1.
Template-Assisted and Self-Activating Clicked Peptide as a Synthetic Mimic of the SH2 Domain.
Abstract
A new synthetic strategy for obtaining artificial receptors that selectively regulate and/or control specific protein/protein interactions was developed based on the template-assisted and the self-activating click reaction applied to a combinatorial library. Synthetic mimics of the Grb2-SH2 domain, examined as a model case, selectively bound to a target signaling protein to induce cytotoxicity and inhibit tumor growth in vivo.
Study on CCR5 analogs and affinity peptides.
Source
Biopharmaceutical Centre, State Key Laboratory of Biocontrol, College of Life Sciences, Sun Yat-Sen University, Guangdong 510275, China.
Abstract
The G protein-coupled receptor of human chemokine receptor 5 (CCR5) is a key target in the human immunodeficiency virus (HIV) infection process due to its major involvement in binding to the HIV type 1 (HIV-1) envelope glycoprotein gp120 and facilitating virus entry into the cells. The identification of naturally occurring CCR5 mutations (especially CCR5 delta-32) has allowed us to address the CCR5 molecule as a promising target to prevent or resist HIV infection in vivo. To obtain high-affinity peptides that can be used to block CCR5, CCR5 analogs with high conformational similarity are required. In this study, two recombinant proteins named CCR5 N-Linker-E2 and CCR5 mN-E1-E2 containing the fragments of the CCR5 N-terminal, the first extracellular loop or the second extracellular loop are cloned from a full-length human CCR5 cDNA. The recombinant human CCR5 analogs with self-cleavage activity of the intein Mxe or Ssp in the vector pTwinI were then produced with a high-yield expression and purification system in Escherichia coli. Experiments of extracellular epitope-activity identification (such as immunoprecipitation and indirective/competitive enzyme-linked immunosorbent assay) confirmed the close similarity between the epitope activity of the CCR5 analogs and that of the natural CCR5, suggesting the applicability of the recombinant CCR5 analogs as antagonists of the chemokine ligands. Subsequent screening of high-affinity peptides from the phage random-peptides library acquired nine polypeptides, which could be used as CCR5 peptide antagonists. The CCR5 analogs and affinity peptides elucidated in this paper provide us with a basis for further study of the mechanism of inhibition of HIV-1 infection.
- PMID:
- 22238429
- [PubMed - as supplied by publisher]
Screening serum biomarker of knee osteoarthritis using a phage display technique.
Abstract
OBJECTIVES:
To screen serum biomarker of knee osteoarthritis (OA) using a phage random peptide library.
DESIGN AND METHODS:
A phage random peptide library of random peptide 12-mers was screened with purified immunoglobulin G (IgG) from sera of knee OA patients. Patients with knee rheumatoid arthritis (RA), hip OA, non-erosive hand OA or erosive hand OA, and healthy volunteers were used as controls.
RESULTS:
A phage clone with inserted peptide TGLESGHGPGDS (named KOA1) showed 90% positive reaction rate with the knee OA patients, significantly higher than that with the knee RA patients (27.8%), the non-erosive hand OA patients (34.3%), the erosive hand OA patients (31.3%) and the healthy controls (12.0%), but not the hip OA patients (82.5%).
CONCLUSIONS:
The novel knee OA mimic peptide KOA1 identified with a random phage display peptide library and sera from knee OA patients could be a potential serum biomarker for knee OA.
Copyright © 2011. Published by Elsevier Inc.
Effects of glucagon-like peptide-1 receptor agonists on weight loss: systematic review and meta-analyses of randomised controlled trials.
Source
Diabetes Research Division, Department of Internal Medicine F, Gentofte Hospital, University of Copenhagen, DK-2900 Hellerup, Denmark.
Abstract
OBJECTIVE:
To determine whether treatment with agonists of glucagon-like peptide-1 receptor (GLP-1R) result in weight loss in overweight or obese patients with or without type 2 diabetes mellitus.
DESIGN:
Systematic review with meta-analyses.
DATA SOURCES:
Electronic searches (Cochrane Library, Medline, Embase, and Web of Science) and manual searches (up to May 2011). Review methods Randomised controlled trials of adult participants with a body mass index of 25 or higher; with or without type 2 diabetes mellitus; and who received exenatide twice daily, exenatide once weekly, or liraglutide once daily at clinically relevant doses for at least 20 weeks. Control interventions assessed were placebo, oral antidiabetic drugs, or insulin.
