uta C.

    2016-01-01

    Introduction The possible role of UCP2 in modulating mitochondrial Ca2+-uptake (mCa2+-uptake) via the mitochondrial calcium uniporter (MCU) is highly controversial. Methods Thus, we analyzed mCa2+-uptake in isolated cardiac mitochondria, MCU single-channel activity in cardiac mitoplasts, dual Ca2+-transients from mitochondrial ((Ca2+)m) and intracellular compartment ((Ca2+)c) in the whole-cell configuration in cardiomyocytes of wild-type (WT) and UCP2-/- mice. Results Isolated mitochondria showed a Ru360 sensitive mCa2+-uptake, which was significantly decreased in UCP2-/- (229.4±30.8 FU vs. 146.3±23.4 FU, P<0.05). Single-channel registrations confirmed a Ru360 sensitive voltage-gated Ca2+-channel in mitoplasts, i.e. mCa1, showing a reduced single-channel activity in UCP2-/- (Po,total: 0.34±0.05% vs. 0.07±0.01%, P<0.05). In UCP2-/- cardiomyocytes (Ca2+)m was decreased (0.050±0.009 FU vs. 0.021±0.005 FU, P<0.05) while (Ca2+)c was unchanged (0.032±0.002 FU vs. 0.028±0.004 FU, P>0.05) and transsarcolemmal Ca2+-influx was inhibited suggesting a possible compensatory mechanism. Additionally, we observed an inhibitory effect of ATP on mCa2+-uptake in WT mitoplasts and (Ca2+)m of cardiomyocytes leading to an increase of (Ca2+)c while no ATP dependent effect was observed in UCP2-/-. Conclusion Our results indicate regulatory effects of UCP2 on mCa2+-uptake. Furthermore, we propose, that previously described inhibitory effects on MCU by ATP may be mediated via UCP2 resulting in changes of excitation contraction coupling. PMID:26849136

  1. NK Cell-Mediated Regulation of Protective Memory Responses against Intracellular Ehrlichial Pathogens

    PubMed Central

    Habib, Samar; El Andaloussi, Abdeljabar; Hisham, Ahmed; Ismail, Nahed

    2016-01-01

    Ehrlichiae are gram-negative obligate intracellular bacteria that cause potentially fatal human monocytic ehrlichiosis. We previously showed that natural killer (NK) cells play a critical role in host defense against Ehrlichia during primary infection. However, the contribution of NK cells to the memory response against Ehrlichia remains elusive. Primary infection of C57BL/6 mice with Ehrlichia muris provides long-term protection against a second challenge with the highly virulent Ixodes ovatus Ehrlichia (IOE), which ordinarily causes fatal disease in naïve mice. Here, we show that the depletion of NK cells in E. muris-primed mice abrogates the protective memory response against IOE. Approximately, 80% of NK cell-depleted E. muris-primed mice succumbed to lethal IOE infection on days 8–10 after IOE infection, similar to naïve mice infected with the same dose of IOE. The lack of a recall response in NK cell-depleted mice correlated with an increased bacterial burden, extensive liver injury, decreased frequency of Ehrlichia-specific IFN-γ-producing memory CD4+ and CD8+ T-cells, and a low titer of Ehrlichia-specific antibodies. Intraperitoneal infection of mice with E. muris resulted in the production of IL-15, IL-12, and IFN-γ as well as an expansion of activated NKG2D+ NK cells. The adoptive transfer of purified E. muris-primed hepatic and splenic NK cells into Rag2-/-Il2rg-/- recipient mice provided protective immunity against challenge with E. muris. Together, these data suggest that E. muris-induced memory-like NK cells, which contribute to the protective, recall response against Ehrlichia. PMID:27092553

  2. The CovS/CovR acid response regulator is required for intracellular survival of group B Streptococcus in macrophages.

    PubMed

    Cumley, Nicola J; Smith, Leanne M; Anthony, Mark; May, Robin C

    2012-05-01

    Group B Streptococcus (GBS) is a leading cause of neonatal meningitis and septicemia. The ability of this organism to survive inside phagocytic cells is poorly understood but thought to be an important step for the establishment of disease in the host. Here, we demonstrate that GBS shows prolonged survival within J774 macrophages and that the capacity to survive is not significantly changed across a diverse range of strains representing different serotypes, multilocus sequence types (MLST), and sites of clinical isolation. Using staining for the lysosome-associated membrane protein (LAMP) and by pharmacological inhibition of phagosome acidification, we demonstrate that streptococci reside in a phagosome and that acidification of the phagosome is required for GBS to survive intracellularly. Moreover, we show that the GBS two-component system CovS/CovR, which is the major acid response regulator in this organism, is required for survival inside the phagosome. PMID:22331428

  3. Critical role for NAD glycohydrolase in regulation of erythropoiesis by hematopoietic stem cells through control of intracellular NAD content.

    PubMed

    Nam, Tae-Sik; Park, Kwang-Hyun; Shawl, Asif Iqbal; Kim, Byung-Ju; Han, Myung-Kwan; Kim, Youngho; Moss, Joel; Kim, Uh-Hyun

    2014-06-01

    NAD glycohydrolases (NADases) catalyze the hydrolysis of NAD to ADP-ribose and nicotinamide. Although many members of the NADase family, including ADP-ribosyltransferases, have been cloned and characterized, the structure and function of NADases with pure hydrolytic activity remain to be elucidated. Here, we report the structural and functional characterization of a novel NADase from rabbit reticulocytes. The novel NADase is a glycosylated, glycosylphosphatidylinositol-anchored cell surface protein exclusively expressed in reticulocytes. shRNA-mediated knockdown of the NADase in bone marrow cells resulted in a reduction of erythroid colony formation and an increase in NAD level. Furthermore, treatment of bone marrow cells with NAD, nicotinamide, or nicotinamide riboside, which induce an increase in NAD content, resulted in a significant decrease in erythroid progenitors. These results indicate that the novel NADase may play a critical role in regulating erythropoiesis of hematopoietic stem cells by modulating intracellular NAD. PMID:24759100

  4. The CovS/CovR Acid Response Regulator Is Required for Intracellular Survival of Group B Streptococcus in Macrophages

    PubMed Central

    Cumley, Nicola J.; Smith, Leanne M.; Anthony, Mark

    2012-01-01

    Group B Streptococcus (GBS) is a leading cause of neonatal meningitis and septicemia. The ability of this organism to survive inside phagocytic cells is poorly understood but thought to be an important step for the establishment of disease in the host. Here, we demonstrate that GBS shows prolonged survival within J774 macrophages and that the capacity to survive is not significantly changed across a diverse range of strains representing different serotypes, multilocus sequence types (MLST), and sites of clinical isolation. Using staining for the lysosome-associated membrane protein (LAMP) and by pharmacological inhibition of phagosome acidification, we demonstrate that streptococci reside in a phagosome and that acidification of the phagosome is required for GBS to survive intracellularly. Moreover, we show that the GBS two-component system CovS/CovR, which is the major acid response regulator in this organism, is required for survival inside the phagosome. PMID:22331428

  5. Aging is a primary risk factor for cardiac arrhythmias: disruption of intracellular Ca2+ regulation as a key suspect.

    PubMed

    Hatch, Fiona; Lancaster, Matthew K; Jones, Sandra A

    2011-08-01

    Aging is an inevitable time-dependent progression associated with a functional decline of the cardiovascular system even in 'healthy' individuals. Age positively correlates with an increasing risk of cardiac problems including arrhythmias. Not only the prevalence but also the severity of arrhythmias escalates with age. The reasons for this are multifactorial but dysregulation of intracellular calcium within the heart is likely to play a key role in initiating and perpetuating these life-threatening events. We now know that several aspects of cardiac calcium regulation significantly change with advancing age - changes that could produce electrical instability. Further development of knowledge of the mechanisms underlying these changes will allow us to reduce what currently is an inevitable increase in the incidence of arrhythmias in the elderly. PMID:21878050

  6. Purification, kinetic properties, and intracellular concentration of SpoIIE, an integral membrane protein that regulates sporulation in Bacillus subtilis.

    PubMed

    Lucet, I; Borriss, R; Yudkin, M D

    1999-05-01

    SpoIIE is a bifunctional protein which controls sigmaF activation and formation of the asymmetric septum in sporulating Bacillus subtilis. The spoIIE gene of B. subtilis has now been overexpressed in Escherichia coli, and SpoIIE has been purified by anion-exchange chromatography and affinity chromatography. Kinetic studies showed that the rate of dephosphorylation of SpoIIAA-P by purified SpoIIE in vitro was 100 times greater, on a molar basis, than the rate of phosphorylation of SpoIIAA by SpoIIAB. The intracellular concentrations of SpoIIE and SpoIIAB were measured by quantitative immunoblotting between 0 and 4 h after the beginning of sporulation. The facts that these concentrations were very similar at hour 2 and that SpoIIE could be readily detected before asymmetric septation suggest that SpoIIE activity may be strongly regulated. PMID:10322028

  7. p120-catenin differentially regulates cell migration by Rho-dependent intracellular and secreted signals

    PubMed Central

    Epifano, Carolina; Megias, Diego; Perez-Moreno, Mirna

    2014-01-01

    The adherens junction protein p120-catenin is implicated in the regulation of cadherin stability, cell migration and inflammatory responses in mammalian epithelial tissues. How these events are coordinated to promote wound repair is not understood. We show that p120 catenin regulates the intrinsic migratory properties of primary mouse keratinocytes, but also influences the migratory behavior of neighboring cells by secreted signals. These events are rooted in the ability of p120-catenin to regulate RhoA GTPase activity, which leads to a two-tiered control of cell migration. One restrains cell motility via an increase in actin stress fibers, reduction in integrin turnover and an increase in the robustness of focal adhesions. The other is coupled to the secretion of inflammatory cytokines including interleukin-24, which causally enhances randomized cell movements. Taken together, our results indicate that p120-RhoA-GTPase-mediated signaling can differentially regulate the migratory behavior of epidermal cells, which has potential implications for chronic wound responses and cancer. PMID:24639556

  8. p120-catenin differentially regulates cell migration by Rho-dependent intracellular and secreted signals.

    PubMed

    Epifano, Carolina; Megias, Diego; Perez-Moreno, Mirna

    2014-05-01

    The adherens junction protein p120-catenin is implicated in the regulation of cadherin stability, cell migration and inflammatory responses in mammalian epithelial tissues. How these events are coordinated to promote wound repair is not understood. We show that p120 catenin regulates the intrinsic migratory properties of primary mouse keratinocytes, but also influences the migratory behavior of neighboring cells by secreted signals. These events are rooted in the ability of p120-catenin to regulate RhoA GTPase activity, which leads to a two-tiered control of cell migration. One restrains cell motility via an increase in actin stress fibers, reduction in integrin turnover and an increase in the robustness of focal adhesions. The other is coupled to the secretion of inflammatory cytokines including interleukin-24, which causally enhances randomized cell movements. Taken together, our results indicate that p120-RhoA-GTPase-mediated signaling can differentially regulate the migratory behavior of epidermal cells, which has potential implications for chronic wound responses and cancer. PMID:24639556

  9. Regulation of BRCA1, BRCA2 and BARD1 intracellular trafficking.

    PubMed

    Henderson, Beric R

    2005-09-01

    The subcellular location and function of many proteins are regulated by nuclear-cytoplasmic shuttling. BRCA1 and BARD1 provide an interesting model system for understanding the influence of protein dimerization on nuclear transport and localization. These proteins function predominantly in the nucleus to regulate cell cycle progression, DNA repair/recombination and gene transcription, and their export to the cytoplasm has been linked to apoptosis. Germ-line mutations in the BRCA1/BRCA2 and BARD1 genes predispose to risk of breast/ovarian cancer, and certain mutations impair protein function and nuclear accumulation. BRCA1 and BARD1 shuttle between the nucleus and cytoplasm; however heterodimerization masks the nuclear export signals located within each protein, causing nuclear retention of the BRCA1-BARD1 complex and potentially influencing its role in DNA repair, cell survival and regulation of centrosome duplication. This review discusses BRCA1, BRCA2 and BARD1 subcellular localization with emphasis on regulation of transport by protein dimerization and its functional implications. PMID:16108063

  10. Structural asymmetry in a conserved signaling system that regulates division, replication, and virulence of an intracellular pathogen

    PubMed Central

    Willett, Jonathan W.; Herrou, Julien; Briegel, Ariane; Rotskoff, Grant; Crosson, Sean

    2015-01-01

    We have functionally and structurally defined an essential protein phosphorelay that regulates expression of genes required for growth, division, and intracellular survival of the global zoonotic pathogen Brucella abortus. Our study delineates phosphoryl transfer through this molecular pathway, which initiates from the sensor kinase CckA and proceeds through the ChpT phosphotransferase to two regulatory substrates: CtrA and CpdR. Genetic perturbation of this system results in defects in cell growth and division site selection, and a specific viability deficit inside human phagocytic cells. Thus, proper control of B. abortus division site polarity is necessary for survival in the intracellular niche. We further define the structural foundations of signaling from the central phosphotransferase, ChpT, to its response regulator substrate, CtrA, and provide evidence that there are at least two modes of interaction between ChpT and CtrA, only one of which is competent to catalyze phosphoryltransfer. The structure and dynamics of the active site on each side of the ChpT homodimer are distinct, supporting a model in which quaternary structure of the 2:2 ChpT–CtrA complex enforces an asymmetric mechanism of phosphoryl transfer between ChpT and CtrA. Our study provides mechanistic understanding, from the cellular to the atomic scale, of a conserved transcriptional regulatory system that controls the cellular and infection biology of B. abortus. More generally, our results provide insight into the structural basis of two-component signal transduction, which is broadly conserved in bacteria, plants, and fungi. PMID:26124143

  11. Structural asymmetry in a conserved signaling system that regulates division, replication, and virulence of an intracellular pathogen.

