LARP1 functions as a molecular switch for mTORC1-mediated translation of an essential class of mRNAs.

PubMed

Hong, Sungki; Freeberg, Mallory A; Han, Ting; Kamath, Avani; Yao, Yao; Fukuda, Tomoko; Suzuki, Tsukasa; Kim, John K; Inoki, Ken

2017-06-26

The RNA binding protein, LARP1, has been proposed to function downstream of mTORC1 to regulate the translation of 5'TOP mRNAs such as those encoding ribosome proteins (RP). However, the roles of LARP1 in the translation of 5'TOP mRNAs are controversial and its regulatory roles in mTORC1-mediated translation remain unclear. Here we show that LARP1 is a direct substrate of mTORC1 and Akt/S6K1. Deep sequencing of LARP1-bound mRNAs reveal that non-phosphorylated LARP1 interacts with both 5' and 3'UTRs of RP mRNAs and inhibits their translation. Importantly, phosphorylation of LARP1 by mTORC1 and Akt/S6K1 dissociates it from 5'UTRs and relieves its inhibitory activity on RP mRNA translation. Concomitantly, phosphorylated LARP1 scaffolds mTORC1 on the 3'UTRs of translationally-competent RP mRNAs to facilitate mTORC1-dependent induction of translation initiation. Thus, in response to cellular mTOR activity, LARP1 serves as a phosphorylation-sensitive molecular switch for turning off or on RP mRNA translation and subsequent ribosome biogenesis.

xRRM

PubMed Central

Singh, Mahavir; Choi, Charles P.; Feigon, Juli

2013-01-01

Genuine La and La-related proteins group 7 (LARP7) bind to the non-coding RNAs transcribed by RNA polymerase III (RNAPIII), which end in UUU-3′OH. The La motif and RRM1 of these proteins (the La module) cooperate to bind the UUU-3′OH, protecting the RNA from degradation, while other domains may be important for RNA folding or other functions. Among the RNAPIII transcripts is ciliate telomerase RNA (TER). p65, a member of the LARP7 family, is an integral Tetrahymena thermophila telomerase holoenzyme protein required for TER biogenesis and telomerase RNP assembly. p65, together with TER and telomerase reverse transcriptase (TERT), form the Tetrahymena telomerase RNP catalytic core. p65 has an N-terminal domain followed by a La module and a C-terminal domain, which binds to the TER stem 4. We recently showed that the p65 C-terminal domain harbors a cryptic, atypical RRM, which uses a unique mode of single- and double-strand RNA binding and is required for telomerase RNP catalytic core assembly. This domain, which we named xRRM, appears to be present in and unique to genuine La and LARP7 proteins. Here we review the structure of the xRRM, discuss how this domain could recognize diverse substrates of La and LARP7 proteins and discuss the functional implications of the xRRM as an RNP chaperone. PMID:23328630

Larp Nb3Sn Quadrupole Magnets for the Lhc Luminosity Upgrade

NASA Astrophysics Data System (ADS)

Ferracin, P.

2010-04-01

The US LHC Accelerator Research Program (LARP) is a collaboration between four US laboratories (BNL, FNAL, LBNL, and SLAC) aimed at contributing to the commissioning and operation of the LHC and conducting R&D on its luminosity upgrade. Within LARP, the Magnet Program's main goal is to demonstrate that Nb3Sn superconducting magnets are a viable option for a future upgrade of the LHC Interaction Regions. Over the past four years, LARP has successfully fabricated and tested several R&D magnets: 1) the subscale quadrupole magnet SQ, to perform technology studies with 300 mm long racetrack coils, 2) the technology quadrupole TQ, to investigate support structure behavior with 1 m long cos 2θ coils, and 3) the long racetrack magnet LR, to test 3.6 m long racetrack coils. The next milestone consists in the fabrication and test of the 3.7 m long quadrupole magnet LQ, with the goal of demonstrating that Nb3Sn technology is mature for use in high energy accelerators. After an overview of design features and test result of the LARP magnets fabricated so far, this paper focuses on the status of the fabrication of LQ: we describe the production of the 3.4 m long cos 2θ coils, and the of the qualification support structure. Finally, the status of the development of the next 1 m long model HQ, conceived to explore stress and field limits of Nb3Sn superconducting, magnets, is presented.

Structural Basis for Telomerase RNA Recognition and RNP Assembly by the Holoenzyme La Family Protein p65

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

Singh, Mahavir; Wang, Zhonghua; Koo, Bon-Kyung

2012-07-01

Telomerase is a ribonucleoprotein complex essential for maintenance of telomere DNA at linear chromosome ends. The catalytic core of Tetrahymena telomerase comprises a ternary complex of telomerase RNA (TER), telomerase reverse transcriptase (TERT), and the essential La family protein p65. NMR and crystal structures of p65 C-terminal domain and its complex with stem IV of TER reveal that RNA recognition is achieved by a combination of single- and double-stranded RNA binding, which induces a 105{sup o} bend in TER. The domain is a cryptic, atypical RNA recognition motif with a disordered C-terminal extension that forms an {alpha} helix in themore » complex necessary for hierarchical assembly of TERT with p65-TER. This work provides the first structural insight into biogenesis and assembly of TER with a telomerase-specific protein. Additionally, our studies define a structurally homologous domain (xRRM) in genuine La and LARP7 proteins and suggest a general mode of RNA binding for biogenesis of their diverse RNA targets.« less

Circular RNA_LARP4 inhibits cell proliferation and invasion of gastric cancer by sponging miR-424-5p and regulating LATS1 expression.

PubMed

Zhang, Jing; Liu, Hui; Hou, Lidan; Wang, Ge; Zhang, Rui; Huang, Yanxia; Chen, Xiaoyu; Zhu, Jinshui

2017-09-11

Non-coding RNAs (ncRNAs) have been shown to regulate gene expression involved in tumor progression of multiple malignancies. Our previous studies indicated that large tumor suppressor kinase 1 (LATS1), a core part of Hippo signaling pathway, functions as a tumor suppressor in gastric cancer (GC). But, the underlying molecular mechanisms by which ncRNAs modulate LATS1 expression in GC remain undetermined. The correlation of LATS1 and has-miR-424-5p (miR-424) expression with clinicopathological characteristics and prognosis of GC patients was analyzed by TCGA RNA-sequencing data. A novel circular RNA_LARP4 (circLARP4) was identified to sponge miR-424 by circRNA expression profile and bioinformatic analysis. The binding site between miR-424 and LATS1 or circLARP4 was verified using dual luciferase assay and RNA immunoprecipitation (RIP) assay. The expression and localization of circLARP4 in GC tissues were investigated by fluorescence in situ hybridization (FISH). MTT, colony formation, Transwell and EdU assays were performed to assess the effects of miR-424 or circLARP4 on cell proliferation and invasion. Increased miR-424 expression or decreased LATS1 expression was associated with pathological stage and unfavorable prognosis of GC patients. Ectopic expression of miR-424 promoted proliferation and invasion of GC cells by targeting LATS1 gene. Furthermore, circLARP4 was mainly localized in the cytoplasm and inhibited biological behaviors of GC cells by sponging miR-424. The expression of circLARP4 was downregulated in GC tissues and represented an independent prognostic factor for overall survival of GC patients. circLARP4 may act as a novel tumor suppressive factor and a potential biomarker in GC.

Protection heater design validation for the LARP magnets using thermal imaging

DOE PAGES

Marchevsky, M.; Turqueti, M.; Cheng, D. W.; ...

2016-03-16

Protection heaters are essential elements of a quench protection scheme for high-field accelerator magnets. Various heater designs fabricated by LARP and CERN have been already tested in the LARP high-field quadrupole HQ and presently being built into the coils of the high-field quadrupole MQXF. In order to compare the heat flow characteristics and thermal diffusion timescales of different heater designs, we powered heaters of two different geometries in ambient conditions and imaged the resulting thermal distributions using a high-sensitivity thermal video camera. We observed a peculiar spatial periodicity in the temperature distribution maps potentially linked to the structure of themore » underlying cable. Two-dimensional numerical simulation of heat diffusion and spatial heat distribution have been conducted, and the results of simulation and experiment have been compared. Imaging revealed hot spots due to a current concentration around high curvature points of heater strip of varying cross sections and visualized thermal effects of various interlayer structural defects. Furthermore, thermal imaging can become a future quality control tool for the MQXF coil heaters.« less

Fabrication of First 4-m Coils for the LARP MQXFA Quadrupole and Assembly in Mirror Structure

DOE PAGES

Holik, Eddie Frank; Ambrosio, Giorgio; Anerella, Michael; ...

2017-01-23

The US LHC Accelerator Research Program is constructing prototype interaction region quadrupoles as part of the US in-kind contribution to the Hi-Lumi LHC project. The low-beta MQXFA Q1/Q3 coils have a 4-m length and a 150 mm bore. The design is first validated on short, one meter models (MQXFS) developed as part of the longstanding Nb3Sn quadrupole R&D by LARP in collaboration with CERN. In parallel, facilities and tooling are being developed and refined at BNL, LBNL, and FNAL to enable long coil production, assembly, and cold testing. Long length scale-up is based on the experience from the LARP 90more » mm aperture (TQ-LQ) and 120 mm aperture (HQ and Long HQ) programs. A 4-m long MQXF practice coil was fabricated, water jet cut and analyzed to verify procedures, parts, and tooling. In parallel, the first complete prototype coil (QXFP01a) was fabricated and assembled in a long magnetic mirror, MQXFPM1, to provide early feedback on coil design and fabrication following the successful experience of previous LARP mirror tests.« less

LARP Long Quadrupole: A "Long" Step Toward an LHC

ScienceCinema

Giorgio Ambrosio

2017-12-09

The beginning of the development of Nb3Sn magnets for particle accelerators goes back to the 1960’s. But only very recently has this development begun to face the challenges of fabricating Nb3Sn magnets which can meet the requirements of modern particle accelerators. LARP (the LHC Accelerator Research Program) is leading this effort focusing on long models of the Interaction Region quadrupoles for a possible luminosity upgrade of the Large Hadron Collider. A major milestone in this development is to test, by the end of 2009, 4m-long quadrupole models, which will be the first Nb3Sn accelerator-type magnets approaching the length of real accelerator magnets. The Long Quadrupoles (LQ) are “Proof-of-Principle” magnets which are to demonstrate that Nb3Sn technology is sufficiently mature for use in high energy particle accelerators. Their design is based on the LARP Technological Quadrupole (TQ) models, under development at FNAL and LBNL, which have design gradients higher than 200 T/m and an aperture of 90 mm. Several challenges must be addressed for the successful fabrication of long Nb3Sn coils and magnets. These challenges and the solutions adopted will be presented together with the main features of the LQ magnets. Several R&D lines are participating to this effort and their contributions will be also presented.

Large Aircraft Robotic Paint Stripping (LARPS) system and the high pressure water process

NASA Astrophysics Data System (ADS)

See, David W.; Hofacker, Scott A.; Stone, M. Anthony; Harbaugh, Darcy

1993-03-01

The aircraft maintenance industry is beset by new Environmental Protection Agency (EPA) guidelines on air emissions, Occupational Safety and Health Administration (OSHA) standards, dwindling labor markets, Federal Aviation Administration (FAA) safety guidelines, and increased operating costs. In light of these factors, the USAF's Wright Laboratory Manufacturing Technology Directorate and the Aircraft Division of the Oklahoma City Air Logistics Center initiated a MANTECH/REPTECH effort to automate an alternate paint removal method and eliminate the current manual methylene chloride chemical stripping methods. This paper presents some of the background and history of the LARPS program, describes the LARPS system, documents the projected operational flow, quantifies some of the projected system benefits and describes the High Pressure Water Stripping Process. Certification of an alternative paint removal method to replace the current chemical process is being performed in two phases: Process Optimization and Process Validation. This paper also presents the results of the Process Optimization for metal substrates. Data on the coating removal rate, residual stresses, surface roughness, preliminary process envelopes, and technical plans for process Validation Testing will be discussed.

Coil End Parts Development Using BEND and Design for MQXF by LARP

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

Yu, Miao; Ambrosio, G.; Bermudez, S. Izquierdo

2016-09-06

End parts are critical components for saddle-shaped coils. They have a structural function where the cables are deformed in order to cross over the magnet aperture. Based on the previous design of the US LARP program for 90 mm aperture quadrupoles (TQ/LQ) and 120 mm aperture quadrupoles (HQ/LHQ) using BEND, the coil ends of the low-β quadruples (MQXF) for the HiLumi LHC upgrade were developed. This paper shows the design of the MQXF coil ends, the analysis of the coil ends during the coil fabrication, the autopsy analysis of the coil ends and the feedback to BEND parameters.

Test results of the LARP Nb$$_3$$Sn quadrupole HQ03a

DOE PAGES

DiMarco, J.; G. Ambrosio; Chlachidze, G.; ...

2016-03-09

The US LHC Accelerator Research Program (LARP) has been developingmore » $$Nb_3Sn$$ quadrupoles of progressively increasing performance for the high luminosity upgrade of the Large Hadron Collider. The 120 mm aperture High-field Quadrupole (HQ) models are the last step in the R&D phase supporting the development of the new IR Quadrupoles (MQXF). Three series of HQ coils were fabricated and assembled in a shell-based support structure, progressively optimizing the design and fabrication process. The final set of coils consistently applied the optimized design solutions, and was assembled in the HQ03a model. Furthermore, this paper reports a summary of the HQ03a test results, including training, mechanical performance, field quality and quench studies.« less

Loss of function mutation in LARP7, chaperone of 7SK ncRNA, causes a syndrome of facial dysmorphism, intellectual disability, and primordial dwarfism.

PubMed

Alazami, Anas M; Al-Owain, Mohammad; Alzahrani, Fatema; Shuaib, Taghreed; Al-Shamrani, Hussain; Al-Falki, Yahya H; Al-Qahtani, Saleh M; Alsheddi, Tarfa; Colak, Dilek; Alkuraya, Fowzan S

2012-10-01

Primordial dwarfism (PD) is a clinically and genetically heterogeneous condition. Various molecular mechanisms are known to underlie the disease including impaired mitotic mechanics, abnormal IGF2 expression, perturbed DNA damage response, defective spliceosomal machinery, and abnormal replication licensing. Here, we describe a syndromic form of PD associated with severe intellectual disability and distinct facial features in a large multiplex Saudi family. Analysis reveals a novel underlying mechanism for PD involving depletion of 7SK, an abundant cellular noncoding RNA (ncRNA), due to mutation of its chaperone LARP7. We show that 7SK levels are tightly linked to LARP7 expression across cell lines, and that this chaperone is ubiquitously expressed in the mouse embryo. The 7SK is known to influence the expression of a wide array of genes through its inhibitory effect on the positive transcription elongation factor b (P-TEFb) as well as its competing role in HMGA1-mediated transcriptional regulation. This study documents a critical role played by ncRNA in human development and adds to the growing list of molecular mechanisms that, when perturbed, converge on the PD phenotype. © 2012 Wiley Periodicals, Inc.

RNA protein interactions governing expression of the most abundant protein in human body, type I collagen.

PubMed

Stefanovic, Branko

2013-01-01

Type I collagen is the most abundant protein in human body. The protein turns over slowly and its replacement synthesis is low. However, in wound healing or in pathological fibrosis the cells can increase production of type I collagen several hundred fold. This increase is predominantly due to posttranscriptional regulation, including increased half-life of collagen messenger RNAs (mRNAs) and their increased translatability. Type I collagen is composed of two α1 and one α2 polypeptides that fold into a triple helix. This stoichiometry is strictly regulated to prevent detrimental synthesis of α1 homotrimers. Collagen polypeptides are co-translationally modified and the rate of modifications is in dynamic equilibrium with the rate of folding, suggesting coordinated translation of collagen α1(I) and α2(I) polypeptides. Collagen α1(I) mRNA has in the 3' untranslated region (UTR) a C-rich sequence that binds protein αCP, this binding stabilizes the mRNA in collagen producing cells. In the 5' UTR both collagen mRNAs have a conserved stem-loop (5' SL) structure. The 5' SL is critical for high collagen expression, knock in mice with disruption of the 5' SL are resistant to liver fibrosis. the 5' SL binds protein LARP6 with strict sequence specificity and high affinity. LARP6 recruits RNA helicase A to facilitate translation initiation and associates collagen mRNAs with vimentin and nonmuscle myosin filaments. Binding to vimentin stabilizes collagen mRNAs, while nonmuscle myosin regulates coordinated translation of α1(I) and α2(I) mRNAs. When nonmuscle myosin filaments are disrupted the cells secrete only α1 homotrimers. Thus, the mechanism governing high collagen expression involves two RNA binding proteins and development of cytoskeletal filaments. Copyright © 2013 John Wiley & Sons, Ltd.

Progress in the Long $${\\rm Nb}_{3}{\\rm Sn}$$ Quadrupole R&D by LARP

DOE PAGES

Ambrosio, G.; Andreev, N.; Anerella, M.; ...

2011-11-14

After the successful test of the first long Nb 3Sn quadrupole (LQS01) the US LHC Accelerator Research Program (LARP, a collaboration of BNL, FNAL, LBNL and SLAC) is assessing training memory, reproducibility, and other accelerator quality features of long Nb 3Sn quadrupole magnets. LQS01b (a reassembly of LQS01 with more uniform and higher pre-stress) was subjected to a full thermal cycle and reached the previous plateau of 222 T/m at 4.5 K in two quenches. A new set of four coils, made of the same type of conductor used in LQS01 (RRP 54/61 by Oxford Superconducting Technology), was assembled inmore » the LQS01 structure and tested at 4.5 K and lower temperatures. The new magnet (LQS02) reached the target gradient (200 T/m) only at 2.6 K and lower temperatures, at intermediate ramp rates. The preliminary test analysis, here reported, showed a higher instability in the limiting coil than in the other coils of LQS01 and LQS02.« less

Primordial dwarfism gene maintains Lin28 expression to safeguard embryonic stem cells from premature differentiation.

PubMed

Dai, Qian; Luan, Guangxin; Deng, Li; Lei, Tingjun; Kang, Han; Song, Xu; Zhang, Yujun; Xiao, Zhi-Xiong; Li, Qintong

2014-05-08

Primordial dwarfism (PD) is characterized by global growth failure, both during embryogenesis and postnatally. Loss-of-function germline mutations in La ribonucleoprotein domain family, member 7 (LAPR7) have recently been linked to PD. Paradoxically, LARP7 deficiency was previously assumed to be associated with increased cell growth and proliferation via activation of positive transcription elongation factor b (P-TEFb). Here, we show that Larp7 deficiency likely does not significantly increase P-TEFb activity. We further discover that Larp7 knockdown does not affect pluripotency but instead primes embryonic stem cells (ESCs) for differentiation via downregulation of Lin28, a positive regulator of organismal growth. Mechanistically, we show that Larp7 interacts with a poly(A) polymerase Star-PAP to maintain Lin28 mRNA stability. We propose that proper regulation of Lin28 and PTEFb is essential for embryonic cells to achieve a sufficient number of cell divisions prior to differentiation and ultimately to maintain proper organismal size. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Interaction between La(III) and proteins on the plasma membrane of horseradish

NASA Astrophysics Data System (ADS)

Yang, Guang-Mei; Chu, Yun-Xia; Lv, Xiao-Fen; Zhou, Qing; Huang, Xiao-Hua

2012-06-01

Lanthanum (La) is an important rare earth element in the ecological environment of plant. The proteins on the plasma membrane control the transport of molecules into and out of cell. It is very important to investigate the effect of La(III) on the proteins on the plasma membrane in the plant cell. In the present work, the interaction between La(III) and proteins on the plasma membrane of horseradish was investigated using optimization of the fluorescence microscopy and fluorescence spectroscopy. It is found that the fluorescence of the complex system of protoplasts and 1-aniline Kenai-8-sulfonic acid in horseradish treated with the low concentration of La(III) is increased compared with that of the control horseradish. The opposite effect is observed in horseradish treated with the high concentration of La(III). These results indicated that the low concentration of La(III) can interact with the proteins on the plasma membrane of horseradish, causing the improvement in the structure of proteins on the plasma membrane. The high concentration of La(III) can also interact with the proteins on the plasma membrane of horseradish, leading to the destruction of the structure of proteins on the plasma membrane. We demonstrate that the proteins on the plasma membrane are the targets of La(III) action on plant cell.

PaLaCe: A Coarse-Grain Protein Model for Studying Mechanical Properties.

PubMed

Pasi, Marco; Lavery, Richard; Ceres, Nicoletta

2013-01-08

We present a coarse-grain protein model PaLaCe (Pasi-Lavery-Ceres) that has been developed principally to allow fast computational studies of protein mechanics and to clarify the links between mechanics and function. PaLaCe uses a two-tier protein representation with one to three pseudoatoms representing each amino acid for the main nonbonded interactions, combined with atomic-scale peptide groups and some side chain atoms to allow the explicit representation of backbone hydrogen bonds and to simplify the treatment of bonded interactions. The PaLaCe force field is composed of physics-based terms, parametrized using Boltzmann inversion of conformational probability distributions derived from a protein structure data set, and iteratively refined to reproduce the experimental distributions. PaLaCe has been implemented in the MMTK simulation package and can be used for energy minimization, normal mode calculations, and molecular or stochastic dynamics. We present simulations with PaLaCe that test its ability to maintain stable structures for folded proteins, reproduce their dynamic fluctuations, and correctly model large-scale, force-induced conformational changes.

Precursor-product discrimination by La protein during tRNA metabolism

PubMed Central

Bayfield, Mark A.; Maraia, Richard J.

2009-01-01

SUMMARY La proteins bind pre-tRNAs at their UUU-3'OH ends, facilitating their maturation. While the mechanism by which La binds pre-tRNA 3' trailers is known, the function of the RNA-binding β-sheet surface of RRM1 is unknown. How La dissociates from UUU-3'OH-containing trailers after 3' processing is also unknown. La preferentially binds pre-tRNAs over processed tRNAs or 3' trailer products through coupled use of two sites: one on the La motif and another on the RRM1 β surface that binds elsewhere on tRNA. Two sites provide stable pre-tRNA binding while processed tRNA and 3' trailer are released from their single sites relatively fast. RRM1 loop-3 mutations decrease affinity for pre-tRNA and tRNA but not UUU-3'OH trailer, and impair tRNA maturation in vivo. We propose that RRM1 functions in activities that are more complex than UUU-3'OH binding. Accordingly, the RRM1 mutations also impair a RNA chaperone activity of La. The results suggest how La distinguishes precursor from product RNAs, allowing it to recycle onto a new pre-tRNA. PMID:19287396

Precursor-product discrimination by La protein during tRNA metabolism.

PubMed

Bayfield, Mark A; Maraia, Richard J

2009-04-01

La proteins bind pre-tRNAs at their UUU-3'OH ends, facilitating their maturation. Although the mechanism by which La binds pre-tRNA 3' trailers is known, the function of the RNA binding beta-sheet surface of the RNA-recognition motif (RRM1) is unknown. How La dissociates from UUU-3'OH-containing trailers after 3' processing is also unknown. Here we show that La preferentially binds pre-tRNAs over processed tRNAs or 3' trailer products through coupled use of two sites: one on the La motif and another on the RRM1 beta-surface that binds elsewhere on tRNA. Two sites provide stable pre-tRNA binding, whereas the processed tRNA and 3' trailer are released from their single sites relatively fast. RRM1 loop-3 mutations decrease affinity for pre-tRNA and tRNA, but not for the UUU-3'OH trailer, and impair tRNA maturation in vivo. We propose that RRM1 functions in activities that are more complex than UUU-3'OH binding. Accordingly, the RRM1 mutations also impair an RNA chaperone activity of La. The results suggest how La distinguishes precursor from product RNAs, allowing it to recycle onto a new pre-tRNA.

Observations on the expression of human papillomavirus major capsid protein in HeLa cells.

PubMed

Xiao, Chang-Yi; Fu, Bing-Bing; Li, Zhi-Ying; Mushtaq, Gohar; Kamal, Mohammad Amjad; Li, Jia-Hua; Tang, Gui-Cheng; Xiao, Shuo-Shuang

2015-01-01

The goal of this study was to identify the nature of the inclusion bodies that have been found in HeLa cells (cervical cancer immortal cell line) by electron microscope and to determine whether the major capsid protein (L1) of human papillomavirus (HPV) can be expressed in HPV-positive uterine cervix cancer cells. HPV L1 protein expression in HeLa cells was detected with anti-HPV L1 multivalent mice monoclonal antibody and rabbit polyclonal anti-HPV L1 antibody by ELISA, light microscope immunohistochemistry, electron microscope immunocytochemistry and Western blotting assays. Reverse transcriptional PCR (RT-PCR) was performed to detect the transcription of L1 mRNA in HeLa cells. The immortalized human keratinocyte HeCat was used as the negative control. HPV L1 proteins reacted positively in the lysate of HeLa cells by ELISA assays. HRP labeled light microscope immunohistochemistry assay showed that there was a strong HPV L1 positive reaction in HeLa cells. Under the electron microscope, irregular shaped inclusion bodies, assembled by many small and uniform granules, had been observed in the cytoplasm of some HeLa cells. These granules could be labeled by the colloidal gold carried by HPV L1 antibody. The Western blotting assay showed that there was a L1 reaction strap at 80-85 kDa in the HeLa cell lysates, hence demonstrating the existence of HPV18 L1 in HeLa cells. RT-PCR assay showed that the L1 mRNA was transcribed in HeLa cells. The inclusion bodies found in the cytoplasm of HeLa cells are composed of HPV18 L1 protein. Since HeLa cell line is a type of cervical cancer cells, this implies that HeLa cells have the ability to express HPV L1 proteins.

Alkaline phosphatase protein increases in response to prednisolone in HeLa cells.

PubMed Central

Hanford, W C; Kottel, R H; Fishman, W H

1981-01-01

Quantification of term-placental alkaline phosphatase isoenzyme protein in HeLa TCRC-1 cells grown in the presence and absence of prednisolone indicates that there is a net increase in amount of enzyme-specific protein in prednisolone-stimulated cells. In a similar analysis of HeLa D98AH2 cells, prednisolone treatment causes the appearance of term-placental alkaline phosphatase protein and the loss of the intestinal isoenzyme protein. These results support the interpretation that the response of these cells to corticosteroids is the net accumulation of alkaline phosphatase protein rather than the modification of pre-existing enzyme to a more active state. Images Fig. 1. Fig. 2. PMID:7340849

Factor H-related proteins.

PubMed

Józsi, Mihály; Meri, Seppo

2014-01-01

Factor H-related proteins (CFHRs) are plasma glycoproteins related in structure and antigenicity to each other and to the complement inhibitory protein factor H. Such proteins are found in most mammals but their number and domain composition vary. This chapter summarizes our current knowledge on the human factor H-related proteins. In contrast to factor H, they have no strong complement inhibitory activity, although for some of them regulatory or complement modulatory activity has been reported. A common feature of CFHRs is that they bind to the C3b component of complement. Novel links between CFHRs and various diseases (C3 glomerulopathies, atypical hemolytic uremic syndrome and age-related macular degeneration) have been revealed in recent years, but we are still far from understanding their biological function.

RNA chaperone activity of human La protein is mediated by variant RNA recognition motif.

PubMed

Naeeni, Amir R; Conte, Maria R; Bayfield, Mark A

2012-02-17

La proteins are conserved factors in eukaryotes that bind and protect the 3' trailers of pre-tRNAs from exonuclease digestion via sequence-specific recognition of UUU-3'OH. La has also been hypothesized to assist pre-tRNAs in attaining their native fold through RNA chaperone activity. In addition to binding polymerase III transcripts, human La has also been shown to enhance the translation of several internal ribosome entry sites and upstream ORF-containing mRNA targets, also potentially through RNA chaperone activity. Using in vitro FRET-based assays, we show that human and Schizosaccharomyces pombe La proteins harbor RNA chaperone activity by enhancing RNA strand annealing and strand dissociation. We use various RNA substrates and La mutants to show that UUU-3'OH-dependent La-RNA binding is not required for this function, and we map RNA chaperone activity to its RRM1 motif including a noncanonical α3-helix. We validate the importance of this α3-helix by appending it to the RRM of the unrelated U1A protein and show that this fusion protein acquires significant strand annealing activity. Finally, we show that residues required for La-mediated RNA chaperone activity in vitro are required for La-dependent rescue of tRNA-mediated suppression via a mutated suppressor tRNA in vivo. This work delineates the structural elements required for La-mediated RNA chaperone activity and provides a basis for understanding how La can enhance the folding of its various RNA targets.

RNA Chaperone Activity of Human La Protein Is Mediated by Variant RNA Recognition Motif*

PubMed Central

Naeeni, Amir R.; Conte, Maria R.; Bayfield, Mark A.

2012-01-01

La proteins are conserved factors in eukaryotes that bind and protect the 3′ trailers of pre-tRNAs from exonuclease digestion via sequence-specific recognition of UUU-3′OH. La has also been hypothesized to assist pre-tRNAs in attaining their native fold through RNA chaperone activity. In addition to binding polymerase III transcripts, human La has also been shown to enhance the translation of several internal ribosome entry sites and upstream ORF-containing mRNA targets, also potentially through RNA chaperone activity. Using in vitro FRET-based assays, we show that human and Schizosaccharomyces pombe La proteins harbor RNA chaperone activity by enhancing RNA strand annealing and strand dissociation. We use various RNA substrates and La mutants to show that UUU-3′OH-dependent La-RNA binding is not required for this function, and we map RNA chaperone activity to its RRM1 motif including a noncanonical α3-helix. We validate the importance of this α3-helix by appending it to the RRM of the unrelated U1A protein and show that this fusion protein acquires significant strand annealing activity. Finally, we show that residues required for La-mediated RNA chaperone activity in vitro are required for La-dependent rescue of tRNA-mediated suppression via a mutated suppressor tRNA in vivo. This work delineates the structural elements required for La-mediated RNA chaperone activity and provides a basis for understanding how La can enhance the folding of its various RNA targets. PMID:22203678

Potential proteins targeted by let-7f-5p in HeLa cells.

PubMed

Wang, Yu; Chen, Xiujuan; Zhang, Yi; Song, Jiandong

2017-07-24

MicroRNAs are a class of small, endogenous, non-coding RNAs mediating posttranscriptional gene silencing. The current authors hypothesized that let-7f-5p is likely involved in cell invasion and proliferation by regulating the expression of target genes. The current study combined let-7f-5p with iTRAQ to assess its effect on gene expression in HeLa cells. Results indicated that 164 proteins were expressed at different levels in HeLa cells overexpressing let-7f-5p and negative controls and that 172 proteins were expressed at different levels in let-7f-5p-silenced HeLa cells and negative controls. Results indicated that let-7f-5p may suppress insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) in HeLa cells.

LaRbp38: A Leishmania amazonensis protein that binds nuclear and kinetoplast DNAs

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

Lira, C.B.B.; Instituto de Biologia, UNICAMP, Campinas, SP; Siqueira Neto, J.L.

Leishmania amazonensis causes a wide spectrum of leishmaniasis. There are no vaccines or adequate treatment for leishmaniasis, therefore there is considerable interest in the identification of new targets for anti-leishmania drugs. The central role of telomere-binding proteins in cell maintenance makes these proteins potential targets for new drugs. In this work, we used a combination of purification chromatographies to screen L. amazonensis proteins for molecules capable of binding double-stranded telomeric DNA. This approach resulted in the purification of a 38 kDa polypeptide that was identified by mass spectrometry as Rbp38, a trypanosomatid protein previously shown to stabilize mitochondrial RNA andmore » to associate with nuclear and kinetoplast DNAs. Western blotting and supershift assays confirmed the identity of the protein as LaRbp38. Competition and chromatin immunoprecipitation assays confirmed that LaRbp38 interacted with kinetoplast and nuclear DNAs in vivo and suggested that LaRbp38 may have dual cellular localization and more than one function.« less

The invasion of tobacco mosaic virus RNA induces endoplasmic reticulum stress-related autophagy in HeLa cells

PubMed Central

Li, Li; Wang, Li; Xiao, Ruijing; Zhu, Guoguo; Li, Yan; Liu, Changxuan; Yang, Ru; Tang, Zhiqing; Li, Jie; Huang, Wei; Chen, Lang; Zheng, Xiaoling; He, Yuling; Tan, Jinquan

2011-01-01

The ability of human cells to defend against viruses originating from distant species has long been ignored. Owing to the pressure of natural evolution and human exploration, some of these viruses may be able to invade human beings. If their ‘fresh’ host had no defences, the viruses could cause a serious pandemic, as seen with HIV, SARS (severe acute respiratory syndrome) and avian influenza virus that originated from chimpanzees, the common palm civet and birds, respectively. It is unknown whether the human immune system could tolerate invasion with a plant virus. To model such an alien virus invasion, we chose TMV (tobacco mosaic virus) and used human epithelial carcinoma cells (HeLa cells) as its ‘fresh’ host. We established a reliable system for transfecting TMV-RNA into HeLa cells and found that TMV-RNA triggered autophagy in HeLa cells as shown by the appearance of autophagic vacuoles, the conversion of LC3-I (light chain protein 3-I) to LC3-II, the up-regulated expression of Beclin1 and the accumulation of TMV protein on autophagosomal membranes. We observed suspected TMV virions in HeLa cells by TEM (transmission electron microscopy). Furthermore, we found that TMV-RNA was translated into CP (coat protein) in the ER (endoplasmic reticulum) and that TMV-positive RNA translocated from the cytoplasm to the nucleolus. Finally, we detected greatly increased expression of GRP78 (78 kDa glucose-regulated protein), a typical marker of ERS (ER stress) and found that the formation of autophagosomes was closely related to the expanded ER membrane. Taken together, our data indicate that HeLa cells used ERS and ERS-related autophagy to defend against TMV-RNA. PMID:21729006

Regulation of post-translational protein arginine methylation during HeLa cell cycle.

PubMed

Kim, Chongtae; Lim, Yongchul; Yoo, Byong Chul; Won, Nam Hee; Kim, Sangduk; Kim, Gieun

2010-09-01

Post-translational arginine methylation which modifies protein-arginyl residues by protein arginine methyltransferase (PRMT) was investigated during synchronized HeLa cell cycle. The lysates of cells synchronized at each stage were subjected to one and/or two dimensional electrophoresis followed by Western immunoblot using against anti-asymmetric-dimethyl-arginine (ASYM24), anti-symmetric-dimethyl-arginine (SYM10), and subclasses of PRMTs, including PRMT1, PRMT3, PRMT4 (CARM1), PRMT5, PRMT6, and PRMT7 antibodies. Proteins with approximate molecular masses of 80 kDa, 68 kDa, and 64 kDa, containing asymmetric-dimethyl-arginine (aDMA) were increased at G0/G1 to G1, which lasted until S phase. In addition, 25 kDa protein of symmetric-dimethyl-arginine (sDMA) was also markedly up-regulated from G0/G1 to G1. The levels of PRMT3, PRMT6 and PRMT7 were concurrently increased during the cell cycle. Two-dimensional gel electrophoresis followed by MALDI-TOF-MS was identified as aDMA-80 kDa and aDMA-68 kDa proteins as heterogeneous nuclear ribonucleoprotein R (hnRNPR), aDMA-64 kDa proteins as cleavage stimulation factor 64 kDa subunit (CstF-64), and sDMA-25 kDa protein as triosephosphate isomerase (TPI). The levels of increased aDMA of hnRNPR were reduced, when HeLa cells were transfected with siRNA for PRMT1, and the aDMA of CstF-64 with siRNA for PRMT3, while depletion of PRMT5 down-regulated sDMA of TPI. Protein arginine dimethylations of hnRNPR, CstF-64, and TPI were regulated during HeLa cell cycle by respective PRMTs. These results suggest that regulation of arginine dimethylation of hnRNPR, CstF-64, and TPI at G0/G1 to G1 are most likely to modulate the cellular growth and proliferation in HeLa cell cycle. Copyright © 2010 Elsevier B.V. All rights reserved.

SUMO-Modification of the La Protein Facilitates Binding to mRNA In Vitro and in Cells.

PubMed

Kota, Venkatesh; Sommer, Gunhild; Durette, Chantal; Thibault, Pierre; van Niekerk, Erna A; Twiss, Jeffery L; Heise, Tilman

2016-01-01

The RNA-binding protein La is involved in several aspects of RNA metabolism including the translational regulation of mRNAs and processing of pre-tRNAs. Besides its well-described phosphorylation by Casein kinase 2, the La protein is also posttranslationally modified by the Small Ubiquitin-like MOdifier (SUMO), but the functional outcome of this modification has not been defined. The objective of this study was to test whether sumoylation changes the RNA-binding activity of La. Therefore, we established an in vitro sumoylation assay for recombinant human La and analyzed its RNA-binding activity by electrophoretic mobility shift assays. We identified two novel SUMO-acceptor sites within the La protein located between the RNA recognition motif 1 and 2 and we demonstrate for the first time that sumoylation facilitates the RNA-binding of La to small RNA oligonucleotides representing the oligopyrimidine tract (TOP) elements from the 5' untranslated regions (UTR) of mRNAs encoding ribosomal protein L22 and L37 and to a longer RNA element from the 5' UTR of cyclin D1 (CCND1) mRNA in vitro. Furthermore, we show by RNA immunoprecipitation experiments that a La mutant deficient in sumoylation has impaired RNA-binding activity in cells. These data suggest that modulating the RNA-binding activity of La by sumoylation has important consequences on its functionality.

SUMO-Modification of the La Protein Facilitates Binding to mRNA In Vitro and in Cells

PubMed Central

Kota, Venkatesh; Sommer, Gunhild; Durette, Chantal; Thibault, Pierre; van Niekerk, Erna A.; Twiss, Jeffery L.

2016-01-01

The RNA-binding protein La is involved in several aspects of RNA metabolism including the translational regulation of mRNAs and processing of pre-tRNAs. Besides its well-described phosphorylation by Casein kinase 2, the La protein is also posttranslationally modified by the Small Ubiquitin-like MOdifier (SUMO), but the functional outcome of this modification has not been defined. The objective of this study was to test whether sumoylation changes the RNA-binding activity of La. Therefore, we established an in vitro sumoylation assay for recombinant human La and analyzed its RNA-binding activity by electrophoretic mobility shift assays. We identified two novel SUMO-acceptor sites within the La protein located between the RNA recognition motif 1 and 2 and we demonstrate for the first time that sumoylation facilitates the RNA-binding of La to small RNA oligonucleotides representing the oligopyrimidine tract (TOP) elements from the 5’ untranslated regions (UTR) of mRNAs encoding ribosomal protein L22 and L37 and to a longer RNA element from the 5’ UTR of cyclin D1 (CCND1) mRNA in vitro. Furthermore, we show by RNA immunoprecipitation experiments that a La mutant deficient in sumoylation has impaired RNA-binding activity in cells. These data suggest that modulating the RNA-binding activity of La by sumoylation has important consequences on its functionality. PMID:27224031

Effects of the TAT peptide orientation and relative location on the protein transduction efficiency.

PubMed

Guo, Qingguo; Zhao, Guojie; Hao, Fengjin; Guan, Yifu

2012-05-01

To understand the protein transduction domain (PTD)-mediated protein transduction behavior and to explore its potential in delivering biopharmaceutic drugs, we prepared four TAT-EGFP conjugates: TAT(+)-EGFP, TAT(-)-EGFP, EGFP-TAT(+) and EGFP-TAT(-), where TAT(+) and TAT(-) represent the original and the reversed TAT sequence, respectively. These four TAT-EGFP conjugates were incubated with HeLa and PC12 cells for in vitro study as well as injected intraperitoneally to mice for in vivo study. Flow cytometric results showed that four TAT-EGFP conjugates were able to traverse HeLa and PC12 cells with almost equal transduction efficiency. The in vivo study showed that the TAT-EGFP conjugates could be delivered into different organs of mice with different transduction capabilities. Bioinformatic analyses and CD spectroscopic data revealed that the TAT peptide has no defined secondary structure, and conjugating the TAT peptide to the EGFP cargo protein would not alter the native structure and the function of the EGFP protein. These results conclude that the sequence orientation, the spatial structure, and the relative location of the TAT peptide have much less effect on the TAT-mediated protein transduction. Thus, the TAT-fused conjugates could be constructed in more convenient and flexible formats for a wide range of biopharmaceutical applications. © 2011 John Wiley & Sons A/S.

Anticancer Activity of a Hexapeptide from Skate (Raja porosa) Cartilage Protein Hydrolysate in HeLa Cells

PubMed Central

Pan, Xin; Zhao, Yu-Qin; Hu, Fa-Yuan; Chi, Chang-Feng; Wang, Bin

2016-01-01

In this study, the hexapeptide Phe-Ile-Met-Gly-Pro-Tyr (FIMGPY), which has a molecular weight of 726.9 Da, was separated from skate (Raja porosa) cartilage protein hydrolysate using ultrafiltration and chromatographic methods, and its anticancer activity was evaluated in HeLa cells. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay indicated that FIMGPY exhibited high, dose-dependent anti-proliferation activities in HeLa cells with an IC50 of 4.81 mg/mL. Acridine orange/ethidium bromide (AO/EB) fluorescence staining and flow cytometry methods confirmed that FIMGPY could inhibit HeLa cell proliferation by inducing apoptosis. Western blot assay revealed that the Bax/Bcl-2 ratio and relative intensity of caspase-3 in HeLa cells treated with 7-mg/mL FIMGPY were 2.63 and 1.83, respectively, significantly higher than those of the blank control (p < 0.01). Thus, FIMGPY could induce apoptosis by upregulating the Bax/Bcl-2 ratio and caspase-3 activation. Using a DNA ladder method further confirmed that the anti-proliferation activity of FIMGPY was attributable to its role in inducing apoptosis. These results suggest that FIMGPY from skate cartilage protein hydrolysate may have applications as functional foods and nutraceuticals for the treatment and prevention of cancer. PMID:27537897

Anticancer Activity of a Hexapeptide from Skate (Raja porosa) Cartilage Protein Hydrolysate in HeLa Cells.

PubMed

Pan, Xin; Zhao, Yu-Qin; Hu, Fa-Yuan; Chi, Chang-Feng; Wang, Bin

2016-08-16

In this study, the hexapeptide Phe-Ile-Met-Gly-Pro-Tyr (FIMGPY), which has a molecular weight of 726.9 Da, was separated from skate (Raja porosa) cartilage protein hydrolysate using ultrafiltration and chromatographic methods, and its anticancer activity was evaluated in HeLa cells. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay indicated that FIMGPY exhibited high, dose-dependent anti-proliferation activities in HeLa cells with an IC50 of 4.81 mg/mL. Acridine orange/ethidium bromide (AO/EB) fluorescence staining and flow cytometry methods confirmed that FIMGPY could inhibit HeLa cell proliferation by inducing apoptosis. Western blot assay revealed that the Bax/Bcl-2 ratio and relative intensity of caspase-3 in HeLa cells treated with 7-mg/mL FIMGPY were 2.63 and 1.83, respectively, significantly higher than those of the blank control (p < 0.01). Thus, FIMGPY could induce apoptosis by upregulating the Bax/Bcl-2 ratio and caspase-3 activation. Using a DNA ladder method further confirmed that the anti-proliferation activity of FIMGPY was attributable to its role in inducing apoptosis. These results suggest that FIMGPY from skate cartilage protein hydrolysate may have applications as functional foods and nutraceuticals for the treatment and prevention of cancer.

Transcription Factor IIB (TFIIB)-Related Protein (pBrp), a Plant-Specific Member of the TFIIB-Related Protein Family

PubMed Central

Lagrange, Thierry; Hakimi, Mohamed-Ali; Pontier, Dominique; Courtois, Florence; Alcaraz, Jean Pierre; Grunwald, Didier; Lam, Eric; Lerbs-Mache, Silva

2003-01-01

Although it is now well documented that metazoans have evolved general transcription factor (GTF) variants to regulate their complex patterns of gene expression, there is so far no information regarding the existence of specific GTFs in plants. Here we report the characterization of a ubiquitously expressed gene that encodes a bona fide novel transcription factor IIB (TFIIB)-related protein in Arabidopsis thaliana. We have shown that this protein is the founding member of a plant-specific TFIIB-related protein family named pBrp (for plant-specific TFIIB-related protein). Surprisingly, in contrast to common GTFs that are localized in the nucleus, the bulk of pBrp proteins are bound to the cytoplasmic face of the plastid envelope, suggesting an organelle-specific function for this novel class of TFIIB-related protein. We show that pBrp proteins harbor conditional proteolytic signals that can target these proteins for rapid turnover by the proteasome-mediated protein degradation pathway. Interestingly, under conditions of proteasome inhibition, pBrp proteins accumulate in the nucleus. Together, our results suggest a possible involvement of these proteins in an intracellular signaling pathway between plastids and the nucleus. Our data provide the first evidence for an organelle-related evolution of the eukaryotic general transcription machinery. PMID:12697827

Proteomic analysis of specific brain proteins in aged SAMP8 mice treated with alpha-lipoic acid: implications for aging and age-related neurodegenerative disorders.

PubMed

Poon, H Fai; Farr, Susan A; Thongboonkerd, Visith; Lynn, Bert C; Banks, William A; Morley, John E; Klein, Jon B; Butterfield, D Allan

2005-01-01

Free radical-mediated damage to neuronal membrane components has been implicated in the etiology of Alzheimer's disease (AD) and aging. The senescence accelerated prone mouse strain 8 (SAMP8) exhibits age-related deterioration in memory and learning along with increased oxidative markers. Therefore, SAMP8 is a suitable model to study brain aging and, since aging is the major risk factor for AD and SAMP8 exhibits many of the biochemical findings of AD, perhaps as a model for and the early phase of AD. Our previous studies reported higher oxidative stress markers in brains of 12-month-old SAMP8 mice when compared to that of 4-month-old SAMP8 mice. Further, we have previously shown that injecting the mice with alpha-lipoic acid (LA) reversed brain lipid peroxidation, protein oxidation, as well as the learning and memory impairments in SAMP8 mice. Recently, we reported the use of proteomics to identify proteins that are expressed differently and/or modified oxidatively in aged SAMP8 brains. In order to understand how LA reverses the learning and memory deficits of aged SAMP8 mice, in the current study, we used proteomics to compare the expression levels and specific carbonyl levels of proteins in brains from 12-month-old SAMP8 mice treated or not treated with LA. We found that the expressions of the three brain proteins (neurofilament triplet L protein, alpha-enolase, and ubiquitous mitochondrial creatine kinase) were increased significantly and that the specific carbonyl levels of the three brain proteins (lactate dehydrogenase B, dihydropyrimidinase-like protein 2, and alpha-enolase) were significantly decreased in the aged SAMP8 mice treated with LA. These findings suggest that the improved learning and memory observed in LA-injected SAMP8 mice may be related to the restoration of the normal condition of specific proteins in aged SAMP8 mouse brain. Moreover, our current study implicates neurofilament triplet L protein, alpha-enolase, ubiquitous mitochondrial

Parathyroid hormone-related protein blood test

MedlinePlus

... gov/ency/article/003691.htm Parathyroid hormone-related protein blood test To use the sharing features on ... page, please enable JavaScript. The parathyroid hormone-related protein (PTH-RP) test measures the level of a ...

Comprehensive circular RNA expression profile in radiation-treated HeLa cells and analysis of radioresistance-related circRNAs.

PubMed

Yu, Duo; Li, Yunfeng; Ming, Zhihui; Wang, Hongyong; Dong, Zhuo; Qiu, Ling; Wang, Tiejun

2018-01-01

Cervical cancer is one of the most common cancers in women worldwide. Malignant tumors develop resistance mechanisms and are less sensitive to or do not respond to irradiation. With the development of high-throughput sequencing technologies, circular RNA (circRNA) has been identified in an increasing number of diseases, especially cancers. It has been reported that circRNA can compete with microRNAs (miRNAs) to change the stability or translation of target RNAs, thus regulating gene expression at the transcriptional level. However, the role of circRNAs in cervical cancer and the radioresistance mechanisms of HeLa cells are unknown. The objective of this study is to investigate the role of circRNAs in radioresistance in HeLa cells. High-throughput sequencing and bioinformatics analysis of irradiated and sham-irradiated HeLa cells. The reliability of high-throughput RNA sequencing was validated using quantitative real-time polymerase chain reaction. The most significant circRNA functions and pathways were selected by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. A circRNA-miRNA-target gene interaction network was used to find circRNAs associated with radioresistance. Moreover, a protein-protein interaction network was constructed to identify radioresistance-related hub proteins. High-throughput sequencing allowed the identification of 16,893 circRNAs involved in the response of HeLa cells to radiation. Compared with the control group, there were 153 differentially expressed circRNAs, of which 76 were up-regulated and 77 were down-regulated. GO covered three domains: biological process (BP), cellular component (CC) and molecular function (MF). The terms assigned to the BP domain were peptidyl-tyrosine dephosphorylation and regulation of cell migration. The identified CC terms were cell-cell adherens junction, nucleoplasm and cytosol, and the identified MF terms were protein binding and protein tyrosine phosphatase activity. The top

Cloning, expression, purification, and activity assay of proteins related to D-lactic acid formation in Lactobacillus rhamnosus.

PubMed

Wang, Xiuwen; Zheng, Zhaojuan; Dou, Peipei; Qin, Jiayang; Wang, Xiaochen; Ma, Cuiqing; Tang, Hongzhi; Xu, Ping

2010-08-01

Two proteins that might be responsible for D-lactic acid (D-LA) formation were screened from the genome database of Lactobacillus rhamnosus GG. The coding genes of the two proteins in L. rhamnosus CASL, ldhD1 and ldhD2, were cloned and expressed in Escherichia coli Rosetta with an inducible expression vector pETDuet-1 (Novagen, Darmstadt, Germany), respectively. The two purified proteins, LdhD-1 and LdhD-2, migrated as a single protein band separately, both corresponding to an apparent molecular mass between 35 kDa and 45 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activities of LdhD-1 and LdhD-2 catalyzing pyruvate to LA were 0.02 U/mg and 0.21 U/mg, respectively. The configuration of LA converted from pyruvate was determined using high-performance liquid chromatography equipped with a chiral column. Only D-LA was detected when LdhD-1 and LdhD-2 were tested. In summary, the two proteins cloned and expressed in this study were most probably responsible for D-LA formation during fermentation of L. rhamnosus CASL.

Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities

PubMed Central

Yang, Li-yun; Liu, Xiao-fang; Yang, Yang; Yang, Lin-lin; Liu, Kai-wen; Tang, Yu-bo; Zhang, Min; Tan, Min-jia; Cheng, Shan-mei; Xu, Ye-chun; Yang, Huai-yu; Liu, Zhi-jie; Song, Gao-jie; Huang, Wei

2017-01-01

CD97 belongs to the adhesion GPCR family characterized by a long ECD linked to the 7TM via a GPCR proteolytic site (GPS) and plays important roles in modulating cell migration and invasion. CD97 (EGF1-5) is a splicing variant of CD97 that recognizes a specific ligand chondroitin sulfate on cell membranes and the extracellular matrix. The aim of this study was to elucidate the extracellular molecular basis of the CD97 EGF1-5 isoform in protein expression, auto-proteolysis and cell adhesion, including epidermal growth factor (EGF)-like domain, GPCR autoproteolysis-inducing (GAIN) domain, as well as GPS mutagenesis and N-glycosylation. Both wild-type (WT) CD97-ECD and its truncated, GPS mutated, PNGase F-deglycosylated, and N-glycosylation site mutated forms were expressed and purified. The auto-proteolysis of the proteins was analyzed with Western blotting and SDS-PAGE. Small angle X-ray scattering (SAXS) and molecular modeling were used to determine a structural profile of the properly expressed receptor. Potential N-glycosylation sites were identified using MS and were modulated with PNGase F digestion and glyco-site mutations. A flow cytometry-based HeLa cell attachment assay was used for all aforementioned CD97 variants to elucidate the molecular basis of CD97-HeLa interactions. A unique concentration-dependent GPS auto-proteolysis was observed in CD97 EGF1-5 isoform with the highest concentration (4 mg/mL) per sample was self-cleaved much faster than the lower concentration (0.1 mg/mL), supporting an intermolecular mechanism of auto-proteolysis that is distinct to the reported intramolecular mechanism for other CD97 isoforms. N-glycosylation affected the auto-proteolysis of CD97 EGF1-5 isoform in a similar way as the other previously reported CD97 isoforms. SAXS data for WT and deglycosylated CD97ECD revealed a spatula-like shape with GAIN and EGF domains constituting the body and handle, respectively. Structural modeling indicated a potential interaction

Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities.

PubMed

Yang, Li-Yun; Liu, Xiao-Fang; Yang, Yang; Yang, Lin-Lin; Liu, Kai-Wen; Tang, Yu-Bo; Zhang, Min; Tan, Min-Jia; Cheng, Shan-Mei; Xu, Ye-Chun; Yang, Huai-Yu; Liu, Zhi-Jie; Song, Gao-Jie; Huang, Wei

2017-01-01

CD97 belongs to the adhesion GPCR family characterized by a long ECD linked to the 7TM via a GPCR proteolytic site (GPS) and plays important roles in modulating cell migration and invasion. CD97 (EGF1-5) is a splicing variant of CD97 that recognizes a specific ligand chondroitin sulfate on cell membranes and the extracellular matrix. The aim of this study was to elucidate the extracellular molecular basis of the CD97 EGF1-5 isoform in protein expression, auto-proteolysis and cell adhesion, including epidermal growth factor (EGF)-like domain, GPCR autoproteolysis-inducing (GAIN) domain, as well as GPS mutagenesis and N-glycosylation. Both wild-type (WT) CD97-ECD and its truncated, GPS mutated, PNGase F-deglycosylated, and N-glycosylation site mutated forms were expressed and purified. The auto-proteolysis of the proteins was analyzed with Western blotting and SDS-PAGE. Small angle X-ray scattering (SAXS) and molecular modeling were used to determine a structural profile of the properly expressed receptor. Potential N-glycosylation sites were identified using MS and were modulated with PNGase F digestion and glyco-site mutations. A flow cytometry-based HeLa cell attachment assay was used for all aforementioned CD97 variants to elucidate the molecular basis of CD97-HeLa interactions. A unique concentration-dependent GPS auto-proteolysis was observed in CD97 EGF1-5 isoform with the highest concentration (4 mg/mL) per sample was self-cleaved much faster than the lower concentration (0.1 mg/mL), supporting an intermolecular mechanism of auto-proteolysis that is distinct to the reported intramolecular mechanism for other CD97 isoforms. N-glycosylation affected the auto-proteolysis of CD97 EGF1-5 isoform in a similar way as the other previously reported CD97 isoforms. SAXS data for WT and deglycosylated CD97ECD revealed a spatula-like shape with GAIN and EGF domains constituting the body and handle, respectively. Structural modeling indicated a potential interaction

Electronegativity and intrinsic disorder of preeclampsia-related proteins.

PubMed

Polanco, Carlos; Castañón-González, Jorge Alberto; Uversky, Vladimir N; Buhse, Thomas; Samaniego Mendoza, José Lino; Calva, Juan J

2017-01-01

Preeclampsia, hemorrhage, and infection are the leading causes of maternal death in underdeveloped countries. Since several proteins associated with preeclampsia are known, we conducted a computational study which evaluated the commonness and potential functionality of intrinsic disorder of these proteins and also made an attempt to characterize their origin. The origin of the preeclampsia-related proteins was assessed with a supervised technique, a Polarity Index Method (PIM), which evaluates the electronegativity of proteins based solely on their sequence. The commonness of intrinsic disorder was evaluated using several disorder predictors from the PONDR family, the charge-hydropathy plot (CH-plot) and cumulative distribution function (CDF) analyses, and using the MobiDB web-based tool, whereas potential functionality of intrinsic disorder was studied with the D2P2 resource and ANCHOR predictor of disorder-based binding sites, and the STRING tool was used to build the interactivity networks of the preeclampsia-related proteins. Peculiarities of the PIM-derived polar profile of the group of preeclampsia-related proteins were then compared with profiles of a group of lipoproteins, antimicrobial peptides, angiogenesis-related proteins, and the intrinsically disordered proteins. Our results showed a high graphical correlation between preeclampsia proteins, lipoproteins, and the angiogenesis proteins. We also showed that many preeclampsia-related proteins contain numerous functional disordered regions. Therefore, these bioinformatics results led us to assume that the preeclampsia proteins are highly associated with the lipoproteins group, and that some preeclampsia-related proteins contain significant amounts of functional disorders.

Ribosome-Inactivating and Related Proteins

PubMed Central

Schrot, Joachim; Weng, Alexander; Melzig, Matthias F.

2015-01-01

Ribosome-inactivating proteins (RIPs) are toxins that act as N-glycosidases (EC 3.2.2.22). They are mainly produced by plants and classified as type 1 RIPs and type 2 RIPs. There are also RIPs and RIP related proteins that cannot be grouped into the classical type 1 and type 2 RIPs because of their different sizes, structures or functions. In addition, there is still not a uniform nomenclature or classification existing for RIPs. In this review, we give the current status of all known plant RIPs and we make a suggestion about how to unify those RIPs and RIP related proteins that cannot be classified as type 1 or type 2 RIPs. PMID:26008228

G2 phase-specific proteins of HeLa cells.

PubMed Central

Al-Bader, A A; Orengo, A; Rao, P N

1978-01-01

The objective of this study was to determine if HeLa cells irreversibly arrested in G2 phase of the cell cycle by a brief exposure to a nitrosourea compound were deficient in certain proteins when compared with G2-synchronized cells. Total cellular proteins of G2-synchronized, G2-arrested, and S phase-synchronized cells were compared by two-dimensional polyacrylamide gel electrophoresis. The S phase cells differed from the G2-synchronized and G2-arrested cells by the absence of about 35 and 25 protein spots, respectively, of a total of nearly 150. At least nine protein spots in the molecular weight range of 4--5 X 10(4) that were present in the G2-synchronized cells were absent in both the G2-arrested and the S phase cells. Thus, these studies suggest that the missing proteins are probably necessary for the transition of cells from G2 phase to mitosis. Supplying the missing proteins to the G2-arrested cells by fusion with G2-synchronized cells facilitated the entry of the former into mitosis. Images PMID:282623

A calmodulin-like protein (LCALA) is a new Leishmania amazonensis candidate for telomere end-binding protein.

