A theoretical approach towards the identification of cleavage-determining amino acid motifs of the 20s proteasome
Identifieur interne : 002559 ( Istex/Corpus ); précédent : 002558; suivant : 002560A theoretical approach towards the identification of cleavage-determining amino acid motifs of the 20s proteasome
Auteurs : Hermann-Georg Holzhütter ; Cornelius Frömmel ; Peter-Michael KloetzelSource :
- Journal of Molecular Biology [ 0022-2836 ] ; 1999.
English descriptors
- KwdEn :
- Teeft :
- Academic press, Acid motifs, Acid residues, Active site, Active sites, Amino, Amino acid, Amino acid motifs, Amino acid residues, Amino acids, Anchor locations, Anchor position, Anchor positions, Anchor residues, Anking, Anking region, Anking regions, Antigen presentation, Antigenic peptides, Appropriate binding conformations, Binding epitope, Bold bars, Bold frames, Carboxyl, Carboxyl site, Cardinal properties, Certain length, Classical protease, Cleavage, Cleavage frequencies, Cleavage frequency, Cleavage mechanism, Cleavage preference, Cleavage preferences, Cleavage probability, Cleavage products, Cleavage rate, Cleavage rates, Cleavage site, Cleavage sites, Conformation, Continuous line, Corresponding property term, Degradation, Degradation pattern, Degradation products, Effective substrate binding, Epitope, Eukaryotic proteasomes, False positives, Febs letters, Fragment, Globular proteins, Grey cells, Hydrophobic residue, Hydrophobic residues, Independent observations, Larger sequence region, Larger weight, Lmp2, Lmp2 subunits, Lmp7 subunits, Major histocompatibility, Molecular ruler, Niedermann, Normalised, Optimal properties, Other cleavage sites, Other hand, Overall cleavage probability, Pattern recognition, Peptide, Peptide bond, Peptide bond cleavage, Peptide bonds, Peptide fragments, Peptide substrate, Peptide substrates, Pituitary multicatalytic proteinase, Possible epitopes, Possible shapes, Potential cleavage site, Predictive capacity, Processive degradation, Properties volume, Property term, Property terms, Property volume, Protease, Proteasome, Proteasome figure, Proteasome table, Proteasomes, Protein substrate, Protein substrates, Proteolytic, Proteolytic fragments, Relative frequencies, Relative orientation, Residual squares, Residue, Residue types, Same residue, Scatter plot, Scissile, Scissile bond, Scissile peptide bond, Selective cleavage, Sequence length, Sequence positions, Sequence window, Short peptides, Shorter fragments, Size distribution, Small number, Subunit, Subunits lmp2, Theoretical approach, Total number, Transfer energy, Tting, Tting round, Tting strategy, Uorgenic peptides, Vertebrate proteasomes, Window method, Yeast proteasome subunit.
Abstract
Abstract: Hitherto the mechanisms controlling the selective cleavage of peptide bonds by the 20 S proteasome have been poorly understood. The observation that peptide bond cleavage may eventually occur at the carboxyl site of either amino acid residue rules out a simple control of cleavage preferences by the P1 residue alone. Here, we follow the rationale that the presence of specific cleavage-determining amino acids motifs (CDAAMs) around the scissile peptide bond are required for the attainment of substrate conformations susceptible to cleavage. We present an exploratory search for these putative motifs based on empirical regression functions relating the cleavage probability for a given peptide bond to some selected side-chain properties of the flanking amino acid residues. Identification of the sequence locations of cleavage-determining residues relative to the scissile bond and of their optimal side-chain properties was carried out by fitting the cleavage probability to (binary) experimental observations on peptide bond cleavage gathered among a set of seven different peptide substrates with known patterns of proteolytic degradation products. In this analysis, all peptide bonds containing the same residue in the P1 position were assumed to be cleaved by the same active sites of the proteasome, and thus to be under control of the same CDAAMs. We arrived at a final set of ten different CDAAMs, accounting for the cleavage of one to five different groups of peptide bonds with an overall predictive correctness of 93%. The CDAAM is composed of two to four “anchor” positions preferentially located between P5 and P5′ around the scissile bond. This implies a length constraint for the usage of cleavage sites, which could considerably suppress the excision of shorter fragments and thus partially explain for the observed preponderance of medium-size cleavage products.
Url:
DOI: 10.1006/jmbi.1998.2530
Links to Exploration step
ISTEX:57AE55468A78910A2AF46546C4D745DF122FD322Le document en format XML
<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title xml:lang="en">A theoretical approach towards the identification of cleavage-determining amino acid motifs of the 20s proteasome</title><author><name sortKey="Holzhutter, Hermann Georg" sort="Holzhutter, Hermann Georg" uniqKey="Holzhutter H" first="Hermann-Georg" last="Holzhütter">Hermann-Georg Holzhütter</name><affiliation><mods:affiliation>E-mail: hergo@rz.hu</mods:affiliation></affiliation><affiliation><mods:affiliation>Institute of Biochemistry Medical Faculty of the Humboldt-University (Charité), Monbijoustr. 2A, Berlin-D 10117, Germany</mods:affiliation></affiliation></author><author><name sortKey="Frommel, Cornelius" sort="Frommel, Cornelius" uniqKey="Frommel C" first="Cornelius" last="Frömmel">Cornelius Frömmel</name><affiliation><mods:affiliation>Institute of Biochemistry Medical Faculty of the Humboldt-University (Charité), Monbijoustr. 2A, Berlin-D 10117, Germany</mods:affiliation></affiliation></author><author><name sortKey="Kloetzel, Peter Michael" sort="Kloetzel, Peter Michael" uniqKey="Kloetzel P" first="Peter-Michael" last="Kloetzel">Peter-Michael Kloetzel</name><affiliation><mods:affiliation>Institute of Biochemistry Medical Faculty of the Humboldt-University (Charité), Monbijoustr. 2A, Berlin-D 10117, Germany</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:57AE55468A78910A2AF46546C4D745DF122FD322</idno><date when="1999" year="1999">1999</date><idno type="doi">10.1006/jmbi.1998.2530</idno><idno type="url">https://api.istex.fr/ark:/67375/6H6-97V86JNT-6/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">002559</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">002559</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">A theoretical approach towards the identification of cleavage-determining amino acid motifs of the 20s proteasome</title><author><name sortKey="Holzhutter, Hermann Georg" sort="Holzhutter, Hermann Georg" uniqKey="Holzhutter H" first="Hermann-Georg" last="Holzhütter">Hermann-Georg Holzhütter</name><affiliation><mods:affiliation>E-mail: hergo@rz.hu</mods:affiliation></affiliation><affiliation><mods:affiliation>Institute of Biochemistry Medical Faculty of the Humboldt-University (Charité), Monbijoustr. 2A, Berlin-D 10117, Germany</mods:affiliation></affiliation></author><author><name sortKey="Frommel, Cornelius" sort="Frommel, Cornelius" uniqKey="Frommel C" first="Cornelius" last="Frömmel">Cornelius Frömmel</name><affiliation><mods:affiliation>Institute of Biochemistry Medical Faculty of the Humboldt-University (Charité), Monbijoustr. 2A, Berlin-D 10117, Germany</mods:affiliation></affiliation></author><author><name sortKey="Kloetzel, Peter Michael" sort="Kloetzel, Peter Michael" uniqKey="Kloetzel P" first="Peter-Michael" last="Kloetzel">Peter-Michael Kloetzel</name><affiliation><mods:affiliation>Institute of Biochemistry Medical Faculty of the Humboldt-University (Charité), Monbijoustr. 