DATA EXTRACTION:
Three authors independently extracted data. We used random effects models for the primary meta-analyses. We also did subgroup, sensitivity, regression, and sequential analyses to evaluate sources of intertrial heterogeneity, bias, and the robustness of results after adjusting for multiple testing and random errors.
RESULTS:
25 trials were included in the analysis. GLP-1R agonist groups achieved a greater weight loss than control groups (weighted mean difference -2.9 kg, 95% confidence interval -3.6 to -2.2; 21 trials, 6411 participants). We found evidence of intertrial heterogeneity, but no evidence of bias or small study effects in regression analyses. The results were confirmed in sequential analyses. We recorded weight loss in the GLP-1R agonist groups for patients without diabetes (-3.2 kg, -4.3 to -2.1; three trials) as well as patients with diabetes (-2.8 kg, -3.4 to -2.3; 18 trials). In the overall analysis, GLP-1R agonists had beneficial effects on systolic and diastolic blood pressure, plasma concentrations of cholesterol, and glycaemic control, but did not have a significant effect on plasma concentrations of liver enzymes. GLP-1R agonists were associated with nausea, diarrhoea, and vomiting, but not with hypoglycaemia.
CONCLUSIONS:
The present review provides evidence that treatment with GLP-1R agonists leads to weight loss in overweight or obese patients with or without type 2 diabetes mellitus.
Development of molecular techniques for imaging and treatment of tumors.
Source
Department of Nuclear Medicine. University Hospital of Heidelberg, Heidelberg, Germany: 2 Clinical Cooperation Unit Nuclear Medicine, DKFZ and University of Heidelberg, Heidelberg, Germany: 3 Department of Radiopharmaceutical Chemistry, DKFZ Heidelberg, Heidelberg, Germany - uwe.haberkorn@med.uni-heidelberg.de.
Abstract
With the advances in molecular biology and biochemistry new imaging and treatment modalities based on the biological properties of tissues have been developed. In oncology, the major progress has been achieved using peptide and antibody targeting vectors. Besides the identification of new target structures, progress in molecular biology also made new techniques for the development of new biomolecules. This relies on the identification of lead compounds and on the screening of various derivatives of these compounds one at a time. The principle of high-troughput methods for the identification of novel high affinity binders is to generate a vast library of possible variants of the molecule of interest and screen the population for the few variants that show the property of interest. The attracting feature of the concept arises from the huge number of candidate molecules that can be used for further evaluation. After the characterization of the structure-function relationships for the lead compounds found in this process further improvement by rational design of analogs can be performed.
Identification and molecular characterization of three new K(+)-channel specific toxins from the Chinese scorpion Mesobuthus martensii Karsch revealing intronic number polymorphism and alternative splicing in duplicated genes.
Source
Department of Biological Science and Technology, School of Environmental Studies & State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences (Wuhan), Wuhan 430074, China.
Abstract
K(+)-channel specific toxins from scorpions are powerful probes used in the structural and functional characterization of different subfamilies of K(+)-channels which are thought to be the most diverse ion channels. However, only a limited number of K(+)-channels toxins have been identified from scorpions so far; moreover, little is known about the mechanisms for the generation of a combinatorial peptide library in a venom gland of a scorpion. Here, we identified and characterized three new K(+)-channels toxin-like peptides from the scorpion Mesobuthus martensii Karsch, which were referred to as BmKcug1, BmKcug2 and BmKcugx, respectively. BmKcug1 and BmKcug2 are two new members of α-KTx1 subfamily, and have been classified as α-KTx1.14 and α-KTx1.15, respectively. BmKcugx represents a new subfamily of K(+)-channel specific toxins which was classified into α-KTx22. BmKcugx was thus classified as α-KTx22.1. Genomic analysis demonstrated that BmKcugx gene has two exons interrupted by an intron inserted in the signal peptide encoding region, whereas BmKcug1a (a close homologue of BmKcug1)/BmKcug2 gene was interrupted by two introns, located within the 5'UTR of the gene and in the signal peptide encoding region, respectively. Transcriptomic analysis for the venom glands of M. martensii Karsch indicated that the abundances of the transcripts of BmKcug1a and BmKcug2 are much higher than that of BmKcugx; it suggests that the intron in 5'UTR could markedly increase the expression level of the K(+)-channel toxins. Alignment of the genomic sequences of BmKcug1a and BmKcug2 revealed that an alternative splicing event occurred at the intron 1-exon 2 junction in the 5'UTR of BmKcug2 transcript.
