DOCSLIB.ORG

Intrinsic Evolutionary Constraints on Protease Structure, Enzyme

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Intrinsic Evolutionary Constraints on Protease Structure, Enzyme Intrinsic evolutionary constraints on protease PNAS PLUS structure, enzyme acylation, and the identity of the catalytic triad Andrew R. Buller and Craig A. Townsend1 Departments of Biophysics and Chemistry, The Johns Hopkins University, Baltimore MD 21218 Edited by David Baker, University of Washington, Seattle, WA, and approved January 11, 2013 (received for review December 6, 2012) The study of proteolysis lies at the heart of our understanding of enzyme evolution remain unanswered. Because evolution oper- biocatalysis, enzyme evolution, and drug development. To un- ates through random forces, rationalizing why a particular out- derstand the degree of natural variation in protease active sites, come occurs is a difficult challenge. For example, the hydroxyl we systematically evaluated simple active site features from all nucleophile of a Ser protease was swapped for the thiol of Cys at serine, cysteine and threonine proteases of independent lineage. least twice in evolutionary history (9). However, there is not This convergent evolutionary analysis revealed several interre- a single example of Thr naturally substituting for Ser in the lated and previously unrecognized relationships. The reactive protease catalytic triad, despite its greater chemical similarity rotamer of the nucleophile determines which neighboring amide (9). Instead, the Thr proteases generate their N-terminal nu- can be used in the local oxyanion hole. Each rotamer–oxyanion cleophile through a posttranslational modification: cis-autopro- hole combination limits the location of the moiety facilitating pro- teolysis (10, 11). These facts constitute clear evidence that there ton transfer and, combined together, fixes the stereochemistry of is a strong selective pressure against Thr in the catalytic triad that catalysis. All proteases that use an acyl-enzyme mechanism natu- is somehow relieved by cis-autoproteolysis. Because the catalytic rally divide into two classes according to which face of the peptide triad is the principal system through which enzymatic rate ac- substrate is attacked during catalysis. We show that each class is celeration has been studied, the inability to rationalize the evo- subject to unique structural constraints that have governed the lutionary selection of the catalytic nucleophile is a fundamental BIOCHEMISTRY convergent evolution of enzyme structure. Using this framework, weakness in our understanding of biocatalysis. Motivated by this we show that the γ-methyl of Thr causes an intrinsic steric clash simple, yet unexplained structure–function relationship, we sought that precludes its use as the nucleophile in the traditional catalytic to answer the question: How do the chemical and structural con- triad. This constraint is released upon autoproteolysis and we pro- straints of proteolysis combine to shape the evolution of a protease pose a molecular basis for the increased enzymatic efficiency in- active site? troduced by the γ-methyl of Thr. Finally, we identify several Two strategies can be undertaken to probe the properties of classes of natural products whose mode of action is sensitive to diverse catalytic architectures. The first is the contemporary ex- the division according to the face of attack identified here. This perimental approach whereby all parameters of interest in an analysis of protease structure and function unifies 50 y of bioca- active site are systematically varied and analyzed in silico for talysis research, providing a framework for the continued study of catalytic viability. This technique was used by Smith et al. to show enzyme evolution and the development of inhibitors with in- that the catalytic triad of esterases adopts a consensus geometry creased selectivity. that minimizes reorganization during the multistep mechanism (12). Computational explorations are unsurpassed in their detail peptidase | antibiotic stereochemistry | N-terminal nucleophile and broad applicability, but hindered by their need to explicitly anticipate the structural and mechanistic diversity generated he acceleration of chemical reactions is essential to all bio- through natural selection. We elected to pursue an alternative Tchemistry. The generation of reactive species by enzymes is strategy to elucidate the constraints that guide active site evo- tightly controlled and limited by the chemical makeup of the 20 proteinogenic amino acids. In isolation, no protein residue har- Significance bors a strong nucleophile at physiological pH. Rather, enzymatic activity arises after protein folding introduces cooperative in- The structure–function relationship of proteases is central to teractions that selectively amplify the reactivity of their otherwise our understanding of biochemistry. Nature has evolved at least weakly active functional groups. The preeminent system through 23 independent solutions to this problem, using an acylation which this phenomenon has