2A, Berlin-D 10117, Germany</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Journal of Molecular Biology</title><title level="j" type="abbrev">YJMBI</title><idno type="ISSN">0022-2836</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1999">1999</date><biblScope unit="volume">286</biblScope><biblScope unit="issue">4</biblScope><biblScope unit="page" from="1251">1251</biblScope><biblScope unit="page" to="1265">1265</biblScope></imprint><idno type="ISSN">0022-2836</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0022-2836</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>CDAAM</term><term>MCR</term><term>SRS</term><term>kinetics</term><term>pattern recognition</term><term>proteasome</term><term>proteolytic cleavage site</term><term>regression analysis</term></keywords><keywords scheme="Teeft" xml:lang="en"><term>Academic press</term><term>Acid motifs</term><term>Acid residues</term><term>Active site</term><term>Active sites</term><term>Amino</term><term>Amino acid</term><term>Amino acid motifs</term><term>Amino acid residues</term><term>Amino acids</term><term>Anchor locations</term><term>Anchor position</term><term>Anchor positions</term><term>Anchor residues</term><term>Anking</term><term>Anking region</term><term>Anking regions</term><term>Antigen presentation</term><term>Antigenic peptides</term><term>Appropriate binding conformations</term><term>Binding epitope</term><term>Bold bars</term><term>Bold frames</term><term>Carboxyl</term><term>Carboxyl site</term><term>Cardinal properties</term><term>Certain length</term><term>Classical protease</term><term>Cleavage</term><term>Cleavage frequencies</term><term>Cleavage frequency</term><term>Cleavage mechanism</term><term>Cleavage preference</term><term>Cleavage preferences</term><term>Cleavage probability</term><term>Cleavage products</term><term>Cleavage rate</term><term>Cleavage rates</term><term>Cleavage site</term><term>Cleavage sites</term><term>Conformation</term><term>Continuous line</term><term>Corresponding property term</term><term>Degradation</term><term>Degradation pattern</term><term>Degradation products</term><term>Effective substrate binding</term><term>Epitope</term><term>Eukaryotic proteasomes</term><term>False positives</term><term>Febs letters</term><term>Fragment</term><term>Globular proteins</term><term>Grey cells</term><term>Hydrophobic residue</term><term>Hydrophobic residues</term><term>Independent observations</term><term>Larger sequence region</term><term>Larger weight</term><term>Lmp2</term><term>Lmp2 subunits</term><term>Lmp7 subunits</term><term>Major histocompatibility</term><term>Molecular ruler</term><term>Niedermann</term><term>Normalised</term><term>Optimal properties</term><term>Other cleavage sites</term><term>Other hand</term><term>Overall cleavage probability</term><term>Pattern recognition</term><term>Peptide</term><term>Peptide bond</term><term>Peptide bond cleavage</term><term>Peptide bonds</term><term>Peptide fragments</term><term>Peptide substrate</term><term>Peptide substrates</term><term>Pituitary multicatalytic proteinase</term><term>Possible epitopes</term><term>Possible shapes</term><term>Potential cleavage site</term><term>Predictive capacity</term><term>Processive degradation</term><term>Properties volume</term><term>Property term</term><term>Property terms</term><term>Property volume</term><term>Protease</term><term>Proteasome</term><term>Proteasome figure</term><term>Proteasome table</term><term>Proteasomes</term><term>Protein substrate</term><term>Protein substrates</term><term>Proteolytic</term><term>Proteolytic fragments</term><term>Relative frequencies</term><term>Relative orientation</term><term>Residual squares</term><term>Residue</term><term>Residue types</term><term>Same residue</term><term>Scatter plot</term><term>Scissile</term><term>Scissile bond</term><term>Scissile peptide bond</term><term>Selective cleavage</term><term>Sequence length</term><term>Sequence positions</term><term>Sequence window</term><term>Short peptides</term><term>Shorter fragments</term><term>Size distribution</term><term>Small number</term><term>Subunit</term><term>Subunits lmp2</term><term>Theoretical approach</term><term>Total number</term><term>Transfer energy</term><term>Tting</term><term>Tting round</term><term>Tting strategy</term><term>Uorgenic peptides</term><term>Vertebrate proteasomes</term><term>Window method</term><term>Yeast proteasome subunit</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: Hitherto the mechanisms controlling the selective cleavage of peptide bonds by the 20 S proteasome have been poorly understood. The observation that peptide bond cleavage may eventually occur at the carboxyl site of either amino acid residue rules out a simple control of cleavage preferences by the P1 residue alone. Here, we follow the rationale that the presence of specific cleavage-determining amino acids motifs (CDAAMs) around the scissile peptide bond are required for the attainment of substrate conformations susceptible to cleavage. We present an exploratory search for these putative motifs based on empirical regression functions relating the cleavage probability for a given peptide bond to some selected side-chain properties of the flanking amino acid residues. Identification of the sequence locations of cleavage-determining residues relative to the scissile bond and of their optimal side-chain properties was carried out by fitting the cleavage probability to (binary) experimental observations on peptide bond cleavage gathered among a set of seven different peptide substrates with known patterns of proteolytic degradation products. In this analysis, all peptide bonds containing the same residue in the P1 position were assumed to be cleaved by the same active sites of the proteasome, and thus to be under control of the same CDAAMs. We arrived at a final set of ten different CDAAMs, accounting for the cleavage of one to five different groups of peptide bonds with an overall predictive correctness of 93%. The CDAAM is composed of two to four “anchor” positions preferentially located between P5 and P5′ around the scissile bond. This implies a length constraint for the usage of cleavage sites, which could considerably suppress the excision of shorter fragments and thus partially explain for the observed preponderance of medium-size cleavage products.</div></front></TEI><istex><corpusName>elsevier</corpusName><keywords><teeft><json:string>proteasome</json:string><json:string>peptide</json:string><json:string>cleavage</json:string><json:string>peptide bonds</json:string><json:string>scissile</json:string><json:string>cleavage probability</json:string><json:string>cleavage sites</json:string><json:string>scissile bond</json:string><json:string>epitope</json:string><json:string>property terms</json:string><json:string>anking</json:string><json:string>subunit</json:string><json:string>amino</json:string><json:string>proteasomes</json:string><json:string>amino acid motifs</json:string><json:string>anchor positions</json:string><json:string>proteolytic fragments</json:string><json:string>tting</json:string><json:string>active sites</json:string><json:string>peptide bond</json:string><json:string>proteolytic</json:string><json:string>carboxyl</json:string><json:string>carboxyl site</json:string><json:string>niedermann</json:string><json:string>normalised</json:string><json:string>anking regions</json:string><json:string>lmp2</json:string><json:string>peptide substrate</json:string><json:string>cleavage preference</json:string><json:string>peptide bond cleavage</json:string><json:string>cleavage frequencies</json:string><json:string>residue</json:string><json:string>amino acid residues</json:string><json:string>bold bars</json:string><json:string>corresponding property term</json:string><json:string>transfer energy</json:string><json:string>anchor residues</json:string><json:string>sequence window</json:string><json:string>small number</json:string><json:string>total number</json:string><json:string>antigenic peptides</json:string><json:string>false positives</json:string><json:string>theoretical approach</json:string><json:string>peptide fragments</json:string><json:string>scissile peptide bond</json:string><json:string>binding epitope</json:string><json:string>proteasome figure</json:string><json:string>same