    PubMed

    Willett, Jonathan W; Herrou, Julien; Briegel, Ariane; Rotskoff, Grant; Crosson, Sean

    2015-07-14

    We have functionally and structurally defined an essential protein phosphorelay that regulates expression of genes required for growth, division, and intracellular survival of the global zoonotic pathogen Brucella abortus. Our study delineates phosphoryl transfer through this molecular pathway, which initiates from the sensor kinase CckA and proceeds through the ChpT phosphotransferase to two regulatory substrates: CtrA and CpdR. Genetic perturbation of this system results in defects in cell growth and division site selection, and a specific viability deficit inside human phagocytic cells. Thus, proper control of B. abortus division site polarity is necessary for survival in the intracellular niche. We further define the structural foundations of signaling from the central phosphotransferase, ChpT, to its response regulator substrate, CtrA, and provide evidence that there are at least two modes of interaction between ChpT and CtrA, only one of which is competent to catalyze phosphoryltransfer. The structure and dynamics of the active site on each side of the ChpT homodimer are distinct, supporting a model in which quaternary structure of the 2:2 ChpT-CtrA complex enforces an asymmetric mechanism of phosphoryl transfer between ChpT and CtrA. Our study provides mechanistic understanding, from the cellular to the atomic scale, of a conserved transcriptional regulatory system that controls the cellular and infection biology of B. abortus. More generally, our results provide insight into the structural basis of two-component signal transduction, which is broadly conserved in bacteria, plants, and fungi. PMID:26124143

  12. The α-Arrestin ARRDC3 Regulates the Endosomal Residence Time and Intracellular Signaling of the β2-Adrenergic Receptor.

    PubMed

    Tian, Xufan; Irannejad, Roshanak; Bowman, Shanna L; Du, Yang; Puthenveedu, Manojkumar A; von Zastrow, Mark; Benovic, Jeffrey L

    2016-07-01

    Arrestin domain-containing protein 3 (ARRDC3) is a member of the mammalian α-arrestin family, which is predicted to share similar tertiary structure with visual-/β-arrestins and also contains C-terminal PPXY motifs that mediate interaction with E3 ubiquitin ligases. Recently, ARRDC3 has been proposed to play a role in regulating the trafficking of G protein-coupled receptors, although mechanistic insight into this process is lacking. Here, we focused on characterizing the role of ARRDC3 in regulating the trafficking of the β2-adrenergic receptor (β2AR). We find that ARRDC3 primarily localizes to EEA1-positive early endosomes and directly interacts with the β2AR in a ligand-independent manner. Although ARRDC3 has no effect on β2AR endocytosis or degradation, it negatively regulates β2AR entry into SNX27-occupied endosomal tubules. This results in delayed recycling of the receptor and a concomitant increase in β2AR-dependent endosomal signaling. Thus, ARRDC3 functions as a switch to modulate the endosomal residence time and subsequent intracellular signaling of the β2AR. PMID:27226565

  13. Metabolic regulation of neutrophil spreading, membrane tubulovesicular extensions (cytonemes) formation and intracellular pH upon adhesion to fibronectin

    SciTech Connect

    Galkina, Svetlana I. . E-mail: galkina@genebee.msu.su; Sud'ina, Galina F.; Klein, Thomas

    2006-08-01

    Circulating leukocytes have a round cell shape and roll along vessel walls. However, metabolic disorders can lead them to adhere to the endothelium and spread (flatten). We studied the metabolic regulation of adhesion, spreading and intracellular pH (pHi) of neutrophils (polymorphonuclear leukocytes) upon adhesion to fibronectin-coated substrata. Resting neutrophils adhered and spread on fibronectin. An increase in pHi accompanied neutrophil spreading. Inhibition of oxidative phosphorylation or inhibition of P- and F-type ATPases affected neither neutrophil spreading nor pHi. Inhibition of glucose metabolism or V-ATPase impaired neutrophil spreading, blocked the increase in the pHi and induced extrusion of membrane tubulovesicular extensions (cytonemes), anchoring cells to substrata. Omission of extracellular Na{sup +} and inhibition of chloride channels caused a similar effect. We propose that these tubulovesicular extensions represent protrusions of exocytotic trafficking, supplying the plasma membrane of neutrophils with ion exchange mechanisms and additional membrane for spreading. Glucose metabolism and V-type ATPase could affect fusion of exocytotic trafficking with the plasma membrane, thus controlling neutrophil adhesive state and pHi. Cl{sup -} efflux through chloride channels and Na{sup +} influx seem to be involved in the regulation of the V-ATPase by carrying out charge compensation for the proton-pumping activity and through V-ATPase in regulation of neutrophil spreading and pHi.

  14. Atypical regulation of G protein-coupled receptor intracellular trafficking by ubiquitination

    PubMed Central

    Dores, Michael R.; Trejo, JoAnn

    2014-01-01

    G protein-coupled receptor (GPCR) signaling is precisely regulated. After activation, GPCRs are desensitized, internalized and either recycled to the cell surface or sorted to lysosomes for degradation. The main route for GPCR lysosomal sorting requires ubiquitination and the endosomal-sorting complex required for transport (ESCRT). Four distinct ESCRT adaptor protein complexes act sequentially to bind and sort ubiquitinated cargo to lysosomes. Several studies now indicate that alternate pathways exist for GPCR lysosomal sorting that require only some components of the ESCRT and autophagy machinery. While direct GPCR ubiquitination is not required for alternate lysosomal sorting, new evidence suggests that ubiquitin may function indirectly to modulate adaptor protein activity. Here, we discuss the atypical regulation of GPCR lysosomal sorting by ubiquitination. PMID:24680429

  15. TRPV4 is endogenously expressed in vertebrate spermatozoa and regulates intracellular calcium in human sperm.

    PubMed

    Kumar, Ashutosh; Majhi, Rakesh Kumar; Swain, Nirlipta; Giri, S C; Kar, Sujata; Samanta, Luna; Goswami, Chandan

    2016-05-13

    Transient Receptor Potential Vanilloid sub-type 4 (TRPV4) is a non-selective cationic channel involved in regulation of temperature, osmolality and different ligand-dependent Ca(2+)-influx. Recently, we have demonstrated that TRPV4 is conserved in all vertebrates. Now we demonstrate that TRPV4 is endogenously expressed in all vertebrate sperm cells ranging from fish to mammals. In human sperm, TRPV4 is present as N-glycosylated protein and its activation induces Ca(2+)-influx. Its expression and localization differs in swim-up and swim-down cells suggesting that TRPV4 is an important determining factor for sperm motility. We demonstrate that pharmacological activation or inhibition of TRPV4 regulates Ca(2+)-wave propagation from head to tail. Such findings may have wide application in male fertility-infertility, contraception and conservation of endangered species as well. PMID:27003252

  16. Transporters involved in regulation of intracellular pH in primary cultured rat brain endothelial cells

    PubMed Central

    Taylor, Caroline J; Nicola, Pieris A; Wang, Shanshan; Barrand, Margery A; Hladky, Stephen B

    2006-01-01

    Fluid secretion across the blood–brain barrier, critical for maintaining the correct fluid balance in the brain, entails net secretion of HCO3−, which is brought about by the combined activities of ion transporters situated in brain microvessels. These same transporters will concomitantly influence intracellular pH (pHi). To analyse the transporters that may be involved in the maintenance of pHi and hence secretion of HCO3−, we have loaded primary cultured endothelial cells derived from rat brain microvessels with the pH indicator BCECF and suspended them in standard NaCl solutions buffered with Hepes or Hepes plus 5% CO2/HCO3−. pHi in the standard solutions showed a slow acidification over at least 30 min, the rate being less in the presence of HCO3− than in its absence. However, after accounting for the difference in buffering, the net rates of acid loading with and without HCO3− were similar. In the nominal absence of HCO3− the rate of acid loading was increased equally by removal of external Na+ or by inhibition of Na+/H+ exchange by ethylisopropylamiloride (EIPA). By contrast, in the presence of HCO3− the increase in the rate of acid loading when Na+ was removed was much larger and the rate was then also significantly greater than the rate observed in the absence of both Na+ and HCO3−. Removal of Cl− in the presence of HCO3− produced an alkalinization followed by a resumption of the slow acid gain. Removal of Na+ following removal of Cl− increased the rate of acid gain. In the presence of HCO3− and initial presence of Na+ and Cl−, DIDS inhibited the changes in pHi produced by removal of either Na+ or Cl−. These are the expected results if these cells possess an AE-like Cl−/HCO3− exchanger, a ‘channel-like’ permeability allowing slow influx of acid (or efflux of HCO3−), a NBC-like Cl−-independent Na+−HCO3− cotransporter, and a NHE-like Na+/H+ exchanger. The in vitro rates of HCO3− loading via the Na+−HCO3

  17. Crosstalk between intracellular and extracellular signals regulating interneuron production, migration and integration into the cortex

    PubMed Central

    Peyre, Elise; Silva, Carla G.; Nguyen, Laurent

    2015-01-01

    During embryogenesis, cortical interneurons are generated by ventral progenitors located in the ganglionic eminences of the telencephalon. They travel along multiple tangential paths to populate the cortical wall. As they reach this structure they undergo intracortical dispersion to settle in their final destination. At the cellular level, migrating interneurons are highly polarized cells that extend and retract processes using dynamic remodeling of microtubule and actin cytoskeleton. Different levels of molecular regulation contribute to interneuron migration. These include: (1) Extrinsic guidance cues distributed along migratory streams that are sensed and integrated by migrating interneurons; (2) Intrinsic genetic programs driven by specific transcription factors that grant specification and set the timing of migration for different subtypes of interneurons; (3) Adhesion molecules and cytoskeletal elements/regulators that transduce molecular signalings into coherent movement. These levels of molecular regulation must be properly integrated by interneurons to allow their migration in the cortex. The aim of this review is to summarize our current knowledge of the interplay between microenvironmental signals and cell autonomous programs that drive cortical interneuron porduction, tangential migration, and intergration in the developing cerebral cortex. PMID:25926769

  18. USP2 regulates the intracellular localization of PER1 and circadian gene expression.

    PubMed

    Yang, Yaoming; Duguay, David; Fahrenkrug, Jan; Cermakian, Nicolas; Wing, Simon S

    2014-08-01

    Endogenous 24-h rhythms in physiology are driven by a network of circadian clocks located in most tissues. The molecular clock mechanism is based on feedback loops involving clock genes and their protein products. Posttranslational modifications, including ubiquitination, are important for regulating the clock feedback mechanism. Recently, we showed that the deubiquitinating enzyme ubiquitin-specific peptidase 2 (USP2) associates with clock proteins and deubiquitinates PERIOD1 (PER1) but does not affect its overall stability. Mice devoid of USP2 display defects in clock function. Here, we show that USP2 regulates nucleocytoplasmic shuttling and nuclear retention of PER1 and its repressive role on the clock transcription factors CLOCK and BMAL1. The rhythm of nuclear entry of PER1 in Usp2 knockout mouse embryonic fibroblasts (MEFs) was advanced but with reduced nuclear accumulation of PER1. Although Per1 mRNA expression rhythm remained intact in the Usp2 KO MEFs, the expression profiles of other core clock genes were altered. This was also true for the expression of clock-controlled genes (e.g., Dbp, Tef, Hlf, E4bp4). A similar phase advance of PER1 nuclear localization rhythm and alteration of clock gene expression profiles were also observed in livers of Usp2 KO mice. Taken together, our results demonstrate a novel function of USP2 in the molecular clock in which it regulates PER1 function by gating its nuclear entry and accumulation. PMID:25238854

  19. Embryonic common snapping turtles (Chelydra serpentina) preferentially regulate intracellular tissue pH during acid-base challenges.

    PubMed

    Shartau, Ryan B; Crossley, Dane A; Kohl, Zachary F; Brauner, Colin J

    2016-07-01

    The nests of embryonic turtles naturally experience elevated CO2 (hypercarbia), which leads to increased blood PCO2  and a respiratory acidosis, resulting in reduced blood pH [extracellular pH (pHe)]. Some fishes preferentially regulate tissue pH [intracellular pH (pHi)] against changes in pHe; this has been proposed to be associated with exceptional CO2 tolerance and has never been identified in amniotes. As embryonic turtles may be CO2 tolerant based on nesting strategy, we hypothesized that they preferentially regulate pHi, conferring tolerance to severe acute acid-base challenges. This hypothesis was tested by investigating pH regulation in common snapping turtles (Chelydra serpentina) reared in normoxia then exposed to hypercarbia (13 kPa PCO2 ) for 1 h at three developmental ages: 70% and 90% of incubation, and yearlings. Hypercarbia reduced pHe but not pHi, at all developmental ages. At 70% of incubation, pHe was depressed by 0.324 pH units while pHi of brain, white muscle and lung increased; heart, liver and kidney pHi remained unchanged. At 90% of incubation, pHe was depressed by 0.352 pH units but heart pHi increased with no change in pHi of other tissues. Yearlings exhibited a pHe reduction of 0.235 pH units but had no changes in pHi of any tissues. The results indicate common snapping turtles preferentially regulate pHi during development, but the degree of response is reduced throughout development. This is the first time preferential pHi regulation has been identified in an amniote. These findings may provide insight into the evolution of acid-base homeostasis during development of amniotes, and vertebrates in general. PMID:27091863

  20. Intracellular pH regulation in resting and contracting segments of rat mesenteric resistance vessels.

    PubMed Central

    Aalkjaer, C; Cragoe, E J

    1988-01-01

    1. The pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF) was used to measure intracellular pH (pHi) in segments of rat resistance vessels (internal diameter about 200 microns) with the vessels mounted in a myograph for simultaneous measurements of isometric contraction. 2. BCECF loaded slowly into the vessels over 1 h and did not affect the maximal contractility of the vessels. There was a loss of dye with time which, however, was very slow when the segments were only excited for 2 s/min, suggesting that the loss was mainly due to dye bleaching with only a very slow leak. 3. The ratio of the emissions (at 540 nm) with excitation at 495 and 450 nm was calibrated in terms of pH using the K+-H+ ionophore nigericin. This calibration gave a pHi value of 7.15 +/- 0.02 (n = 20), suggesting that hydrogen ions are not in electrochemical equilibrium in these vascular smooth muscles which have a membrane potential of about -60 mV. 4. Addition of 10 mM-NH4Cl caused a transient alkalinization and wash-out of 10 mM-NH4Cl a transient acidification. Increasing CO2 with maintained bicarbonate caused a rapid acidification followed by an incomplete recovery. Removal of CO2 and bicarbonate (HEPES-buffered solution) with constant extracellular pH caused a transient alkalinization but steady-state pHi was not significantly altered. 5. In bicarbonate-free buffer the Na+-H+ exchange blocker 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and sodium-free conditions caused a slow acidification. In bicarbonate buffer (PSS) EIPA had no detectable effect after 10 min but the anion exchange blocker diisothio-cyanatostilbenedisulphonic acid (DIDS) caused a small acidification over that time course. 6. The rate of recovery after an acid load was about 50% lower in HEPES buffer compared to PSS and it was inhibited by EIPA. In PSS amiloride and EIPA each had a small inhibitory effect on the pH recovery after an acid load. DIDS also inhibited the recovery from an acid load