PubMed

Morea, Edna G O; Viviescas, Maria Alejandra; Fernandes, Carlos A H; Matioli, Fabio F; Lira, Cristina B B; Fernandez, Maribel F; Moraes, Barbara S; da Silva, Marcelo S; Storti, Camila B; Fontes, Marcos R M; Cano, Maria Isabel N

2017-11-01

Leishmania spp. telomeres are composed of 5'-TTAGGG-3' repeats associated with proteins. We have previously identified LaRbp38 and LaRPA-1 as proteins that bind the G-rich telomeric strand. At that time, we had also partially characterized a protein: DNA complex, named LaGT1, but we could not identify its protein component. Using protein-DNA interaction and competition assays, we confirmed that LaGT1 is highly specific to the G-rich telomeric single-stranded DNA. Three protein bands, with LaGT1 activity, were isolated from affinity-purified protein extracts in-gel digested, and sequenced de novo using mass spectrometry analysis. In silico analysis of the digested peptide identified them as a putative calmodulin with sequences identical to the T. cruzi calmodulin. In the Leishmania genome, the calmodulin ortholog is present in three identical copies. We cloned and sequenced one of the gene copies, named it LCalA, and obtained the recombinant protein. Multiple sequence alignment and molecular modeling showed that LCalA shares homology to most eukaryotes calmodulin. In addition, we demonstrated that LCalA is nuclear, partially co-localizes with telomeres and binds in vivo the G-rich telomeric strand. Recombinant LCalA can bind specifically and with relative affinity to the G-rich telomeric single-strand and to a 3'G-overhang, and DNA binding is calcium dependent. We have described a novel candidate component of Leishmania telomeres, LCalA, a nuclear calmodulin that binds the G-rich telomeric strand with high specificity and relative affinity, in a calcium-dependent manner. LCalA is the first reported calmodulin that binds in vivo telomeric DNA. Copyright © 2017 Elsevier B.V. All rights reserved.

Protein function prediction using neighbor relativity in protein-protein interaction network.

PubMed

Moosavi, Sobhan; Rahgozar, Masoud; Rahimi, Amir

2013-04-01

There is a large gap between the number of discovered proteins and the number of functionally annotated ones. Due to the high cost of determining protein function by wet-lab research, function prediction has become a major task for computational biology and bioinformatics. Some researches utilize the proteins interaction information to predict function for un-annotated proteins. In this paper, we propose a novel approach called "Neighbor Relativity Coefficient" (NRC) based on interaction network topology which estimates the functional similarity between two proteins. NRC is calculated for each pair of proteins based on their graph-based features including distance, common neighbors and the number of paths between them. In order to ascribe function to an un-annotated protein, NRC estimates a weight for each neighbor to transfer its annotation to the unknown protein. Finally, the unknown protein will be annotated by the top score transferred functions. We also investigate the effect of using different coefficients for various types of functions. The proposed method has been evaluated on Saccharomyces cerevisiae and Homo sapiens interaction networks. The performance analysis demonstrates that NRC yields better results in comparison with previous protein function prediction approaches that utilize interaction network. Copyright © 2012 Elsevier Ltd. All rights reserved.

Autodisplay of the La/SSB protein on LPS-free E. coli for the diagnosis of Sjögren's syndrome.

PubMed

Yoo, Gu; Dilkaute, Carina; Bong, Ji-Hong; Song, Hyun-Woo; Lee, Misu; Kang, Min-Jung; Jose, Joachim; Pyun, Jae-Chul

2017-05-01

The objective of this study was to present an immunoassay for the diagnosis of Sjögren's syndrome based on the autodisplayed La/SSB protein on the outer membrane of intact E. coli (strain UT-5600) and LPS-free E. coli (ClearColi™). As the first step, an autodisplay vector (pCK002) was transfected into intact E. coli and LPS-free E. coli for comparison of efficiency of autdisplay of La/SSB. The maximal level of La/SSB expression was estimated to be similar for LPS-free E. coli and intact E. coli at different optimal induction periods. Intact E. coli was found to grow twofold faster than LPS-free E. coli, and the maximal level of expression for LPS-free E. coli was obtained with a longer induction period. When the zeta potential was measured, both intact E. coli and LPS-free E. coli showed negative values, and the autodisplay of negatively charged La/SSB protein (pI<7) on the outer membrane of intact E. coli and LPS-free E. coli resulted in a slight change in zeta potential values. E. coli with autodisplayed La/SSB protein was used for an immunoassay of anti-La/SSB antibodies for the diagnosis of Sjögren's syndrome. The surface of E. coli with the autodisplayed antigen was modified with rabbit serum and papain to prevent false positive signals because of nonspecific binding of unrelated antibodies from human serum. LPS-free E. coli with autodisplayed La/SSB protein yielded sensitivity and selectivity of 81.6% and 78.6%, respectively. The Bland-Altman test showed that the immunoassays based on LPS-free E. coli and intact E. coli with autodisplayed La/SSB protein were statistically equivalent to a clinical immunoassay for detection of anti-La/SSB antibodies (confidence coefficient 95%). Copyright © 2017 Elsevier Inc. All rights reserved.

The new platinum-based anticancer agent LA-12 induces retinol binding protein 4 in vivo

PubMed Central

2011-01-01

Background The initial pharmacokinetic study of a new anticancer agent (OC-6-43)-bis(acetato)(1-adamantylamine)amminedichloroplatinum (IV) (LA-12) was complemented by proteomic screening of rat plasma. The objective of the study was to identify new LA-12 target proteins that serve as markers of LA-12 treatment, response and therapy monitoring. Methods Proteomic profiles were measured by surface-enhanced laser desorption-ionization time-of-flight mass spectrometry (SELDI-TOF MS) in 72 samples of rat plasma randomized according to LA-12 dose and time from administration. Correlation of 92 peak clusters with platinum concentration was evaluated using Spearman correlation analysis. Results We identified Retinol-binding protein 4 (RBP4) whose level correlated with LA-12 level in treated rats. Similar results were observed in randomly selected patients involved in Phase I clinical trials. Conclusions RBP4 induction is in agreement with known RBP4 regulation by amantadine and cisplatin. Since retinol metabolism is disrupted in many cancers and inversely associates with malignancy, these data identify a potential novel mechanism for the action of LA-12 and other similar anti-cancer drugs. PMID:22040120

CMP‑N‑acetylneuraminic acid synthetase interacts with fragile X related protein 1.

PubMed

Ma, Yun; Tian, Shuai; Wang, Zongbao; Wang, Changbo; Chen, Xiaowei; Li, Wei; Yang, Yang; He, Shuya

2016-08-01

Fragile X mental retardation protein (FMRP), fragile X related 1 protein (FXR1P) and FXR2P are the members of the FMR protein family. These proteins contain two KH domains and a RGG box, which are characteristic of RNA binding proteins. The absence of FMRP, causes fragile X syndrome (FXS), the leading cause of hereditary mental retardation. FXR1P is expressed throughout the body and important for normal muscle development, and its absence causes cardiac abnormality. To investigate the functions of FXR1P, a screen was performed to identify FXR1P‑interacting proteins and determine the biological effect of the interaction. The current study identified CMP‑N‑acetylneuraminic acid synthetase (CMAS) as an interacting protein using the yeast two‑hybrid system, and the interaction between FXR1P and CMAS was validated in yeast using a β‑galactosidase assay and growth studies with selective media. Furthermore, co‑immunoprecipitation was used to analyze the FXR1P/CMAS association and immunofluorescence microscopy was performed to detect expression and intracellular localization of the proteins. The results of the current study indicated that FXR1P and CMAS interact, and colocalize in the cytoplasm and the nucleus of HEK293T and HeLa cells. Accordingly, a fragile X related 1 (FXR1) gene overexpression vector was constructed to investigate the effect of FXR1 overexpression on the level of monosialotetrahexosylganglioside 1 (GM1). The results of the current study suggested that FXR1P is a tissue‑specific regulator of GM1 levels in SH‑SY5Y cells, but not in HEK293T cells. Taken together, the results initially indicate that FXR1P interacts with CMAS, and that FXR1P may enhance the activation of sialic acid via interaction with CMAS, and increase GM1 levels to affect the development of the nervous system, thus providing evidence for further research into the pathogenesis of FXS.

Predicting disease-related proteins based on clique backbone in protein-protein interaction network.

PubMed

Yang, Lei; Zhao, Xudong; Tang, Xianglong

2014-01-01

Network biology integrates different kinds of data, including physical or functional networks and disease gene sets, to interpret human disease. A clique (maximal complete subgraph) in a protein-protein interaction network is a topological module and possesses inherently biological significance. A disease-related clique possibly associates with complex diseases. Fully identifying disease components in a clique is conductive to uncovering disease mechanisms. This paper proposes an approach of predicting disease proteins based on cliques in a protein-protein interaction network. To tolerate false positive and negative interactions in protein networks, extending cliques and scoring predicted disease proteins with gene ontology terms are introduced to the clique-based method. Precisions of predicted disease proteins are verified by disease phenotypes and steadily keep to more than 95%. The predicted disease proteins associated with cliques can partly complement mapping between genotype and phenotype, and provide clues for understanding the pathogenesis of serious diseases.

Loss of Selenium-Binding Protein 1 Decreases Sensitivity to Clastogens and Intracellular Selenium Content in HeLa Cells.

PubMed

Zhao, Changhui; Zeng, Huawei; Wu, Ryan T Y; Cheng, Wen-Hsing

2016-01-01

Selenium-binding protein 1 (SBP1) is not a selenoprotein but structurally binds selenium. Loss of SBP1 during carcinogenesis usually predicts poor prognosis. Because genome instability is a hallmark of cancer, we hypothesize that SBP1 sequesters cellular selenium and sensitizes cancer cells to DNA-damaging agents. To test this hypothesis, we knocked down SBP1 expression in HeLa cervical cancer cells by employing a short hairpin RNA (shRNA) approach. Reduced sensitivity to hydrogen peroxide, paraquat and camptothecin, reactive oxygen species content, and intracellular retention of selenium after selenomethionine treatment were observed in SBP1 shRNA HeLa cells. Results from Western analyses showed that treatment of HeLa cells with selenomethionine resulted in increased SBP1 protein expression in a dose-dependent manner. Knockdown of SBP1 rendered HeLa cells increased expression of glutathione peroxidase-1 but not glutathione peroxidase-4 protein levels and accelerated migration from a wound. Altogether, SBP1 retains supplemental selenium and sensitizes HeLa cancer cells to clastogens, suggesting a new cancer treatment strategy by sequestering selenium through SBP1.

bmp15l, figla, smc1bl, and larp6l are preferentially expressed in germ cells in Atlantic salmon (Salmo salar L.).

PubMed

Kleppe, Lene; Edvardsen, Rolf Brudvik; Furmanek, Tomasz; Andersson, Eva; Juanchich, Amélie; Wargelius, Anna

2017-01-01

Atlantic salmon is a valuable commercial aquaculture species that would benefit economically and environmentally by controlling precocious puberty and preventing escapees from reproducing with wild populations. One solution to both these challenges is the production of sterile individuals by inhibiting the formation of germ cells, but achieving this requires more information on the specific factors that control germ cell formation. Here, we identified and characterized novel factors that are preferentially expressed in Atlantic salmon germ cells by screening for gonad-specific genes using available adult multi-tissue transcriptomes. We excluded genes with expression in tissues other than gonads based on quantity of reads, and then a subset of genes was selected for verification in a multi-tissue PCR screen. Four gonad-specific genes (bmp15l, figla, smc1bl, and larp6l) were chosen for further characterization, namely: germ cell specificity, investigated by comparing mRNA abundance in wild-type and germ cell-free gonads by quantitative real-time PCR, and cellular location, visualized by in situ hybridization. All four genes were expressed in both testis and ovary, and preferentially within the germ cells of both sexes. These genes may be essential players in salmon germ cell development, and could be important for future studies aiming to understand and control reproduction. Mol. Reprod. Dev. 84: 76-87, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Propofol Inhibits HeLa Cells by Impairing Autophagic Flux via AMP-Activated Protein Kinase (AMPK) Activation and Endoplasmic Reticulum Stress Regulated by Calcium.

PubMed

Chen, Xi; Li, Kai; Zhao, Guoqing

2018-04-18

BACKGROUND Propofol has antitumor effects against various cancers. However, the mechanism of action of propofol in HeLa human cervical cancer cells has not been elucidated. MATERIAL AND METHODS We treated HeLa human cervical cancer cells with different concentrations of propofol. Cell viability was evaluated with Cell Counting Kit-8 and apoptosis was analyzed by annexin V-fluorescein isothiocyanate and propidium iodide staining and flow cytometry. Autophagosome formation was evaluated based on microtubule-associated protein light chain (LC)3 conversion and light chain 3 puncta formation. Autophagosome clearance was assessed according to p62 protein level and autolysosome generation. RESULTS We found that propofol decreased cell viability and increased autophagosome generation in HeLa cells. Autophagosome formation was evaluated based on LC3 conversion and LC3 puncta formation. Autophagosome clearance was assessed according to p62 protein level. The AMPK/mTOR signaling pathway was found to be activated in propofol-induced autophagosome accumulation. Fluorescence analysis using LysoTracker dye revealed that propofol blocked autophagosome-lysosome fusion. Administration of rapamycin increased autophagosome clearance in propofol-treated HeLa cells. Additionally, propofol induced endoplasmic reticulum (ER) stress and disrupted intracellular Ca2+ balance, thereby enhancing autophagosome accumulation. Suppressing ER stress by treatment with tauroursodeoxycholic acid (TUDCA) enhanced these effects, suggesting that the cytotoxicity of propofol is related to induction of ER stress. CONCLUSIONS This study is the first to provide evidence that propofol-mediated autophagy regulation is an underlying part of the mechanism by which propofol regulates HeLa cells progression.

DITOP: drug-induced toxicity related protein database.

PubMed

Zhang, Jing-Xian; Huang, Wei-Juan; Zeng, Jing-Hua; Huang, Wen-Hui; Wang, Yi; Zhao, Rui; Han, Bu-Cong; Liu, Qing-Feng; Chen, Yu-Zong; Ji, Zhi-Liang

2007-07-01

Drug-induced toxicity related proteins (DITRPs) are proteins that mediate adverse drug reactions (ADRs) or toxicities through their binding to drugs or reactive metabolites. Collection of these proteins facilitates better understanding of the molecular mechanisms of drug-induced toxicity and the rational drug discovery. Drug-induced toxicity related protein database (DITOP) is such a database that is intending to provide comprehensive information of DITRPs. Currently, DITOP contains 1501 records, covering 618 distinct literature-reported DITRPs, 529 drugs/ligands and 418 distinct toxicity terms. These proteins were confirmed experimentally to interact with drugs or their reactive metabolites, thus directly or indirectly cause adverse effects or toxicities. Five major types of drug-induced toxicities or ADRs are included in DITOP, which are the idiosyncratic adverse drug reactions, the dose-dependent toxicities, the drug-drug interactions, the immune-mediated adverse drug effects (IMADEs) and the toxicities caused by genetic susceptibility. Molecular mechanisms underlying the toxicity and cross-links to related resources are also provided while available. Moreover, a series of user-friendly interfaces were designed for flexible retrieval of DITRPs-related information. The DITOP can be accessed freely at http://bioinf.xmu.edu.cn/databases/ADR/index.html. Supplementary data are available at Bioinformatics online.

Curcumin causes DNA damage and affects associated protein expression in HeLa human cervical cancer cells.

PubMed

Shang, Hung-Sheng; Chang, Chuan-Hsun; Chou, Yu-Ru; Yeh, Ming-Yang; Au, Man-Kuan; Lu, Hsu-Feng; Chu, Yung-Lin; Chou, Hsiao-Min; Chou, Hsiu-Chen; Shih, Yung-Luen; Chung, Jing-Gung

2016-10-01

Cervical cancer is one of the most common cancers in women worldwide and it is a prominent cause of cancer mortality. Curcumin is one of the major compounds from Turmeric and has been shown to induce cytotoxic cell death in human cervical cancer cells. However, there is no study to show curcumin induced DNA damage action via the effect on the DNA damage and repair protein in cervical cancer cells in detail. In this study, we investigated whether or not curcumin induced cell death via DNA damage, chromatin condensation in human cervical cancer HeLa cells by using comet assay and DAPI staining, respectively, we found that curcumin induced cell death through the induction of DNA damage, and chromatin condensation. Western blotting and confocal laser microscopy examination were used to examine the effects of curcumin on protein expression associated with DNA damage, repair and translocation of proteins. We found that curcumin at 13 µM increased the protein levels associated with DNA damage and repair, such as O6-methylguanine-DNA methyltransferase, early-onset breast cancer 1 (BRCA1), mediator of DNA damage checkpoint 1, p-p53 and p-H2A.XSer140 in HeLa cells. Results from confocal laser systems microscopy indicated that curcumin increased the translocation of p-p53 and p-H2A.XSer140 from cytosol to nuclei in HeLa cells. In conclusion, curcumin induced cell death in HeLa cells via induction of DNA damage, and chromatin condensation in vitro.

Molecular cloning of ADIR, a novel interferon responsive gene encoding a protein related to the torsins.

PubMed

Dron, Michel; Meritet, Jean François; Dandoy-Dron, Françoise; Meyniel, Jean-Philippe; Maury, Chantal; Tovey, Michael G

2002-03-01

The expression of the previously uncharacterized gene Adir (for ATP dependent interferon responsive gene) was increased by 5- to 15-fold in tissue of the oral cavity or in spleen and liver of mice treated orally or intraperitoneally with IFN-alpha, and in mouse cells treated in vitro with IFN-alpha or IFN-gamma. The level of Adir mRNA was also increased 20- to 40-fold in the brains of animals infected with encephalomyocarditis virus. Adir is expressed ubiquitously in mouse tissues as 1.9-, 2.4-, and 3.5-kb mRNA transcripts encoding a 385-amino-acid protein with a conserved ATP binding domain containing typical nucleotide and Mg(2+) binding sites. We also characterized the human ortholog, ADIR, which is located on chromosome 1q25-q31 and contains six exons encoding a 397-amino-acid protein with 80% homology to the mouse protein. A single 2.3-kb mRNA was detected in all human tissues examined, except for placenta, which also contained a 1.25-kb tissue-specific transcript generated by alternative splicing and encoding a putative 336-amino-acid protein. Although ADIR exhibits low homology to DYT1 and TOR1B, the deduced ADIR protein sequences are highly homologous to torsin A and torsin B and more distantly related to members of the Clp/HSP100 family of proteins, suggesting that ADIR, like torsins, is related to the AAA chaperone-like family of ATPases. An ADIR-EGFP fusion protein expressed in HeLa cells was shown to be associated with the endoplasmic reticulum.

La protein and its associated small nuclear and nucleolar precursor RNAs.

PubMed

Maraia, Richard J; Intine, Robert V

2002-01-01

After transcription by RNA polymerase (pol) III, nascent Pol III transcripts pass through RNA processing, modification, and transport machineries as part of their posttranscriptional maturation process. The first factor to interact with Pol III transcripts is La protein, which binds principally via its conserved N-terminal domain (NTD), to the UUU-OH motif that results from transcription termination. This review includes a sequence Logo of the most conserved region of La and its refined modeling as an RNA recognition motif (RRM). La protects RNAs from 3' exonucleolytic digestion and also contributes to their nuclear retention. The variety of modifications found on La-associated RNAs is reviewed in detail and considered in the contexts of how La may bind the termini of structured RNAs without interfering with recognition by modification enzymes, and its ability to chaperone RNAs through multiple parts of their maturation pathways. The CTD of human La recognizes the 5' end region of nascent RNA in a manner that is sensitive to serine 366 phosphorylation. Although the CTD can control pre-tRNA cleavage by RNase P, a rate-limiting step in tRNASerUGA maturation, the extent to which it acts in the maturation pathway(s) of other transcripts is unknown but considered here. Evidence that a fraction of La resides in the nucleolus together with recent findings that several Pol III transcripts pass through the nucleolus is also reviewed. An imminent goal is to understand how the bipartite RNA binding, intracellular trafficking, and signal transduction activities of La are integrated with the maturation pathways of the various RNAs with which it associates.

SPATA4 Counteracts Etoposide-Induced Apoptosis via Modulating Bcl-2 Family Proteins in HeLa Cells.

PubMed

Jiang, Junjun; Li, Liyuan; Xie, Mingchao; Fuji, Ryosuke; Liu, Shangfeng; Yin, Xiaobei; Li, Genlin; Wang, Zhao

2015-01-01

Spermatogenesis associated 4 (SPATA4) is a testis-specific gene first cloned by our laboratory, and plays an important role in maintaining the physiological function of germ cells. Accumulated evidence suggests that SPATA4 might be associated with apoptosis. Here we established HeLa cells that stably expressed SPATA4 to investigate the function of SPATA4 in apoptosis. SPATA4 protected HeLa cells from etoposide-induced apoptosis through the mitochondrial apoptotic pathway, in the way that SPATA4 suppressed decrease of the mitochondrial membrane potential, the release of cytochrome c, and subsequent activation of caspase-9 and -3. We further demonstrated that SPATA4 upregulated anti-apoptotic members of Bcl-2 family proteins, Bcl-2, and downregulated the pro-apoptotic member of Bcl-2 family proteins, Bax. Knockdown of SPATA4 in HeLa/SPATA4 cells could partially rescue expression levels of bcl-2 and bax. In conclusion, SPATA4 protects HeLa cells against etoposide-induced apoptosis through the mitochondrial apoptotic pathway. Our findings provide further evidence that SPATA4 plays a role in regulating apoptosis.

A new carbamidemethyl-linked lanthanoid chelating tag for PCS NMR spectroscopy of proteins in living HeLa cells.

PubMed

Hikone, Yuya; Hirai, Go; Mishima, Masaki; Inomata, Kohsuke; Ikeya, Teppei; Arai, Souichiro; Shirakawa, Masahiro; Sodeoka, Mikiko; Ito, Yutaka

2016-10-01

Structural analyses of proteins under macromolecular crowding inside human cultured cells by in-cell NMR spectroscopy are crucial not only for explicit understanding of their cellular functions but also for applications in medical and pharmaceutical sciences. In-cell NMR experiments using human cultured cells however suffer from low sensitivity, thus pseudocontact shifts from protein-tagged paramagnetic lanthanoid ions, analysed using sensitive heteronuclear two-dimensional correlation NMR spectra, offer huge potential advantage in obtaining structural information over conventional NOE-based approaches. We synthesised a new lanthanoid-chelating tag (M8-CAM-I), in which the eight-fold, stereospecifically methylated DOTA (M8) scaffold was retained, while a stable carbamidemethyl (CAM) group was introduced as the functional group connecting to proteins. M8-CAM-I successfully fulfilled the requirements for in-cell NMR: high-affinity to lanthanoid, low cytotoxicity and the stability under reducing condition inside cells. Large PCSs for backbone N-H resonances observed for M8-CAM-tagged human ubiquitin mutant proteins, which were introduced into HeLa cells by electroporation, demonstrated that this approach readily provides the useful information enabling the determination of protein structures, relative orientations of domains and protein complexes within human cultured cells.

Actin, actin-binding proteins, and actin-related proteins in the nucleus.

PubMed

Kristó, Ildikó; Bajusz, Izabella; Bajusz, Csaba; Borkúti, Péter; Vilmos, Péter

2016-04-01

Extensive research in the past decade has significantly broadened our view about the role actin plays in the life of the cell and added novel aspects to actin research. One of these new aspects is the discovery of the existence of nuclear actin which became evident only recently. Nuclear activities including transcriptional activation in the case of all three RNA polymerases, editing and nuclear export of mRNAs, and chromatin remodeling all depend on actin. It also became clear that there is a fine-tuned equilibrium between cytoplasmic and nuclear actin pools and that this balance is ensured by an export-import system dedicated to actin. After over half a century of research on conventional actin and its organizing partners in the cytoplasm, it was also an unexpected finding that the nucleus contains more than 30 actin-binding proteins and new classes of actin-related proteins which are not able to form filaments but had evolved nuclear-specific functions. The actin-binding and actin-related proteins in the nucleus have been linked to RNA transcription and processing, nuclear transport, and chromatin remodeling. In this paper, we attempt to provide an overview of the wide range of information that is now available about actin, actin-binding, and actin-related proteins in the nucleus.

Identification and characterization of a HeLa nuclear protein that specifically binds to the trans-activation-response (TAR) element of human immunodeficiency virus.

PubMed Central

Marciniak, R A; Garcia-Blanco, M A; Sharp, P A

1990-01-01

Human immunodeficiency virus type 1 RNAs contain a sequence, trans-activation-response (TAR) element, which is required for tat protein-mediated trans-activation of viral gene expression. We have identified a nuclear protein from extracts of HeLa cells that binds to the TAR element RNA in a sequence-specific manner. The binding of this 68-kDa polypeptide was detected by UV cross-linking proteins to TAR element RNA transcribed in vitro. Competition experiments were performed by using a partially purified preparation of the protein to quantify the relative binding affinities of TAR element RNA mutants. The binding affinity of the TAR mutants paralleled the reported ability of those mutants to support tat trans-activation in vivo. We propose that this cellular protein moderates TAR activity in vivo. Images PMID:2333305

Investigation of Cu-, Zn- and Fe-containing human brain proteins using isotopic-enriched tracers by LA-ICP-MS and MALDI-FT-ICR-MS

NASA Astrophysics Data System (ADS)

Becker, J. Susanne; Zoriy, Miroslav; Pickhardt, Carola; Przybylski, Michael; Becker, J. Sabine

2005-04-01

Identification of metal-containing proteins and determination of Cu, Fe, Zn concentration in very small protein volumes is of increasing importance in protein research. Proteins containing metal ions were analyzed directly and simultaneously in separated protein spots in two-dimensional gels (2D gels) by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) as an element mass spectrometric technique. In order to study the formation of proteins containing Cu, Zn and Fe in a human brain sample, isotopic-enriched tracers (54Fe, 65Cu and 67Zn) were doped to two-dimensional gels of separated Alzheimer-diseased brain proteins after two-dimensional (2D) gel electrophoresis. The protein spots were screened systematically by LA-ICP-MS with respect to these metal ion intensities. 54Fe/56Fe, 65Cu/63Cu and 67Zn/64Zn isotope ratios in metal-containing proteins were measured directly by LA-ICP-MS. The isotope ratio measurements obtained by LA-ICP-MS indicate certain protein spots with a natural isotope composition of Cu, Zn and/or Fe. These proteins already contained the metal investigated in the original proteins and are stable enough to survive the reducing conditions during gel electrophoresis. On the other hand, proteins with a changed isotope ratio of metals in comparison to the isotope ratio in nature demonstrate the accumulation of tracers within the protein complexes during the tracer experiments in 2D gels. The identification of singular protein spots from Alzheimer-diseased brain separated by 2D gel electrophoresis was attempted by biopolymer mass spectrometry using MALDI-FTICR-MS after excision from the 2D gel and tryptic digestion.

A protein relational database and protein family knowledge bases to facilitate structure-based design analyses.

PubMed

Mobilio, Dominick; Walker, Gary; Brooijmans, Natasja; Nilakantan, Ramaswamy; Denny, R Aldrin; Dejoannis, Jason; Feyfant, Eric; Kowticwar, Rupesh K; Mankala, Jyoti; Palli, Satish; Punyamantula, Sairam; Tatipally, Maneesh; John, Reji K; Humblet, Christine

2010-08-01

The Protein Data Bank is the most comprehensive source of experimental macromolecular structures. It can, however, be difficult at times to locate relevant structures with the Protein Data Bank search interface. This is particularly true when searching for complexes containing specific interactions between protein and ligand atoms. Moreover, searching within a family of proteins can be tedious. For example, one cannot search for some conserved residue as residue numbers vary across structures. We describe herein three databases, Protein Relational Database, Kinase Knowledge Base, and Matrix Metalloproteinase Knowledge Base, containing protein structures from the Protein Data Bank. In Protein Relational Database, atom-atom distances between protein and ligand have been precalculated allowing for millisecond retrieval based on atom identity and distance constraints. Ring centroids, centroid-centroid and centroid-atom distances and angles have also been included permitting queries for pi-stacking interactions and other structural motifs involving rings. Other geometric features can be searched through the inclusion of residue pair and triplet distances. In Kinase Knowledge Base and Matrix Metalloproteinase Knowledge Base, the catalytic domains have been aligned into common residue numbering schemes. Thus, by searching across Protein Relational Database and Kinase Knowledge Base, one can easily retrieve structures wherein, for example, a ligand of interest is making contact with the gatekeeper residue.

Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells

PubMed Central

Zhen, Hong; Huang, Ming; Zheng, Xi; Feng, Lixing; Jiang, Baohong; Yang, Min; Wu, Wanying; Liu, Xuan; Guo, Dean

2016-01-01

Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K

Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.

PubMed

Yue, Qingxi; Zhen, Hong; Huang, Ming; Zheng, Xi; Feng, Lixing; Jiang, Baohong; Yang, Min; Wu, Wanying; Liu, Xuan; Guo, Dean

2016-01-01

Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K

Metal ions induced heat shock protein response by elevating superoxide anion level in HeLa cells transformed by HSE-SEAP reporter gene.

PubMed

Yu, Zhanjiang; Yang, Xiaoda; Wang, Kui

2006-06-01

The aim of this work is to define the relationship between heat shock protein (HSP) and reactive oxygen species (ROS) in the cells exposed to different concentrations of metal ions, and to evaluate a new method for tracing the dynamic levels of cellular reactive oxygen species using a HSE-SEAP reporter gene. The expression of heat shock protein was measured using a secreted alkaline phosphatase (SEAP) reporter gene transformed into HeLa cell strain, the levels of superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)) were determined by NBT reduction assay and DCFH staining flow cytometry (FCM), respectively. The experimental results demonstrated that the expression of heat shock protein induced by metal ions was linearly related to the cellular superoxide anion level before cytotoxic effects were observed, but not related to the cellular hydrogen peroxide level. The experimental results suggested that metal ions might induce heat shock protein by elevating cellular superoxide anion level, and thus the expression of heat shock protein indicated by the HSE-SEAP reporter gene can be an effective model for monitoring the dynamic level of superoxide anion and early metal-induced oxidative stress/cytotoxicity.

Separate RNA-binding surfaces on the multifunctional La protein mediate distinguishable activities in tRNA maturation.

PubMed

Huang, Ying; Bayfield, Mark A; Intine, Robert V; Maraia, Richard J

2006-07-01

By sequence-specific binding to 3' UUU-OH, the La protein shields precursor (pre)-RNAs from 3' end digestion and is required to protect defective pre-transfer RNAs from decay. Although La is comprised of a La motif and an RNA-recognition motif (RRM), a recent structure indicates that the RRM beta-sheet surface is not involved in UUU-OH recognition, raising questions as to its function. Progressively defective suppressor tRNAs in Schizosaccharomyces pombe reveal differential sensitivities to La and Rrp6p, a 3' exonuclease component of pre-tRNA decay. 3' end protection is compromised by mutations to the La motif but not the RRM surface. The most defective pre-tRNAs require a second activity of La, in addition to 3' protection, that requires an intact RRM surface. The two activities of La in tRNA maturation map to its two conserved RNA-binding surfaces and suggest a modular model that has implications for its other ligands.

Phosphorylated Akt Protein at Ser473 Enables HeLa Cells to Tolerate Nutrient-Deprived Conditions

PubMed

Fathy, Moustafa; Awale, Suresh; Nikaido, Toshio

2017-12-29

Background: Despite angiogenesis, many tumours remain hypovascular and starved of nutrients while continuing to grow rapidly. The specific biochemical mechanisms associated with starvation resistance, austerity, may be new biological characters of cancer that are critical for cancer progression. Objective: This study aim was to investigate the effect of nutrient starvation on HeLa cells and the possible mechanism by which the cells are able to tolerate nutrient-deprived conditions. Methods: Nutrient starvation was achieved by culturing HeLa cells in nutrient-deprived medium (NDM) and cell survival was estimated by using cell counting kit-8. The effect of starvation on cell cycle distribution and the quantitative analysis of apoptotic cells were investigated by flow cytometry using propidium iodide staining. Western blotting was used to detect the expression levels of Akt and phosphorylated Akt at Ser473 (Ser473p-Akt) proteins. Results: HeLa cells displayed extremely long survival when cultured in NDM. The percentage of apoptotic HeLa cells was significantly increased by starvation in a time-dependent manner. A significant increase in the expression of Ser473p-Akt protein after starvation was also observed. Furthermore, it was found that Akt inhibitor III molecule inhibited the cells proliferation in a concentration- and time-dependent manner. Conclusion: Results of the present study provide evidence that Akt activation may be implicated in the tolerance of HeLa cells for nutrient starvation and may help to suggest new therapeutic strategies designed to prevent austerity of cervical cancer cells through inhibition of Akt activation. Creative Commons Attribution License

ABA, porphyrins and plant TSPO-related protein.

PubMed

Guillaumot, Damien; Guillon, Stéphanie; Morsomme, Pierre; Batoko, Henri

2009-11-01

We have shown that, unexpectedly, AtTSPO (Arabidopsis thaliana TSPO-related protein) is an endoplasmic reticulum and Golgi-localized membrane protein in plant cells.(1) This localization contrasts with that of mammalian 18-kDa translocator protein (at least for the mostly studied isoform, 18-kDa TSPO), a mitochondrial outer membrane protein (reviewed in ref. 2). Whereas the potential functions of 18-kDa TSPO are well documented, involved mainly in mitochondrial physiology,(2) and its interest as drugs target is been explored,(3) the roles of TSPO-related proteins in plant growth and development are yet to be specified. AtTSPO is expressed in dry seeds and can be induced in vegetative tissues by osmotic and salt stress or abscisic acid (ABA) treatment. Moreover, it was shown that the ABA-dependent induction is transient, and that boosting tetrapyrroles biosynthesis through 5-aminolevulinic acid (ALA) feeding enhanced downregulation of AtTSPO, suggesting an inherent post-translational regulation mechanism also involving ABA and likely porphyrins. We present additional evidence that ABA can help stabilize constitutively expressed AtTSPO and that ALA feeding to knockout mutant seeds, induces substantial germination delay. Here we discuss the possible link between ABA and tetrapyrroles in AtTSPO expression and post-translational regulation.

AtLa1 protein initiates IRES-dependent translation of WUSCHEL mRNA and regulates the stem cell homeostasis of Arabidopsis in response to environmental hazards.

PubMed

Cui, Yuchao; Rao, Shaofei; Chang, Beibei; Wang, Xiaoshuang; Zhang, Kaidian; Hou, Xueliang; Zhu, Xueyi; Wu, Haijun; Tian, Zhaoxia; Zhao, Zhong; Yang, Chengwei; Huang, Tao

2015-10-01

Plant stem cells are hypersensitive to environmental hazards throughout their life cycle, but the mechanism by which plants safeguard stem cell homeostasis in response to environmental hazards is largely unknown. The homeodomain transcription factor WUSCHEL (WUS) protein maintains the stem cell pool in the shoot apical meristem of Arabidopsis. Here, we demonstrate that the translation of WUS mRNA is directed by an internal ribosomal entry site (IRES) located in the 5'-untranslated region. The AtLa1 protein, an RNA-binding factor, binds to the 5'-untranslated region and initiates the IRES-dependent translation of WUS mRNA. Knockdown of AtLa1 expression represses the WUS IRES-dependent translation and leads to the arrest of growth and development. The AtLa1 protein is mainly located in the nucleoplasm. However, environmental hazards promote the nuclear-to-cytoplasmic translocation of the AtLa1 protein, which further enhances the IRES-dependent translation of WUS mRNA. Genetic evidence indicates that the WUS protein increases the tolerance of the shoot apical meristem to environmental hazards. Based on these results, we conclude that the stem cell niche in Arabidopsis copes with environmental hazards by enhancing the IRES-dependent translation of WUS mRNA under the control of the AtLa1 protein. © 2015 John Wiley & Sons Ltd.

Cellular La protein shields nonsegmented negative-strand RNA viral leader RNA from RIG-I and enhances virus growth by diverse mechanisms.

PubMed

Bitko, Vira; Musiyenko, Alla; Bayfield, Mark A; Maraia, Richard J; Barik, Sailen

2008-08-01

The La antigen (SS-B) associates with a wide variety of cellular and viral RNAs to affect gene expression in multiple systems. We show that La is the major cellular protein found to be associated with the abundant 44-nucleotide viral leader RNA (leRNA) early after infection with respiratory syncytial virus (RSV), a nonsegmented negative-strand RNA virus. Consistent with this, La redistributes from the nucleus to the cytoplasm in RSV-infected cells. Upon RNA interference knockdown of La, leRNA is redirected to associate with the RNA-binding protein RIG-I, a known activator of interferon (IFN) gene expression, and this is accompanied by the early induction of IFN mRNA. These results suggest that La shields leRNA from RIG-I, abrogating the early viral activation of type I IFN. We mapped the leRNA binding function to RNA recognition motif 1 of La and showed that while wild-type La greatly enhanced RSV growth, a La mutant defective in RSV leRNA binding also did not support RSV growth. Comparative studies of RSV and Sendai virus and the use of IFN-negative Vero cells indicated that La supports the growth of nonsegmented negative-strand RNA viruses by both IFN suppression and a potentially novel IFN-independent mechanism.

Endoplasmic reticulum stress causes autophagy and apoptosis leading to cellular redistribution of the autoantigens Ro/Sjögren's syndrome-related antigen A (SSA) and La/SSB in salivary gland epithelial cells.

PubMed

Katsiougiannis, S; Tenta, R; Skopouli, F N

2015-08-01

The aim of this study was to examine the levels of endoplasmic reticulum (ER) stress in minor salivary glands, to investigate the interplay between ER stress-induced autophagy and apoptosis in human salivary gland (HSG) cells and to test the effect of ER stress-induced apoptosis on the cellular redistribution of the two major Sjögren's syndrome (SS) autoantigens Ro/Sjögren's syndrome-related antigen A (SSA) and La/Sjögren's syndrome-related antigen B (SSB). Minor salivary gland biopsies from SS patients and sicca controls were examined by immunohistochemistry for the expression of 78 kDa glucose-regulated protein/binding immunoglobulin protein (GRP78/BiP) as an indicator of unfolded protein response (UPR). HSG cells were treated with thapsigargin (TG) and cell viability, autophagy and apoptosis were assessed. Immunoblot was applied to detect the conversion of LC3I to LC3II and the protein levels of GRP78/BiP and X-box binding protein-1 (XBP-1). Apoptosis was evaluated by a single-stranded DNA enzyme-linked immunosorbent assay (ELISA). Ro/SSA and La/SSB localization was visualized using immunofluorescence. GRP78/BiP was expressed by acinar and ductal epithelial cells in salivary glands of patients and sicca controls. TG treatment induced autophagy, as indicated by enhanced protein expression of LC3II. The protein levels of UPR marker XBP-1 were increased after TG treatment, while GRP78/BiP levels were decreased. TG treatment resulted in induction of HSG apoptosis. Ro/SSA and La/SSB autoantigens were localized predominantly to the cytoplasm in resting cells, while they were redistributed to cell membrane and blebs in the apoptotic cells. In conclusion, ER stress is activated in minor salivary gland epithelial cells from SS patients and controls. ER stress-induced apoptosis in HSG cells leads to cell surface and apoptotic blebs relocalization of Ro/SSA and La/SSB autoantigens. © 2015 British Society for Immunology.

Current overview of allergens of plant pathogenesis related protein families.

PubMed

Sinha, Mau; Singh, Rashmi Prabha; Kushwaha, Gajraj Singh; Iqbal, Naseer; Singh, Avinash; Kaushik, Sanket; Kaur, Punit; Sharma, Sujata; Singh, Tej P

2014-01-01

Pathogenesis related (PR) proteins are one of the major sources of plant derived allergens. These proteins are induced by the plants as a defense response system in stress conditions like microbial and insect infections, wounding, exposure to harsh chemicals, and atmospheric conditions. However, some plant tissues that are more exposed to environmental conditions like UV irradiation and insect or fungal attacks express these proteins constitutively. These proteins are mostly resistant to proteases and most of them show considerable stability at low pH. Many of these plant pathogenesis related proteins are found to act as food allergens, latex allergens, and pollen allergens. Proteins having similar amino acid sequences among the members of PR proteins may be responsible for cross-reactivity among allergens from diverse plants. This review analyzes the different pathogenesis related protein families that have been reported as allergens. Proteins of these families have been characterized in regard to their biological functions, amino acid sequence, and cross-reactivity. The three-dimensional structures of some of these allergens have also been evaluated to elucidate the antigenic determinants of these molecules and to explain the cross-reactivity among the various allergens.

Resource for structure related information on transmembrane proteins

NASA Astrophysics Data System (ADS)

Tusnády, Gábor E.; Simon, István

Transmembrane proteins are involved in a wide variety of vital biological processes including transport of water-soluble molecules, flow of information and energy production. Despite significant efforts to determine the structures of these proteins, only a few thousand solved structures are known so far. Here, we review the various resources for structure-related information on these types of proteins ranging from the 3D structure to the topology and from the up-to-date databases to the various Internet sites and servers dealing with structure prediction and structure analysis. Abbreviations: 3D, three dimensional; PDB, Protein Data Bank; TMP, transmembrane protein.

Inhibition of protein synthesis in intact HeLa cells by Shigella dysenteriae 1 toxin.

PubMed

Brown, J E; Rothman, S W; Doctor, B P

1980-07-01

Shiga toxin purified to near homogeneity from cell lysates of Shigella dysenteriae 1 inhibited protein and deoxyribonucle acid syntheses in intact HeLa cells. Inhibition was dependent on toxin concentration and time of incubation. A minimal latent period of 30 min was observed with saturating doses of toxin. Ribonucleic acid synthesis, uptake of alpha-aminoisobutyric acid, and maintenance of intracellular K+ concentrations were not affected until well after maximal inhibition of protein and deoxyribonucleic acid syntheses. The inhibitory effect of toxin was sensitive to heat inactivation and was prevented by antibody neutralization. Several cytotoxic components were separated by polyacrylamide gel electrophoresis of the purified toxin preparations; all inhibited protein and deoxyribonucleic acid syntheses equally.

Assembly Tests of the First Nb 3 Sn Low-Beta Quadrupole Short Model for the Hi-Lumi LHC

DOE PAGES

Pan, H.; Felice, H.; Cheng, D. W.; ...

2016-01-18

In preparation for the high-luminosity upgrade of the Large Hadron Collider (LHC), the LHC Accelerator Research Program (LARP) in collaboration with CERN is pursuing the development of MQXF: a 150-mm-aperture high-field Nb3Sn quadrupole magnet. Moreover, the development phase starts with the fabrication and test of several short models (1.2-m magnetic length) and will continue with the development of several long prototypes. All of them are mechanically supported using a shell-based support structure, which has been extensively demonstrated on several R&D models within LARP. The first short model MQXFS-AT has been assembled at LBNL with coils fabricated by LARP and CERN.more » In our paper, we summarize the assembly process and show how it relies strongly on experience acquired during the LARP 120-mm-aperture HQ magnet series. We also present comparison between strain gauges data and finite-element model analysis. Finally, we present the implication of the MQXFS-AT experience on the design of the long prototype support structure.« less

Hypergravity signal transduction in HeLa cells with concomitant phosphorylation of proteins immunoprecipitated with anti-microtubule-associated protein antibodies

NASA Technical Reports Server (NTRS)

Kumei, Yasuhiro; Whitson, Peggy A.; Sato, Atsushige; Cintron, Nitza M.

1991-01-01

It is shown that hypergravity (35g) stimulates the production of inositol 1,4,5-trisphosphate (IP3) and decreases adenosine 3-prime,5-prime-cyclic monophosphate (cAMP) levels in HeLa cells. It is proposed that IP3 and cAMP may act as second messengers in hypergravity signal transduction. Phosphorylation of microtubule-associated proteins in both the detergent-soluble and -insoluble fractions suggests that cytoskeletal structures may be influenced by gravity.

Structural studies of human glioma pathogenesis-related protein 1

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

Asojo, Oluwatoyin A., E-mail: oasojo@unmc.edu; Koski, Raymond A.; Bonafé, Nathalie

2011-10-01

Structural analysis of a truncated soluble domain of human glioma pathogenesis-related protein 1, a membrane protein implicated in the proliferation of aggressive brain cancer, is presented. Human glioma pathogenesis-related protein 1 (GLIPR1) is a membrane protein that is highly upregulated in brain cancers but is barely detectable in normal brain tissue. GLIPR1 is composed of a signal peptide that directs its secretion, a conserved cysteine-rich CAP (cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 proteins) domain and a transmembrane domain. GLIPR1 is currently being investigated as a candidate for prostate cancer gene therapy and for glioblastoma targeted therapy. Crystal structuresmore » of a truncated soluble domain of the human GLIPR1 protein (sGLIPR1) solved by molecular replacement using a truncated polyalanine search model of the CAP domain of stecrisp, a snake-venom cysteine-rich secretory protein (CRISP), are presented. The correct molecular-replacement solution could only be obtained by removing all loops from the search model. The native structure was refined to 1.85 Å resolution and that of a Zn{sup 2+} complex was refined to 2.2 Å resolution. The latter structure revealed that the putative binding cavity coordinates Zn{sup 2+} similarly to snake-venom CRISPs, which are involved in Zn{sup 2+}-dependent mechanisms of inflammatory modulation. Both sGLIPR1 structures have extensive flexible loop/turn regions and unique charge distributions that were not observed in any of the previously reported CAP protein structures. A model is also proposed for the structure of full-length membrane-bound GLIPR1.« less

CK2 is responsible for phosphorylation of human La protein serine-366 and can modulate rpL37 5'-terminal oligopyrimidine mRNA metabolism.

PubMed

Schwartz, Elena I; Intine, Robert V; Maraia, Richard J

2004-11-01

La protein binds precursors to 5S rRNA, tRNAs, and other transcripts that contain 3' UUU-OH and also promotes their maturation in the nucleus. Separate from this function, human La has been shown to positively modulate the translation of mRNAs that contain complex 5' regulatory motifs that direct internal initiation of translation. Nonphosphorylated La (npLa) inhibits pre-tRNA processing, while phosphorylation of human La serine-366 (S(366)) promotes pre-tRNA processing. npLa was found specifically associated with a class of mRNAs that have unusually short 5' untranslated regions comprised of terminal oligopyrimidine (5'TOP) tracts and that encode ribosomal proteins and translation elongation factors. Although La S(366) represents a CK2 phosphorylation site, there was no evidence that CK2 phosphorylates it in vivo. We used the CK2-specific inhibitor, 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), and antisense-mediated knockdown to demonstrate that CK2 is responsible for La S(366) phosphorylation in vivo. Hypophosphorylation was not associated with significant change in total La levels or proteolytic cleavage. Quantitative reverse transcription-PCR revealed increased association of the 5'TOP-mRNA encoding ribosomal protein L37 (rpL37) with La after TBB treatment. Transfection revealed more rpL37 mRNA associated with nonphosphorylatable La A(366) than with La S(366), concomitant with La A(366)-specific shift of a fraction of L37 mRNA off polysomes. The data indicate that CK2 phosphorylates La S(366) in vivo, that this limits 5'TOP mRNA binding, and that increasing npLa leads to greater association with potentially negative effects on TOP mRNA translation. Consistent with data that indicate that phosphorylation reverses negative effects of npLa on tRNA production, the present data suggest that CK2 phosphorylation of La can affect production of the translational machinery.

Prediction of the Ebola Virus Infection Related Human Genes Using Protein-Protein Interaction Network.

PubMed

Cao, HuanHuan; Zhang, YuHang; Zhao, Jia; Zhu, Liucun; Wang, Yi; Li, JiaRui; Feng, Yuan-Ming; Zhang, Ning

2017-01-01

Ebola hemorrhagic fever (EHF) is caused by Ebola virus (EBOV). It is reported that human could be infected by EBOV with a high fatality rate. However, association factors between EBOV and host still tend to be ambiguous. According to the "guilt by association" (GBA) principle, proteins interacting with each other are very likely to function similarly or the same. Based on this assumption, we tried to obtain EBOV infection-related human genes in a protein-protein interaction network using Dijkstra algorithm. We hope it could contribute to the discovery of novel effective treatments. Finally, 15 genes were selected as potential EBOV infection-related human genes. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

The selectivity of protein-imprinted gels and its relation to protein properties: A computer simulation study.

PubMed

Yankelov, Rami; Yungerman, Irena; Srebnik, Simcha

2017-07-01

Polymer-based protein recognition systems have enormous potential within clinical and diagnostic fields due to their reusability, biocompatibility, ease of manufacturing, and potential specificity. Imprinted polymer matrices have been extensively studied and applied as a simple technique for creating artificial polymer-based recognition gels for a target molecule. Although this technique has been proven effective when targeting small molecules (such as drugs), imprinting of proteins have so far resulted in materials with limited selectivity due to the large molecular size of the protein and aqueous environment. Using coarse-grained molecular simulation, we investigate the relation between protein makeup, polymer properties, and the selectivity of imprinted gels. Nonspecific binding that results in poor selectivity is shown to be strongly dependent on surface chemistry of the template and competitor proteins as well as on polymer chemistry. Residence time distributions of proteins diffusing within the gels provide a transparent picture of the relation between polymer constitution, protein properties, and the nonspecific interactions with the imprinted gel. The pronounced effect of protein surface chemistry on imprinted gel specificity is demonstrated. Copyright © 2017 John Wiley & Sons, Ltd.

The octamer-binding proteins form multi-protein--DNA complexes with the HSV alpha TIF regulatory protein.

PubMed Central

Kristie, T M; LeBowitz, J H; Sharp, P A

1989-01-01

The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation of at least two DNA--protein complexes on this regulatory element. Both of these complexes contain the ubiquitous Oct-1 protein, whose POU domain alone is sufficient to allow assembly of the alpha TIF-dependent complexes. A second member of the POU domain family, the lymphoid specific Oct-2 protein, can also be assembled into similar complexes at high concentrations of alpha TIF protein. These complexes contain at least two cellular proteins in addition to Oct-1. One of these proteins is present in both insect and HeLa cells and probably recognizes sequences in the cis element. The second cellular protein, only present in HeLa cells, probably binds by protein-protein interactions. Images PMID:2556266

The octamer-binding proteins form multi-protein--DNA complexes with the HSV alpha TIF regulatory protein.

PubMed

Kristie, T M; LeBowitz, J H; Sharp, P A

1989-12-20

The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation of at least two DNA--protein complexes on this regulatory element. Both of these complexes contain the ubiquitous Oct-1 protein, whose POU domain alone is sufficient to allow assembly of the alpha TIF-dependent complexes. A second member of the POU domain family, the lymphoid specific Oct-2 protein, can also be assembled into similar complexes at high concentrations of alpha TIF protein. These complexes contain at least two cellular proteins in addition to Oct-1. One of these proteins is present in both insect and HeLa cells and probably recognizes sequences in the cis element. The second cellular protein, only present in HeLa cells, probably binds by protein-protein interactions.

Nasal protein profiles in work-related asthma caused by different exposures.

PubMed

Suojalehto, H; Lindström, I; Wolff, H; Puustinen, A

2018-03-01

The mechanisms of work-related asthma (WRA) are incompletely delineated. Nasal cell samples may be informative about processes in the lower airways. Our aim was to determine the nasal protein expression profiles of WRA caused by different kind of exposures. We collected nasal brush samples from 82 nonsmoking participants, including healthy controls and WRA patients exposed to (i) protein allergens, (ii) isocyanates and (iii) welding fumes the day after relevant exposure. The proteome changes in samples were analysed by two-dimensional difference gel electrophoresis, and the differentially regulated proteins found were identified by mass spectrometry. Immunological comparison was carried out using Western blot. We detected an average of 2500 spots per protein gel. Altogether, 228 protein spots were chosen for identification, yielding 77 different proteins. Compared to the controls, exposure to protein allergens had the largest effects on the proteome. Hierarchical clustering revealed that protein allergen- and isocyanate-related asthma had similar profiles, whereas asthma related to welding fumes differed. The highly overrepresented functional categories in the asthma groups were defence response, protease inhibitor activity, inflammatory and calcium signalling, complement activation and cellular response to oxidative stress. Immunological analysis confirmed the found abundance differences in galectin 10 and protein S100-A9 between the groups. Work-related asthma patients exposed to protein allergens and isocyanates elicit similar nasal proteome responses and the profiles of welders and healthy controls were alike. Revealed biological activities of the protein expression changes are associated with allergic inflammation and asthma. © 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

Proteomic Analysis of Sauvignon Blanc Grape Skin, Pulp and Seed and Relative Quantification of Pathogenesis-Related Proteins.

PubMed

Tian, Bin; Harrison, Roland; Morton, James; Deb-Choudhury, Santanu

2015-01-01

Thaumatin-like proteins (TLPs) and chitinases are the main constituents of so-called protein hazes which can form in finished white wine and which is a great concern of winemakers. These soluble pathogenesis-related (PR) proteins are extracted from grape berries. However, their distribution in different grape tissues is not well documented. In this study, proteins were first separately extracted from the skin, pulp and seed of Sauvignon Blanc grapes, followed by trypsin digestion and analysis by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Proteins identified included 75 proteins from Sauvignon Blanc grape skin, 63 from grape pulp and 35 from grape seed, mostly functionally classified as associated with metabolism and energy. Some were present exclusively in specific grape tissues; for example, proteins involved in photosynthesis were only detected in grape skin and proteins found in alcoholic fermentation were only detected in grape pulp. Moreover, proteins identified in grape seed were less diverse than those identified in grape skin and pulp. TLPs and chitinases were identified in both Sauvignon Blanc grape skin and pulp, but not in the seed. To relatively quantify the PR proteins, the protein extracts of grape tissues were seperated by HPLC first and then analysed by SDS-PAGE. The results showed that the protein fractions eluted at 9.3 min and 19.2 min under the chromatographic conditions of this study confirmed that these corresponded to TLPs and chitinases seperately. Thus, the relative quantification of TLPs and chitinases in protein extracts was carried out by comparing the area of corresponding peaks against the area of a thamautin standard. The results presented in this study clearly demonstrated the distribution of haze-forming PR proteins in grape berries, and the relative quantification of TLPs and chitinases could be applied in fast tracking of changes in PR proteins during grape growth and determination of PR

Proteomic Analysis of Sauvignon Blanc Grape Skin, Pulp and Seed and Relative Quantification of Pathogenesis-Related Proteins

PubMed Central

Tian, Bin; Harrison, Roland; Morton, James; Deb-Choudhury, Santanu

2015-01-01

Thaumatin-like proteins (TLPs) and chitinases are the main constituents of so-called protein hazes which can form in finished white wine and which is a great concern of winemakers. These soluble pathogenesis-related (PR) proteins are extracted from grape berries. However, their distribution in different grape tissues is not well documented. In this study, proteins were first separately extracted from the skin, pulp and seed of Sauvignon Blanc grapes, followed by trypsin digestion and analysis by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Proteins identified included 75 proteins from Sauvignon Blanc grape skin, 63 from grape pulp and 35 from grape seed, mostly functionally classified as associated with metabolism and energy. Some were present exclusively in specific grape tissues; for example, proteins involved in photosynthesis were only detected in grape skin and proteins found in alcoholic fermentation were only detected in grape pulp. Moreover, proteins identified in grape seed were less diverse than those identified in grape skin and pulp. TLPs and chitinases were identified in both Sauvignon Blanc grape skin and pulp, but not in the seed. To relatively quantify the PR proteins, the protein extracts of grape tissues were seperated by HPLC first and then analysed by SDS-PAGE. The results showed that the protein fractions eluted at 9.3 min and 19.2 min under the chromatographic conditions of this study confirmed that these corresponded to TLPs and chitinases seperately. Thus, the relative quantification of TLPs and chitinases in protein extracts was carried out by comparing the area of corresponding peaks against the area of a thamautin standard. The results presented in this study clearly demonstrated the distribution of haze-forming PR proteins in grape berries, and the relative quantification of TLPs and chitinases could be applied in fast tracking of changes in PR proteins during grape growth and determination of PR

Quantitative Evaluation of Serum Proteins Uncovers a Protein Signature Related to Maturity-Onset Diabetes of the Young (MODY).

PubMed

Tuerxunyiming, Muhadasi; Xian, Feng; Zi, Jin; Yimamu, Yilihamujiang; Abuduwayite, Reshalaiti; Ren, Yan; Li, Qidan; Abudula, Abulizi; Liu, SiQi; Mohemaiti, Patamu

2018-01-05

Maturity-onset diabetes of the young (MODY) is an inherited monogenic type of diabetes. Genetic mutations in MODY often cause nonsynonymous changes that directly lead to the functional distortion of proteins and the pathological consequences. Herein, we proposed that the inherited mutations found in a MODY family could cause a disturbance of protein abundance, specifically in serum. The serum samples were collected from a Uyghur MODY family through three generations, and the serum proteins after depletion treatment were examined by quantitative proteomics to characterize the MODY-related serum proteins followed by verification using target quantification of proteomics. A total of 32 serum proteins were preliminarily identified as the MODY-related. Further verification test toward the individual samples demonstrated the 12 candidates with the significantly different abundance in the MODY patients. A comparison of the 12 proteins among the sera of type 1 diabetes, type 2 diabetes, MODY, and healthy subjects was conducted and revealed a protein signature related with MODY composed of the serum proteins such as SERPINA7, APOC4, LPA, C6, and F5.

CK2 Is Responsible for Phosphorylation of Human La Protein Serine-366 and Can Modulate rpL37 5′-Terminal Oligopyrimidine mRNA Metabolism

PubMed Central

Schwartz, Elena I.; Intine, Robert V.; Maraia, Richard J.

2004-01-01

La protein binds precursors to 5S rRNA, tRNAs, and other transcripts that contain 3′ UUU-OH and also promotes their maturation in the nucleus. Separate from this function, human La has been shown to positively modulate the translation of mRNAs that contain complex 5′ regulatory motifs that direct internal initiation of translation. Nonphosphorylated La (npLa) inhibits pre-tRNA processing, while phosphorylation of human La serine-366 (S366) promotes pre-tRNA processing. npLa was found specifically associated with a class of mRNAs that have unusually short 5′ untranslated regions comprised of terminal oligopyrimidine (5′TOP) tracts and that encode ribosomal proteins and translation elongation factors. Although La S366 represents a CK2 phosphorylation site, there was no evidence that CK2 phosphorylates it in vivo. We used the CK2-specific inhibitor, 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), and antisense-mediated knockdown to demonstrate that CK2 is responsible for La S366 phosphorylation in vivo. Hypophosphorylation was not associated with significant change in total La levels or proteolytic cleavage. Quantitative reverse transcription-PCR revealed increased association of the 5′TOP-mRNA encoding ribosomal protein L37 (rpL37) with La after TBB treatment. Transfection revealed more rpL37 mRNA associated with nonphosphorylatable La A366 than with La S366, concomitant with La A366-specific shift of a fraction of L37 mRNA off polysomes. The data indicate that CK2 phosphorylates La S366 in vivo, that this limits 5′TOP mRNA binding, and that increasing npLa leads to greater association with potentially negative effects on TOP mRNA translation. Consistent with data that indicate that phosphorylation reverses negative effects of npLa on tRNA production, the present data suggest that CK2 phosphorylation of La can affect production of the translational machinery. PMID:15485924

Carboxyl methylation of Ras-related proteins during signal transduction in neutrophils.

PubMed

Philips, M R; Pillinger, M H; Staud, R; Volker, C; Rosenfeld, M G; Weissmann, G; Stock, J B

1993-02-12

In human neutrophils, as in other cell types, Ras-related guanosine triphosphate-binding proteins are directed toward their regulatory targets in membranes by a series of posttranslational modifications that include methyl esterification of a carboxyl-terminal prenylcysteine residue. In intact cells and in a reconstituted in vitro system, the amount of carboxyl methylation of Ras-related proteins increased in response to the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP). Activation of Ras-related proteins by guanosine-5'-O-(3-thiotriphosphate) had a similar effect and induced translocation of p22rac2 from cytosol to plasma membrane. Inhibitors of prenylcysteine carboxyl methylation effectively blocked neutrophil responses to FMLP. These findings suggest a direct link between receptor-mediated signal transduction and the carboxyl methylation of Ras-related proteins.