residue</json:string><json:string>cleavage rates</json:string><json:string>cleavage frequency</json:string><json:string>amino acids</json:string><json:string>protein substrates</json:string><json:string>protease</json:string><json:string>degradation</json:string><json:string>conformation</json:string><json:string>cleavage rate</json:string><json:string>eukaryotic proteasomes</json:string><json:string>other hand</json:string><json:string>molecular ruler</json:string><json:string>size distribution</json:string><json:string>property term</json:string><json:string>major histocompatibility</json:string><json:string>peptide substrates</json:string><json:string>amino acid</json:string><json:string>effective substrate binding</json:string><json:string>sequence positions</json:string><json:string>cleavage mechanism</json:string><json:string>optimal properties</json:string><json:string>window method</json:string><json:string>protein substrate</json:string><json:string>anchor position</json:string><json:string>residue types</json:string><json:string>independent observations</json:string><json:string>fragment</json:string><json:string>subunits lmp2</json:string><json:string>academic press</json:string><json:string>scatter plot</json:string><json:string>hydrophobic residue</json:string><json:string>cleavage site</json:string><json:string>other cleavage sites</json:string><json:string>pattern recognition</json:string><json:string>pituitary multicatalytic proteinase</json:string><json:string>processive degradation</json:string><json:string>cardinal properties</json:string><json:string>degradation pattern</json:string><json:string>selective cleavage</json:string><json:string>overall cleavage probability</json:string><json:string>possible shapes</json:string><json:string>lmp7 subunits</json:string><json:string>lmp2 subunits</json:string><json:string>residual squares</json:string><json:string>short peptides</json:string><json:string>properties volume</json:string><json:string>cleavage preferences</json:string><json:string>larger weight</json:string><json:string>acid residues</json:string><json:string>vertebrate proteasomes</json:string><json:string>shorter fragments</json:string><json:string>tting strategy</json:string><json:string>active site</json:string><json:string>acid motifs</json:string><json:string>certain length</json:string><json:string>anking region</json:string><json:string>uorgenic peptides</json:string><json:string>tting round</json:string><json:string>predictive capacity</json:string><json:string>hydrophobic residues</json:string><json:string>potential cleavage site</json:string><json:string>property volume</json:string><json:string>grey cells</json:string><json:string>cleavage products</json:string><json:string>bold frames</json:string><json:string>anchor locations</json:string><json:string>continuous line</json:string><json:string>relative frequencies</json:string><json:string>sequence length</json:string><json:string>appropriate binding conformations</json:string><json:string>larger sequence region</json:string><json:string>relative orientation</json:string><json:string>degradation products</json:string><json:string>classical protease</json:string><json:string>globular proteins</json:string><json:string>yeast proteasome subunit</json:string><json:string>febs letters</json:string><json:string>antigen presentation</json:string><json:string>possible epitopes</json:string><json:string>proteasome table</json:string></teeft></keywords><author><json:item><name>Hermann-Georg Holzhütter</name><affiliations><json:string>E-mail: hergo@rz.hu</json:string><json:string>Institute of Biochemistry Medical Faculty of the Humboldt-University (Charité), Monbijoustr. 2A, Berlin-D 10117, Germany</json:string></affiliations></json:item><json:item><name>Cornelius Frömmel</name><affiliations><json:string>Institute of Biochemistry Medical Faculty of the Humboldt-University (Charité), Monbijoustr. 2A, Berlin-D 10117, Germany</json:string></affiliations></json:item><json:item><name>Peter-Michael Kloetzel</name><affiliations><json:string>Institute of Biochemistry Medical Faculty of the Humboldt-University (Charité), Monbijoustr. 2A, Berlin-D 10117, Germany</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Regular article</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>proteasome</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>proteolytic cleavage site</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>kinetics</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>pattern recognition</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>regression analysis</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>CDAAM : cleavage-determining amino acid motif</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>SRS : sum of residual squares</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>MCR : misclassification rate</value></json:item></subject><arkIstex>ark:/67375/6H6-97V86JNT-6</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Abstract: Hitherto the mechanisms controlling the selective cleavage of peptide bonds by the 20 S proteasome have been poorly understood. The observation that peptide bond cleavage may eventually occur at the carboxyl site of either amino acid residue rules out a simple control of cleavage preferences by the P1 residue alone. Here, we follow the rationale that the presence of specific cleavage-determining amino acids motifs (CDAAMs) around the scissile peptide bond are required for the attainment of substrate conformations susceptible to cleavage. We present an exploratory search for these putative motifs based on empirical regression functions relating the cleavage probability for a given peptide bond to some selected side-chain properties of the flanking amino acid residues. Identification of the sequence locations of cleavage-determining residues relative to the scissile bond and of their optimal side-chain properties was carried out by fitting the cleavage probability to (binary) experimental observations on peptide bond cleavage gathered among a set of seven different peptide substrates with known patterns of proteolytic degradation products. In this analysis, all peptide bonds containing the same residue in the P1 position were assumed to be cleaved by the same active sites of the proteasome, and thus to be under control of the same CDAAMs. We arrived at a final set of ten different CDAAMs, accounting for the cleavage of one to five different groups of peptide bonds with an overall predictive correctness of 93%. The CDAAM is composed of two to four “anchor” positions preferentially located between P5 and P5′ around the scissile bond. This implies a length constraint for the usage of cleavage sites, which could considerably suppress the excision of shorter fragments and thus partially explain for the observed preponderance of medium-size cleavage products.</abstract><qualityIndicators><score>10</score><pdfWordCount>8195</pdfWordCount><pdfCharCount>49812</pdfCharCount><pdfVersion>1.3</pdfVersion><pdfPageCount>15</pdfPageCount><pdfPageSize>530 x 782 pts</pdfPageSize><refBibsNative>true</refBibsNative><abstractWordCount>286</abstractWordCount><abstractCharCount>1893</abstractCharCount><keywordCount>9</keywordCount></qualityIndicators><title>A theoretical approach towards the identification of cleavage-determining amino acid motifs of the 20s proteasome</title><pmid><json:string>10047495</json:string></pmid><pii><json:string>S0022-2836(98)92530-X</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Journal of Molecular Biology</title><language><json:string>unknown</json:string></language><publicationDate>1999</publicationDate><issn><json:string>0022-2836</json:string></issn><pii><json:string>S0022-2836(00)X0432-9</json:string></pii><volume>286</volume><issue>4</issue><pages><first>1251</first><last>1265</last></pages><genre><json:string>journal</json:string></genre></host><namedEntities><unitex><date><json:string>1263</json:string><json:string>1999</json:string></date><geogName></geogName><orgName><json:string>Germany Hitherto</json:string></orgName><orgName_funder></orgName_funder><orgName_provider></orgName_provider><persName><json:string>Val Trp</json:string><json:string>Lys Leu</json:string><json:string>Lys Met</json:string><json:string>Cornelius Frommel</json:string></persName><placeName></placeName><ref_url><json:string>http://www.idealibrary.com</json:string></ref_url><ref_bibl><json:string>Dick et al., 1996</json:string><json:string>Niedermann et al., 1996</json:string><json:string>Hubbard et al., 1994</json:string><json:string>Orlowski et al., 1993</json:string><json:string>Sijts et al.