Copyright © 2012. Published by Elsevier Inc.
A novel mouse CD133 binding-peptide screened by phage display inhibits cancer cell motility in vitro.
Source
Department of Pathology and Key Laboratory of Molecule Tumor Pathology of Guangdong Province, Southern Medical University, Guangzhou, 510515, Guangdong, China, Sunjinmin198206@163.com.
Abstract
Increased expression of CD133 (Prominin-1), an important cancer stem cell-associated marker, has been observed in the cancer stem cells of a variety of human and mouse cancers. However, no natural ligand of CD133 has yet been identified and little is known about its function. In the present study, LS-7 (amino acid sequence: LQNAPRS), a specific binding peptide targeting mouse CD133, was screened and identified for the first time by phage-displayed peptide librarytechnology. The in vitro and in vivo affinity and specificity of LS-7 were determined, and MTT, adhesion, and migration assays were performed to evaluate the effects of LS-7 on the biological behaviors of cancer cells. To determine which signaling pathways are affected by LS-7, HMGB1, S-100A4, CXCR7, uPAR, AMFR, STAT3, and c-Met gene and protein expression were evaluated by RT-PCR and Western blot. Flow cytometry and immunofluorescence assays showed specific, high-affinity binding of the peptide to mCD133 in vitro. Confocal microscopy confirmed the co-localization of LS-7 positive cells and CD133-positive cells. Migration and wound-healing assays showed that LS-7 significantly inhibited the migration of colon and breast cancer cells in a concentration-dependent manner. In vivo experiments also confirmed the high specificity and affinity of LS-7 to mCD133. RT-PCR and Western blot showed that the expressions of only c-Met and STAT3 decreased obviously in colon and breast cancer cells exposed to LS-7. These findings may provide a novel tool for anti-motility and anti-metastasis strategies in cancer research and cancer stem cell therapy.
Characterization of potential elastase inhibitor-peptides regulated by a molecular switch for wound dressings applications.
Source
Center of Chemistry, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
Abstract
Elastase plays an important role in wound healing process, degrading damaged tissue and allowing complete tissue recovery. The levels of human neutrophil elastase (HNE) are usually controlled by endogenous inhibitors. However, in the presence of high levels of elastase, like the ones present in chronic wounds, the inhibitors cannot overcome this overproduction and the enzyme starts to degrade the surrounding healthy tissue. In this work we report the development of a molecular switch to control the elastase activity in the exudate of non-healing chronic wounds. A peptide library was generated and screened in a microarray format for protein kinase-mediated phosphorylation. Two peptides were identified as casein kinase Iδ (CKI) substrates: KRCCPDTCGIKCL and its analogous peptide KRMMPDTMGIKML, with cysteine residues replaced by methionine residues. These peptides were studied in solution, both in the phosphorylated and non-phosphorylated forms as potential inhibitors for elastase. The obtained results show that the reversible process of phosphorylation/dephosphorylation results in differential inhibitory activity of the peptides. Thus the reversible process of phosphorylation/dephosphorylation can be used as a kind of molecular switch to control elastase activity. Degradation studies reveal that both the inhibitor-peptides and CKI are degraded by elastase. These results envisage the safe utilisation of these inhibitor-peptides together with CKI in the formulation of wound dressings.
Copyright © 2011 Elsevier Inc. All rights reserved.
A comprehensive library of blocked dipeptides reveals intrinsic backbone conformational propensities of unfolded proteins.
Source
Department of Chemistry, Korea University, Seoul 136-701, Korea.
Abstract
Despite prolonged scientific efforts to elucidate the intrinsic peptide backbone preferences of amino-acids based on understanding of intermolecular forces, many open questions remain, particularly concerning neighboring peptideinteraction effects on the backbone conformational distribution of short peptides and unfolded proteins. Here, we show that spectroscopic studies of a complete library of 400 dipeptides reveal that, irrespective of side-chain properties, the backbone conformation distribution is narrow and they adopt polyproline II and β-strand, indicating the importance of backbone peptide solvation and electronic effects. By directly comparing the dipeptide circular dichroism and NMR results with those of unfolded proteins, the comprehensive dipeptides form a complete set of structural motifs of unfolded proteins. We thus anticipate that the present dipeptide library with spectroscopic data can serve as a useful database for understanding the nature of unfolded protein structures and for further refinements of molecular mechanical parameters. Proteins 2011; © 2011 Wiley Periodicals, Inc.