Up-regulation of glutathione-related genes, enzyme activities and transport proteins in human cervical cancer cells treated with doxorubicin.

PubMed

Drozd, Ewa; Krzysztoń-Russjan, Jolanta; Marczewska, Jadwiga; Drozd, Janina; Bubko, Irena; Bielak, Magda; Lubelska, Katarzyna; Wiktorska, Katarzyna; Chilmonczyk, Zdzisław; Anuszewska, Elżbieta; Gruber-Bzura, Beata

2016-10-01

Doxorubicin (DOX), one of the most effective anticancer drugs, acts in a variety of ways including DNA damage, enzyme inhibition and generation of reactive oxygen species. Glutathione (GSH) and glutathione-related enzymes including: glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferases (GST) may play a role in adaptive detoxification processes in response to the oxidative stress, thus contributing to drug resistance phenotype. In this study, we investigated effects of DOX treatment on expression and activity of GSH-related enzymes and multidrug resistance-associated proteins in cultured human cervical cancer cells displaying different resistance against this drug (HeLa and KB-V1). Determination of expression level of genes encoding GST isoforms and MRP proteins (GCS, GPX, GSR, GSTA1-3, GSTM1, GSTP1, ABCC1-3, MGST1-3) was performed using StellARray™ Technology. Enzymatic activities of GPX and GSR were measured using biochemical methods. Expression of MRP1 was examined by immunofluorescence microscopy. This study showed that native expression levels of GSTM1 and GSTA3 were markedly higher in KB-V1 cells (2000-fold and 200-fold) compared to HeLa cells. Resistant cells have also shown significantly elevated expression of GSTA1 and GSTA2 genes (200-fold and 50-fold) as a result of DOX treatment. In HeLa cells, exposure to DOX increased expression of all genes: GSTM1 (7-fold) and GSTA1-3 (550-fold, 150-fold and 300-fold). Exposure to DOX led to the slight increase of GCS expression as well as GPX activity in KB-V1 cells, while in HeLa cells it did not. Expression of ABCC1 (MRP1) was not increased in any of the tested cell lines. Our results indicate that expression of GSTM1 and GSTA1-3 genes is up-regulated by DOX treatment and suggest that activity of these genes may be associated with drug resistance of the tested cells. At the same time, involvement of MRP1 in DOX resistance in the given experimental conditions is unlikely

Requirement of T-lymphokine-activated killer cell-originated protein kinase for TRAIL resistance of human HeLa cervical cancer cells

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

Kwon, Hyeok-Ran; Lee, Ki Won; Dong, Zigang

2010-01-01

T-lymphokine-activated killer cell-originated protein kinase (TOPK) appears to be highly expressed in various cancer cells and to play an important role in maintaining proliferation of cancer cells. However, the underlying mechanism by which TOPK regulates growth of cancer cells remains elusive. Here we report that upregulated endogenous TOPK augments resistance of cancer cells to apoptosis induced by tumor necrosis factor-related apoptosis inducing ligand (TRAIL). Stable knocking down of TOPK markedly increased TRAIL-mediated apoptosis of human HeLa cervical cancer cells, as compared with control cells. Caspase 8 or caspase 3 activities in response to TRAIL were greatly incremented in TOPK-depleted cells.more » Ablation of TOPK negatively regulated TRAIL-mediated NF-{kappa}B activity. Furthermore, expression of NF-{kappa}B-dependent genes, FLICE-inhibitory protein (FLIP), inhibitor of apoptosis protein 1 (c-IAP1), or X-linked inhibitor of apoptosis protein (XIAP) was reduced in TOPK-depleted cells. Collectively, these findings demonstrated that TOPK contributed to TRAIL resistance of cancer cells via NF-{kappa}B activity, suggesting that TOPK might be a potential molecular target for successful cancer therapy using TRAIL.« less

Mineral-related proteins of sea urchin teeth: Lytechinus variegatus.

PubMed

Veis, Arthur; Barss, Joseph; Dahl, Thomas; Rahima, Mohammed; Stock, Stuart

2002-12-01

Sea urchins have a set of five continuously growing teeth, each of which has a very complex structure. The mineral phase is calcite of varying Mg content, depending on the location within a tooth. The calcium carbonate is present in amorphous, plate-like and rod-like forms. It has been hypothesized that the mineral deposition is a matrix-mediated process, similar to that in vertebrate bone and tooth, wherein certain macromolecules within the organic matrix of the mineralized tissue play an important role in nucleating and controlling the growth habit of the mineral crystals. It has also been hypothesized that the mineral-related macromolecules involved in urchin teeth might bear a direct evolutionary relationship to those of the vertebrate tooth. These hypotheses are explored here by examining the pattern and nature of the mineral distribution, using microCT of intact teeth, and the nature of the mineral-related matrix proteins. The mineral-related proteins were extracted and fractionated by anion exchange chromatography. The relationship of certain fractions to vertebrate matrix proteins was established by immunoblots using antibodies to vertebrate tooth proteins. The antibodies were then used to localize the proteins within the teeth, by immunocytochemistry and histology with specific staining. The microCT data on mineral density has been correlated with the patterns of cellular migration and mineral deposition within the tooth as it grows. It appears that the mineralization within the different tooth compartments might take place under the influence of different matrix proteins. Further studies are in progress to more completely describe the vertebrate-invertebrate immunologically cross-reactive proteins of the urchin teeth. Copyright 2002 Wiley-Liss, Inc.

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

PubMed

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

2009-10-02

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

Exploration of Spinal Cord Aging-Related Proteins Using a Proteomics Approach.

PubMed

Kamiya, Koshiro; Furuya, Takeo; Hashimoto, Masayuki; Mannoji, Chikato; Inada, Taigo; Ota, Mitsutoshi; Maki, Satoshi; Ijima, Yasushi; Saito, Junya; Kitamura, Mitsuhiro; Ohtori, Seiji; Orita, Sumihisa; Inage, Kazuhide; Yamazaki, Masashi; Koda, Masao

2017-01-01

How aging affects the spinal cord at a molecular level is unclear. The aim of this study was to explore spinal cord aging-related proteins that may be involved in pathological mechanisms of age-related changes in the spinal cord. Spinal cords of 2-year-old and 8-week-old female Sprague-Dawley rats were dissected from the animals. Protein samples were subjected to 2-dimentional polyacrylamide gel electrophoresis followed by mass spectrometry. Screened proteins were further investigated with immunohistochemistry and Western blotting. Among the screened proteins, we selected α-crystallin B-subunit (αB-crystallin) and peripherin for further investigation because these proteins were previously reported to be related to central nervous system pathologies. Immunohistochemistry and Western blotting revealed significant upregulation of αB-crystallin and peripherin expression in aged rat spinal cord. Further exploration is needed to elucidate the precise mechanism and potential role of these upregulated proteins in spinal cord aging processes.

Exploration of Spinal Cord Aging–Related Proteins Using a Proteomics Approach

PubMed Central

Kamiya, Koshiro; Furuya, Takeo; Hashimoto, Masayuki; Mannoji, Chikato; Inada, Taigo; Ota, Mitsutoshi; Maki, Satoshi; Ijima, Yasushi; Saito, Junya; Kitamura, Mitsuhiro; Ohtori, Seiji; Orita, Sumihisa; Inage, Kazuhide; Yamazaki, Masashi; Koda, Masao

2017-01-01

How aging affects the spinal cord at a molecular level is unclear. The aim of this study was to explore spinal cord aging–related proteins that may be involved in pathological mechanisms of age-related changes in the spinal cord. Spinal cords of 2-year-old and 8-week-old female Sprague-Dawley rats were dissected from the animals. Protein samples were subjected to 2-dimentional polyacrylamide gel electrophoresis followed by mass spectrometry. Screened proteins were further investigated with immunohistochemistry and Western blotting. Among the screened proteins, we selected α-crystallin B-subunit (αB-crystallin) and peripherin for further investigation because these proteins were previously reported to be related to central nervous system pathologies. Immunohistochemistry and Western blotting revealed significant upregulation of αB-crystallin and peripherin expression in aged rat spinal cord. Further exploration is needed to elucidate the precise mechanism and potential role of these upregulated proteins in spinal cord aging processes. PMID:28634429

Nanoparticles-cell association predicted by protein corona fingerprints

NASA Astrophysics Data System (ADS)

Palchetti, S.; Digiacomo, L.; Pozzi, D.; Peruzzi, G.; Micarelli, E.; Mahmoudi, M.; Caracciolo, G.

2016-06-01

In a physiological environment (e.g., blood and interstitial fluids) nanoparticles (NPs) will bind proteins shaping a ``protein corona'' layer. The long-lived protein layer tightly bound to the NP surface is referred to as the hard corona (HC) and encodes information that controls NP bioactivity (e.g. cellular association, cellular signaling pathways, biodistribution, and toxicity). Decrypting this complex code has become a priority to predict the NP biological outcomes. Here, we use a library of 16 lipid NPs of varying size (Ø ~ 100-250 nm) and surface chemistry (unmodified and PEGylated) to investigate the relationships between NP physicochemical properties (nanoparticle size, aggregation state and surface charge), protein corona fingerprints (PCFs), and NP-cell association. We found out that none of the NPs' physicochemical properties alone was exclusively able to account for association with human cervical cancer cell line (HeLa). For the entire library of NPs, a total of 436 distinct serum proteins were detected. We developed a predictive-validation modeling that provides a means of assessing the relative significance of the identified corona proteins. Interestingly, a minor fraction of the HC, which consists of only 8 PCFs were identified as main promoters of NP association with HeLa cells. Remarkably, identified PCFs have several receptors with high level of expression on the plasma membrane of HeLa cells.In a physiological environment (e.g., blood and interstitial fluids) nanoparticles (NPs) will bind proteins shaping a ``protein corona'' layer. The long-lived protein layer tightly bound to the NP surface is referred to as the hard corona (HC) and encodes information that controls NP bioactivity (e.g. cellular association, cellular signaling pathways, biodistribution, and toxicity). Decrypting this complex code has become a priority to predict the NP biological outcomes. Here, we use a library of 16 lipid NPs of varying size (Ø ~ 100-250 nm) and surface

Lamprey Prohibitin2 Arrest G2/M Phase Transition of HeLa Cells through Down-regulating Expression and Phosphorylation Level of Cell Cycle Proteins.

PubMed

Shi, Ying; Guo, Sicheng; Wang, Ying; Liu, Xin; Li, Qingwei; Li, Tiesong

2018-03-02

Prohibitin 2(PHB2) is a member of the SFPH trans-membrane family proteins. It is a highly conserved and functionally diverse protein that plays an important role in preserving the structure and function of the mitochondria. In this study, the lamprey PHB2 gene was expressed in HeLa cells to investigate its effect on cell proliferation. The effect of Lm-PHB2 on the proliferation of HeLa cells was determined by treating the cells with pure Lm-PHB2 protein followed by MTT assay. Using the synchronization method with APC-BrdU and PI double staining revealed rLm-PHB2 treatment induced the decrease of both S phase and G0/G1 phase and then increase of G2/M phase. Similarly, cells transfected with pEGFP-N1-Lm-PHB2 also exhibited remarkable reduction in proliferation. Western blot and quantitative real-time PCR(qRT-PCR) assays suggested that Lm-PHB2 caused cell cycle arrest in HeLa cells through inhibition of CDC25C and CCNB1 expression. According to our western blot analysis, Lm-PHB2 was also found to reduce the expression level of Wee1 and PLK1 and the phosphorylation level of CCNB1, CDC25C and CDK1 in HeLa cells. Lamprey prohibitin 2 could arrest G2/M phase transition of HeLa cells through down-regulating expression and phosphorylation level of cell cycle proteins.

Dynamin-Related Protein 1 Translocates from the Cytosol to Mitochondria during UV-Induced Apoptosis

NASA Astrophysics Data System (ADS)

Zhang, Zhenzhen; Wu, Shengnan; Feng, Jie

2011-01-01

Mitochondria are dynamic structures that frequently divide and fuse with one another to form interconnecting network. This network disintegrates into punctiform organelles during apoptosis. However, the mechanisms involved in these processes are still not well characterized. In this study, we investigate the role of dynamin-related protein 1 (Drp1), a large GTPase that mediates outer mitochondrial membrane fission, in mitochondrial dynamics in response to UV irradiation in human lung adenocarcinoma cells (ASTC-α-1) and HeLa cells. Using time-lapse fluorescent imaging, we find that Drp1 primarily distributes in cytosol under physiological conditions. After UV treatment, Drp1 translocates from cytosol to mitochondria, indicating the enhancement of Drp1 mitochondrial accumulation. Our results suggest that Drp1 is involved in the regulation of transition from an interconnecting network to a punctiform mitochondrial phenotype during UV-induced apoptosis.

The pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is a fatty acid-binding protein

PubMed Central

Darwiche, Rabih; Mène-Saffrané, Laurent; Gfeller, David; Asojo, Oluwatoyin A.; Schneiter, Roger

2017-01-01

Members of the CAP superfamily (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins), also known as SCP superfamily (sperm-coating proteins), have been implicated in many physiological processes, including immune defenses, venom toxicity, and sperm maturation. Their mode of action, however, remains poorly understood. Three proteins of the CAP superfamily, Pry1, -2, and -3 (pathogen related in yeast), are encoded in the Saccharomyces cerevisiae genome. We have shown previously that Pry1 binds cholesterol in vitro and that Pry function is required for sterol secretion in yeast cells, indicating that members of this superfamily may generally bind sterols or related small hydrophobic compounds. On the other hand, tablysin-15, a CAP protein from the horsefly Tabanus yao, has been shown to bind leukotrienes and free fatty acids in vitro. Therefore, here we assessed whether the yeast Pry1 protein binds fatty acids. Computational modeling and site-directed mutagenesis indicated that the mode of fatty acid binding is conserved between tablysin-15 and Pry1. Pry1 bound fatty acids with micromolar affinity in vitro, and its function was essential for fatty acid export in cells lacking the acyl-CoA synthetases Faa1 and Faa4. Fatty acid binding of Pry1 is independent of its capacity to bind sterols, and the two sterol- and fatty acid-binding sites are nonoverlapping. These results indicate that some CAP family members, such as Pry1, can bind different lipids, particularly sterols and fatty acids, at distinct binding sites, suggesting that the CAP domain may serve as a stable, secreted protein domain that can accommodate multiple ligand-binding sites. PMID:28365570

Specific binding of a HeLa cell nuclear protein to RNA sequences in the human immunodeficiency virus transactivating region.

PubMed Central

Gaynor, R; Soultanakis, E; Kuwabara, M; Garcia, J; Sigman, D S

1989-01-01

The transactivator protein, tat, encoded by the human immunodeficiency virus is a key regulator of viral transcription. Activation by the tat protein requires sequences downstream of the transcription initiation site called the transactivating region (TAR). RNA derived from the TAR is capable of forming a stable stem-loop structure and the maintenance of both the stem structure and the loop sequences located between +19 and +44 is required for complete in vivo activation by tat. Gel retardation assays with RNA from both wild-type and mutant TAR constructs generated in vitro with SP6 polymerase indicated specific binding of HeLa nuclear proteins to the TAR. To characterize this RNA-protein interaction, a method of chemical "imprinting" has been developed using photoactivated uranyl acetate as the nucleolytic agent. This reagent nicks RNA under physiological conditions at all four nucleotides in a reaction that is independent of sequence and secondary structure. Specific interaction of cellular proteins with TAR RNA could be detected by enhanced cleavages or imprints surrounding the loop region. Mutations that either disrupted stem base-pairing or extensively changed the primary sequence resulted in alterations in the cleavage pattern of the TAR RNA. Structural features of the TAR RNA stem-loop essential for tat activation are also required for specific binding of the HeLa cell nuclear protein. Images PMID:2544877

[Astragalus polysaccharide may increase sensitivity of cervical cancer HeLa cells to cisplatin by regulating cell autophagy].

PubMed

Zhai, Qiu-Li; Hu, Xiang-Dan; Xiao, Jing; Yu, Dong-Qing

2018-02-01

This study aimed to investigate the possible sensitivity of Astragalus polysaccharides, in order to improve the chemosensitivity of cervical cancer HeLa cells to cisplatin by regulating the cell autophagy, and explore its possible mechanism. In this study, HeLa cells were divided into control group, cisplatin group, Astragalus polysaccharide group, and Astragalus polysaccharide combined with cisplatin group. MTT assay was used to detect the proliferation of cervical cancer HeLa cells. Flow cytometry was used to detect the apoptosis and cycle of HeLa cells in each experimental group. RT-PCR was used to detect the mRNA expression of autophagy-related proteins beclin1, LC3Ⅱ and p62. The expression levels of autophagy-related proteins beclin1, LC3Ⅱ, LC3Ⅰ and p62 were detected by WB method. MTT results showed that compared with the control group, the proliferation of HeLa cells was significantly inhibited in each administration group( P <0.05), and the inhibitory effect of the combination group was more significant( P <0.01). The apoptotic rate of HeLa cells was significantly increased( P <0.05), and the apoptotic rate of the combination group was significantly increased( P <0.01) compared with the control group( P <0.05).In conclusion, G₀/G₁ phase showed the most significant differences between the two groups. RT-PCR and WB results showed that the gene and protein expressions of beclin1 and LC3Ⅱ were up-regulated, while the gene and protein expressions of p62 were down-regulated compared with the control group. The above-mentioned changes in the combination group were more significant. Through the analysis of the above experimental results, it is speculated that Astragalus polysaccharides may increase the sensitivity of cervical cancer HeLa cells to cisplatin by regulating the cell autophagy. Its possible mechanism of action is correlated with the up-regulation of autophagy-related proteins beclin1, the promote the conversion from LC3Ⅰ to LC3Ⅱ, the down

Glycolysis-related proteins are broad spectrum vaccine candidates against aquacultural pathogens.

PubMed

Liu, Xiaohong; Sun, Jiamin; Wu, Haizhen

2017-07-05

Reverse vaccinology (RV) has become a popular method for developing vaccines. Although Edwardsiella tarda is deemed to be an important fish pathogen, so far, no reports have used a genome-based approach to screen vaccine candidates against E. tarda. In the current study, protective antigens of E. tarda were screened using RV. Large-scale cloning, expression and purification of potential candidates were carried out, and their immunoprotective potential was evaluated. A candidate fructose-bisphosphate aldolase (FBA) exhibited broad spectrum protection, as did another glycolysis-related protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which we reported previously, indicating the potential of other glycolysis-related proteins of E. tarda as broad spectrum protective antigens. In total, half (5 out 10) of these proteins showed prominent immunoprotective potential. Therefore, we suggest that glycolysis-related proteins are a class of potential broad spectrum protective antigens and that these proteins should be preferentially selected. Copyright © 2017 Elsevier Ltd. All rights reserved.

Models for evaluation of relative immunogenic potential of protein particles in biopharmaceutical protein formulations.

PubMed

Johnson, Richard; Jiskoot, Wim

2012-10-01

An immune response to a therapeutic protein that compromises the biopharmaceutical activity or cross-reacts with an endogenous protein is a serious clinical event. The role of protein aggregates and particles in biopharmaceutical formulations in mediating this immune response has gained considerable attention over the recent past. Model systems that could consistently and reliably predict the relative immunogenicity of biopharmaceutical protein formulations would be extremely valuable. Several approaches have been developed in an attempt to provide this insight, including in silico algorithms, in vitro tests utilizing human leukocytes and in vivo animal models. This commentary provides an update of these various approaches as well as the author's perspectives on the pros and cons of these different methods. Copyright © 2012 Wiley Periodicals, Inc.

Lamprey immune protein-1 (LIP-1) from Lampetra japonica induces cell cycle arrest and cell death in HeLa cells.

PubMed

Chi, Xiaoyuan; Su, Peng; Bi, Dan; Tai, Zhao; Li, Yingying; Pang, Yue; Li, Qingwei

2018-04-01

The lamprey (Lampetra japonica), a representative of the jawless vertebrates, is the oldest extant species in the world. LIP-1, which has a jacalin-like domain and an aerolysin pore-forming domain, has previously been identified in Lampetra japonica. However, the structure and function of the LIP-1 protein have not been described. In this study, the LIP-1 gene was overexpressed in HeLa cells and H293T cells. The results showed that the overexpression of LIP-1 in HeLa cells significantly elevated LDH release (P < 0.05), phosphatidylserine exposure and ROS accumulation. The overexpression of LIP-1 also had remarkable effects on the organelles in HeLa cells, while it had no effect on H293T cell organelles. Array data indicated that overexpression of LIP-1 primarily upregulated P53 signaling pathways in HeLa cells. Cell cycle assay results confirmed that LIP-1 caused arrest in the G 2 /M phase of the cell cycle in HeLa cells. In summary, our findings provide insights into the function and characterization of LIP-1 genes in vertebrates and establish the foundation for further research into the biological function of LIP-1. Our observations suggest that this lamprey protein has the potential for use in new applications in the medical field. Copyright © 2018. Published by Elsevier Ltd.

Closely related dermatophyte species produce different patterns of secreted proteins.

PubMed

Giddey, Karin; Favre, Bertrand; Quadroni, Manfredo; Monod, Michel

2007-02-01

Dermatophytes are the most common infectious agents responsible for superficial mycosis in humans and animals. Various species in this group of fungi show overlapping characteristics. We investigated the possibility that closely related dermatophyte species with different behaviours secrete distinct proteins when grown in the same culture medium. Protein patterns from culture filtrates of several strains of the same species were very similar. In contrast, secreted protein profiles from various species were different, and so a specific signature could be associated with each of the six analysed species. In particular, protein patterns were useful to distinguish Trichophyton tonsurans from Trichophyton equinum, which cannot be differentiated by ribosomal DNA sequencing. The secreted proteases Sub2, Sub6 and Sub7 of the subtilisin family, as well as Mep3 and Mep4 of the fungalisin family were identified. SUB6, SUB7, MEP3 and MEP4 genes were cloned and sequenced. Although the protein sequence of each protease was highly conserved across species, their level of secretion by the various species was not equivalent. These results suggest that a switch of habitat could be related to a differential expression of genes encoding homologous secreted proteins.

Identification of the G13 (cAMP-response-element-binding protein-related protein) gene product related to activating transcription factor 6 as a transcriptional activator of the mammalian unfolded protein response.

PubMed

Haze, K; Okada, T; Yoshida, H; Yanagi, H; Yura, T; Negishi, M; Mori, K

2001-04-01

Eukaryotic cells control the levels of molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) by a transcriptional induction process termed the unfolded protein response (UPR). The mammalian UPR is mediated by the cis-acting ER stress response element consisting of 19 nt (CCAATN(9)CCACG), the CCACG part of which is considered to provide specificity. We recently identified the basic leucine zipper (bZIP) protein ATF6 as a mammalian UPR-specific transcription factor; ATF6 is activated by ER stress-induced proteolysis and binds directly to CCACG. Here we report that eukaryotic cells express another bZIP protein closely related to ATF6 in both structure and function. This protein encoded by the G13 (cAMP response element binding protein-related protein) gene is constitutively synthesized as a type II transmembrane glycoprotein anchored in the ER membrane and processed into a soluble form upon ER stress as occurs with ATF6. The proteolytic processing of ATF6 and the G13 gene product is accompanied by their relocation from the ER to the nucleus; their basic regions seem to function as a nuclear localization signal. Overexpression of the soluble form of the G13 product constitutively activates the UPR, whereas overexpression of a mutant lacking the activation domain exhibits a strong dominant-negative effect. Furthermore, the soluble forms of ATF6 and the G13 gene product are unable to bind to several point mutants of the cis-acting ER stress response element in vitro that hardly respond to ER stress in vivo. We thus concluded that the two related bZIP proteins are crucial transcriptional regulators of the mammalian UPR, and propose calling the ATF6 gene product ATF6alpha and the G13 gene product ATF6beta.

Huntingtin-associated protein-1 (HAP1) regulates endocytosis and interacts with multiple trafficking-related proteins.

PubMed

Mackenzie, Kimberly D; Lim, Yoon; Duffield, Michael D; Chataway, Timothy; Zhou, Xin-Fu; Keating, Damien J

2017-07-01

Huntingtin-associated protein 1 (HAP1) was initially identified as a binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking, cell signalling and receptor internalization. In this study, a proteomics approach was used for the identification of novel HAP1-interacting partners to attempt to shed light on the physiological function of HAP1. Using affinity chromatography with HAP1-GST protein fragments bound to Sepharose columns, this study identified a number of trafficking-related proteins that bind to HAP1. Interestingly, many of the proteins that were identified by mass spectrometry have trafficking-related functions and include the clathrin light chain B and Sec23A, an ER to Golgi trafficking vesicle coat component. Using co-immunoprecipitation and GST-binding assays the association between HAP1 and clathrin light chain B has been validated in vitro. This study also finds that HAP1 co-localizes with clathrin light chain B. In line with a physiological function of the HAP1-clathrin interaction this study detected a dramatic reduction in vesicle retrieval and endocytosis in adrenal chromaffin cells. Furthermore, through examination of transferrin endocytosis in HAP1 -/- cortical neurons, this study has determined that HAP1 regulates neuronal endocytosis. In this study, the interaction between HAP1 and Sec23A was also validated through endogenous co-immunoprecipitation in rat brain homogenate. Through the identification of novel HAP1 binding partners, many of which have putative trafficking roles, this study provides us with new insights into the mechanisms underlying the important physiological function of HAP1 as an intracellular trafficking protein through its protein-protein interactions. Copyright © 2017 Elsevier Inc. All rights reserved.

Cilia/Ift protein and motor -related bone diseases and mouse models.

PubMed

Yuan, Xue; Yang, Shuying

2015-01-01

Primary cilia are essential cellular organelles projecting from the cell surface to sense and transduce developmental signaling. They are tiny but have complicated structures containing microtubule (MT)-based internal structures (the axoneme) and mother centriole formed basal body. Intraflagellar transport (Ift) operated by Ift proteins and motors are indispensable for cilia formation and function. Mutations in Ift proteins or Ift motors cause various human diseases, some of which have severe bone defects. Over the last few decades, major advances have occurred in understanding the roles of these proteins and cilia in bone development and remodeling by examining cilia/Ift protein-related human diseases and establishing mouse transgenic models. In this review, we describe current advances in the understanding of the cilia/Ift structure and function. We further summarize cilia/Ift-related human diseases and current mouse models with an emphasis on bone-related phenotypes, cilia morphology, and signaling pathways.

PPI-IRO: a two-stage method for protein-protein interaction extraction based on interaction relation ontology.

PubMed

Li, Chuan-Xi; Chen, Peng; Wang, Ru-Jing; Wang, Xiu-Jie; Su, Ya-Ru; Li, Jinyan

2014-01-01

Mining Protein-Protein Interactions (PPIs) from the fast-growing biomedical literature resources has been proven as an effective approach for the identification of biological regulatory networks. This paper presents a novel method based on the idea of Interaction Relation Ontology (IRO), which specifies and organises words of various proteins interaction relationships. Our method is a two-stage PPI extraction method. At first, IRO is applied in a binary classifier to determine whether sentences contain a relation or not. Then, IRO is taken to guide PPI extraction by building sentence dependency parse tree. Comprehensive and quantitative evaluations and detailed analyses are used to demonstrate the significant performance of IRO on relation sentences classification and PPI extraction. Our PPI extraction method yielded a recall of around 80% and 90% and an F1 of around 54% and 66% on corpora of AIMed and BioInfer, respectively, which are superior to most existing extraction methods.

Protein Simulation Data in the Relational Model.

PubMed

Simms, Andrew M; Daggett, Valerie

2012-10-01

High performance computing is leading to unprecedented volumes of data. Relational databases offer a robust and scalable model for storing and analyzing scientific data. However, these features do not come without a cost-significant design effort is required to build a functional and efficient repository. Modeling protein simulation data in a relational database presents several challenges: the data captured from individual simulations are large, multi-dimensional, and must integrate with both simulation software and external data sites. Here we present the dimensional design and relational implementation of a comprehensive data warehouse for storing and analyzing molecular dynamics simulations using SQL Server.

Protein Simulation Data in the Relational Model

PubMed Central

Simms, Andrew M.; Daggett, Valerie

2011-01-01

High performance computing is leading to unprecedented volumes of data. Relational databases offer a robust and scalable model for storing and analyzing scientific data. However, these features do not come without a cost—significant design effort is required to build a functional and efficient repository. Modeling protein simulation data in a relational database presents several challenges: the data captured from individual simulations are large, multi-dimensional, and must integrate with both simulation software and external data sites. Here we present the dimensional design and relational implementation of a comprehensive data warehouse for storing and analyzing molecular dynamics simulations using SQL Server. PMID:23204646

The sterol-binding activity of PATHOGENESIS-RELATED PROTEIN 1 reveals the mode of action of an antimicrobial protein.

PubMed

Gamir, Jordi; Darwiche, Rabih; Van't Hof, Pieter; Choudhary, Vineet; Stumpe, Michael; Schneiter, Roger; Mauch, Felix

2017-02-01

Pathogenesis-related proteins played a pioneering role 50 years ago in the discovery of plant innate immunity as a set of proteins that accumulated upon pathogen challenge. The most abundant of these proteins, PATHOGENESIS-RELATED 1 (PR-1) encodes a small antimicrobial protein that has become, as a marker of plant immune signaling, one of the most referred to plant proteins. The biochemical activity and mode of action of PR-1 proteins has remained elusive, however. Here, we provide genetic and biochemical evidence for the capacity of PR-1 proteins to bind sterols, and demonstrate that the inhibitory effect on pathogen growth is caused by the sequestration of sterol from pathogens. In support of our findings, sterol-auxotroph pathogens such as the oomycete Phytophthora are particularly sensitive to PR-1, whereas sterol-prototroph fungal pathogens become highly sensitive only when sterol biosynthesis is compromised. Our results are in line with previous findings showing that plants with enhanced PR-1 expression are particularly well protected against oomycete pathogens. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Fast electron transfer through a single molecule natively structured redox protein

NASA Astrophysics Data System (ADS)

Della Pia, Eduardo Antonio; Chi, Qijin; MacDonald, J. Emyr; Ulstrup, Jens; Jones, D. Dafydd; Elliott, Martin

2012-10-01

The electron transfer properties of proteins are normally measured as molecularly averaged ensembles. Through these and related measurements, proteins are widely regarded as macroscopically insulating materials. Using scanning tunnelling microscopy (STM), we present new measurements of the conductance through single-molecules of the electron transfer protein cytochrome b562 in its native conformation, under pseudo-physiological conditions. This is achieved by thiol (SH) linker pairs at opposite ends of the molecule through protein engineering, resulting in defined covalent contact between a gold surface and a platinum-iridium STM tip. Two different orientations of the linkers were examined: a long-axis configuration (SH-LA) and a short-axis configuration (SH-SA). In each case, the molecular conductance could be `gated' through electrochemical control of the heme redox state. Reproducible and remarkably high conductance was observed in this relatively complex electron transfer system, with single-molecule conductance values peaking around 18 nS and 12 nS for the SH-SA and SH-LA cytochrome b562 molecules near zero electrochemical overpotential. This strongly points to the important role of the heme co-factor bound to the natively structured protein. We suggest that the two-step model of protein electron transfer in the STM geometry requires a multi-electron transfer to explain such a high conductance. The model also yields a low value for the reorganisation energy, implying that solvent reorganisation is largely absent.The electron transfer properties of proteins are normally measured as molecularly averaged ensembles. Through these and related measurements, proteins are widely regarded as macroscopically insulating materials. Using scanning tunnelling microscopy (STM), we present new measurements of the conductance through single-molecules of the electron transfer protein cytochrome b562 in its native conformation, under pseudo-physiological conditions. This is

Low Proportion of Dietary Plant Protein among Athletes with Premenstrual Syndrome-Related Performance Impairment.

PubMed

Yamada, Keiko; Takeda, Takashi

2018-02-01

Premenstrual syndrome (PMS) is psychosomatic disorder that are limited to the late luteal phase in the menstrual cycle. PMS could impair athletic performance. To investigate associations between proportions of dietary plant and animal protein and PMS-related impairment of athletic performance, we surveyed 135 female athletes aged 18-23 years attending Kindai University. Participants belonged to authorized university clubs, all of which have high rankings in Japanese university sports. Participants completed self-administered questionnaires on diet history, demographics, and PMS-related impairment of athletic performance. Total protein, animal protein, and plant protein intake were examined, and the proportion of dietary plant protein was calculated for each participant. We divided athletes into two groups: those without PMS-related impairment of athletic performance (n = 117) and those with PMS-related performance impairment (n = 18). A t-test was used to compare mean values and multivariable adjusted mean values between groups; adjustment variables were energy intake, body mass index, and daily training duration. Total protein intake was not significantly different between the groups. However, athletes whose performance was affected by PMS reported higher intake of animal protein (mean 50.6 g) than athletes whose performance was unaffected by PMS (mean 34.9 g). Plant protein intake was lower among athletes with PMS-related impairment (mean 25.4 g) than among athletes without impairment (mean 26.9 g). The proportion of dietary plant protein was lower among athletes with PMS-related impairment (39.3%) than those without impairment (45.9%). A low proportion of dietary plant protein may cause PMS-related athletic impairment among athletes.

JAM related proteins in mucosal homeostasis and inflammation

PubMed Central

Luissint, Anny-Claude; Nusrat, Asma; Parkos, Charles A.

2014-01-01

Mucosal surfaces are lined by epithelial cells that form a physical barrier protecting the body against external noxious substances and pathogens. At a molecular level, the mucosal barrier is regulated by tight junctions (TJs) that seal the paracellular space between adjacent epithelial cells. Transmembrane proteins within TJs include Junctional Adhesion Molecules (JAMs) that belong to the CTX (Cortical Thymocyte marker for Xenopus) family of proteins. JAM family encompasses three classical members (JAM-A, -B and –C) and related molecules including JAM4, JAM-Like protein (JAM-L), Coxsackie and Adenovirus Receptor (CAR), CAR-Like Membrane Protein (CLMP) and Endothelial cell-Selective Adhesion Molecule (ESAM). JAMs have multiple functions that include regulation of endothelial and epithelial paracellular permeability, leukocyte recruitment during inflammation, angiogenesis, cell migration and proliferation. In this review, we summarize the current knowledge regarding the roles of the JAM family members in the regulation of mucosal homeostasis and leukocyte trafficking with a particular emphasis on barrier function and its perturbation during pathological inflammation. PMID:24667924

Expression of Lipid Metabolism-Related Proteins in Metastatic Breast Cancer.

PubMed

Jung, Yoon Yang; Kim, Hye Min; Koo, Ja Seung

2015-01-01

The tumor biology of metastatic breast cancers differ according to the metastatic sites, and the features of cancer metabolism may also be different. The aim of this study is to investigate the expression of lipid metabolism-related proteins in metastatic breast cancer according to metastatic site and discuss the clinical significance thereof. Immunohistochemical staining for lipid metabolism-related proteins [fatty acid synthase (FASN), hormone-sensitive lipase (HSL), carnitine palmitoyltransferase IA (CPT-1A), acyl-CoA oxidase 1 (ACOX1), fatty acid binding protein 4 (FABP4,) and perilipin 1 (PLIN1)] was performed using a tissue microarray of 149 cases of metastatic breast cancer (bone metastasis = 39, brain metastasis = 37, liver metastasis = 21, and lung metastasis = 52). The expression levels of ACOX1 (p = 0.009) and FASN (p = 0.007) varied significantly according to metastatic site, with the highest expression in brain metastasis and the lowest expression in liver metastasis. ACOX1 positivity (p = 0.005) and FASN positivity (p = 0.003) correlated with HER-2 positivity. The expression of FASN was significantly higher in HER-2 type breast cancer, and lower in luminal A and TNBC type breast cancer (p<0.001). Among lipid metabolism-related proteins, PLIN1 positivity was found to be an independent poor prognostic factor on multivariate analysis (Hazard ratio: 4.979, 95% CI: 1.054-22.59, p = 0.043). Different expression levels of lipid metabolism-related proteins were observed according to metastatic site. The expression of ACOX1 and FASN was highest in brain metastasis. These results suggest that the metastatic site should be considered when using lipid metabolism inhibitors for targeted therapy.

Relating protein conformational changes to packing efficiency and disorder

PubMed Central

Bhardwaj, Nitin; Gerstein, Mark

2009-01-01

Changes in protein conformation play key roles in facilitating various biochemical processes, ranging from signaling and phosphorylation to transport and catalysis. While various factors that drive these motions such as environmental changes and binding of small molecules are well understood, specific causative effects on the structural features of the protein due to these conformational changes have not been studied on a large scale. Here, we study protein conformational changes in relation to two key structural metrics: packing efficiency and disorder. Packing has been shown to be crucial for protein stability and function by many protein design and engineering studies. We study changes in packing efficiency during conformational changes, thus extending the analysis from a static context to a dynamic perspective and report some interesting observations. First, we study various proteins that adopt alternate conformations and find that tendencies to show motion and change in packing efficiency are correlated: residues that change their packing efficiency show larger motions. Second, our results suggest that residues that show higher changes in packing during motion are located on the changing interfaces which are formed during these conformational changes. These changing interfaces are slightly different from shear or static interfaces that have been analyzed in previous studies. Third, analysis of packing efficiency changes in the context of secondary structure shows that, as expected, residues buried in helices show the least change in packing efficiency, whereas those embedded in bends are most likely to change packing. Finally, by relating protein disorder to motions, we show that marginally disordered residues which are ordered enough to be crystallized but have sequence patterns indicative of disorder show higher dislocation and a higher change in packing than ordered ones and are located mostly on the changing interfaces. Overall, our results demonstrate

SH2/SH3 adaptor proteins can link tyrosine kinases to a Ste20-related protein kinase, HPK1.

PubMed

Anafi, M; Kiefer, F; Gish, G D; Mbamalu, G; Iscove, N N; Pawson, T

1997-10-31

Ste20-related protein kinases have been implicated as regulating a range of cellular responses, including stress-activated protein kinase pathways and the control of cytoskeletal architecture. An important issue involves the identities of the upstream signals and regulators that might control the biological functions of mammalian Ste20-related protein kinases. HPK1 is a protein-serine/threonine kinase that possesses a Ste20-like kinase domain, and in transfected cells activates a protein kinase pathway leading to the stress-activated protein kinase SAPK/JNK. Here we have investigated candidate upstream regulators that might interact with HPK1. HPK1 possesses an N-terminal catalytic domain and an extended C-terminal tail with four proline-rich motifs. The SH3 domains of Grb2 bound in vitro to specific proline-rich motifs in the HPK1 tail and functioned synergistically to direct the stable binding of Grb2 to HPK1 in transfected Cos1 cells. Epidermal growth factor (EGF) stimulation did not affect the binding of Grb2 to HPK1 but induced recruitment of the Grb2.HPK1 complex to the autophosphorylated EGF receptor and to the Shc docking protein. Several activated receptor and cytoplasmic tyrosine kinases, including the EGF receptor, stimulated the tyrosine phosphorylation of the HPK1 serine/threonine kinase. These results suggest that HPK1, a mammalian Ste20-related protein-serine/threonine kinase, can potentially associate with protein-tyrosine kinases through interactions mediated by SH2/SH3 adaptors such as Grb2. Such interaction may provide a possible mechanism for cross-talk between distinct biochemical pathways following the activation of tyrosine kinases.

Performance of the first short model 150 mm aperture Nb$$_3$$Sn Quadrupole MQXFS for the High- Luminosity LHC upgrade

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

Chlachidze, G.; et al.

2016-08-30

The US LHC Accelerator Research Program (LARP) and CERN combined their efforts in developing Nb3Sn magnets for the High-Luminosity LHC upgrade. The ultimate goal of this collaboration is to fabricate large aperture Nb3Sn quadrupoles for the LHC interaction regions (IR). These magnets will replace the present 70 mm aperture NbTi quadrupole triplets for expected increase of the LHC peak luminosity by a factor of 5. Over the past decade LARP successfully fabricated and tested short and long models of 90 mm and 120 mm aperture Nb3Sn quadrupoles. Recently the first short model of 150 mm diameter quadrupole MQXFS was builtmore » with coils fabricated both by the LARP and CERN. The magnet performance was tested at Fermilab’s vertical magnet test facility. This paper reports the test results, including the quench training at 1.9 K, ramp rate and temperature dependence studies.« less

LC3/GABARAP family proteins: autophagy-(un)related functions.

PubMed

Schaaf, Marco B E; Keulers, Tom G; Vooijs, Marc A; Rouschop, Kasper M A

2016-12-01

From yeast to mammals, autophagy is an important mechanism for sustaining cellular homeostasis through facilitating the degradation and recycling of aged and cytotoxic components. During autophagy, cargo is captured in double-membraned vesicles, the autophagosomes, and degraded through lysosomal fusion. In yeast, autophagy initiation, cargo recognition, cargo engulfment, and vesicle closure is Atg8 dependent. In higher eukaryotes, Atg8 has evolved into the LC3/GABARAP protein family, consisting of 7 family proteins [LC3A (2 splice variants), LC3B, LC3C, GABARAP, GABARAPL1, and GABARAPL2]. LC3B, the most studied family protein, is associated with autophagosome development and maturation and is used to monitor autophagic activity. Given the high homology, the other LC3/GABARAP family proteins are often presumed to fulfill similar functions. Nevertheless, substantial evidence shows that the LC3/GABARAP family proteins are unique in function and important in autophagy-independent mechanisms. In this review, we discuss the current knowledge and functions of the LC3/GABARAP family proteins. We focus on processing of the individual family proteins and their role in autophagy initiation, cargo recognition, vesicle closure, and trafficking, a complex and tightly regulated process that requires selective presentation and recruitment of these family proteins. In addition, functions unrelated to autophagy of the LC3/GABARAP protein family members are discussed.-Schaaf, M. B. E., Keulers, T. G, Vooijs, M. A., Rouschop, K. M. A. LC3/GABARAP family proteins: autophagy-(un)related functions. © FASEB.

Yeast hnRNP-related proteins contribute to the maintenance of telomeres

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

Lee-Soety, Julia Y., E-mail: jlee04@sju.edu; Jones, Jennifer; MacGibeny, Margaret A.

Highlights: Black-Right-Pointing-Pointer Yeast hnRNP-related proteins are able to prevent faster senescence in telomerase-null cells. Black-Right-Pointing-Pointer The conserved RRMs in Npl3 are important for telomere maintenance. Black-Right-Pointing-Pointer Human hnRNP A1 is unable to complement the lack of NPL3 in yeast. Black-Right-Pointing-Pointer Npl3 and Cbc2 may work as telomere capping proteins. -- Abstract: Telomeres protect the ends of linear chromosomes, which if eroded to a critical length can become uncapped and lead to replicative senescence. Telomerase maintains telomere length in some cells, but inappropriate expression facilitates the immortality of cancer cells. Recently, proteins involved in RNA processing and ribosome assembly, such asmore » hnRNP (heterogeneous nuclear ribonucleoprotein) A1, have been found to participate in telomere maintenance in mammals. The Saccharomyces cerevisiae protein Npl3 shares significant amino acid sequence similarities with hnRNP A1. We found that deleting NPL3 accelerated the senescence of telomerase null cells. The highly conserved RNA recognition motifs (RRM) in Npl3 appear to be important for preventing faster senescence. Npl3 preferentially binds telomere sequences in vitro, suggesting that Npl3 may affect telomeres directly. Despite similarities between the two proteins, human hnRNP A1 is unable to complement the lack of Npl3 to rescue accelerated senescence in tlc1 npl3 cells. Deletion of CBC2, which encodes another hnRNP-related protein that associates with Npl3, also accelerates senescence. Potential mechanisms by which hnRNP-related proteins maintain telomeres are discussed.« less

La Piedad Michoacán Mexico Virus V protein antagonizes type I interferon response by binding STAT2 protein and preventing STATs nuclear translocation

PubMed Central

Pisanelli, Giuseppe; Laurent-Rolle, Maudry; Manicassamy, Balaji; Belicha-Villanueva, Alan; Morrison, Juliet; Lozano-Dubernard, Bernardo; Castro-Peralta, Felipa; Iovane, Giuseppe; García-Sastre, Adolfo

2017-01-01

La Piedad Michoacán Mexico Virus (LPMV) is a member of the Rubulavirus genus within the Paramyxoviridae family. LPMV is the etiologic agent of “blue eye disease”, causing a significant disease burden in swine in Mexico with long-term implications for the agricultural industry. This virus mainly affects piglets and is characterized by meningoencephalitis and respiratory distress. It also affects adult pigs, causing reduced fertility and abortions in females, and orchitis and epididymitis in males. Viruses of the Paramyxoviridae family evade the innate immune response by targeting components of the interferon (IFN) signaling pathway. The V protein, expressed by most paramyxoviruses, is a well-characterized IFN signaling antagonist. Until now, there were no reports on the role of the LPMV-V protein in inhibiting the IFN response. In this study we demonstrate that LPMV-V protein antagonizes type I but not type II IFN signaling by binding STAT2, a component of the type I IFN cascade. Our results indicate that the last 18 amino acids of LPMV-V protein are required for binding to STAT2 in human and swine cells. While LPMV-V protein does not affect the protein levels of STAT1 or STAT2, it does prevent the IFN-induced phosphorylation and nuclear translocation of STAT1 and STAT2 thereby inhibiting cellular responses to IFN α/β PMID:26546155

Delivery of the autofluorescent protein R-phycoerythrin by calcium phosphate nanoparticles into four different eukaryotic cell lines (HeLa, HEK293T, MG-63, MC3T3): Highly efficient, but leading to endolysosomal proteolysis in HeLa and MC3T3 cells.

PubMed

Kopp, Mathis; Rotan, Olga; Papadopoulos, Chrisovalantis; Schulze, Nina; Meyer, Hemmo; Epple, Matthias

2017-01-01

Nanoparticles can be used as carriers to transport biomolecules like proteins and synthetic molecules across the cell membrane because many molecules are not able to cross the cell membrane on their own. The uptake of nanoparticles together with their cargo typically occurs via endocytosis, raising concerns about the possible degradation of the cargo in the endolysosomal system. As the tracking of a dye-labelled protein during cellular uptake and processing is not indicative of the presence of the protein itself but only for the fluorescent label, a label-free tracking was performed with the red-fluorescing model protein R-phycoerythrin (R-PE). Four different eukaryotic cell lines were investigated: HeLa, HEK293T, MG-63, and MC3T3. Alone, the protein was not taken up by any cell line; only with the help of calcium phosphate nanoparticles, an efficient uptake occurred. After the uptake into HeLa cells, the protein was found in early endosomes (shown by the marker EEA1) and lysosomes (shown by the marker Lamp1). There, it was still intact and functional (i.e. properly folded) as its red fluorescence was detected. However, a few hours after the uptake, proteolysis started as indicated by the decreasing red fluorescence intensity in the case of HeLa and MC3T3 cells. 12 h after the uptake, the protein was almost completely degraded in HeLa cells and MC3T3 cells. In HEK293T cells and MG-63 cells, no degradation of the protein was observed. In the presence of Bafilomycin A1, an inhibitor of acidification and protein degradation in lysosomes, the fluorescence of R-PE remained intact over the whole observation period in the four cell lines. These results indicate that despite an efficient nanoparticle-mediated uptake of proteins by cells, a rapid endolysosomal degradation may prevent the desired (e.g. therapeutic) effect of a protein inside a cell.

Effect of solvation-related interaction on the low-temperature dynamics of proteins

NASA Astrophysics Data System (ADS)

Zuo, Guanghong; Wang, Jun; Qin, Meng; Xue, Bin; Wang, Wei

2010-03-01

The effect of solvation-related interaction on the low-temperature dynamics of proteins is studied by taking into account the desolvation barriers in the interactions of native contacts. It is found out that about the folding transition temperature, the protein folds in a cooperative manner, and the water molecules are expelled from the hydrophobic core at the final stage in the folding process. At low temperature, however, the protein would generally be trapped in many metastable conformations with some water molecules frozen inside the protein. The desolvation takes an important role in these processes. The number of frozen water molecules and that of frozen states of proteins are further analyzed with the methods based on principal component analysis (PCA) and the clustering of conformations. It is found out that both the numbers of frozen water molecules and the frozen states of the protein increase quickly below a certain temperature. Especially, the number of frozen states of the protein increases exponentially following the decrease in the temperature, which resembles the basic features of glassy dynamics. Interestingly, it is observed that the freezing of water molecules and that of protein conformations happen at almost the same temperature. This suggests that the solvation-related interaction performs an important role for the low-temperature dynamics of the model protein.

The Diversity of Yellow-Related Proteins in Sand Flies (Diptera: Psychodidae)

PubMed Central

Sima, Michal; Novotny, Marian; Pravda, Lukas; Sumova, Petra; Rohousova, Iva; Volf, Petr

2016-01-01

Yellow-related proteins (YRPs) present in sand fly saliva act as affinity binders of bioamines, and help the fly to complete a bloodmeal by scavenging the physiological signals of damaged cells. They are also the main antigens in sand fly saliva and their recombinant form is used as a marker of host exposure to sand flies. Moreover, several salivary proteins and plasmids coding these proteins induce strong immune response in hosts bitten by sand flies and are being used to design protecting vaccines against Leishmania parasites. In this study, thirty two 3D models of different yellow-related proteins from thirteen sand fly species of two genera were constructed based on the known protein structure from Lutzomyia longipalpis. We also studied evolutionary relationships among species based on protein sequences as well as sequence and structural variability of their ligand-binding site. All of these 33 sand fly YRPs shared a similar structure, including a unique tunnel that connects the ligand-binding site with the solvent by two independent paths. However, intraspecific modifications found among these proteins affects the charges of the entrances to the tunnel, the length of the tunnel and its hydrophobicity. We suggest that these structural and sequential differences influence the ligand-binding abilities of these proteins and provide sand flies with a greater number of YRP paralogs with more nuanced answers to bioamines. All these characteristics allow us to better evaluate these proteins with respect to their potential use as part of anti-Leishmania vaccines or as an antigen to measure host exposure to sand flies. PMID:27812196

Defense-related proteins involved in sugarcane responses to biotic stress

PubMed Central

Souza, Thais P.; Dias, Renata O.; Silva-Filho, Marcio C.

2017-01-01

Abstract Sugarcane is one of the most important agricultural crops in the world. However, pathogen infection and herbivore attack cause constant losses in yield. Plants respond to pathogen infection by inducing the expression of several protein types, such as glucanases, chitinases, thaumatins, peptidase inhibitors, defensins, catalases and glycoproteins. Proteins induced by pathogenesis are directly or indirectly involved in plant defense, leading to pathogen death or inducing other plant defense responses. Several of these proteins are induced in sugarcane by different pathogens or insects and have antifungal or insecticidal activity. In this review, defense-related proteins in sugarcane are described, with their putative mechanisms of action, pathogen targets and biotechnological perspectives. PMID:28222203

Suppression of NYVAC Infection in HeLa Cells Requires RNase L but Is Independent of Protein Kinase R Activity

PubMed Central

Fernández-Escobar, Mercedes; Nájera, José Luis; Baldanta, Sara; Rodriguez, Dolores; Way, Michael; Esteban, Mariano

2015-01-01

Protein kinase R (PKR) and RNase L are host cell components that function to contain viral spread after infections. In this study, we analyzed the role of both proteins in the abortive infection of human HeLa cells with the poxvirus strain NYVAC, for which an inhibition of viral A27L and B5R gene expression is described. Specifically, the translation of these viral genes is independent of PKR activation, but their expression is dependent on the RNase L activity. PMID:26656695

FYVE-dependent endosomal targeting of an arrestin-related protein in amoeba.

PubMed

Guetta, Dorian; Langou, Karine; Grunwald, Didier; Klein, Gérard; Aubry, Laurence

2010-12-13

Visual and β-arrestins are scaffolding proteins involved in the regulation of receptor-dependent intracellular signaling and their trafficking. The arrestin superfamilly includes several arrestin domain-containing proteins and the structurally related protein Vps26. In Dictyostelium discoideum, the arrestin-domain containing proteins form a family of six members, namely AdcA to -F. In contrast to canonical arrestins, Dictyostelium Adc proteins show a more complex architecture, as they possess, in addition to the arrestin core, other domains, such as C2, FYVE, LIM, MIT and SAM, which potentially mediate selective interactions with either lipids or proteins. A detailed analysis of AdcA has been performed. AdcA extends on both sides of the arrestin core, in particular by a FYVE domain which mediates selective interactions with PI(3)P, as disclosed by intrinsic fluorescence measurements and lipid overlay assays. Localization studies showed an enrichment of tagged- and endogenous AdcA on the rim of early macropinosomes and phagosomes. This vesicular distribution relies on a functional FYVE domain. Our data also show that the arrestin core binds the ADP-ribosylation factor ArfA, the unique amoebal Arf member, in its GDP-bound conformation. This work describes one of the 6 arrestin domain-containing proteins of Dictyostelium, a novel and atypical member of the arrestin clan. It provides the basis for a better understanding of arrestin-related protein involvement in trafficking processes and for further studies on the expanding roles of arrestins in eukaryotes.

Evaluation of the site specific protein glycation and antioxidant capacity of rare sugar-protein/peptide conjugates.

PubMed

Sun, Yuanxia; Hayakawa, Shigeru; Ogawa, Masahiro; Izumori, Ken

2005-12-28

Protein-sugar conjugates generated in nonenzymatic glycation of alpha-lactalbumin (LA) with rare sugars [D-allose (All) and D-psicose (Psi)] and alimentary sugars as controls [D-glucose (Glc) and D-fructose (Fru)] were qualitatively determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Mass spectra revealed that the extent of glycation at lysine residues on LA with D-aldose molecules was very much higher than that of glycation with d-ketose molecules. To identify the specific site of glycation, the peptide mapping was established from protease V8 digestion, using a combination of computational cutting of proteins and MALDI-TOF-MS. As compared to peptide mapping, three and seven glycation sites were located in the primary structure of LA-ketose and LA-aldose conjugates, respectively. On the other hand, the antioxidant activities of protein-sugar conjugates and their peptic hydrolysates were investigated by 1,1-diphenyl-2-picrylhydrazyl radical scavenging method. The antioxidant activities of proteins/peptides glycated with rare sugars were significantly higher than those modified with the control sugars. The results indicated that the glycation degree and position were not markedly different between rare sugar and corresponding control sugar, but the antioxidant properties of protein and its hydrolysate were significantly enhanced by modifying with rare sugar.

Ubiquilin overexpression reduces GFP-polyalanine-induced protein aggregates and toxicity

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

Wang Hongmin; Monteiro, Mervyn J.

2007-08-01

Several human disorders are associated with an increase in a continuous stretch of alanine amino acids in proteins. These so-called polyalanine expansion diseases share many similarities with polyglutamine-related disorders, including a length-dependent reiteration of amino acid induction of protein aggregation and cytotoxicity. We previously reported that overexpression of ubiquilin reduces protein aggregates and toxicity of expanded polyglutamine proteins. Here, we demonstrate a similar role for ubiquilin toward expanded polyalanine proteins. Overexpression of ubiquilin-1 in HeLa cells reduced protein aggregates and the cytotoxicity associated with expression of a transfected nuclear-targeted GFP-fusion protein containing 37-alanine repeats (GFP-A37), in a dose dependent manner.more » Ubiquilin coimmunoprecipitated more with GFP proteins containing a 37-polyalanine tract compared to either 7 (GFP-A7), or no alanine tract (GFP). Moreover, overexpression of ubiquilin suppressed the increased vulnerability of HeLa cell lines stably expressing the GFP-A37 fusion protein to oxidative stress-induced cell death compared to cell lines expressing GFP or GFP-A7 proteins. By contrast, siRNA knockdown of ubiquilin expression in the GFP-A37 cell line was associated with decreased cellular proliferation, and increases in GFP protein aggregates, nuclear fragmentation, and cell death. Our results suggest that boosting ubiquilin levels in cells might provide a universal and attractive strategy to prevent toxicity of proteins containing reiterative expansions of amino acids involved in many human diseases.« less

Effect of preoperative pelvic floor muscle therapy with biofeedback versus standard care on stress urinary incontinence and quality of life in men undergoing laparoscopic radical prostatectomy: a randomised control trial.

PubMed

Dijkstra-Eshuis, Joke; Van den Bos, Tine W L; Splinter, Rosa; Bevers, Rob F M; Zonneveld, Willemijn C G; Putter, Hein; Pelger, Rob C M; Voorham-van der Zalm, Petra J

2015-02-01

Laparoscopic radical prostatectomy (LARP) may cause stress urinary incontinence (SUI). This study reports the effects of preoperative pelvic floor muscle therapy (PFMT) on SUI and quality of life (QoL) in men undergoing LARP. In this single-center randomized controlled trial, 122 patients undergoing LARP were assigned to an intervention group of PFMT with biofeedback once a week preoperatively, with 4 weeks' follow-up or to a control group receiving standard care. Randomization and allocation to the trial group were carried out by a central computer system. The primary analysis was based on 121 (n = 65; n = 56), comparing SUI rates and QoL in the two groups in a 1-year follow-up. Validated questionnaires, the Pelvic Floor Inventories (PeLFls), the King's Health Questionnaire (KHQ), the International Prostate Symptom Score (IPSS), a bladder diary, a 24-hr pad test and pelvic floor examination were used. Continence was defined as no leakage at all. All analyses were performed according to intention-to-treat. One hundred twenty-two patients were randomized, 19 patients were excluded from analysis because of early drop-out. There were no significant differences between both groups in the incidence of SUI and QoL based on the KHQ, IPSS, and pad tests (P ≥ 0.05). In all patients continence was achieved in 77.2% at 1 year postoperatively. Preoperative PFMT does not appear to be effective in the prevention of SUI and QoL following LARP. © 2014 Wiley Periodicals, Inc.

Relating the effects of protein type and content in increased-protein cheese pies to consumers' perception of satiating capacity.

PubMed

Marcano, J; Varela, P; Fiszman, S

2015-02-01

Since proteins have been shown to have the highest satiation-inducing effects of all the macronutrients, increasing the protein level is one of the main strategies for designing foods with enhanced satiating capacity. However, few studies analyze the effect that protein addition has on the texture and flavor characteristics of the target food item to relate it to the expected satiating capacity it elicits. The present work studied cheese pies with three levels of soy and whey proteins. Since the protein level altered the rheological behavior of the batters before baking and the texture of the baked pies, the feasibility of adding several protein levels for obtaining a range of final products was investigated. A check-all-that-apply questionnaire containing 32 sensory and non-sensory characteristics of the samples was given to consumers (n = 131) who also scored the perceived samples' satiating capacity. The results showed that the type and content of protein contributed distinctive sensory characteristics to the samples that could be related to their satiating capacity perception. Harder and drier samples (high protein levels) were perceived as more satiating with less perceptible sweet and milky cheese pie characteristic flavors. Soy contributed an off-flavour. These results will contribute to a better understanding of the interrelation of all these factors, aiding the development of highly palatable solid foods with enhanced satiating capacities.