</json:string><json:string>Heinemeyer, 1993</json:string><json:string>Kramer et al., 1998</json:string><json:string>Niedermann et al., 1995</json:string><json:string>Jentsch & Schenker, 1995</json:string><json:string>Seemuller et al., 1996</json:string><json:string>Dick et al., 1994</json:string><json:string>[0,1]</json:string><json:string>Hochstrasser, 1995</json:string><json:string>Arendt & Hochstrasser, 1997</json:string><json:string>Schneider et al., 1993</json:string><json:string>Eggers et al., 1995</json:string><json:string>Motoc & Marshall, 1985</json:string><json:string>Shimbara et al., 1998</json:string><json:string>Groll et al., 1997</json:string><json:string>Enenkel, 1994</json:string><json:string>Frommel, 1984</json:string><json:string>Lowe et al., 1995</json:string><json:string>Ehring et al., 1995</json:string><json:string>Theobald et al., 1998</json:string><json:string>Wenzel et al., 1994</json:string><json:string>Lehner & Cresswell, 1996</json:string><json:string>Ossendorp et al., 1996</json:string><json:string>Brannigan et al., 1995</json:string><json:string>Djaballah et al., 1992</json:string><json:string>Goldberg et al., 1995</json:string><json:string>Kuckelkorn et al., 1995</json:string><json:string>Fontana, 1989</json:string><json:string>Orlowski & Michaud, 1989</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/6H6-97V86JNT-6</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - biochemistry & molecular biology</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - biomedical research</json:string><json:string>3 - biochemistry & molecular biology</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Molecular Biology</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences biologiques fondamentales et appliquees. psychologie</json:string></inist></categories><publicationDate>1999</publicationDate><copyrightDate>1999</copyrightDate><doi><json:string>10.1006/jmbi.1998.2530</json:string></doi><id>57AE55468A78910A2AF46546C4D745DF122FD322</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-97V86JNT-6/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-97V86JNT-6/bundle.zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/6H6-97V86JNT-6/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">A theoretical approach towards the identification of cleavage-determining amino acid motifs of the 20s proteasome</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher scheme="https://scientific-publisher.data.istex.fr">ELSEVIER</publisher><availability><licence><p>©1999 Academic Press</p></licence><p scheme="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</p></availability><date>1999</date></publicationStmt><notesStmt><note type="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="journal" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note><note>Edited by R. Huber</note><note type="content">Section title: Regular article</note><note type="content">Figure 1: Proteins with experimentally determined degradation patterns used in the theoretical analysis. Cleavage sites were identified by inspection of the N and C-terminal ends of the proteolytic fragments generated after various time intervals of digestion by the 20 S proteasome. Cleaved peptide bond are marked by bold bars above the sequence. (a) Oxidised insulin B chain (Ehring et al., 1995). Cleavage sites were identified after pooling of all peptide fragments generated by IFN-γ-stimulated T1 or T2 proteasomes after 15 minutes and four hours of digestion. The incorporation of LMP2/7 did not alter the time-dependent degradation pattern compared with the wild-type proteasome. (b) HBVcAg131-162 (Sijtset al., unpublished). Synthetic 32-mer corresponding to HBVcAg131-162 and covering an MHC class I-presented hepatitis B virus CTL epitope (STLPETTVVRR). (c) pp89 (Kuckelkorn et al., 1995). Synthetic 25-mer which corresponds to a sequence region of the murine cytomegalovirus IE pp89 containing the antigenic nonamer region (YPHFMPTNL). Cleavage sites were identified after pooling of all peptide fragments generated by purified 20 S proteasomes from T2 cells (lacking LMP2 and LMP7 subunits), T2/LMP2 cells (possessing LMP2 subunits), T2/LMP7 cells (possessing LMP7 subunits) and T2/LMP2+7 cells (possessing the LMP2 subunits). (d) OvaY51–71 (Niedermann et al., 1995). Synthetic 22-mer corresponding to the region 51-71 of ovalbumin and containing a marginally immunogenic H-2 Kbbinding epitope (KVVRFDKL). Peptides were analysed after a six hour digest. (e) OvaY249-269 (Niedermann et al., 1995). Synthetic 22-mer corresponding to the region 249–269 of ovalbumin and containing the immunogenic H-2 Kb binding epitope (SIINFEKL). Peptides were analysed after a six hour digest. (f) OvaY239-281 (Niedermann et al., 1995). Synthetic 44-mer corresponding to the region 239-281 of ovalbumin and containing the immunogenic H-2 Kb binding epitope (SIINFEKL). Cleavage sites were identified after lumping together all peptide fragments generated after one hour and 15 hour digestions. (g) p53wt (Theobald et al., 1998). Synthetic 27-mer corresponding to the region 256–282 of the p53wt protein. Peptides were analysed after a 24 hour digestion. The bold bars below the sequences indicate the cleavage sites predicted by our method. A peptide was classified as cleaved if the value of the cleavage probability defined inequation (1) was larger than 0.5.</note><note type="content">Figure 2: Scatter plot: cleavage frequencies versus cleavage rates. For definitions and numerical values, refer to Table 2.</note><note type="content">Figure 3: Possible shapes of a single property termp = exp−E−E ̃/√σ2(cf.equation (2)). The values of the side-chain property E are normalised to the range [0,1], for a missing residue the value is put at −1. (a)Ẽ = −1, σ = 0.3, a missing residue favours binding. (b)Ẽ = 0,σ = 0.3, small values of the side-chain property are favourable for cleavage.(c)Ẽ = 0.5, σ=0.1, medium values of the side-chain property are optimal for cleavage.(d)Ẽ = 1, σ=0.3, large values of the side-chain property are favourable for cleavage. (e)Ẽ = 0.3, σ = 5.0, large weight parameter σ, no impact of the side-chain property on cleavage.