been studied is the Ser-His-Asp mechanism. We examined the structures of these proteases, catalytic triad of the Ser proteases (1). Several distinct molecular using a new framework to characterize the geometric rela- strategies have been identified that contribute to rate enhance- tionships within each active site. This analysis revealed the ment. Substrate binding increases the local concentration of orientation of the base determines the stereochemistry of ca- interacting species, general acids and bases facilitate proton talysis and elucidated why threonine does not substitute for transfer, and a high-energy step can be split into multiple lower- serine in the catalytic triad. These observations explain how energy steps (2). These distinct molecular strategies may be the absolute stereostructures of natural protease inhibitors unified through the formalism of transition-state theory (3) and prevent off-target inhibition and serve as boundary conditions the idea that the active site is electrostatically preorganized for to enzyme design. transition-state stabilization (4, 5). Although the biophysics of rate acceleration are intricate and sensitive to even minor struc- Author contributions: A.R.B. and C.A.T. designed research; A.R.B. performed research; tural perturbations, evolution has converged on a catalytic triad A.R.B. and C.A.T. analyzed data; and A.R.B. and C.A.T. wrote the paper. (or diad) with a reactive Ser, Cys, or Thr nucleophile more than The authors declare no conflict of interest. 25 separate times to facilitate central biochemical reactions such This article is a PNAS Direct Submission. as hydrolysis (6), transacylation (7), and phosphorylation (8). 1To whom correspondence should be addressed. E-mail: [email protected]. Our understanding of biochemistry has progressed into an This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. increasingly complex picture but many basic questions about 1073/pnas.1221050110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1221050110 PNAS | Published online February 4, 2013 | E653–E661 Downloaded by guest on September 27, 2021 lution that is not computational, but observational. Studying the the energetics of hydrolysis are different from enzyme acylation, large set of naturally occurring structures limits analyses to coarser they share many structural constraints. As is shown in this in- features, but greatly increases the universality of the results. This vestigation, it is sufficient to consider only the acylation step of the technique requires only a diverse set of enzyme structures and reaction, as displayed in Fig. 1, to rationalize much of the ob- a rudimentary understanding of organic and biochemistry. By servable convergent evolution of proteases. recognizing the constraints imposed by bond geometries and Evolution has selected for many different residues to activate steric clashes, it is easy to compare the potential catalytic archi- the nucleophile, but the cooperative interactions that engender tectures for a given reaction. reactivity are effectively “tuned” differently for each individual Results from the veritable explosion of gene sequencing and enzyme. This principle was first tested by the chemical swapping protein structure determination have identified at least 23 folds of the hydroxyl nucleophile of subtilisin for a thiol and observing related through convergent evolution that support proteolysis loss of proteolytic activity (16, 17). More exhaustive studies using through an acyl-enzyme intermediate (9). We cross-compared site-directed mutagenesis of both subtilisin and trypsin showed the active site architecture among these “analogous” enzymes that alteration of any component of the catalytic triad results in (13), each of which has been optimized for catalysis through a large decrease in enzyme efficiency (18, 19). Interestingly, the → natural selection, to interrogate the entire known structural di- Ser Thr variant of trypsin created a worse enzyme than simul- versity that supports efficient proteolysis. We first identified key taneously mutating all three residues of the catalytic triad. parameters that define active site architecture, such as the ori- Conversely, when the N-terminal Thr nucleophile of the pro- β entation of substrate binding, the φ, ψ angles of the nucleophile, teasome -subunit was mutated to Ser, there was a reduction in and the stereochemistry of catalysis. Analysis of all Ser, Cys, and its catalytic power (20). The juxtaposition of these results and the γ Thr proteases of independent lineage showed they were all inability to rationalize the opposite effects
Load more

Recommended publications
  • The Secretory Proprotein Convertase Neural Apoptosis-Regulated Convertase 1 (NARC-1): Liver Regeneration and Neuronal Differentiation