Composition-related structural transition of random peptides: insight into the boundary between intrinsically disordered proteins and folded proteins.

PubMed

Kang, Wen-Bin; He, Chuan; Liu, Zhen-Xing; Wang, Jun; Wang, Wei

2018-05-16

Previous studies based on bioinformatics showed that there is a sharp distinction of structural features and residue composition between the intrinsically disordered proteins and the folded proteins. What induces such a composition-related structural transition? How do various kinds of interactions work in such processes? In this work, we investigate these problems based on a survey on peptides randomly composed of charged residues (including glutamic acids and lysines) and the residues with different hydrophobicity, such as alanines, glycines, or phenylalanines. Based on simulations using all-atom model and replica-exchange Monte Carlo method, a coil-globule transition is observed for each peptide. The corresponding transition temperature is found to be dependent on the contents of the hydrophobic and charged residues. For several cases, when the mean hydrophobicity is larger than a certain threshold, the transition temperature is higher than the room temperature, and vise versa. These thresholds of hydrophobicity and net charge are quantitatively consistent with the border line observed from the study of bioinformatics. These results outline the basic physical reasons for the compositional distinction between the intrinsically disordered proteins and the folded proteins. Furthermore, the contributions of various interactions to the structural variation of peptides are analyzed based on the contact statistics and the charge-pattern dependence of the gyration radii of the peptides. Our observations imply that the hydrophobicity contributes essentially to such composition-related transitions. Thus, we achieve a better understanding on composition-structure relation of the natural proteins and the underlying physics.

Hyper-O-GlcNAcylation inhibits the induction of heat shock protein 70 (Hsp 70) by sodium arsenite in HeLa cells.

PubMed

Miura, Yuri; Sato, Takatoshi; Sakurai, Yoko; Sakai, Ryo; Hiraoka, Wakako; Endo, Tamao

2014-01-01

O-Linked β-N-acetylglucosamine-modification (O-GlcNAcylation) is a reversible, post-translational, and regulatory modification of nuclear, mitochondrial, and cytoplasmic proteins that is responsive to cellular stress. However, the role of O-GlcNAcylation in the induction of heat shock proteins (Hsps) by arsenite remains unclear. We used O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenyl carbamate (PUGNAc), an inhibitor of O-GlcNAcase, and glucosamine (GlcN), an enhancer of the hexosamine biosynthesis pathway, or O-GlcNAc transferase (OGT) short interfering RNA (siRNA) to enhance or suppress cellular O-GlcNAcylation levels, respectively, in HeLa cells. The exposure to arsenite increased O-GlcNAcylation and Hsp 70 levels in HeLa cells. However, the pre-treatment with PUGNAc or GlcN, which enhanced O-GlcNAcylation levels, decreased the arsenite-induced expression of Hsp 70. The pre-treatment with OGT siRNA, which suppressed O-GlcNAcylation levels, did not affect the induction of Hsp 70. We then examined the effects of O-GlcNAcylation on the nuclear translocation and phosphorylation of heat shock factor 1 (HSF1), and found that neither the nuclear translocation nor phosphorylation of HSF1 was regulated by O-GlcNAcylation. Finally, Hsp 70 mRNA expression was induced by arsenite, whereas the addition of PUGNAc slightly suppressed its induction. These results indicate that O-GlcNAcylation is related to arsenite-induced Hsp 70 expression, and demonstrated that hyper-O-GlcNAcylation inhibited the induction of Hsp 70 via transcriptional factors instead of HSF1.

La Piedad Michoacán Mexico Virus V protein antagonizes type I interferon response by binding STAT2 protein and preventing STATs nuclear translocation.

PubMed

Pisanelli, Giuseppe; Laurent-Rolle, Maudry; Manicassamy, Balaji; Belicha-Villanueva, Alan; Morrison, Juliet; Lozano-Dubernard, Bernardo; Castro-Peralta, Felipa; Iovane, Giuseppe; García-Sastre, Adolfo

2016-02-02

La Piedad Michoacán Mexico Virus (LPMV) is a member of the Rubulavirus genus within the Paramyxoviridae family. LPMV is the etiologic agent of "blue eye disease", causing a significant disease burden in swine in Mexico with long-term implications for the agricultural industry. This virus mainly affects piglets and is characterized by meningoencephalitis and respiratory distress. It also affects adult pigs, causing reduced fertility and abortions in females, and orchitis and epididymitis in males. Viruses of the Paramyxoviridae family evade the innate immune response by targeting components of the interferon (IFN) signaling pathway. The V protein, expressed by most paramyxoviruses, is a well-characterized IFN signaling antagonist. Until now, there were no reports on the role of the LPMV-V protein in inhibiting the IFN response. In this study we demonstrate that LPMV-V protein antagonizes type I but not type II IFN signaling by binding STAT2, a component of the type I IFN cascade. Our results indicate that the last 18 amino acids of LPMV-V protein are required for binding to STAT2 in human and swine cells. While LPMV-V protein does not affect the protein levels of STAT1 or STAT2, it does prevent the IFN-induced phosphorylation and nuclear translocation of STAT1 and STAT2 thereby inhibiting cellular responses to IFN α/β. Copyright © 2015 Elsevier B.V. All rights reserved.

The impact of carbon-13 and deuterium on relative quantification of proteins using stable isotope diethyl labeling.

PubMed

Koehler, Christian J; Arntzen, Magnus Ø; Thiede, Bernd

2015-05-15

Stable isotopic labeling techniques are useful for quantitative proteomics. A cost-effective and convenient method for diethylation by reductive amination was established. The impact using either carbon-13 or deuterium on quantification accuracy and precision was investigated using diethylation. We established an effective approach for stable isotope labeling by diethylation of amino groups of peptides. The approach was validated using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and nanospray liquid chromatography/electrospray ionization (nanoLC/ESI)-ion trap/orbitrap for mass spectrometric analysis as well as MaxQuant for quantitative data analysis. Reaction conditions with low reagent costs, high yields and minor side reactions were established for diethylation. Furthermore, we showed that diethylation can be applied to up to sixplex labeling. For duplex experiments, we compared diethylation in the analysis of the proteome of HeLa cells using acetaldehyde-(13) C(2)/(12) C(2) and acetaldehyde-(2) H(4)/(1) H(4). Equal numbers of proteins could be identified and quantified; however, (13) C(4)/(12) C(4) -diethylation revealed a lower variance of quantitative peptide ratios within proteins resulting in a higher precision of quantified proteins and less falsely regulated proteins. The results were compared with dimethylation showing minor effects because of the lower number of deuteriums. The described approach for diethylation of primary amines is a cost-effective and accurate method for up to sixplex relative quantification of proteomes. (13) C(4)/(12) C(4) -diethylation enables duplex quantification based on chemical labeling without using deuterium which reduces identification of false-negatives and increases the quality of the quantification results. Copyright © 2015 John Wiley & Sons, Ltd.

Induction of ketosis in rats fed low-carbohydrate, high-fat diets depends on the relative abundance of dietary fat and protein.

PubMed

Bielohuby, Maximilian; Menhofer, Dominik; Kirchner, Henriette; Stoehr, Barbara J M; Müller, Timo D; Stock, Peggy; Hempel, Madlen; Stemmer, Kerstin; Pfluger, Paul T; Kienzle, Ellen; Christ, Bruno; Tschöp, Matthias H; Bidlingmaier, Martin

2011-01-01

Low-carbohydrate/high-fat diets (LC-HFDs) in rodent models have been implicated with both weight loss and as a therapeutic approach to treat neurological diseases. LC-HFDs are known to induce ketosis; however, systematic studies analyzing the impact of the macronutrient composition on ketosis induction and weight loss success are lacking. Male Wistar rats were pair-fed for 4 wk either a standard chow diet or one of three different LC-HFDs, which only differed in the relative abundance of fat and protein (percentages of fat/protein in dry matter: LC-75/10; LC-65/20; LC-55/30). We subsequently measured body composition by nuclear magnetic resonance (NMR), analyzed blood chemistry and urine acetone content, evaluated gene expression changes of key ketogenic and gluconeogenic genes, and measured energy expenditure (EE) and locomotor activity (LA) during the first 4 days and after 3 wk on the respective diets. Compared with chow, rats fed with LC-75/10, LC-65/20, and LC-55/30 gained significantly less body weight. Reductions in body weight were mainly due to lower lean body mass and paralleled by significantly increased fat mass. Levels of β-hydroxybutyate were significantly elevated feeding LC-75/10 and LC-65/20 but decreased in parallel to reductions in dietary fat. Acetone was about 16-fold higher with LC-75/10 only (P < 0.001). In contrast, rats fed with LC-55/30 were not ketotic. Serum fibroblast growth factor-21, hepatic mRNA expression of hydroxymethylglutaryl-CoA-lyase, peroxisome proliferator-activated receptor-γ coactivator-1α, and peroxisome proliferator-activated receptor-γ coactivator-1β were increased with LC-75/10 only. Expression of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase was downregulated by 50-70% in LC-HF groups. Furthermore, EE and LA were significantly decreased in all groups fed with LC-HFDs after 3 wk on the diets. In rats, the absence of dietary carbohydrates per se does not induce ketosis. LC-HFDs must be high in fat

Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages

PubMed Central

Liang, Xue-hai; Sun, Hong; Shen, Wen; Crooke, Stanley T.

2015-01-01

Although the RNase H-dependent mechanism of inhibition of gene expression by chemically modified antisense oligonucleotides (ASOs) has been well characterized, little is known about the interactions between ASOs and intracellular proteins that may alter cellular localization and/or potency of ASOs. Here, we report the identification of 56 intracellular ASO-binding proteins using multi-step affinity selection approaches. Many of the tested proteins had no significant effect on ASO activity; however, some proteins, including La/SSB, NPM1, ANXA2, VARS and PC4, appeared to enhance ASO activities, likely through mechanisms related to subcellular distribution. VARS and ANXA2 co-localized with ASOs in endocytic organelles, and reduction in the level of VARS altered lysosome/ASO localization patterns, implying that these proteins may facilitate ASO release from the endocytic pathway. Depletion of La and NPM1 reduced nuclear ASO levels, suggesting potential roles in ASO nuclear accumulation. On the other hand, Ku70 and Ku80 proteins inhibited ASO activity, most likely by competition with RNase H1 for ASO/RNA duplex binding. Our results demonstrate that phosphorothioate-modified ASOs bind a set of cellular proteins that affect ASO activity via different mechanisms. PMID:25712094

Oxysterol-binding protein-related protein (ORP) 9 is a PDK-2 substrate and regulates Akt phosphorylation.

PubMed

Lessmann, Eva; Ngo, Mike; Leitges, Michael; Minguet, Susana; Ridgway, Neale D; Huber, Michael

2007-02-01

The oxysterol-binding protein and oxysterol-binding protein-related protein family has been implicated in lipid transport and metabolism, vesicle trafficking and cell signaling. While investigating the phosphorylation of Akt/protein kinase B in stimulated bone marrow-derived mast cells, we observed that a monoclonal antibody directed against phospho-S473 Akt cross-reacted with oxysterol-binding protein-related protein 9 (ORP9). Further analysis revealed that mast cells exclusively express ORP9S, an N-terminal truncated version of full-length ORP9L. A PDK-2 consensus phosphorylation site in ORP9L and OPR9S at S287 (VPEFS(287)Y) was confirmed by site-directed mutagenesis. In contrast to Akt, increased phosphorylation of ORP9S S287 in stimulated mast cells was independent of phosphatidylinositol 3-kinase but sensitive to inhibition of conventional PKC isotypes. PKC-beta dependence was confirmed by lack of ORP9S phosphorylation at S287 in PKC-beta-deficient, but not PKC-alpha-deficient, mast cells. Moreover, co-immunoprecipitation of PKC-beta and ORP9S, and in vitro phosphorylation of ORP9S in this complex, argued for direct phosphorylation of ORP9S by PKC-beta, introducing ORP9S as a novel PKC-beta substrate. Akt was also detected in a PKC-beta/ORP9S immune complex and phosphorylation of Akt on S473 was delayed in PKC-deficient mast cells. In HEK293 cells, RNAi experiments showed that depletion of ORP9L increased Akt S473 phosphorylation 3-fold without affecting T308 phosphorylation in the activation loop. Furthermore, mammalian target of rapamycin was implicated in ORP9L phosphorylation in HEK293 cells. These studies identify ORP9 as a PDK-2 substrate and negative regulator of Akt phosphorylation at the PDK-2 site.

Silencing of DNA-PKcs alters the transcriptional profile of certain signal transduction genes related to proliferation and differentiation in HeLa cells.

PubMed

An, Jing; Xu, Qin-Zhi; Sui, Jian-Li; Bai, Bei; Zhou, Ping-Kun

2005-09-01

DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a member of a sub-family of phosphoinositol 3-kinases, has been reported overexpressed in various human cancers, but its significance is unclear. In the present study, we generated the stable cell line HeLa(siRNAH1) of silenced DNA-PKcs by transfecting HeLa cells with the siRNA construct targeting the catalytic motif of DNA-PKcs. The expression of DNA-PKcs was markedly suppressed in HeLa(siRNAH1) cells, and eventuating in increased cellular sensitivity to ionizing radiation as well as cisplatin. Microarray assay was used to explore the transcriptional profiling of signal transduction-associated genes. The results demonstrated that 15 genes were up-regulated and eight were down-regulated in HeLa(siRNAH1) as compared with the HeLa(control) cells that transfected with non-specific siRNA construct. Seven of the up-regulated genes are associated with the interferon-signaling events, the others function in the BMP signal pathway, or as regulators of cell cycle and differentiation. The down-regulated genes include IL8, IL10RA, DAPK3, and those involved in nuclear factor of activated T cells (NFAT) signal pathway and endocrine responsiveness. Using the NFAT-driving secreted alkaline phosphatase reporter expression system, we further confirmed that NFAT transcriptional activity was markedly minimized after silencing DNA-PKcs. These results demonstrated that inactivation of DNA-PKcs altered the transcriptional level of certain signal transduction-associated genes related to proliferation and differentiation.

Identification of a crustacean β-1,3-glucanase related protein as a pattern recognition protein in antibacterial response.

PubMed

Chai, Lian-Qin; Meng, Jing-Hui; Gao, Jie; Xu, Yi-Hui; Wang, Xian-Wei

2018-06-02

Prophenoloxidase (proPO) activating system is an important immune response for arthropods. β-1, 3-glucanase related protein (previously named as lipopolysaccharide and β-1, 3-glucan binding protein (LGBP) in crustaceans) is a typical pattern recognition receptor family involved in the proPO activation by recognizing the invading microbes. In this study, we pay special attention to a bacteria-induced β-1,3-glucanase related protein from red swamp crayfish Procambarus clarkii, an important aquaculture specie in China. This protein, designated PcBGRP, was found a typical member of crustacean BGRP family with the glucanase-related domain and the characteristic motifs. PcBGRP was expressed in hemcoyes and hepatopancreas, and its expression could be induced by the carbohydrate and bacteria stimulants. The induction by lipopolysaccharide (LPS) and β-1,3-glucan (βG) was more significant than by peptidoglycan (PG). The response of PcBGRP to the native Gram-negative bacterial pathogen Aeromonas hydrophila was more obvious than to Gram-positive bacteria. Using RNA interference and recombinant protein, PcBGRP was found to protect crayfish from A. hydrophila infection revealed by the survival test and morphological analysis. A mechanism study found PcBGRP could bind LPS and βG in a dose-dependent manner, and the LPS recognizing ability determined the Gram-negative bacterium binding activity of PcBGRP. PcBGRP was found to enhance the PO activation both in vitro and in vivo, and the protective role was related to the PO activating ability of PcBGRP. This study emphasized the role of BGRP family in crustacean immune response, and provided new insight to the immunity of red swamp crayfish which suffered serious disease during the aquaculture in China. Copyright © 2018 Elsevier Ltd. All rights reserved.

A topologically related singularity suggests a maximum preferred size for protein domains.

PubMed

Zbilut, Joseph P; Chua, Gek Huey; Krishnan, Arun; Bossa, Cecilia; Rother, Kristian; Webber, Charles L; Giuliani, Alessandro

2007-02-15

A variety of protein physicochemical as well as topological properties, demonstrate a scaling behavior relative to chain length. Many of the scalings can be modeled as a power law which is qualitatively similar across the examples. In this article, we suggest a rational explanation to these observations on the basis of both protein connectivity and hydrophobic constraints of residues compactness relative to surface volume. Unexpectedly, in an examination of these relationships, a singularity was shown to exist near 255-270 residues length, and may be associated with an upper limit for domain size. Evaluation of related G-factor data points to a wide range of conformational plasticity near this point. In addition to its theoretical importance, we show by an application of CASP experimental and predicted structures, that the scaling is a practical filter for protein structure prediction. 2006 Wiley-Liss, Inc.

Matrix Metallopeptidase 14 Plays an Important Role in Regulating Tumorigenic Gene Expression and Invasion Ability of HeLa Cells.

PubMed

Zhang, Ying-Hui; Wang, Juan-Juan; Li, Min; Zheng, Han-Xi; Xu, Lan; Chen, You-Guo

2016-03-01

The objectives of this study were to investigate the functional effect of matrix metallopeptidase 14 (MMP14) on cell invasion in cervical cancer cells (HeLa line) and to study the underlying molecular mechanisms. Expression vector of short hairpin RNA targeting MMP14 was treated in HeLa cells, and then, transfection efficiency was verified by a florescence microscope. Transwell assay was used to investigate cell invasion ability in HeLa cells. Quantitative polymerase chain reaction and Western blotting analysis were used to detect the expression of MMP14 and relative factors in messenger RNA and protein levels, respectively. Matrix metallopeptidase 14 short hairpin RNA expression vector transfection obviously decreased MMP14 expression in messenger RNA and protein levels. Down-regulation of MMP14 suppressed invasion ability of HeLa cells and reduced transforming growth factor β1 and vascular endothelial growth factor B expressions. Furthermore, MMP14 knockdown decreased bone sialoprotein and enhanced forkhead box protein L2 expression in both RNA and protein levels. Matrix metallopeptidase 14 plays an important role in regulating invasion of HeLa cells. Matrix metallopeptidase 14 knockdown contributes to attenuating the malignant phenotype of cervical cancer cell.

Kelch-like ECH-associated Protein 1-dependent Nuclear Factor-E2-related Factor 2 Activation in Relation to Antioxidation Induced by Sevoflurane Preconditioning.

PubMed

Cai, Min; Tong, Li; Dong, Beibei; Hou, Wugang; Shi, Likai; Dong, Hailong

2017-03-01

The authors have reported that antioxidative effects play a crucial role in the volatile anesthetic-induced neuroprotection. Accumulated evidence shows that endogenous antioxidation could be up-regulated by nuclear factor-E2-related factor 2 through multiple pathways. However, whether nuclear factor-E2-related factor 2 activation is modulated by sevoflurane preconditioning and, if so, what is the signaling cascade underlying upstream of this activation are still unknown. Sevoflurane preconditioning in mice was performed with sevoflurane (2.5%) 1 h per day for five consecutive days. Focal cerebral ischemia/reperfusion injury was induced by middle cerebral artery occlusion. Expression of nuclear factor-E2-related factor 2, kelch-like ECH-associated protein 1, manganese superoxide dismutase, thioredoxin-1, and nicotinamide adenine dinucleotide phosphate quinolone oxidoreductase-1 was detected (n = 6). The antioxidant activities and oxidative product expression were also examined. To determine the role of kelch-like ECH-associated protein 1 inhibition-dependent nuclear factor-E2-related factor 2 activation in sevoflurane preconditioning-induced neuroprotection, the kelch-like ECH-associated protein 1-nuclear factor-E2-related factor 2 signal was modulated by nuclear factor-E2-related factor 2 knockout, kelch-like ECH-associated protein 1 overexpression lentivirus, and kelch-like ECH-associated protein 1 deficiency small interfering RNA (n = 8). The infarct volume, neurologic scores, and cellular apoptosis were assessed. Sevoflurane preconditioning elicited neuroprotection and increased nuclear factor-E2-related factor 2 nuclear translocation, which in turn up-regulated endogenous antioxidation and reduced oxidative injury. Sevoflurane preconditioning reduced kelch-like ECH-associated protein 1 expression. Nuclear factor-E2-related factor 2 ablation abolished neuroprotection and reversed sevoflurane preconditioning by mediating the up-regulation of antioxidants. Kelch

Posttranslational modification of autophagy-related proteins in macroautophagy

PubMed Central

Xie, Yangchun; Kang, Rui; Sun, Xiaofang; Zhong, Meizuo; Huang, Jin; Klionsky, Daniel J.; Tang, Daolin

2014-01-01

Macroautophagy is an intracellular catabolic process involved in the formation of multiple membrane structures ranging from phagophores to autophagosomes and autolysosomes. Dysfunction of macroautophagy is implicated in both physiological and pathological conditions. To date, 38 autophagy-related (ATG) genes have been identified as controlling these complicated membrane dynamics during macroautophagy in yeast; approximately half of these genes are clearly conserved up to human, and there are additional genes whose products function in autophagy in higher eukaryotes that are not found in yeast. The function of the ATG proteins, in particular their ability to interact with a number of macroautophagic regulators, is modulated by posttranslational modifications (PTMs) such as phosphorylation, glycosylation, ubiquitination, acetylation, lipidation, and proteolysis. In this review, we summarize our current knowledge of the role of ATG protein PTMs and their functional relevance in macroautophagy. Unraveling how these PTMs regulate ATG protein function during macroautophagy will not only reveal fundamental mechanistic insights into the regulatory process, but also provide new therapeutic targets for the treatment of autophagy-associated diseases. PMID:25484070

Protein phosphorylations in poliovirus infected cells.

PubMed

James, L A; Tershak, D R

1981-01-01

In vivo phosphorylation of proteins that are associated with polysomes of poliovirus-infected VERO (African green monkey kidney) and HeLa (Henrietta Lacks) cells differed from phosphorylations observed with uninfected cells that were fed fresh medium. With both types of cells infection stimulated phosphorylation of proteins with molecular weights of 40 000-41 000, 39 000, 34 000, 32 000, and 24 000. Similarities of phosphorylations in VERO and HeLa cells suggest that they are a specific consequence of infection and might serve a regulatory function during protein synthesis.

Arrestin-related proteins mediate pH signaling in fungi.

PubMed

Herranz, Silvia; Rodríguez, José M; Bussink, Henk-Jan; Sánchez-Ferrero, Juan C; Arst, Herbert N; Peñalva, Miguel A; Vincent, Olivier

2005-08-23

Metazoan arrestins bind to seven-transmembrane (7TM) receptors to regulate function. Aspergillus nidulans PalF, a protein involved in the fungal ambient pH signaling pathway, contains arrestin N-terminal and C-terminal domains and binds strongly to two different regions within the C-terminal cytoplasmic tail of the 7TM, putative pH sensor PalH. Upon exposure to alkaline ambient pH, PalF is phosphorylated and, like mammalian beta-arrestins, ubiquitinated in a signal-dependent and 7TM protein-dependent manner. Substitution in PalF of a highly conserved arrestin N-terminal domain Ser residue prevents PalF-PalH interaction and pH signaling in vivo. Thus, PalF is the first experimentally documented fungal arrestin-related protein, dispelling the notion that arrestins are restricted to animal proteomes. Epistasis analyses demonstrate that PalF posttranslational modification is partially dependent on the 4TM protein PalI but independent of the remaining pH signal transduction pathway proteins PalA, PalB, and PalC, yielding experimental evidence bearing on the order of participation of the six components of the pH signal transduction pathway. Our data strongly implicate PalH as an ambient pH sensor, possibly with the cooperation of PalI.

Summary of Test Results of MQXFS1 - The First Short Model 150 mm Aperture $$Nb_3Sn$$ Quadrupole for the High-Luminosity

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

Stoynev, S.; et al.

The development ofmore » $$Nb_3Sn$$ quadrupole magnets for the High-Luminosity LHC upgrade is a joint venture between the US LHC Accelerator Research Program (LARP)* and CERN with the goal of fabricating large aperture quadrupoles for the LHC in-teraction regions (IR). The inner triplet (low-β) NbTi quadrupoles in the IR will be replaced by the stronger Nb3Sn magnets boosting the LHC program of having 10-fold increase in integrated luminos-ity after the foreseen upgrades. Previously LARP conducted suc-cessful tests of short and long models with up to 120 mm aperture. The first short 150 mm aperture quadrupole model MQXFS1 was assembled with coils fabricated by both CERN and LARP. The magnet demonstrated strong performance at the Fermilab’s verti-cal magnet test facility reaching the LHC operating limits. This paper reports the latest results from MQXFS1 tests with changed pre-stress levels. The overall magnet performance, including quench training and memory, ramp rate and temperature depend-ence, is also summarized.« less

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

PubMed Central

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

2012-01-01

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

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

PubMed

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

2012-06-01

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

Sources of Dietary Protein in Relation to Blood Pressure in a General Dutch Population

PubMed Central

Altorf - van der Kuil, Wieke; Engberink, Mariëlle F.; Vedder, Moniek M.; Boer, Jolanda M. A.; Verschuren, W. M. Monique; Geleijnse, Johanna M.

2012-01-01

Background Little is known about the relation of different dietary protein types with blood pressure (BP). We examined whether intake of total, plant, animal, dairy, meat, and grain protein was related to BP in a cross sectional cohort of 20,820 Dutch adults, aged 20–65 y and not using antihypertensive medication. Design Mean BP levels were calculated in quintiles of energy-adjusted protein with adjustment for age, sex, BMI, education, smoking, and intake of energy, alcohol, and other nutrients including protein from other sources. In addition, mean BP difference after substitution of 3 en% carbohydrates or MUFA with protein was calculated. Results Total protein and animal protein were not associated with BP (ptrend = 0.62 and 0.71 respectively), both at the expense of carbohydrates and MUFA. Systolic BP was 1.8 mmHg lower (ptrend<0.01) in the highest (>36 g/d) than in the lowest (<27 g/d) quintile of plant protein. This inverse association was present both at the expense of carbohydrates and MUFA and more pronounced in individuals with untreated hypertension (−3.6 mmHg) than in those with normal (+0.1 mmHg) or prehypertensive BP (−0.3 mmHg; pinteraction<0.01). Meat and grain protein were not related to BP. Dairy protein was directly associated with systolic BP (+1.6 mmHg, ptrend<0.01), but not with diastolic BP (ptrend = 0.24). Conclusions Total protein and animal protein were not associated with BP in this general untreated Dutch population. Plant protein may be beneficial to BP, especially in people with elevated BP. However, because high intake of plant protein may be a marker of a healthy diet and lifestyle in general, confirmation from randomized controlled trials is warranted. PMID:22347387

Proteomic analysis identifies insulin-like growth factor-binding protein-related protein-1 as a podocyte product.

PubMed

Matsumoto, Takayuki; Hess, Sonja; Kajiyama, Hiroshi; Sakairi, Toru; Saleem, Moin A; Mathieson, Peter W; Nojima, Yoshihisa; Kopp, Jeffrey B

2010-10-01

The podocyte secretory proteome may influence the phenotype of adjacent podocytes, endothelial cells, parietal epithelial cells, and tubular epithelial cells but has not been systematically characterized. We have initiated studies to characterize this proteome, with the goal of further understanding the podocyte cell biology. We cultured differentiated conditionally immortalized human podocytes and subjected the proteins in conditioned medium to mass spectrometry. At a false discovery rate of <3%, we identified 111 candidates from conditioned medium, including 44 proteins that have signal peptides or are described as secreted proteins in the UniProt database. As validation, we confirmed that one of these proteins, insulin-like growth factor-binding protein-related protein-1 (IGFBP-rP1), was expressed in mRNA and protein of cultured podocytes. In addition, transforming growth factor-β1 stimulation increased IGFBP-rP1 in conditioned medium. We analyzed IGFBP-rP1 glomerular expression in a mouse model of human immunodeficiency virus-associated nephropathy. IGFBP-rP1 was absent from podocytes of normal mice and was expressed in podocytes and pseudocrescents of transgenic mice, where it was coexpressed with desmin, a podocyte injury marker. We conclude that IGFBP-rP1 may be a product of injured podocytes. Further analysis of the podocyte secretory proteome may identify biomarkers of podocyte injury.

Protein level affects the relative lysine requirement of growing rainbow trout (Oncorhynchus mykiss) fry.

PubMed

Bodin, Noelie; Govaerts, Bernadette; Abboudi, Tarik; Detavernier, Christel; De Saeger, Sarah; Larondelle, Yvan; Rollin, Xavier

2009-07-01

The effect of two digestible protein levels (310 and 469 g/kg DM) on the relative lysine (Lys; g Lys/kg DM or g Lys/100 g protein) and the absolute Lys (g Lys intake/kg 0.75 per d) requirements was studied in rainbow trout fry using a dose-response trial. At each protein level, sixteen isoenergetic (22-23 MJ digestible energy/kg DM) diets were tested, involving a full range (2-70 g/kg DM) of sixteen Lys levels. Each diet was given to one group of sixty rainbow trout fry (mean initial body weight 0.78 g) reared at 15 degrees C for 31 feeding d. The Lys requirements were estimated based on the relationships between weight, protein, and Lys gains (g/kg 0.75 per d) and Lys concentration (g/kg DM or g/100 g protein) or Lys intake (g/kg 0.75 per d), using the broken-line model (BLM) and the non-linear four-parameter saturation kinetics model (SKM-4). Both the model and the response criterion chosen markedly impacted the relative Lys requirement. The relative Lys requirement for Lys gain of rainbow trout estimated with the BLM (and SKM-4 at 90 % of the maximum response) increased from 16.8 (19.6) g/kg DM at a low protein level to 23.4 (24.5) g/kg DM at a high protein level. However, the dietary protein content affected neither the absolute Lys requirement nor the relative Lys requirement expressed as g Lys/100 g protein nor the Lys requirement for maintenance (21 mg Lys/kg 0.75 per d).

Refolding of SDS-Unfolded Proteins by Nonionic Surfactants.

PubMed

Kaspersen, Jørn Døvling; Søndergaard, Anne; Madsen, Daniel Jhaf; Otzen, Daniel E; Pedersen, Jan Skov

2017-04-25

The strong and usually denaturing interaction between anionic surfactants (AS) and proteins/enzymes has both benefits and drawbacks: for example, it is put to good use in electrophoretic mass determinations but limits enzyme efficiency in detergent formulations. Therefore, studies of the interactions between proteins and AS as well as nonionic surfactants (NIS) are of both basic and applied relevance. The AS sodium dodecyl sulfate (SDS) denatures and unfolds globular proteins under most conditions. In contrast, NIS such as octaethylene glycol monododecyl ether (C 12 E 8 ) and dodecyl maltoside (DDM) protect bovine serum albumin (BSA) from unfolding in SDS. Membrane proteins denatured in SDS can also be refolded by addition of NIS. Here, we investigate whether globular proteins unfolded by SDS can be refolded upon addition of C 12 E 8 and DDM. Four proteins, BSA, α-lactalbumin (αLA), lysozyme, and β-lactoglobulin (βLG), were studied by small-angle x-ray scattering and both near- and far-UV circular dichroism. All proteins and their complexes with SDS were attempted to be refolded by the addition of C 12 E 8 , while DDM was additionally added to SDS-denatured αLA and βLG. Except for αLA, the proteins did not interact with NIS alone. For all proteins, the addition of NIS to the protein-SDS samples resulted in extraction of the SDS from the protein-SDS complexes and refolding of βLG, BSA, and lysozyme, while αLA changed to its NIS-bound state instead of the native state. We conclude that NIS competes with globular proteins for association with SDS, making it possible to release and refold SDS-denatured proteins by adding sufficient amounts of NIS, unless the protein also interacts with NIS alone. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Upregulated expression of La ribonucleoprotein domain family member 6 and collagen type I gene following water-filtered broad-spectrum near-infrared irradiation in a 3-dimensional human epidermal tissue culture model as revealed by microarray analysis.

PubMed

Tanaka, Yohei; Nakayama, Jun

2018-05-01

Water-filtered broad-spectrum near-infrared irradiation can induce various biological effects, as our previous clinical, histological, and biochemical investigations have shown. However, few studies that examined the changes thus induced in gene expression. The aim was to investigate the changes in gene expression in a 3-dimensional reconstructed epidermal tissue culture exposed to water-filtered broad-spectrum near-infrared irradiation. DNA microarray and quantitative real-time polymerase chain reaction (PCR) analysis was used to assess gene expression levels in a 3-dimensional reconstructed epidermal model composed of normal human epidermal cells exposed to water-filtered broad-spectrum near-infrared irradiation. The water filter allowed 1000-1800 nm wavelengths and excluded 1400-1500 nm wavelengths, and cells were exposed to 5 or 10 rounds of near-infrared irradiation at 10 J/cm 2 . A DNA microarray with over 50 000 different probes showed 18 genes that were upregulated or downregulated by at least twofold after irradiation. Quantitative real-time PCR revealed that, relative to control cells, the gene encoding La ribonucleoprotein domain family member 6 (LARP6), which regulates collagen expression, was significantly and dose-dependently upregulated (P < 0.05) by water-filtered broad-spectrum near-infrared exposure. Gene encoding transcripts of collagen type I were significantly upregulated compared with controls (P < 0.05). This study demonstrates the ability of water-filtered broad-spectrum near-infrared irradiation to stimulate the production of type I collagen. © 2017 The Australasian College of Dermatologists.

Ran-binding protein 5 (RanBP5) is related to the nuclear transport factor importin-beta but interacts differently with RanBP1.

PubMed Central

Deane, R; Schäfer, W; Zimmermann, H P; Mueller, L; Görlich, D; Prehn, S; Ponstingl, H; Bischoff, F R

1997-01-01

We report the identification and characterization of a novel 124-kDa Ran binding protein, RanBP5. This protein is related to importin-beta, the key mediator of nuclear localization signal (NLS)-dependent nuclear transport. RanBP5 was identified by two independent methods: it was isolated from HeLa cells by using its interaction with RanGTP in an overlay assay to monitor enrichment, and it was also found by the yeast two-hybrid selection method with RanBP1 as bait. RanBP5 binds to RanBP1 as part of a trimeric RanBP1-Ran-RanBP5 complex. Like importin-beta, RanBP5 strongly binds the GTP-bound form of Ran, stabilizing it against both intrinsic and RanGAP1-induced GTP hydrolysis and also against nucleotide exchange. The GAP resistance of the RanBP5-RanGTP complex can be relieved by RanBP1, which might reflect an in vivo role for RanBP1. RanBP5 is a predominantly cytoplasmic protein that can bind to nuclear pore complexes. We propose that RanBP5 is a mediator of a nucleocytoplasmic transport pathway that is distinct from the importin-alpha-dependent import of proteins with a classical NLS. PMID:9271386

Expression of Iron-Related Proteins Differentiate Non-Cancerous and Cancerous Breast Tumors

PubMed Central

Pizzamiglio, Sara; De Bortoli, Maida; Taverna, Elena; Signore, Michele; Veneroni, Silvia; Chi-shing Cho, William; Orlandi, Rosaria; Verderio, Paolo; Bongarzone, Italia

2017-01-01

We have previously reported hepcidin and ferritin increases in the plasma of breast cancer patients, but not in patients with benign breast disease. We hypothesized that these differences in systemic iron homeostasis may reflect alterations in different iron-related proteins also play a key biochemical and regulatory role in breast cancer. Thus, here we explored the expression of a bundle of molecules involved in both iron homeostasis and tumorigenesis in tissue samples. Enzyme-linked immunosorbent assay (ELISA) or reverse-phase protein array (RPPA), were used to measure the expression of 20 proteins linked to iron processes in 24 non-cancerous, and 56 cancerous, breast tumors. We found that cancerous tissues had higher level of hepcidin than benign lesions (p = 0.012). The univariate analysis of RPPA data highlighted the following seven proteins differentially expressed between non-cancerous and cancerous breast tissue: signal transducer and transcriptional activator 5 (STAT5), signal transducer and activator of transcription 3 (STAT3), bone morphogenetic protein 6 (BMP6), cluster of differentiation 74 (CD74), transferrin receptor (TFRC), inhibin alpha (INHA), and STAT5_pY694. These findings were confirmed for STAT5, STAT3, BMP6, CD74 and INHA when adjusting for age. The multivariate statistical analysis indicated an iron-related 10-protein panel effective in separating non-cancerous from cancerous lesions including STAT5, STAT5_pY694, myeloid differentiation factor 88 (MYD88), CD74, iron exporter ferroportin (FPN), high mobility group box 1 (HMGB1), STAT3_pS727, TFRC, ferritin heavy chain (FTH), and ferritin light chain (FTL). Our results showed an association between some iron-related proteins and the type of tumor tissue, which may provide insight in strategies for using iron chelators to treat breast cancer. PMID:28216608

PPInterFinder--a mining tool for extracting causal relations on human proteins from literature.

PubMed

Raja, Kalpana; Subramani, Suresh; Natarajan, Jeyakumar

2013-01-01

One of the most common and challenging problem in biomedical text mining is to mine protein-protein interactions (PPIs) from MEDLINE abstracts and full-text research articles because PPIs play a major role in understanding the various biological processes and the impact of proteins in diseases. We implemented, PPInterFinder--a web-based text mining tool to extract human PPIs from biomedical literature. PPInterFinder uses relation keyword co-occurrences with protein names to extract information on PPIs from MEDLINE abstracts and consists of three phases. First, it identifies the relation keyword using a parser with Tregex and a relation keyword dictionary. Next, it automatically identifies the candidate PPI pairs with a set of rules related to PPI recognition. Finally, it extracts the relations by matching the sentence with a set of 11 specific patterns based on the syntactic nature of PPI pair. We find that PPInterFinder is capable of predicting PPIs with the accuracy of 66.05% on AIMED corpus and outperforms most of the existing systems. DATABASE URL: http://www.biomining-bu.in/ppinterfinder/

Perovskite-related LaTiO3.41.

PubMed

Daniels, Peter; Lichtenberg, Frank; van Smaalen, Sander

2003-02-01

Crystals of pentalanthanum pentatitanium heptadecaoxide (La(5)Ti(5)O(17) with 0.3% oxygen excess, or LaTiO(3.41)) have been synthesized by floating-zone melting, and the structure has been solved using single-crystal X-ray diffraction intensities. The monoclinic (P2(1)/c) structure consists of perovskite-like slabs of vertex-sharing TiO(6) octahedra, which are separated by additional oxygen layers. The slabs are five octahedra wide. Due to the adjustment of the TiO(6) octahedra to meet the coordination requirements of the La(3+) cations, a superstructure develops along the a axis.

Pollution Atmosphérique et Santé: Corrélation ou Causalité? Le Cas de la Relation entre l'Exposition aux Particules et la Mortalité Cardio-pulmonaire.

PubMed

Dab, William; Ségala, Claire; Dor, Frédéric; Festy, Bernard; Lameloise, Philippe; Moullec, Yvon Le; Tertre, Alain Le; Médina, Sylvia; Quénel, Philippe; Wallaert, Benoît; Zmirou, Et Denis

2001-02-01

De nombreuses études épidémiologiques ont observé dans des contextes différents une faible relation à court terme entre les particules et la mortalité cardio-pulmonaire, même quand les normes de qualité de l'air n'étaient pas dépassées. La causalité de cette relation est un enjeu de santé publique en raison de l'importance de la population exposée. Notre objectif est de faire l'inventaire critique des arguments utilisés dans 15 revues de la littérature publiées. Nous expliquons l'importance de distinguer la validité de la causalité et analysons de façon systématique les différents critères de jugement dans le contexte des études écologiques temporelles. Notre conclusion est que la relation observée est valide et que la plupart des critères de causalité sont respectés. Diminuer le niveau d'exposition des populations aux particules est souhaitable. En Europe, en agissant à la source, notamment sur les émissions Diesel, on diminuera aussi d'autres polluants qui peuvent jouer un rôle sanitaire. Aux États-Unis, la situation est plus complexe car les particules sont surtout secondaires. Il est également indispensable de poursuivre les recherches pour mieux connaître les déterminants des expositions globales des individus et mieux comprendre le rôle toxique des différents facteurs physico-chimiques des particules.

Terminating protein ubiquitination: Hasta la vista, ubiquitin.

PubMed

Stringer, Daniel K; Piper, Robert C

2011-09-15

Ubiquitination is a post-translational modification that generally directs proteins for degradation by the proteasome or by lysosomes. However, ubiquitination has been implicated in many other cellular processes, including transcriptional regulation, DNA repair, regulation of protein-protein interactions and association with ubiquitin-binding scaffolds. Ubiquitination is a dynamic process. Ubiquitin is added to proteins by E3 ubiquitin ligases as a covalent modification to one or multiple lysine residues as well as non-lysine amino acids. Ubiquitin itself contains seven lysines, each of which can also be ubiquitinated, leading to polyubiquitin chains that are best characterized for linkages occurring through K48 and K63. Ubiquitination can also be reversed by the action of deubiquitination enzymes (DUbs). Like E3 ligases, DUbs play diverse and critical roles in cells. ( 1) Ubiquitin is expressed as a fusion protein, as a linear repeat or as a fusion to ribosomal subunits, and DUbs are necessary to liberate free ubiquitin, making them the first enzyme of the ubiquitin cascade. Proteins destined for degradation by the proteasome or by lysosomes are deubiquitinated prior to their degradation, which allows ubiquitin to be recycled by the cell, contributing to the steady-state pool of free ubiquitin. Proteins destined for degradation by lysosomes are also acted upon by both ligases and DUbs. Deubiquitination can also act as a means to prevent protein degradation, and many proteins are thought to undergo rounds of ubiquitination and deubiquitination, ultimately resulting in either the degradation or stabilization of those proteins. Despite years of study, examining the effects of the ubiquitination of proteins remains quite challenging. This is because the methods that are currently being employed to study ubiquitination are limiting. Here, we briefly examine current strategies to study the effects of ubiquitination and describe an additional novel approach that we have

Cell protein cross-linking by erbstatin and related compounds | Center for Cancer Research

Cancer.gov

The scheme depicts a possible mechanism of cross-linking by erbstatin and related analogues. A mechanism of action is proposed which involves initial oxidation to reactive quinone intermediates that subsequently cross-link protein nucleophiles via multiple 1,4-Michael-type additions. Similar alkylation of protein by protein-tyrosine kinase inhibitors, such as herbimycin A, has

Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages.

PubMed

Liang, Xue-hai; Sun, Hong; Shen, Wen; Crooke, Stanley T

2015-03-11

Although the RNase H-dependent mechanism of inhibition of gene expression by chemically modified antisense oligonucleotides (ASOs) has been well characterized, little is known about the interactions between ASOs and intracellular proteins that may alter cellular localization and/or potency of ASOs. Here, we report the identification of 56 intracellular ASO-binding proteins using multi-step affinity selection approaches. Many of the tested proteins had no significant effect on ASO activity; however, some proteins, including La/SSB, NPM1, ANXA2, VARS and PC4, appeared to enhance ASO activities, likely through mechanisms related to subcellular distribution. VARS and ANXA2 co-localized with ASOs in endocytic organelles, and reduction in the level of VARS altered lysosome/ASO localization patterns, implying that these proteins may facilitate ASO release from the endocytic pathway. Depletion of La and NPM1 reduced nuclear ASO levels, suggesting potential roles in ASO nuclear accumulation. On the other hand, Ku70 and Ku80 proteins inhibited ASO activity, most likely by competition with RNase H1 for ASO/RNA duplex binding. Our results demonstrate that phosphorothioate-modified ASOs bind a set of cellular proteins that affect ASO activity via different mechanisms. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Gene promoter methylation and protein expression of BRMS1 in uterine cervix in relation to high-risk human papilloma virus infection and cancer.

PubMed

Panagopoulou, Maria; Lambropoulou, Maria; Balgkouranidou, Ioanna; Nena, Evangelia; Karaglani, Makrina; Nicolaidou, Christina; Asimaki, Anthi; Konstantinidis, Theocharis; Constantinidis, Theodoros C; Kolios, George; Kakolyris, Stylianos; Agorastos, Theodoros; Chatzaki, Ekaterini

2017-04-01

Cervical cancer is strongly related to certain high-risk types of human papilloma virus infection. Breast cancer metastasis suppressor 1 (BRMS1) is a tumor suppressor gene, its expression being regulated by DNA promoter methylation in several types of cancers. This study aims to evaluate the methylation status of BRMS1 promoter in relation to high-risk types of human papilloma virus infection and the development of pre-cancerous lesions and describe the pattern of BRMS1 protein expression in normal, high-risk types of human papilloma virus-infected pre-cancerous and malignant cervical epithelium. We compared the methylation status of BRMS1 in cervical smears of 64 women with no infection by high-risk types of human papilloma virus to 70 women with proven high-risk types of human papilloma virus infection, using real-time methylation-specific polymerase chain reaction. The expression of BRMS1 protein was described by immunohistochemistry in biopsies from cervical cancer, pre-cancerous lesions, and normal cervices. Methylation of BRMS1 promoter was detected in 37.5% of women with no high-risk types of human papilloma virus infection and was less frequent in smears with high-risk types of human papilloma virus (11.4%) and in women with pathological histology (cervical intraepithelial neoplasia) (11.9%). Methylation was detected also in HeLa cervical cancer cells. Immunohistochemistry revealed nuclear BRMS1 protein staining in normal high-risk types of human papilloma virus-free cervix, in cervical intraepithelial neoplasias, and in malignant tissues, where staining was occasionally also cytoplasmic. In cancer, expression was stronger in the more differentiated cancer blasts. In conclusion, BRMS1 promoter methylation and aberrant protein expression seem to be related to high-risk types of human papilloma virus-induced carcinogenesis in uterine cervix and is worthy of further investigation.

Dynamin-Related Protein 1 and Mitochondrial Fragmentation in Neurodegenerative Diseases

PubMed Central

Reddy, P. Hemachandra; Reddy, Tejaswini P.; Manczak, Maria; Calkins, Marcus J.; Shirendeb, Ulziibat; Mao, Peizhong

2010-01-01

The purpose of this article is to review the recent developments of abnormal mitochondrial dynamics, mitochondrial fragmentation, and neuronal damage in neurodegenerative diseases, including Alzheimer’s, Parkinson’s, Huntington’s, and amyotrophic lateral sclerosis. The GTPase family of proteins, including fission proteins, dynamin related protein 1 (Drp1), mitochondrial fission 1 (Fis1), and fusion proteins (Mfn1, Mfn2 and Opa1) are essential to maintain mitochondrial fission and fusion balance, and to provide necessary adenosine triphosphate to neurons. Among these, Drp1 is involved in several important aspects of mitochondria, including shape, size, distribution, remodeling, and maintenance of X in mammalian cells. In addition, recent advancements in molecular, cellular, electron microscopy, and confocal imaging studies revealed that Drp1 is associated with several cellular functions, including mitochondrial and peroxisomal fragmentation, phosphorylation, SUMOylation, ubiquitination, and cell death. In the last two decades, tremendous progress has been made in researching mitochondrial dynamics, in yeast, worms, and mammalian cells; and this research has provided evidence linking Drp1 to neurodegenerative diseases. Researchers in the neurodegenerative disease field are beginning to recognize the possible involvement of Drp1 in causing mitochondrial fragmentation and abnormal mitochondrial dynamics in neurodegenerative diseases. This article summarizes research findings relating Drp1 to mitochondrial fission and fusion, in yeast, worms, and mammals. Based on findings from the Reddy laboratory and others’, we propose that mutant proteins of neurodegenerative diseases, including AD, PD, HD, and ALS, interact with Drp1, activate mitochondrial fission machinery, fragment mitochondria excessively, and impair mitochondrial transport and mitochondrial dynamics, ultimately causing mitochondrial dysfunction and neuronal damage. PMID:21145355

Hypoxia Strongly Affects Mitochondrial Ribosomal Proteins and Translocases, as Shown by Quantitative Proteomics of HeLa Cells.

PubMed

Bousquet, Paula A; Sandvik, Joe Alexander; Arntzen, Magnus Ø; Jeppesen Edin, Nina F; Christoffersen, Stine; Krengel, Ute; Pettersen, Erik O; Thiede, Bernd

2015-01-01

Hypoxia is an important and common characteristic of many human tumors. It is a challenge clinically due to the correlation with poor prognosis and resistance to radiation and chemotherapy. Understanding the biochemical response to hypoxia would facilitate the development of novel therapeutics for cancer treatment. Here, we investigate alterations in gene expression in response to hypoxia by quantitative proteome analysis using stable isotope labeling with amino acids in cell culture (SILAC) in conjunction with LCMS/MS. Human HeLa cells were kept either in a hypoxic environment or under normoxic conditions. 125 proteins were found to be regulated, with maximum alteration of 18-fold. In particular, three clusters of differentially regulated proteins were identified, showing significant upregulation of glycolysis and downregulation of mitochondrial ribosomal proteins and translocases. This interaction is likely orchestrated by HIF-1. We also investigated the effect of hypoxia on the cell cycle, which shows accumulation in G1 and a prolonged S phase under these conditions. Implications. This work not only improves our understanding of the response to hypoxia, but also reveals proteins important for malignant progression, which may be targeted in future therapies.

Inflammation-initiating illnesses, inflammation-related proteins, and cognitive impairment in extremely preterm infants.

PubMed

O'Shea, T Michael; Shah, Bhavesh; Allred, Elizabeth N; Fichorova, Raina N; Kuban, Karl C K; Dammann, Olaf; Leviton, Alan

2013-03-01

Neonatal inflammation is associated with perinatal brain damage. We evaluated to what extent elevated blood levels of inflammation-related proteins supplement information about the risk of impaired early cognitive function provided by inflammation-related illnesses. From 800 infants born before the 28th week of gestation, we collected blood spots on days 1, 7 and 14, for analysis of 25 inflammation-related proteins, and data about culture-positive bacteremia, necrotizing enterocolitis (Bell stage IIIb), and isolated perforation of the intestine, during the first two weeks, and whether they were ventilated on postnatal day 14. We considered a protein to be persistently or recurrently elevated if its concentration was in the top quartile (for gestational age and day blood was collected) on two separate days one week apart. We assessed the children at 2 years of age with the Bayley Mental Development Index (MDI). The combinations of NEC and ventilation on day 14, and of bacteremia and ventilation on day 14 consistently provided information about elevated risk of MDI <55, regardless of whether or not a variable for an elevated protein concentration was included in the model. A variable for a persistently or recurrently elevated concentration of each of the following proteins provided additional information about an increased risk of MDI <55: CRP, SAA, IL-6, TNF-alpha, IL-8, MIP-1beta, ICAM-1, E-SEL, and IGFBP-1. We conclude that elevated blood concentrations of inflammation-related proteins provide information about the risk of impaired cognitive function at age 2 years that supplements information provided by inflammation-associated illnesses. Copyright © 2013 Elsevier Inc. All rights reserved.

The maize pathogenesis-related PRms protein localizes to plasmodesmata in maize radicles.

PubMed Central

Murillo, I; Cavallarin, L; San Segundo, B

1997-01-01

Pathogenesis-related (PR) proteins are plant proteins induced in response to infection by pathogens. In this study, an antibody raised against the maize PRms protein was used to localize the protein in fungal-infected maize radicles. The PRms protein was found to be localized at the contact areas between parenchyma cells of the differentiating protoxylem elements. By using immunoelectron microscopy, we found that these immunoreactive regions correspond to plasmodesmal regions. This was also true for the parenchyma cells filling the central pith of the vascular cylinder, although PRms mRNA accumulation was not detected in these cells. These findings suggest that for one cell type, the parenchyma cells of the central pith, the protein is imported rather than synthesized. The localization of the PRms protein indicates the possible existence of mechanisms for sorting of plant proteins to plasmodesmata and suggests that this protein may have a specialized function in the plant defense response. These findings are discussed with respect to the structure and function of plasmodesmata in cell-to-cell communication processes in higher plants. PMID:9061947

Clinical spectrum and diagnostic value of antibodies against the potassium channel-related protein complex☆

PubMed Central

Montojo, M.T.; Petit-Pedrol, M.; Graus, F.; Dalmau, J.

2016-01-01

Introduction Antibodies against a protein complex that includes voltage-gated potassium channels (VGKC) have been reported in patients with limbic encephalitis, peripheral nerve hyperexcitability, Morvan's syndrome, and a large variety of neurological syndromes. Review summary In this article, a review is presented of the syndromes associated with antibodies against VGKC-related proteins and the main antigens of this protein complex, the proteins LGI1 (leucine rich glioma inactivated protein 1) and Caspr2 (contactin-associated protein-like 2). The conceptual problems and clinical implications of the description of antibodies against VGKC-related proteins other than LGI1 and Caspr2 are also discussed. Although initial studies indicated the occurrence of antibodies against VGKC, recent investigations have shown that the main antigens are a neuronal secreted protein known as LGI1 which modulates synaptic excitability, and a protein called Caspr2 located on the cell surface and processes of neurons of different brain regions, and at the juxtaparanodal region of myelinated axons. While antibodies against LGI1 preferentially associate with classical limbic encephalitis, antibodies against Caspr2 associate with a wider spectrum of symptoms, including Morvan's syndrome, peripheral nerve hyperexcitability or neuromyotonia, and limbic or more extensive encephalitis. In addition there are reports of patients with antibodies against VGKC-related proteins that are different from LGI1 or Caspr2. In these cases, the identity and location of the antigens are unknown, the syndrome association is not specific, and the response to treatment uncertain. Conclusions The discovery of antigens such as LGI1 and Caspr2 has resulted in a clinical and molecular definition of the broad group of diseases previously attributed to antibodies against VGKC. Considering the literature that describes the presence of antibodies against VGKC other than LGI1 and Caspr2 proteins, we propose a practical

The reducibility of heLa cell viability by Sargassum polycystum extracts

NASA Astrophysics Data System (ADS)

Firdaus, M.; Setijawati, D.; Islam, I.; Nursyam, H.; Kartikaningsih, H.; Yufidasari, H. S.; Prihanto, A. A.; Nurdiani, R.; Jaziri, A. A.

2018-04-01

Cervical cancer is the second largest cause of death-related cancer in women. The efficacy of cancer drugs is still low. Bioactive of brown seaweed has been studied by in vitro and in vivo as anticancer. The aim of this study was to evaluate the cytotoxicity of Sargassum polycystum extracts on HeLa cell, to recognize bioactive on extract and estimate the interaction between the bioactive and target protein. S. polycystum was found from Talango Island waters and HeLa cell was obtained from Indonesian Science Institute. Sample was extracted by ethanol, ethyl acetate and hexane, concentrated and finally, extracts were assayed on HeLa cell. The viability of this cell was quantified on ELISA-Reader. The bioactive compounds of the extract were elucidated by GC-MS. The interaction between bioactive and target protein was evaluated by using in silico method. The result showed that the lowest viability of HeLa cell on n-hexane extracts treatment. The n-hexane extract of this seaweed contained benzenepropanoic acid. This compound reduced HeLa cell viability by reducing of thrombin concentration. In conclusion, the benzene propanoic acid of S. polycystum was the cytotoxic agent and it is potential agent for anti-cervical cancer.

Protective Immunogenicity of Group A Streptococcal M-Related Proteins

PubMed Central

Niedermeyer, Shannon E.; Agbaosi, Tina; Hysmith, Nicholas D.; Penfound, Thomas A.; Hohn, Claudia M.; Pullen, Matthew; Bright, Michael I.; Murrell, Daniel S.; Shenep, Lori E.; Courtney, Harry S.

2015-01-01

Many previous studies have focused on the surface M proteins of group A streptococci (GAS) as virulence determinants and protective antigens. However, the majority of GAS isolates express M-related protein (Mrp) in addition to M protein, and both have been shown to be required for optimal virulence. In the current study, we evaluated the protective immunogenicity of Mrp to determine its potential as a vaccine component that may broaden the coverage of M protein-based vaccines. Sequence analyses of 33 mrp genes indicated that there are three families of structurally related Mrps (MrpI, MrpII, and MrpIII). N-terminal peptides of Mrps were cloned, expressed, and purified from M type 2 (M2) (MrpI), M4 (MrpII), and M49 (MrpIII) GAS. Rabbit antisera against the Mrps reacted at high titers with the homologous Mrp, as determined by enzyme-linked immunosorbent assay, and promoted bactericidal activity against GAS emm types expressing Mrps within the same family. Mice passively immunized with rabbit antisera against MrpII were protected against challenge infections with M28 GAS. Assays for Mrp antibodies in serum samples from 281 pediatric subjects aged 2 to 16 indicated that the Mrp immune response correlated with increasing age of the subjects. Affinity-purified human Mrp antibodies promoted bactericidal activity against a number of GAS representing different emm types that expressed an Mrp within the same family but showed no activity against emm types expressing an Mrp from a different family. Our results indicate that Mrps have semiconserved N-terminal sequences that contain bactericidal epitopes which are immunogenic in humans. These findings may have direct implications for the development of GAS vaccines. PMID:25630406

Analysis of the interaction with the hepatitis C virus mRNA reveals an alternative mode of RNA recognition by the human La protein.

PubMed

Martino, Luigi; Pennell, Simon; Kelly, Geoff; Bui, Tam T T; Kotik-Kogan, Olga; Smerdon, Stephen J; Drake, Alex F; Curry, Stephen; Conte, Maria R

2012-02-01

Human La protein is an essential factor in the biology of both coding and non-coding RNAs. In the nucleus, La binds primarily to 3' oligoU containing RNAs, while in the cytoplasm La interacts with an array of different mRNAs lacking a 3' UUU(OH) trailer. An example of the latter is the binding of La to the IRES domain IV of the hepatitis C virus (HCV) RNA, which is associated with viral translation stimulation. By systematic biophysical investigations, we have found that La binds to domain IV using an RNA recognition that is quite distinct from its mode of binding to RNAs with a 3' UUU(OH) trailer: although the La motif and first RNA recognition motif (RRM1) are sufficient for high-affinity binding to 3' oligoU, recognition of HCV domain IV requires the La motif and RRM1 to work in concert with the atypical RRM2 which has not previously been shown to have a significant role in RNA binding. This new mode of binding does not appear sequence specific, but recognizes structural features of the RNA, in particular a double-stranded stem flanked by single-stranded extensions. These findings pave the way for a better understanding of the role of La in viral translation initiation.

Website on Protein Interaction and Protein Structure Related Work

NASA Technical Reports Server (NTRS)

Samanta, Manoj; Liang, Shoudan; Biegel, Bryan (Technical Monitor)

2003-01-01

In today's world, three seemingly diverse fields - computer information technology, nanotechnology and biotechnology are joining forces to enlarge our scientific knowledge and solve complex technological problems. Our group is dedicated to conduct theoretical research exploring the challenges in this area. The major areas of research include: 1) Yeast Protein Interactions; 2) Protein Structures; and 3) Current Transport through Small Molecules.