</note><note type="content">Figure 4: Illustration of the expanding window fitting strategy for the Arg bonds. The various sequence windows used are marked by bold-line frames. Each box within a frame refers to a single sequence position. The two parameters for the volume property term are given in the left-hand panel, and the two parameters for the transfer energy property term are given in the right-hand panel of the box; the upper number is the optimal value of the property indicated and the lower number the variance. The largest possible initial sequence window was n = 2 because of the condition 8 n≤number of observations (=16 for the group of Arg peptide bonds, cf. Figure 5). All property terms except those for the P1 position were regarded as potentially significant, and thus included when fitting the cleavage probability function to the observed cleavage classifications. After this initial fit, all property terms were evaluated according to the estimated variances σ. A property term was designated as non-significant, i.e. possessing no significant discriminating power for cleavage classification, if its variance σ exceeded the cut-off value σc = 5. Once proved to be non-significant, the corresponding property term was excluded from all further fitting rounds, i.e. the value of this property term was put at unity and the parameters were not further included in the minimisation procedure. Property terms meeting the condition σ<σc were evaluated as potentially significant (marked in grey) and remained included during the next fitting round. In the example the property terms pV3, pV1′, pT1′ and pT2′ were identified as non-significant after initial fitting. Excluding these four terms from further fitting increased the degrees of freedom by 4, and thus allowed the sequence window to expand to a size of n = 3. This enlarged window covered the sequence positions P4 to P3′ and comprised eight potentially significant property terms, four of which (pV4, pT4, pV3′ and pT3′) have newly entered the window, and the four others (pT3, pV2, pT2 and pV2′) survived the preceding round. Evaluation of the fit proved five property terms to be non-significant and thus allowed the sequence window to expand again. From the eight property terms of the initial window, only pT2 remained within the set of potentially significant property terms after the second fitting round. After nine rounds of fitting and dropping of non-significant property terms, the maximum window size of n = 10 was reached and the final set of three significant property terms pV8, pT5′ and pV6′ was established. Inset: decline of the least-square sum (bars) and the number of misclassifications with increasing window size. The abrupt decline at nw = 7 is due to the involvement of the strongly discriminating position P8.</note><note type="content">Figure 5: Cleavage-determining amino acid motifs for Arg bonds as identified by the expanding window method. (a) Observed and predicted cleavage classifications for the 16 Arg bonds contained in the database. The flanking sequences within a window of length ten around the scissile bond Arg:X are shown. The predicted anchor locations at P8, P5′ and P6′ are marked in grey. A bond was assessed as potential cleavage site if the value of the cleavage probability CP (given in the last column) was above the cut-off value 0.5. (b) The relevant property terms pV8, pT5′ and pV6′ as a function of the corresponding (normalised) side-chain property. The sharply centred term pV8 indicates a high degree of selectivity of the proteasome for substrates with medium-sized residues at P8. The two other terms express the fact that a sufficiently long upstream sequence segment is required in a peptide substrate in order to be cleaved whereby there seems to be no further restraint for the type of residue to be accepted at P5′ and P6′. (c) Bar diagrams visualising the predicted impact of the residue types present at the three anchor positions P8, P5′ and P6′ on cleavage probability. The black spot of a column designates the “optimal” residue in this position according to the estimated parameters of the corresponding property term. The dark grey part of a column designates residues (among those listed on the vertical axis with monotone increasing or decreasing side-chain property) for which the corresponding property term has values between 0.9 and 1.0 and which thus favour cleavage. Residue types associated with values of the property terms in the range 0.5-0.9 match with the light grey part of a column. Residues giving rise to property terms <0.5 and thus preventing cleavage match with the filled part of a column. From the diagram it can be determined that peptides comprising at least six residues beyond the scissile bond towards the C-terminal end and a medium-sized residue (preferentially Cys, Thr, Val) at P8 are favourite candidates for the cleavage of Arg bonds.</note><note type="content">Figure 6: Cleavage-determining amino acid profiles. Locations of the anchor residues are marked by grey cells in the right panel. Grey cells indicated by an x refer to unspecific anchor positions which do not exhibit any selectivity for specific residue types, but simply mark the minimum sequence length required for effective substrate binding. The other symbols provide a gross characterisation of the side-chain property which when present at the indicated anchor position favour cleavage. The bold frames indicate anchoring regions spanned by those anchor positions in the N and C-terminal flanks being at the greatest distance from the scissile bond and thus defining the minimum sequence segment for cleavage.</note><note type="content">Figure 7: Observed cleavage sites of the JAK1 21-mer (Dick et al., 1996) and cleavage sites predicted on the basis of the CDAAMs (SYFPEITHI: H-2 Kd binding epitope). The pattern of observed cleavage sites (bold bars above the sequence) of this peptide substrate has not been included in the database used for the establishment of the CDAMMs. The bold bars below the sequence mark the cleavage sites predicted by our method. A peptide was classified “cleaved” if the value of the cleavage probability defined in equation (1)was larger than 0.5. The overall predictive correctness for this example is 80%, i.e. there are four misclassifications out of 20 cases comprising three false positives (predicted but not observed cleavages at P1 residues W-4, F10, V-17) and one false negative (observed but not predicted cleavage at P1 residue N-6).</note><note type="content">Figure 8: Size distribution of cleavage products and of cleavage site distances. Continuous line, the relative frequencies of sequence length enclosed by arbitrary cleavage sites. Filled bars, the relative frequencies of sequence length enclosed by cleavage sites which do not mutually extinct because of overlapping anchoring regions. Open bars, the relative length frequencies (normalised to unity) observed among hydrolytic fragments generated from the proteins listed in Figure 2.</note><note type="content">Table 1: Cleavage frequencies and cleavage rates at P1 residues</note><note type="content">Table 2: Side-chain properties (normalised to unity) used for the calculation of cleavage probabilities</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">A theoretical approach towards the identification of cleavage-determining amino acid motifs of the 20s proteasome</title><author xml:id="author-0000"><persName><forename type="first">Hermann-Georg</forename><surname>Holzhütter</surname></persName><email>hergo@rz.hu</email><note type="correspondence"><p>Corresponding author</p></note><affiliation>Institute of Biochemistry Medical Faculty of the Humboldt-University (Charité), Monbijoustr. 2A, Berlin-D 10117, Germany</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Cornelius</forename><surname>Frömmel</surname></persName><affiliation>Institute of Biochemistry Medical Faculty of the Humboldt-University (Charité), Monbijoustr. 2A, Berlin-D 10117, Germany</affiliation></author><author xml:id="author-0002"><persName><forename type="first">Peter-Michael</forename><surname>Kloetzel</surname></persName><affiliation>Institute of Biochemistry Medical Faculty of the Humboldt-University (Charité), Monbijoustr. 