    The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): Liver regeneration and neuronal differentiation Nabil G. Seidah*†, Suzanne Benjannet*, Louise Wickham*, Jadwiga Marcinkiewicz*, Ste´phanie Be´langer Jasmin‡, Stefano Stifani‡, Ajoy Basak§, Annik Prat*, and Michel Chre´ tien§ *Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, QC, H2W 1R7 Canada; ‡Montreal Neurological Institute, McGill University, Montreal, QC, H3A 2B4 Canada; and §Regional Protein Chemistry Center and Diseases of Aging Unit, Ottawa Health Research Institute, Ottawa Hospital, Civic Campus, 725 Parkdale Avenue, Ottawa, ON, K1Y 4E9 Canada Edited by Donald F. Steiner, University of Chicago, Chicago, IL, and approved December 5, 2002 (received for review September 10, 2002) Seven secretory mammalian kexin-like subtilases have been iden- LP251 (Eli Lilly, patent no. WO 02͞14358 A2) recently cloned tified that cleave a variety of precursor proteins at monobasic and by two pharmaceutical companies. NARC-1 was identified via dibasic residues. The recently characterized pyrolysin-like subtilase the cloning of cDNAs up-regulated after apoptosis induced by SKI-1 cleaves proproteins at nonbasic residues. In this work we serum deprivation in primary cerebellar neurons, whereas LP251 describe the properties of a proteinase K-like subtilase, neural was discovered via global cloning of secretory proteins. Aside apoptosis-regulated convertase 1 (NARC-1), representing the ninth from the fact that NARC-1 mRNA is expressed in liver ϾϾ member of the secretory subtilase family. Biosynthetic and micro- testis Ͼ kidney and that the gene localizes to human chromo- sequencing analyses of WT and mutant enzyme revealed that some 1p33-p34.3, no information is available on NARC-1 ac- human and mouse pro-NARC-1 are autocatalytically and intramo- tivity, cleavage specificity, cellular and tissue expression, and lecularly processed into NARC-1 at the (Y,I)VV(V,L)(L,M)2 motif, a biological function.
    [Show full text]
  • The Global Architecture Shaping the Heterogeneity and Tissue-Dependency of the MHC Class I Immunopeptidome Is Evolutionarily Conserved
    bioRxiv preprint doi: https://doi.org/10.1101/2020.09.28.317750; this version posted September 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The Global Architecture Shaping the Heterogeneity and Tissue-Dependency of the MHC Class I Immunopeptidome is Evolutionarily Conserved Authors Peter Kubiniok†1, Ana Marcu†2,3, Leon Bichmann†2,4, Leon Kuchenbecker4, Heiko Schuster1,5, David Hamelin1, Jérome Despault1, Kevin Kovalchik1, Laura Wessling1, Oliver Kohlbacher4,7,8,9,10 Stefan Stevanovic2,3,6, Hans-Georg Rammensee2,3,6, Marian C. Neidert11, Isabelle Sirois1, Etienne Caron1,12* Affiliations *Corresponding and Leading author: Etienne Caron ([email protected]) †Equal contribution to this work 1CHU Sainte-Justine Research Center, Montreal, QC H3T 1C5, Canada 2Department of Immunology, Interfaculty Institute for Cell Biology, University of Tübingen, Tübingen, Baden-Württemberg, 72076, Germany. 3Cluster of Excellence iFIT (EXC 2180) "Image-Guided and Functionally Instructed Tumor Therapies", University of Tübingen, Tübingen, Baden-Württemberg, 72076, Germany. 4Applied Bioinformatics, Dept. of Computer Science, University of Tübingen, Tübingen, Baden- Württemberg, 72074, Germany. 5Immatics Biotechnologies GmbH, Tübingen, 72076, Baden-Württemberg, Germany. 6DKFZ Partner Site Tübingen, German Cancer Consortium (DKTK), Tübingen, Baden- Württemberg, 72076, Germany. 7Institute for Bioinformatics and Medical Informatics,
    [Show full text]
  • NIH Public Access Author Manuscript Biochemistry
    NIH Public Access Author Manuscript Biochemistry. Author manuscript; available in PMC 2010 June 23. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Biochemistry. 2009 June 23; 48(24): 5731±5737. doi:10.1021/bi9003099. Neisseria gonorrhoeae Penicillin-Binding Protein 3 Demonstrates a Pronounced Preference for Nε-Acylated Substrates† Sridhar Peddi‡,§, Robert A. Nicholas∥, and William G. Gutheil‡,* ‡Division of Pharmaceutical Sciences, University of Missouri-Kansas City, 5005 Rockhill Road, Kansas City, Missouri 64110. ∥Department of Pharmacology, CB#7365, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7365. Abstract Penicillin-binding proteins (PBPs) are bacterial enzymes involved in the final stages of cell wall biosynthesis, and are the lethal targets of β-lactam antibiotics. Despite their importance, their roles in cell wall biosynthesis remain enigmatic. A series of eight substrates, based on variation of the pentapeptide Boc-L-Ala-γ-D-Glu-L-Lys-D-Ala-D-Ala, were synthesized to test specificity for three features of PBP substrates: 1) the presence or absence of an Nε-acyl group, 2) the presence of D- IsoGln in place of γ-D-Glu, and 3) the presence or absence of the N-terminal L-Ala residue. The capacity of these peptides to serve as substrates for Neisseria gonorrhoeae (NG) PBP3 was assessed. NG PBP3 demonstrated good catalytic efficiency (2.5 × 105 M−1sec−1) with the best of these substrates, with a pronounced preference (50-fold) for Nε-acylated substrates over Nε-nonacylated substrates. This observation suggests that NG PBP3 is specific for the ∼D-Ala-D-Ala moiety of pentapeptides engaged in cross-links in the bacterial cell wall, such that NG PBP3 would act after transpeptidase-catalyzed reactions generate the acylated amino group required for its specificity.