Characterization of the Sterol and Phosphatidylinositol 4-Phosphate Binding Properties of Golgi-Associated OSBP-Related Protein 9 (ORP9)

PubMed Central

Liu, Xinwei; Ridgway, Neale D.

2014-01-01

Oxysterol binding protein (OSBP) and OSBP-related proteins (ORPS) have a conserved lipid-binding fold that accommodates cholesterol, oxysterols and/or phospholipids. The diversity of OSBP/ORPs and their potential ligands has complicated the analysis of transfer and signalling properties of this mammalian gene family. In this study we explored the use of the fluorescent sterol cholestatrienol (CTL) to measure sterol binding by ORP9 and competition by other putative ligands. Relative to cholesterol, CTL and dehydroergosterol (DHE) were poor ligands for OSBP. In contrast, both long (ORP9L) and short (ORP9S) variants of ORP9 rapidly extracted CTL, and to a lesser extent DHE, from liposomes. ORP9L and ORP9S also extracted [32P]phosphatidylinositol 4-phosphate (PI-4P) from liposomes, which was inhibited by mutating two conserved histidine residues (HH488,489AA) at the entrance to the binding pocket but not by a mutation in the lid region that inhibited cholesterol binding. Results of direct binding and competition assays showed that phosphatidylserine was poorly extracted from liposomes by ORP9 compared to CTL and PI-4P. ORP9L and PI-4P did not co-localize in the trans-Golgi/TGN of HeLa cells, and siRNA silencing of ORP9L expression did not affect PI-4P distribution in the Golgi apparatus. However, transient overexpression of ORP9L or ORP9S in CHO cells, but not the corresponding PI-4P binding mutants, prevented immunostaining of Golgi-associated PI-4P. The apparent sequestration of Golgi PI-4P by ORP9S was identified as a possible mechanism for its growth inhibitory effects. These studies identify ORP9 as a dual sterol/PI-4P binding protein that could regulate PI-4P in the Golgi apparatus. PMID:25255026

Characterization of the sterol and phosphatidylinositol 4-phosphate binding properties of Golgi-associated OSBP-related protein 9 (ORP9).

PubMed

Liu, Xinwei; Ridgway, Neale D

2014-01-01

Oxysterol binding protein (OSBP) and OSBP-related proteins (ORPS) have a conserved lipid-binding fold that accommodates cholesterol, oxysterols and/or phospholipids. The diversity of OSBP/ORPs and their potential ligands has complicated the analysis of transfer and signalling properties of this mammalian gene family. In this study we explored the use of the fluorescent sterol cholestatrienol (CTL) to measure sterol binding by ORP9 and competition by other putative ligands. Relative to cholesterol, CTL and dehydroergosterol (DHE) were poor ligands for OSBP. In contrast, both long (ORP9L) and short (ORP9S) variants of ORP9 rapidly extracted CTL, and to a lesser extent DHE, from liposomes. ORP9L and ORP9S also extracted [32P]phosphatidylinositol 4-phosphate (PI-4P) from liposomes, which was inhibited by mutating two conserved histidine residues (HH488,489AA) at the entrance to the binding pocket but not by a mutation in the lid region that inhibited cholesterol binding. Results of direct binding and competition assays showed that phosphatidylserine was poorly extracted from liposomes by ORP9 compared to CTL and PI-4P. ORP9L and PI-4P did not co-localize in the trans-Golgi/TGN of HeLa cells, and siRNA silencing of ORP9L expression did not affect PI-4P distribution in the Golgi apparatus. However, transient overexpression of ORP9L or ORP9S in CHO cells, but not the corresponding PI-4P binding mutants, prevented immunostaining of Golgi-associated PI-4P. The apparent sequestration of Golgi PI-4P by ORP9S was identified as a possible mechanism for its growth inhibitory effects. These studies identify ORP9 as a dual sterol/PI-4P binding protein that could regulate PI-4P in the Golgi apparatus.

Protein v. carbohydrate intake differentially affects liking- and wanting-related brain signalling.

PubMed

Born, Jurriaan M; Martens, Mieke J I; Lemmens, Sofie G T; Goebel, Rainer; Westerterp-Plantenga, Margriet S

2013-01-28

Extreme macronutrient intakes possibly lead to different brain signalling. The aim of the present study was to determine the effects of ingesting high-protein v. high-carbohydrate food on liking and wanting task-related brain signalling (TRS) and subsequent macronutrient intake. A total of thirty female subjects (21.6 (SD 2.2) years, BMI 25.0 (SD 3.7) kg/m²) completed four functional MRI scans: two fasted and two satiated on two different days. During the scans, subjects rated all food items for liking and wanting, thereby choosing the subsequent meal. The results show that high-protein (PROT) v. high-carbohydrate (CARB) conditions were generated using protein or carbohydrate drinks at the first meal. Energy intake and hunger were recorded. PROT (protein: 53.7 (SD 2.1) percentage of energy (En%); carbohydrate: 6.4 (SD 1.3) En%) and CARB conditions (protein: 11.8 (SD 0.6) En%; carbohydrate: 70.0 (SD 2.4) En%) were achieved during the first meal, while the second meals were not different between the conditions. Hunger, energy intake, and behavioural liking and wanting ratings were decreased after the first meal (P< 0.001). Comparing the first with the second meal, the macronutrient content changed: carbohydrate -26.9 En% in the CARB condition, protein -37.8 En% in the PROT condition. After the first meal in the CARB condition, wanting TRS was increased in the hypothalamus. After the first meal in the PROT condition, liking TRS was decreased in the putamen (P< 0.05). The change in energy intake from the first to the second meal was inversely related to the change in liking TRS in the striatum and hypothalamus in the CARB condition and positively related in the PROT condition (P< 0.05). In conclusion, wanting and liking TRS were affected differentially with a change in carbohydrate or protein intake, underscoring subsequent energy intake and shift in macronutrient composition.

Relations de Dispersion et Diffusion des Glueballs et des Mesons dans la Theorie de Jauge U(1)(2+1) Compacte

NASA Astrophysics Data System (ADS)

Ahmed, Chaara El Mouez

Nous avons etudie les relations de dispersion et la diffusion des glueballs et des mesons dans le modele U(1)_{2+1} compact. Ce modele a ete souvent utilise comme un simple modele de la chromodynamique quantique (QCD), parce qu'il possede le confinement ainsi que les etats de glueballs. Par contre, sa structure mathematique est beaucoup plus simple que la QCD. Notre methode consiste a diagonaliser l'Hamiltonien de ce modele dans une base appropriee de graphes et sur reseau impulsion, afin de generer les relations de dispersion des glueballs et des mesons. Pour la diffusion, nous avons utilise la methode dependante du temps pour calculer la matrice S et la section efficace de diffusion des glueballs et des mesons. Les divers resultats obtenus semblent etre en accord avec les travaux anterieurs de Hakim, Alessandrini et al., Irving et al., qui eux, utilisent plutot la theorie des perturbations en couplage fort, et travaillent sur un reseau espace-temps.

On the nature of a glassy state of matter in a hydrated protein: Relation to protein function.

PubMed

Teeter, M M; Yamano, A; Stec, B; Mohanty, U

2001-09-25

Diverse biochemical and biophysical experiments indicate that all proteins, regardless of size or origin, undergo a dynamic transition near 200 K. The cause of this shift in dynamic behavior, termed a "glass transition," and its relation to protein function are important open questions. One explanation postulated for the transition is solidification of correlated motions in proteins below the transition. We verified this conjecture by showing that crambin's radius of gyration (Rg) remains constant below approximately 180 K. We show that both atom position and dynamics of protein and solvent are physically coupled, leading to a novel cooperative state. This glassy state is identified by negative slopes of the Debye-Waller (B) factor vs. temperature. It is composed of multisubstate side chains and solvent. Based on generalization of Adam-Gibbs' notion of a cooperatively rearranging region and decrease of the total entropy with temperature, we calculate the slope of the Debye-Waller factor. The results are in accord with experiment.

Quantity of protein deposited on hydrogel contact lenses and its relation to visible protein deposits.

PubMed

Myers, R I; Larsen, D W; Tsao, M; Castellano, C; Becherer, L D; Fontana, F; Ghormley, N R; Meier, G

1991-10-01

The purposes of this study were to determine if the quantity of protein deposited (QPD) upon hydrogel lenses was affected by enzymatic cleaning and to test the potential relation between QPD and visible protein deposition (VPD) and change. Seventy-four contact lens patients classified as "heavy depositors" wore new lenses for an average of 80 (SD = 32) days. Cleaning and disinfection solutions varied. One lens was cleaned weekly by a papain enzymatic treatment. The distribution of QPD measurements was bimodal and was related to the FDA material for nonionic, low water content lenses (FDA Materials Group no. 1). The mean deposition was 45 micrograms/cm2 (N = 112) compared with that of ionic, high water content lenses (FDA Materials Group no. 4), which was 1010 micrograms/cm2 (N = 30). VPD distributions were the same for the FDA Group no. 1 and no. 4 lenses. Enzymatic treatment did not significantly reduce QPD; however, enzymatic treatment did reduce VPD. Thus QPD and VPD are independent phenomena and possible reasons for this are given.

Molecular Basis for Transmission Barrier and Interference between Closely Related Prion Proteins in Yeast*

PubMed Central

Afanasieva, Evgenia G.; Kushnirov, Vitaly V.; Tuite, Mick F.; Ter-Avanesyan, Michael D.

2011-01-01

Replicating amyloids, called prions, are responsible for transmissible neurodegenerative diseases in mammals and some heritable phenotypes in fungi. The transmission of prions between species is usually inhibited, being highly sensitive to small differences in amino acid sequence of the prion-forming proteins. To understand the molecular basis of this prion interspecies barrier, we studied the transmission of the [PSI+] prion state from Sup35 of Saccharomyces cerevisiae to hybrid Sup35 proteins with prion-forming domains from four other closely related Saccharomyces species. Whereas all the hybrid Sup35 proteins could adopt a prion form in S. cerevisiae, they could not readily acquire the prion form from the [PSI+] prion of S. cerevisiae. Expression of the hybrid Sup35 proteins in S. cerevisiae [PSI+] cells often resulted in frequent loss of the native [PSI+] prion. Furthermore, all hybrid Sup35 proteins showed different patterns of interaction with the native [PSI+] prion in terms of co-polymerization, acquisition of the prion state, and induced prion loss, all of which were also dependent on the [PSI+] variant. The observed loss of S. cerevisiae [PSI+] can be related to inhibition of prion polymerization of S. cerevisiae Sup35 and formation of a non-heritable form of amyloid. We have therefore identified two distinct molecular origins of prion transmission barriers between closely sequence-related prion proteins: first, the inability of heterologous proteins to co-aggregate with host prion polymers, and second, acquisition by these proteins of a non-heritable amyloid fold. PMID:21454674

Ro small cytoplasmic ribonucleoproteins are a subclass of La ribonucleoproteins: further characterization of the Ro and La small ribonucleoproteins from uninfected mammalian cells.

PubMed Central

Hendrick, J P; Wolin, S L; Rinke, J; Lerner, M R; Steitz, J A

1981-01-01

Small ribonucleic acid (RNA)-protein complexes precipitated by anti-Ro and anti-La antibodies from lupus patients have been examined with emphasis on their RNA components. In both ribonucleoprotein (RNP) classes, the numbers of different RNA molecules and their sequences vary between mouse and human cells. The complex mixtures of La RNAs include two previously sequenced 4.5S RNAs from mouse cells and 5S ribosomal RNA-like molecules from both mouse and human cells. All Ro and La RNAs possess 5-triphosphates. Some La RNAs have internal modifications typical of transfer RNAs. The Ro RNPs are quite stable and are localized by immunofluorescence in the cell cytoplasm, whereas the majority of the La RNPs turn over rapidly and reside in the nucleus. Despite these differences, reconstitution experiments show that the Ro particles carry the La as well as the Ro determinant. Studies using a nuclear transcription system demonstrate that most of the La RNAs are synthesized by RNA polymerase III. The possibility that the La protein(s) functions in the transcription or maturation of all RNA polymerase III transcripts is discussed. Images PMID:6180298

Comparisons of MRI images, and auditory-related and vocal-related protein expressions in the brain of echolocation bats and rodents.

PubMed

Hsiao, Chun-Jen; Hsu, Chih-Hsiang; Lin, Ching-Lung; Wu, Chung-Hsin; Jen, Philip Hung-Sun

2016-08-17

Although echolocating bats and other mammals share the basic design of laryngeal apparatus for sound production and auditory system for sound reception, they have a specialized laryngeal mechanism for ultrasonic sound emissions as well as a highly developed auditory system for processing species-specific sounds. Because the sounds used by bats for echolocation and rodents for communication are quite different, there must be differences in the central nervous system devoted to producing and processing species-specific sounds between them. The present study examines the difference in the relative size of several brain structures and expression of auditory-related and vocal-related proteins in the central nervous system of echolocation bats and rodents. Here, we report that bats using constant frequency-frequency-modulated sounds (CF-FM bats) and FM bats for echolocation have a larger volume of midbrain nuclei (inferior and superior colliculi) and cerebellum relative to the size of the brain than rodents (mice and rats). However, the former have a smaller volume of the cerebrum and olfactory bulb, but greater expression of otoferlin and forkhead box protein P2 than the latter. Although the size of both midbrain colliculi is comparable in both CF-FM and FM bats, CF-FM bats have a larger cerebrum and greater expression of otoferlin and forkhead box protein P2 than FM bats. These differences in brain structure and protein expression are discussed in relation to their biologically relevant sounds and foraging behavior.

Heat-induced Protein Structure and Subfractions in Relation to Protein Degradation Kinetics and Intestinal Availability in Dairy Cattle

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

Doiron, K.; Yu, P; McKinnon, J

2009-01-01

The objectives of this study were to reveal protein structures of feed tissues affected by heat processing at a cellular level, using the synchrotron-based Fourier transform infrared microspectroscopy as a novel approach, and quantify protein structure in relation to protein digestive kinetics and nutritive value in the rumen and intestine in dairy cattle. The parameters assessed included (1) protein structure a-helix to e-sheet ratio; (2) protein subfractions profiles; (3) protein degradation kinetics and effective degradability; (4) predicted nutrient supply using the intestinally absorbed protein supply (DVE)/degraded protein balance (OEB) system for dairy cattle. In this study, Vimy flaxseed protein wasmore » used as a model feed protein and was autoclave-heated at 120C for 20, 40, and 60 min in treatments T1, T2, and T3, respectively. The results showed that using the synchrotron-based Fourier transform infrared microspectroscopy revealed and identified the heat-induced protein structure changes. Heating at 120C for 40 and 60 min increased the protein structure a-helix to e-sheet ratio. There were linear effects of heating time on the ratio. The heating also changed chemical profiles, which showed soluble CP decreased upon heating with concomitant increases in nonprotein nitrogen, neutral, and acid detergent insoluble nitrogen. The protein subfractions with the greatest changes were PB1, which showed a dramatic reduction, and PB2, which showed a dramatic increase, demonstrating a decrease in overall protein degradability. In situ results showed a reduction in rumen-degradable protein and in rumen-degradable dry matter without differences between the treatments. Intestinal digestibility, determined using a 3-step in vitro procedure, showed no changes to rumen undegradable protein. Modeling results showed that heating increased total intestinally absorbable protein (feed DVE value) and decreased degraded protein balance (feed OEB value), but there were no

Gene Expression Analysis Reveals the Concurrent Activation of Proapoptotic and Antioxidant-Defensive Mechanisms in Flavokawain B-Treated Cervical Cancer HeLa Cells.

PubMed

Yeap, Swee Keong; Abu, Nadiah; Akthar, Nadeem; Ho, Wan Yong; Ky, Huynh; Tan, Sheau Wei; Alitheen, Noorjahan Banu; Kamarul, Tunku

2017-09-01

Flavokawain B (FKB) is known to possess promising anticancer abilities. This is demonstrated in various cancer cell lines including HeLa cells. Cervical cancer is among the most widely diagnosed cancer among women today. Though FKB has been shown to be effective in treating cancer cells, the exact molecular mechanism is still unknown. This study is aimed at understanding the effects of FKB on HeLa cells using a microarray-based mRNA expression profiling and proteome profiling of stress-related proteins. The results of this study suggest that FKB induced cell death through p21-mediated cell cycle arrest and activation of p38. However, concurrent activation of antioxidant-related pathways and iron sequestration pathway followed by activation of ER-resident stress proteins clearly indicate that FKB failed to induce apoptosis in HeLa cells via oxidative stress. This effect implies that the protection of HeLa cells by FKB from H 2 O 2 -induced cell death is via neutralization of reactive oxygen species.

High Dietary Protein Intake and Protein-Related Acid Load on Bone Health.

PubMed

Cao, Jay J

2017-12-01

Consumption of high-protein diets is increasingly popular due to the benefits of protein on preserving lean mass and controlling appetite and satiety. The paper is to review recent clinical research assessing dietary protein on calcium metabolism and bone health. Epidemiological studies show that long-term, high-protein intake is positively associated with bone mineral density and reduced risk of bone fracture incidence. Short-term interventional studies demonstrate that a high-protein diet does not negatively affect calcium homeostasis. Existing evidence supports that the negative effects of the acid load of protein on urinary calcium excretion are offset by the beneficial skeletal effects of high-protein intake. Future research should focus on the role and the degree of contribution of other dietary and physiological factors, such as intake of fruits and vegetables, in reducing the acid load and further enhancing the anabolic effects of protein on the musculoskeletal system.

76 FR 63316 - Prospective Grant of Exclusive License: Secreted Frizzled Related Protein-1 (sFRP-1) and...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-10-12

... Exclusive License: Secreted Frizzled Related Protein-1 (sFRP-1) and derivatives thereof and their Use In... belonging to the patent families having HHS Reference Numbers E-160-1997/0,/1,/2 and/3; E-014-2000/0; and E... Related Protein-1 (sFRP-1). sFRP-1, also known as SARP-2 (Secreted Apoptosis Related Protein-2). The IP...

Noninvasive imaging of protein-protein interactions in living animals

NASA Astrophysics Data System (ADS)

Luker, Gary D.; Sharma, Vijay; Pica, Christina M.; Dahlheimer, Julie L.; Li, Wei; Ochesky, Joseph; Ryan, Christine E.; Piwnica-Worms, Helen; Piwnica-Worms, David

2002-05-01

Protein-protein interactions control transcription, cell division, and cell proliferation as well as mediate signal transduction, oncogenic transformation, and regulation of cell death. Although a variety of methods have been used to investigate protein interactions in vitro and in cultured cells, none can analyze these interactions in intact, living animals. To enable noninvasive molecular imaging of protein-protein interactions in vivo by positron-emission tomography and fluorescence imaging, we engineered a fusion reporter gene comprising a mutant herpes simplex virus 1 thymidine kinase and green fluorescent protein for readout of a tetracycline-inducible, two-hybrid system in vivo. By using micro-positron-emission tomography, interactions between p53 tumor suppressor and the large T antigen of simian virus 40 were visualized in tumor xenografts of HeLa cells stably transfected with the imaging constructs. Imaging protein-binding partners in vivo will enable functional proteomics in whole animals and provide a tool for screening compounds targeted to specific protein-protein interactions in living animals.

Eukaryotic elongation factor 2 kinase regulates the synthesis of microtubule-related proteins in neurons.

PubMed

Kenney, Justin W; Genheden, Maja; Moon, Kyung-Mee; Wang, Xuemin; Foster, Leonard J; Proud, Christopher G

2016-01-01

Modulation of the elongation phase of protein synthesis is important for numerous physiological processes in both neurons and other cell types. Elongation is primarily regulated via eukaryotic elongation factor 2 kinase (eEF2K). However, the consequence of altering eEF2K activity on the synthesis of specific proteins is largely unknown. Using both pharmacological and genetic manipulations of eEF2K combined with two protein-labeling techniques, stable isotope labeling of amino acids in cell culture and bio-orthogonal non-canonical amino acid tagging, we identified a subset of proteins whose synthesis is sensitive to inhibition of eEF2K in murine primary cortical neurons. Gene ontology (GO) analyses indicated that processes related to microtubules are particularly sensitive to eEF2K inhibition. Our findings suggest that eEF2K likely contributes to neuronal function by regulating the synthesis of microtubule-related proteins. Modulation of the elongation phase of protein synthesis is important for numerous physiological processes in neurons. Here, using labeling of new proteins coupled with proteomic techniques in primary cortical neurons, we find that the synthesis of microtubule-related proteins is up-regulated by inhibition of elongation. This suggests that translation elongation is a key regulator of cytoskeletal dynamics in neurons. © 2015 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.

Clinical spectrum and diagnostic value of antibodies against the potassium channel related protein complex.

PubMed

Montojo, M T; Petit-Pedrol, M; Graus, F; Dalmau, J

2015-06-01

Antibodies against a protein complex that includes voltage-gated potassium channels (VGKC) have been reported in patients with limbic encephalitis, peripheral nerve hyperexcitability, Morvan's syndrome, and a large variety of neurological syndromes. In this article, a review is presented of the syndromes associated with antibodies against VGKC-related proteins and the main antigens of this protein complex, the proteins LGI1 (leucine rich glioma inactivated protein 1) and Caspr2 (contactin-associated protein-like 2). The conceptual problems and clinical implications of the description of antibodies against VGKC-related proteins other than LGI1 and Caspr2 are also discussed. Although initial studies indicated the occurrence of antibodies against VGKC, recent investigations have shown that the main antigens are a neuronal secreted protein known as LGI1 which modulates synaptic excitability, and a protein called Caspr2 located on the cell surface and processes of neurons of different brain regions, and at the juxtaparanodal region of myelinated axons. While antibodies against LGI1 preferentially associate with classical limbic encephalitis, antibodies against Caspr2 associate with a wider spectrum of symptoms, including Morvan's syndrome, peripheral nerve hyperexcitability or neuromyotonia, and limbic or more extensive encephalitis. In addition there are reports of patients with antibodies against VGKC-related proteins that are different from LGI1 or Caspr2. In these cases, the identity and location of the antigens are unknown, the syndrome association is not specific, and the response to treatment uncertain. The discovery of antigens such as LGI1 and Caspr2 has resulted in a clinical and molecular definition of the broad group of diseases previously attributed to antibodies against VGKC. Considering the literature that describes the presence of antibodies against VGKC other than LGI1 and Caspr2 proteins, we propose a practical algorithm for the diagnosis and treatment

A close relative of the nuclear, chromosomal high-mobility group protein HMG1 in yeast mitochondria.

PubMed Central

Diffley, J F; Stillman, B

1991-01-01

ABF2 (ARS-binding factor 2), a small, basic DNA-binding protein that binds specifically to the autonomously replicating sequence ARS1, is located primarily in the mitochondria of the yeast Saccharomyces cerevisiae. The abundance of ABF2 and the phenotype of abf2- null mutants argue that this protein plays a key role in the structure, maintenance, and expression of the yeast mitochondrial genome. The predicted amino acid sequence of ABF2 is closely related to the high-mobility group proteins HMG1 and HMG2 from vertebrate cell nuclei and to several other DNA-binding proteins. Additionally, ABF2 and the other HMG-related proteins are related to a globular domain from the heat shock protein hsp70 family. ABF2 interacts with DNA both nonspecifically and in a specific manner within regulatory regions, suggesting a mechanism whereby it may aid in compacting the mitochondrial genome without interfering with expression. Images PMID:1881919

High dietary protein intake and protein-related acid load on bone health

USDA-ARS?s Scientific Manuscript database

Protein is an essential nutrient for humans and is required for maintaining optimal bone structure and growth. Consumption of high protein diets in excess of the Recommended Dietary Allowance of (0.8 g protein/kg body weight/d) is increasingly popular due to the benefits of protein on preserving lea...

Analysis of metal-binding proteins separated by non-denaturating gel electrophoresis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS).

PubMed

Becker, J Susanne; Mounicou, Sandra; Zoriy, Miroslav V; Becker, J Sabine; Lobinski, Ryszard

2008-09-15

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) have become established as very efficient and sensitive biopolymer and elemental mass spectrometric techniques for studying metal-binding proteins (metalloproteins) in life sciences. Protein complexes present in rat tissues (liver and kidney) were separated in their native state in the first dimension by blue native gel electrophoresis (BN-PAGE). Essential and toxic metals, such as zinc, copper, iron, nickel, chromium, cadmium and lead, were detected by scanning the gel bands using quadrupole LA-ICP-MS with and without collision cell as a microanalytical technique. Several proteins were identified by using MALDI-TOF-MS together with a database search. For example, on one protein band cut from the BN-PAGE gel and digested with the enzyme trypsin, two different proteins - protein FAM44B and cathepsin B precursor - were identified. By combining biomolecular and elemental mass spectrometry, it was possible to characterize and identify selected metal-binding rat liver and kidney tissue proteins.

Age-related carbonylation of fibrocartilage structural proteins drives tissue degenerative modification.

PubMed

Scharf, Brian; Clement, Cristina C; Yodmuang, Supansa; Urbanska, Aleksandra M; Suadicani, Sylvia O; Aphkhazava, David; Thi, Mia M; Perino, Giorgio; Hardin, John A; Cobelli, Neil; Vunjak-Novakovic, Gordana; Santambrogio, Laura

2013-07-25

Aging-related oxidative stress has been linked to degenerative modifications in different organs and tissues. Using redox proteomic analysis and illustrative tandem mass spectrometry mapping, we demonstrate oxidative posttranslational modifications in structural proteins of intervertebral discs (IVDs) isolated from aging mice. Increased protein carbonylation was associated with protein fragmentation and aggregation. Complementing these findings, a significant loss of elasticity and increased stiffness was measured in fibrocartilage from aging mice. Studies using circular dichroism and intrinsic tryptophan fluorescence revealed a significant loss of secondary and tertiary structures of purified collagens following oxidation. Collagen unfolding and oxidation promoted both nonenzymatic and enzymatic degradation. Importantly, induction of oxidative modification in healthy fibrocartilage recapitulated the biochemical and biophysical modifications observed in the aging IVD. Together, these results suggest that protein carbonylation, glycation, and lipoxidation could be early events in promoting IVD degenerative changes. Copyright © 2013 Elsevier Ltd. All rights reserved.

Prion Protein M129V Polymorphism Affects Retrieval-Related Brain Activity

ERIC Educational Resources Information Center

Buchmann, Andreas; Mondadori, Christian R. A.; Hanggi, Jurgen; Aerni, Amanda; Vrticka, Pascal; Luechinger, Roger; Boesiger, Peter; Hock, Christoph; Nitsch, Roger M.; de Quervain, Dominique J.-F.; Papassotiropoulos, Andreas; Henke, Katharina

2008-01-01

The prion protein Met129Val polymorphism has recently been related to human long-term memory with carriers of either the 129[superscript MM] or the 129[superscript MV] genotype recalling 17% more words than 129[superscript VV] carriers at 24 h following learning. Here, we sampled genotype differences in retrieval-related brain activity at 30 min…

Omega-3 Fatty Acid Docosahexaenoic Acid Increases SorLA/LR11, a Sorting Protein with Reduced Expression in Sporadic Alzheimer’s Disease (AD): Relevance to AD Prevention

PubMed Central

Ma, Qiu-Lan; Teter, Bruce; Ubeda, Oliver J.; Morihara, Takashi; Dhoot, Dilsher; Nyby, Michael D.; Tuck, Michael L.; Frautschy, Sally A.; Cole, Greg M.

2008-01-01

Environmental and genetic factors, notably ApoE4, contribute to the etiology of late-onset Alzheimer’s disease (LOAD). Reduced mRNA and protein for an apolipoprotein E (ApoE) receptor family member, SorLA (LR11) has been found in LOAD but not early-onset AD, suggesting that LR11 loss is not secondary to pathology. LR11 is a neuronal sorting protein that reduces amyloid precursor protein (APP) trafficking to secretases that generate β-amyloid (Aβ). Genetic polymorphisms that reduce LR11 expression are associated with increased AD risk. However these polymorphisms account for only a fraction of cases with LR11 deficits, suggesting involvement of environmental factors. Because lipoprotein receptors are typically lipid-regulated, we postulated that LR11 is regulated by docosahexaenoic acid (DHA), an essential ω-3 fatty acid related to reduced AD risk and reduced Aβ accumulation. In this study, we report that DHA significantly increases LR11 in multiple systems, including primary rat neurons, aged non-Tg mice and an aged DHA-depleted APPsw AD mouse model. DHA also increased LR11 in a human neuronal line. In vivo elevation of LR11 was also observed with dietary fish oil in young rats with insulin resistance, a model for type II diabetes, another AD risk factor. These data argue that DHA induction of LR11 does not require DHA-depleting diets and is not age dependent. Because reduced LR11 is known to increase Aβ production and may be a significant genetic cause of LOAD, our results indicate that DHA increases in SorLA/LR11 levels may play an important role in preventing LOAD. PMID:18160637

The etiology of human age-related cataract. Proteins don't last forever.

PubMed

Truscott, Roger J W; Friedrich, Michael G

2016-01-01

It is probable that the great majority of human cataract results from the spontaneous decomposition of long-lived macromolecules in the human lens. Breakdown/reaction of long-lived proteins is of primary importance and recent proteomic analysis has enabled the identification of the particular crystallins, and their exact sites of amino acid modification. Analysis of proteins from cataractous lenses revealed that there are sites on some structural proteins that show a consistently greater degree of deterioration than age-matched normal lenses. The most abundant posttranslational modification of aged lens proteins is racemization. Deamidation, truncation and crosslinking, each arising from the spontaneous breakdown of susceptible amino acids within proteins, are also present. Fundamental to an understanding of nuclear cataract etiology, it is proposed that once a certain degree of modification at key sites occurs, that protein-protein interactions are disrupted and lens opacification ensues. Since long-lived proteins are now recognized to be present in many other sites of the body, such as the brain, the information gleaned from detailed analyses of degraded proteins from aged lenses will apply more widely to other age-related human diseases. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease. Copyright © 2015 Elsevier B.V. All rights reserved.

Performance benchmarking of four cell-free protein expression systems.

PubMed

Gagoski, Dejan; Polinkovsky, Mark E; Mureev, Sergey; Kunert, Anne; Johnston, Wayne; Gambin, Yann; Alexandrov, Kirill

2016-02-01

Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species-independent translation initiation sequence to express and characterize 87 N-terminally GFP-tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems. Using a combination of single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full-length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species-particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE-produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins. © 2015 Wiley Periodicals, Inc.

Cysteine-rich domains related to Frizzled receptors and Hedgehog-interacting proteins

PubMed Central

Pei, Jimin; Grishin, Nick V

2012-01-01

Frizzled and Smoothened are homologous seven-transmembrane proteins functioning in the Wnt and Hedgehog signaling pathways, respectively. They harbor an extracellular cysteine-rich domain (FZ-CRD), a mobile evolutionary unit that has been found in a number of other metazoan proteins and Frizzled-like proteins in Dictyostelium. Domains distantly related to FZ-CRDs, in Hedgehog-interacting proteins (HHIPs), folate receptors and riboflavin-binding proteins (FRBPs), and Niemann-Pick Type C1 proteins (NPC1s), referred to as HFN-CRDs, exhibit similar structures and disulfide connectivity patterns compared with FZ-CRDs. We used computational analyses to expand the homologous set of FZ-CRDs and HFN-CRDs, providing a better understanding of their evolution and classification. First, FZ-CRD-containing proteins with various domain compositions were identified in several major eukaryotic lineages including plants and Chromalveolata, revealing a wider phylogenetic distribution of FZ-CRDs than previously recognized. Second, two new and distinct groups of highly divergent FZ-CRDs were found by sensitive similarity searches. One of them is present in the calcium channel component Mid1 in fungi and the uncharacterized FAM155 proteins in metazoans. Members of the other new FZ-CRD group occur in the metazoan-specific RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) proteins that are putative tumor suppressors acting as inhibitors of matrix metalloproteases. Finally, sequence and three-dimensional structural comparisons helped us uncover a divergent HFN-CRD in glypicans, which are important morphogen-binding heparan sulfate proteoglycans. Such a finding reinforces the evolutionary ties between the Wnt and Hedgehog signaling pathways and underscores the importance of gene duplications in creating essential signaling components in metazoan evolution. PMID:22693159

Some physical approaches to protein folding

NASA Astrophysics Data System (ADS)

Bascle, J.; Garel, T.; Orland, H.

1993-02-01

To understand how a protein folds is a problem which has important biological implications. In this article, we would like to present a physics-oriented point of view, which is twofold. First of all, we introduce simple statistical mechanics models which display, in the thermodynamic limit, folding and related transitions. These models can be divided into (i) crude spin glass-like models (with their Mattis analogs), where one may look for possible correlations between the chain self-interactions and the folded structure, (ii) glass-like models, where one emphasizes the geometrical competition between one- or two-dimensional local order (mimicking α helix or β sheet structures), and the requirement of global compactness. Both models are too simple to predict the spatial organization of a realistic protein, but are useful for the physicist and should have some feedback in other glassy systems (glasses, collapsed polymers .... ). These remarks lead us to the second physical approach, namely a new Monte-Carlo method, where one grows the protein atom-by-atom (or residue-by-residue), using a standard form (CHARMM .... ) for the total energy. A detailed comparison with other Monte-Carlo schemes, or Molecular Dynamics calculations, is then possible; we will sketch such a comparison for poly-alanines. Our twofold approach illustrates some of the difficulties one encounters in the protein folding problem, in particular those associated with the existence of a large number of metastable states. Le repliement des protéines est un problème qui a de nombreuses implications biologiques. Dans cet article, nous présentons, de deux façons différentes, un point de vue de physicien. Nous introduisons tout d'abord des modèles simples de mécanique statistique qui exhibent, à la limite thermodynamique, des transitions de repliement. Ces modèles peuvent être divisés en (i) verres de spin (éventuellement à la Mattis), où l'on peut chercher des corrélations entre les

The 29-kDa proteins phosphorylated ion thrombin-activated human platelets are forms of the estrogen receptor-related 27-kDa heat shock protein

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

Mendelsohn, M.E.; Yan Zhu; O'Neill, S.

Thrombin plays a critical role in platelet activation, hemostasis, and thrombosis. Cellular activation by thrombin leads to the phosphorylation of multiple proteins, most of which are unidentified. The authors have characterized several 29-kDa proteins that are rapidly phosphorylated following exposure of intact human platelets to thrombin. A murine monoclonal antibody raised to an unidentified estrogen receptor-related 29-kDa protein selectively recognized these proteins as well as a more basic, unphosphorylated 27-kDa protein. Cellular activation by thrombin led to a marked shift in the proportion of protein from the 27-kDa unphosphorylated form to the 29-kDa phosphoprotein species. Using this antibody, they isolatedmore » and sequenced a human cDNA clone encoding a protein that was identical to the mammalian 27-kDa heat shock protein (HSP27), a protein of uncertain function that is known to be phosphorylated to several forms and to be transcriptionally induced by estrogen. The 29-kDa proteins were confirmed to be phosphorylated forms of HSP27 by immunoprecipitation studies. Thus, the estrogen receptor-related protein is HSP27, and the three major 20-kDa proteins phosphorylated in thrombin-activated platelets are forms of HSP27. These data suggest a role for HSP27 in the signal transduction events of platelet activation.« less

Incorporating significant amino acid pairs and protein domains to predict RNA splicing-related proteins with functional roles

NASA Astrophysics Data System (ADS)

Hsu, Justin Bo-Kai; Huang, Kai-Yao; Weng, Tzu-Ya; Huang, Chien-Hsun; Lee, Tzong-Yi

2014-01-01

Machinery of pre-mRNA splicing is carried out through the interaction of RNA sequence elements and a variety of RNA splicing-related proteins (SRPs) (e.g. spliceosome and splicing factors). Alternative splicing, which is an important post-transcriptional regulation in eukaryotes, gives rise to multiple mature mRNA isoforms, which encodes proteins with functional diversities. However, the regulation of RNA splicing is not yet fully elucidated, partly because SRPs have not yet been exhaustively identified and the experimental identification is labor-intensive. Therefore, we are motivated to design a new method for identifying SRPs with their functional roles in the regulation of RNA splicing. The experimentally verified SRPs were manually curated from research articles. According to the functional annotation of Splicing Related Gene Database, the collected SRPs were further categorized into four functional groups including small nuclear Ribonucleoprotein, Splicing Factor, Splicing Regulation Factor and Novel Spliceosome Protein. The composition of amino acid pairs indicates that there are remarkable differences among four functional groups of SRPs. Then, support vector machines (SVMs) were utilized to learn the predictive models for identifying SRPs as well as their functional roles. The cross-validation evaluation presents that the SVM models trained with significant amino acid pairs and functional domains could provide a better predictive performance. In addition, the independent testing demonstrates that the proposed method could accurately identify SRPs in mammals/plants as well as effectively distinguish between SRPs and RNA-binding proteins. This investigation provides a practical means to identifying potential SRPs and a perspective for exploring the regulation of RNA splicing.

Incorporating significant amino acid pairs and protein domains to predict RNA splicing-related proteins with functional roles.

PubMed

Hsu, Justin Bo-Kai; Huang, Kai-Yao; Weng, Tzu-Ya; Huang, Chien-Hsun; Lee, Tzong-Yi

2014-01-01

Machinery of pre-mRNA splicing is carried out through the interaction of RNA sequence elements and a variety of RNA splicing-related proteins (SRPs) (e.g. spliceosome and splicing factors). Alternative splicing, which is an important post-transcriptional regulation in eukaryotes, gives rise to multiple mature mRNA isoforms, which encodes proteins with functional diversities. However, the regulation of RNA splicing is not yet fully elucidated, partly because SRPs have not yet been exhaustively identified and the experimental identification is labor-intensive. Therefore, we are motivated to design a new method for identifying SRPs with their functional roles in the regulation of RNA splicing. The experimentally verified SRPs were manually curated from research articles. According to the functional annotation of Splicing Related Gene Database, the collected SRPs were further categorized into four functional groups including small nuclear Ribonucleoprotein, Splicing Factor, Splicing Regulation Factor and Novel Spliceosome Protein. The composition of amino acid pairs indicates that there are remarkable differences among four functional groups of SRPs. Then, support vector machines (SVMs) were utilized to learn the predictive models for identifying SRPs as well as their functional roles. The cross-validation evaluation presents that the SVM models trained with significant amino acid pairs and functional domains could provide a better predictive performance. In addition, the independent testing demonstrates that the proposed method could accurately identify SRPs in mammals/plants as well as effectively distinguish between SRPs and RNA-binding proteins. This investigation provides a practical means to identifying potential SRPs and a perspective for exploring the regulation of RNA splicing.

PPInterFinder—a mining tool for extracting causal relations on human proteins from literature

PubMed Central

Raja, Kalpana; Subramani, Suresh; Natarajan, Jeyakumar

2013-01-01

One of the most common and challenging problem in biomedical text mining is to mine protein–protein interactions (PPIs) from MEDLINE abstracts and full-text research articles because PPIs play a major role in understanding the various biological processes and the impact of proteins in diseases. We implemented, PPInterFinder—a web-based text mining tool to extract human PPIs from biomedical literature. PPInterFinder uses relation keyword co-occurrences with protein names to extract information on PPIs from MEDLINE abstracts and consists of three phases. First, it identifies the relation keyword using a parser with Tregex and a relation keyword dictionary. Next, it automatically identifies the candidate PPI pairs with a set of rules related to PPI recognition. Finally, it extracts the relations by matching the sentence with a set of 11 specific patterns based on the syntactic nature of PPI pair. We find that PPInterFinder is capable of predicting PPIs with the accuracy of 66.05% on AIMED corpus and outperforms most of the existing systems. Database URL: http://www.biomining-bu.in/ppinterfinder/ PMID:23325628

ECM Proteins Glycosylation and Relation to Diabetes

NASA Astrophysics Data System (ADS)

Pernodet, Nadine; Bloomberg, Ayla; Sood, Vandana; Slutsky, Lenny; Ge, Shouren; Clark, Richard; Rafailovich, Miriam

2004-03-01

The chemical modification and crosslinking of proteins by sugar glycosylation contribute to the aging of tissue proteins, and acceleration of this reaction during hyperglycemia is implicated in the pathogenesis of diabetic complications, such as disorder of the wound healing. Advanced glycation endproducts (AGEs) formation and protein crosslinking are irreversible processes that alter the structural and functional properties of proteins, lipid components and nucleic acids. And the mechanism, by which it happens, is not clear. Fibrinogen and fibronectin are plasma proteins, which play a major role in human wound healing. Fibrinogen converts to an insoluble fibrin "gel" following a cut, which eventually forms a clot to prevent blood loss, to direct cell adhesion and migration for forming scars. Fibronectin is a critical protein for cell adhesion and migration in wound healing. The effects of glucose on the binding of these plasma proteins from the extra cellular matrix (ECM) were followed at different concentrations by atomic force microscopy and lateral force modulation to measure the mechanical response of the samples. Glucose solutions (1, 2, and 3mg/mL) were incubated with the protein (100 mg/ml) and silicon (Si) substrates spun with sulfonated polystyrene (SPS) 28% for five days. Data showed that not only the organization of the protein on the surface was affected but also its mechanical properties. At 3 mg/mL glucose, Fn fibers were observed to be harder than those of the control, in good agreement with our hypothesis that glycosylation hardens tissues by crosslinking of proteins in the ECM and might cause fibers to break more easily.

Effect of ethephon on protein degradation and the accumulation of pathogensis-related (PR) proteins in tomato leaf discs. [Lycopersicon esculentum

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

Vera, P.; Conejero, V.

The effect of ethephon (2-chloroetylphosphonic acid) on the degradation of proteins and on the induction of Lycopersicon esculentum pathogenesis-related (PR) proteins was studied in tomato leaf discs. The rate of ribulose, -1,5-bisphosphate carboxylase/oxygenase (Rubisco) degradation was maximal in discs after 48 hours of incubation with 1 millimolar ethephon, leading to complete disappearance of Rubisco after 96 hours. This effect was correlated with an increase in PR protein synthesis and the induction of the previously reported alkaline proteolytic enzyme PR-P69. In vivo pulse-chase experiments demonstrated that ethephon not only affected Rubisco content but that of many other {sup 35}S-labeled proteins asmore » well, indicating that ethylene activates a general and nonspecific mechanism of protein degradation. This effect was partially inhibited in vivo by the action of pCMB, a selective inhibitor of cysteine-proteinases such as P69. These data reinforce the hypothesis that P69 and perhaps other PR proteins are involved in the mechanism of accelerated protein degradation activated by ethylene.« less

Expression of bovine non-classical major histocompatibility complex class I proteins in mouse P815 and human K562 cells.

PubMed

Parasar, Parveen; Wilhelm, Amanda; Rutigliano, Heloisa M; Thomas, Aaron J; Teng, Lihong; Shi, Bi; Davis, William C; Suarez, Carlos E; New, Daniel D; White, Kenneth L; Davies, Christopher J

2016-08-01

Major histocompatibility complex class I (MHC-I) proteins can be expressed as cell surface or secreted proteins. To investigate whether bovine non-classical MHC-I proteins are expressed as cell surface or secreted proteins, and to assess the reactivity pattern of monoclonal antibodies with non-classical MHC-I isoforms, we expressed the MHC proteins in murine P815 and human K562 (MHC-I deficient) cells. Following antibiotic selection, stably transfected cell lines were stained with H1A or W6/32 antibodies to detect expression of the MHC-I proteins by flow cytometry. Two non-classical proteins (BoLA-NC1*00501 and BoLA-NC3*00101) were expressed on the cell surface in both cell lines. Surprisingly, the BoLA-NC4*00201 protein was expressed on the cell membrane of human K562 but not mouse P815 cells. Two non-classical proteins (BoLA-NC1*00401, which lacks a transmembrane domain, and BoLA-NC2*00102) did not exhibit cell surface expression. Nevertheless, Western blot analyses demonstrated expression of the MHC-I heavy chain in all transfected cell lines. Ammonium-sulfate precipitation of proteins from culture supernatants showed that BoLA-NC1*00401 was secreted and that all surface expressed proteins where shed from the cell membrane by the transfected cells. Interestingly, the surface expressed MHC-I proteins were present in culture supernatants at a much higher concentration than BoLA-NC1*00401. This comprehensive study shows that bovine non-classical MHC-I proteins BoLA-NC1*00501, BoLA-NC3*00101, and BoLA-NC4*00201 are expressed as surface isoforms with the latter reaching the cell membrane only in K562 cells. Furthermore, it demonstrated that BoLA-NC1*00401 is a secreted isoform and that significant quantities of membrane associated MHC-I proteins can be shed from the cell membrane. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Determination of Protein by Fluorescence Enhancement of Curcumin in Lanthanum-Curcumin-Sodium Dodecyl Benzene Sulfonate-Protein System

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

Wang, Feng; Huang, Wei; Zhang, Yunfeng

2011-01-01

We found that the fluorescence intensity of the lanthanum (La(3+))-curcumin (CU) complex can be highly enhanced by proteins in the presence of sodium dodecyl benzene sulphonate (SDBS). Based on this finding, a new fluorimetric method for the determination of protein was developed. Under optimized conditions, the enhanced intensities of fluorescence are quantitatively in proportion to the concentrations of proteins in the range 0.0080-20.0 g mL(-1) for bovine serum albumin (BSA) and 0.00080-20.0 g mL(-1) for human serum albumin (HSA) with excitation of 425 nm, and 0.00020-20.0 g mL(-1) for bovine serum albumin (BSA) and 0.00080-20.0 g mL(-1)for human serum albuminmore » (HSA) with excitation of 280 nm, while corresponding qualitative detection limits (S/N 3) are as low as 5.368, 0.573, 0.049, 0.562 g mL(-1), respectively. Study on reaction mechanism reveals that proteins can bind with La(3+), CU and SDBS through self-assembling function with electrostatic attraction, hydrogen bonding, hydrophobic interaction and van der Waals forces, etc. The proteins form a supermolecular association with multilayer structure, in which La(3+)-CU is clamped between BSA and SDBS. The unique high fluorescence enhancement of CU is resulted through synergic effects of favorable hydrophobic microenvironment provided by BSA and SDBS, and efficient intermolecular energy transfer among BSA, SDBS and CU. In energy transfer process, La(3+) plays a crucial role because it not only shortens the distance between SDBS and CU, but also acts as a "bridge" for transferring the energy from BSA to CU.« less

Differentiation of the Ribosomal Protein Compositions in the Genus Escherichia and Its Related Bacteria

PubMed Central

Osawa, Syozo; Itoh, Takuzi; Otaka, Eiko

1971-01-01

Compositions of the ribosomal proteins of 60 bacterial strains belonging to the genus Escherichia and its related genera were examined by use of a column of carboxymethyl cellulose. The ribosomes were classified into seven groups and were further differentiated into several types (subgroups) according to their protein compositions. It was shown that ribosomal protein composition is a useful characteristic for studies of bacterial taxonomy. PMID:5563866

Activation of multiple mitogen-activated protein kinases by recombinant calcitonin gene-related peptide receptor.

PubMed

Parameswaran, N; Disa, J; Spielman, W S; Brooks, D P; Nambi, P; Aiyar, N

2000-02-18

Calcitonin gene-related peptide is a 37-amino-acid neuropeptide and a potent vasodilator. Although calcitonin gene-related peptide has been shown to have a number of effects in a variety of systems, the mechanisms of action and the intracellular signaling pathways, especially the regulation of mitogen-activated protien kinase (MAPK) pathway, is not known. In the present study we investigated the role of calcitonin gene-related peptide in the regulation of MAPKs in human embryonic kidney (HEK) 293 cells stably transfected with a recombinant porcine calcitonin gene-related peptide-1 receptor. Calcitonin gene-related peptide caused a significant dose-dependent increase in cAMP response and the effect was inhibited by calcitonin gene-related peptide(8-37), the calcitonin gene-related peptide-receptor antagonist. Calcitonin gene-related peptide also caused a time- and concentration-dependent increase in extracellular signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (P38 MAPK) activities, with apparently no significant change in cjun-N-terminal kinase (JNK) activity. Forskolin, a direct activator of adenylyl cyclase also stimulated ERK and P38 activities in these cells suggesting the invovement of cAMP in this process. Calcitonin gene-related peptide-stimulated ERK and P38 MAPK activities were inhibited significantly by calcitonin gene-related peptide receptor antagonist, calcitonin gene-related peptide-(8-37) suggesting the involvement of calcitonin gene-related peptide-1 receptor. Preincubation of the cells with the cAMP-dependent protein kinase inhibitor, H89 [¿N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride¿] inhibited calcitonin gene-related peptide-mediated activation of ERK and p38 kinases. On the other hand, preincubation of the cells with wortmannin ¿[1S-(1alpha,6balpha,9abeta,11alpha, 11bbeta)]-11-(acetyloxy)-1,6b,7,8,9a,10,11, 11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3, 2-de]indeno[4,5-h]-2

The Primary Role of Fibrinogen-Related Proteins in Invertebrates Is Defense, Not Coagulation

PubMed Central

Hanington, Patrick C.; Zhang, Si-Ming

2010-01-01

In vertebrates, the conversion of fibrinogen into fibrin is an essential process that underlies the establishment of the supporting protein framework required for coagulation. In invertebrates, fibrinogen-domain-containing proteins play a role in the defense response generated against pathogens; however, they do not function in coagulation, suggesting that this role has been recently acquired. Molecules containing fibrinogen motifs have been identified in numerous invertebrate organisms, and most of these molecules known to date have been linked to defense. Moreover, recent genome projects of invertebrate animals have revealed surprisingly high numbers of fibrinogen-like loci in their genomes, suggesting important and perhaps diverse functions of fibrinogen-like proteins in invertebrates. The ancestral role of molecules containing fibrinogen-related domains (FReDs) with immunity is the focus of this review, with emphasis on specific FReDs called fibrinogen-related proteins (FREPs) identified from the schistosome-transmitting mollusc Biomphalaria glabrata. Herein, we outline the range of invertebrate organisms FREPs can be found in, and detail the roles these molecules play in defense and protection against infection. PMID:21063081

Domain fusion analysis by applying relational algebra to protein sequence and domain databases.

PubMed

Truong, Kevin; Ikura, Mitsuhiko

2003-05-06

Domain fusion analysis is a useful method to predict functionally linked proteins that may be involved in direct protein-protein interactions or in the same metabolic or signaling pathway. As separate domain databases like BLOCKS, PROSITE, Pfam, SMART, PRINTS-S, ProDom, TIGRFAMs, and amalgamated domain databases like InterPro continue to grow in size and quality, a computational method to perform domain fusion analysis that leverages on these efforts will become increasingly powerful. This paper proposes a computational method employing relational algebra to find domain fusions in protein sequence databases. The feasibility of this method was illustrated on the SWISS-PROT+TrEMBL sequence database using domain predictions from the Pfam HMM (hidden Markov model) database. We identified 235 and 189 putative functionally linked protein partners in H. sapiens and S. cerevisiae, respectively. From scientific literature, we were able to confirm many of these functional linkages, while the remainder offer testable experimental hypothesis. Results can be viewed at http://calcium.uhnres.utoronto.ca/pi. As the analysis can be computed quickly on any relational database that supports standard SQL (structured query language), it can be dynamically updated along with the sequence and domain databases, thereby improving the quality of predictions over time.

Domain fusion analysis by applying relational algebra to protein sequence and domain databases

PubMed Central

Truong, Kevin; Ikura, Mitsuhiko

2003-01-01

Background Domain fusion analysis is a useful method to predict functionally linked proteins that may be involved in direct protein-protein interactions or in the same metabolic or signaling pathway. As separate domain databases like BLOCKS, PROSITE, Pfam, SMART, PRINTS-S, ProDom, TIGRFAMs, and amalgamated domain databases like InterPro continue to grow in size and quality, a computational method to perform domain fusion analysis that leverages on these efforts will become increasingly powerful. Results This paper proposes a computational method employing relational algebra to find domain fusions in protein sequence databases. The feasibility of this method was illustrated on the SWISS-PROT+TrEMBL sequence database using domain predictions from the Pfam HMM (hidden Markov model) database. We identified 235 and 189 putative functionally linked protein partners in H. sapiens and S. cerevisiae, respectively. From scientific literature, we were able to confirm many of these functional linkages, while the remainder offer testable experimental hypothesis. Results can be viewed at . Conclusion As the analysis can be computed quickly on any relational database that supports standard SQL (structured query language), it can be dynamically updated along with the sequence and domain databases, thereby improving the quality of predictions over time. PMID:12734020

PACSY, a relational database management system for protein structure and chemical shift analysis.

PubMed

Lee, Woonghee; Yu, Wookyung; Kim, Suhkmann; Chang, Iksoo; Lee, Weontae; Markley, John L

2012-10-01

PACSY (Protein structure And Chemical Shift NMR spectroscopY) is a relational database management system that integrates information from the Protein Data Bank, the Biological Magnetic Resonance Data Bank, and the Structural Classification of Proteins database. PACSY provides three-dimensional coordinates and chemical shifts of atoms along with derived information such as torsion angles, solvent accessible surface areas, and hydrophobicity scales. PACSY consists of six relational table types linked to one another for coherence by key identification numbers. Database queries are enabled by advanced search functions supported by an RDBMS server such as MySQL or PostgreSQL. PACSY enables users to search for combinations of information from different database sources in support of their research. Two software packages, PACSY Maker for database creation and PACSY Analyzer for database analysis, are available from http://pacsy.nmrfam.wisc.edu.

PACSY, a relational database management system for protein structure and chemical shift analysis

PubMed Central

Lee, Woonghee; Yu, Wookyung; Kim, Suhkmann; Chang, Iksoo

2012-01-01

PACSY (Protein structure And Chemical Shift NMR spectroscopY) is a relational database management system that integrates information from the Protein Data Bank, the Biological Magnetic Resonance Data Bank, and the Structural Classification of Proteins database. PACSY provides three-dimensional coordinates and chemical shifts of atoms along with derived information such as torsion angles, solvent accessible surface areas, and hydrophobicity scales. PACSY consists of six relational table types linked to one another for coherence by key identification numbers. Database queries are enabled by advanced search functions supported by an RDBMS server such as MySQL or PostgreSQL. PACSY enables users to search for combinations of information from different database sources in support of their research. Two software packages, PACSY Maker for database creation and PACSY Analyzer for database analysis, are available from http://pacsy.nmrfam.wisc.edu. PMID:22903636

Gene Expression Analysis Reveals the Concurrent Activation of Proapoptotic and Antioxidant-Defensive Mechanisms in Flavokawain B–Treated Cervical Cancer HeLa Cells

PubMed Central

Yeap, Swee Keong; Abu, Nadiah; Akthar, Nadeem; Ho, Wan Yong; Ky, Huynh; Tan, Sheau Wei; Alitheen, Noorjahan Banu; Kamarul, Tunku

2016-01-01

Flavokawain B (FKB) is known to possess promising anticancer abilities. This is demonstrated in various cancer cell lines including HeLa cells. Cervical cancer is among the most widely diagnosed cancer among women today. Though FKB has been shown to be effective in treating cancer cells, the exact molecular mechanism is still unknown. This study is aimed at understanding the effects of FKB on HeLa cells using a microarray-based mRNA expression profiling and proteome profiling of stress-related proteins. The results of this study suggest that FKB induced cell death through p21-mediated cell cycle arrest and activation of p38. However, concurrent activation of antioxidant-related pathways and iron sequestration pathway followed by activation of ER-resident stress proteins clearly indicate that FKB failed to induce apoptosis in HeLa cells via oxidative stress. This effect implies that the protection of HeLa cells by FKB from H2O2–induced cell death is via neutralization of reactive oxygen species. PMID:27458249

The Saccharomyces cerevisiae enolase-related regions encode proteins that are active enolases.

PubMed

Kornblatt, M J; Richard Albert, J; Mattie, S; Zakaib, J; Dayanandan, S; Hanic-Joyce, P J; Joyce, P B M

2013-02-01

In addition to two genes (ENO1 and ENO2) known to code for enolase (EC4.2.1.11), the Saccharomyces cerevisiae genome contains three enolase-related regions (ERR1, ERR2 and ERR3) which could potentially encode proteins with enolase function. Here, we show that products of these genes (Err2p and Err3p) have secondary and quaternary structures similar to those of yeast enolase (Eno1p). In addition, Err2p and Err3p can convert 2-phosphoglycerate to phosphoenolpyruvate, with kinetic parameters similar to those of Eno1p, suggesting that these proteins could function as enolases in vivo. To address this possibility, we overexpressed the ERR2 and ERR3 genes individually in a double-null yeast strain lacking ENO1 and ENO2, and showed that either ERR2 or ERR3 could complement the growth defect in this strain when cells are grown in medium with glucose as the carbon source. Taken together, these data suggest that the ERR genes in Saccharomyces cerevisiae encode a protein that could function in glycolysis as enolase. The presence of these enolase-related regions in Saccharomyces cerevisiae and their absence in other related yeasts suggests that these genes may play some unique role in Saccharomyces cerevisiae. Further experiments will be required to determine whether these functions are related to glycolysis or other cellular processes. Copyright © 2012 John Wiley & Sons, Ltd.

A microscopic insight from conformational thermodynamics to functional ligand binding in proteins.

PubMed

Sikdar, Samapan; Chakrabarti, J; Ghosh, Mahua

2014-12-01

We show that the thermodynamics of metal ion-induced conformational changes aid to understand the functions of protein complexes. This is illustrated in the case of a metalloprotein, alpha-lactalbumin (aLA), a divalent metal ion binding protein. We use the histograms of dihedral angles of the protein, generated from all-atom molecular dynamics simulations, to calculate conformational thermodynamics. The thermodynamically destabilized and disordered residues in different conformational states of a protein are proposed to serve as binding sites for ligands. This is tested for β-1,4-galactosyltransferase (β4GalT) binding to the Ca(2+)-aLA complex, in which the binding residues are known. Among the binding residues, the C-terminal residues like aspartate (D) 116, glutamine (Q) 117, tryptophan (W) 118 and leucine (L) 119 are destabilized and disordered and can dock β4GalT onto Ca(2+)-aLA. No such thermodynamically favourable binding residues can be identified in the case of the Mg(2+)-aLA complex. We apply similar analysis to oleic acid binding and predict that the Ca(2+)-aLA complex can bind to oleic acid through the basic histidine (H) 32 of the A2 helix and the hydrophobic residues, namely, isoleucine (I) 59, W60 and I95, of the interfacial cleft. However, the number of destabilized and disordered residues in Mg(2+)-aLA are few, and hence, the oleic acid binding to Mg(2+)-bound aLA is less stable than that to the Ca(2+)-aLA complex. Our analysis can be generalized to understand the functionality of other ligand bound proteins.

A longitudinal analysis of circulating stress-related proteins and chronic ethanol self-administration in cynomolgus macaques

PubMed Central

Helms, Christa M.; Messaoudi, Ilhem; Jeng, Sophia; Freeman, Willard M.; Vrana, Kent E.; Grant, Kathleen A.