  1. Intra-cellular mechanism of Anti-Müllerian hormone (AMH) in regulation of follicular development.

    PubMed

    Hayes, Emily; Kushnir, Vitaly; Ma, Xiaoting; Biswas, Anindita; Prizant, Hen; Gleicher, Norbert; Sen, Aritro

    2016-09-15

    Anti-Müllerian hormone (AMH) is a member of the transforming growth factor-β superfamily and plays a crucial role in testicular and ovarian functions. In clinical practice, AMH is used as a diagnostic and/or prognostic marker in women in association with ovulation induction and in various pathophysiological conditions. Despite widespread clinical use of AMH, our mechanistic understanding of AMH actions in regulating follicular development is limited. Using a mouse model, we in this study report that in vivo AMH treatment while stalls follicular development and inhibits ovulation, also prevents follicular atresia. We further show that these AMH actions are mediated through induction of two miRNAs, miR-181a and miR-181b, which regulate various aspects of FSH signaling and follicular growth, ultimately affecting downstream gene expression and folliculogenesis. We also report that in this mouse model AMH pre-treatment prior to superovulation improves oocyte yield. These studies, therefore, offer new mechanistic insight into AMH actions in folliculogenesis and point toward potential utilization of AMH as a therapeutic agent. PMID:27235859

  2. Hollow spherical nucleic acids for intracellular gene regulation based upon biocompatible silica shells.

    PubMed

    Young, Kaylie L; Scott, Alexander W; Hao, Liangliang; Mirkin, Sarah E; Liu, Guoliang; Mirkin, Chad A

    2012-07-11

    Cellular transfection of nucleic acids is necessary for regulating gene expression through antisense or RNAi pathways. The development of spherical nucleic acids (SNAs, originally gold nanoparticles functionalized with synthetic oligonucleotides) has resulted in a powerful set of constructs that are able to efficiently transfect cells and regulate gene expression without the use of auxiliary cationic cocarriers. The gold core in such structures is primarily used as a template to arrange the nucleic acids into a densely packed and highly oriented form. In this work, we have developed methodology for coating the gold particle with a shell of silica, modifying the silica with a layer of oligonucleotides, and subsequently oxidatively dissolving the gold core with I(2). The resulting hollow silica-based SNAs exhibit cooperative binding behavior with respect to complementary oligonucleotides and cellular uptake properties comparable to their gold-core SNA counterparts. Importantly, they exhibit no cytotoxicity and have been used to effectively silence the eGFP gene in mouse endothelial cells through an antisense approach. PMID:22725653

  3. Role of Sodium Bicarbonate Cotransporters in Intracellular pH Regulation and Their Regulatory Mechanisms in Human Submandibular Glands.

    PubMed

    Namkoong, Eun; Shin, Yong-Hwan; Bae, Jun-Seok; Choi, Seulki; Kim, Minkyoung; Kim, Nahyun; Hwang, Sung-Min; Park, Kyungpyo

    2015-01-01

    Sodium bicarbonate cotransporters (NBCs) are involved in the pH regulation of salivary glands. However, the roles and regulatory mechanisms among different NBC isotypes have not been rigorously evaluated. We investigated the roles of two different types of NBCs, electroneutral (NBCn1) and electrogenic NBC (NBCe1), with respect to pH regulation and regulatory mechanisms using human submandibular glands (hSMGs) and HSG cells. Intracellular pH (pHi) was measured and the pHi recovery rate from cell acidification induced by an NH4Cl pulse was recorded. Subcellular localization and protein phosphorylation were determined using immunohistochemistry and co-immunoprecipitation techniques. We determined that NBCn1 is expressed on the basolateral side of acinar cells and the apical side of duct cells, while NBCe1 is exclusively expressed on the apical membrane of duct cells. The pHi recovery rate in hSMG acinar cells, which only express NBCn1, was not affected by pre-incubation with 5 μM PP2, an Src tyrosine kinase inhibitor. However, in HSG cells, which express both NBCe1 and NBCn1, the pHi recovery rate was inhibited by PP2. The apparent difference in regulatory mechanisms for NBCn1 and NBCe1 was evaluated by artificial overexpression of NBCn1 or NBCe1 in HSG cells, which revealed that the pHi recovery rate was only inhibited by PP2 in cells overexpressing NBCe1. Furthermore, only NBCe1 was significantly phosphorylated and translocated by NH4Cl, which was inhibited by PP2. Our results suggest that both NBCn1 and NBCe1 play a role in pHi regulation in hSMG acinar cells, and also that Src kinase does not regulate the activity of NBCn1. PMID:26375462

  4. Neprilysin and Aβ Clearance: Impact of the APP Intracellular Domain in NEP Regulation and Implications in Alzheimer’s Disease

    PubMed Central

    Grimm, Marcus O. W.; Mett, Janine; Stahlmann, Christoph P.; Haupenthal, Viola J.; Zimmer, Valerie C.; Hartmann, Tobias

    2013-01-01

    One of the characteristic hallmarks of Alzheimer’s disease (AD) is an accumulation of amyloid β (Aβ) leading to plaque formation and toxic oligomeric Aβ complexes. Besides the de novo synthesis of Aβ caused by amyloidogenic processing of the amyloid precursor protein (APP), Aβ levels are also highly dependent on Aβ degradation. Several enzymes are described to cleave Aβ. In this review we focus on one of the most prominent Aβ degrading enzymes, the zinc-metalloprotease Neprilysin (NEP). In the first part of the review we discuss beside the general role of NEP in Aβ degradation the alterations of the enzyme observed during normal aging and the progression of AD. In vivo and cell culture experiments reveal that a decreased NEP level results in an increased Aβ level and vice versa. In a pathological situation like AD, it has been reported that NEP levels and activity are decreased and it has been suggested that certain polymorphisms in the NEP gene result in an increased risk for AD. Conversely, increasing NEP activity in AD mouse models revealed an improvement in some behavioral tests. Therefore it has been suggested that increasing NEP might be an interesting potential target to treat or to be protective for AD making it indispensable to understand the regulation of NEP. Interestingly, it is discussed that the APP intracellular domain (AICD), one of the cleavage products of APP processing, which has high similarities to Notch receptor processing, might be involved in the transcriptional regulation of NEP. However, the mechanisms of NEP regulation by AICD, which might be helpful to develop new therapeutic strategies, are up to now controversially discussed and summarized in the second part of this review. In addition, we review the impact of AICD not only in the transcriptional regulation of NEP but also of further genes. PMID:24391587

  5. Role of Sodium Bicarbonate Cotransporters in Intracellular pH Regulation and Their Regulatory Mechanisms in Human Submandibular Glands

    PubMed Central

    Namkoong, Eun; Shin, Yong-Hwan; Bae, Jun-Seok; Choi, Seulki; Kim, Minkyoung; Kim, Nahyun; Hwang, Sung-Min; Park, Kyungpyo

    2015-01-01

    Sodium bicarbonate cotransporters (NBCs) are involved in the pH regulation of salivary glands. However, the roles and regulatory mechanisms among different NBC isotypes have not been rigorously evaluated. We investigated the roles of two different types of NBCs, electroneutral (NBCn1) and electrogenic NBC (NBCe1), with respect to pH regulation and regulatory mechanisms using human submandibular glands (hSMGs) and HSG cells. Intracellular pH (pHi) was measured and the pHi recovery rate from cell acidification induced by an NH4Cl pulse was recorded. Subcellular localization and protein phosphorylation were determined using immunohistochemistry and co-immunoprecipitation techniques. We determined that NBCn1 is expressed on the basolateral side of acinar cells and the apical side of duct cells, while NBCe1 is exclusively expressed on the apical membrane of duct cells. The pHi recovery rate in hSMG acinar cells, which only express NBCn1, was not affected by pre-incubation with 5 μM PP2, an Src tyrosine kinase inhibitor. However, in HSG cells, which express both NBCe1 and NBCn1, the pHi recovery rate was inhibited by PP2. The apparent difference in regulatory mechanisms for NBCn1 and NBCe1 was evaluated by artificial overexpression of NBCn1 or NBCe1 in HSG cells, which revealed that the pHi recovery rate was only inhibited by PP2 in cells overexpressing NBCe1. Furthermore, only NBCe1 was significantly phosphorylated and translocated by NH4Cl, which was inhibited by PP2. Our results suggest that both NBCn1 and NBCe1 play a role in pHi regulation in hSMG acinar cells, and also that Src kinase does not regulate the activity of NBCn1. PMID:26375462

  6. Monoamine oxidase: an important intracellular regulator of gastrin release in the rat.

    PubMed

    Dial, E J; Huang, J; Delansorne, R; Lichtenberger, L M

    1986-04-01

    The role of monoamine oxidase (MAO) in the meal-induced or amino acid-induced release of gastrin was investigated. Rats that were pretreated with the nonspecific MAO inhibitor nialamide (200 mg/kg) showed a greater rise in meal-induced serum gastrin than did untreated controls. In vitro experiments demonstrated that gastrin secretion from dispersed antral G cells in response to a stimulatory dose of phenylalanine or methylbenzylamine (10 mM) was markedly enhanced if the cells were treated with nialamide. Studies with the more specific MAO inhibitors clorgyline and deprenyl indicated that antral mucosa contained predominantly type A activity. Inhibition of MAO type A with clorgyline, both in vivo and in vitro, resulted in a greater release of gastrin after stimulation by a meal or phenylalanine. It is concluded that MAO may play an important role in the regulation of gastrin release from the G cell by partially controlling the level of amines within the cell. PMID:3081396

  7. Nonsecreted cytoplasmic alpha-fetoprotein: a newly discovered role in intracellular signaling and regulation. An update and commentary.

    PubMed

    Mizejewski, G J

    2015-12-01

    The concept of a non-secreted cytoplasmic-bound form of alpha-fetoprotein is not a new notion in AFP biological activities. Cytoplasmic AFP (CyAFP) is a long known but forgotten protein in search of a function other than a histochemical biomarker. In this report, CyAFP is presented as an "old" protein with a newly described intracellular function. In 1976, CyAFP was shown to be a product of hepatoma cells utilizing 14Cleucine incorporation and demonstrated by autoradiographic procedures. The synthesis of CyAFP without secretion was demonstrated to occur in both malignant and non-malignant cells encompassing hepatomas, ascite fluid cells, immature rodent uterus, MCF-7 breast cancers, and cytosols from human breast cancer patients. Using computer protein matching and alignments in AFP versus members of the nuclear receptor superfamily, a consecutive series of leucine zipper (heptad) repeats in AFP was previously reported, suggesting the possibility for protein-to-protein interactions. The potential for heptad heterodimerization between protein-binding partners provided the rationale for proposing that CyAFP might have the capability to form molecular hetero-complexes with cytoplasmic based transcription factors. More recent investigations have now provided experimental evidence that CyAFP is capable of colocalizing and interacting with transcription-associated factors. Such proteins can modulate intracellular signaling leading to regulation of transcription factors and initiation of growth in human cancer cells. Although circulating serum AFP is known as a growth-enhancing factor during development, cytoplasmic AFP has a lethal role in the oncogenesis, growth, and metastasis of adult liver cancer. PMID:26162540

  8. Regulation of the collagenase-3 receptor and its role in intracellular ligand processing in rat osteoblastic cells

    NASA Technical Reports Server (NTRS)

    Walling, H. W.; Chan, P. T.; Omura, T. H.; Barmina, O. Y.; Fiacco, G. J.; Jeffrey, J. J.; Partridge, N. C.

    1998-01-01

    We have previously described a specific, saturable receptor for rat collagenase-3 in the rat osteosarcoma cell line, UMR 106-01. Binding of rat collagenase-3 to this receptor is coupled to the internalization and eventual degradation of the enzyme and correlates with observed extracellular levels of the enzyme. In this study we have shown that decreased binding, internalization, and degradation of 125I-rat collagenase-3 were observed in cells after 24 h of parathyroid hormone treatment; these activities returned to control values after 48 h and were increased substantially (twice control levels) after 96 h of treatment with the hormone. Subcellular fractionation studies to identify the route of uptake and degradation of collagenase-3 localized intracellular accumulation of 125I-rat collagenase-3 initially in Golgi-associated lysosomes and later in secondary lysosomes. Maximal lysosomal accumulation of the radiolabel and stimulation of general lysosomal activity occurred after 72 h of parathyroid hormone treatment. Preventing fusion of endosomes with lysosomes (by temperature shift, colchicine, or monensin) resulted in no internalized 125I-collagenase-3 in either lysosomal fraction. Treatment of UMR cells with the above agents or ammonium chloride decreased excretion of 125I-labeled degradation products of collagenase-3. These experiments demonstrated that degradation of collagenase-3 required receptor-mediated endocytosis and sequential processing by endosomes and lysosomes. Thus, parathyroid hormone regulates the expression and synthesis of collagenase-3 as well as the abundance and functioning of the collagenase-3 receptor and the intracellular degradation of its ligand. The coordinate changes in the secretion of collagenase-3 and expression of the receptor determine the net abundance of the enzyme in the extracellular space.