    [Show full text]
  • Chymotrypsin-Like Peptidases in Insects
    Chymotrypsin-like peptidases in insects Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) Fachbereich Biologie/Chemie der Universität Osnabrück vorgelegt von Gunnar Bröhan Osnabrück, Mai 2010 TABLE OF CONTENTS I Table of contents 1. Introduction 1 1.1. Serine endopeptidases 1 1.2. The structure of S1A chymotrypsin-like peptidases 2 1.3. Catalytic mechanism of chymotrypsin-like peptidases 6 1.4. Insect chymotrypsin-like peptidases 9 1.4.1. Chymotrypsin-like peptidases in insect immunity 9 1.4.2. Role of chymotrypsin-like peptidases in digestion 14 1.4.3. Involvement of chymotrypsin-like peptidases in molt 16 1.5. Objective of the work 18 2. Material and Methods 20 2.1. Material 20 2.1.1. Culture Media 20 2.1.2. Insects 20 2.2. Molecular biological methods 20 2.2.1. Tissue preparations for total RNA isolation 20 2.2.2. Total RNA isolation 21 2.2.3. Reverse transcription 21 2.2.4. Quantification of nucleic acids 21 2.2.5. Chemical competent Escherichia coli 21 2.2.6. Ligation and transformation in E. coli 21 2.2.7. Preparation of plasmid DNA 22 2.2.8. Restriction enzyme digestion of DNA 22 2.2.9. DNA gel-electrophoresis and DNA isolation 22 2.2.10. Polymerase-chain-reaction based methods 23 2.2.10.1. RACE-PCR 23 2.2.10.2. Quantitative Realtime PCR 23 2.2.10.3. Megaprimer PCR 24 2.2.11. Cloning of insect CTLPs 25 2.2.12. Syntheses of Digoxigenin-labeled DNA and RNA probes 26 2.2.13.
    [Show full text]
  • Molecular Markers of Serine Protease Evolution
    The EMBO Journal Vol. 20 No. 12 pp. 3036±3045, 2001 Molecular markers of serine protease evolution Maxwell M.Krem and Enrico Di Cera1 ment and specialization of the catalytic architecture should correspond to signi®cant evolutionary transitions in the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St Louis, history of protease clans. Evolutionary markers encoun- MO 63110-1093, USA tered in the sequences contributing to the catalytic apparatus would thus give an account of the history of 1Corresponding author e-mail: [email protected] an enzyme family or clan and provide for comparative analysis with other families and clans. Therefore, the use The evolutionary history of serine proteases can be of sequence markers associated with active site structure accounted for by highly conserved amino acids that generates a model for protease evolution with broad form crucial structural and chemical elements of applicability and potential for extension to other classes of the catalytic apparatus. These residues display non- enzymes. random dichotomies in either amino acid choice or The ®rst report of a sequence marker associated with serine codon usage and serve as discrete markers for active site chemistry was the observation that both AGY tracking changes in the active site environment and and TCN codons were used to encode active site serines in supporting structures. These markers categorize a variety of enzyme families (Brenner, 1988). Since serine proteases of the chymotrypsin-like, subtilisin- AGY®TCN interconversion is an uncommon event, it like and a/b-hydrolase fold clans according to phylo- was reasoned that enzymes within the same family genetic lineages, and indicate the relative ages and utilizing different active site codons belonged to different order of appearance of those lineages.