Copyright © 2011 Wiley Periodicals, Inc.
Current advances in peptide and small molecule microarray technologies.
Source
Department of Biological Science, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore.
Abstract
Microarrays offer a compact solution for massively parallel screening. In recent years, microarrays have branched away from the exclusive pursuit of small molecule 'hits' in target centric screens, towards the sophisticated dissection of disease biology and comparative profiling of cellular states. This has led to innovative and instructive ways in which the platform may be deployed, providing new-found methods with which to harness the throughput achievable. Library design and diversity continues to drive success with peptide and small molecule microarrays. Newer synthesis and immobilization strategies extend the already wide repertoire of fabrication methods available. Herein we describe the latest advances in the small molecule and peptide microarray arena, which herald even more exciting breakthroughs in the coming decade.
Copyright © 2011 Elsevier Ltd. All rights reserved.
Radionuclide Therapy in Neuroendocrine Tumours: A Systematic Review.
Source
Department of Nuclear Medicine, Hamilton Health Sciences & St. Joseph's Healthcare Hamilton, Hamilton, Ontario, Canada.
Abstract
The purpose of this systematic review was to investigate the effects of therapeutic radiopharmaceuticals in patients with different types of advanced neuroendocrine tumour (NETs). A literature search was carried out in MEDLINE and EMBASE from January 1998 to November 2010. The Cochrane Library (to Issue 10, 2010) and the Standards and Guidelines Evidence Inventory of Cancer Guidelines, including over 1100 English-language cancer guidelines from January 2003 to June 2010, were also checked. No existing systematic reviews or clinical practice guidelines based on a systematic review or randomised controlled trials focusing on this topic were found. Twenty-four fully published articles were abstracted and summarised: 16 articles focused on five peptide receptor radionuclide therapy ((111)In-DTPAOC, (90)Y-DOTALAN, (90)Y-DOTATOC, (90)Y-DOTATATE, and (177)Lu-DOTATATE) and eight focused on (131)I-MIBG treatment. Limited evidence from a historical comparison of studies in one centre supported that (177)Lu-DOTATATE might be associated with greater clinical outcomes compared with (90)Y-DOTATOC or (111)In-DTPAOC. The severe toxicities for (177)Lu-DOTATATE included hepatic insufficiency in 0.6%, myelodysplastic syndrome in 0.8% and renal insufficiency in 0.4% of patients in this study. Insufficient evidence suggested efficacy of (131)I-MIBG in adult NET patients, but the overall tumour response rate from (131)I-MIBG was 27-75% for malignant neuroblastoma, paraganglioma or pheochromocytoma. Haematological toxicities were the main severe side-effects after (131)I-MIBG and 4% of patients developed secondary malignancies in one study. To date, peptide receptor radionuclide therapy seems to be an acceptable option and is relatively safe in adult advanced NET patients with receptor uptake positive on scintigraphy, but patients' renal function must be monitored. (131)I-MIBG may be effective for malignant neuroblastoma, paraganglioma or pheochromocytoma, but its side-effects need to be considered. No strong evidence exists to support that one therapeutic radiopharmaceutical is more effective than others. Well-designed and good-quality randomised controlled trials are required on this research topic.
Copyright © 2011 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.
Pepitome: evaluating improved spectral library search for identification complementarity and quality assessment.
Abstract
Spectral libraries have emerged as a viable alternative to protein sequence databases for peptide identification. These libraries contain previously detected peptide sequences and their corresponding tandem mass spectra (MS/MS). Search engines can then identify peptides by comparing experimental MS/MS scans to those in the library. Many of these algorithms employ the dot product score for measuring the quality of a spectrum-spectrum match (SSM). This scoring system does not offer a clear statistical interpretation and ignores fragment ion m/z discrepancies in the scoring. We developed a new spectral library search engine, Pepitome, which employs statistical systems for scoring SSMs. Pepitome outperformed the leading library search tool, SpectraST, when analyzing data sets acquired on three different mass spectrometry platforms. We characterized the reliability of spectral library searches by confirming shotgun proteomics identifications through RNA-Seq data. Applying spectral library and database searches on the same sample revealed their complementary nature. Pepitome identifications enabled the automation of quality analysis and quality control (QA/QC) for shotgun proteomics data acquisition pipelines.