  9. Aphid amino acid transporter regulates glutamine supply to intracellular bacterial symbionts.

    PubMed

    Price, Daniel R G; Feng, Honglin; Baker, James D; Bavan, Selvan; Luetje, Charles W; Wilson, Alex C C

    2014-01-01

    Endosymbiotic associations have played a major role in evolution. However, the molecular basis for the biochemical interdependence of these associations remains poorly understood. The aphid-Buchnera endosymbiosis provides a powerful system to elucidate how these symbioses are regulated. In aphids, the supply of essential amino acids depends on an ancient nutritional symbiotic association with the gamma-proteobacterium Buchnera aphidicola. Buchnera cells are densely packed in specialized aphid bacteriocyte cells. Here we confirm that five putative amino acid transporters are highly expressed and/or highly enriched in Acyrthosiphon pisum bacteriocyte tissues. When expressed in Xenopus laevis oocytes, two bacteriocyte amino acid transporters displayed significant levels of glutamine uptake, with transporter ACYPI001018, LOC100159667 (named here as Acyrthosiphon pisum glutamine transporter 1, ApGLNT1) functioning as the most active glutamine transporter. Transporter ApGLNT1 has narrow substrate selectivity, with high glutamine and low arginine transport capacity. Notably, ApGLNT1 has high binding affinity for arginine, and arginine acts as a competitive inhibitor for glutamine transport. Using immunocytochemistry, we show that ApGLNT1 is localized predominantly to the bacteriocyte plasma membrane, a location consistent with the transport of glutamine from A. pisum hemolymph to the bacteriocyte cytoplasm. On the basis of functional transport data and localization, we propose a substrate feedback inhibition model in which the accumulation of the essential amino acid arginine in A. pisum hemolymph reduces the transport of the precursor glutamine into bacteriocytes, thereby regulating amino acid biosynthesis in the bacteriocyte. Structural similarities in the arrangement of hosts and symbionts across endosymbiotic systems suggest that substrate feedback inhibition may be mechanistically important in other endosymbioses. PMID:24367072

  10. The cowpox virus SPI-3 and myxoma virus SERP1 serpins are not functionally interchangeable despite their similar proteinase inhibition profiles in vitro.

    PubMed

    Wang, Y X; Turner, P C; Ness, T L; Moon, K B; Schoeb, T R; Moyer, R W

    2000-07-01

    The myxoma virus (MYX) serpin SERP1 is a secreted glycoprotein with anti-inflammatory activity that is required for full MYX virulence in vivo. The cowpox virus (CPV) serpin SPI-3 (vaccinia virus ORF K2L) is a nonsecreted glycoprotein that blocks cell-cell fusion, independent of serpin activity, and is not required for virulence of vaccinia virus or CPV in mice. Although SPI-3 has only 29% overall identity to SERP1, both serpins have arginine at the P1 position in the reactive center loop, and SPI-3 has a proteinase inhibitory profile strikingly similar to that of SERP1 [Turner, P. C., Baquero, M. T., Yuan, S., Thoennes, S. R., and Moyer, R. W. (2000) Virology 272, 267-280]. To determine whether SPI-3 and SERP1 were functionally equivalent, a CPV variant was constructed where the SPI-3 gene was deleted and replaced with the SERP1 gene regulated by the SPI-3 promoter. Cells infected with CPVDeltaSPI-3::SERP1 secrete SERP1 and show extensive fusion, suggesting that SERP1 is unable to functionally substitute for SPI-3 in fusion inhibition. In the reciprocal experiment, both copies of SERP1 were deleted from MYX and replaced with SPI-3 under the control of the SERP1 promoter. Cells infected with the MYXDeltaSERP1::SPI-3 recombinant unexpectedly secreted SPI-3, suggesting either that the cellular secretory pathway is enhanced by MYX or that CPV encodes a protein that prevents SPI-3 secretion. MYXDeltaSERP1::SPI-3 was as attenuated in rabbits as MYXDeltaSERP1::lacZ, indicating that SPI-3 cannot substitute for SERP1 in MYX pathogenesis. PMID:10873771

  11. The Potential of Vitamin D-Regulated Intracellular Signaling Pathways as Targets for Myeloid Leukemia Therapy

    PubMed Central

    Gocek, Elzbieta; Studzinski, George P.

    2015-01-01

    The current standard regimens for the treatment of acute myeloid leukemia (AML) are curative in less than half of patients; therefore, there is a great need for innovative new approaches to this problem. One approach is to target new treatments to the pathways that are instrumental to cell growth and survival with drugs that are less harmful to normal cells than to neoplastic cells. In this review, we focus on the MAPK family of signaling pathways and those that are known to, or potentially can, interact with MAPKs, such as PI3K/AKT/FOXO and JAK/STAT. We exemplify the recent studies in this field with specific relevance to vitamin D and its derivatives, since they have featured prominently in recent scientific literature as having anti-cancer properties. Since microRNAs also are known to be regulated by activated vitamin D, this is also briefly discussed here, as are the implications of the emerging acquisition of transcriptosome data and potentiation of the biological effects of vitamin D by other compounds. While there are ongoing clinical trials of various compounds that affect signaling pathways, more studies are needed to establish the clinical utility of vitamin D in the treatment of cancer. PMID:26239344

  12. Mitofusin 2 decreases intracellular lipids in macrophages by regulating peroxisome proliferator-activated receptor-γ

    SciTech Connect

    Liu, Chun; Ge, Beihai; He, Chao; Zhang, Yi; Liu, Xiaowen; Liu, Kejian; Qian, Cuiping; Zhang, Yu; Peng, Wenzhong; Guo, Xiaomei

    2014-07-18

    Highlights: • Mfn2 decreases cellular lipid accumulation by activating cholesterol transporters. • PPARγ is involved in the Mfn2-mediated increase of cholesterol transporter expressions. • Inactivation of ERK1/2 and p38 is involved in Mfn2-induced PPARγ expression. - Abstract: Mitofusin 2 (Mfn2) inhibits atherosclerotic plaque formation, but the underlying mechanism remains elusive. This study aims to reveal how Mfn2 functions in the atherosclerosis. Mfn2 expression was found to be significantly reduced in arterial atherosclerotic lesions of both mice and human compared with healthy counterparts. Here, we observed that Mfn2 increased cellular cholesterol transporter expression in macrophages by upregulating peroxisome proliferator-activated receptor-γ, an effect achieved at least partially by inhibiting extracellular signal-regulated kinase1/2 (ERK1/2) and p38 mitogen-activated protein kinases (MAPKs) pathway. These findings provide insights into potential mechanisms of Mfn2-mediated alterations in cholesterol transporter expression, which may have significant implications for the treatment of atherosclerotic heart disease.

  13. Induction of DKK1 by ox-LDL negatively regulates intracellular lipid accumulation in macrophages.

    PubMed

    Zhang, Yu; Ge, Cheng; Wang, Lin; Liu, Xinxin; Chen, Yifei; Li, Mengmeng; Zhang, Mei

    2015-01-01

    Dickkopf1 (DKK1), a canonical Wnt/β-catenin pathway antagonist, is closely associated with cardiovascular disease and adipogenesis. We performed an in vitro study to determine whether oxidized low-density lipoprotein (ox-LDL) increased the expression of DKK1 in macrophages and whether β-catenin and liver X receptor α (LXRα) were involved in this regulation. Induction of DKK1 expression by ox-LDL decreased the level of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) via a Wnt/β-catenin pathway and increased ATP-binding cassette transporter A/G1 (ABCA/G1) levels via a signal transducer and activator of transcription 3 (STAT3) pathway. Lower LOX-1 and higher ABCA/G1 levels inhibited cholesterol loading in macrophages. In conclusion, ox-LDL may induce DKK1 expression in macrophages to inhibit the accumulation of lipids through a mechanism that involves downregulation of LOX-1-mediated lipid uptake and upregulation of ABCA/G1-dependent cholesterol efflux. PMID:25436422

  14. Inhibition of epithelial Na+ currents by intracellular domains of the cystic fibrosis transmembrane conductance regulator.

    PubMed

    Kunzelmann, K; Kiser, G L; Schreiber, R; Riordan, J R

    1997-01-01

    Cystic fibrosis is characterized by an impaired cyclic adenosine 3,5-monophosphate (cAMP) activated Cl- conductance in parallel with an enhanced amiloride sensitive Na+ conductance (ENaC) of the respiratory epithelium. Very recently, acute downregulation of ENaC by the cystic fibrosis transmembrane conductance regulator (CFTR) was demonstrated in several studies. The mechanism, however, by which CFTR exerts its inhibitory effect on ENaC remains obscure. We demonstrate that cytosolic domains of human CFTR are sufficient to induce inhibition of rat epithelial Na+ currents (rENaC) when coexpressed in Xenopus oocytes and stimulated with 3-isobutyl-1-methylxanthine (IBMX). Moreover, mutations of CFTR, which occur in cystic fibrosis, abolish CFTR-dependent downregulation of rENaC. Yeast two hybrid analysis of CFTR domains and rENaC subunits suggest direct interaction between the proteins. Enhanced Na+ transport as found in the airways of cystic fibrosis patients is probably due to a lack of CFTR dependent downregulation of ENaC. PMID:9009227

  15. An intracellular trafficking pathway in the seminiferous epithelium regulating spermatogenesis: A biochemical and molecular perspective*

    PubMed Central

    Cheng, C. Yan; Mruk, Dolores D.

    2009-01-01

    During spermatogenesis, fully developed spermatids (i.e., spermatozoa) at the luminal edge of the seminiferous epithelium undergo ‘spermiation’ at stage VIII of the seminiferous epithelial cycle. This is manifested by the disruption of the apical ectoplasmic specialization (apical ES) so that spermatozoa can enter the tubule lumen and to complete their maturation in the epididymis. At the same time, the blood-testis barrier (BTB) located near the basement membrane undergoes extensive restructuring to allow transit of preleptotene spermatocytes so that post-meiotic germ cells complete their development behind the BTB. While spermiation and BTB restructuring take place concurrently at opposite ends of the Sertoli cell epithelium, the biochemical mechanism(s) by which they are coordinated were not known until recently. Studies have shown that fragments of laminin chains are generated from the laminin/integrin protein complex at the apical ES via the action of MMP-2 (matrix metalloprotease-2) at spermiation. These peptides serve as the local autocrine factors to ‘destabilize’ the BTB. These laminin peptides also exert their effects on hemidesmosome which, in turn, further potentiates BTB restructuring. Thus, a novel apical ES-BTB-hemidesmosome regulatory loop is operating in the seminiferous epithelium to coordinate these two crucial cellular events of spermatogenesis. This functional loop is further assisted by the Par3/Par6-based polarity protein complex in coordination with cytokines and testosterone at the BTB. Herein, we provide a critical review based on the latest findings in the field regarding the regulation of these cellular events. These recent findings also open up a new window for investigators studying blood-tissue barriers. PMID:19622063

  16. Serpine1 Mediates Porphyromonas gingivalis Induced Insulin Secretion in the Pancreatic Beta Cell Line MIN6

    PubMed Central

    Bhat, Uppoor G.; Watanabe, Keiko

    2015-01-01

    Periodontitis is an inflammatory disease resulting in destruction of gingiva and alveolar bone caused by an exuberant host immunological response to periodontal pathogens. Results from a number of epidemiological studies indicate a close association between diabetes and periodontitis. Results from cross-sectional studies indicate that subjects with periodontitis have a higher odds ratio of developing insulin resistance (IR). However, the mechanisms by which periodontitis influences the development of diabetes are not known. Results from our previous studies using an animal model of periodontitis suggest that periodontitis accelerates the onset of hyperinsulinemia and IR. In addition, LPS from a periodontal pathogen, Porphyromonas gingivalis (Pg), stimulates Serpine1 expression in the pancreatic beta cell line MIN6. Based on these observations, we hypothesized that a periodontal pathogen induces hyperinsulinemia and Serpine1 may be involved in this process. To test this hypothesis, we co-incubated Pg with the pancreatic beta cell line MIN6 and measured the effect on insulin secretion by MIN6 cells. We further determined the involvement of Serpine1 in insulin secretion by downregulating Serpine1 expression. Our results indicated that Pg stimulated insulin secretion by approximately 3.0 fold under normoglycemic conditions. In a hyperglycemic state, Pg increased insulin secretion by 1.5 fold. Pg significantly upregulated expression of the Serpine1 gene and this was associated with increased secretion of insulin by MIN6 cells. However, cells with downregulated Serpine1 expression were resistant to Pg stimulated insulin secretion under normoglycemic conditions. We conclude that the periodontal pathogen, Pg, induced insulin secretion by MIN6 cells and this induction was, in part, Serpine1 dependent. Thus, Serpine1 may play a pivotal role in insulin secretion during the accelerated development of hyperinsulinemia and the resulting IR in the setting of periodontitis. PMID

  17. Volatile profiling reveals intracellular metabolic changes in Aspergillus parasiticus: veA regulates branched chain amino acid and ethanol metabolism

    PubMed Central

    2010-01-01

    Background Filamentous fungi in the genus Aspergillus produce a variety of natural products, including aflatoxin, the most potent naturally occurring carcinogen known. Aflatoxin biosynthesis, one of the most highly characterized secondary metabolic pathways, offers a model system to study secondary metabolism in eukaryotes. To control or customize biosynthesis of natural products we must understand how secondary metabolism integrates into the overall cellular metabolic network. By applying a metabolomics approach we analyzed volatile compounds synthesized by Aspergillus parasiticus in an attempt to define the association of secondary metabolism with other metabolic and cellular processes. Results Volatile compounds were examined using solid phase microextraction - gas chromatography/mass spectrometry. In the wild type strain Aspergillus parasiticus SU-1, the largest group of volatiles included compounds derived from catabolism of branched chain amino acids (leucine, isoleucine, and valine); we also identified alcohols, esters, aldehydes, and lipid-derived volatiles. The number and quantity of the volatiles produced depended on media composition, time of incubation, and light-dark status. A block in aflatoxin biosynthesis or disruption of the global regulator veA affected the volatile profile. In addition to its multiple functions in secondary metabolism and development, VeA negatively regulated catabolism of branched chain amino acids and synthesis of ethanol at the transcriptional level thus playing a role in controlling carbon flow within the cell. Finally, we demonstrated that volatiles generated by a veA disruption mutant are part of the complex regulatory machinery that mediates the effects of VeA on asexual conidiation and sclerotia formation. Conclusions 1) Volatile profiling provides a rapid, effective, and powerful approach to identify changes in intracellular metabolic networks in filamentous fungi. 2) VeA coordinates the biosynthesis of secondary

  18. Fasting and postprandial regulation of the intracellular localization of adiponectin and of adipokines secretion by dietary fat in rats

    PubMed Central

    Olivares-García, V; Torre-Villalvazo, I; Velázquez-Villegas, L; Alemán, G; Lara, N; López-Romero, P; Torres, N; Tovar, A R; Díaz-Villaseñor, A

    2015-01-01

    Background/Objective: Dietary fat sources modulate fasting serum concentration of adipokines, particularly adiponectin. However, previous studies utilized obese animals in which adipose tissue function is severely altered. Thus, the present study aimed to assess the postprandial regulation of adipokine secretion in nonobese rats that consumed high-fat diet (HFD) composed of different types of fat for a short time. Methods: The rats were fed a control diet or a HFD containing coconut, safflower or soybean oil (rich in saturated fatty acid, monounsaturated fatty acid or polyunsaturated fatty acid, respectively) for 21 days. The serum concentrations of adiponectin, leptin, retinol, retinol-binding protein-4 (RBP-4), visfatin and resistin were determined at fasting and after refeeding. Adiponectin multimerization and intracellular localization, as well as the expression of endoplasmic reticulum (ER) chaperones and transcriptional regulators, were evaluated in epididymal white adipose tissue. Results: In HFD-fed rats, serum adiponectin was significantly decreased 30 min after refeeding. With coconut oil, all three multimeric forms were reduced; with safflower oil, only the high-molecular-weight (HMW) and medium-molecular-weight (MMW) forms were decreased; and with soybean oil, only the HMW form was diminished. These reductions were due not to modifications in mRNA abundance or adiponectin multimerization but rather to an increment in intracellular localization at the ER and plasma membrane. Thus, when rats consumed a HFD, the type of dietary fat differentially affected the abundance of endoplasmic reticulum resident protein 44 kDa (ERp44), sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor-γ (PPARγ) mRNAs, all of which are involved in the post-translational processing of adiponectin required for its secretion. Leptin, RBP-4, resistin and visfatin serum concentrations did not change during fasting, whereas modest alterations were observed after

  19. Role of H(+)-pyrophosphatase activity in the regulation of intracellular pH in a scuticociliate parasite of turbot: Physiological effects.