    [Show full text]
  • Structural and Biochemical Characterizations of Three Potential Drug Targets from Pathogens
    Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 2020 Structural and Biochemical Characterizations of Three Potential Drug Targets from Pathogens LU LU ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-513-1148-7 UPPSALA urn:nbn:se:uu:diva-435815 2021 Dissertation presented at Uppsala University to be publicly examined in Room A1:111a, BMC, Husargatan 3, Uppsala, Friday, 16 April 2021 at 13:15 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Christian Cambillau. Abstract Lu, L. 2021. Structural and Biochemical Characterizations of Three Potential Drug Targets from Pathogens. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 2020. 91 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-1148-7. As antibiotic resistance of various pathogens emerged globally, the need for new effective drugs with novel modes of action became urgent. In this thesis, we focus on infectious diseases, e.g. tuberculosis, malaria, and nosocomial infections, and the corresponding causative pathogens, Mycobacterium tuberculosis, Plasmodium falciparum, and the Gram-negative ESKAPE pathogens that underlie so many healthcare-acquired diseases. Following the same- target-other-pathogen (STOP) strategy, we attempted to comprehensively explore the properties of three promising drug targets. Signal peptidase I (SPase I), existing both in Gram-negative and Gram-positive bacteria, as well as in parasites, is vital for cell viability, due to its critical role in signal peptide cleavage, thus, protein maturation, and secreted protein transport. Three factors, comprising essentiality, a unique mode of action, and easy accessibility, make it an attractive drug target.
    [Show full text]
  • Characterisation of Aspergillus Niger Prolyl Aminopeptidase
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Wageningen University & Research Publications Mol Gen Genomics (2005) 272: 673–679 DOI 10.1007/s00438-004-1094-5 ORIGINAL PAPER Danie¨lle E. J. W. Basten Æ Antoine P. H. A. Moers Albert J. J. van. Ooyen Æ Peter J. Schaap Characterisation of Aspergillus niger prolyl aminopeptidase Received: 29 April 2004 / Accepted: 16 November 2004 / Published online: 15 January 2005 Ó Springer-Verlag 2005 Abstract We have cloned a gene (papA) that encodes a ases and tripeptidases and finally by carboxypeptidases prolyl aminopeptidase from Aspergillus niger. Homolo- and aminopeptidases. The turnover of proteins by pro- gous genes are present in the genomes of the Eurotiales teases provides a ready pool of amino acids as precur- A. nidulans, A. fumigatus and Talaromyces emersonii, sors for the synthesis of new proteins (Bennet and Klich but the gene is not present in the genome of the yeast 1992). Saccharomyces cerevisiae. Cell extracts of strains over- Proteases normally do not hydrolyse bonds adjacent expressing the gene under the control of its own pro- to proline residues. Instead a specialised group of en- moter showed a fourfold to sixfold increase in prolyl zymes has evolved that hydrolyses these bonds. Their aminopeptidase activity, but no change in phenylalanine activity depends on both the isomeric state of the proline or leucine aminopeptidase activity. The overexpressed residue and its position in the peptide chain (Vanhoof enzyme was subsequently purified and characterised. et al. 1995; Cunningham and O’Connor 1997). Proline The enzyme specifically removes N-terminal proline and aminopeptidases (Pap, prolyl iminopeptidase, EC hydroxyproline residues from peptides.