2011-01-01

Background Alcoholics have alterations in endocrine and immune function and increased susceptibility to stress-related disorders. A longitudinal analysis of chronic ethanol intake on homeostatic mechanisms is, however, incompletely characterized in primates. Methods Plasma proteins (n = 60; Luminex) and hormones (adrenocorticotropic hormone, ACTH; cortisol) were repeatedly measured in adult male cynomolgus monkeys (Macaca fascicularis, n = 10) during a 32-month experimental protocol at baseline, during induction of water and ethanol (4% w/v in water) self-administration, after 4 months and after 12 months of 22-h daily concurrent access to ethanol and water. Results Significant changes were observed in ACTH, cortisol and 45/60 plasma proteins: a majority (28/45) were suppressed as a function of ethanol self-administration, eight proteins were elevated and nine showed biphasic changes. Cortisol and ACTH were greatest during induction, and correlations between these hormones and plasma proteins varied across the experiment. Pathway analyses implicated nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) as possible mediators of ethanol-induced effects on immune-related proteins in primates. Conclusion Chronic ethanol consumption in primates leads to an allostatic state of physiological compromise with respect to circulating immune- and stress-related proteins in NF-κB- and STAT/JAK-related pathways in correlation with altered endocrine activity. PMID:22141444

Equilibrium fluctuation relations for voltage coupling in membrane proteins.

PubMed

Kim, Ilsoo; Warshel, Arieh

2015-11-01

A general theoretical framework is developed to account for the effects of an external potential on the energetics of membrane proteins. The framework is based on the free energy relation between two (forward/backward) probability densities, which was recently generalized to non-equilibrium processes, culminating in the work-fluctuation theorem. Starting from the probability densities of the conformational states along the "voltage coupling" reaction coordinate, we investigate several interconnected free energy relations between these two conformational states, considering voltage activation of ion channels. The free energy difference between the two conformational states at zero (depolarization) membrane potential (i.e., known as the chemical component of free energy change in ion channels) is shown to be equivalent to the free energy difference between the two "equilibrium" (resting and activated) conformational states along the one-dimensional voltage couplin reaction coordinate. Furthermore, the requirement that the application of linear response approximation to the free energy functionals of voltage coupling should satisfy the general free energy relations, yields a novel closed-form expression for the gating charge in terms of other basic properties of ion channels. This connection is familiar in statistical mechanics, known as the equilibrium fluctuation-response relation. The theory is illustrated by considering the coupling of a unit charge to the external voltage in the two sites near the surface of membrane, representing the activated and resting states. This is done using a coarse-graining (CG) model of membrane proteins, which includes the membrane, the electrolytes and the electrodes. The CG model yields Marcus-type voltage dependent free energy parabolas for the response of the electrostatic environment (electrolytes etc.) to the transition from the initial to the final configuratinal states, leading to equilibrium free energy difference and free

Predicting network modules of cell cycle regulators using relative protein abundance statistics.

PubMed

Oguz, Cihan; Watson, Layne T; Baumann, William T; Tyson, John J

2017-02-28

Parameter estimation in systems biology is typically done by enforcing experimental observations through an objective function as the parameter space of a model is explored by numerical simulations. Past studies have shown that one usually finds a set of "feasible" parameter vectors that fit the available experimental data equally well, and that these alternative vectors can make different predictions under novel experimental conditions. In this study, we characterize the feasible region of a complex model of the budding yeast cell cycle under a large set of discrete experimental constraints in order to test whether the statistical features of relative protein abundance predictions are influenced by the topology of the cell cycle regulatory network. Using differential evolution, we generate an ensemble of feasible parameter vectors that reproduce the phenotypes (viable or inviable) of wild-type yeast cells and 110 mutant strains. We use this ensemble to predict the phenotypes of 129 mutant strains for which experimental data is not available. We identify 86 novel mutants that are predicted to be viable and then rank the cell cycle proteins in terms of their contributions to cumulative variability of relative protein abundance predictions. Proteins involved in "regulation of cell size" and "regulation of G1/S transition" contribute most to predictive variability, whereas proteins involved in "positive regulation of transcription involved in exit from mitosis," "mitotic spindle assembly checkpoint" and "negative regulation of cyclin-dependent protein kinase by cyclin degradation" contribute the least. These results suggest that the statistics of these predictions may be generating patterns specific to individual network modules (START, S/G2/M, and EXIT). To test this hypothesis, we develop random forest models for predicting the network modules of cell cycle regulators using relative abundance statistics as model inputs. Predictive performance is assessed by the

Screening and identification of resistance related proteins from apple leaves inoculated with Marssonina coronaria (EII. & J. J. Davis)

PubMed Central

2014-01-01

Background Apple, an invaluable fruit crop worldwide, is often prone to infection by pathogenic fungi. Identification of potentially resistance-conferring apple proteins is one of the most important aims for studying apple resistance mechanisms and promoting the development of disease-resistant apple strains. In order to find proteins which promote resistance to Marssonina coronaria, a deadly pathogen which has been related to premature apple maturation, proteomes from apple leaves inoculated with M. coronaria were characterized at 3 and 6 days post-inoculation by two dimensional electrophoresis (2-DE). Results Overall, 59 differentially accumulated protein spots between inoculation and non-inoculation were successfully identified and aligned as 35 different proteins or protein families which involved in photosynthesis, amino acid metabolism, transport, energy metabolism, carbohydrate metabolism, binding, antioxidant, defense and stress. Quantitative real-time PCR (qRT-PCR) was also used to examine the change of some defense and stress related genes abundance under inoculated conditions. Conclusions In a conclusion, different proteins in response to Marssonina coronaria were identified by proteomic analysis. Among of these proteins, there are some PR proteins, for example class III endo-chitinase, beta-1,3-glucanase and thaumatine-like protein, and some antioxidant related proteins including aldo/keto reductase AKR, ascorbate peroxidase and phi class glutathione S-transferase protein that were associated with disease resistance. The transcription levels of class III endo-chitinase (L13) and beta-1, 3-glucanase (L17) have a good relation with the abundance of the encoded protein’s accumulation, however, the mRNA abundance of thaumatine-like protein (L22) and ascorbate peroxidase (L28) are not correlated with their protein abundance of encoded protein. To elucidate the resistant mechanism, the data in the present study will promote us to investigate further the

Equilibrium Fluctuation Relations for Voltage Coupling in Membrane Proteins

PubMed Central

Kim, Ilsoo; Warshel, Arieh

2015-01-01

A general theoretical framework is developed to account for the effects of an external potential on the energetics of membrane proteins. The framework is based on the free energy relation between two (forward/backward) probability densities, which was recently generalized to non-equilibrium processes, culminating in the work-fluctuation theorem. Starting from the probability densities of the conformational states along the reaction coordinate of “voltage coupling”, we investigate several interconnected free energy relations between these two conformational states, considering voltage activation of ion channels. The free energy difference at zero membrane potential (i.e., between the two “non-equilibrium” conformational states) is shown to be equivalent to the free energy difference between the two “equilibrium” conformational states along the one-dimensional reaction coordinate of voltage coupling. Furthermore, the requirement that the application of linear response approximation to the free energy functions (free energies) of voltage coupling should satisfy the general free energy relations, yields a novel expression for the gating charge in terms of other experimentally measurable quantities. This connection is familiar in statistical mechanics, known as the equilibrium fluctuation-response relation. The theory is illustrated by considering the movement of a unit charge within the membrane under the influence of an external potential, using a coarse-graining (CG) model of membrane proteins, which includes the membrane, the electrolytes and the electrodes. The CG model yields Marcus–type voltage dependent free energy parabolas for the two conformational states, which allow for quantitative estimations of an equilibrium free energy difference, a free energy of barrier, and the voltage dependency of channel activation (Q-V curve) for the unit charge movement. In addition, our analysis offers a quantitative rationale for the correlation between the free

Intrinsic fluorescence of protein in turbid media using empirical relation based on Monte Carlo lookup table

NASA Astrophysics Data System (ADS)

Einstein, Gnanatheepam; Udayakumar, Kanniyappan; Aruna, Prakasarao; Ganesan, Singaravelu

2017-03-01

Fluorescence of Protein has been widely used in diagnostic oncology for characterizing cellular metabolism. However, the intensity of fluorescence emission is affected due to the absorbers and scatterers in tissue, which may lead to error in estimating exact protein content in tissue. Extraction of intrinsic fluorescence from measured fluorescence has been achieved by different methods. Among them, Monte Carlo based method yields the highest accuracy for extracting intrinsic fluorescence. In this work, we have attempted to generate a lookup table for Monte Carlo simulation of fluorescence emission by protein. Furthermore, we fitted the generated lookup table using an empirical relation. The empirical relation between measured and intrinsic fluorescence is validated using tissue phantom experiments. The proposed relation can be used for estimating intrinsic fluorescence of protein for real-time diagnostic applications and thereby improving the clinical interpretation of fluorescence spectroscopic data.

Identification of related gene/protein names based on an HMM of name variations.

PubMed

Yeganova, L; Smith, L; Wilbur, W J

2004-04-01

Gene and protein names follow few, if any, true naming conventions and are subject to great variation in different occurrences of the same name. This gives rise to two important problems in natural language processing. First, can one locate the names of genes or proteins in free text, and second, can one determine when two names denote the same gene or protein? The first of these problems is a special case of the problem of named entity recognition, while the second is a special case of the problem of automatic term recognition (ATR). We study the second problem, that of gene or protein name variation. Here we describe a system which, given a query gene or protein name, identifies related gene or protein names in a large list. The system is based on a dynamic programming algorithm for sequence alignment in which the mutation matrix is allowed to vary under the control of a fully trainable hidden Markov model.

Pathogenesis-Related Proteins of Tomato 1

PubMed Central

Vera, Pablo; Conejero, Vicente

1988-01-01

An endoproteinase induced by citrus exocortis viroid has been purified from tomato (Lycopersicon esculentum Mill, cv “Rutgers”) leaves. The proteinase corresponds to one of the major pathogenesis-related proteins of tomato plants and was designated proteinase P-69 as it has a molecular weight of 69,000 to 70,000. The proteinase was purified in four steps: (NH4)2SO4 fractionation, chromatography on Bio-Gel P-60, DEAE-Sepharose chromatography, and casein-Sepharose affinity chromatography. The proteinase had a pH optimum of 8.5 to 9.0 when assayed with either fluorescein thiocarbamoyl derivative (FTC)-casein or FTC-ribulose 1,5-bisphosphate carboxylase/oxygenase as substrates. The proteinase activity was inhibited by pCMB and strongly activated by calcium and magnesium ions as well as by DTT. When analyzed by electrofocusing, the activity showed a pI around 9.0. Images Fig. 4 Fig. 8 PMID:16666127

Macroevolutionary trends of atomic composition and related functional group proportion in eukaryotic and prokaryotic proteins.

PubMed

Zhang, Yu-Juan; Yang, Chun-Lin; Hao, You-Jin; Li, Ying; Chen, Bin; Wen, Jian-Fan

2014-01-25

To fully explore the trends of atomic composition during the macroevolution from prokaryote to eukaryote, five atoms (oxygen, sulfur, nitrogen, carbon, hydrogen) and related functional groups in prokaryotic and eukaryotic proteins were surveyed and compared. Genome-wide analysis showed that eukaryotic proteins have more oxygen, sulfur and nitrogen atoms than prokaryotes do. Clusters of Orthologous Groups (COG) analysis revealed that oxygen, sulfur, carbon and hydrogen frequencies are higher in eukaryotic proteins than in their prokaryotic orthologs. Furthermore, functional group analysis demonstrated that eukaryotic proteins tend to have higher proportions of sulfhydryl, hydroxyl and acylamino, but lower of sulfide and carboxyl. Taken together, an apparent trend of increase was observed for oxygen and sulfur atoms in the macroevolution; the variation of oxygen and sulfur compositions and their related functional groups in macroevolution made eukaryotic proteins carry more useful functional groups. These results will be helpful for better understanding the functional significances of atomic composition evolution. Copyright © 2013 Elsevier B.V. All rights reserved.

Multivariate Modeling of Proteins Related to Trapezius Myalgia, a Comparative Study of Female Cleaners with or without Pain

PubMed Central

Hadrevi, Jenny; Ghafouri, Bijar; Larsson, Britt; Gerdle, Björn; Hellström, Fredrik

2013-01-01

The prevalence of chronic trapezius myalgia is high in women with high exposure to awkward working positions, repetitive movements and movements with high precision demands. The mechanisms behind chronic trapezius myalgia are not fully understood. The purpose of this study was to explore the differences in protein content between healthy and myalgic trapezius muscle using proteomics. Muscle biopsies from 12 female cleaners with work-related trapezius myalgia and 12 pain free female cleaners were obtained from the descending part of the trapezius. Proteins were separated with two-dimensional differential gel electrophoresis (2D-DIGE) and selected proteins were identified with mass spectrometry. In order to discriminate the two groups, quantified proteins were fitted to a multivariate analysis: partial least square discriminate analysis. The model separated 28 unique proteins which were related to glycolysis, the tricaboxylic acid cycle, to the contractile apparatus, the cytoskeleton and to acute response proteins. The results suggest altered metabolism, a higher abundance of proteins related to inflammation in myalgic cleaners compared to healthy, and a possible alteration of the contractile apparatus. This explorative proteomic screening of proteins related to chronic pain in the trapezius muscle provides new important aspects of the pathophysiology behind chronic trapezius myalgia. PMID:24023854

Multivariate modeling of proteins related to trapezius myalgia, a comparative study of female cleaners with or without pain.

PubMed

Hadrevi, Jenny; Ghafouri, Bijar; Larsson, Britt; Gerdle, Björn; Hellström, Fredrik

2013-01-01

The prevalence of chronic trapezius myalgia is high in women with high exposure to awkward working positions, repetitive movements and movements with high precision demands. The mechanisms behind chronic trapezius myalgia are not fully understood. The purpose of this study was to explore the differences in protein content between healthy and myalgic trapezius muscle using proteomics. Muscle biopsies from 12 female cleaners with work-related trapezius myalgia and 12 pain free female cleaners were obtained from the descending part of the trapezius. Proteins were separated with two-dimensional differential gel electrophoresis (2D-DIGE) and selected proteins were identified with mass spectrometry. In order to discriminate the two groups, quantified proteins were fitted to a multivariate analysis: partial least square discriminate analysis. The model separated 28 unique proteins which were related to glycolysis, the tricaboxylic acid cycle, to the contractile apparatus, the cytoskeleton and to acute response proteins. The results suggest altered metabolism, a higher abundance of proteins related to inflammation in myalgic cleaners compared to healthy, and a possible alteration of the contractile apparatus. This explorative proteomic screening of proteins related to chronic pain in the trapezius muscle provides new important aspects of the pathophysiology behind chronic trapezius myalgia.

RELATIVE ACTIN NUCLEATION PROMOTION EFFICIENCY BY WASP AND WAVE PROTEINS IN ENDOTHELIAL CELLS

PubMed Central

Kang, Hyeran; Wang, Jingjing; Longley, Sarah J.; Tang, Jay X.; Shaw, Sunil K.

2010-01-01

The mammalian genome encodes multiple WASP1 (Wiskott-Aldrich Syndrome Protein)/WAVE (WASP-family Verprolin homologous) proteins. Members of this family interact with the Arp (actin related protein) 2/3 complex to promote growth of a branched actin network near the plasma membrane or the surface of moving cargos. Arp2/3 mediated branching can further lead to formation of comet tails (actin rockets). Despite their similar domain structure, different WASP/WAVE family members fulfill unique functions that depend on their subcellular location and activity levels. We measured the relative efficiency of actin nucleation promotion of full length WASP/WAVE proteins in a cytoplasmic extract from primary human umbilical vein endothelial cells (HUVEC). In this assay WAVE2 and WAVE3 complexes showed higher nucleation efficiency than WAVE1 and N-WASP, indicating distinct cellular controls for different family members. Previously, WASP and N-WASP were the only members that were known to stimulate comet formation. We observed that in addition to N-WASP, WAVE3 also induced short actin tails, and the other WAVEs induced formation of asymmetric actin shells. Differences in shape and structure of actin-based growth may reflect varying ability of WASP/WAVE proteins to break symmetry of the actin shell, possibly by differential recruitment of actin bundling or severing (pruning or debranching) factors. PMID:20816932

Collagenase-3 binds to a specific receptor and requires the low density lipoprotein receptor-related protein for internalization

NASA Technical Reports Server (NTRS)

Barmina, O. Y.; Walling, H. W.; Fiacco, G. J.; Freije, J. M.; Lopez-Otin, C.; Jeffrey, J. J.; Partridge, N. C.

1999-01-01

We have previously identified a specific receptor for collagenase-3 that mediates the binding, internalization, and degradation of this ligand in UMR 106-01 rat osteoblastic osteosarcoma cells. In the present study, we show that collagenase-3 binding is calcium-dependent and occurs in a variety of cell types, including osteoblastic and fibroblastic cells. We also present evidence supporting a two-step mechanism of collagenase-3 binding and internalization involving both a specific collagenase-3 receptor and the low density lipoprotein receptor-related protein. Ligand blot analysis shows that (125)I-collagenase-3 binds specifically to two proteins ( approximately 170 kDa and approximately 600 kDa) present in UMR 106-01 cells. Western blotting identified the 600-kDa protein as the low density lipoprotein receptor-related protein. Our data suggest that the 170-kDa protein is a specific collagenase-3 receptor. Low density lipoprotein receptor-related protein-null mouse embryo fibroblasts bind but fail to internalize collagenase-3, whereas UMR 106-01 and wild-type mouse embryo fibroblasts bind and internalize collagenase-3. Internalization, but not binding, is inhibited by the 39-kDa receptor-associated protein. We conclude that the internalization of collagenase-3 requires the participation of the low density lipoprotein receptor-related protein and propose a model in which the cell surface interaction of this ligand requires a sequential contribution from two receptors, with the collagenase-3 receptor acting as a high affinity primary binding site and the low density lipoprotein receptor-related protein mediating internalization.

Tardigrade workbench: comparing stress-related proteins, sequence-similar and functional protein clusters as well as RNA elements in tardigrades.

PubMed

Förster, Frank; Liang, Chunguang; Shkumatov, Alexander; Beisser, Daniela; Engelmann, Julia C; Schnölzer, Martina; Frohme, Marcus; Müller, Tobias; Schill, Ralph O; Dandekar, Thomas

2009-10-12

Tardigrades represent an animal phylum with extraordinary resistance to environmental stress. To gain insights into their stress-specific adaptation potential, major clusters of related and similar proteins are identified, as well as specific functional clusters delineated comparing all tardigrades and individual species (Milnesium tardigradum, Hypsibius dujardini, Echiniscus testudo, Tulinus stephaniae, Richtersius coronifer) and functional elements in tardigrade mRNAs are analysed. We find that 39.3% of the total sequences clustered in 58 clusters of more than 20 proteins. Among these are ten tardigrade specific as well as a number of stress-specific protein clusters. Tardigrade-specific functional adaptations include strong protein, DNA- and redox protection, maintenance and protein recycling. Specific regulatory elements regulate tardigrade mRNA stability such as lox P DICE elements whereas 14 other RNA elements of higher eukaryotes are not found. Further features of tardigrade specific adaption are rapidly identified by sequence and/or pattern search on the web-tool tardigrade analyzer http://waterbear.bioapps.biozentrum.uni-wuerzburg.de. The work-bench offers nucleotide pattern analysis for promotor and regulatory element detection (tardigrade specific; nrdb) as well as rapid COG search for function assignments including species-specific repositories of all analysed data. Different protein clusters and regulatory elements implicated in tardigrade stress adaptations are analysed including unpublished tardigrade sequences.

Overexpression of the RD RNA binding protein in hepatitis C virus-related hepatocellular carcinoma.

PubMed

Iida, Michihisa; Iizuka, Norio; Tsunedomi, Ryouichi; Tsutsui, Masahiro; Yoshida, Shin; Maeda, Yoshinari; Tokuhisa, Yoshihiro; Sakamoto, Kazuhiko; Yoshimura, Kiyoshi; Tamesa, Takao; Oka, Masaaki

2012-08-01

Hepatocellular carcinoma (HCC) often exhibits a poor prognosis due to metastatic spread caused by portal vein invasion (PVI). In the present study, we attempted to identify a novel therapeutic target related to PVI of HCC. Based on pooled genomic data, we identified RD RNA binding protein (RDBP), a member of the negative elongation factor (NELF) transcription elongation regulatory complex, to be preferentially overexpressed in HCC with PVI. We used quantitative reverse transcription polymerase chain reaction (RT-PCR) and immuno-histochemical analyses to investigate the relationship between RDBP mRNA and protein with metastatic potential in sample sets of hepatitis C virus (HCV)-related HCC and corresponding non-HCC liver tissues. We also used the small interfering RNA technique to examine the role of RDBP in invasion and proliferation of HCC cells in vitro. Our data showed that both mRNA and protein levels of RDBP were significantly higher in HCC compared to non-HCC liver tissue, and that these levels were also significantly higher in HCC with PVI compared to HCC without PVI. Multivariate analysis revealed that RDBP protein levels were an independent risk factor for early intrahepatic recurrence of HCC within 2 years of surgery. Knockdown of RDBP protein significantly inhibited the proliferation and invasion of cells in vitro. These data demonstrate that RDBP is related to the metastatic potential of HCC, suggesting a possible candidate for prevention of HCC cell metastasis.

Distribution of language-related Cntnap2 protein in neural circuits critical for vocal learning.

PubMed

Condro, Michael C; White, Stephanie A

2014-01-01

Variants of the contactin associated protein-like 2 (Cntnap2) gene are risk factors for language-related disorders including autism spectrum disorder, specific language impairment, and stuttering. Songbirds are useful models for study of human speech disorders due to their shared capacity for vocal learning, which relies on similar cortico-basal ganglia circuitry and genetic factors. Here we investigate Cntnap2 protein expression in the brain of the zebra finch, a songbird species in which males, but not females, learn their courtship songs. We hypothesize that Cntnap2 has overlapping functions in vocal learning species, and expect to find protein expression in song-related areas of the zebra finch brain. We further expect that the distribution of this membrane-bound protein may not completely mirror its mRNA distribution due to the distinct subcellular localization of the two molecular species. We find that Cntnap2 protein is enriched in several song control regions relative to surrounding tissues, particularly within the adult male, but not female, robust nucleus of the arcopallium (RA), a cortical song control region analogous to human layer 5 primary motor cortex. The onset of this sexually dimorphic expression coincides with the onset of sensorimotor learning in developing males. Enrichment in male RA appears due to expression in projection neurons within the nucleus, as well as to additional expression in nerve terminals of cortical projections to RA from the lateral magnocellular nucleus of the nidopallium. Cntnap2 protein expression in zebra finch brain supports the hypothesis that this molecule affects neural connectivity critical for vocal learning across taxonomic classes. Copyright © 2013 Wiley Periodicals, Inc.

Structure–Function Relationships of Pre-Fibrillar Protein Assemblies in Alzheimer's Disease and Related Disorders

PubMed Central

Rahimi, F.; Shanmugam, A.; Bitan, G.

2010-01-01

Several neurodegenerative diseases, including Alzheimer's, Parkinson's, Huntington's and prion diseases, are characterized pathognomonically by the presence of intra- and/or extracellular lesions containing proteinaceous aggregates, and by extensive neuronal loss in selective brain regions. Related non-neuropathic systemic diseases, e.g., light-chain and senile systemic amyloidoses, and other organ-specific diseases, such as dialysis-related amyloidosis and type-2 diabetes mellitus, also are characterized by deposition of aberrantly folded, insoluble proteins. It is debated whether the hallmark pathologic lesions are causative. Substantial evidence suggests that these aggregates are the end state of aberrant protein folding whereas the actual culprits likely are transient, pre-fibrillar assemblies preceding the aggregates. In the context of neurodegenerative amyloidoses, the proteinaceous aggregates may eventuate as potentially neuroprotective sinks for the neurotoxic, oligomeric protein assemblies. The pre-fibrillar, oligomeric assemblies are believed to initiate the pathogenic mechanisms that lead to synaptic dysfunction, neuronal loss, and disease-specific regional brain atrophy. The amyloid β-protein (Aβ), which is believed to cause Alzheimer's disease (AD), is considered an archetypal amyloidogenic protein. Intense studies have led to nominal, functional, and structural descriptions of oligomeric Aβ assemblies. However, the dynamic and metastable nature of Aβ oligomers renders their study difficult. Different results generated using different methodologies under different experimental settings further complicate this complex area of research and identification of the exact pathogenic assemblies in vivo seems daunting. Here we review structural, functional, and biological experiments used to produce and study pre-fibrillar Aβ assemblies, and highlight similar studies of proteins involved in related diseases. We discuss challenges that contemporary

Myotubularin-related protein 7 inhibits insulin signaling in colorectal cancer

PubMed Central

Gutting, Tobias; Friedrich, Teresa; Gaiser, Timo; Magdeburg, Julia; Kienle, Peter; Ruh, Hermelindis; Hopf, Carsten; Behrens, Hans-Michael; Röcken, Christoph; Hanoch, Tamar; Seger, Rony; Ebert, Matthias P.A.; Burgermeister, Elke

2016-01-01

Phosphoinositide (PIP) phosphatases such as myotubularins (MTMs) inhibit growth factor receptor signaling. However, the function of myotubularin-related protein 7 (MTMR7) in cancer is unknown. We show that MTMR7 protein was down-regulated with increasing tumor grade (G), size (T) and stage (UICC) in patients with colorectal cancer (CRC) (n=1786). The presence of MTMR7 in the stroma correlated with poor prognosis, whereas MTMR7 expression in the tumor was not predictive for patients' survival. Insulin reduced MTMR7 protein levels in human CRC cell lines, and CRC patients with type 2 diabetes mellitus (T2DM) or loss of imprinting (LOI) of insulin-like growth factor 2 (IGF2) had an increased risk for MTMR7 loss. Mechanistically, MTMR7 lowered PIPs and inhibited insulin-mediated AKT-ERK1/2 signaling and proliferation in human CRC cell lines. MTMR7 provides a novel link between growth factor signaling and cancer, and may thus constitute a potential marker or drug target for human CRC. PMID:27409167

How Do Rab Proteins Determine Golgi Structure?

PubMed Central

Liu, Shijie; Storrie, Brian

2015-01-01

Rab proteins, small GTPases, are key regulators of mammalian Golgi apparatus organization. Based on the effect of Rab activation state, Rab proteins fall into two functional classes. In Class1, inactivation induces Golgi ribbon fragmentation and/or redistribution of Golgi enzymes to the ER, while overexpression of wild type or activation has little, if any, effect on Golgi ribbon organization. In Class 2, the reverse is true. We give emphasis to Rab6, the most abundant Golgi-associated Rab protein. Rab6 depletion in HeLa cells causes an increase in Golgi cisternal number, longer, more continuous cisternae, and a pronounced accumulation of vesicles; the effect of Rab6 on Golgi ribbon organization is probably through regulation of vesicle transport. In effector studies, motor proteins and their regulators are found to be key Rab6 effectors. A related Rab, Rab41, affects Golgi ribbon organization in a contrasting manner. The balance between minus- and plus-end directed motor recruitment may well be the major Rab-dependent factor in Golgi ribbon organization. PMID:25708460

BAD, a Proapoptotic Protein, Escapes ERK/RSK Phosphorylation in Deguelin and siRNA-Treated HeLa Cells

PubMed Central

Hafeez, Samra; Urooj, Mahwish; Saleem, Shamiala; Gillani, Zeeshan; Shaheen, Sumaira; Qazi, Mahmood Husain; Naseer, Muhammad Imran; Iqbal, Zafar; Ansari, Shakeel Ahmed; Haque, Absarul; Asif, Muhammad; Mir, Manzoor Ahmad; Ali, Ashraf; Pushparaj, Peter Natesan; Jamal, Mohammad Sarwar; Rasool, Mahmood

2016-01-01

This study has been undertaken to explore the therapeutic effects of deguelin and specific siRNAs in HeLa cells. The data provided clearly show the silencing of ERK 1/2 with siRNAs and inhibition of ERK1/2 with deguelin treatment in HeLa cells. Additionally, we are providing information that deguelin binds directly to anti-apoptotic Bcl-2, Bcl-xl and Mcl-1 in the hydrophobic grooves, thereby releasing BAD and BAX from dimerization with these proteins. This results in increased apoptotic activity through the intrinsic pathway involved in rupture of mitochondrial membrane and release of cytochrome C. Evidence for inhibition of ERK1/2 by deguelin and escape of BAD phosphorylation at serine 112 through ERK/RSK pathway has been further fortified by obtaining similar results by silencing ERK 1/2 each with specific siRNAs. Increase in BAD after treatment with deguelin or siRNAs has been interpreted to mean that deguelin acts through several alternative pathways and therefore can be used as effective therapeutic agent. PMID:26745145

Molecular and Insecticidal Characterization of a Novel Cry-Related Protein from Bacillus Thuringiensis Toxic against Myzus persicae

PubMed Central

Palma, Leopoldo; Muñoz, Delia; Berry, Colin; Murillo, Jesús; Ruiz de Escudero, Iñigo; Caballero, Primitivo

2014-01-01

This study describes the insecticidal activity of a novel Bacillus thuringiensis Cry-related protein with a deduced 799 amino acid sequence (~89 kDa) and ~19% pairwise identity to the 95-kDa-aphidicidal protein (sequence number 204) from patent US 8318900 and ~40% pairwise identity to the cancer cell killing Cry proteins (parasporins Cry41Ab1 and Cry41Aa1), respectively. This novel Cry-related protein contained the five conserved amino acid blocks and the three conserved domains commonly found in 3-domain Cry proteins. The protein exhibited toxic activity against the green peach aphid, Myzus persicae (Sulzer) (Homoptera: Aphididae) with the lowest mean lethal concentration (LC50 = 32.7 μg/mL) reported to date for a given Cry protein and this insect species, whereas it had no lethal toxicity against the Lepidoptera of the family Noctuidae Helicoverpa armigera (Hübner), Mamestra brassicae (L.), Spodoptera exigua (Hübner), S. frugiperda (J.E. Smith) and S. littoralis (Boisduval), at concentrations as high as ~3.5 μg/cm2. This novel Cry-related protein may become a promising environmentally friendly tool for the biological control of M. persicae and possibly also for other sap sucking insect pests. PMID:25384108

Identification and preliminary characterization of a protein motif related to the zinc finger.

PubMed Central

Lovering, R; Hanson, I M; Borden, K L; Martin, S; O'Reilly, N J; Evan, G I; Rahman, D; Pappin, D J; Trowsdale, J; Freemont, P S

1993-01-01

We have identified a protein motif, related to the zinc finger, which defines a newly discovered family of proteins. The motif was found in the sequence of the human RING1 gene, which is proximal to the major histocompatibility complex region on chromosome six. We propose naming this motif the "RING finger" and it is found in 27 proteins, all of which have putative DNA binding functions. We have synthesized a peptide corresponding to the RING1 motif and examined a number of properties, including metal and DNA binding. We provide evidence to support the suggestion that the RING finger motif is the DNA binding domain of this newly defined family of proteins. Images Fig. 1 Fig. 4 PMID:7681583

DNA Stimulates ATP-Dependent Proteolysis and Protein-Dependent ATPase Activity of Protease La from Escherichia coli

NASA Astrophysics Data System (ADS)

Chung, Chin Ha; Goldberg, Alfred L.

1982-02-01

The product of the lon gene in Escherichia coli is an ATP-dependent protease, protease La, that also binds strongly to DNA. Addition of double-stranded or single-stranded DNA to the protease in the presence of ATP was found to stimulate the hydrolysis of casein or globin 2- to 7-fold, depending on the DNA concentration. Native DNA from several sources (plasmid pBR322, phage T7, or calf thymus) had similar effects, but after denaturation the DNA was 20-100% more effective than the native form. Although poly(rA), globin mRNA, and various tRNAs did not stimulate proteolysis, poly(rC) and poly(rU) were effective. Poly(dT) was stimulatory but (dT)10 was not. In the presence of DNA as in its absence, proteolysis required concomitant ATP hydrolysis, and the addition of DNA also enhanced ATP hydrolysis by protease La 2-fold, but only in the presence of casein. At much higher concentrations, DNA inhibited proteolysis as well as ATP cleavage. Thus, association of this enzyme with DNA may regulate the degradation of cell proteins in vivo.

LDL receptor-related protein 1 regulates the abundance of diverse cell-signaling proteins in the plasma membrane proteome.

PubMed

Gaultier, Alban; Simon, Gabriel; Niessen, Sherry; Dix, Melissa; Takimoto, Shinako; Cravatt, Benjamin F; Gonias, Steven L

2010-12-03

LDL receptor-related protein 1 (LRP1) is an endocytic receptor, reported to regulate the abundance of other receptors in the plasma membrane, including uPAR and tissue factor. The goal of this study was to identify novel plasma membrane proteins, involved in cell-signaling, that are regulated by LRP1. Membrane protein ectodomains were prepared from RAW 264.7 cells in which LRP1 was silenced and control cells using protease K. Peptides were identified by LC-MS/MS. By analysis of spectral counts, 31 transmembrane and secreted proteins were regulated in abundance at least 2-fold when LRP1 was silenced. Validation studies confirmed that semaphorin4D (Sema4D), plexin domain-containing protein-1 (Plxdc1), and neuropilin-1 were more abundant in the membranes of LRP1 gene-silenced cells. Regulation of Plxdc1 by LRP1 was confirmed in CHO cells, as a second model system. Plxdc1 coimmunoprecipitated with LRP1 from extracts of RAW 264.7 cells and mouse liver. Although Sema4D did not coimmunoprecipitate with LRP1, the cell-surface level of Sema4D was increased by RAP, which binds to LRP1 and inhibits binding of other ligands. These studies identify Plxdc1, Sema4D, and neuropilin-1 as novel LRP1-regulated cell-signaling proteins. Overall, LRP1 emerges as a generalized regulator of the plasma membrane proteome.

Expression of the Grb2-related protein of the lymphoid system in B cell subsets enhances B cell antigen receptor signaling through mitogen-activated protein kinase pathways.

PubMed

Yankee, Thomas M; Solow, Sasha A; Draves, Kevin D; Clark, Edward A

2003-01-01

Adapter proteins play a critical role in regulating signals triggered by Ag receptor cross-linking. These small molecules link receptor proximal events with downstream signaling pathways. In this study, we explore the expression and function of the Grb2-related protein of the lymphoid system (GrpL)/Grb2-related adaptor downstream of Shc adapter protein in human B cells. GrpL is expressed in naive B cells and is down-regulated following B cell Ag receptor ligation. By contrast, germinal center and memory B cells express little or no GrpL. Using human B cell lines, we detected constitutive interactions between GrpL and B cell linker protein, Src homology (SH)2 domain-containing leukocyte protein of 76 kDa, hemopoietic progenitor kinase 1, and c-Cbl. The N-terminal SH3 domain of GrpL binds c-Cbl while the C-terminal SH3 domain binds B cell linker protein and SH2 domain-containing leukocyte protein of 76 kDa. Exogenous expression of GrpL in a GrpL-negative B cell line leads to enhanced Ag receptor-induced extracellular signal-related kinase and p38 mitogen-activated protein kinase phosphorylation. Thus, GrpL expression in human B cell subsets appears to regulate Ag receptor-mediated signaling events.

Biotechnological production of inducible defense-related proteins in edible radish (raphanus sativus) found in Nepal.

PubMed

Khanal, Praval; Karmacharya, Anil; Sharma, Shishir; Nepal, Ashwini K; Shrestha, Kanti

2014-01-01

Fungal infection in plant leads to use of many hazardous antifungal chemicals. Alternative to these chemicals, defense related antifungal proteins can be used in case of fungal diseases. An experiment was done in two varieties of edible radish (Raphanus sativus var. Pyuthane Raato and Raphanus sativus var. all season) with aims to produce defense protein within the plant, to identify and perform molecular characterization of those antifungal proteins. The next aim was to compare the antifungal property of those proteins with commercially available synthetic pesticides. Both varieties of radish were infected with fungi (Alternaria alternata and Fusarium oxysporum). Protein samples were isolated from leaves following the standard protocol as described for β-glucuronidase (GUS) assay and were run along with the standard protein marker of 10-250kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to identify and molecularly characterize them. An additional band in the range of 37-50kDa was observed in the fungal infected samples, which was not seen on uninfected samples. The antifungal assay was carried out for every sample in 96 wells microtitre plate. The extracted protein samples from fungal inoculated plants showed the significant inhibition of fungal growth compared to other samples. On the basis of molecular weight and their antifungal properties, the protein samples from the fungal infected plant were found to be PR2 (Glucanase) and PR3 (Chitinase). Defense related proteins were successfully produced in two varieties of radish found in Nepal. The use of such biologically produced proteins may reduce the use of biologically harmful synthetic pesticides.

Arabidopsis dynamin-related protein 1A polymers bind, but do not tubulate, liposomes

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

Backues, Steven K.; Bednarek, Sebastian Y., E-mail: sybednar@wisc.edu

2010-03-19

The Arabidopsis dynamin-related protein 1A (AtDRP1A) is involved in endocytosis and cell plate maturation in Arabidopsis. Unlike dynamin, AtDRP1A does not have any recognized membrane binding or protein-protein interaction domains. We report that GTPase active AtDRP1A purified from Escherichia coli as a fusion to maltose binding protein forms homopolymers visible by negative staining electron microscopy. These polymers interact with protein-free liposomes whose lipid composition mimics that of the inner leaflet of the Arabidopsis plasma membrane, suggesting that lipid-binding may play a role in AtDRP1A function. However, AtDRP1A polymers do not appear to assemble and disassemble in a dynamic fashion andmore » do not have the ability to tubulate liposomes in vitro, suggesting that additional factors or modifications are necessary for AtDRP1A's in vivo function.« less

Toward rules relating zinc finger protein sequences and DNA binding site preferences.

PubMed

Desjarlais, J R; Berg, J M

1992-08-15

Zinc finger proteins of the Cys2-His2 type consist of tandem arrays of domains, where each domain appears to contact three adjacent base pairs of DNA through three key residues. We have designed and prepared a series of variants of the central zinc finger within the DNA binding domain of Sp1 by using information from an analysis of a large data base of zinc finger protein sequences. Through systematic variations at two of the three contact positions (underlined), relatively specific recognition of sequences of the form 5'-GGGGN(G or T)GGG-3' has been achieved. These results provide the basis for rules that may develop into a code that will allow the design of zinc finger proteins with preselected DNA site specificity.

Le rôle du soutien parental dans la relation entre la victimisation homophobe, l’homophobie intériorisée et la détresse psychologique chez les jeunes de minorités sexuelles (JMS) : une approche de médiation modérée

PubMed Central

Bergeron, Félix-Antoine; Blais, Martin; Hébert, Martine

2016-01-01

Résumé Cet article explore le rôle modérateur du soutien parental dans les relations entre la victimisation homophobe, l’homophobie intériorisée et la détresse psychologique chez des jeunes de minorités sexuelles (JMS), que l’on dit aussi lesbiennes, gais, bisexuels ou en questionnement. Il vise à 1) documenter la prévalence des différentes formes de victimisation homophobe vécue par les JMS, et ce, selon le genre et l’âge et 2) à explorer l’effet modérateur du soutien parental dans la relation entre la victimisation homophobe, l’homophobie intériorisée et la détresse psychologique. Un échantillon de 228 JMS âgés de 14 à 22 ans, non exclusivement hétérosexuels, recrutés en milieu communautaire dans le cadre de l’enquête sur le Parcours Amoureux des Jeunes (PAJ) du Québec a été analysé. L’impact de la victimisation homophobe, du soutien parental, de l’homophobie intériorisée sur la détresse psychologique est exploré par un modèle de régression linéaire avec effets de médiation modérée. Le rôle modérateur du soutien parental est confirmé dans la relation entre la victimisation homophobe et la détresse psychologique. Ces variables peuvent constituer des leviers pour prévenir les effets négatifs des préjudices homophobes sur la santé mentale des JMS. PMID:26966851

Proteomic analysis of stress-related proteins and metabolic pathways in Picea asperata somatic embryos during partial desiccation.

PubMed

Jing, Danlong; Zhang, Jianwei; Xia, Yan; Kong, Lisheng; OuYang, Fangqun; Zhang, Shougong; Zhang, Hanguo; Wang, Junhui

2017-01-01

Partial desiccation treatment (PDT) stimulates germination and enhances the conversion of conifer somatic embryos. To better understand the mechanisms underlying the responses of somatic embryos to PDT, we used proteomic and physiological analyses to investigate these responses during PDT in Picea asperata. Comparative proteomic analysis revealed that, during PDT, stress-related proteins were mainly involved in osmosis, endogenous hormones, antioxidative proteins, molecular chaperones and defence-related proteins. Compared with those in cotyledonary embryos before PDT, these stress-related proteins remained at high levels on days 7 (D7) and 14 (D14) of PDT. The proteins that differentially accumulated in the somatic embryos on D7 were mapped to stress and/or stimuli. They may also be involved in the glyoxylate cycle and the chitin metabolic process. The most significant difference in the differentially accumulated proteins occurred in the metabolic pathways of photosynthesis on D14. Furthermore, in accordance with the changes in stress-related proteins, analyses of changes in water content, abscisic acid, indoleacetic acid and H 2 O 2 levels in the embryos indicated that PDT is involved in water-deficit tolerance and affects endogenous hormones. Our results provide insight into the mechanisms responsible for the transition from morphologically mature to physiologically mature somatic embryos during the PDT process in P. asperata. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

[Effects of UO-126 on proliferation and fbw7 expression of HeLa cells].

PubMed

Sun, Di; Shen, Yi; Wang, Shao-hua; Xiang, Zi-wu; Xie, Ying-shan; Jiang, Xin

2010-02-01

To observe the effects of UO-126 on the expression of F-box and WD repeat domain-containing protein 7(FBW7)and on the proliferation of human cervical cancer cell lines (HeLa cells). HeLa cells were treated with different concentrations of UO-126, MTT assay was used to observe the proliferation of HeLa cells. Immunofluorescence showed the location and expression of FBW7 in HeLa cells. The mRNA and protein expression of FBW7 were detected by RT-PCR and Western blot before and after mitogen-activated protein kinases (MAPK)signal was blocked by UO-126 a MAPK inhibitor. MTT results showed that the concentration range of MAPK signaling pathway inhibitor UO-126 inhibited the proliferation of HeLa cells in a concentration-and time-dependent manner(P<0.05). Immunofluorescence showed that the expression of positive FBW7 had increased after HeLa cells were treated with UO-126. RT-PCR and Western blot exhibited that the FBW7 mRNA and protein expression had significantly increased before and after HeLa cells were treated with UO-126(P<0.05). UO-126 could inhibit HeLa cells proliferation, FBW7 lied downstream of MAPK signaling pathway.

A systems biology analysis of the changes in gene expression via silencing of HPV-18 E1 expression in HeLa cells.

PubMed

Castillo, Andres; Wang, Lu; Koriyama, Chihaya; Eizuru, Yoshito; Jordan, King; Akiba, Suminori

2014-10-01

Previous studies have reported the detection of a truncated E1 mRNA generated from HPV-18 in HeLa cells. Although it is unclear whether a truncated E1 protein could function as a replicative helicase for viral replication, it would still retain binding sites for potential interactions with different host cell proteins. Furthermore, in this study, we found evidence in support of expression of full-length HPV-18 E1 mRNA in HeLa cells. To determine whether interactions between E1 and cellular proteins play an important role in cellular processes other than viral replication, genome-wide expression profiles of HPV-18 positive HeLa cells were compared before and after the siRNA knockdown of E1 expression. Differential expression and gene set enrichment analysis uncovered four functionally related sets of genes implicated in host defence mechanisms against viral infection. These included the toll-like receptor, interferon and apoptosis pathways, along with the antiviral interferon-stimulated gene set. In addition, we found that the transcriptional coactivator E1A-binding protein p300 (EP300) was downregulated, which is interesting given that EP300 is thought to be required for the transcription of HPV-18 genes in HeLa cells. The observed changes in gene expression produced via the silencing of HPV-18 E1 expression in HeLa cells indicate that in addition to its well-known role in viral replication, the E1 protein may also play an important role in mitigating the host's ability to defend against viral infection.

Tardigrade workbench: comparing stress-related proteins, sequence-similar and functional protein clusters as well as RNA elements in tardigrades

PubMed Central

2009-01-01

Background Tardigrades represent an animal phylum with extraordinary resistance to environmental stress. Results To gain insights into their stress-specific adaptation potential, major clusters of related and similar proteins are identified, as well as specific functional clusters delineated comparing all tardigrades and individual species (Milnesium tardigradum, Hypsibius dujardini, Echiniscus testudo, Tulinus stephaniae, Richtersius coronifer) and functional elements in tardigrade mRNAs are analysed. We find that 39.3% of the total sequences clustered in 58 clusters of more than 20 proteins. Among these are ten tardigrade specific as well as a number of stress-specific protein clusters. Tardigrade-specific functional adaptations include strong protein, DNA- and redox protection, maintenance and protein recycling. Specific regulatory elements regulate tardigrade mRNA stability such as lox P DICE elements whereas 14 other RNA elements of higher eukaryotes are not found. Further features of tardigrade specific adaption are rapidly identified by sequence and/or pattern search on the web-tool tardigrade analyzer http://waterbear.bioapps.biozentrum.uni-wuerzburg.de. The work-bench offers nucleotide pattern analysis for promotor and regulatory element detection (tardigrade specific; nrdb) as well as rapid COG search for function assignments including species-specific repositories of all analysed data. Conclusion Different protein clusters and regulatory elements implicated in tardigrade stress adaptations are analysed including unpublished tardigrade sequences. PMID:19821996

Global, quantitative and dynamic mapping of protein subcellular localization.

PubMed

Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg Hh

2016-06-09

Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology.

Predicting Protein Relationships to Human Pathways through a Relational Learning Approach Based on Simple Sequence Features.

PubMed

García-Jiménez, Beatriz; Pons, Tirso; Sanchis, Araceli; Valencia, Alfonso

2014-01-01

Biological pathways are important elements of systems biology and in the past decade, an increasing number of pathway databases have been set up to document the growing understanding of complex cellular processes. Although more genome-sequence data are becoming available, a large fraction of it remains functionally uncharacterized. Thus, it is important to be able to predict the mapping of poorly annotated proteins to original pathway models. We have developed a Relational Learning-based Extension (RLE) system to investigate pathway membership through a function prediction approach that mainly relies on combinations of simple properties attributed to each protein. RLE searches for proteins with molecular similarities to specific pathway components. Using RLE, we associated 383 uncharacterized proteins to 28 pre-defined human Reactome pathways, demonstrating relative confidence after proper evaluation. Indeed, in specific cases manual inspection of the database annotations and the related literature supported the proposed classifications. Examples of possible additional components of the Electron transport system, Telomere maintenance and Integrin cell surface interactions pathways are discussed in detail. All the human predicted proteins in the 2009 and 2012 releases 30 and 40 of Reactome are available at http://rle.bioinfo.cnio.es.

Visualisation of variable binding pockets on protein surfaces by probabilistic analysis of related structure sets.

PubMed

Ashford, Paul; Moss, David S; Alex, Alexander; Yeap, Siew K; Povia, Alice; Nobeli, Irene; Williams, Mark A

2012-03-14

Protein structures provide a valuable resource for rational drug design. For a protein with no known ligand, computational tools can predict surface pockets that are of suitable size and shape to accommodate a complementary small-molecule drug. However, pocket prediction against single static structures may miss features of pockets that arise from proteins' dynamic behaviour. In particular, ligand-binding conformations can be observed as transiently populated states of the apo protein, so it is possible to gain insight into ligand-bound forms by considering conformational variation in apo proteins. This variation can be explored by considering sets of related structures: computationally generated conformers, solution NMR ensembles, multiple crystal structures, homologues or homology models. It is non-trivial to compare pockets, either from different programs or across sets of structures. For a single structure, difficulties arise in defining particular pocket's boundaries. For a set of conformationally distinct structures the challenge is how to make reasonable comparisons between them given that a perfect structural alignment is not possible. We have developed a computational method, Provar, that provides a consistent representation of predicted binding pockets across sets of related protein structures. The outputs are probabilities that each atom or residue of the protein borders a predicted pocket. These probabilities can be readily visualised on a protein using existing molecular graphics software. We show how Provar simplifies comparison of the outputs of different pocket prediction algorithms, of pockets across multiple simulated conformations and between homologous structures. We demonstrate the benefits of use of multiple structures for protein-ligand and protein-protein interface analysis on a set of complexes and consider three case studies in detail: i) analysis of a kinase superfamily highlights the conserved occurrence of surface pockets at the active

Perilipin-related protein regulates lipid metabolism in C. elegans.

PubMed

Chughtai, Ahmed Ali; Kaššák, Filip; Kostrouchová, Markéta; Novotný, Jan Philipp; Krause, Michael W; Saudek, Vladimír; Kostrouch, Zdenek; Kostrouchová, Marta

2015-01-01

Perilipins are lipid droplet surface proteins that contribute to fat metabolism by controlling the access of lipids to lipolytic enzymes. Perilipins have been identified in organisms as diverse as metazoa, fungi, and amoebas but strikingly not in nematodes. Here we identify the protein encoded by the W01A8.1 gene in Caenorhabditis elegans as the closest homologue and likely orthologue of metazoan perilipin. We demonstrate that nematode W01A8.1 is a cytoplasmic protein residing on lipid droplets similarly as human perilipins 1 and 2. Downregulation or elimination of W01A8.1 affects the appearance of lipid droplets resulting in the formation of large lipid droplets localized around the dividing nucleus during the early zygotic divisions. Visualization of lipid containing structures by CARS microscopy in vivo showed that lipid-containing structures become gradually enlarged during oogenesis and relocate during the first zygotic division around the dividing nucleus. In mutant embryos, the lipid containing structures show defective intracellular distribution in subsequent embryonic divisions and become gradually smaller during further development. In contrast to embryos, lipid-containing structures in enterocytes and in epidermal cells of adult animals are smaller in mutants than in wild type animals. Our results demonstrate the existence of a perilipin-related regulation of fat metabolism in nematodes and provide new possibilities for functional studies of lipid metabolism.

Risk prediction for malignant conversion of oral epithelial dysplasia by hypoxia related protein expression.

PubMed

Zhang, Xianglan; Han, Seonhui; Han, Hye-Yeon; Ryu, Mi Heon; Kim, Ki-Yeol; Choi, Eun-Joo; Cha, In-Ho; Kim, Jin

2013-08-01

Increased aerobic glycolysis is a unique finding in cancers and hypoxia-related proteins are associated with aerobic glycolysis. Therefore, we aimed to investigate whether hypoxia-related proteins can be predictive markers for malignant conversion of oral premalignant lesions with epithelial dysplasia (OED). Expression of HIF-1α, Glut-1 and CA9 were detected in clinical samples of eight normal oral mucosa, 85 transitional areas of oral squamous cell carcinoma (OSCC) and 28 OED with or without malignant conversion using immunohistochemistry and were also comparatively detected in immortalised human oral keratinocyte (IHOK) and OSCC cell lines under hypoxia using immunoblotting. Sequential expression of HIF-1α, Glut-1 and CA9 was found both in transitional areas of OSCC and cell lines of IHOK and OSCC under hypoxia, supporting hypoxia-aerobic glycolysis-acidosis axis. Expression of all proteins showed significant association with malignant conversion of OED and CA9 was an independent risk factor of malignant transformation of OED. But the predictability of malignant transformation was improved when all three proteins were applied together. High expression of CA9 was an independent predictive marker of malignant conversion. Moreover, the combined application of these three proteins may be useful to assess the risk of malignant conversion of OED.

Biotechnological Production of Inducible Defense-Related Proteins in Edible Radish (Raphanus Sativus) Found in Nepal

PubMed Central

Khanal, Praval; Karmacharya, Anil; Sharma, Shishir; Nepal, Ashwini K.; Shrestha, Kanti

2014-01-01

Background: Fungal infection in plant leads to use of many hazardous antifungal chemicals. Alternative to these chemicals, defense related antifungal proteins can be used in case of fungal diseases. Aims: An experiment was done in two varieties of edible radish (Raphanus sativus var. Pyuthane Raato and Raphanus sativus var. all season) with aims to produce defense protein within the plant, to identify and perform molecular characterization of those antifungal proteins. The next aim was to compare the antifungal property of those proteins with commercially available synthetic pesticides. Methods: Both varieties of radish were infected with fungi (Alternaria alternata and Fusarium oxysporum). Protein samples were isolated from leaves following the standard protocol as described for β-glucuronidase (GUS) assay and were run along with the standard protein marker of 10-250kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to identify and molecularly characterize them. Results: An additional band in the range of 37-50kDa was observed in the fungal infected samples, which was not seen on uninfected samples. The antifungal assay was carried out for every sample in 96 wells microtitre plate. The extracted protein samples from fungal inoculated plants showed the significant inhibition of fungal growth compared to other samples. On the basis of molecular weight and their antifungal properties, the protein samples from the fungal infected plant were found to be PR2 (Glucanase) and PR3 (Chitinase). Conclusion: Defense related proteins were successfully produced in two varieties of radish found in Nepal. The use of such biologically produced proteins may reduce the use of biologically harmful synthetic pesticides. PMID:24596739

Comparative study of label and label-free techniques using shotgun proteomics for relative protein quantification.

PubMed

Sjödin, Marcus O D; Wetterhall, Magnus; Kultima, Kim; Artemenko, Konstantin

2013-06-01

The analytical performance of three different strategies, iTRAQ (isobaric tag for relative and absolute quantification), dimethyl labeling (DML) and label free (LF) for relative protein quantification using shotgun proteomics have been evaluated. The methods have been explored using samples containing (i) Bovine proteins in known ratios and (ii) Bovine proteins in known ratios spiked into Escherichia coli. The latter case mimics the actual conditions in a typical biological sample with a few differentially expressed proteins and a bulk of proteins with unchanged ratios. Additionally, the evaluation was performed on both QStar and LTQ-FTICR mass spectrometers. LF LTQ-FTICR was found to have the highest proteome coverage while the highest accuracy based on the artificially regulated proteins was found for DML LTQ-FTICR (54%). A varying linearity (k: 0.55-1.16, r(2): 0.61-0.96) was shown for all methods within selected dynamic ranges. All methods were found to consistently underestimate Bovine protein ratios when matrix proteins were added. However, LF LTQ-FTICR was more tolerant toward a compression effect. A single peptide was demonstrated to be sufficient for a reliable quantification using iTRAQ. A ranking system utilizing several parameters important for quantitative proteomics demonstrated that the overall performance of the five different methods was; DML LTQ-FTICR>iTRAQ QStar>LF LTQ-FTICR>DML QStar>LF QStar. Copyright © 2013 Elsevier B.V. All rights reserved.

Expression and Functional Characterization of two Pathogenesis-Related Protein 10 Genes from Zea mays

USDA-ARS?s Scientific Manuscript database

Pathogenesis-related protein 10 (PR10) is one of seventeen PR protein families and plays important roles in plant response to biotic and abiotic stresses. A novel PR10 gene (ZmPR10.1), which shares 89.8% and 85.7% identity to the previous ZmPR10 at the nucleotide and amino acid sequence level, respe...

Surface expression of heterogeneous nuclear RNA binding protein M4 on Kupffer cell relates to its function as a carcinoembryonic antigen receptor.

PubMed

Bajenova, Olga; Stolper, Eugenia; Gapon, Svetlana; Sundina, Natalia; Zimmer, Regis; Thomas, Peter

2003-11-15

Elevated concentrations of carcinoembryonic antigen (CEA) in the blood are associated with the development of hepatic metastases from colorectal cancers. Clearance of circulating CEA occurs through endocytosis by liver macrophages, Kupffer cells. Previously we identified heterogeneous nuclear ribonucleoproteins M4 (hnRNP M4) as a receptor (CEAR) for CEA. HnRNP M4 has two isoform proteins (p80, p76), the full-length hnRNP M4 (CEARL) and a truncated form (CEARS) with a deletion of 39 amino acids between RNA binding domains 1 and 2, generated by alternative splicing. The present study was undertaken to clarify any isoform-specific differences in terms of their function as CEA receptor and localization. We develop anti-CEAR isoform-specific antibodies and show that both CEAR splicing isoforms are expressed on the surface of Kupffer cells and can function as CEA receptor. Alternatively, in P388D1 macrophages CEARS protein has nuclear and CEARL has cytoplasmic localization. In MIP101 colon cancer and HeLa cells the CEARS protein is localized to the nucleus and CEARL to the cytoplasm. These findings imply that different functions are assigned to CEAR isoforms depending on the cell type. The search of 39 amino acids deleted region against the Prosite data base revealed the presence of N-myristylation signal PGGPGMITIP that may be involved in protein targeting to the plasma membrane. Overall, this report demonstrates that the cellular distribution, level of expression, and relative amount of CEARL and CEARS isoforms determine specificity for CEA binding and the expression of alternative spliced forms of CEAR is regulated in a tissue-specific manner.

Functional modules by relating protein interaction networks and gene expression.

PubMed

Tornow, Sabine; Mewes, H W

2003-11-01

Genes and proteins are organized on the basis of their particular mutual relations or according to their interactions in cellular and genetic networks. These include metabolic or signaling pathways and protein interaction, regulatory or co-expression networks. Integrating the information from the different types of networks may lead to the notion of a functional network and functional modules. To find these modules, we propose a new technique which is based on collective, multi-body correlations in a genetic network. We calculated the correlation strength of a group of genes (e.g. in the co-expression network) which were identified as members of a module in a different network (e.g. in the protein interaction network) and estimated the probability that this correlation strength was found by chance. Groups of genes with a significant correlation strength in different networks have a high probability that they perform the same function. Here, we propose evaluating the multi-body correlations by applying the superparamagnetic approach. We compare our method to the presently applied mean Pearson correlations and show that our method is more sensitive in revealing functional relationships.

Functional modules by relating protein interaction networks and gene expression

PubMed Central

Tornow, Sabine; Mewes, H. W.

2003-01-01

Genes and proteins are organized on the basis of their particular mutual relations or according to their interactions in cellular and genetic networks. These include metabolic or signaling pathways and protein interaction, regulatory or co-expression networks. Integrating the information from the different types of networks may lead to the notion of a functional network and functional modules. To find these modules, we propose a new technique which is based on collective, multi-body correlations in a genetic network. We calculated the correlation strength of a group of genes (e.g. in the co-expression network) which were identified as members of a module in a different network (e.g. in the protein interaction network) and estimated the probability that this correlation strength was found by chance. Groups of genes with a significant correlation strength in different networks have a high probability that they perform the same function. Here, we propose evaluating the multi-body correlations by applying the superparamagnetic approach. We compare our method to the presently applied mean Pearson correlations and show that our method is more sensitive in revealing functional relationships. PMID:14576317

The ubiquitin-related protein PLIC-1 regulates heterotrimeric G protein function through association with Gβγ

PubMed Central

N'Diaye, Elsa-Noah; Brown, Eric J.