    PubMed

    Mallo, Natalia; Lamas, Jesús; de Felipe, Ana-Paula; Sueiro, Rosa-Ana; Fontenla, Francisco; Leiro, José-Manuel

    2016-10-01

    The scuticociliatosis is a very serious disease that affects the cultured turbot, and whose causal agent is the anphizoic and marine euryhaline ciliate Philasterides dicentrarchi. Several protozoans possess acidic organelles that contain high concentrations of pyrophosphate (PPi), Ca(2+) and other elements with essential roles in vesicular trafficking, pH homeostasis and osmoregulation. P. dicentrarchi possesses a pyrophosphatase (H(+)-PPase) that pumps H(+) through the membranes of vacuolar and alveolar sacs. These compartments share common features with the acidocalcisomes described in other parasitic protozoa (e.g. acid content and Ca(2+) storage). We evaluated the effects of Ca(2+) and ATP on H (+)-PPase activity in this ciliate and analyzed their role in maintaining intracellular pH homeostasis and osmoregulation, by the addition of PPi and inorganic molecules that affect osmolarity. Addition of PPi led to acidification of the intracellular compartments, while the addition of ATP, CaCl2 and bisphosphonates analogous of PPi and Ca(2+) metabolism regulators led to alkalinization and a decrease in H(+)-PPase expression in trophozoites. Addition of NaCl led to proton release, intracellular Ca(2+) accumulation and downregulation of H(+)-PPase expression. We conclude that the regulation of the acidification of intracellular compartments may be essential for maintaining the intracellular pH homeostasis necessary for survival of ciliates and their adaptation to salt stress, which they will presumably face during the endoparasitic phase, in which the salinity levels are lower than in their natural environment. PMID:27480055

  20. Regulation of L-type calcium current by intracellular magnesium in rat cardiac myocytes

    PubMed Central

    Wang, Min; Tashiro, Michiko; Berlin, Joshua R

    2004-01-01

    calcineurin. Thus, physiologically relevant [Mg2+]i modulates ICa by counteracting the effects of Ca2+ channel phosphorylation and by an unknown [Ca2+]i-dependent mechanism. The magnitude of these effects suggests that changes in [Mg2+]i could be critical in regulating l-type channel gating. PMID:14617671

  1. Three-dimensional structure of a schistosome serpin revealing an unusual configuration of the helical subdomain

    SciTech Connect

    Granzin, Joachim; Huang, Ying; Topbas, Celalettin; Huang, Wenying; Wu, Zhiping; Misra, Saurav; Hazen, Stanley L.; Blanton, Ronald E.; Lee, Xavier; Weiergräber, Oliver H.

    2012-06-01

    The crystal structure of ShSPI, a serpin from the blood fluke S. haematobium, reveals some peculiar features of the helical subdomain which have not been observed previously in the serpin superfamily. Parasitic organisms are constantly challenged by the defence mechanisms of their respective hosts, which often depend on serine protease activities. Consequently, protease inhibitors such as those belonging to the serpin superfamily have emerged as protective elements that support the survival of the parasites. This report describes the crystal structure of ShSPI, a serpin from the trematode Schistosoma haematobium. The protein is exposed on the surface of invading cercaria as well as of adult worms, suggesting its involvement in the parasite–host interaction. While generally conforming to the well established serpin fold, the structure reveals several distinctive features, mostly concerning the helical subdomain of the protein. It is proposed that these peculiarities are related to the unique biological properties of a small serpin subfamily which is conserved among pathogenic schistosomes.

  2. Lysosome-associated membrane proteins (LAMPs) regulate intracellular positioning of mitochondria in MC3T3-E1 cells.

    PubMed

    Rajapakshe, Anupama R; Podyma-Inoue, Katarzyna A; Terasawa, Kazue; Hasegawa, Katsuya; Namba, Toshimitsu; Kumei, Yasuhiro; Yanagishita, Masaki; Hara-Yokoyama, Miki

    2015-02-01

    The intracellular positioning of both lysosomes and mitochondria meets the requirements of degradation and energy supply, which are respectively the two major functions for cellular maintenance. The positioning of both lysosomes and mitochondria is apparently affected by the nutrient status of the cells. However, the mechanism coordinating the positioning of the organelles has not been sufficiently elucidated. Lysosome-associated membrane proteins-1 and -2 (LAMP-1 and LAMP-2) are highly glycosylated proteins that are abundant in lysosomal membranes. In the present study, we demonstrated that the siRNA-mediated downregulation of LAMP-1, LAMP-2 or their combination enhanced the perinuclear localization of mitochondria, in the pre-osteoblastic cell line MC3T3-E1. On the other hand, in the osteocytic cell line MLO-Y4, in which both the lysosomes and mitochondria originally accumulate in the perinuclear region and mitochondria also fill dendrites, the effect of siRNA of LAMP-1 or LAMP-2 was barely observed. LAMPs are not directly associated with mitochondria, and there do not seem to be any accessory molecules commonly required to recruit the motor proteins to lysosomes and mitochondria. Our results suggest that LAMPs may regulate the positioning of lysosomes and mitochondria. A possible mechanism involving the indirect and context-dependent action of LAMPs is discussed. PMID:25246127

  3. Proton/l-Glutamate Symport and the Regulation of Intracellular pH in Isolated Mesophyll Cells 1

    PubMed Central

    Snedden, Wayne A.; Chung, Induk; Pauls, Randy H.; Bown, Alan W.

    1992-01-01

    Addition of l-[U-14C]glutamate to a suspension of mechanically isolated asparagus (Asparagus sprengeri Regel) mesophyll cells results in (a) alkalinization of the medium, (b) uptake of l-[U-14C]glutamate, and (c) efflux of [14C]4-aminobutyrate, a product of glutamate decarboxylation. All three phenomena were eliminated by treatment with 1 millimolar aminooxyacetate. In vitro glutamate decarboxylase (GAD) assays showed that (a) 2 millimolar aminooxyacetate eliminated enzyme activity, (b) activity was pyridoxal phosphate-dependent, and (c) activity exhibited a sharp pH optimum at 6.0 that decreased to 20% of optimal activity at pH 5.0 and 7.0. Addition of 1.5 millimolar sodium butyrate or sodium acetate to cell suspensions caused immediate alkalinization of the medium followed by a resumption of acidification of the medium at a rate approximately double the initial rate. The data indicate that (a) continued H+/l-glutamate contransport is dependent upon GAD activity, (b) the pH-dependent properties of GAD are consistent with a role in a metabolic pH-stat, and (c) the regulation of intracellular pH during H+/l-Glu symport may involve both H+ consumption during 4-aminobutyrate production and ATP-driven H+ efflux. PMID:16668938

  4. Arv1 regulates PM and ER membrane structure and homeostasis but is dispensable for intracellular sterol transport

    PubMed Central

    Georgiev, Alexander G.; Johansen, Jesper; Ramanathan, Vidhya D.; Sere, Yves Y.; Beh, Christopher T.; Menon, Anant K.

    2013-01-01

    The pan-eukaryotic endoplasmic reticulum (ER) membrane protein Arv1 has been suggested to play a role in intracellular sterol transport. We tested this proposal by comparing sterol traffic in wild-type and Arv1-deficient Saccharomyces cerevisiae. We used fluorescence microscopy to track the retrograde movement of exogenously supplied dehydroergosterol (DHE) from the plasma membrane (PM) to the ER and lipid droplets and high performance liquid chromatography to quantify, in parallel, the transport-coupled formation of DHE esters. Metabolic labeling and subcellular fractionation were used to assay anterograde transport of ergosterol from the ER to the PM. We report that sterol transport between the ER and PM is unaffected by Arv1 deficiency. Instead, our results indicate differences in ER morphology and the organization of the PM lipid bilayer between wild-type and arv1Δ cells suggesting a distinct role for Arv1 in membrane homeostasis. In arv1Δ cells, specific defects affecting single C-terminal transmembrane domain proteins suggest that Arv1 might regulate membrane insertion of tail-anchored proteins involved in membrane homoeostasis. PMID:23668914

  5. Idh1 protects murine hepatocytes from endotoxin-induced oxidative stress by regulating the intracellular NADP(+)/NADPH ratio.

    PubMed

    Itsumi, M; Inoue, S; Elia, A J; Murakami, K; Sasaki, M; Lind, E F; Brenner, D; Harris, I S; Chio, I I C; Afzal, S; Cairns, R A; Cescon, D W; Elford, A R; Ye, J; Lang, P A; Li, W Y; Wakeham, A; Duncan, G S; Haight, J; You-Ten, A; Snow, B; Yamamoto, K; Ohashi, P S; Mak, T W

    2015-11-01

    Isocitrate dehydrogenase-1 (Idh1) is an important metabolic enzyme that produces NADPH by converting isocitrate to α-ketoglutarate. Idh1 is known to reduce reactive oxygen species (ROS) induced in cells by treatment with lipopolysaccharide (LPS) in vitro. Here, we used Idh1-deficient knockout (Idh1 KO) mice to investigate the role of Idh1 in antioxidant defense in vivo. Idh1 KO mice showed heightened susceptibility to death induced by LPS and exhibited increased serum levels of inflammatory cytokines such as tumor necrosis factor-α and interleukin-6. The serum of LPS-injected Idh1 KO mice also contained elevated levels of AST, a marker of inflammatory liver damage. Furthermore, after LPS injection, livers of Idh1 KO mice showed histological evidence of elevated oxidative DNA damage compared with livers of wild-type (WT) mice. Idh1 KO livers showed a faster and more pronounced oxidative stress than WT livers. In line with that, Idh1 KO hepatocytes showed higher ROS levels and an increase in the NADP(+)/NADPH ratio when compared with hepatocytes isolated from WT mice. These results suggest that Idh1 has a physiological function in protecting cells from oxidative stress by regulating the intracellular NADP(+)/NADPH ratio. Our findings suggest that stimulation of Idh1 activity may be an effective therapeutic strategy for reducing oxidative stress during inflammatory responses, including the early stages of septic shock. PMID:25882048

  6. Identification of the nuclear export signals that regulate the intracellular localization of the mouse CMP-sialic acid synthetase

    SciTech Connect

    Fujita, Akiko; Sato, Chihiro; Kitajima, Ken. E-mail: kitajima@agr.nagoya-u.ac.jp

    2007-03-30

    The CMP-sialic acid synthetase (CSS) catalyzes the activation of sialic acid (Sia) to CMP-Sia which is a donor substrate of sialyltransferases. The vertebrate CSSs are usually localized in nucleus due to the nuclear localization signal (NLS) on the molecule. In this study, we first point out that a small, but significant population of the mouse CMP-sialic acid synthetase (mCSS) is also present in cytoplasm, though mostly in nucleus. As a mechanism for the localization in cytoplasm, we first identified two nuclear export signals (NESs) in mCSS, based on the localization studies of the potential NES-deleted mCSS mutants as well as the potential NES-tagged eGFP proteins. These two NESs are conserved among mammalian and fish CSSs, but not present in the bacterial or insect CSS. These results suggest that the intracellular localization of vertebrate CSSs is regulated by not only the NLS, but also the NES sequences.

  7. [Phagocytosis of Mycobacterium leprae down-regulates anti-microbial activity of murine macrophages against Mycobacterium intracellulare].

    PubMed

    Tatano, Yutaka; Sano, Chiaki; Emori, Masako; Saito, Hajime; Sato, Katsumasa; Shimizu, Toshiaki; Tomioka, Haruaki

    2012-09-01

    Patients with highly bacillated lepromatous leprosy (LL) essentially lack T cell-mediated immune responses specific to Mycobacterium leprae (ML) antigens, resulting in severely impaired host resistance to leprosy bacilli. Such type of immune unresponsiveness characteristic of LL patients is mainly attributable to markedly depressed T cell ability to activate/expand in response to ML antigens. In this study, we examined profiles of antimycobacterial activity of macrophages, which phagocytized leprosy bacilli, because there is another possibility that, in LL patients, host macrophages in the leprosy lesions are impaired in their antimicrobial activity due to their interaction with infected leprosy bacilli, particularly cellular events through binding with and/or internalization of the pathogens, thereby causing the reduction in host resistance to ML pathogens. The present study indicated the following. First, the anti-M. avium complex activity of murine peritoneal macrophages was significantly reduced when they had phagocytosed heat-killed leprosy bacilli. Second, infection of macrophages with leprosy bacilli did not affect macrophage-mediated suppressor activity against T cell proliferative response to Concanavalin A. These findings indicate that macrophage's intracellular signaling pathways that are up-regulated in response to phagocytosis of leprosy bacilli are linked to the signaling cascades participating in macrophage antimicrobial functions, but not cross-talk with those allowing the expression of macrophage's suppressor activity against T cell functions. PMID:23012845

  8. FGF23 is a novel regulator of intracellular calcium and cardiac contractility in addition to cardiac hypertrophy

    PubMed Central

    Touchberry, Chad D.; Green, Troy M.; Tchikrizov, Vladimir; Mannix, Jaimee E.; Mao, Tiffany F.; Carney, Brandon W.; Girgis, Magdy; Vincent, Robert J.; Wetmore, Lori A.; Dawn, Buddhadeb; Bonewald, Lynda F.; Stubbs, Jason R.