    [Show full text]
  • Cellular and Molecular Signatures in the Disease Tissue of Early
    Cellular and Molecular Signatures in the Disease Tissue of Early Rheumatoid Arthritis Stratify Clinical Response to csDMARD-Therapy and Predict Radiographic Progression Frances Humby1,* Myles Lewis1,* Nandhini Ramamoorthi2, Jason Hackney3, Michael Barnes1, Michele Bombardieri1, Francesca Setiadi2, Stephen Kelly1, Fabiola Bene1, Maria di Cicco1, Sudeh Riahi1, Vidalba Rocher-Ros1, Nora Ng1, Ilias Lazorou1, Rebecca E. Hands1, Desiree van der Heijde4, Robert Landewé5, Annette van der Helm-van Mil4, Alberto Cauli6, Iain B. McInnes7, Christopher D. Buckley8, Ernest Choy9, Peter Taylor10, Michael J. Townsend2 & Costantino Pitzalis1 1Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK. Departments of 2Biomarker Discovery OMNI, 3Bioinformatics and Computational Biology, Genentech Research and Early Development, South San Francisco, California 94080 USA 4Department of Rheumatology, Leiden University Medical Center, The Netherlands 5Department of Clinical Immunology & Rheumatology, Amsterdam Rheumatology & Immunology Center, Amsterdam, The Netherlands 6Rheumatology Unit, Department of Medical Sciences, Policlinico of the University of Cagliari, Cagliari, Italy 7Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow G12 8TA, UK 8Rheumatology Research Group, Institute of Inflammation and Ageing (IIA), University of Birmingham, Birmingham B15 2WB, UK 9Institute of
    [Show full text]
  • Serine Proteases with Altered Sensitivity to Activity-Modulating
    (19) & (11) EP 2 045 321 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 08.04.2009 Bulletin 2009/15 C12N 9/00 (2006.01) C12N 15/00 (2006.01) C12Q 1/37 (2006.01) (21) Application number: 09150549.5 (22) Date of filing: 26.05.2006 (84) Designated Contracting States: • Haupts, Ulrich AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 51519 Odenthal (DE) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • Coco, Wayne SK TR 50737 Köln (DE) •Tebbe, Jan (30) Priority: 27.05.2005 EP 05104543 50733 Köln (DE) • Votsmeier, Christian (62) Document number(s) of the earlier application(s) in 50259 Pulheim (DE) accordance with Art. 76 EPC: • Scheidig, Andreas 06763303.2 / 1 883 696 50823 Köln (DE) (71) Applicant: Direvo Biotech AG (74) Representative: von Kreisler Selting Werner 50829 Köln (DE) Patentanwälte P.O. Box 10 22 41 (72) Inventors: 50462 Köln (DE) • Koltermann, André 82057 Icking (DE) Remarks: • Kettling, Ulrich This application was filed on 14-01-2009 as a 81477 München (DE) divisional application to the application mentioned under INID code 62. (54) Serine proteases with altered sensitivity to activity-modulating substances (57) The present invention provides variants of ser- screening of the library in the presence of one or several ine proteases of the S1 class with altered sensitivity to activity-modulating substances, selection of variants with one or more activity-modulating substances. A method altered sensitivity to one or several activity-modulating for the generation of such proteases is disclosed, com- substances and isolation of those polynucleotide se- prising the provision of a protease library encoding poly- quences that encode for the selected variants.
    [Show full text]
  • Supplemental Materials Supplemental Table 1
    Electronic Supplementary Material (ESI) for RSC Advances. This journal is © The Royal Society of Chemistry 2016 Supplemental Materials Supplemental Table 1. The differentially expressed proteins from rat pancreas identified by proteomics (SAP vs. SO) No. Protein name Gene name ratio P value 1 Metallothionein Mt1m 3.35 6.34E-07 2 Neutrophil antibiotic peptide NP-2 Defa 3.3 8.39E-07 3 Ilf2 protein Ilf2 3.18 1.75E-06 4 Numb isoform o/o rCG 3.12 2.73E-06 5 Lysozyme Lyz2 3.01 5.63E-06 6 Glucagon Gcg 2.89 1.17E-05 7 Serine protease HTRA1 Htra1 2.75 2.97E-05 8 Alpha 2 macroglobulin cardiac isoform (Fragment) 2.75 2.97E-05 9 Myosin IF (Predicted) Myo1f 2.65 5.53E-05 10 Neuroendocrine secretory protein 55 Gnas 2.61 7.60E-05 11 Matrix metallopeptidase 8 Mmp8 2.57 9.47E-05 12 Protein Tnks1bp1 Tnks1bp1 2.53 1.22E-04 13 Alpha-parvin Parva 2.47 