2A, Berlin-D 10117, Germany</affiliation></author><idno type="istex">57AE55468A78910A2AF46546C4D745DF122FD322</idno><idno type="ark">ark:/67375/6H6-97V86JNT-6</idno><idno type="DOI">10.1006/jmbi.1998.2530</idno><idno type="PII">S0022-2836(98)92530-X</idno></analytic><monogr><title level="j">Journal of Molecular Biology</title><title level="j" type="abbrev">YJMBI</title><idno type="pISSN">0022-2836</idno><idno type="PII">S0022-2836(00)X0432-9</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1999"></date><biblScope unit="volume">286</biblScope><biblScope unit="issue">4</biblScope><biblScope unit="page" from="1251">1251</biblScope><biblScope unit="page" to="1265">1265</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1999</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: Hitherto the mechanisms controlling the selective cleavage of peptide bonds by the 20 S proteasome have been poorly understood. The observation that peptide bond cleavage may eventually occur at the carboxyl site of either amino acid residue rules out a simple control of cleavage preferences by the P1 residue alone. Here, we follow the rationale that the presence of specific cleavage-determining amino acids motifs (CDAAMs) around the scissile peptide bond are required for the attainment of substrate conformations susceptible to cleavage. We present an exploratory search for these putative motifs based on empirical regression functions relating the cleavage probability for a given peptide bond to some selected side-chain properties of the flanking amino acid residues. Identification of the sequence locations of cleavage-determining residues relative to the scissile bond and of their optimal side-chain properties was carried out by fitting the cleavage probability to (binary) experimental observations on peptide bond cleavage gathered among a set of seven different peptide substrates with known patterns of proteolytic degradation products. In this analysis, all peptide bonds containing the same residue in the P1 position were assumed to be cleaved by the same active sites of the proteasome, and thus to be under control of the same CDAAMs. We arrived at a final set of ten different CDAAMs, accounting for the cleavage of one to five different groups of peptide bonds with an overall predictive correctness of 93%. The CDAAM is composed of two to four “anchor” positions preferentially located between P5 and P5′ around the scissile bond. This implies a length constraint for the usage of cleavage sites, which could considerably suppress the excision of shorter fragments and thus partially explain for the observed preponderance of medium-size cleavage products.</p></abstract><textClass><keywords scheme="keyword"><list><head>article-category</head><item><term>Regular article</term></item></list></keywords></textClass><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Keywords</head><item><term>proteasome</term></item><item><term>proteolytic cleavage site</term></item><item><term>kinetics</term></item><item><term>pattern recognition</term></item><item><term>regression analysis</term></item></list></keywords></textClass><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Abbreviations</head><item><term>CDAAM</term><term>cleavage-determining amino acid motif</term></item><item><term>SRS</term><term>sum of residual squares</term></item><item><term>MCR</term><term>misclassification rate</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="1998-12-30">Modified</change><change when="1999">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-97V86JNT-6/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="GR1"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="GR2"></istex:entity><istex:entity SYSTEM="gr3" NDATA="IMAGE" name="GR3"></istex:entity><istex:entity SYSTEM="gr4" NDATA="IMAGE" name="GR4"></istex:entity><istex:entity SYSTEM="gr5" NDATA="IMAGE" name="GR5"></istex:entity><istex:entity SYSTEM="gr6" NDATA="IMAGE" name="GR6"></istex:entity><istex:entity SYSTEM="gr7" NDATA="IMAGE" name="GR7"></istex:entity><istex:entity SYSTEM="gr8" NDATA="IMAGE" name="GR8"></istex:entity><istex:entity SYSTEM="fx1" NDATA="IMAGE" name="FX1"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>YJMBI</jid><aid>92530</aid><ce:pii>S0022-2836(98)92530-X</ce:pii><ce:doi>10.1006/jmbi.1998.2530</ce:doi><ce:copyright type="full-transfer" year="1999">Academic Press</ce:copyright><ce:doctopics><ce:doctopic><ce:text>Regular article</ce:text></ce:doctopic></ce:doctopics></item-info><head><ce:dochead><ce:textfn>Regular article</ce:textfn></ce:dochead><ce:title>A theoretical approach towards the identification of cleavage-determining amino acid motifs of the 20s proteasome<ce:cross-ref refid="FN1"><ce:sup>1</ce:sup></ce:cross-ref><ce:footnote id="FN1"><ce:label>1</ce:label><ce:note-para><ce:bold><ce:italic>Edited by R. Huber</ce:italic></ce:bold></ce:note-para></ce:footnote></ce:title><ce:author-group><ce:author><ce:indexed-name>Holzhutter</ce:indexed-name><ce:given-name>Hermann-Georg</ce:given-name><ce:surname>Holzhütter</ce:surname><ce:cross-ref refid="COR1">*</ce:cross-ref><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref><ce:e-address>hergo@rz.hu</ce:e-address></ce:author><ce:author><ce:indexed-name>Frommel</ce:indexed-name><ce:given-name>Cornelius</ce:given-name><ce:surname>Frömmel</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Peter-Michael</ce:given-name><ce:surname>Kloetzel</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref></ce:author><ce:affiliation id="AFF1"><ce:label>a</ce:label><ce:textfn>Institute of Biochemistry Medical Faculty of the Humboldt-University (Charité), Monbijoustr. 2A, Berlin-D 10117, Germany</ce:textfn></ce:affiliation><ce:correspondence id="COR1"><ce:label>*</ce:label><ce:text>Corresponding author</ce:text></ce:correspondence></ce:author-group><ce:date-received day="7" month="10" year="1998"></ce:date-received><ce:date-revised day="30" month="12" year="1998"></ce:date-revised><ce:date-accepted day="30" month="12" year="1998"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>Hitherto the mechanisms controlling the selective cleavage of peptide bonds by the 20 S proteasome have been poorly understood. The observation that peptide bond cleavage may eventually occur at the carboxyl site of either amino acid residue rules out a simple control of cleavage preferences by the P1 residue alone. Here, we follow the rationale that the presence of specific cleavage-determining amino acids motifs (CDAAMs) around the scissile peptide bond are required for the attainment of substrate conformations susceptible to cleavage. We present an exploratory search for these putative motifs based on empirical regression functions relating the cleavage probability for a given peptide bond to some selected side-chain properties of the flanking amino acid residues. Identification of the sequence locations of cleavage-determining residues relative to the scissile bond and of their optimal side-chain properties was carried out by fitting the cleavage probability to (binary) experimental observations on peptide bond cleavage gathered among a set of seven different peptide substrates with known patterns of proteolytic degradation products. In this analysis, all peptide bonds containing the same residue in the P1 position were assumed to be cleaved by the same active sites of the proteasome, and thus to be under control of the same CDAAMs. We arrived at a final set of ten different CDAAMs, accounting for the cleavage of one to five different groups of peptide bonds with an overall predictive correctness of 93%. The CDAAM is composed of two to four “anchor” positions preferentially located between P5 and P5′ around the scissile bond. This implies a length constraint for the usage of cleavage sites, which could considerably suppress the excision of shorter fragments and thus partially explain for the observed preponderance of medium-size cleavage products.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>proteasome</ce:text></ce:keyword><ce:keyword><ce:text>proteolytic cleavage site</ce:text></ce:keyword><ce:keyword><ce:text>kinetics</ce:text></ce:keyword><ce:keyword><ce:text>pattern recognition</ce:text></ce:keyword><ce:keyword><ce:text>regression analysis</ce:text></ce:keyword></ce:keywords><ce:keywords class="abr"><ce:section-title>Abbreviations</ce:section-title><ce:keyword><ce:text>CDAAM</ce:text><ce:keyword><ce:text>cleavage-determining amino acid motif</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>SRS</ce:text><ce:keyword><ce:text>sum of residual squares</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>MCR</ce:text><ce:keyword><ce:text>misclassification rate</ce:text></ce:keyword></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>A theoretical approach towards the identification of cleavage-determining amino acid motifs of the 20s proteasome</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>A theoretical approach towards the identification of cleavage-determining amino acid motifs of the 20s proteasome</title></titleInfo><name type="personal"><namePart type="given">Hermann-Georg</namePart><namePart type="family">Holzhütter</namePart><affiliation>E-mail: hergo@rz.hu</affiliation><affiliation>Institute of Biochemistry Medical Faculty of the Humboldt-University (Charité), Monbijoustr. 