Characterization of Xyn10J, a Novel Family 10 Xylanase from a Compost Metagenomic Library.
Source
Department of Agricultural Chemistry, Sunchon National University, Suncheon, 540-742, Republic of Korea.
Abstract
A gene encoding an extracellular xylanase was cloned from a compost metagenomic library. The xylanase gene, xyn10J, was 1,137 bp in length and was predicted to encode a protein of 378 amino acid residues with a putative signalpeptide of 27 amino acid residues. The molecular mass of the mature Xyn10J was calculated to be 39,882 Da with a pI of 6.09. Xyn10J had a motif GVKVHFTEMDI characteristic of most members of glycosyl hydrolase family 10. The amino acid sequence of Xyn10J showed 60.0% identity to that of XynH, a xylanase from an uncultured soil bacterium and 55% identity to XylC of Cellvibrio mixtus. Site-directed mutagenesis of the expected active site based on the sequence analysis indicated that an aspartic acid residue (Asp207), in addition to the identified catalytic residues Glu165 and Glu270, plays a crucial role for the catalytic activity. The purified Xyn10J had a mass of about 40 kDa and was optimally active at pH 7.0 and 40 °C. Xyn10J hydrolyzed beechwood xylan > birchwood xylan > oat spelt xylan > arabinoxylan. Xyn10J hydrolyzed xylotetraose and xylohexaose exclusively to xylobiose, xylopentaose, and xylotriose mainly to xylobiose with transglycosylation activity. The saccharification of reed (Phragmites communis) powder by commercial enzymes was significantly increased by the addition of a small amount of Xyn10J to the commercial preparation. Xyn10J is the first xylanase screened directly from a compost metagenomic library, and the enzyme has the potential to be used in the conversion of biomass to fermentable sugars for biofuel production.
GuiTope: An Application for Mapping Random-Sequence Peptides to Protein Sequences.
Abstract
ABSTRACT:
BACKGROUND:
Random-sequence peptide libraries are a commonly used tool to identify novel ligands for binding antibodies, other proteins, and small molecules. It is often of interest to compare the selected peptide sequences to the natural protein binding partners to infer the exact binding site or the importance of particular residues. The ability to search a set of sequences for similarity to a set of peptides may sometimes enable the prediction of an antibody epitope or a novel binding partner. We have developed a software application designed specifically for this task.
RESULTS:
GuiTope provides a graphical user interface for aligning peptide sequences to protein sequences. All alignment parameters are accessible to the user including the ability to specify the underlying amino acid frequency in the peptide library, which often differs significantly from the frequencies assumed by popular alignment programs. It also includes a novel feature to align di-peptide inversions, which we have found improves the accuracy of antibody epitope prediction from peptide microarray data and shows utility in analyzing phage display datasets. Finally, GuiTope can randomly select peptides from a given library to estimate a null distribution of scores to calculate statistical significance.
CONCLUSIONS:
GuiTope provides a convenient method for comparing selected peptide sequences to protein sequences, including flexible alignment parameters, novel alignment features, ability to search a database, and statistical significance of results. The latest version of the software available as an executable (for PC) at www.immunosignature.com/software and ongoing updates and source code will be available at sourceforge.net.
Characterization of the grain-specific lipid transfer protein TdPR61 from wheat, and its promoter activity in wheat, barley, and rice.
Source
Australian Centre for Plant Functional Genomics, University of Adelaide, Hartley Grove, Urrbrae, South Australia 5064, Australia.
Abstract
The TaPR61 gene from bread wheat encodes a lipid transfer protein (LTP) with a hydrophobic signal peptide, predicted to direct the TaPR61 protein to the apoplast. Modelling of TaPR61 revealed the presence of an internal cavity which can accommodate at least two lipid molecules. The full-length gene, including the promoter sequence of a TaPR61 orthologue, was cloned from a BAC library of Triticum durum. Quantitative RT-PCR analysis revealed the presence of TaPR61 and TdPR61 mainly in grain. A transcriptional TdPR61 promoter-GUS fusion was stably transformed into wheat, barley, and rice. The strongest GUS expression in all three plants was found in the endosperm transfer cells, the embryo surrounding region (ESR), and in the embryo. The promoter is strong and has similar but not identical spatial patterns of activity in wheat, barley, and rice. These results suggest that the TdPR61 promoter will be a useful tool for improving grain quality by manipulating the quality and quantity of nutrient/lipid uptake to the endosperm and embryo. Mapping of regions important for the promoter function using transient expression assays in developing embryos resulted in the identification of two segments important for promoter activation in embryos. The putative cis-elements from the distal segment were used as bait in a yeast 1-hybrid (Y1H) screen of a cDNA library prepared from the liquid part of the wheat multinucleate syncytium. A transcription factor isolated in the screen is similar to BES1/BLZ1 from Arabidopsis, which is known to be a key transcriptional regulator of the brassinosteroid signalling pathway.
