Determination of peptide substrate specificity for mu-calpain by a peptide library-based approach: the importance of primed side interactions.
Identifieur interne : 002987 ( Main/Curation ); précédent : 002986; suivant : 002988Determination of peptide substrate specificity for mu-calpain by a peptide library-based approach: the importance of primed side interactions.
Auteurs : Dominic Cuerrier [Canada] ; Tudor Moldoveanu ; Peter L. DaviesSource :
- The Journal of biological chemistry [ 0021-9258 ] ; 2005.
English descriptors
- KwdEn :
- Acetylation, Amino Acid Sequence, Binding Sites, Biotinylation, Calpain (metabolism), Chromatography, Gel, Consensus Sequence, Fluorescence Resonance Energy Transfer, Kinetics, Peptide Fragments (chemistry), Peptide Fragments (metabolism), Peptide Library, Peptides (chemistry), Peptides (metabolism), Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Substrate Specificity.
- MESH :
- chemical , chemistry : Peptide Fragments, Peptides.
- chemical , metabolism : Calpain, Peptide Fragments, Peptides.
- Acetylation, Amino Acid Sequence, Binding Sites, Biotinylation, Chromatography, Gel, Consensus Sequence, Fluorescence Resonance Energy Transfer, Kinetics, Peptide Library, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Substrate Specificity.
Abstract
Calpains are proteases that catalyze the limited cleavage of target proteins in response to Ca(2+) signaling. Because of their involvement in pathological conditions such as post-ischemic injury and Alzheimer and Parkinson disease, calpains form a class of pharmacologically significant targets for inhibition. We have determined the sequence preference for the hydrolysis of peptide substrates of the ubiquitous mu-calpain isoform by a peptide library-based approach using the proteolytic core of mu-calpain (muI-II). The approach, first described by Turk et al. (Turk, B. E., Huang, L. L., Piro, E. T., and Cantley, L. C. (2001) Nat. Biotechnol. 19, 661-667), involved the digestion of an N-terminally acetylated degenerate peptide library in conjunction with Edman sequencing to determine the specificity for residues found at primed positions. The cleavage consensus for these positions was then used to design a second, partially degenerate library, to determine specificity at unprimed positions. We have improved upon the original methodology by using a degenerate peptide dendrimer for determination of specificity at unprimed positions. By using this modified approach, the complete cleavage specificity profile for muI-II was determined for all positions flanking the cleaved peptide. A previously known preference of calpains for hydrophobic amino acids at unprimed positions was confirmed. In addition, a novel residue specificity for primed positions was revealed to highlight the importance of these sites for substrate recognition. The optimal primed site motif (MER) was shown to be capable of directing cleavage to a specific peptide bond. Accordingly, we designed a fluorescent resonance energy transfer-based substrate with optimal cleavage motifs on the primed and non-primed sides (PLFAER). The mu-calpain core shows a far greater turnover rate for our substrate than for those based on the cleavage site of alpha-spectrin or the proteolytic sequence consensus compiled from substrate alignments.
DOI: 10.1074/jbc.M506870200
PubMed: 16216885
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pubmed:16216885Le document en format XML
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Davies</name></author></analytic><series><title level="j">The Journal of biological chemistry</title><idno type="ISSN">0021-9258</idno><imprint><date when="2005" type="published">2005</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Acetylation</term><term>Amino Acid Sequence</term><term>Binding Sites</term><term>Biotinylation</term><term>Calpain (metabolism)</term><term>Chromatography, Gel</term><term>Consensus Sequence</term><term>Fluorescence Resonance Energy Transfer</term><term>Kinetics</term><term>Peptide Fragments (chemistry)</term><term>Peptide Fragments (metabolism)</term><term>Peptide Library</term><term>Peptides (chemistry)</term><term>Peptides (metabolism)</term><term>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</term><term>Substrate Specificity</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Peptide Fragments</term><term>Peptides</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Calpain</term><term>Peptide Fragments</term><term>Peptides</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Acetylation</term><term>Amino Acid Sequence</term><term>Binding Sites</term><term>Biotinylation</term><term>Chromatography, Gel</term><term>Consensus Sequence</term><term>Fluorescence Resonance Energy Transfer</term><term>Kinetics</term><term>Peptide Library</term><term>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</term><term>Substrate Specificity</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Calpains are proteases that catalyze the limited cleavage of target proteins in response to Ca(2+) signaling. Because of their involvement in pathological conditions such as post-ischemic injury and Alzheimer and Parkinson disease, calpains form a class of pharmacologically significant targets for inhibition. We have determined the sequence preference for the hydrolysis of peptide substrates of the ubiquitous mu-calpain isoform by a peptide library-based approach using the proteolytic core of mu-calpain (muI-II). The approach, first described by Turk et al. (Turk, B. E., Huang, L. L., Piro, E. T., and Cantley, L. C. (2001) Nat. Biotechnol. 19, 661-667), involved the digestion of an N-terminally acetylated degenerate peptide library in conjunction with Edman sequencing to determine the specificity for residues found at primed positions. The cleavage consensus for these positions was then used to design a second, partially degenerate library, to determine specificity at unprimed positions. We have improved upon the original methodology by using a degenerate peptide dendrimer for determination of specificity at unprimed positions. By using this modified approach, the complete cleavage specificity profile for muI-II was determined for all positions flanking the cleaved peptide. A previously known preference of calpains for hydrophobic amino acids at unprimed positions was confirmed. In addition, a novel residue specificity for primed positions was revealed to highlight the importance of these sites for substrate recognition. The optimal primed site motif (MER) was shown to be capable of directing cleavage to a specific peptide bond. Accordingly, we designed a fluorescent resonance energy transfer-based substrate with optimal cleavage motifs on the primed and non-primed sides (PLFAER). The mu-calpain core shows a far greater turnover rate for our substrate than for those based on the cleavage site of alpha-spectrin or the proteolytic sequence consensus compiled from substrate alignments.</div></front></TEI></record>Pour manipuler ce document sous Unix (Dilib)
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