2003-01-01

PLIC-1, a newly described ubiquitin-related protein, inhibited both Jurkat migration toward SDF-1α and A431 wound healing, but the closely related PLIC-2 did not. PLIC-1 prevented the SDF-1α–induced activation of phospholipase C, decreased ligand-induced internalization of SDF-1α receptor CXCR4 and inhibited chemotaxis signaled by a transfected Gi-coupled receptor. However, PLIC-1 had no effect on Gs-mediated adenylyl cyclase activation, and inhibited only the Gβγ-dependent component of Gq-initiated increase in [Ca2+]i, which is consistent with selective inhibition of Gβγ function. PLIC-1 colocalized with G proteins in lamellae and pseudopods, and precipitated Gβγ in pull downs. Interaction with Gβγ did not require PLIC-1's ubiquitin-like or ubiquitin-associated domains, and proteasome inhibition had no effect on SDF-1α activation of phospholipase C, indicating that PLIC-1's inhibition of Gβγ did not result from effects on proteasome function. Thus, PLIC-1 inhibits Gi signaling by direct association with Gβγ; because it also interacts with CD47, a modulator of integrin function, it likely has a role integrating adhesion and signaling components of cell migration. PMID:14662753

Identification of genes related to proliferative diabetic retinopathy through RWR algorithm based on protein-protein interaction network.

PubMed

Zhang, Jian; Suo, Yan; Liu, Min; Xu, Xun

2018-06-01

Proliferative diabetic retinopathy (PDR) is one of the most common complications of diabetes and can lead to blindness. Proteomic studies have provided insight into the pathogenesis of PDR and a series of PDR-related genes has been identified but are far from fully characterized because the experimental methods are expensive and time consuming. In our previous study, we successfully identified 35 candidate PDR-related genes through the shortest-path algorithm. In the current study, we developed a computational method using the random walk with restart (RWR) algorithm and the protein-protein interaction (PPI) network to identify potential PDR-related genes. After some possible genes were obtained by the RWR algorithm, a three-stage filtration strategy, which includes the permutation test, interaction test and enrichment test, was applied to exclude potential false positives caused by the structure of PPI network, the poor interaction strength, and the limited similarity on gene ontology (GO) terms and biological pathways. As a result, 36 candidate genes were discovered by the method which was different from the 35 genes reported in our previous study. A literature review showed that 21 of these 36 genes are supported by previous experiments. These findings suggest the robustness and complementary effects of both our efforts using different computational methods, thus providing an alternative method to study PDR pathogenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Whey protein fractionation using supercritical carbon dioxide

USDA-ARS?s Scientific Manuscript database

Sweet whey, a coproduct of the cheesemaking process, can be concentrated using ultrafiltration and ion-exchange to produce whey protein isolates (WPI). WPI contains approximately 32% alpha-lactalbumin (alpha-LA) and 61% beta-lactoglobulin (beta-LG), plus a small amount of minor whey proteins. Whil...

Plasma Protein Pentosidine and Carboxymethyllysine, Biomarkers for Age-related Macular Degeneration*

PubMed Central

Ni, Jiaqian; Yuan, Xianglin; Gu, Jiayin; Yue, Xiuzhen; Gu, Xiaorong; Nagaraj, Ram H.; Crabb, John W.

2009-01-01

Age-related macular degeneration (AMD) causes severe vision loss in the elderly; early identification of AMD risk could help slow or prevent disease progression. Toward the discovery of AMD biomarkers, we quantified plasma protein Nε-carboxymethyllysine (CML) and pentosidine from 58 AMD and 32 control donors. CML and pentosidine are advanced glycation end products that are abundant in Bruch membrane, the extracellular matrix separating the retinal pigment epithelium from the blood-bearing choriocapillaris. We measured CML and pentosidine by LC-MS/MS and LC-fluorometry, respectively, and found higher mean levels of CML (∼54%) and pentosidine (∼64%) in AMD (p < 0.0001) relative to normal controls. Plasma protein fructosyl-lysine, a marker of early glycation, was found by amino acid analysis to be in equal amounts in control and non-diabetic AMD donors, supporting an association between AMD and increased levels of CML and pentosidine independent of other diseases like diabetes. Carboxyethylpyrrole (CEP), an oxidative modification from docosahexaenoate-containing lipids and also abundant in AMD Bruch membrane, was elevated ∼86% in the AMD cohort, but autoantibody titers to CEP, CML, and pentosidine were not significantly increased. Compellingly higher mean levels of CML and pentosidine were present in AMD plasma protein over a broad age range. Receiver operating curves indicate that CML, CEP adducts, and pentosidine alone discriminated between AMD and control subjects with 78, 79, and 88% accuracy, respectively, whereas CML in combination with pentosidine provided ∼89% accuracy, and CEP plus pentosidine provided ∼92% accuracy. Pentosidine levels appeared slightly altered in AMD patients with hypertension and cardiovascular disease, indicating further studies are warranted. Overall this study supports the potential utility of plasma protein CML and pentosidine as biomarkers for assessing AMD risk and susceptibility, particularly in combination with CEP

Electrophoresis and spectrometric analyses of adaptation-related proteins in thermally stressed Chromobacterium violaceum.

PubMed

Cordeiro, I B; Castro, D P; Nogueira, P P O; Angelo, P C S; Nogueira, P A; Gonçalves, J F C; Pereira, A M R F; Garcia, J S; Souza, G H M F; Arruda, M A Z; Eberlin, M N; Astolfi-Filho, S; Andrade, E V; López-Lozano, J L

2013-10-29

Chromobacterium violaceum is a Gram-negative proteobacteria found in water and soil; it is widely distributed in tropical and subtropical regions, such as the Amazon rainforest. We examined protein expression changes that occur in C. violaceum at different growth temperatures using electrophoresis and mass spectrometry. The total number of spots detected was 1985; the number ranged from 99 to 380 in each assay. The proteins that were identified spectrometrically were categorized as chaperones, proteins expressed exclusively under heat stress, enzymes involved in the respiratory and fermentation cycles, ribosomal proteins, and proteins related to transport and secretion. Controlling inverted repeat of chaperone expression and inverted repeat DNA binding sequences, as well as regions recognized by sigma factor 32, elements involved in the genetic regulation of the bacterial stress response, were identified in the promoter regions of several of the genes coding proteins, involved in the C. violaceum stress response. We found that 30 °C is the optimal growth temperature for C. violaceum, whereas 25, 35, and 40 °C are stressful temperatures that trigger the expression of chaperones, superoxide dismutase, a probable small heat shock protein, a probable phasing, ferrichrome-iron receptor protein, elongation factor P, and an ornithine carbamoyltransferase catabolite. This information improves our comprehension of the mechanisms involved in stress adaptation by C. violaceum.

Protein markers for identification of Yersinia pestis and their variation related to culture

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

Wunschel, David S.; Engelmann, Heather E.; Victry, Kristin D.

2013-12-11

The detection of high consequence pathogens, such as Yersinia pestis, is well established in biodefense laboratories for bioterror situations. Laboratory protocols are well established using specified culture media and a growth temperature of 37 °C for expression of specific antigens. Direct detection of Y. pestis protein markers, without prior culture, depends on their expression. Unfortunately protein expression can be impacted by the culture medium which cannot be predicted ahead of time. Furthermore, higher biomass yields are obtained at the optimal growth temperature (i.e. 28 °C–30 °C) and therefore are more likely to be used for bulk production. Analysis of Y.more » pestis grown on several types of media at 30 °C showed that several protein markers were found to be differentially detected in different media. Analysis of the identified proteins against a comprehensive database provided an additional level of organism identification. Peptides corresponding to variable regions of some proteins could separate large groups of strains and aid in organism identification. This work illustrates the need to understand variability of protein expression for detection targets. The potential for relating expression changes of known proteins to specific media factors, even in nutrient rich and chemically complex culture medium, may provide the opportunity to draw forensic information from protein profiles.« less

Signaling Pathways Related to Protein Synthesis and Amino Acid Concentration in Pig Skeletal Muscles Depend on the Dietary Protein Level, Genotype and Developmental Stages

PubMed Central

Liu, Yingying; Li, Fengna; Kong, Xiangfeng; Tan, Bie; Li, Yinghui; Duan, Yehui; Blachier, François; Hu, Chien-An A.; Yin, Yulong

2015-01-01

Muscle growth is regulated by the homeostatic balance of the biosynthesis and degradation of muscle proteins. To elucidate the molecular interactions among diet, pig genotype, and physiological stage, we examined the effect of dietary protein concentration, pig genotype, and physiological stages on amino acid (AA) pools, protein deposition, and related signaling pathways in different types of skeletal muscles. The study used 48 Landrace pigs and 48 pure-bred Bama mini-pigs assigned to each of 2 dietary treatments: lower/GB (Chinese conventional diet)- or higher/NRC (National Research Council)-protein diet. Diets were fed from 5 weeks of age to respective market weights of each genotype. Samples of biceps femoris muscle (BFM, type I) and longissimus dorsi muscle (LDM, type II) were collected at nursery, growing, and finishing phases according to the physiological stage of each genotype, to determine the AA concentrations, mRNA levels for growth-related genes in muscles, and protein abundances of mechanistic target of rapamycin (mTOR) signaling pathway. Our data showed that the concentrations of most AAs in LDM and BFM of pigs increased (P<0.05) gradually with increasing age. Bama mini-pigs had generally higher (P<0.05) muscle concentrations of flavor-related AA, including Met, Phe, Tyr, Pro, and Ser, compared with Landrace pigs. The mRNA levels for myogenic determining factor, myogenin, myocyte-specific enhancer binding factor 2 A, and myostatin of Bama mini-pigs were higher (P<0.05) than those of Landrace pigs, while total and phosphorylated protein levels for protein kinase B, mTOR, and p70 ribosomal protein S6 kinases (p70S6K), and ratios of p-mTOR/mTOR, p-AKT/AKT, and p-p70S6K/p70S6K were lower (P<0.05). There was a significant pig genotype-dependent effect of dietary protein on the levels for mTOR and p70S6K. When compared with the higher protein-NRC diet, the lower protein-GB diet increased (P<0.05) the levels for mTOR and p70S6K in Bama mini-pigs, but

Gadd45 proteins: Relevance to aging, longevity and age-related pathologies

PubMed Central

Moskalev, Alexey A.; Smit-McBride, Zeljka; Shaposhnikov, Mikhail V.; Plyusnina, Ekaterina N.; Zhavoronkov, Alex; Budovsky, Arie; Tacutu, Robi; Fraifeld, Vadim E.

2013-01-01

The Gadd45 proteins have been intensively studied, in view of their important role in key cellular processes. Indeed, the Gadd45 proteins stand at the crossroad of the cell fates by controlling the balance between cell (DNA) repair, eliminating (apoptosis) or preventing the expansion of potentially dangerous cells (cell cycle arrest, cellular senescence), and maintaining the stem cell pool. However, the biogerontological aspects have not thus far received sufficient attention. Here we analyzed the pathways and modes of action by which Gadd45 members are involved in aging, longevity and age-related diseases. Because of their pleiotropic action, a decreased inducibility of Gadd45 members may have far-reaching consequences including genome instability, accumulation of DNA damage, and disorders in cellular homeostasis – all of which may eventually contribute to the aging process and age-related disorders (promotion of tumorigenesis, immune disorders, insulin resistance and reduced responsiveness to stress). Most recently, the dGadd45 gene has been identified as a longevity regulator in Drosophila. Although further wide-scale research is warranted, it is becoming increasingly clear that Gadd45s are highly relevant to aging, age-related diseases (ARDs) and to the control of life span, suggesting them as potential therapeutic targets in ARDs and pro-longevity interventions. PMID:21986581

The protein type within a hypocaloric diet affects obesity-related inflammation: the RESMENA project.

PubMed

Lopez-Legarrea, Patricia; de la Iglesia, Rocio; Abete, Itziar; Navas-Carretero, Santiago; Martinez, J Alfredo; Zulet, M Angeles

2014-04-01

The aim of this study was to compare the effect of two energy-restricted, differing with regard to protein content, on the inflammation state of obese individuals with features of metabolic syndrome. Ninety-six participants completed an 8-wk randomized intervention trial that compared the RESMENA diet (-30% energy, with 30% energy from protein) with a control diet (-30% energy, with 15% energy from protein) that was based on American Heart Association criteria. The mean body weight losses were 7.09 ± 0.82 kg and 6.73 ± 0.71 kg, respectively, with no differences seen between the groups. The endpoint inflammation score-which was based on high-sensitivity C-reactive protein, interleukin-6, tumor necrosis factor-α, and plasminogen activator inhibitor-1 levels-was significantly lower (P = 0.012) in the low-protein group (6.81 ± 2.32 versus 7.94 ± 1.94). The linear regression analyses revealed that total protein intake was positively associated with inflammation (P = 0.007) as well as with animal protein (P = 0.025) and meat protein (P = 0.015), but neither vegetable- nor fish-derived proteins were found to influence inflammatory status. Our results suggest that the type of protein consumed (more than the total protein consumed) within an energy-restricted diet influences the inflammation status associated with obesity-related comorbidities. Copyright © 2014 Elsevier Inc. All rights reserved.

Mifepristone sensitizing cisplatin for cervical adenocarcinoma HeLa cell sensitivity to chemotherapy and its mechanism.

PubMed

Li, Caihong; Ye, Hong

2013-01-01

The study was designed to investigate proliferation inhibition for cervical adenocarcinoma HeLa cell treated with cisplatin combined with mifepristone and access its possible mechanism. HeLa cell was processed by different concentrations of mifepristone, cisplatin, and their combination respectively. Cell's proliferation inhibition rate and induction apoptosis ability were detected by MTT assay, FCM; the expression of P53, survivin and HPV E6 protein were measured by Western Blot. The results showed that cisplatin inhibits proliferation of HeLa cells in different concentrations (p <0.01). Mifepristone had no effect on HeLa cell proliferation inhibition rate during 24 and 48 hours (p > 0.05). Mifepristone at low concentrations (< or = 10 micromol/l) combined with cisplatin can significantly enhance the inhibitory effect of cisplatin on HeLa cell line. Flow cytometry showed that mifepristone at low concentrations (< or = 10 micromol/l) combined with cisplatin can induce apparent apoptosis of HeLa cell line in concentration dependent manner. Western blotting demonstrated that the expression of P53 protein increased and the expression of HPV E6 survivin protein decreased in HeLa cells treated with MIF at low concentrations (< or = 10 micromol/l) combined with cisplatin. Mifepristone at low concentrations (< or = 10 micromol/1) can enhance chemosensitivity and capability of inducing apoptosis of cisplatin to HeLa cells. The strengthening effect of growth inhibition and chemosensitivity to cisplatin of mifepristone are associated with down-regulating HPV E6 survivin protein and upregulating p53 protein.

Synthesis and structural characterization of carboxyethylpyrrole-modified proteins: mediators of age-related macular degeneration.

PubMed

Lu, Liang; Gu, Xiaorong; Hong, Li; Laird, James; Jaffe, Keeve; Choi, Jaewoo; Crabb, John; Salomon, Robert G

2009-11-01

Protein modifications in which the epsilon-amino group of lysyl residues is incorporated into a 2-(omega-carboxyethyl)pyrrole (CEP) are mediators of age-related macular degeneration (AMD). They promote both angiogenesis into the retina ('wet AMD') and geographic retinal atrophy ('dry AMD'). Blood levels of CEPs are biomarkers for clinical prognosis of the disease. To enable mechanistic studies of their role in promoting AMD, for example, through the activation of B- and T-cells, interaction with receptors, or binding with complement proteins, we developed an efficient synthesis of CEP derivatives, that is especially effective for proteins. The structures of tryptic peptides derived from CEP-modified proteins were also determined. A key finding is that 4,7-dioxoheptanoic acid 9-fluorenylmethyl ester reacts with primary amines to provide 9-fluorenylmethyl esters of CEP-modified proteins that can be deprotected in situ with 1,8-diazabicyclo[5.4.0]undec-7-ene without causing protein denaturation. The introduction of multiple CEP-modifications with a wide variety of CEP:protein ratios is readily achieved using this strategy.

Global, quantitative and dynamic mapping of protein subcellular localization

PubMed Central

Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg HH

2016-01-01

Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology. DOI: http://dx.doi.org/10.7554/eLife.16950.001 PMID:27278775

Histone deacetylase-related protein inhibits AES-mediated neuronal cell death by direct interaction.

PubMed

Zhang, Xiaoguang; Chen, Hsin-Mei; Jaramillo, Eduardo; Wang, Lulu; D'Mello, Santosh R

2008-08-15

Histone deacetylase-related protein (HDRP), an alternatively spliced and truncated form of histone deacetylase-9 that lacks a C-terminal catalytic domain, protects neurons from death. In an effort to understand the mechanism by which HDRP mediates its neuroprotective effect, we screened for proteins in the brain that interact with HDRP by using a yeast two-hybrid assay. One of the HDRP-interacting proteins identified in this screen was amino enhancer of split (AES), a 197-amino acid protein belonging to the Groucho family. Interaction between HDRP and AES was verified by in vitro binding assays, coimmunoprecipitation, and colocalization studies. To investigate the significance of the HDRP-AES association to the regulation of neuronal survival, we used cultured cerebellar granule neurons, which undergo apoptosis when treated with low potassium (LK) medium. We found that in contrast to HDRP, whose expression is markedly reduced by LK treatment, AES expression was not appreciably altered. Forced expression of AES in healthy neurons results in cell death, an action that is blocked by the coexpression of HDRP. AES is a truncated version of larger Groucho-related proteins, one of which is transducin-like enhancer of split (TLE)-1. We found that the expression of TLE1 is reduced in LK-treated neurons and the forced expression of TLE1 blocks LK-induced neuronal death as well as death induced by AES. Our results show that AES has apoptotic activity in neurons and suggest that neuroprotection by HDRP is mediated by the inhibition of this activity through direct interaction.

Differential protein-coding gene and long noncoding RNA expression in smoking-related lung squamous cell carcinoma.

PubMed

Li, Shicheng; Sun, Xiao; Miao, Shuncheng; Liu, Jia; Jiao, Wenjie

2017-11-01

Cigarette smoking is one of the greatest preventable risk factors for developing cancer, and most cases of lung squamous cell carcinoma (lung SCC) are associated with smoking. The pathogenesis mechanism of tumor progress is unclear. This study aimed to identify biomarkers in smoking-related lung cancer, including protein-coding gene, long noncoding RNA, and transcription factors. We selected and obtained messenger RNA microarray datasets and clinical data from the Gene Expression Omnibus database to identify gene expression altered by cigarette smoking. Integrated bioinformatic analysis was used to clarify biological functions of the identified genes, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, the construction of a protein-protein interaction network, transcription factor, and statistical analyses. Subsequent quantitative real-time PCR was utilized to verify these bioinformatic analyses. Five hundred and ninety-eight differentially expressed genes and 21 long noncoding RNA were identified in smoking-related lung SCC. GO and KEGG pathway analysis showed that identified genes were enriched in the cancer-related functions and pathways. The protein-protein interaction network revealed seven hub genes identified in lung SCC. Several transcription factors and their binding sites were predicted. The results of real-time quantitative PCR revealed that AURKA and BIRC5 were significantly upregulated and LINC00094 was downregulated in the tumor tissues of smoking patients. Further statistical analysis indicated that dysregulation of AURKA, BIRC5, and LINC00094 indicated poor prognosis in lung SCC. Protein-coding genes AURKA, BIRC5, and LINC00094 could be biomarkers or therapeutic targets for smoking-related lung SCC. © 2017 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

Mammalian Peptidylglycine α-Amidating Monooxygenase mRNA Expression Can Be Modulated by the La Autoantigen

PubMed Central

Brenet, Fabienne; Dussault, Nadège; Borch, Jonas; Ferracci, Géraldine; Delfino, Christine; Roepstorff, Peter; Miquelis, Raymond; Ouafik, L'Houcine

2005-01-01

Peptidylglycine α-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal α-amidation of peptidylglycine substrates, yielding amidated products. We have previously reported a putative regulatory RNA binding protein (PAM mRNA-BP) that binds specifically to the 3′ untranslated region (UTR) of PAM-mRNA. Here, the PAM mRNA-BP was isolated and revealed to be La protein using affinity purification onto a 3′ UTR PAM RNA, followed by tandem mass spectrometry identification. We determined that the core binding sequence is approximately 15-nucleotides (nt) long and is located 471 nt downstream of the stop codon. Moreover, we identified the La autoantigen as a protein that specifically binds the 3′ UTR of PAM mRNA in vivo and in vitro. Furthermore, La protein overexpression caused a nuclear retention of PAM mRNAs and resulted in the down-regulation of endogenous PAM activity. Most interestingly, the nuclear retention of PAM mRNA is lost upon expressing the La proteins that lack a conserved nuclear retention element, suggesting a direct association between PAM mRNA and La protein in vivo. Reporter assays using a chimeric mRNA that combined luciferase and the 3′ UTR of PAM mRNA demonstrated a decrease of the reporter activity due to an increase in the nuclear localization of reporter mRNAs, while the deletion of the 15-nt La binding site led to their clear-cut cytoplasmic relocalization. The results suggest an important role for the La protein in the modulation of PAM expression, possibly by mechanisms that involve a nuclear retention and perhaps a processing of pre-PAM mRNA molecules. PMID:16107699

Roles of dynamin-related protein 1 in the regulation of mitochondrial fission and apoptosis in response to UV stimuli

NASA Astrophysics Data System (ADS)

Zhang, Zhenzhen; Feng, Jie; Wu, Shengnan

2011-03-01

Mitochondria are dynamic structures that frequently divide and fuse with one another to form interconnecting network. This network disintegrates into punctiform organelles during apoptosis. However, it remains unclear whether this event has a significant impact on the rate of cell death or only accompanies apoptosis as an epiphenomenon. In this study, we investigate the role of dynamin-related protein 1 (Drp1), a large GTPase that mediates outer mitochondrial membrane fission, in mitochondrial morphology and apoptosis in response to UV irradiation in human lung adenocarcinoma cells (ASTC-a-1) and HeLa cells. Using time-lapse fluorescent imaging, we find that Drp1 primarily distributes in cytosol under physiological conditions. After UV treatment, Drp1 translocates from cytosol to mitochondria, indicating the enhancement of Drp1 mitochondrial accumulation. Down-regulation of Drp1 by shRNA inhibits UV-induced apoptosis. Our results suggest that Drp1 is involved in the regulation of transition from a reticulo-tubular to a punctiform mitochondrial phenotype and mitochondrial fission plays an important role in UV-induced apoptosis.

Characterization of pancreatic lipase-related protein 2 isolated from human pancreatic juice.

PubMed

De Caro, Josiane; Sias, Barbara; Grandval, Philippe; Ferrato, Francine; Halimi, Hubert; Carrière, Frédéric; De Caro, Alain

2004-09-01

Human pancreatic lipase-related protein 2 (HPLRP2) was identified for the first time in pancreatic juice using specific anti-peptide antibodies and purified to homogeneity. Antibodies were raised in the rabbit using a synthetic peptide from the HPLRP2 protein sequence deduced from cDNA. Western blotting analysis showed that these antibodies did not react with classical human pancreatic lipase (HPL) or human pancreatic lipase-related protein 1 (HPLRP1) but cross-reacted with native rat PLRP2 (RPLRP2), as well as with recombinant rat and guinea-pig PLRP2 (GPLRP2). Immunoaffinity chromatography was performed on immobilized anti-recombinant HPLRP2 polyclonal antibodies to purify native HPLRP2 after conventional chromatographic steps including gel filtration and chromatrography on an anion-exchanger. The substrate specificity of HPLRP2 was investigated using various triglycerides, phospholipids and galactolipids as substrates. The lipase activity on triglycerides was inhibited by bile salts and weakly restored by colipase. The phospholipase activity of HPLRP2 on phospholipid micelles was very low. A significant level of galactolipase activity was measured using monogalactosyldiglyceride monomolecular films. These data suggest that the main physiological function of HPLRP2 is the hydrolysis of galactolipids, which are the main lipids present in vegetable food.

Reaching the Overlooked Student in Physical Education

ERIC Educational Resources Information Center

Esslinger, Keri; Esslinger, Travis; Bagshaw, Jarad

2015-01-01

This article describes the use of live action role-playing, or "LARPing," as a non-traditional activity that has the potential to reach students who are not interested in traditional physical education.

Novel Biosensor of Membrane Protein Proximity Based on Fluorogen Activated Proteins.

PubMed

Vasilev, Kalin V; Gallo, Eugenio; Shank, Nathaniel; Jarvik, Jonathan W

2016-01-01

We describe a novel biosensor system for reporting proximity between cell surface proteins in live cultured cells. The biosensor takes advantage of recently developed fluorogen-activating proteins (FAPs) that display fluorescence only when bound to otherwise-nonfluorescent fluorogen molecules. To demonstrate feasibility for the approach, two recombinant rapamycin-binding proteins were expressed as single-pass plasma membrane proteins in HeLa cells; one of the proteins (scAvd- FRB) carried an extracellular avidin tag; the other (HL1-TO1-FKBP) carried an extracellular FAP. Cells were incubated with a membrane-impermeable bivalent ligand (biotin-PEG2000-DIR) consisting of biotin joined to a dimethyl-indole red (DIR) fluorogen by a polyethylene glycol linker, thus tethering the fluorogen to the scAvd-FRB fusion protein. Addition of rapamycin, which promotes FKBP-FRB dimerization and thereby brings the FAP in close proximity to the tethered fluorogen, led to a significant increase in DIR fluorescence. We call the new proximity assay TEFLA, for tethered fluorogen assay.

Proteomic Analysis Revealed the Important Role of Vimentin in Human Cervical Carcinoma HeLa Cells Treated With Gambogic Acid*

PubMed Central

Yue, Qingxi; Feng, Lixing; Cao, Biyin; Liu, Miao; Zhang, Dongmei; Wu, Wanying; Jiang, Baohong; Yang, Min; Liu, Xuan; Guo, Dean

2016-01-01

Gambogic acid (GA) is an anticancer agent in phase IIb clinical trial in China. In HeLa cells, GA inhibited cell proliferation, induced cell cycle arrest at G2/M phase and apoptosis, as showed by results of MTT assay and flow cytometric analysis. Possible target-related proteins of GA were searched using comparative proteomic analysis (2-DE) and nine proteins at early (3 h) stage together with nine proteins at late (24 h) stage were found. Vimentin was the only target-related protein found at both early and late stage. Results of both 2-DE analysis and Western blotting assay suggested cleavage of vimentin induced by GA. MS/MS analysis of cleaved vimentin peptides indicated possible cleavage sites of vimentin at or near ser51 and glu425. Results of targeted proteomic analysis showed that GA induced change in phosphorylation state of the vimentin head domain (aa51–64). Caspase inhibitors could not abrogate GA-induced cleavage of vimentin. Over-expression of vimentin ameliorated cytotoxicity of GA in HeLa cells. The GA-activated signal transduction, from p38 MAPK, heat shock protein 27 (HSP27), vimentin, dysfunction of cytoskeleton, to cell death, was predicted and then confirmed. Results of animal study showed that GA treatment inhibited tumor growth in HeLa tumor-bearing mice and cleavage of vimentin could be observed in tumor xenografts of GA-treated animals. Results of immunohistochemical staining also showed down-regulated vimentin level in tumor xenografts of GA-treated animals. Furthermore, compared with cytotoxicity of GA in HeLa cells, cytotoxicity of GA in MCF-7 cells with low level of vimentin was weaker whereas cytotoxicity of GA in MG-63 cells with high level of vimentin was stronger. These results indicated the important role of vimentin in the cytotoxicity of GA. The effects of GA on vimentin and other epithelial-to-mesenchymal transition (EMT) markers provided suggestion for better usage of GA in clinic. PMID:26499837

Global versus Local Regulatory Roles for Lrp-Related Proteins: Haemophilus influenzae as a Case Study

PubMed Central

Friedberg, Devorah; Midkiff, Michael; Calvo, Joseph M.

2001-01-01

Lrp (leucine-responsive regulatory protein) plays a global regulatory role in Escherichia coli, affecting expression of dozens of operons. Numerous lrp-related genes have been identified in different bacteria and archaea, including asnC, an E. coli gene that was the first reported member of this family. Pairwise comparisons of amino acid sequences of the corresponding proteins shows an average sequence identity of only 29% for the vast majority of comparisons. By contrast, Lrp-related proteins from enteric bacteria show more than 97% amino acid identity. Is the global regulatory role associated with E. coli Lrp limited to enteric bacteria? To probe this question we investigated LrfB, an Lrp-related protein from Haemophilus influenzae that shares 75% sequence identity with E. coli Lrp (highest sequence identity among 42 sequences compared). A strain of H. influenzae having an lrfB null allele grew at the wild-type growth rate but with a filamentous morphology. A comparison of two-dimensional (2D) electrophoretic patterns of proteins from parent and mutant strains showed only two differences (comparable studies with lrp+ and lrp E. coli strains by others showed 20 differences). The abundance of LrfB in H. influenzae, estimated by Western blotting experiments, was about 130 dimers per cell (compared to 3,000 dimers per E. coli cell). LrfB expressed in E. coli replaced Lrp as a repressor of the lrp gene but acted only to a limited extent as an activator of the ilvIH operon. Thus, although LrfB resembles Lrp sufficiently to perform some of its functions, its low abundance is consonant with a more local role in regulating but a few genes, a view consistent with the results of the 2D electrophoretic analysis. We speculate that an Lrp having a global regulatory role evolved to help enteric bacteria adapt to their ecological niches and that it is unlikely that Lrp-related proteins in other organisms have a broad regulatory function. PMID:11395465

Fabrication and Analysis of 150-mm-Aperture Nb 3Sn MQXF Coils

DOE PAGES

Holik, E. F.; Ambrosio, G.; Anerella, M.; ...

2016-01-12

The U.S. LHC Accelerator Research Program (LARP) and CERN are combining efforts for the HiLumi-LHC upgrade to design and fabricate 150-mm-aperture, interaction region quadrupoles with a nominal gradient of 130 T/m using Nb 3Sn. To successfully produce the necessary long MQXF triplets, the HiLumi-LHC collaboration is systematically reducing risk and design modification by heavily relying upon the experience gained from the successful 120-mm-aperture LARP HQ program. First generation MQXF short (MQXFS) coils were predominately a scaling up of the HQ quadrupole design allowing comparable cable expansion during Nb 3Sn formation heat treatment and increased insulation fraction for electrical robustness. Amore » total of 13 first generation MQXFS coils were fabricated between LARP and CERN. Systematic differences in coil size, coil alignment symmetry, and coil length contraction during heat treatment are observed and likely due to slight variances in tooling and insulation/cable systems. Analysis of coil cross sections indicate that field-shaping wedges and adjacent coil turns are systematically displaced from the nominal location and the cable is expanding less than nominally designed. Lastly, a second generation MQXF coil design seeks to correct the expansion and displacement discrepancies by increasing insulation and adding adjustable shims at the coil pole and midplanes to correct allowed magnetic field harmonics.« less

Computational prediction of protein interactions related to the invasion of erythrocytes by malarial parasites.

PubMed

Liu, Xuewu; Huang, Yuxiao; Liang, Jiao; Zhang, Shuai; Li, Yinghui; Wang, Jun; Shen, Yan; Xu, Zhikai; Zhao, Ya

2014-11-30

The invasion of red blood cells (RBCs) by malarial parasites is an essential step in the life cycle of Plasmodium falciparum. Human-parasite surface protein interactions play a critical role in this process. Although several interactions between human and parasite proteins have been discovered, the mechanism related to invasion remains poorly understood because numerous human-parasite protein interactions have not yet been identified. High-throughput screening experiments are not feasible for malarial parasites due to difficulty in expressing the parasite proteins. Here, we performed computational prediction of the PPIs involved in malaria parasite invasion to elucidate the mechanism by which invasion occurs. In this study, an expectation maximization algorithm was used to estimate the probabilities of domain-domain interactions (DDIs). Estimates of DDI probabilities were then used to infer PPI probabilities. We found that our prediction performance was better than that based on the information of D. melanogaster alone when information related to the six species was used. Prediction performance was assessed using protein interaction data from S. cerevisiae, indicating that the predicted results were reliable. We then used the estimates of DDI probabilities to infer interactions between 490 parasite and 3,787 human membrane proteins. A small-scale dataset was used to illustrate the usability of our method in predicting interactions between human and parasite proteins. The positive predictive value (PPV) was lower than that observed in S. cerevisiae. We integrated gene expression data to improve prediction accuracy and to reduce false positives. We identified 80 membrane proteins highly expressed in the schizont stage by fast Fourier transform method. Approximately 221 erythrocyte membrane proteins were identified using published mass spectral datasets. A network consisting of 205 interactions was predicted. Results of network analysis suggest that SNARE proteins of

Effect of Lactobacillus johnsonii La1 on immune function and serum albumin in aged and malnourished aged mice.

PubMed

Kaburagi, Tomoko; Yamano, Toshihiko; Fukushima, Yoichi; Yoshino, Haruka; Mito, Natsuko; Sato, Kazuto

2007-04-01

Protein-energy malnutrition (PEM) is a serious nutritional problem that causes immune dysfunction in elderly people. Probiotic lactic acid bacteria may potentially modify immunity; however, there is little evidence to elucidate the influence of these bacteria on PEM in the elderly. The immune modulation effects of lactic acid bacterium Lactobacillus johnsonii La1 (La1) were examined in aged mice and aged mice with PEM. Twenty-month-old male 57BL6/n mice (n = 28) were divided into four groups and received the following diet for 14 d: a complete diet (20% protein) without Lal (control) or with Lal or a low-protein diet (5% protein) to induce PEM, with or without La1. All mice were immunized with diphtheria toxin (DT) with alfacalciferol at 7 d and sacrificed 14 d after starting the experimental diets. Serum albumin concentrations and body weight, both of which were reduced by the low-protein diet, were ameliorated by La1 intake and were the same as in mice receiving the control diet. Anti-DT immunoglobulin (Ig) A in fecal extract was increased by La1 intake in mice receiving the complete and low-protein diets. Serum anti-DT IgA, IgG, splenocyte proliferation, and CD8(+) T cells were reduced by the low-protein diet and restored by La1 intake. La1 enhances intestinal IgA production and helps recover nutritional status and systemic immune responses in aged mice with PEM. It is possible that La1 may contribute to immune system recovery in immunocompromised hosts such as elderly humans with PEM.

Exercise training decreases activation of the mitochondrial fission protein dynamin-related protein-1 in insulin-resistant human skeletal muscle.

PubMed

Fealy, Ciaran E; Mulya, Anny; Lai, Nicola; Kirwan, John P

2014-08-01

Defects in mitochondrial dynamics, the processes of fission, fusion, and mitochondrial autophagy, may contribute to metabolic disease including type 2 diabetes. Dynamin-related protein-1 (Drp1) is a GTPase protein that plays a central role in mitochondrial fission. We hypothesized that aerobic exercise training would decrease Drp1 Ser(616) phosphorylation and increase fat oxidation and insulin sensitivity in obese (body mass index: 34.6 ± 0.8 kg/m(2)) insulin-resistant adults. Seventeen subjects performed supervised exercise for 60 min/day, 5 days/wk at 80-85% of maximal heart rate for 12 wk. Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp, and fat oxidation was determined by indirect calorimetry. Skeletal muscle biopsies were obtained from the vastus lateralis muscle before and after the 12-wk program. The exercise intervention increased insulin sensitivity 2.1 ± 0.2-fold (P < 0.01) and fat oxidation 1.3 ± 0.3-fold (P < 0.01). Phosphorylation of Drp1 at Ser(616) was decreased (pre vs. post: 0.81 ± 0.15 vs. 0.58 ± 0.14 arbitrary units; P < 0.05) following the intervention. Furthermore, reductions in Drp1 Ser(616) phosphorylation were negatively correlated with increases in fat oxidation (r = -0.58; P < 0.05) and insulin sensitivity (rho = -0.52; P < 0.05). We also examined expression of genes related to mitochondrial dynamics. Dynamin1-like protein (DNM1L; P < 0.01), the gene that codes for Drp1, and Optic atrophy 1 (OPA1; P = 0.05) were significantly upregulated following the intervention, while there was a trend towards an increase in expression of both mitofusin protein MFN1 (P = 0.08) and MFN2 (P = 0.07). These are the first data to suggest that lifestyle-mediated improvements in substrate metabolism and insulin sensitivity in obese insulin-resistant adults may be regulated through decreased activation of the mitochondrial fission protein Drp1. Copyright © 2014 the American Physiological Society.

The mechanism of lauric acid-modified protein nanocapsules escape from intercellular trafficking vesicles and its implication for drug delivery.

PubMed

Jiang, Lijuan; Liang, Xin; Liu, Gan; Zhou, Yun; Ye, Xinyu; Chen, Xiuli; Miao, Qianwei; Gao, Li; Zhang, Xudong; Mei, Lin

2018-11-01

Protein nanocapsules have exhibited promising potential applications in the field of protein drug delivery. A major issue with various promising nano-sized biotherapeutics including protein nanocapsules is that owing to their particle size they are subject to cellular uptake via endocytosis, and become entrapped and then degraded within endolysosomes, which can significantly impair their therapeutic efficacy. In addition, many nano-sized biotherapeutics could be also sequestered by autophagosomes and degraded through the autolysosomal pathway. Thus, a limiting step in achieving an effective protein therapy is to facilitate the endosomal escape and auto-lysosomal escape to ensure cytosolic delivery of the protein drugs. Here, we prepared a protein nanocapsule based on BSA (nBSA) and the BSA nanocapsules modified with a bilayer of lauric acid (LA-nBSA) to investigate the escape effects from the endosome and autophagosome. The size distribution of nBSA and LA-nBSA analyzed using DLS presents a uniform diameter centered at 10 nm and 16 nm. The data also showed that FITC-labeled nBSA and LA-nBSA were taken up by the cells mainly through Arf-6-dependent endocytosis and Rab34-mediated macropinocytosis. In addition, LA-nBSA could efficiently escape from endosomal before the degradation in endo-lysosomes. Autophagy could also sequester the LA-nBSA through p62 autophagosome vesicles. These two types of nanocapsules underwent different intracellular destinies and lauric acid (LA) coating played a vital role in intracellular particle retention. In conclusion, the protein nanocapsules modified with LA could enhance the protein nanocapsules escape from intercellular trafficking vesicles, and protect the protein from degradation by the lysosomes.

Sperm-binding fibronectin type II-module proteins are genetically linked and functionally related.

PubMed

Ekhlasi-Hundrieser, Mahnaz; Schäfer, Bettina; Philipp, Ute; Kuiper, Heidi; Leeb, Tosso; Mehta, Meenal; Kirchhoff, Christiane; Töpfer-Petersen, Edda

2007-05-01

Fibronectin type II (Fn2) module-containing proteins in the male genital tract are characterized by different numbers of Fn2 modules. Predominantly two classes exist which are distinct by having either two or four Fn2 modules. Minor variants with three Fn2 modules were also found in the human and the porcine epididymis. To reveal their relationship, mRNAs and proteins of representatives of these classes were studied in human, in Sus scrofa, and in rodents. Adult boars expressed members of both classes, i.e. ELSPBP1 and pB1, in subsequent regions of the epididymis, and both were under androgenic control. Human and rodent epididymides, on the other hand, alternatively contained only representatives of one of these two classes, i.e. ELSPBP1 in the human and two different pB1-related counterparts in rodents. ELSPBP1 and pB1-related genomic sequences were closely linked in chromosomal regions HSA 19q and SSC 6 q11-q21; conserved synteny between these regions is well established. On the other hand, in a syntenic region on mouse chromosome 7, ELSPBP1-related sequences were lacking. Tight binding to the sperm membrane via a choline-mediated mechanism was a common feature of the two classes of Fn2-module proteins, suggesting related function(s). However, differences in their regionalized expression patterns along the male genital tract as well as in association sites on the sperm surface suggested a species-specific sequential order in sperm binding.

Protein Secondary Structures (alpha-helix and beta-sheet) at a Cellular Levle and Protein Fractions in Relation to Rumen Degradation Behaviours of Protein: A New Approach

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

Yu,P.

2007-01-01

Studying the secondary structure of proteins leads to an understanding of the components that make up a whole protein, and such an understanding of the structure of the whole protein is often vital to understanding its digestive behaviour and nutritive value in animals. The main protein secondary structures are the {alpha}-helix and {beta}-sheet. The percentage of these two structures in protein secondary structures influences protein nutritive value, quality and digestive behaviour. A high percentage of {beta}-sheet structure may partly cause a low access to gastrointestinal digestive enzymes, which results in a low protein value. The objectives of the present studymore » were to use advanced synchrotron-based Fourier transform IR (S-FTIR) microspectroscopy as a new approach to reveal the molecular chemistry of the protein secondary structures of feed tissues affected by heat-processing within intact tissue at a cellular level, and to quantify protein secondary structures using multicomponent peak modelling Gaussian and Lorentzian methods, in relation to protein digestive behaviours and nutritive value in the rumen, which was determined using the Cornell Net Carbohydrate Protein System. The synchrotron-based molecular chemistry research experiment was performed at the National Synchrotron Light Source at Brookhaven National Laboratory, US Department of Energy. The results showed that, with S-FTIR microspectroscopy, the molecular chemistry, ultrastructural chemical make-up and nutritive characteristics could be revealed at a high ultraspatial resolution ({approx}10 {mu}m). S-FTIR microspectroscopy revealed that the secondary structure of protein differed between raw and roasted golden flaxseeds in terms of the percentages and ratio of {alpha}-helixes and {beta}-sheets in the mid-IR range at the cellular level. By using multicomponent peak modelling, the results show that the roasting reduced (P <0.05) the percentage of {alpha}-helixes (from 47.1% to 36.1%: S

A novel method for preparation of HAMLET-like protein complexes.

PubMed

Permyakov, Sergei E; Knyazeva, Ekaterina L; Leonteva, Marina V; Fadeev, Roman S; Chekanov, Aleksei V; Zhadan, Andrei P; Håkansson, Anders P; Akatov, Vladimir S; Permyakov, Eugene A

2011-09-01

Some natural proteins induce tumor-selective apoptosis. α-Lactalbumin (α-LA), a milk calcium-binding protein, is converted into an antitumor form, called HAMLET/BAMLET, via partial unfolding and association with oleic acid (OA). Besides triggering multiple cell death mechanisms in tumor cells, HAMLET exhibits bactericidal activity against Streptococcus pneumoniae. The existing methods for preparation of active complexes of α-LA with OA employ neutral pH solutions, which greatly limit water solubility of OA. Therefore these methods suffer from low scalability and/or heterogeneity of the resulting α-LA - OA samples. In this study we present a novel method for preparation of α-LA - OA complexes using alkaline conditions that favor aqueous solubility of OA. The unbound OA is removed by precipitation under acidic conditions. The resulting sample, bLA-OA-45, bears 11 OA molecules and exhibits physico-chemical properties similar to those of BAMLET. Cytotoxic activities of bLA-OA-45 against human epidermoid larynx carcinoma and S. pneumoniae D39 cells are close to those of HAMLET. Treatment of S. pneumoniae with bLA-OA-45 or HAMLET induces depolarization and rupture of the membrane. The cells are markedly rescued from death upon pretreatment with an inhibitor of Ca(2+) transport. Hence, the activation mechanisms of S. pneumoniae death are analogous for these two complexes. The developed express method for preparation of active α-LA - OA complex is high-throughput and suited for development of other protein complexes with low-molecular-weight amphiphilic substances possessing valuable cytotoxic properties. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Expression of Tau Pathology-Related Proteins in Different Brain Regions: A Molecular Basis of Tau Pathogenesis.

PubMed

Hu, Wen; Wu, Feng; Zhang, Yanchong; Gong, Cheng-Xin; Iqbal, Khalid; Liu, Fei

2017-01-01

Microtubule-associated protein tau is hyperphosphorylated and aggregated in affected neurons in Alzheimer disease (AD) brains. The tau pathology starts from the entorhinal cortex (EC), spreads to the hippocampus and frontal and temporal cortices, and finally to all isocortex areas, but the cerebellum is spared from tau lesions. The molecular basis of differential vulnerability of different brain regions to tau pathology is not understood. In the present study, we analyzed brain regional expressions of tau and tau pathology-related proteins. We found that tau was hyperphosphorylated at multiple sites in the frontal cortex (FC), but not in the cerebellum, from AD brain. The level of tau expression in the cerebellum was about 1/4 of that seen in the frontal and temporal cortices in human brain. In the rat brain, the expression level of tau with three microtubule-binding repeats (3R-tau) was comparable in the hippocampus, EC, FC, parietal-temporal cortex (PTC), occipital-temporal cortex (OTC), striatum, thalamus, olfactory bulb (OB) and cerebellum. However, the expression level of 4R-tau was the highest in the EC and the lowest in the cerebellum. Tau phosphatases, kinases, microtubule-related proteins and other tau pathology-related proteins were also expressed in a region-specific manner in the rat brain. These results suggest that higher levels of tau and tau kinases in the EC and low levels of these proteins in the cerebellum may accounts for the vulnerability and resistance of these representative brain regions to the development of tau pathology, respectively. The present study provides the regional expression profiles of tau and tau pathology-related proteins in the brain, which may help understand the brain regional vulnerability to tau pathology in neurodegenerative tauopathies.

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria†

PubMed Central

Deutscher, Josef; Francke, Christof; Postma, Pieter W.

2006-01-01

The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens. PMID:17158705

Surface properties of heat-induced soluble soy protein aggregates of different molecular masses.

PubMed

Guo, Fengxian; Xiong, Youling L; Qin, Fang; Jian, Huajun; Huang, Xiaolin; Chen, Jie

2015-02-01

Suspensions (2% and 5%, w/v) of soy protein isolate (SPI) were heated at 80, 90, or 100 °C for different time periods to produce soluble aggregates of different molecular sizes to investigate the relationship between particle size and surface properties (emulsions and foams). Soluble aggregates generated in these model systems were characterized by gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Heat treatment increased surface hydrophobicity, induced SPI aggregation via hydrophobic interaction and disulfide bonds, and formed soluble aggregates of different sizes. Heating of 5% SPI always promoted large-size aggregate (LA; >1000 kDa) formation irrespective of temperature, whereas the aggregate size distribution in 2% SPI was temperature dependent: the LA fraction progressively rose with temperature (80→90→100 °C), corresponding to the attenuation of medium-size aggregates (MA; 670 to 1000 kDa) initially abundant at 80 °C. Heated SPI with abundant LA (>50%) promoted foam stability. LA also exhibited excellent emulsifying activity and stabilized emulsions by promoting the formation of small oil droplets covered with a thick interfacial protein layer. However, despite a similar influence on emulsion stability, MA enhanced foaming capacity but were less capable of stabilizing emulsions than LA. The functionality variation between heated SPI samples is clearly related to the distribution of aggregates that differ in molecular size and surface activity. The findings may encourage further research to develop functional SPI aggregates for various commercial applications. © 2015 Institute of Food Technologists®

Processus ultra-rapides associés à la dynamique d'émission de la protéine GFP

NASA Astrophysics Data System (ADS)

Didier, P.; Guidoni, L.; Schwalbach, G.; Bigot, J.-Y.

2002-06-01

La protéine GFP (Green Fluorescent Protein) est un marqueur très efficace, utilisable en milieu vivant. La spectroscopie femtoseconde est particulièrement bien adaptée pour comprendre les mécanismes d'émission de cette protéine, étant donné la rapidité des processus de transfert mis en jeu. Nous-présentons des résultats sur la dynamique spectro-temporelle d'émission du mutant GFPuv résolue à l'échelle de la centaine de femtosecondes. Une transition Raman à 3300 cm^{-1} ainsi que la dynamique d'etablissement du gain avec un temps caractéristique d'environ 1.5 ps ont été mis en évidence.

The first report of prion-related protein gene (PRNT) polymorphisms in goat.

PubMed

Kim, Yong-Chan; Jeong, Byung-Hoon

2017-06-01

Prion protein is encoded by the prion protein gene (PRNP). Polymorphisms of several members of the prion gene family have shown association with prion diseases in several species. Recent studies on a novel member of the prion gene family in rams have shown that prion-related protein gene (PRNT) has a linkage with codon 26 of prion-like protein (PRND). In a previous study, codon 26 polymorphism of PRND has shown connection with PRNP haplotype which is strongly associated with scrapie vulnerability. In addition, the genotype of a single nucleotide polymorphism (SNP) at codon 26 of PRND is related to fertilisation capacity. These findings necessitate studies on the SNP of PRNT gene which is connected with PRND. In goat, several polymorphism studies have been performed for PRNP, PRND, and shadow of prion protein gene (SPRN). However, polymorphism on PRNT has not been reported. Hence, the objective of this study was to determine the genotype and allelic distribution of SNPs of PRNT in 238 Korean native goats and compare PRNT DNA sequences between Korean native goats and several ruminant species. A total of five SNPs, including PRNT c.-114G > T, PRNT c.-58A > G in the upstream of PRNT gene, PRNT c.71C > T (p.Ala24Val) and PRNT c.102G > A in the open reading frame (ORF) and c.321C > T in the downstream of PRNT gene, were found in this study. All five SNPs of caprine PRNT gene in Korean native goat are in complete linkage disequilibrium (LD) with a D' value of 1.0. Interestingly, comparative sequence analysis of the PRNT gene revealed five mismatches between DNA sequences of Korean native goats and those of goats deposited in the GenBank. Korean native black goats also showed 5 mismatches in PRNT ORF with cattle. To the best of our knowledge, this is the first genetic research of the PRNT gene in goat.

Constitutive expression of human pancreatic lipase-related protein 1 in Pichia pastoris.

PubMed

Aloulou, Ahmed; Grandval, Philippe; De Caro, Josiane; De Caro, Alain; Carrière, Frédéric

2006-06-01

High-level constitutive expression of the human pancreatic lipase-related protein 1 (HPLRP1) was achieved using the methylotrophic yeast Pichia pastoris. The HPLRP1 cDNA, including its original leader sequence, was subcloned into the pGAPZB vector and further integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase (GAP) constitutive promoter. A major protein with a molecular mass of 50 kDa was found to be secreted into the culture medium and was identified using anti-HPLRP1 polyclonal antibodies as HPLRP1 recombinant protein. The level of expression reached 100-120 mg of HPLRP1 per liter of culture medium after 40 h, as attested by specific and quantitative enzyme-linked immunosorbent assay. A single cation-exchange chromatography sufficed to obtain a highly purified recombinant HPLRP1 after direct batch adsorption onto S-Sepharose of the HPLRP1 present in the culture medium, at pH 5.5. N-terminal sequencing and mass spectrometry analysis were carried out to monitor the production of the mature protein and to confirm that its signal peptide was properly processed.

Discovery of new candidate genes related to brain development using protein interaction information.

PubMed

Chen, Lei; Chu, Chen; Kong, Xiangyin; Huang, Tao; Cai, Yu-Dong

2015-01-01

Human brain development is a dramatic process composed of a series of complex and fine-tuned spatiotemporal gene expressions. A good comprehension of this process can assist us in developing the potential of our brain. However, we have only limited knowledge about the genes and gene functions that are involved in this biological process. Therefore, a substantial demand remains to discover new brain development-related genes and identify their biological functions. In this study, we aimed to discover new brain-development related genes by building a computational method. We referred to a series of computational methods used to discover new disease-related genes and developed a similar method. In this method, the shortest path algorithm was executed on a weighted graph that was constructed using protein-protein interactions. New candidate genes fell on at least one of the shortest paths connecting two known genes that are related to brain development. A randomization test was then adopted to filter positive discoveries. Of the final identified genes, several have been reported to be associated with brain development, indicating the effectiveness of the method, whereas several of the others may have potential roles in brain development.

Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination.

PubMed

Mbantenkhu, MacMillan; Wang, Xiaowen; Nardozzi, Jonathan D; Wilkens, Stephan; Hoffman, Elizabeth; Patel, Anamika; Cosgrove, Michael S; Chen, Xin Jie

2011-12-09

Homologous recombination is a conserved molecular process that has primarily evolved for the repair of double-stranded DNA breaks and stalled replication forks. However, the recombination machinery in mitochondria is poorly understood. Here, we show that the yeast mitochondrial nucleoid protein, Mgm101, is related to the Rad52-type recombination proteins that are widespread in organisms from bacteriophage to humans. Mgm101 is required for repeat-mediated recombination and suppression of mtDNA fragmentation in vivo. It preferentially binds to single-stranded DNA and catalyzes the annealing of ssDNA precomplexed with the mitochondrial ssDNA-binding protein, Rim1. Transmission electron microscopy showed that Mgm101 forms large oligomeric rings of ∼14-fold symmetry and highly compressed helical filaments. Specific mutations affecting ring formation reduce protein stability in vitro. The data suggest that the ring structure may provide a scaffold for stabilization of Mgm101 by preventing the aggregation of the otherwise unstable monomeric conformation. Upon binding to ssDNA, Mgm101 is remobilized from the rings to form distinct nucleoprotein filaments. These studies reveal a recombination protein of likely bacteriophage origin in mitochondria and support the notion that recombination is indispensable for mtDNA integrity.

Structure and stability insights into tumour suppressor p53 evolutionary related proteins.

PubMed

Pagano, Bruno; Jama, Abdullah; Martinez, Pierre; Akanho, Ester; Bui, Tam T T; Drake, Alex F; Fraternali, Franca; Nikolova, Penka V

2013-01-01

The p53 family of genes and their protein products, namely, p53, p63 and p73, have over one billion years of evolutionary history. Advances in computational biology and genomics are enabling studies of the complexities of the molecular evolution of p53 protein family to decipher the underpinnings of key biological conditions spanning from cancer through to various metabolic and developmental disorders and facilitate the design of personalised medicines. However, a complete understanding of the inherent nature of the thermodynamic and structural stability of the p53 protein family is still lacking. This is due, to a degree, to the lack of comprehensive structural information for a large number of homologous proteins and to an incomplete knowledge of the intrinsic factors responsible for their stability and how these might influence function. Here we investigate the thermal stability, secondary structure and folding properties of the DNA-binding domains (DBDs) of a range of proteins from the p53 family using biophysical methods. While the N- and the C-terminal domains of the p53 family show sequence diversity and are normally targets for post-translational modifications and alternative splicing, the central DBD is highly conserved. Together with data obtained from Molecular Dynamics simulations in solution and with structure based homology modelling, our results provide further insights into the molecular properties of evolutionary related p53 proteins. We identify some marked structural differences within the p53 family, which could account for the divergence in biological functions as well as the subtleties manifested in the oligomerization properties of this family.

Collagen-binding proteins of Streptococcus mutans and related streptococci.

PubMed

Avilés-Reyes, A; Miller, J H; Lemos, J A; Abranches, J

2017-04-01

The ability of Streptococcus mutans to interact with collagen through the expression of collagen-binding proteins (CBPs) bestows this oral pathogen with an alternative to the sucrose-dependent mechanism of colonization classically attributed to caries development. Based on the abundance and distribution of collagen throughout the human body, stringent adherence to this molecule grants S. mutans with the opportunity to establish infection at different host sites. Surface proteins, such as SpaP, WapA, Cnm and Cbm, have been shown to bind collagen in vitro, and it has been suggested that these molecules play a role in colonization of oral and extra-oral tissues. However, robust collagen binding is not achieved by all strains of S. mutans, particularly those that lack Cnm or Cbm. These observations merit careful dissection of the contribution from these different CBPs towards tissue colonization and virulence. In this review, we will discuss the current understanding of mechanisms used by S. mutans and related streptococci to colonize collagenous tissues, and the possible contribution of CBPs to infections in different sites of the host. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Space-related pharma-motifs for fast search of protein binding motifs and polypharmacological targets

PubMed Central

2012-01-01

Background To discover a compound inhibiting multiple proteins (i.e. polypharmacological targets) is a new paradigm for the complex diseases (e.g. cancers and diabetes). In general, the polypharmacological proteins often share similar local binding environments and motifs. As the exponential growth of the number of protein structures, to find the similar structural binding motifs (pharma-motifs) is an emergency task for drug discovery (e.g. side effects and new uses for old drugs) and protein functions. Results We have developed a Space-Related Pharmamotifs (called SRPmotif) method to recognize the binding motifs by searching against protein structure database. SRPmotif is able to recognize conserved binding environments containing spatially discontinuous pharma-motifs which are often short conserved peptides with specific physico-chemical properties for protein functions. Among 356 pharma-motifs, 56.5% interacting residues are highly conserved. Experimental results indicate that 81.1% and 92.7% polypharmacological targets of each protein-ligand complex are annotated with same biological process (BP) and molecular function (MF) terms, respectively, based on Gene Ontology (GO). Our experimental results show that the identified pharma-motifs often consist of key residues in functional (active) sites and play the key roles for protein functions. The SRPmotif is available at http://gemdock.life.nctu.edu.tw/SRP/. Conclusions SRPmotif is able to identify similar pharma-interfaces and pharma-motifs sharing similar binding environments for polypharmacological targets by rapidly searching against the protein structure database. Pharma-motifs describe the conservations of binding environments for drug discovery and protein functions. Additionally, these pharma-motifs provide the clues for discovering new sequence-based motifs to predict protein functions from protein sequence databases. We believe that SRPmotif is useful for elucidating protein functions and drug discovery

Space-related pharma-motifs for fast search of protein binding motifs and polypharmacological targets.

PubMed

Chiu, Yi-Yuan; Lin, Chun-Yu; Lin, Chih-Ta; Hsu, Kai-Cheng; Chang, Li-Zen; Yang, Jinn-Moon

2012-01-01

To discover a compound inhibiting multiple proteins (i.e. polypharmacological targets) is a new paradigm for the complex diseases (e.g. cancers and diabetes). In general, the polypharmacological proteins often share similar local binding environments and motifs. As the exponential growth of the number of protein structures, to find the similar structural binding motifs (pharma-motifs) is an emergency task for drug discovery (e.g. side effects and new uses for old drugs) and protein functions. We have developed a Space-Related Pharmamotifs (called SRPmotif) method to recognize the binding motifs by searching against protein structure database. SRPmotif is able to recognize conserved binding environments containing spatially discontinuous pharma-motifs which are often short conserved peptides with specific physico-chemical properties for protein functions. Among 356 pharma-motifs, 56.5% interacting residues are highly conserved. Experimental results indicate that 81.1% and 92.7% polypharmacological targets of each protein-ligand complex are annotated with same biological process (BP) and molecular function (MF) terms, respectively, based on Gene Ontology (GO). Our experimental results show that the identified pharma-motifs often consist of key residues in functional (active) sites and play the key roles for protein functions. The SRPmotif is available at http://gemdock.life.nctu.edu.tw/SRP/. SRPmotif is able to identify similar pharma-interfaces and pharma-motifs sharing similar binding environments for polypharmacological targets by rapidly searching against the protein structure database. Pharma-motifs describe the conservations of binding environments for drug discovery and protein functions. Additionally, these pharma-motifs provide the clues for discovering new sequence-based motifs to predict protein functions from protein sequence databases. We believe that SRPmotif is useful for elucidating protein functions and drug discovery.

The p38 mitogen-activated protein kinase signaling pathway is involved in regulating low-density lipoprotein receptor-related protein 1-mediated β-amyloid protein internalization in mouse brain.