Molecular characterization and tissue expression profile of three novel ovine genes: ATP5O, NDUFA12 and UQCRH from muscle full-length cDNA library of black-boned sheep.
Source
Yunnan Key Laboratory of Animal Nutrition and Feed Science, Yunnan Agricultural University, Kunming, 650201, China.
Abstract
Three novel ovine genes were obtained from muscle full-length cDNA library of black-boned sheep. Sequence analysis revealed that nucleotide sequences of these genes were not homologous to any of the known sheep or goat genes, but these genes have high similarity to ATP synthase subunit O (ATP5O), NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 12 (NDUFA12) and ubiquinol-cytochrome c reductase hinge protein (UQCRH) genes of other mammal animals (accession number: FJ546085, FJ546078 and FJ546083). The alignment analysis showed that the ovine ATP5O, NDUFA12 and UQCRH genes and proteins have closer genetic relationships with the ATP5O, NDUFA12 and UQCRH genes and proteins from cattle. Conserved domain prediction showed that these three genes included OSCP, NDUFA12 superfamily and UCR-hinge superfamily domains respectively. The deduced sequence of ATP5O, NDUFA12 and UQCRH protein had 213, 145 and 91 amino acid residues, with a molecular weight of approximately 23419.66, 17089.50 and 10657.75 Da and a theoretical isoelectric point of 9.90, 9.68 and 4.45. The secondary structure prediction revealed that 60% helix structure in ATP5O, 60% coils in NDUFA12 and no strand in UQCRH. One potential signalpeptide structure in ATP5O protein were found. NDUFA12 and UQCRH have the extremely low possibility of signal peptides. Meanwhile, RasMol was used for visualizing the PDB files generated by Swiss-Model in cartoon or three-dimensional format. ATP5O and UQCRH protein were modeled by Swiss-Model. Tissue expression profile indicated that the ovine ATP5O, NDUFA12 and UQCRH genes could be expressed in all detected tissues including muscles, heart, liver, spleen, lung, kidney and adipose tissues, but the expression abundance of these genes were various in the different tissues. Our experiment supplied the primary foundation for further researches on these three ovine genes.
Identification of putative cathepsin S in mangrove red snapper Lutjanus argentimaculatus and its role in antigen presentation.
Source
The Division of Ocean Science and Technology, Graduate School at Shenzhen, Tsinghua University, Shenzhen, PR China.
Abstract
Cathepsin S (CTSS) is a key enzyme employed in the histocompatibility complex (MHC) class II-restricted antigens, which are presented by processing class II-associated invariant chains and loaded antigen peptides into class II molecules. To date, little is known about the character and function of CTSS in fish. In the present study, we screened and identified a CTSS cDNA sequence from the mangrove red snapper head kidney cDNA library. The full-length CTSS cDNA contained 1339-bp nucleotide acids encoding 337 amino acids. The sequence shared high identity and similarity with other known cathepsins, especially CTSS (about 56-78% and 79-89%, respectively). Like other cathepsins, the deduced peptide consisted of regions with N-terminal signal peptides, propeptides, and mature peptides. A typical ERWNIN motif in L-like cathepsins and three conservative catalytic activity sites forming a catalytic triad active center were respectively identified in the pro-peptide and mature peptide regions of CTSS. Phylogenetic analysis revealed that mangrove red snapper CTSS was located in the CTSS clade belonging to the L-like cathepsin group, and evolved from the same ancestry. To further characterize the biological activity of the putative CTSS of mangrove snapper, CTSS was expressed in Escherichia coli M15 strains. Like other mammalian CTSS, the recombinant CTSS (rCTSS) had autocatalytic activation properties, can remove pro-peptides, and can release active mature peptides. Active CTSS had the ability to catalyze Z-Phe-Arg-AMC substrates in acidic conditions (pH 5.0) and weak alkaline environments (pH 7.5); this activity could be blocked by the cysteine protease inhibitor E-64. Active CTSS can process recombinant Ii chains (invariant chains) in a stepwise manner in vitro. The results indicate that mangrove red snapper CTSS is a lysosomal cysteine protease family member with a key role in antigen processing in fish.