    2013-01-01

    Fibroblast growth factor 23 (FGF23) is a hormone released primarily by osteocytes that regulates phosphate and vitamin D metabolism. Recent observational studies in humans suggest that circulating FGF23 is independently associated with cardiac hypertrophy and increased mortality, but it is unknown whether FGF23 can directly alter cardiac function. We found that FGF23 significantly increased cardiomyocyte cell size in vitro, the expression of gene markers of cardiac hypertrophy, and total protein content of cardiac muscle. In addition, FGFR1 and FGFR3 mRNA were the most abundantly expressed FGF receptors in cardiomyocytes, and the coreceptor α-klotho was expressed at very low levels. We tested an animal model of chronic kidney disease (Col4a3−/− mice) that has elevated serum FGF23. We found elevations in common hypertrophy gene markers in Col4a3−/− hearts compared with wild type but did not observe changes in wall thickness or cell size by week 10. However, the Col4a3−/− hearts did show reduced fractional shortening (−17%) and ejection fraction (−11%). Acute exposure of primary cardiomyocytes to FGF23 resulted in elevated intracellular Ca2+ ([Ca2+]i; F/Fo + 86%) which was blocked by verapamil pretreatment. FGF23 also increased ventricular muscle strip contractility (67%), which was inhibited by FGF receptor antagonism. We hypothesize that although FGF23 can acutely increase [Ca2+]i, chronically this may lead to decreases in contractile function or stimulate cardiac hypertrophy, as observed with other stress hormones. In conclusion, FGF23 is a novel bone/heart endocrine factor and may be an important mediator of cardiac Ca2+ regulation and contractile function during chronic kidney disease. PMID:23443925

  9. The interaction between AMPKβ2 and the PP1-targeting subunit R6 is dynamically regulated by intracellular glycogen content.

    PubMed

    Oligschlaeger, Yvonne; Miglianico, Marie; Dahlmans, Vivian; Rubio-Villena, Carla; Chanda, Dipanjan; Garcia-Gimeno, Maria Adelaida; Coumans, Will A; Liu, Yilin; Voncken, J Willem; Luiken, Joost J F P; Glatz, Jan F C; Sanz, Pascual; Neumann, Dietbert

    2016-04-01

    AMP-activated protein kinase (AMPK) is a metabolic stress-sensing kinase. We previously showed that glucose deprivation induces autophosphorylation of AMPKβ at Thr-148, which prevents the binding of AMPK to glycogen. Furthermore, in MIN6 cells, AMPKβ1 binds to R6 (PPP1R3D), a glycogen-targeting subunit of protein phosphatase type 1 (PP1), thereby regulating the glucose-induced inactivation of AMPK. In the present study, we further investigated the interaction of R6 with AMPKβ and the possible dependency on Thr-148 phosphorylation status. Yeast two-hybrid (Y2H) analyses and co-immunoprecipitation (IP) of the overexpressed proteins in human embryonic kidney (HEK) 293T) cells revealed that both AMPKβ1 and AMPK-β2 wild-type (WT) isoforms bind to R6. The AMPKβ-R6 interaction was stronger with the muscle-specific AMPKβ2-WT and required association with the substrate-binding motif of R6. When HEK293T cells or C2C12 myotubes were cultured in high-glucose medium, AMPKβ2-WT and R6 weakly interacted. In contrast, glycogen depletion significantly enhanced this protein interaction. Mutation of AMPKβ2 Thr-148 prevented the interaction with R6 irrespective of the intracellular glycogen content. Treatment with the AMPK activator oligomycin enhanced the AMPKβ2-R6 interaction in conjunction with increased Thr-148 phosphorylation in cells grown in low-glucose medium. These data are in accordance with R6 binding directly to AMPKβ2 when both proteins detach from the diminishing glycogen particle, which is simultaneous with increased AMPKβ2 Thr-148 autophosphorylation. Such a model points to a possible control of AMPK by PP1-R6 upon glycogen depletion in muscle. PMID:26831516

  10. Cyclic ADP ribose is a novel regulator of intracellular Ca2+ oscillations in human bone marrow mesenchymal stem cells

    PubMed Central

    Tao, Rong; Sun, Hai-Ying; Lau, Chu-Pak; Tse, Hung-Fat; Lee, Hon-Cheung; Li, Gui-Rong

    2011-01-01

    Abstract Bone marrow mesenchymal stem cells (MSCs) are a promising cell source for regenerative medicine. However, the cellular biology of these cells is not fully understood. The present study characterizes the cyclic ADP-ribose (cADPR)-mediated Ca2+ signals in human MSCs and finds that externally applied cADPR can increase the frequency of spontaneous intracellular Ca2+ (Ca2+i) oscillations. The increase was abrogated by a specific cADPR antagonist or an inositol trisphosphate receptor (IP3R) inhibitor, but not by ryanodine. In addition, the cADPR-induced increase of Ca2+i oscillation frequency was prevented by inhibitors of nucleoside transporter or by inhibitors of the transient receptor potential cation melastatin-2 (TRPM2) channel. RT-PCR revealed mRNAs for the nucleoside transporters, concentrative nucleoside transporters 1/2 and equilibrative nucleoside transporters 1/3, IP3R1/2/3 and the TRPM2 channel, but not those for ryanodine receptors and CD38 in human MSCs. Knockdown of the TRPM2 channel by specific short interference RNA abolished the effect of cADPR on the Ca2+i oscillation frequency, and prevented the stimulation of proliferation by cADPR. Moreover, cADPR remarkably increased phosphorylated extracellular-signal-regulated kinases 1/2 (ERK1/2), but not Akt or p38 mitogen-activated protein kinase (MAPK). However, cADPR had no effect on adipogenesis or osteogenesis in human MSCs. Our results indicate that cADPR is a novel regulator of Ca2+i oscillations in human MSCs. It permeates the cell membrane through the nucleoside transporters and increases Ca2+ oscillation via activation of the TRPM2 channel, resulting in enhanced phosphorylation of ERK1/2 and, thereby, stimulation of human MSC proliferation. This study delineates an alternate signalling pathway of cADPR that is distinct from its well-established role of serving as a Ca2+ messenger for mobilizing the internal Ca2+ stores. Whether cADPR can be used clinically for stimulating marrow function in

  11. Regulation of MMP-1 expression in response to hypoxia is dependent on the intracellular redox status of metastatic bladder cancer cells.

    PubMed

    Shin, Dong Hui; Dier, Usawadee; Melendez, Juan Andres; Hempel, Nadine

    2015-12-01

    High steady-state reactive oxygen species (ROS) production has been implicated with metastatic disease progression. We provide new evidence that this increased intracellular ROS milieu uniquely predisposes metastatic tumor cells to hypoxia-mediated regulation of the matrix metalloproteinase MMP-1. Using a cell culture metastatic progression model we previously reported that steady-state intracellular H2O2 levels are elevated in highly metastatic 253J-BV bladder cancer cells compared to their non-metastatic 253J parental cells. 253J-BV cells display higher basal MMP-1 expression, which is further enhanced under hypoxic conditions (1% O2). This hypoxia-mediated MMP-1 increase was not observed in the non-metastatic 253J cells. Hypoxia-induced MMP-1 increases are accompanied by the stabilization of hypoxia-inducible transcription factors (HIFs)-1α and HIF-2α, and a rise in intracellular ROS in metastatic 253J-BV cells. RNA interference studies show that hypoxia-mediated MMP-1 expression is primarily dependent on the presence of HIF-2α. Further, hypoxia promotes migration and spheroid outgrowth of only the metastatic 253J-BV cells and not the parental 253J cells. The observed HIF stabilization, MMP-1 expression and migration under hypoxia are dependent on increases in intracellular ROS, as these effects are attenuated by treatment with the antioxidant N-acetyl-L-cysteine. These data show that ROS play an important role in hypoxia-mediated MMP-1 expression and that an elevated intracellular redox environment, as observed in metastasis, predisposes tumor cells to an enhanced hypoxic response. It further supports the notion that metastatic tumor cells are uniquely able to utilize intracellular increases in ROS to drive pro-metastatic signaling events and highlights the important interplay between ROS and hypoxia in malignancy. PMID:26343184

  12. Regulation of intracellular pH in cardiac muscle during cell shrinkage and swelling in anisosmolar solutions.

    PubMed

    Whalley, D W; Hemsworth, P D; Rasmussen, H H

    1994-02-01

    The effect on intracellular pH (pHi) of exposure to solutions of progressively increasing osmolarity from 418 to 620 mosM and to hyposmolar solutions (240 mosM) was examined in guinea pig ventricular muscle using ion-selective microelectrodes. Exposure of tissue to 418 mosM Tyrode solution (100 mM sucrose added) produced an intracellular alkalosis of approximately 0.1 U, whereas exposure to 620 mosM solution (300 mM sucrose added) caused an intracellular acidosis of approximately 0.1 U. The maximal rate of recovery of pHi from acidosis induced by an NH4Cl prepulse increased progressively as extracellular osmolarity was raised from 310 to 620 mosM. This suggests that the acidosis observed at steady state in 620 mosM solution is not due to inhibition of the Na(+)-H+ exchanger. In the presence of 10 microM ryanodine, exposure to 620 mosM solution produced a sustained intracellular alkalosis. We suggest that the decrease in pHi during exposure to 620 mosM solution is due, at least in part, to the acidifying influence of Ca2+ release from the sarcoplasmic reticulum. This decrease in pHi is expected to contribute to the negative inotrop reported in studies of cardiac contractility in markedly hyperosmolar solutions. There was no change in pHi when tissue was exposed to hyposmolar solution. However, the maximal rate of recovery of pHi from acidosis was slower in hyposmolar than in isosmolar solution, despite a concomitant decrease in the intracellular buffer capacity. This suggests that osmotic cell swelling results in inhibition of the sarcolemmal Na(+)-H+ exchanger. PMID:8141367

  13. Increased surfactant protein D fails to improve bacterial clearance and inflammation in serpinB1-/- mice.

    PubMed

    Stolley, J Michael; Gong, Dapeng; Farley, Kalamo; Zhao, Picheng; Cooley, Jessica; Crouch, Erika C; Benarafa, Charaf; Remold-O'Donnell, Eileen

    2012-12-01

    Previously, we described the protective role of the neutrophil serine protease inhibitor serpinB1 in preventing early mortality of Pseudomonas aeruginosa lung infection by fostering bacterial clearance and limiting inflammatory cytokines and proteolytic damage. Surfactant protein D (SP-D), which maintains the antiinflammatory pulmonary environment and mediates bacterial removal, was degraded in infected serpinB1-deficient mice. Based on the hypothesis that increased SP-D would rescue or mitigate the pathological effects of serpinB1 deletion, we generated two serpinB1(-/-) lines overexpressing lung-specific rat SP-D and inoculated the mice with P. aeruginosa. Contrary to predictions, bacterial counts in the lungs of SP-D(low)serpinB1(-/-) and SP-D(high) serpinB1(-/-) mice were 4 logs higher than wild-type and not different from serpinB1(-/-) mice. SP-D overexpression also failed to mitigate inflammation (TNF-α), lung injury (free protein, albumin), or excess neutrophil death (free myeloperoxidase, elastase). These pathological markers were higher for infected SP-D(high)serpinB1(-/-) mice than for serpinB1(-/-) mice, although the differences were not significant after controlling for multiple comparisons. The failure of transgenic SP-D to rescue antibacterial defense of serpinB1-deficient mice occurred despite 5-fold or 20-fold increased expression levels, largely normal structure, and dose-dependent bacteria-aggregating activity. SP-D of infected wild-type mice was intact in 43-kD monomers by reducing SDS-PAGE. By contrast, proteolytic fragments of 35, 17, and 8 kD were found in infected SP-D(low)serpinB1(-/-), SP-D(high) serpinB1(-/-) mice, and serpinB1(-/-) mice. Thus, although therapies to increase lung concentration of SP-D may have beneficial applications, the findings suggest that therapy with SP-D may not be beneficial for lung inflammation or infection if the underlying clinical condition includes excess proteolysis. PMID:23024061

  14. SET Protein Interacts with Intracellular Domains of the Gonadotropin-releasing Hormone Receptor and Differentially Regulates Receptor Signaling to cAMP and Calcium in Gonadotrope Cells*

    PubMed Central

    Avet, Charlotte; Garrel, Ghislaine; Denoyelle, Chantal; Laverrière, Jean-Noël; Counis, Raymond; Cohen-Tannoudji, Joëlle; Simon, Violaine

    2013-01-01

    In mammals, the receptor of the neuropeptide gonadotropin-releasing hormone (GnRHR) is unique among the G protein-coupled receptor (GPCR) family because it lacks the carboxyl-terminal tail involved in GPCR desensitization. Therefore, mechanisms involved in the regulation of GnRHR signaling are currently poorly known. Here, using immunoprecipitation and GST pull-down experiments, we demonstrated that SET interacts with GnRHR and targets the first and third intracellular loops. We delineated, by site-directed mutagenesis, SET binding sites to the basic amino acids 66KRKK69 and 246RK247, located next to sequences required for receptor signaling. The impact of SET on GnRHR signaling was assessed by decreasing endogenous expression of SET with siRNA in gonadotrope cells. Using cAMP and calcium biosensors in gonadotrope living cells, we showed that SET knockdown specifically decreases GnRHR-mediated mobilization of intracellular cAMP, whereas it increases its intracellular calcium signaling. This suggests that SET influences signal transfer between GnRHR and G proteins to enhance GnRHR signaling to cAMP. Accordingly, complexing endogenous SET by introduction of the first intracellular loop of GnRHR in αT3-1 cells significantly reduced GnRHR activation of the cAMP pathway. Furthermore, decreasing SET expression prevented cAMP-mediated GnRH stimulation of Gnrhr promoter activity, highlighting a role of SET in gonadotropin-releasing hormone regulation of gene expression. In conclusion, we identified SET as the first direct interacting partner of mammalian GnRHR and showed that SET contributes to a switch of GnRHR signaling toward the cAMP pathway. PMID:23233674

  15. Intracellular proteoglycans.

    PubMed Central

    Kolset, Svein Olav; Prydz, Kristian; Pejler, Gunnar

    2004-01-01

    Proteoglycans (PGs) are proteins with glycosaminoglycan chains, are ubiquitously expressed and have a wide range of functions. PGs in the extracellular matrix and on the cell surface have been the subject of extensive structural and functional studies. Less attention has so far been given to PGs located in intracellular compartments, although several reports suggest that these have biological functions in storage granules, the nucleus and other intracellular organelles. The purpose of this review is, therefore, to present some of these studies and to discuss possible functions linked to PGs located in different intracellular compartments. Reference will be made to publications relevant for the topics we present. It is beyond the scope of this review to cover all publications on PGs in intracellular locations. PMID:14759226

  16. Overexpression and altered nucleocytoplasmic distribution of Anopheles ovalbumin-like SRPN10 serpins in Plasmodium-infected midgut cells.