2A, Berlin-D 10117, Germany</affiliation><description>Corresponding author</description><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Cornelius</namePart><namePart type="family">Frömmel</namePart><affiliation>Institute of Biochemistry Medical Faculty of the Humboldt-University (Charité), Monbijoustr. 2A, Berlin-D 10117, Germany</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Peter-Michael</namePart><namePart type="family">Kloetzel</namePart><affiliation>Institute of Biochemistry Medical Faculty of the Humboldt-University (Charité), Monbijoustr. 2A, Berlin-D 10117, Germany</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1999</dateIssued><dateModified encoding="w3cdtf">1998-12-30</dateModified><copyrightDate encoding="w3cdtf">1999</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: Hitherto the mechanisms controlling the selective cleavage of peptide bonds by the 20 S proteasome have been poorly understood. The observation that peptide bond cleavage may eventually occur at the carboxyl site of either amino acid residue rules out a simple control of cleavage preferences by the P1 residue alone. Here, we follow the rationale that the presence of specific cleavage-determining amino acids motifs (CDAAMs) around the scissile peptide bond are required for the attainment of substrate conformations susceptible to cleavage. We present an exploratory search for these putative motifs based on empirical regression functions relating the cleavage probability for a given peptide bond to some selected side-chain properties of the flanking amino acid residues. Identification of the sequence locations of cleavage-determining residues relative to the scissile bond and of their optimal side-chain properties was carried out by fitting the cleavage probability to (binary) experimental observations on peptide bond cleavage gathered among a set of seven different peptide substrates with known patterns of proteolytic degradation products. In this analysis, all peptide bonds containing the same residue in the P1 position were assumed to be cleaved by the same active sites of the proteasome, and thus to be under control of the same CDAAMs. We arrived at a final set of ten different CDAAMs, accounting for the cleavage of one to five different groups of peptide bonds with an overall predictive correctness of 93%. The CDAAM is composed of two to four “anchor” positions preferentially located between P5 and P5′ around the scissile bond. This implies a length constraint for the usage of cleavage sites, which could considerably suppress the excision of shorter fragments and thus partially explain for the observed preponderance of medium-size cleavage products.</abstract><note type="footnote">Edited by R. Huber</note><note type="content">Section title: Regular article</note><note type="content">Figure 1: Proteins with experimentally determined degradation patterns used in the theoretical analysis. Cleavage sites were identified by inspection of the N and C-terminal ends of the proteolytic fragments generated after various time intervals of digestion by the 20 S proteasome. Cleaved peptide bond are marked by bold bars above the sequence. (a) Oxidised insulin B chain (Ehring et al., 1995). Cleavage sites were identified after pooling of all peptide fragments generated by IFN-γ-stimulated T1 or T2 proteasomes after 15 minutes and four hours of digestion. The incorporation of LMP2/7 did not alter the time-dependent degradation pattern compared with the wild-type proteasome. (b) HBVcAg131-162 (Sijtset al., unpublished). Synthetic 32-mer corresponding to HBVcAg131-162 and covering an MHC class I-presented hepatitis B virus CTL epitope (STLPETTVVRR). (c) pp89 (Kuckelkorn et al., 1995). Synthetic 25-mer which corresponds to a sequence region of the murine cytomegalovirus IE pp89 containing the antigenic nonamer region (YPHFMPTNL). Cleavage sites were identified after pooling of all peptide fragments generated by purified 20 S proteasomes from T2 cells (lacking LMP2 and LMP7 subunits), T2/LMP2 cells (possessing LMP2 subunits), T2/LMP7 cells (possessing LMP7 subunits) and T2/LMP2+7 cells (possessing the LMP2 subunits). (d) OvaY51–71 (Niedermann et al., 1995). Synthetic 22-mer corresponding to the region 51-71 of ovalbumin and containing a marginally immunogenic H-2 Kbbinding epitope (KVVRFDKL). Peptides were analysed after a six hour digest. (e) OvaY249-269 (Niedermann et al., 1995). Synthetic 22-mer corresponding to the region 249–269 of ovalbumin and containing the immunogenic H-2 Kb binding epitope (SIINFEKL). Peptides were analysed after a six hour digest. (f) OvaY239-281 (Niedermann et al., 1995). Synthetic 44-mer corresponding to the region 239-281 of ovalbumin and containing the immunogenic H-2 Kb binding epitope (SIINFEKL). Cleavage sites were identified after lumping together all peptide fragments generated after one hour and 15 hour digestions. (g) p53wt (Theobald et al., 1998). Synthetic 27-mer corresponding to the region 256–282 of the p53wt protein. Peptides were analysed after a 24 hour digestion. The bold bars below the sequences indicate the cleavage sites predicted by our method. A peptide was classified as cleaved if the value of the cleavage probability defined inequation (1) was larger than 0.5.</note><note type="content">Figure 2: Scatter plot: cleavage frequencies versus cleavage rates. For definitions and numerical values, refer to Table 2.</note><note type="content">Figure 3: Possible shapes of a single property termp = exp−E−E ̃/√σ2(cf.equation (2)). The values of the side-chain property E are normalised to the range [0,1], for a missing residue the value is put at −1. (a)Ẽ = −1, σ = 0.3, a missing residue favours binding. (b)Ẽ = 0,σ = 0.3, small values of the side-chain property are favourable for cleavage.(c)Ẽ = 0.5, σ=0.1, medium values of the side-chain property are optimal for cleavage.(d)Ẽ = 1, σ=0.3, large values of the side-chain property are favourable for cleavage. (e)Ẽ = 0.3, σ = 5.0, large weight parameter σ, no impact of the side-chain property on cleavage.</note><note type="content">Figure 4: Illustration of the expanding window fitting strategy for the Arg bonds. The various sequence windows used are marked by bold-line frames. Each box within a frame refers to a single sequence position. The two parameters for the volume property term are given in the left-hand panel, and the two parameters for the transfer energy property term are given in the right-hand panel of the box; the upper number is the optimal value of the property indicated and the lower number the variance. The largest possible initial sequence window was n = 2 because of the condition 8 n≤number of observations (=16 for the group of Arg peptide bonds, cf. Figure 5). All property terms except those for the P1 position were regarded as potentially significant, and thus included when fitting the cleavage probability function to the observed cleavage classifications. After this initial fit, all property terms were evaluated according to the estimated variances σ. A property term was designated as non-significant, i.e. possessing no significant discriminating power for cleavage classification, if its variance σ exceeded the cut-off value σc = 5. Once proved to be non-significant, the corresponding property term was excluded from all further fitting rounds, i.e. the value of this property term was put at unity and the parameters were not further included in the minimisation procedure. Property terms meeting the condition σ<σc were