Copyright © 2011. Published by Elsevier Ltd.
Asymmetric proteome equalization of the skeletal muscle proteome using a combinatorial hexapeptide library.
Source
Protein Function Group, Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.
Abstract
Immobilized combinatorial peptide libraries have been advocated as a strategy for equalization of the dynamic range of a typical proteome. The technology has been applied predominantly to blood plasma and other biological fluids such as urine, but has not been used extensively to address the issue of dynamic range in tissue samples. Here, we have applied the combinatorial library approach to the equalization of a tissue where there is also a dramatic asymmetry in the range of abundances of proteins; namely, the soluble fraction of skeletal muscle. We have applied QconCAT and label-free methodology to the quantification of the proteins that bind to the beads as the loading is progressively increased. Although some equalization is achieved, and the most abundant proteins no longer dominate the proteome analysis, at high protein loadings a new asymmetry of protein expression is reached, consistent with the formation of complex assembles of heat shock proteins, cytoskeletal elements and other proteins on the beads. Loading at different ionic strength values leads to capture of different subpopulations of proteins, but does not completely eliminate the bias in protein accumulation. These assemblies may impair the broader utility of combinatorial library approaches to the equalization of tissue proteomes. However, the asymmetry in equalization is manifest at either low and high ionic strength values but manipulation of the solvent conditions may extend the capacity of the method.
Nonplanar peptide bonds in proteins are common and conserved but not biased toward active sites.
Source
Department of Biochemistry and Biophysics, Oregon State University, 2011 Agriculture and Life Sciences Building, Corvallis, OR 97331.
Abstract
The planarity of peptide bonds is an assumption that underlies decades of theoretical modeling of proteins. Peptidebonds strongly deviating from planarity are considered very rare features of protein structure that occur for functional reasons. Here, empirical analyses of atomic-resolution protein structures reveal that trans peptide groups can vary by more than 25° from planarity and that the true extent of nonplanarity is underestimated even in 1.2 Å resolution structures. Analyses as a function of the ϕ,ψ-backbone dihedral angles show that the expected value deviates by ± 8° from planar as a systematic function of conformation, but that the large majority of variation in planarity depends on tertiary effects. Furthermore, we show that those peptide bonds in proteins that are most nonplanar, deviating by over 20° from planarity, are not strongly associated with active sites. Instead, highly nonplanar peptides are simply integral components of protein structure related to local and tertiary structural features that tend to be conserved among homologs. To account for the systematic ϕ,ψ-dependent component of nonplanarity, we present a conformation-dependent library that can be used in crystallographic refinement and predictive protein modeling.
Engineering Antibody Fitness and Function Using Membrane-Anchored Display of Correctly Folded Proteins.
Source
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.
Abstract
A hallmark of the bacterial twin-arginine translocation (Tat) pathway is its ability to export folded proteins. Here, we discovered that overexpressed Tat substrate proteins form two distinct, long-lived translocation intermediates that are readily detected by immunolabeling methods. Formation of the early translocation intermediate Ti-1, which exposes the N- and C-termini to the cytoplasm, did not require an intact Tat translocase, a functional Tat signal peptide, or a correctly folded substrate. In contrast, formation of the later translocation intermediate, Ti-2, which exhibits a bitopic topology with the N-terminus in the cytoplasm and C-terminus in the periplasm, was much more particular, requiring an intact translocase, a functional signal peptide, and a correctly folded substrate protein. The ability to directly detect Ti-2 intermediates was subsequently exploited for a new protein engineering technology called MAD-TRAP (membrane-anchored display for Tat-based recognition of associating proteins). Through the use of just two rounds of mutagenesis and screening with MAD-TRAP, the intracellular folding and antigen-binding activity of a human single-chain antibody fragment were simultaneously improved. This approach has several advantages for library screening, including the unique involvement of the Tat folding quality control mechanism that ensures only native-like proteins are displayed, thus eliminating poorly folded sequences from the screening process.
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