PubMed

Ma, Kai-Ge; Lv, Jia; Hu, Xiao-Dan; Shi, Li-Li; Chang, Ke-Wei; Chen, Xin-Lin; Qian, Yi-Hua; Yang, Wei-Na; Qu, Qiu-Min

2016-07-01

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. Recently, increasing evidence suggests that intracellular β-amyloid protein (Aβ) alone plays a pivotal role in the progression of AD. Therefore, understanding the signaling pathway and proteins that control Aβ internalization may provide new insight for regulating Aβ levels. In the present study, the regulation of Aβ internalization by p38 mitogen-activated protein kinases (MAPK) through low-density lipoprotein receptor-related protein 1 (LRP1) was analyzed in vivo. The data derived from this investigation revealed that Aβ1-42 were internalized by neurons and astrocytes in mouse brain, and were largely deposited in mitochondria and lysosomes, with some also being found in the endoplasmic reticulum. Aβ1-42-LRP1 complex was formed during Aβ1-42 internalization, and the p38 MAPK signaling pathway was activated by Aβ1-42 via LRP1. Aβ1-42 and LRP1 were co- localized in the cells of parietal cortex and hippocampus. Furthermore, the level of LRP1-mRNA and LRP1 protein involved in Aβ1-42 internalization in mouse brain. The results of this investigation demonstrated that Aβ1-42 induced an LRP1-dependent pathway that related to the activation of p38 MAPK resulting in internalization of Aβ1-42. These results provide evidence supporting a key role for the p38 MAPK signaling pathway which is involved in the regulation of Aβ1-42 internalization in the parietal cortex and hippocampus of mouse through LRP1 in vivo. Copyright © 2016 Elsevier Ltd. All rights reserved.

[The effect of UV-irradiation on telomerase activity and other stress-related proteins in human lens epithelial cells].

PubMed

Wu, Zhi-hong; Zhang, Jin-song

2005-05-01

To investigate the changes and the role of telomerase activity and other stress-related proteins in the process of UV-induced DNA damage and repair in human lens epithelial cells. Human lens epithelial cells were irradiated at UV-doses 0.0 (control group) and 0.5, 1.5, 2.5, 3.5, 5.0, 7.5, 10.0 mJ/cm(2) (treated 1-7 group). Telomerase activity was determined by Telomerase Repeat Amplification Protocol-Enzyme Linked Immunosorbent Assay (TRAP-ELISA), p53, growth arrest and DNA damage inducible (GADD45), proliferating cell nuclear antigen (PCNA) and p16 protein levels were analyzed by Western blotting. Telomerase activity in control group and treated 1-7 group showed increased tendency, the differences of telomerase activity in 8 groups were significantly (P < 0.01). The expression of p53, GADD45, PCNA, p16 proteins showed increased tendency in experimental group, comparing with the control group, there were significant difference (P < 0.01). During UV-induced DNA damage and repair in human lens epithelial cells, telomerase activity was upregulated and the expression of stress-related proteins levels was increased. Upregulated telomerase activity may play both a protective and a proliferative role in human lens epithelial cells. Increased stress-related proteins level is critic in UV-induced DNA damage and repair in human lens epithelial. Increased telomerase activity is associated with increased levels of the stress-related proteins.

Proliferating fibroblasts and HeLa cells co-cultured in vitro reciprocally influence growth patterns, protein expression, chromatin features and cell survival.

PubMed

Delinasios, John G; Angeli, Flora; Koumakis, George; Kumar, Shant; Kang, Wen-Hui; Sica, Gigliola; Iacopino, Fortunata; Lama, Gina; Lamprecht, Sergio; Sigal-Batikoff, Ina; Tsangaris, George T; Farfarelos, Christos D; Farfarelos, Maria C; Vairaktaris, Eleftherios; Vassiliou, Stavros; Delinasios, George J

2015-04-01

if fibroblast proliferation is blocked by contact inhibition of growth at confluency, or by omitting replacement of the nutrient medium. The present observations show that: (a) interaction between proliferating fibroblasts and HeLa cells in vitro drastically influences each other's protein expression, growth pattern, chromatin features and survival; (b) these functions depend on the fibroblast/HeLa ratio, cell topology (cell-cell contact and the architectural pattern developed during co-culture) and frequent medium change, as prerequisites for fibroblast proliferation; (c) this co-culture model is useful in the study of the complex processes within the tumour microenvironment, as well as the in vitro reproduction and display of several phenomena conventionally seen in tumour cytological sections, such as desmoplasia, apoptosis, nuclear abnormalities; and (d) overgrown fibroblasts adhering to the boundaries of HeLa colonies produce and secrete lipid droplets. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Protein complex formation and intranuclear dynamics of NAC1 in cancer cells.

PubMed

Nakayama, Naomi; Kato, Hiroaki; Sakashita, Gyosuke; Nariai, Yuko; Nakayama, Kentaro; Kyo, Satoru; Urano, Takeshi

2016-09-15

Nucleus accumbens-associated protein 1 (NAC1) is a cancer-related transcription regulator protein that is also involved in the pluripotency and differentiation of embryonic stem cells. NAC1 is overexpressed in various carcinomas including ovarian, cervical, breast, and pancreatic carcinomas. NAC1 knock-down was previously shown to result in the apoptosis of ovarian cancer cell lines and to rescue their sensitivity to chemotherapy, suggesting that NAC1 may be a potential therapeutic target, but protein complex formation and the dynamics of intranuclear NAC1 in cancer cells remain poorly understood. In this study, analysis of HeLa cell lysates by fast protein liquid chromatography (FPLC) on a sizing column showed that the NAC1 peak corresponded to an apparent molecular mass of 300-500 kDa, which is larger than the estimated molecular mass (58 kDa) of the protein. Furthermore, live cell photobleaching analyses with green fluorescent protein (GFP)-fused NAC1 proteins revealed the intranuclear dynamics of NAC1. Collectively our results demonstrate that NAC1 forms a protein complex to function as a transcriptional regulator in cancer cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Interactions between whey proteins and salivary proteins as related to astringency of whey protein beverages at low pH.

PubMed

Ye, A; Streicher, C; Singh, H

2011-12-01

Whey protein beverages have been shown to be astringent at low pH. In the present study, the interactions between model whey proteins (β-lactoglobulin and lactoferrin) and human saliva in the pH range from 7 to 2 were investigated using particle size, turbidity, and ζ-potential measurements and sodium dodecyl sulfate-PAGE. The correlation between the sensory results of astringency and the physicochemical data was discussed. Strong interactions between β-lactoglobulin and salivary proteins led to an increase in the particle size and turbidity of mixtures of both unheated and heated β-lactoglobulin and human saliva at pH ∼3.4. However, the large particle size and high turbidity that occurred at pH 2.0 were the result of aggregation of human salivary proteins. The intense astringency in whey protein beverages may result from these increases in particle size and turbidity at these pH values and from the aggregation and precipitation of human salivary proteins alone at pH <3.0. The involvement of salivary proteins in the interaction is a key factor in the perception of astringency in whey protein beverages. At any pH, the increases in particle size and turbidity were much smaller in mixtures of lactoferrin and saliva, which suggests that aggregation and precipitation may not be the only mechanism linked to the perception of astringency in whey protein. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Interaction of the Sliding Clamp β-Subunit and Hda, a DnaA-Related Protein

PubMed Central

Kurz, Mareike; Dalrymple, Brian; Wijffels, Gene; Kongsuwan, Kritaya

2004-01-01

In Escherichia coli, interactions between the replication initiation protein DnaA, the β subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and β proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with β in vitro. A new β-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified β-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind β. A 10-amino-acid peptide containing the E. coli Hda β-binding motif was shown to compete with Hda for binding to β in an Hda-β interaction assay. These results establish that the interaction of Hda with β is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication. PMID:15150238

Interaction of the sliding clamp beta-subunit and Hda, a DnaA-related protein.

PubMed

Kurz, Mareike; Dalrymple, Brian; Wijffels, Gene; Kongsuwan, Kritaya

2004-06-01

In Escherichia coli, interactions between the replication initiation protein DnaA, the beta subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and beta proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with beta in vitro. A new beta-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified beta-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind beta. A 10-amino-acid peptide containing the E. coli Hda beta-binding motif was shown to compete with Hda for binding to beta in an Hda-beta interaction assay. These results establish that the interaction of Hda with beta is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication.

De novo design of RNA-binding proteins with a prion-like domain related to ALS/FTD proteinopathies.

PubMed

Mitsuhashi, Kana; Ito, Daisuke; Mashima, Kyoko; Oyama, Munenori; Takahashi, Shinichi; Suzuki, Norihiro

2017-12-04

Aberrant RNA-binding proteins form the core of the neurodegeneration cascade in spectrums of disease, such as amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Six ALS-related molecules, TDP-43, FUS, TAF15, EWSR1, heterogeneous nuclear (hn)RNPA1 and hnRNPA2 are RNA-binding proteins containing candidate mutations identified in ALS patients and those share several common features, including harboring an aggregation-prone prion-like domain (PrLD) containing a glycine/serine-tyrosine-glycine/serine (G/S-Y-G/S)-motif-enriched low-complexity sequence and rich in glutamine and/or asparagine. Additinally, these six molecules are components of RNA granules involved in RNA quality control and become mislocated from the nucleus to form cytoplasmic inclusion bodies (IBs) in the ALS/FTD-affected brain. To reveal the essential mechanisms involved in ALS/FTD-related cytotoxicity associated with RNA-binding proteins containing PrLDs, we designed artificial RNA-binding proteins harboring G/S-Y-G/S-motif repeats with and without enriched glutamine residues and nuclear-import/export-signal sequences and examined their cytotoxicity in vitro. These proteins recapitulated features of ALS-linked molecules, including insoluble aggregation, formation of cytoplasmic IBs and components of RNA granules, and cytotoxicity instigation. These findings indicated that these artificial RNA-binding proteins mimicked features of ALS-linked molecules and allowed the study of mechanisms associated with gain of toxic functions related to ALS/FTD pathogenesis.

Identification of a follistatin-related protein from the tick Haemaphysalis longicornis and its effect on tick oviposition.

PubMed

Zhou, Jinlin; Liao, Min; Hatta, Takeshi; Tanaka, Miho; Xuan, Xuenan; Fujisaki, Kozo

2006-05-10

The identification of ovary-associated molecules will lead to a better understanding of the physiology of tick reproduction and vector-pathogen interactions. A gene encoding a follistatin-related protein (FRP) was obtained by random sequencing from the ovary cDNA library of the tick Haemaphysalis longicornis. The full-length cDNA is 1157 bp, including an intact ORF encoding an expected protein with 289 amino acids. Three distinct domains were present in the deduced amino acids, namely, the follistatin-like domain, KAZAL, and two calcium-binding motifs, EFh. The sequence shows homology with the follistatin-related protein (FRP), which was thought to play some roles in the negative regulation of cellular growth. RT-PCR showed that the gene was expressed throughout the developing stages and mainly in the ovary as well as in fat body, hemocytes, salivary glands, and midgut. This gene was expressed in GST-fused recombinant protein with an expected size. The mouse antiserum against the recombinant protein recognized a 56-kDa native protein in both tick ovary and hemolymph. The recombinant proteins were found to have binding activity for both activin A and bone morphogenetic protein-2 (BMP-2). Silencing of FRP by RNAi showed a decrease in tick oviposition, which is consistent with the effect of a recombinant protein vaccine on the adult tick. These results showed that the tick FRP might be involved in tick oviposition. This is the first report of a member of follistatin family proteins in Chelicerata, which include ticks, spiders, and scorpions.

Human pancreatic lipase-related protein 2 is a galactolipase.

PubMed

Sias, Barbara; Ferrato, Francine; Grandval, Philippe; Lafont, Dominique; Boullanger, Paul; De Caro, Alain; Leboeuf, Bernard; Verger, Robert; Carrière, Frédéric

2004-08-10

Human pancreatic lipase-related protein 2 (HPLRP2) was found to be expressed in the pancreas, but its biochemical properties were not investigated in detail. A recombinant HPLRP2 was produced in insect cells and the yeast Pichia pastoris and purified by cation exchange chromatography. Its substrate specificity was investigated using pH-stat and monomolecular film techniques and various lipid substrates (triglycerides, diglycerides, phospholipids, and galactolipids). Lipase activity of HPLRP2 on trioctanoin was inhibited by bile salts and poorly restored by adding colipase. In vivo, HPLRP2 therefore seems unlikely to show any lipase activity on dietary fat. In human pancreatic lipase (HPL), residues R256, D257, Y267, and K268 are involved in the stabilization of the open conformation of the lid domain, which interacts with colipase. These residues are not conserved in HPLRP2. When the corresponding mutations (R256G, D257G, Y267F, and K268E) are introduced into HPL, the effects of colipase are drastically reduced in the presence of bile salts. This may explain why colipase has such weak effects on HPLRP2. HPLRP2 displayed a very low level of activity on phospholipid micelles and monomolecular films. Its activity on monogalactosyldiglyceride monomolecular film, which was much higher, was similar to the activity of guinea pig pancreatic lipase related-protein 2, which shows the highest galactolipase activity ever measured. The physiological role of HPLRP2 suggested by the present results is the digestion of galactolipids, the most abundant lipids occurring in plant cells, and therefore, in the vegetables that are part of the human diet.

Fukutin-related protein localizes to the Golgi apparatus and mutations lead to mislocalization in muscle in vivo.

PubMed

Keramaris-Vrantsis, Elizabeth; Lu, Pei J; Doran, Timothy; Zillmer, Allen; Ashar, Jignya; Esapa, Christopher T; Benson, Matthew A; Blake, Derek J; Rosenfeld, Jeffrey; Lu, Qi L

2007-10-01

Mutations in the fukutin-related protein gene (FKRP) are associated with a spectrum of diseases from mild limb-girdle muscular dystrophy type 2I to severe congenital muscular dystrophy type 1C, muscle-eye-brain disease (MEB), and Walker-Warburg syndrome (WWS). The effect of mutations on the transportation of the mutant proteins may constitute the underlying mechanisms for the pathogenesis of these diseases. Here we examined the subcellular localization of mouse and human normal and mutant FKRP proteins in cells and in muscle in vivo. Both normal human and mouse FKRPs localize in part of the Golgi apparatus in muscle fibers. Mutations in the FKRP gene invariably altered the localization of the protein, leading to endoplasmic reticulum retention within cells and diminished Golgi localization in muscle fibers. Our results therefore suggest that an individual missense point mutation can confer at least two independent effects on the protein, causing (1) reduction or loss of the presumed glycosyltransferase activity directly and (2) mislocalization that could further alter the function of the protein. The complexity of the effect of individual missense point mutations may partly explain the wide variation of the FKRP-related myopathies.

Glutamate Induced Thermal Equilibrium Intermediate and Counteracting Effect on Chemical Denaturation of Proteins.

PubMed

Anumalla, Bramhini; Prabhu, N Prakash

2018-01-25

When organisms are subjected to stress conditions, one of their adaptive responses is accumulation of small organic molecules called osmolytes. These osmolytes affect the structure and stability of the biological macromolecules including proteins. The present study examines the effect of a negatively charged amino acid osmolyte, glutamate (Glu), on two model proteins, ribonuclease A (RNase A) and α-lactalbumin (α-LA), which have positive and negative surface charges at pH 7, respectively. These proteins follow two-state unfolding transitions during both heat and chemical induced denaturation processes. The addition of Glu stabilizes the proteins against temperature and induces an early equilibrium intermediate during unfolding. The stability is found to be enthalpy-driven, and the free energy of stabilization is more for α-LA compared to RNase A. The decrease in the partial molar volume and compressibility of both of the proteins in the presence of Glu suggests that the proteins attain a more compact state through surface hydration which could provide a more stable conformation. This is also supported by molecule dynamic simulation studies which demonstrate that the water density around the proteins is increased upon the addition of Glu. Further, the intermediates could be completely destabilized by lower concentrations (∼0.5 M) of guanidinium chloride and salt. However, urea subverts the Glu-induced intermediate formed by α-LA, whereas it only slightly destabilizes in the case of RNase A which has a positive surface charge and could possess charge-charge interactions with Glu. This suggests that, apart from hydration, columbic interactions might also contribute to the stability of the intermediate. Gdm-induced denaturation of RNase A and α-LA in the absence and the presence of Glu at different temperatures was carried out. These results also show the Glu-induced stabilization of both of the proteins; however, all of the unfolding transitions followed two

Relation between native ensembles and experimental structures of proteins

PubMed Central

Best, Robert B.; Lindorff-Larsen, Kresten; DePristo, Mark A.; Vendruscolo, Michele

2006-01-01

Different experimental structures of the same protein or of proteins with high sequence similarity contain many small variations. Here we construct ensembles of “high-sequence similarity Protein Data Bank” (HSP) structures and consider the extent to which such ensembles represent the structural heterogeneity of the native state in solution. We find that different NMR measurements probing structure and dynamics of given proteins in solution, including order parameters, scalar couplings, and residual dipolar couplings, are remarkably well reproduced by their respective high-sequence similarity Protein Data Bank ensembles; moreover, we show that the effects of uncertainties in structure determination are insufficient to explain the results. These results highlight the importance of accounting for native-state protein dynamics in making comparisons with ensemble-averaged experimental data and suggest that even a modest number of structures of a protein determined under different conditions, or with small variations in sequence, capture a representative subset of the true native-state ensemble. PMID:16829580

High-pressure-induced interactions between milk fat globule membrane proteins and skim milk proteins in whole milk.

PubMed

Ye, A; Anema, S G; Singh, H

2004-12-01

The association of beta-lactoglobulin (beta-LG) and alpha-lactalbumin (alpha-LA) with milk fat globule membrane (MFGM), when whole milk was treated by high pressure in the range 100 to 800 MPa, was investigated using sodium dodecyl sulfate (SDS)-PAGE under reducing and nonreducing conditions. In SDS-PAGE under reducing conditions, beta-LG was observed in the MFGM material isolated from milk treated at 100 to 800 MPa for 30 min, and small amounts of alpha-LA and kappa-casein were also observed at pressures >600 MPa for 30 min. However, these proteins were not observed in SDS-PAGE under nonreducing conditions. These results indicate that beta-LG and alpha-LA associated with MFGM proteins via disulfide bonds during the high-pressure treatment of whole milk. The amount of beta-LG associated with the MFGM increased with an increase in pressure up to 800 MPa and with increasing time of pressure treatment. The maximum value for beta-LG association with the MFGM was approximately 0.75 mg/g of fat. Of the major original MFGM proteins, no change in butyrophilin was observed during the high-pressure treatment of whole milk, whereas xanthine oxidase was reduced to some extent beyond 400 MPa. In contrast to the behavior during heat treatment, PAS 6 and PAS 7 were stable during high-pressure treatment, and they remained associated with the MFGM.

Photo-reduction on the rupture of disulfide bonds and the related protein assembling

NASA Astrophysics Data System (ADS)

Wang, Wei

It has been found that many proteins can self-assemble into nanoscale assemblies when they unfold or partially unfold under harsh conditions, such as low pH, high temperature, or the presence of denaturants, and so on. These nanoscale assemblies can have some applications such as the drug-delivery systems (DDSs). Here we report a study that a very physical way, the UV illumination, can be used to facilitate the formation of protein fibrils and nanoparticles under native conditions by breaking disulfide bonds in some disulfide-containing proteins. By controlling the intensity of UV light and the illumination time, we realized the preparation of self-assembly nanoparticles which encapsulate the anticancer drug doxorubicin (DOX) and can be used as the DDS for inhibiting the growth of tumor. The formation of fibrillary assemblies was also observed. The rupture of disulfide bonds through photo-reduction process due to the effect of tryptophan and tyrosine was studied, and the physical mechanism of the assembling of the related disulfide-containing proteins was also discussed. We thank the financial support from NSF of China and the 973 project.

Relation ofSpodoptera eridania choice to tannins and protein oflotus corniculatus.

PubMed

Briggs, M A

1990-05-01

Plant secondary compounds such as tannins may influence herbivore choice. To determine if herbivory was influenced by tannin concentration,Spodoptera eridania larvae were given a choice ofLotus corniculatus plants whose chemical profiles were altered by fertilization. Herbivores chose plants that had been grown with symbiotic nitrogen fixation as their only nitrogen source more often than fertilized plants. Choice was related to protein concentration, but not to tannin concentration.

[Apoptosis and expression of apoptosis-related proteins in experimental different denervated guinea-pig facial muscle].

PubMed

Hui, Lian; Wei, Hong-Quan; Li, Xiao-Tian; Guan, Chao; Ren, Zhong

2005-02-01

To study apoptosis and expression of apoptosis-related proteins in experimental different denervated guinea-pig facial muscle. An experimental model was established with guinea pigs by compressing the facial nerve 30 second (reinnervated group) and resecting the facial nerve (denervated group). TUNEL method and immunohistochemical technique (SABC) were applied to detect the apoptosis and expression of apoptosis-related proteins bcl-2 and bax from 1st to 8th week after operation. Experimentally denervated facial muscle revealed consistently increase of DNA fragmentation, average from(34.4 +/- 4.6)% to (38.2 +/- 10.6)%, from 1st week to 8th week after operation; Reinnervated facial muscle showed a temporal increase of DNA fragmentation, and then the muscle fiber nuclei revealed decreased DNA fragmentation along with the function of facial nerve recovered, latterly normal, average from (32.0 +/- 8.03)% to (5.6 +/- 3.5)%, from 1st week to 8th week after operation. In denervated group, bcl-2 and bax were expressed strongly; in reinnervated group, bcl-2 expressed consistently, but bax disappeared latterly along with the function of facial nerve recovered. Expression of DNA fragmentation and apoptosis-related proteins in denervated muscle are general reaction to denervation. bcl-2 can prevent early apoptotic muscle fiber to survival until reinnervation. It is concluded that proteins control apoptosis may give information for possible therapeutic interventions to reduce the rate of muscle fiber death in denervated atrophy in absence of effective primary treatment.

Age-related spatial learning impairment is unrelated to spinophilin immunoreactive spine number and protein levels in rat hippocampus.

PubMed

Calhoun, Michael E; Fletcher, Bonnie R; Yi, Stella; Zentko, Diana C; Gallagher, Michela; Rapp, Peter R

2008-08-01

Age-related impairments in hippocampus-dependent learning and memory tasks are not associated with a loss of hippocampal neurons, but may be related to alterations in synaptic integrity. Here we used stereological techniques to estimate spine number in hippocampal subfields using immunostaining for the spine-associated protein, spinophilin, as a marker. Quantification of the immunoreactive profiles was performed using the optical disector/fractionator technique. Aging was associated with a modest increase in spine number in the molecular layer of the dentate gyrus and CA1 stratum lacunosum-moleculare. By comparison, spinophilin protein levels in the hippocampus, measured by Western blot analysis, failed to differ as a function of age. Neither the morphological nor the protein level data were correlated with spatial learning ability across individual aged rats. The results extend current evidence on synaptic integrity in the aged brain, indicating that a substantial loss of dendritic spines and spinophilin protein in the hippocampus are unlikely to contribute to age-related impairment in spatial learning.

Molecular comparison of the structural proteins encoding gene clusters of two related Lactobacillus delbrueckii bacteriophages.

PubMed Central

Vasala, A; Dupont, L; Baumann, M; Ritzenthaler, P; Alatossava, T

1993-01-01

Virulent phage LL-H and temperate phage mv4 are two related bacteriophages of Lactobacillus delbrueckii. The gene clusters encoding structural proteins of these two phages have been sequenced and further analyzed. Six open reading frames (ORF-1 to ORF-6) were detected. Protein sequencing and Western immunoblotting experiments confirmed that ORF-3 (g34) encoded the main capsid protein Gp34. The presence of a putative late promoter in front of the phage LL-H g34 gene was suggested by primer extension experiments. Comparative sequence analysis between phage LL-H and phage mv4 revealed striking similarities in the structure and organization of this gene cluster, suggesting that the genes encoding phage structural proteins belong to a highly conservative module. Images PMID:8497043

Purine inhibitors of protein kinases, G proteins and polymerases

DOEpatents

Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

2001-07-03

The present invention relates to purine analogs that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such purine analogs to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

Serine protease-related proteins in the malaria mosquito, Anopheles gambiae.

PubMed

Cao, Xiaolong; Gulati, Mansi; Jiang, Haobo

2017-09-01

Insect serine proteases (SPs) and serine protease homologs (SPHs) participate in digestion, defense, development, and other physiological processes. In mosquitoes, some clip-domain SPs and SPHs (i.e. CLIPs) have been investigated for possible roles in antiparasitic responses. In a recent test aimed at improving quality of gene models in the Anopheles gambiae genome using RNA-seq data, we observed various discrepancies between gene models in AgamP4.5 and corresponding sequences selected from those modeled by Cufflinks, Trinity and Bridger. Here we report a comparative analysis of the 337 SP-related proteins in A. gambiae by examining their domain structures, sequence diversity, chromosomal locations, and expression patterns. One hundred and ten CLIPs contain 1 to 5 clip domains in addition to their protease domains (PDs) or non-catalytic, protease-like domains (PLDs). They are divided into five subgroups: CLIPAs (22) are clip 1-5 -PLD; CLIPBs (29), CLIPCs (12) and CLIPDs (14) are mainly clip-PD; most CLIPEs (33) have a domain structure of PD/PLD-PLD-clip-PLD 0-1 . While expression of the CLIP genes in group-1 is generally low and detected in various tissue- and stage-specific RNA-seq libraries, some putative GPs/GPHs (i.e. single domain gut SPs/SPHs) in group-2 are highly expressed in midgut, whole larva or whole adult libraries. In comparison, 46 SPs, 26 SPHs, and 37 multi-domain SPs/SPHs (i.e. PD/PLD-PLD ≥1 ) in group-3 do not seem to be specifically expressed in digestive tract. There are 16 SPs and 2 SPH containing other types of putative regulatory domains (e.g. LDLa, CUB, Gd). Of the 337 SP and SPH genes, 159 were sorted into 46 groups (2-8 members/group) based on similar phylogenetic tree position, chromosomal location, and expression profile. This information and analysis, including improved gene models and protein sequences, constitute a solid foundation for functional analysis of the SP-related proteins in A. gambiae. Copyright © 2017 Elsevier Ltd

["Protein of senility" CCL11, "protein of juvenility" GDF11 and their role in age-related pathology].

PubMed

Khavinson, V Kh; Kuznik, B I; Ryzhak, G A; Linkova, N S; Kozina, L S; Sall, T S

2016-01-01

The paper presents the latest literature data on the structure and functions of «protein of juvenility» - CCL11 and «protein of senility» - GDF11. Chemokine CCL11 injected to young animals has been shown to lead to degenerative changes in the central nervous system (CNS), disturb cognitive functions and impede tissue regeneration. CCL11 concentration increases dramatically in schizophrenia, Alzheimer's disease, neuro-inflammatory disorders, cerebral malaria, drug addiction, as well as in atherosclerosis, periodontal disease, macular degeneration, cancer and other pathologies. In contrast to CCL11, differentiation growth factor 11 (GDF11), being administered to old mice, eliminates age-associated hypertrophy of the heart, improves muscle tone and prevents degenerative changes in the CNS, improves cognitive functions and enhances tissue regeneration. Its concentration decreases in cardiovascular disease, osteoporosis, and other «diseases of old age». At the same time, the higher the GDF11 level in the blood, the milder myocardial infarction, stroke and other age-related diseases of the cardiovascular system.

Complement factor H-related proteins in IgA nephropathy-sometimes a gentle nudge does the trick.

PubMed

Thurman, Joshua M; Laskowski, Jennifer

2017-10-01

Complement activation probably contributes to glomerular inflammation and damage in IgA nephropathy. In this issue, 2 groups report that levels of factor H-related protein 1 are elevated in patients with IgA nephropathy and correlate with disease progression. These studies provide new evidence that the complement cascade is important to the pathogenesis of this disease. These results also suggest that factor H-related protein 1 levels may be useful for identifying those patients at high risk of disease progression. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Isolation and characterization of adrenoleukodystrophy protein (ALDP) related sequences in the human genome

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

Geraghty, M.T.; Stetten, G.; Kearns, W.

1994-09-01

X-linked adrenoleukodystrophy (ALD) is a disorder of peroxisomal {beta}-oxidation of very long chain fatty acids. It presents either as progressive dementia in childhood or as progressive paraparesis in later years. Adrenal insufficiency occurs in both phenotypes. The gene of the ALD protein has been mapped to Xq28 and has recently been cloned and characterized. The ALD protein has significant homology to the peroxisomal membrane protein, PMP70 and belongs to the ATP binding cassette superfamily of transporters. We screened a human genomic library with an ALDP cDNA and isolated 5 different but highly similar clones containing sequences corresponding to the 3{prime}more » end of the ALDP gene. Comparison of the sequences over the region corresponding to exon 9 through the 3{prime} end of the ALDP gene reveals {approximately}96% nucleotide identity in both exonic and intronic regions. Splice sites and open reading frames are maintained. Using both FISH and human-rodent DNA mapping panels, we positively assign these ALDP-related sequences to chromosomes 2, 16 and 22, and provisionally to 1 and 20. Southern blot of primate DNA probed with a partial ALDP cDNA (exon 2-10) shows that expansion of ALDP-related sequences occurred in higher primates (chimp, gorilla and human). Although Northern blots show multiple ALDP-hybridizing transcripts in certain tissues, we have no evidence to date for expression of these ALDP-related sequences. In conclusion, our data show there has been an unusual and recent dispersal to multiple chromosomes of structural gene sequences related to the ALDP gene. The functional significance of these sequences remains to be determined but their existence complicates PCR and mutation analysis of the ALDP gene.« less

Biologically active protein fragments containing specific binding regions of serum albumin or related proteins

NASA Technical Reports Server (NTRS)

Carter, Daniel C. (Inventor)

1998-01-01

In accordance with the present invention, biologically active protein fragments can be constructed which contain only those specific portions of the serum albumin family of proteins such as regions known as subdomains IIA and IIIA which are primarily responsible for the binding properties of the serum albumins. The artificial serums that can be prepared from these biologically active protein fragments are advantageous in that they can be produced much more easily than serums containing the whole albumin, yet still retain all or most of the original binding potential of the full albumin proteins. In addition, since the protein fragment serums of the present invention can be made from non-natural sources using conventional recombinant DNA techniques, they are far safer than serums containing natural albumin because they do not carry the potentially harmful viruses and other contaminants that will be found in the natural substances.

Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays

PubMed Central

Popescu, Sorina C.; Popescu, George V.; Bachan, Shawn; Zhang, Zimei; Seay, Montrell; Gerstein, Mark; Snyder, Michael; Dinesh-Kumar, S. P.

2007-01-01

Calmodulins (CaMs) are the most ubiquitous calcium sensors in eukaryotes. A number of CaM-binding proteins have been identified through classical methods, and many proteins have been predicted to bind CaMs based on their structural homology with known targets. However, multicellular organisms typically contain many CaM-like (CML) proteins, and a global identification of their targets and specificity of interaction is lacking. In an effort to develop a platform for large-scale analysis of proteins in plants we have developed a protein microarray and used it to study the global analysis of CaM/CML interactions. An Arabidopsis thaliana expression collection containing 1,133 ORFs was generated and used to produce proteins with an optimized medium-throughput plant-based expression system. Protein microarrays were prepared and screened with several CaMs/CMLs. A large number of previously known and novel CaM/CML targets were identified, including transcription factors, receptor and intracellular protein kinases, F-box proteins, RNA-binding proteins, and proteins of unknown function. Multiple CaM/CML proteins bound many binding partners, but the majority of targets were specific to one or a few CaMs/CMLs indicating that different CaM family members function through different targets. Based on our analyses, the emergent CaM/CML interactome is more extensive than previously predicted. Our results suggest that calcium functions through distinct CaM/CML proteins to regulate a wide range of targets and cellular activities. PMID:17360592

Cocaine-induced behavioral sensitization decreases the expression of endocannabinoid signaling-related proteins in the mouse hippocampus.

PubMed

Blanco, Eduardo; Galeano, Pablo; Palomino, Ana; Pavón, Francisco J; Rivera, Patricia; Serrano, Antonia; Alen, Francisco; Rubio, Leticia; Vargas, Antonio; Castilla-Ortega, Estela; Decara, Juan; Bilbao, Ainhoa; de Fonseca, Fernando Rodríguez; Suárez, Juan

2016-03-01

In the reward mesocorticolimbic circuits, the glutamatergic and endocannabinoid systems are implicated in neurobiological mechanisms underlying cocaine addiction. However, the involvement of both systems in the hippocampus, a critical region to process relational information relevant for encoding drug-associated memories, in cocaine-related behaviors remains unknown. In the present work, we studied whether the hippocampal gene/protein expression of relevant glutamate signaling components, including glutamate-synthesizing enzymes and metabotropic and ionotropic receptors, and the hippocampal gene/protein expression of cannabinoid type 1 (CB1) receptor and endocannabinoid metabolic enzymes were altered following acute and/or repeated cocaine administration resulting in conditioned locomotion and locomotor sensitization. Results showed that acute cocaine administration induced an overall down-regulation of glutamate-related gene expression and, specifically, a low phosphorylation level of GluA1. In contrast, locomotor sensitization to cocaine produced an up-regulation of several glutamate receptor-related genes and, specifically, an increased protein expression of the GluN1 receptor subunit. Regarding the endocannabinoid system, acute and repeated cocaine administration were associated with an increased gene/protein expression of CB1 receptors and a decreased gene/protein expression of the endocannabinoid-synthesis enzymes N-acyl phosphatidylethanolamine D (NAPE-PLD) and diacylglycerol lipase alpha (DAGLα). These changes resulted in an overall decrease in endocannabinoid synthesis/degradation ratios, especially NAPE-PLD/fatty acid amide hydrolase and DAGLα/monoacylglycerol lipase, suggesting a reduced endocannabinoid production associated with a compensatory up-regulation of CB1 receptor. Overall, these findings suggest that repeated cocaine administration resulting in locomotor sensitization induces a down-regulation of the endocannabinoid signaling that could

Prostate Cancer Cell Growth: Stimulatory Role of Neurotensin and Mechanism of Inhibition by Flavonoids as Related to Protein Kinase C

DTIC Science & Technology

2009-01-01

Stimulatory Role of Neurotensin and Mechanism of Inhibition by Flavonoids as Related to Protein Kinase C PRINCIPAL INVESTIGATOR: Robert E. Carraway...CONTRACT NUMBER Prostate Cancer Cell Growth: Stimulatory Role of Neurotensin and Mechanism of Inhibition by Flavonoids as Related to Protein Kinase C...relationship between neurotensin (NT) and protein kinases and to investigate the mechanism by which flavonoids (FLAV) inhibit NT growth signaling in PC3

Proteomic investigation into betulinic acid-induced apoptosis of human cervical cancer HeLa cells.

PubMed

Xu, Tao; Pang, Qiuying; Zhou, Dong; Zhang, Aiqin; Luo, Shaman; Wang, Yang; Yan, Xiufeng

2014-01-01

Betulinic acid is a pentacyclic triterpenoid that exhibits anticancer functions in human cancer cells. This study provides evidence that betulinic acid is highly effective against the human cervical cancer cell line HeLa by inducing dose- and time-dependent apoptosis. The apoptotic process was further investigated using a proteomics approach to reveal protein expression changes in HeLa cells following betulinic acid treatment. Proteomic analysis revealed that there were six up- and thirty down-regulated proteins in betulinic acid-induced HeLa cells, and these proteins were then subjected to functional pathway analysis using multiple analysis software. UDP-glucose 6-dehydrogenase, 6-phosphogluconate dehydrogenase decarboxylating, chain A Horf6-a novel human peroxidase enzyme that involved in redox process, was found to be down-regulated during the apoptosis process of the oxidative stress response pathway. Consistent with our results at the protein level, an increase in intracellular reactive oxygen species was observed in betulinic acid-treated cells. The proteins glucose-regulated protein and cargo-selection protein TIP47, which are involved in the endoplasmic reticulum pathway, were up-regulated by betulinic acid treatment. Meanwhile, 14-3-3 family proteins, including 14-3-3β and 14-3-3ε, were down-regulated in response to betulinic acid treatment, which is consistent with the decrease in expression of the target genes 14-3-3β and 14-3-3ε. Furthermore, it was found that the antiapoptotic bcl-2 gene was down-regulated while the proapoptotic bax gene was up-regulated after betulinic acid treatment in HeLa cells. These results suggest that betulinic acid induces apoptosis of HeLa cells by triggering both the endoplasmic reticulum pathway and the ROS-mediated mitochondrial pathway.

Multivariate proteomic profiling identifies novel accessory proteins of coated vesicles

PubMed Central

Antrobus, Robin; Hirst, Jennifer; Bhumbra, Gary S.; Kozik, Patrycja; Jackson, Lauren P.; Sahlender, Daniela A.

2012-01-01

Despite recent advances in mass spectrometry, proteomic characterization of transport vesicles remains challenging. Here, we describe a multivariate proteomics approach to analyzing clathrin-coated vesicles (CCVs) from HeLa cells. siRNA knockdown of coat components and different fractionation protocols were used to obtain modified coated vesicle-enriched fractions, which were compared by stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative mass spectrometry. 10 datasets were combined through principal component analysis into a “profiling” cluster analysis. Overall, 136 CCV-associated proteins were predicted, including 36 new proteins. The method identified >93% of established CCV coat proteins and assigned >91% correctly to intracellular or endocytic CCVs. Furthermore, the profiling analysis extends to less well characterized types of coated vesicles, and we identify and characterize the first AP-4 accessory protein, which we have named tepsin. Finally, our data explain how sequestration of TACC3 in cytosolic clathrin cages causes the severe mitotic defects observed in auxilin-depleted cells. The profiling approach can be adapted to address related cell and systems biological questions. PMID:22472443

Effect of a nutrient mixture on the localization of extracellular matrix proteins in HeLa human cervical cancer xenografts in female nude mice.

PubMed

Roomi, M Waheed; Cha, John; Kalinovsky, Tatiana; Roomi, Nusrath; Niedzwiecki, Aleksandra; Rath, Matthias

2015-09-01

Cervical cancer is one of the most commonly diagnosed cancers and a significant cause of mortality in women worldwide. Although cervical cancer is fully treatable in the early stages, once it has metastasized, patient outcome is poor. The objective of the present study was to investigate the effect of dietary supplementation with a nutrient mixture (NM) containing lysine, ascorbic acid, proline, green tea extract and other micronutrients on the expression of extracellular matrix (ECM) proteins in HeLa cell xenografts in nude female mice. After housing for 1 week, female athymic nude mice between 5 and 6 weeks of age (n=12) were inoculated subcutaneously with 3×10 6 HeLa cells in phosphate-buffered saline and Matrigel and randomly divided into two groups. These were the control group, in which the mice were fed with regular mouse chow, and the NM group, in which the mice were fed with the regular diet supplemented with 0.5% NM (w/w). After 4 weeks, the tumors were excised and processed for histology. Tumor growth was evaluated and the tumors were stained for the ECM proteins collagen I, collagen IV, fibronectin, laminin, periodic acid-Schiff (PAS) and elastin. NM strongly inhibited (by 59%, P=0.001) the growth of HeLa xenografts in nude mice. Tumors from control mice exhibited little to no collagen I expression either internally or in the fibrous capsule, while tumors from the NM group expressed collagen I in the fibrous capsule and within the tumor. Tumors from the control group showed diffuse cytoplasmic and capsular collagen IV with abundant nucleated cells. NM treatment substantially increased collagen IV production and induced a dense fibrous network of collagen IV with chambers that surrounded live nucleated cells and large amounts of necrotic cell debris. Tumors from the mice fed with the NM exhibited a well-defined border of fibronectin in the capsule and intense areas of staining internally whereas control group tumors showed less overall fibronectin with

Relative size selection of a conjugated polyelectrolyte in virus-like protein structures.

PubMed

Brasch, Melanie; Cornelissen, Jeroen J L M

2012-02-01

A conjugated polyelectrolyte poly[(2-methoxy-5-propyloxy sulfonate)-phenyl-ene vinylene] (MPS-PPV) drives the assembly of virus capsid proteins to form single virus-like particles (VLPs) and aggregates with more than two VLPs, with a relative selection of high molecular weight polymer in the latter. This journal is © The Royal Society of Chemistry 2012

Age-Related Changes in the Expression of the Circadian Clock Protein PERIOD in Drosophila Glial Cells

PubMed Central

Long, Dani M.; Giebultowicz, Jadwiga M.

2018-01-01

Circadian clocks consist of molecular negative feedback loops that coordinate physiological, neurological, and behavioral variables into “circa” 24-h rhythms. Rhythms in behavioral and other circadian outputs tend to weaken during aging, as evident in progressive disruptions of sleep-wake cycles in aging organisms. However, less is known about the molecular changes in the expression of clock genes and proteins that may lead to the weakening of circadian outputs. Western blot studies have demonstrated that the expression of the core clock protein PERIOD (PER) declines in the heads of aged Drosophila melanogaster flies. This age-related decline in PER does not occur in the central pacemaker neurons but has been demonstrated so far in retinal photoreceptors. Besides photoreceptors, clock proteins are also expressed in fly glia, which play important roles in neuronal homeostasis and are further categorized into subtypes based on morphology and function. While previous studies of mammalian glial cells have demonstrated the presence of functional clocks in astrocytes and microglia, it is not known which glial cell types in Drosophila express clock proteins and how their expression may change in aged individuals. Here, we conducted immunocytochemistry experiments to identify which glial subtypes express PER protein suggestive of functional circadian clocks. Glial cell subtypes that showed night-time accumulation and day-time absence in PER consistent with oscillations reported in the pacemaker neurons were selected to compare the level of PER protein between young and old flies. Our data demonstrate that some glial subtypes show rhythmic PER expression and the relative PER levels become dampened with advanced age. Identification of glial cell types that display age-related dampening of PER levels may help to understand the cellular changes that contribute to the loss of homeostasis in the aging brain. PMID:29375400

Power blue and green laser diodes and their applications

NASA Astrophysics Data System (ADS)

Hager, Thomas; Strauß, Uwe; Eichler, Christoph; Vierheilig, Clemens; Tautz, Sönke; Brüderl, Georg; Stojetz, Bernhard; Wurm, Teresa; Avramescu, Adrian; Somers, André; Ristic, Jelena; Gerhard, Sven; Lell, Alfred; Morgott, Stefan; Mehl, Oliver

2013-03-01

InGaN based green laser diodes with output powers up to 50mW are now well established for variety of applications ranging from leveling to special lighting effects and mobile projection of 12lm brightness. In future the highest market potential for visible single mode profile lasers might be laser projection of 20lm. Therefore direct green single-mode laser diodes with higher power are required. We found that self heating was the limiting factor for higher current operation. We present power-current characteristics of improved R and D samples with up to 200mW in cw-operation. An optical output power of 100mW is reached at 215mA, a current level which is suitable for long term operation. Blue InGaN laser diodes are also the ideal source for phosphor based generation of green light sources of high luminance. We present a light engine based on LARP (Laser Activated Remote Phosphor) which can be used in business projectors of several thousand lumens on screen. We discuss the advantages of a laser based systems in comparison with LED light engines. LARP requires highly efficient blue power laser diodes with output power above 1W. Future market penetration of LARP will require lower costs. Therefore we studied new designs for higher powers levels. R and D chips with power-current characteristics up to 4W in continuous wave operation on C-mount at 25°C are presented.

A flow-proteometric platform for analyzing protein concentration (FAP): Proof of concept for quantification of PD-L1 protein in cells and tissues.

PubMed

Chou, Chao-Kai; Huang, Po-Jung; Tsou, Pei-Hsiang; Wei, Yongkun; Lee, Heng-Huan; Wang, Ying-Nai; Liu, Yen-Liang; Shi, Colin; Yeh, Hsin-Chih; Kameoka, Jun; Hung, Mien-Chie

2018-05-29

Protein expression level is critically related to the cell physiological function. However, current methodologies such as Western blot (WB) and Immunohistochemistry (IHC) in analyzing the protein level are rather semi-quantitative and without the information of actual protein concentration. We have developed a microfluidic technique termed a "flow-proteometric platform for analyzing protein concentration (FAP)" that can measure the concentration of a target protein in cells or tissues without the requirement of a calibration standard, e.g., the purified target molecules. To validate our method, we tested a number of control samples with known target protein concentrations and showed that the FAP measurement resulted in concentrations that well matched the actual concentrations in the control samples (coefficient of determination [R 2 ], 0.998), demonstrating a dynamic range of concentrations from 0.13 to 130 pM of a detection for 2 min. We successfully determined a biomarker protein (for predicting the treatment response of cancer immune check-point therapy) PD-L1 concentration in cancer cell lines (HeLa PD-L1 and MDA-MB-231) and breast cancer patient tumor tissues without any prior process of sample purification and standard line construction. Therefore, FAP is a simple, faster, and reliable method to measure the protein concentration in cells and tissues, which can support the conventional methods such as WB and IHC to determine the actual protein level. Copyright © 2018 Elsevier B.V. All rights reserved.

Rapid increase of inositol 1,4,5-trisphosphate in the HeLa cells after hypergravity exposure

NASA Technical Reports Server (NTRS)

Kumei, Yasuhiro; Whitson, Peggy A.; Cintron, Nitza M.; Sato, Atsushige

1990-01-01

The IP3 level in HeLa cells has been elevated through the application in hypergravity in a time-dependent manner. The data obtained for the hydrolytic products of PIP2, IP3, and DG are noted to modulate c-myc gene expression. It is also established that the cAMP accumulation by the IBMX in hypergravity-exposed cells was suppressed relative to the control. In light of IP3 increase and cAMP decrease results, a single GTP-binding protein may play a role in the hypergravity signal transduction of HeLa cells by stimulating PLC while inhibiting adenylate cyclase.

Usher syndrome protein network functions in the retina and their relation to other retinal ciliopathies.

PubMed

Sorusch, Nasrin; Wunderlich, Kirsten; Bauss, Katharina; Nagel-Wolfrum, Kerstin; Wolfrum, Uwe

2014-01-01

The human Usher syndrome (USH) is the most frequent cause of combined hereditary deaf-blindness. USH is genetically and clinically heterogeneous: 15 chromosomal loci assigned to 3 clinical types, USH1-3. All USH1 and 2 proteins are organized into protein networks by the scaffold proteins harmonin (USH1C), whirlin (USH2D) and SANS (USH1G). This has contributed essentially to our current understanding of the USH protein function in the eye and the ear and explains why defects in proteins of different families cause very similar phenotypes. Ongoing in depth analyses of USH protein networks in the eye indicated cytoskeletal functions as well as roles in molecular transport processes and ciliary cargo delivery in photoreceptor cells. The analysis of USH protein networks revealed molecular links of USH to other ciliopathies, including non-syndromic inner ear defects and isolated retinal dystrophies but also to kidney diseases and syndromes like the Bardet-Biedl syndrome. These findings provide emerging evidence that USH is a ciliopathy molecularly related to other ciliopathies, which opens an avenue for common therapy strategies to treat these diseases.

Lack of A-factor production induces the expression of nutrient scavenging and stress-related proteins in Streptomyces griseus.

PubMed

Birkó, Zsuzsanna; Swiatek, Magdalena; Szájli, Emília; Medzihradszky, Katalin F; Vijgenboom, Erik; Penyige, András; Keseru, Judit; van Wezel, Gilles P; Biró, Sándor

2009-10-01

The small gamma-butyrolactone A-factor is an important autoregulatory signaling molecule for the soil-inhabiting streptomycetes. Starvation is a major trigger for development, and nutrients are provided by degradation of the vegetative mycelium via a process of programmed cell death, reusing proteins, nucleic acids, and cell wall material. The A-factor regulon includes many extracellular hydrolases. Here we show via proteomics analysis that many nutrient-scavenging and stress-related proteins were overexpressed in an A-factor non-producing mutant of Streptomyces griseus B-2682. Transcript analysis showed that this is primarily due to differential transcription of the target genes during early development. The targets include proteins relating to nutrient stress and environmental stress and an orthologue of the Bacillus sporulation control protein Spo0M. The enhanced expression of these proteins underlines the stress that is generated by the absence of A-factor. Wild-type developmental gene expression was restored to the A-factor non-producing mutant by the signaling protein Factor C in line with our earlier observation that Factor C triggers A-factor production.

Zac1, an Sp1-like protein, regulates human p21{sup WAF1/Cip1} gene expression in HeLa cells

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

Liu, Pei-Yao; Hsieh, Tsai-Yuan; Liu, Shu-Ting

2011-12-10

Zac1 functions as both a transcription factor and a transcriptional cofactor for p53, nuclear receptors (NRs) and NR coactivators. Zac1 might also act as a transcriptional repressor via the recruitment of histone deacetylase 1 (HDAC1). The ability of Zac1 to interact directly with GC-specific elements indicates that Zac1 possibly binds to Sp1-responsive elements. In the present study, our data show that Zac1 is able to interact directly with the Sp1-responsive element in the p21{sup WAF1/Cip1} gene promoter and enhance the transactivation activity of Sp1 through direct physical interaction. Our data further demonstrate that Zac1 might enhance Sp1-specific promoter activity bymore » interacting with the Sp1-responsive element, affecting the transactivation activity of Sp1 via a protein-protein interaction, or competing the HDAC1 protein away from the pre-existing Sp1/HDAC1 complex. Finally, the synergistic regulation of p21{sup WAF1/Cip1} gene expression by Zac1 and Sp1 is mediated by endogenous p53 protein and p53-responsive elements in HeLa cells. Our work suggests that Zac1 might serve as an Sp1-like protein that directly interacts with the Sp1-responsive element to oligomerize with and/or to coactivate Sp1.« less

Refining the treatment of membrane proteins by coarse-grained models.

PubMed

Vorobyov, Igor; Kim, Ilsoo; Chu, Zhen T; Warshel, Arieh

2016-01-01

Obtaining a quantitative description of the membrane proteins stability is crucial for understanding many biological processes. However the advance in this direction has remained a major challenge for both experimental studies and molecular modeling. One of the possible directions is the use of coarse-grained models but such models must be carefully calibrated and validated. Here we use a recent progress in benchmark studies on the energetics of amino acid residue and peptide membrane insertion and membrane protein stability in refining our previously developed coarse-grained model (Vicatos et al., Proteins 2014;82:1168). Our refined model parameters were fitted and/or tested to reproduce water/membrane partitioning energetics of amino acid side chains and a couple of model peptides. This new model provides a reasonable agreement with experiment for absolute folding free energies of several β-barrel membrane proteins as well as effects of point mutations on a relative stability for one of those proteins, OmpLA. The consideration and ranking of different rotameric states for a mutated residue was found to be essential to achieve satisfactory agreement with the reference data. © 2015 Wiley Periodicals, Inc.

Intake of Meat Proteins Substantially Increased the Relative Abundance of Genus Lactobacillus in Rat Feces

PubMed Central

Zhu, Yingying; Lin, Xisha; Li, He; Li, Yingqiu; Shi, Xuebin; Zhao, Fan; Xu, Xinglian; Li, Chunbao; Zhou, Guanghong

2016-01-01

Diet has been shown to have a critical influence on gut bacteria and host health, and high levels of red meat in diet have been shown to increase colonic DNA damage and thus be harmful to gut health. However, previous studies focused more on the effects of meat than of meat proteins. In order to investigate whether intake of meat proteins affects the composition and metabolic activities of gut microbiota, feces were collected from growing rats that were fed with either meat proteins (from beef, pork or fish) or non-meat proteins (casein or soy) for 14 days. The resulting composition of gut microbiota was profiled by sequencing the V4-V5 region of the 16S ribosomal RNA genes and the short chain fatty acids (SCFAs) were analyzed using gas chromatography. The composition of gut microbiota and SCFA levels were significantly different between the five diet groups. At a recommended dose of 20% protein in the diet, meat protein-fed rats had a higher relative abundance of the beneficial genus Lactobacillus, but lower levels of SCFAs and SCFA-producing bacteria including Fusobacterium, Bacteroides and Prevotella, compared with the soy protein-fed group. Further work is needed on the regulatory pathways linking dietary protein intake to gut microbiota. PMID:27042829

Intake of Meat Proteins Substantially Increased the Relative Abundance of Genus Lactobacillus in Rat Feces.

PubMed

Zhu, Yingying; Lin, Xisha; Li, He; Li, Yingqiu; Shi, Xuebin; Zhao, Fan; Xu, Xinglian; Li, Chunbao; Zhou, Guanghong

2016-01-01

Diet has been shown to have a critical influence on gut bacteria and host health, and high levels of red meat in diet have been shown to increase colonic DNA damage and thus be harmful to gut health. However, previous studies focused more on the effects of meat than of meat proteins. In order to investigate whether intake of meat proteins affects the composition and metabolic activities of gut microbiota, feces were collected from growing rats that were fed with either meat proteins (from beef, pork or fish) or non-meat proteins (casein or soy) for 14 days. The resulting composition of gut microbiota was profiled by sequencing the V4-V5 region of the 16S ribosomal RNA genes and the short chain fatty acids (SCFAs) were analyzed using gas chromatography. The composition of gut microbiota and SCFA levels were significantly different between the five diet groups. At a recommended dose of 20% protein in the diet, meat protein-fed rats had a higher relative abundance of the beneficial genus Lactobacillus, but lower levels of SCFAs and SCFA-producing bacteria including Fusobacterium, Bacteroides and Prevotella, compared with the soy protein-fed group. Further work is needed on the regulatory pathways linking dietary protein intake to gut microbiota.

The Collagen Binding Proteins of Streptococcus mutans and Related Streptococci

PubMed Central

Avilés-Reyes, Alejandro; Miller, James H.; Lemos, José A.; Abranches, Jacqueline

2016-01-01

Summary The ability of Streptococcus mutans to interact with collagen through the expression of collagen-binding proteins (CBPs) bestows this oral pathogen with an alternative to the sucrose-dependent mechanism of colonization classically attributed to caries development. Based on the abundance and distribution of collagen throughout the human body, stringent adherence to this molecule grants S. mutans with the opportunity to establish infection at different host sites. Surface proteins, such as SpaP, WapA, Cnm and Cbm, have been shown to bind collagen in vitro, and it has been suggested that these molecules play a role in colonization of oral and extra-oral tissues. However, robust collagen binding is not achieved by all strains of S. mutans, particularly those that lack Cnm or Cbm. These observations merit careful dissection of the contribution from these different CBPs towards tissue colonization and virulence. In this review, we will discuss the current understanding of mechanisms utilized by S. mutans and related streptococci to colonize collagenous tissues, and the possible contribution of CBPs to infections in different sites of the host. PMID:26991416

Proteome-Level Analysis of Metabolism- and Stress-Related Proteins during Seed Dormancy and Germination in Gnetum parvifolium.

PubMed

Chang, Ermei; Deng, Nan; Zhang, Jin; Liu, Jianfeng; Chen, Lanzhen; Zhao, Xiulian; Abbas, M; Jiang, Zeping; Shi, Shengqing

2018-03-21

Gnetum parvifolium is a rich source of materials for traditional medicines, food, and oil, but little is known about the mechanism underlying its seed dormancy and germination. In this study, we analyzed the proteome-level changes in its seeds during germination using isobaric tags for relative and absolute quantitation. In total, 1,040 differentially expressed proteins were identified, and cluster analysis revealed the distinct time points during which signal transduction and oxidation-reduction activity changed. Gene Ontology analysis showed that "carbohydrate metabolic process" and "response to oxidative stress" were the main enriched terms. Proteins associated with starch degradation and antioxidant enzymes were important for dormancy-release, while proteins associated with energy metabolism and protein synthesis were up-regulated during germination. Moreover, protein-interaction networks were mainly associated with heat-shock proteins. Furthermore, in accord with changes in the energy metabolism- and antioxidant-related proteins, indole-3-acetic acid, Peroxidase, and soluble sugar content increased, and the starch content decreased in almost all six stages of dormancy and germination analyzed (S1-S6). The activity of superoxide dismutase, abscisic acid, and malondialdehyde content increased in the dormancy stages (S1-S3) and then decreased in the germination stages (S4-S6). Our results provide new insights into G. parvifolium seed dormancy and germination at the proteome and physiological levels, with implications for improving seed propagation.

Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses

DOE PAGES

Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; ...

2015-09-04

The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. Furthemore, these analyses, which were confirmed usingmore » bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. Our analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases.« less

Identification of the proteins related to SET-mediated hepatic cytotoxicity of trichloroethylene by proteomic analysis.

PubMed

Ren, Xiaohu; Yang, Xifei; Hong, Wen-Xu; Huang, Peiwu; Wang, Yong; Liu, Wei; Ye, Jinbo; Huang, Haiyan; Huang, Xinfeng; Shen, Liming; Yang, Linqing; Zhuang, Zhixiong; Liu, Jianjun

2014-05-16

Trichloroethylene (TCE) is an effective solvent for a variety of organic materials. Since the wide use of TCE as industrial degreasing of metals, adhesive paint and polyvinyl chloride production, TCE has turned into an environmental and occupational toxicant. Exposure to TCE could cause severe hepatotoxicity; however, the toxic mechanisms of TCE remain poorly understood. Recently, we reported that SET protein mediated TCE-induced cytotoxicity in L-02 cells. Here, we further identified the proteins related to SET-mediated hepatic cytotoxicity of TCE using the techniques of DIGE (differential gel electrophoresis) and MALDI-TOF-MS/MS. Among the 20 differential proteins identified, 8 were found to be modulated by SET in TCE-induced cytotoxicity and three of them (cofilin-1, peroxiredoxin-2 and S100-A11) were validated by Western-blot analysis. The functional analysis revealed that most of the identified SET-modulated proteins are apoptosis-associated proteins. These data indicated that these proteins may be involved in SET-mediated hepatic cytotoxicity of TCE in L-02 cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

A major stylar matrix polypeptide (sp41) is a member of the pathogenesis-related proteins superclass.

PubMed Central

Ori, N; Sessa, G; Lotan, T; Himmelhoch, S; Fluhr, R

1990-01-01

A novel stylar-specific glycosylated protein, sp41, was characterized. Sp41 constitutes greater than 12% of the transmitting tract tissue soluble proteins and is mainly localized in the extracellular matrix. Two cDNA clones corresponding to sp41 mRNA were isolated and sequenced. The decoded sequences are, respectively, 80% and 49% homologous to acidic and basic pathogen-induced (1-3)-beta-glucanases of the leaf. Thus a subfamily of (1-3)-beta-glucanase pathogenesis-related (PR) proteins constitutes one of the major stylar matrix proteins. The accumulation of sp41 transcripts in normally developing and elicitor-treated styles and leaves was followed using an RNase protection assay. During development sp41 transcript accumulation starts well after carpel differentiation. It is first detected in styles at 8 days before anthesis. The maximal level of accumulation is reached during anthesis. Elicitor-treated styles do not accumulate the leaf-type (1-3)-beta-glucanase transcript, although they retain the capacity to synthesize leaf-type pathogenesis-related proteins such as the pathogen-induced acidic chitinase. The developmental regulation of sp41 expression points to a role for them in the normal processes of flowering and reproductive physiology. Images Fig.1 Fig.2 Fig.5 Fig.6 Fig.7 Fig.8 Fig.9 PMID:2120041