    PubMed

    Danielli, Alberto; Barillas-Mury, Carolina; Kumar, Sanjeev; Kafatos, Fotis C; Loukeris, Thanasis G

    2005-02-01

    The design of effective, vector-based malaria transmission blocking strategies relies on a thorough understanding of the molecular and cellular interactions that occur during the parasite sporogonic cycle in the mosquito. During Plasmodium berghei invasion, transcription from the SRPN10 locus, encoding four serine protease inhibitors of the ovalbumin family, is strongly induced in the mosquito midgut. Herein we demonstrate that intense induction as well as redistribution of SRPN10 occurs specifically in the parasite-invaded midgut epithelial cells. Quantitative analysis establishes that in response to epithelial invasion, SRPN10 translocates from the nucleus to the cytoplasm and this is followed by strong SRPN10 overexpression. The invaded cells exhibit signs of apoptosis, suggesting a link between this type of intracellular serpin and epithelial damage. The SRPN10 gene products constitute a novel, robust and cell-autonomous marker of midgut invasion by ookinetes. The SRPN10 dynamics at the subcellular level confirm and further elaborate the 'time bomb' model of P. berghei invasion in both Anopheles stephensi and Anopheles gambiae. In contrast, this syndrome of responses is not elicited by mutant P. berghei ookinetes lacking the major ookinete surface proteins, P28 and P25. Molecular markers with defined expression patterns, in combination with mutant parasite strains, will facilitate dissection of the molecular mechanisms underlying vector competence and development of effective transmission blocking strategies. PMID:15659062

  17. The C-terminal tail of protein kinase D2 and protein kinase D3 regulates their intracellular distribution

    SciTech Connect

    Papazyan, Romeo; Rozengurt, Enrique; Rey, Osvaldo . E-mail: orey@mednet.ucla.edu

    2006-04-14

    We generated a set of GFP-tagged chimeras between protein kinase D2 (PKD2) and protein kinase D3 (PKD3) to examine in live cells the contribution of their C-terminal region to their intracellular localization. We found that the catalytic domain of PKD2 and PKD3 can localize to the nucleus when expressed without other kinase domains. However, when the C-terminal tail of PKD2 was added to its catalytic domain, the nuclear localization of the resulting protein was inhibited. In contrast, the nuclear localization of the CD of PKD3 was not inhibited by its C-terminal tail. Furthermore, the exchange of the C-terminal tail of PKD2 and PKD3 in the full-length proteins was sufficient to exchange their intracellular localization. Collectively, these data demonstrate that the short C-terminal tail of these kinases plays a critical role in determining their cytoplasmic/nuclear localization.

  18. Signaling Microdomains Regulate Inositol 1,4,5-Trisphosphate-Mediated Intracellular Calcium Transients in Cultured Neurons

    PubMed Central

    Jacob, Simon N.; Choe, Chi-Un; Uhlen, Per; DeGray, Brenda; Yeckel, Mark F.; Ehrlich, Barbara E.

    2010-01-01

    Ca2+signals in neurons use specific temporal and spatial patterns to encode unambiguous information about crucial cellular functions. To understand the molecular basis for initiation and propagation of inositol 1,4,5-trisphosphate (InsP3)-mediated intracellular Ca2+ signals, we correlated the subcellular distribution of components of the InsP3 pathway with measurements of agonist-induced intracellular Ca2+ transients in cultured rat hippocampal neurons and pheochromocytoma cells. We found specialized domains with high levels of phosphatidylinositol-4-phosphate kinase (PIPKIγ) and chromogranin B (CGB), proteins acting synergistically to increase InsP3 pumps in the plasma membrane (PMCA) and sarco-endoplasmic reticulum receptor (InsP3R) activity and sensitivity. In contrast, Ca2+ as well as buffers that antagonize the rise in intracellular Ca2+ were distributed uniformly. By pharmacologically blocking phosphatidylinositol-4-kinase and PIPKIγ or disrupting the CGB–InsP3R interaction by transfecting an interfering polypeptide fragment, we produced major changes in the initiation site and kinetics of the Ca2+signal. This study shows that a limited number of proteins can reassemble to form unique, spatially restricted signaling domains to generate distinctive signals in different regions of the same neuron. The finding that the subcellular location of initiation sites and protein microdomains was cell type specific will help to establish differences in spatiotemporal Ca2+signaling in different types of neurons. PMID:15772345

  19. TRIM30α Is a Negative-Feedback Regulator of the Intracellular DNA and DNA Virus-Triggered Response by Targeting STING

    PubMed Central

    Yang, Bo; Yan, Shanshan; Zhou, Haiyan; He, Lan; Lin, Guomei; Lian, Zhexiong; Jiang, Zhengfan; Sun, Bing

    2015-01-01

    Uncontrolled immune responses to intracellular DNA have been shown to induce autoimmune diseases. Homeostasis regulation of immune responses to cytosolic DNA is critical for limiting the risk of autoimmunity and survival of the host. Here, we report that the E3 ubiquitin ligase tripartite motif protein 30α (TRIM30α) was induced by herpes simplex virus type 1 (HSV-1) infection in dendritic cells (DCs). Knockdown or genetic ablation of TRIM30α augmented the type I IFNs and interleukin-6 response to intracellular DNA and DNA viruses. Trim30α-deficient mice were more resistant to infection by DNA viruses. Biochemical analyses showed that TRIM30α interacted with the stimulator of interferon genes (STING), which is a critical regulator of the DNA-sensing response. Overexpression of TRIM30α promoted the degradation of STING via K48-linked ubiquitination at Lys275 through a proteasome-dependent pathway. These findings indicate that E3 ligase TRIM30α is an important negative-feedback regulator of innate immune responses to DNA viruses by targeting STING. PMID:26114947

  20. Isolation and molecular characterization of a major hemolymph serpin from the triatomine, Panstrongylus megistus

    PubMed Central

    2014-01-01

    Background Chagas disease kills 2.5 thousand people per year of 15 million persons infected in Latin America. The disease is caused by the protozoan, Trypanosome cruzi, and vectored by triatomine insects, including Panstrongylus megistus, an important vector in Brazil. Medicines treating Chagas disease have unpleasant side effects and may be ineffective, therefore, alternative control techniques are required. Knowledge of the T. cruzi interactions with the triatomine host needs extending and new targets/strategies for control identified. Serine and cysteine peptidases play vital roles in protozoan life cycles including invasion and entry of T. cruzi into host cells. Peptidase inhibitors are, therefore, promising targets for disease control. Methods SDS PAGE and chromatograpy detected and isolated a P. megistus serpin which was peptide sequenced by mass spectrometry. A full amino acid sequence was obtained from the cDNA and compared with other insect serpins. Reverse transcription PCR analysis measured serpin transcripts of P. megistus tissues with and without T. cruzi infection. Serpin homology modeling used the Swiss Model and Swiss-PDB viewer programmes. Results The P. megistus serpin (PMSRP1) has a ca. 40 kDa molecular mass with 404 amino acid residues. A reactive site loop contains a highly conserved hinge region but, based on sequence alignment, the normal cleavage site for serine proteases at P1-P1′ was translocated to the putative position P4′-P5′. A small peptide obtained corresponded to the C-terminal 40 amino acid region. The secondary structure of PMSRP1 indicated nine α-helices and three β-sheets, similar to other serpins. PMSRP1 transcripts occurred in all tested tissues but were highest in the fat body and hemocytes. Levels of mRNA encoding PMSRP1 were significantly modulated in the hemocytes and stomach by T. cruzi infection indicating a role for PMSRP1 in the parasite interactions with P. megistus. Conclusions For the first time, a

  1. The putative role of Rhipicephalus microplus salivary serpins in the tick-host relationship.

    PubMed

    Tirloni, Lucas; Kim, Tae Kwon; Coutinho, Mariana Loner; Ali, Abid; Seixas, Adriana; Termignoni, Carlos; Mulenga, Albert; da Silva Vaz, Itabajara

    2016-04-01

    Inflammation and hemostasis are part of the host's first line of defense to tick feeding. These systems are in part serine protease mediated and are tightly controlled by their endogenous inhibitors, in the serpin superfamily (serine protease inhibitors). From this perspective ticks are thought to use serpins to evade host defenses during feeding. The cattle tick Rhipicephalus microplus encodes at least 24 serpins, of which RmS-3, RmS-6, and RmS-17 were previously identified in saliva of this tick. In this study, we screened inhibitor functions of these three saliva serpins against a panel of 16 proteases across the mammalian defense pathway. Our data confirm that Pichia pastoris-expressed rRmS-3, rRmS-6, and rRmS-17 are likely inhibitors of pro-inflammatory and pro-coagulant proteases. We show that rRmS-3 inhibited chymotrypsin and cathepsin G with stoichiometry of inhibition (SI) indices of 1.8 and 2.0, and pancreatic elastase with SI higher than 10. Likewise, rRmS-6 inhibited trypsin with SI of 2.6, chymotrypsin, factor Xa, factor XIa, and plasmin with SI higher than 10, while rRmS-17 inhibited trypsin, cathepsin G, chymotrypsin, plasmin, and factor XIa with SI of 1.6, 2.6, 2.7, 3.4, and 9.0, respectively. Additionally, we observed the formation of irreversible complexes between rRmS-3 and chymotrypsin, rRmS-6/rRmS-17 and trypsin, and rRmS-3/rRmS-17 and cathepsin G, which is consistent with typical mechanism of inhibitory serpins. In blood clotting assays, rRmS-17 delayed plasma clotting by 60 s in recalcification time assay, while rRmS-3 and rRmS-6 did not have any effect. Consistent with inhibitor function profiling data, 2.0 μM rRmS-3 and rRmS-17 inhibited cathepsin G-activated platelet aggregation in a dose-responsive manner by up to 96% and 95% respectively. Of significant interest, polyclonal antibodies blocked inhibitory functions of the three serpins. Also notable, antibodies to Amblyomma americanum, Ixodes scapularis, and Rhipicephalus sanguineus

  2. Evaluation of potential implication of membrane estrogen binding sites on ERE-dependent transcriptional activity and intracellular estrogen receptor-alpha regulation in MCF-7 breast cancer cells.

    PubMed

    Seo, Hye Sook; Leclercq, Guy

    2002-01-01

    The potential involvement of membrane estrogen binding sites in the induction of ERE-dependent transcriptional activity as well as in the regulation of intracellular estrogen receptor alpha (ER-alpha) level under estradiol (E2) stimulation was investigated. Our approach relied upon the use of two DCC-treated E2-BSA (bovine serum albumin) solutions (E2-6-BSA and E2-17-BSA). The absence of detectable free E2 in these solutions was established. Both E2-BSA conjugates led to a transient dose-dependent stimulation of the expression of ERE-luciferase (LUC) reporter gene in MVLN cells (MCF-7 cells stably transfected with a pVit-tk-LUC reporter plasmid), a property not recorded with free E2, which maintained enhanced transcriptional activity during the whole experiment. A very low concentration of E2 (10 pM) synergistically acted with E2-BSA conjugates. Hence, ERE-dependent transcriptional activity induced by these conjugates appeared to result from their known interactions with membrane estrogen binding sites. Anti-estrogens (AEs: 4-OH-TAM and RU 58,668), which antagonize genomic ER responses, abrogated the luciferase activity induced by E2-BSA conjugates, confirming a potential relationship between membrane-related signals and intracellular ER. Moreover, induction of luciferase was recorded when the cells were exposed to IBMX (3-isobutyl-1-methylxanthine) and cyclic nucleotides (cAMP/cGMP), suggesting the implication of the latter in the signal transduction pathway leading to the expression of the reporter gene. Growth factors (IGF-I, EGF and TGF-alpha) also slightly stimulated luciferase and synergistically acted with 10 pM E2, or 1 microM E2-BSA conjugates, in agreement with the concept of a cross-talk between steroids and peptides acting on the cell membrane. Remarkably, E2-BSA conjugates, IBMX and all investigated growth factors failed to down-regulate intracellular ER in MCF-7 cells, indicating the need for a direct intracellular interaction of the ligand with the

  3. SERPINE2 is a possible candidate promotor for lymph node metastasis in testicular cancer

    SciTech Connect

    Nagahara, Akira; Nakayama, Masashi; Oka, Daizo; Tsuchiya, Mutsumi; Kawashima, Atsunari; Mukai, Masatoshi; Nakai, Yasutomo; Takayama, Hitoshi; Nishimura, Kazuo; Jo, Yoshimasa; Nagai, Atsushi; Okuyama, Akihiko; Nonomura, Norio

    2010-01-22

    Testicular germ cell tumors (TGCTs) commonly metastasize to the lymph node or lung. However, it remains unclear which genes are associated with TGCT metastasis. The aim of this study was to identify gene(s) that promoted human TGCT metastasis. We intraperitoneally administered conditioned medium (CM) from JKT-1, a cell-line from a human testicular seminoma, or JKT-HM, a JKT-1 cell sub-line with high metastatic potential, into mice with JKT-1 xenografts. Administration of CM from JKT-HM significantly promoted lymph node metastasis. A cDNA microarray analysis showed that JKT-HM cells highly expressed the Serpine peptidase inhibitor, clade E, member 2 (SERPINE2), which encodes a secreted protein. Administration of CM from SERPINE2-silenced JKT-HM cells inhibited lymph node metastasis in the xenograft model, compared with administration of CM from JKT-HM cells. There was no significant difference in xenograft volume. Moreover, administration of CM from SERPINE2-over-expressing JKT-1 was likely to promote lymph node metastasis in the xenograft model. There was no difference in the in vitro proliferation or migration of JKT-1 cells cultured with CM from JKT-HM cells, compared to that with CM from JKT-1. There was no promotion of proliferation or lymphangiogenesis in the xenografts, as measured by Ki-67 and LYVE-1 immunohistochemistry, respectively. Although we could not clarify how SERPINE2 promoted lymph node metastasis, it may be a promoter in the development of lymph node metastasis in the human seminoma cells in a mouse xenograft model.