1.78E-04 14 C4b-binding protein alpha chain C4bpa 2.42 2.53E-04 15 Protein KTI12 homolog Kti12 2.41 2.74E-04 16 Protein Rab11fip5 Rab11fip5 2.41 2.84E-04 17 Protein Mcpt1l3 Mcpt1l3 2.33 4.43E-04 18 Phospholipase B-like 1 Plbd1 2.33 4.76E-04 Aldehyde dehydrogenase (NAD), cytosolic 19 2.32 4.93E-04 (Fragments) 20 Protein Dpy19l2 Dpy19l2 2.3 5.68E-04 21 Regenerating islet-derived 3 alpha, isoform CRA_a Reg3a 2.27 6.74E-04 22 60S acidic ribosomal protein P1 Rplp1 2.26 7.22E-04 23 Serum albumin Alb 2.25 7.98E-04 24 Ribonuclease 4 Rnase4 2.24 8.25E-04 25 Cct-5 protein (Fragment) Cct5 2.24 8.52E-04 26 Protein S100-A9 S100a9 2.22 9.71E-04 27 Creatine kinase M-type Ckm 2.21 1.00E-03 28 Protein Larp4b Larp4b 2.18 1.25E-03
    [Show full text]
  • Leishmania (L.) Amazonensis Peptidase Activities Inside the Living Cells and in Their Lysates
    Molecular & Biochemical Parasitology 184 (2012) 82–89 Contents lists available at SciVerse ScienceDirect Molecular & Biochemical Parasitology Leishmania (L.) amazonensis peptidase activities inside the living cells and in their lysates a a b c a Elide E. Caroselli , Diego M. Assis , Clara L. Barbiéri , Wagner A.S. Júdice , Maria A. Juliano , d a,∗ Marcos L. Gazarini , Luiz Juliano a Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, SP, Brazil b Department of Microbiology, Immunology and Parasitology, Escola Paulista de Medicina, Universidade Federal de São Paulo, SP, Brazil c Centro Interdisciplinar de Investigac¸ ão Bioquímica, Universidade de Mogi das Cruzes, Av. Dr. Cândido Xavier de Almeida Souza 200, 08780-911 Mogi das Cruzes, Brazil d Department of Biosciences, Universidade Federal de São Paulo, Santos, Brazil a r t i c l e i n f o a b s t r a c t Article history: In this study we investigated the peptidase activity in Leishmania (L.) amazonensis live amastigote by con- Received 24 November 2011 focal microscopy using peptidyl-MCA as substrates, the hydrolysis of which releases the MCA fluorophore Received in revised form 13 March 2012 inside the cells. Cell pre-treatment with peptidase inhibitors indicated the presence of cysteine and ser- Accepted 27 April 2012 ine peptidases. It was noteworthy that Leishmania amastigotes incorporate only substrates (Z-FR-MCA, Available online 6 May 2012 Z-RR-MCA) or inhibitors (E64, TLCK) containing positively charged groups. The peptidase activities in the supernatants of amastigotes and promastigotes lysates were also evaluated with the same peptidyl-MCA Keywords: substrates and inhibitors in the pH range 4.5–9.0.
    [Show full text]
  • Supplemental Methods
    Supplemental Methods: Sample Collection Duplicate surface samples were collected from the Amazon River plume aboard the R/V Knorr in June 2010 (4 52.71’N, 51 21.59’W) during a period of high river discharge. The collection site (Station 10, 4° 52.71’N, 51° 21.59’W; S = 21.0; T = 29.6°C), located ~ 500 Km to the north of the Amazon River mouth, was characterized by the presence of coastal diatoms in the top 8 m of the water column. Sampling was conducted between 0700 and 0900 local time by gently impeller pumping (modified Rule 1800 submersible sump pump) surface water through 10 m of tygon tubing (3 cm) to the ship's deck where it then flowed through a 156 µm mesh into 20 L carboys. In the lab, cells were partitioned into two size fractions by sequential filtration (using a Masterflex peristaltic pump) of the pre-filtered seawater through a 2.0 µm pore-size, 142 mm diameter polycarbonate (PCTE) membrane filter (Sterlitech Corporation, Kent, CWA) and a 0.22 µm pore-size, 142 mm diameter Supor membrane filter (Pall, Port Washington, NY). Metagenomic and non-selective metatranscriptomic analyses were conducted on both pore-size filters; poly(A)-selected (eukaryote-dominated) metatranscriptomic analyses were conducted only on the larger pore-size filter (2.0 µm pore-size). All filters were immediately submerged in RNAlater (Applied Biosystems, Austin, TX) in sterile 50 mL conical tubes, incubated at room temperature overnight and then stored at -80oC until extraction. Filtration and stabilization of each sample was completed within 30 min of water collection.
    [Show full text]