evaluated as potentially significant (marked in grey) and remained included during the next fitting round. In the example the property terms pV3, pV1′, pT1′ and pT2′ were identified as non-significant after initial fitting. Excluding these four terms from further fitting increased the degrees of freedom by 4, and thus allowed the sequence window to expand to a size of n = 3. This enlarged window covered the sequence positions P4 to P3′ and comprised eight potentially significant property terms, four of which (pV4, pT4, pV3′ and pT3′) have newly entered the window, and the four others (pT3, pV2, pT2 and pV2′) survived the preceding round. Evaluation of the fit proved five property terms to be non-significant and thus allowed the sequence window to expand again. From the eight property terms of the initial window, only pT2 remained within the set of potentially significant property terms after the second fitting round. After nine rounds of fitting and dropping of non-significant property terms, the maximum window size of n = 10 was reached and the final set of three significant property terms pV8, pT5′ and pV6′ was established. Inset: decline of the least-square sum (bars) and the number of misclassifications with increasing window size. The abrupt decline at nw = 7 is due to the involvement of the strongly discriminating position P8.</note><note type="content">Figure 5: Cleavage-determining amino acid motifs for Arg bonds as identified by the expanding window method. (a) Observed and predicted cleavage classifications for the 16 Arg bonds contained in the database. The flanking sequences within a window of length ten around the scissile bond Arg:X are shown. The predicted anchor locations at P8, P5′ and P6′ are marked in grey. A bond was assessed as potential cleavage site if the value of the cleavage probability CP (given in the last column) was above the cut-off value 0.5. (b) The relevant property terms pV8, pT5′ and pV6′ as a function of the corresponding (normalised) side-chain property. The sharply centred term pV8 indicates a high degree of selectivity of the proteasome for substrates with medium-sized residues at P8. The two other terms express the fact that a sufficiently long upstream sequence segment is required in a peptide substrate in order to be cleaved whereby there seems to be no further restraint for the type of residue to be accepted at P5′ and P6′. (c) Bar diagrams visualising the predicted impact of the residue types present at the three anchor positions P8, P5′ and P6′ on cleavage probability. The black spot of a column designates the “optimal” residue in this position according to the estimated parameters of the corresponding property term. The dark grey part of a column designates residues (among those listed on the vertical axis with monotone increasing or decreasing side-chain property) for which the corresponding property term has values between 0.9 and 1.0 and which thus favour cleavage. Residue types associated with values of the property terms in the range 0.5-0.9 match with the light grey part of a column. Residues giving rise to property terms <0.5 and thus preventing cleavage match with the filled part of a column. From the diagram it can be determined that peptides comprising at least six residues beyond the scissile bond towards the C-terminal end and a medium-sized residue (preferentially Cys, Thr, Val) at P8 are favourite candidates for the cleavage of Arg bonds.</note><note type="content">Figure 6: Cleavage-determining amino acid profiles. Locations of the anchor residues are marked by grey cells in the right panel. Grey cells indicated by an x refer to unspecific anchor positions which do not exhibit any selectivity for specific residue types, but simply mark the minimum sequence length required for effective substrate binding. The other symbols provide a gross characterisation of the side-chain property which when present at the indicated anchor position favour cleavage. The bold frames indicate anchoring regions spanned by those anchor positions in the N and C-terminal flanks being at the greatest distance from the scissile bond and thus defining the minimum sequence segment for cleavage.</note><note type="content">Figure 7: Observed cleavage sites of the JAK1 21-mer (Dick et al., 1996) and cleavage sites predicted on the basis of the CDAAMs (SYFPEITHI: H-2 Kd binding epitope). The pattern of observed cleavage sites (bold bars above the sequence) of this peptide substrate has not been included in the database used for the establishment of the CDAMMs. The bold bars below the sequence mark the cleavage sites predicted by our method. A peptide was classified “cleaved” if the value of the cleavage probability defined in equation (1)was larger than 0.5. The overall predictive correctness for this example is 80%, i.e. there are four misclassifications out of 20 cases comprising three false positives (predicted but not observed cleavages at P1 residues W-4, F10, V-17) and one false negative (observed but not predicted cleavage at P1 residue N-6).</note><note type="content">Figure 8: Size distribution of cleavage products and of cleavage site distances. Continuous line, the relative frequencies of sequence length enclosed by arbitrary cleavage sites. Filled bars, the relative frequencies of sequence length enclosed by cleavage sites which do not mutually extinct because of overlapping anchoring regions. Open bars, the relative length frequencies (normalised to unity) observed among hydrolytic fragments generated from the proteins listed in Figure 2.</note><note type="content">Table 1: Cleavage frequencies and cleavage rates at P1 residues</note><note type="content">Table 2: Side-chain properties (normalised to unity) used for the calculation of cleavage probabilities</note><subject><genre>article-category</genre><topic>Regular article</topic></subject><subject lang="en"><genre>Keywords</genre><topic>proteasome</topic><topic>proteolytic cleavage site</topic><topic>kinetics</topic><topic>pattern recognition</topic><topic>regression analysis</topic></subject><subject lang="en"><genre>Abbreviations</genre><topic>CDAAM : cleavage-determining amino acid motif</topic><topic>SRS : sum of residual squares</topic><topic>MCR : misclassification rate</topic></subject><relatedItem type="host"><titleInfo><title>Journal of Molecular Biology</title></titleInfo><titleInfo type="abbreviated"><title>YJMBI</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1999</dateIssued></originInfo><identifier type="ISSN">0022-2836</identifier><identifier type="PII">S0022-2836(00)X0432-9</identifier><part><date>1999</date><detail type="volume"><number>286</number><caption>vol.</caption></detail><detail type="issue"><number>4</number><caption>no.</caption></detail><extent unit="issue-pages"><start>973</start><end>1265</end></extent><extent unit="pages"><start>1251</start><end>1265</end></extent></part></relatedItem><identifier type="istex">57AE55468A78910A2AF46546C4D745DF122FD322</identifier><identifier type="ark">ark:/67375/6H6-97V86JNT-6</identifier><identifier type="DOI">10.1006/jmbi.1998.2530</identifier><identifier type="PII">S0022-2836(98)92530-X</identifier><accessCondition type="use and reproduction" contentType="copyright">©1999 Academic Press</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource><recordOrigin>Academic Press, ©1999</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-97V86JNT-6/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/MersV1/Data/Istex/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 002559 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Istex/Corpus/biblio.hfd -nk 002559 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Sante
|area= MersV1
|flux= Istex
|étape= Corpus
|type= RBID
|clé= ISTEX:57AE55468A78910A2AF46546C4D745DF122FD322
|texte= A theoretical approach towards the identification of cleavage-determining amino acid motifs of the 20s proteasome
}}
| This area was generated with Dilib version V0.6.33. |  |