  4. pH-regulated activation and release of a bacteria-associated phospholipase C during intracellular infection by Listeria monocytogenes.

    PubMed

    Marquis, H; Hager, E J

    2000-01-01

    Listeria monocytogenes grows in the cytosol of mammalian cells and spreads from cell to cell without exiting the intracellular milieu. During cell-cell spread, bacteria become transiently entrapped in double-membrane vacuoles. Escape from these vacuoles is mediated in part by a bacterial phospholipase C (PC-PLC), whose activation requires cleavage of an N-terminal peptide. PC-PLC activation occurs in the acidified vacuolar environment. In this study, the pH-dependent mechanism of PC-PLC activation was investigated by manipulating the intracellular pH of the host. PC-PLC secreted into infected cells was immunoprecipitated, and both forms of the protein were identified by SDS-PAGE fluorography. PC-PLC activation occurred at pH 7.0 and lower, but not at pH 7.3. Total amounts of PC-PLC secreted into infected cells increased several-fold over controls within 5 min of a decrease in intracellular pH, and the active form of PC-PLC was the most abundant species detected. Bacterial release of active PC-PLC was dependent on Mpl, a bacterial metalloprotease that processes the proform (proPC-PLC), and did not require de novo protein synthesis. The amount of proPC-PLC released in response to a decrease in pH was the same in wild-type and Mpl-minus-infected cells. Immunofluorescence detection of PC-PLC in infected cells was performed. When fixed and permeabilized infected cells were treated with a bacterial cell wall hydrolase, over 97% of wild-type and Mpl-minus bacteria stained positively for PC-PLC, in contrast to less than 5% in untreated cells. These results indicate that intracellular bacteria carry pools of proPC-PLC. Upon cell-cell spread, a decrease in vacuolar pH triggers Mpl activation of proPC-PLC, resulting in bacterial release of active PC-PLC. PMID:10652090

  5. Bayesian phylogeny analysis of vertebrate serpins illustrates evolutionary conservation of the intron and indels based six groups classification system from lampreys for ∼500 MY

    PubMed Central

    2015-01-01

    The serpin superfamily is characterized by proteins that fold into a conserved tertiary structure and exploits a sophisticated and irreversible suicide-mechanism of inhibition. Vertebrate serpins are classified into six groups (V1–V6), based on three independent biological features—genomic organization, diagnostic amino acid sites and rare indels. However, this classification system was based on the limited number of mammalian genomes available. In this study, several non-mammalian genomes are used to validate this classification system using the powerful Bayesian phylogenetic method. This method supports the intron and indel based vertebrate classification and proves that serpins have been maintained from lampreys to humans for about 500 MY. Lampreys have fewer than 10 serpins, which expand into 36 serpins in humans. The two expanding groups V1 and V2 have SERPINB1/SERPINB6 and SERPINA8/SERPIND1 as the ancestral serpins, respectively. Large clusters of serpins are formed by local duplications of these serpins in tetrapod genomes. Interestingly, the ancestral HCII/SERPIND1 locus (nested within PIK4CA) possesses group V4 serpin (A2APL1, homolog of α2-AP/SERPINF2) of lampreys; hence, pointing to the fact that group V4 might have originated from group V2. Additionally in this study, details of the phylogenetic history and genomic characteristics of vertebrate serpins are revisited. PMID:26157611

  6. E-selectin ligand–1 regulates growth plate homeostasis in mice by inhibiting the intracellular processing and secretion of mature TGF-β

    PubMed Central

    Yang, Tao; Mendoza-Londono, Roberto; Lu, Huifang; Tao, Jianning; Li, Kaiyi; Keller, Bettina; Jiang, Ming Ming; Shah, Rina; Chen, Yuqing; Bertin, Terry K.; Engin, Feyza; Dabovic, Branka; Rifkin, Daniel B.; Hicks, John; Jamrich, Milan; Beaudet, Arthur L.; Lee, Brendan

    2010-01-01

    The majority of human skeletal dysplasias are caused by dysregulation of growth plate homeostasis. As TGF-β signaling is a critical determinant of growth plate homeostasis, skeletal dysplasias are often associated with dysregulation of this pathway. The context-dependent action of TFG-β signaling is tightly controlled by numerous mechanisms at the extracellular level and downstream of ligand-receptor interactions. However, TGF-β is synthesized as an inactive precursor that is cleaved to become mature in the Golgi apparatus, and the regulation of this posttranslational intracellular processing and trafficking is much less defined. Here, we report that a cysteine-rich protein, E-selectin ligand–1 (ESL-1), acts as a negative regulator of TGF-β production by binding TGF-β precursors in the Golgi apparatus in a cell-autonomous fashion, inhibiting their maturation. Furthermore, ESL-1 inhibited the processing of proTGF-β by a furin-like protease, leading to reduced secretion of mature TGF-β by primary mouse chondrocytes and HEK293 cells. In vivo loss of Esl1 in mice led to increased TGF-β/SMAD signaling in the growth plate that was associated with reduced chondrocyte proliferation and delayed terminal differentiation. Gain-of-function and rescue studies of the Xenopus ESL-1 ortholog in the context of early embryogenesis showed that this regulation of TGF-β/Nodal signaling was evolutionarily conserved. This study identifies what we believe to be a novel intracellular mechanism for regulating TGF-β during skeletal development and homeostasis. PMID:20530870

  7. Subcellular localization of CrmA: identification of a novel leucine-rich nuclear export signal conserved in anti-apoptotic serpins.

    PubMed Central

    Rodriguez, Jose A; Span, Simone W; Kruyt, Frank A E; Giaccone, Giuseppe

    2003-01-01

    The cowpox virus-encoded anti-apoptotic protein cytokine response modifier A (CrmA) is a member of the serpin family that specifically inhibits the cellular proteins caspase 1, caspase 8 and granzyme B. In this study, we have used Flag- and yellow fluorescent protein (YFP)-tagged versions of CrmA to investigate the mechanisms that regulate its subcellular localization. We show that CrmA can actively enter and exit the nucleus and we demonstrate the role of the nuclear export receptor CRM1 in this shuttling process. CrmA contains a novel leucine-rich nuclear export signal (NES) that is functionally conserved in the anti-apoptotic cellular serpin PI-9. Besides this leucine-rich export signal, additional sequences mapping to a 103-amino-acid region flanking the NES contribute to the CRM1-dependent nuclear export of CrmA. Although YFP-tagged CrmA is primarily located in the cytoplasm, shifting its localization to be predominantly nuclear by fusion of a heterologous nuclear localization signal did not impair its ability to prevent Fas-induced apoptosis. We propose that nucleocytoplasmic shuttling would allow CrmA to efficiently target cellular pro-apoptotic proteins not only in the cytoplasm, but also in the nucleus, and thus to carry out its anti-apoptotic function in both compartments. PMID:12667137

  8. Intracellular pH and its relationship to regulation of ion transport in normal and cystic fibrosis human nasal epithelia.

    PubMed Central

    Willumsen, N J; Boucher, R C

    1992-01-01

    1. Intracellular pH (pHi) of cultured human airway epithelial cells from normal and cystic fibrosis (CF) subjects were measured with double-barrelled pH-sensitive liquid exchanger microelectrodes. The cells, which were grown to confluence on a permeable collagen matrix support, were mounted in a modified miniature Ussing chamber. All studies were conducted under open circuit conditions. Values are given as means +/- S.E.M. and n refers to the number of preparations. 2. Normal preparations (n = 15) were characterized by a transepithelial potential difference (Vt) of -18 +/- 2 mV, an apical membrane potential (Va) of -19 +/- 2 mV, a basolateral membrane potential (Vb) of -37 +/- 2 mV, a transepithelial resistance (Rt) of 253 +/- 15 omega cm2, a fractional apical membrane resistance (fRa) of 0.40 +/- 0.04 and an equivalent short circuit current (Ieq) of -73 +/- 7 microA cm-2. 3. CF preparations (n = 13) were characterized by a Vt of -46 +/- 7 mV, a Va of 3 +/- 5 mV, a Vb of -43 +/- 3 mV, Rt of 373 +/- 47 omega cm2, fRa of 0.44 +/- 0.04 and an Ieq of -130 +/- 16 microA cm-2. All parameters except Vb and fRa were significantly different (P < 0.025) from those of normal preparations. 4. Despite large differences in electrochemical driving force for proton flow across the apical cell membranes between normal and CF preparations (-4 +/- 3 mV and 20 +/- 7 mV, respectively), pHi was similar (7.15 +/- 0.02 and 7.11 +/- 0.05, respectively). The driving force across the basolateral membrane was similar in normal and CF preparations (22 +/- 3 and 26 +/- 3 mV, respectively). 5. Intracellular alkalinization achieved by removal of CO2 from the luminal Ringer solution or by luminal ammonium prepulse led to stimulation of Ieq in both normal (from -58 to -70 microA cm-2, n = 4; P < 0.05) and CF (from -144 to -163 microA cm-2, n = 4; P < 0.005) preparations. The increase in Ieq was associated with a reduction of Rt, increase in fRa, and hyperpolarization of Vb. All changes in

  9. Altered Expression of Brain Proteinase-Activated Receptor-2, Trypsin-2 and Serpin Proteinase Inhibitors in Parkinson's Disease.

    PubMed

    Hurley, Michael J; Durrenberger, Pascal F; Gentleman, Steve M; Walls, Andrew F; Dexter, David T

    2015-09-01

    Neuroinflammation is thought to contribute to cell death in neurodegenerative disorders, but the factors involved in the inflammatory process are not completely understood. Proteinase-activated receptor-2 (PAR2) expression in brain is increased in Alzheimer's disease and multiple sclerosis, but the status of PAR2 in Parkinson's disease is unknown. This study examined expression of PAR2 and endogenous proteinase activators (trypsin-2, mast cell tryptase) and proteinase inhibitors (serpin-A5, serpin-A13) in areas vulnerable and resistant to neurodegeneration in Parkinson's disease at different Braak α-synuclein stages of the disease in post-mortem brain. In normal aged brain, expression of PAR-2, trypsin-2, and serpin-A5 and serpin-A13 was found in neurons and microglia, and alterations in the amount of immunoreactivity for these proteins were found in some brain regions. Namely, there was a decrease in neurons positive for serpin-A5 in the dorsal motor nucleus, and serpin-A13 expression was reduced in the locus coeruleus and primary motor cortex, while expression of PAR2, trypsin-2 and both serpins was reduced in neurons within the substantia nigra. There was an increased number of microglia that expressed serpin-A5 in the dorsal motor nucleus of vagus and elevated numbers of microglia that expressed serpin-A13 in the substantia nigra of late Parkinson's disease cases. The number of microglia that expressed trypsin-2 increased in primary motor cortex of incidental Lewy body disease cases. Analysis of Parkinson's disease cases alone indicated that serpin-A5 and serpin-A13, and trypsin-2 expression in midbrain and cerebral cortex was different in cases with a high incidence of L-DOPA-induced dyskinesia and psychosis compared to those with low levels of these treatment-induced side effects. This study showed that there was altered expression in brain of PAR2 and some proteins that can control its function in Parkinson's disease. Given the role of PAR2 in

  10. Histidine-domain-containing protein tyrosine phosphatase regulates platelet-derived growth factor receptor intracellular sorting and degradation.

    PubMed

    Ma, Haisha; Wardega, Piotr; Mazaud, David; Klosowska-Wardega, Agnieszka; Jurek, Aleksandra; Engström, Ulla; Lennartsson, Johan; Heldin, Carl-Henrik

    2015-11-01

    Histidine domain-containing protein tyrosine phosphatase (HD-PTP) is a putative phosphatase that has been shown to affect the signaling and downregulation of certain receptor tyrosine kinases. To investigate if HD-PTP affects platelet-derived growth factor receptor β (PDGFRβ) signaling, we employed the overexpression of HA-tagged HD-PTP, as well as siRNA-mediated and lentivirus shRNA-mediated silencing of HD-PTP in NIH3T3 cells. We found that HD-PTP was recruited to the PDGFRβ in a ligand-dependent manner. Depletion of HD-PTP resulted in an inability of PDGF-BB to promote tyrosine phosphorylation of the ubiquitin ligases c-Cbl and Cbl-b, with a concomitant missorting and reduction of the degradation of activated PDGFRβ. In contrast, ligand-induced internalization of PDGFRβ was unaffected by HD-PTP silencing. Furthermore, the levels of STAM and Hrs of the ESCRT0 machinery were decreased, and immunofluorescence staining showed that in HD-PTP-depleted cells, PDGFRβ accumulated in large aberrant intracellular structures. After the reduction of HD-PTP expression, an NIH3T3-derived cell line that has autocrine PDGF-BB signaling (sis-3T3) showed increased ability of anchorage-independent growth. However, exogenously added PDGF-BB promoted efficient additional colony formation in control cells, but was not able to do so in HD-PTP-depleted cells. Furthermore, cells depleted of HD-PTP migrated faster than control cells. In summary, HD-PTP affects the intracellular sorting of activated PDGFRβ and the migration, proliferation and tumorigenicity of cells stimulated by PDGF. PMID:26232618