Determination of Peptide Substrate Specificity for μ-Calpain by a Peptide Library-based Approach

Calpains are proteases that catalyze the limited cleavage of target proteins in response to Ca2+ 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 μ-calpain isoform by a peptide library-based approach using the proteolytic core of μ-calpain (μI-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 μI-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 μ-calpain core shows a far greater turnover rate for our substrate than for those based on the cleavage site of α-spectrin or the proteolytic sequence consensus compiled from substrate alignments. Calpains are proteases that catalyze the limited cleavage of target proteins in response to Ca2+ 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 μ-calpain isoform by a peptide library-based approach using the proteolytic core of μ-calpain (μI-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 μI-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 μ-calpain core shows a far greater turnover rate for our substrate than for those based on the cleavage site of α-spectrin or the proteolytic sequence consensus compiled from substrate alignments. Calpains (clan CA, family C2 in the MEROPS data base (1Barrett A.J. Curr. Opin. Drug Discovery Dev. 2004; 7: 334-341PubMed Google Scholar)), a family of calcium-activated intracellular proteases, are found in animals, plants, and possibly bacteria (2Sorimachi H. Suzuki K. J. Biochem. (Tokyo). 2001; 129: 653-664Crossref PubMed Scopus (246) Google Scholar). They convert intracellular calcium signals (3Carafoli E. Santella L. Branca D. Brini M. Crit. Rev. Biochem. Mol. Biol. 2001; 36: 107-260Crossref PubMed Scopus (434) Google Scholar) into a proteolytic signal by catalyzing the limited cleavage of target proteins (4Goll D.E. Thompson V.F. Li H. Wei W. Cong J. Physiol. Rev. 2003; 83: 731-801Crossref PubMed Scopus (2378) Google Scholar, 5Croall D.E. DeMartino G.N. Physiol. Rev. 1991; 71: 813-847Crossref PubMed Scopus (782) Google Scholar). Among the known cellular substrates of calpain are numerous cytoskeletal proteins, as well as some receptors and integral membrane proteins like the Na+/Ca2+ exchanger, NCX-3 (6Bano D. Young K.W. Guerin C.J. Lefeuvre R. Rothwell N.J. Naldini L. Rizzuto R. Carafoli E. Nicotera P. Cell. 2005; 120: 275-285Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar). Calpains must be strictly regulated because they catalyze irreversible processing in the cell. In one scenario, calpains localize to the plasma membrane under activating conditions (7Gil-Parrado S. Popp O. Knoch T.A. Zahler S. Bestvater F. Felgentrager M. Holloschi A. Fernandez-Montalvan A. Auerswald E.A. Fritz H. Fuentes-Prior P. Machleidt W. Spiess E. J. Biol. Chem. 2003; 278: 16336-16346Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 8Sakai K. Hayashi M. Kawashima S. Akanuma H. Biochim. Biophys. Acta. 1989; 985: 51-54Crossref PubMed Scopus (11) Google Scholar). This placement may act to position calpains where they can respond to brief calcium influxes from the opening of calcium channels, resulting in very localized and transient activity. Deactivation of calpain can come about in several ways: binding to the endogenous calpain inhibitor calpastatin (9Wendt A. Thompson V.F. Goll D.E. Biol. Chem. 2004; 385: 465-472Crossref PubMed Scopus (179) Google Scholar); autoproteolytic inactivation; or simply the dissipation of local high calcium levels. Unregulated intracellular calcium influx leads to hyperactivation of calpain and is associated with a large numbers of pathologies such as post-ischemic injury (10Kambe A. Yokota M. Saido T.C. Satokata I. Fujikawa H. Tabuchi S. Kamitani H. Watanabe T. Brain Res. 2005; 1040: 36-43Crossref PubMed Scopus (16) Google Scholar). Calpains are thus of great biomedical and therapeutic interest, and there is a need for calpain-specific inhibitors that will not affect other cysteine proteases. The active site of any enzyme serves the dual purpose of binding substrates and performing catalysis. For proteases, improvements in the selectivity constant (kcat/Km) for small substrates are mainly driven though improvements in Km. Optimized cleavage sequences are therefore strongly linked to an optimized binding and are valuable for the design of sensitive and specific peptidomimetic substrates, as well for the design of lead compounds in drug and inhibitor design (12Richardson P.L. Curr Pharm Des. 2002; 8: 2559-2581Crossref PubMed Scopus (33) Google Scholar, 13Sasaki T. Kikuchi T. Fukui I. Murachi T. J. Biochem. (Tokyo). 1986; 99: 173-179Crossref PubMed Scopus (33) Google Scholar). Because of the broad substrate specificity of calpains, the determination of their optimal cleavage sequence has been elusive. This is reflected in the paucity of sensitive substrates and highly specific, active site-directed The of residues that known calpain cleavage sites that the calpains can a of their active site binding P. P. A. A. A. F. P. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, K. Scholar). have been to determine the cleavage specificity of calpains P. P. A. A. A. F. P. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Kikuchi T. Murachi T. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Res. PubMed Scopus Google Scholar, 36: PubMed Scopus Google Scholar, T. K. K. J. Biochem. (Tokyo). PubMed Scopus Google Scholar, D. K. B. PubMed Scopus (11) Google Scholar, K. Akanuma H. K. Kawashima S. J. Biochem. (Tokyo). PubMed Scopus Google Scholar, S. J. C. P. K. Biol. Chem. 1986; PubMed Scopus Google Scholar). of compounds a of unprimed of substrates of proteases residue positions to the cleaved as positions and those to the cleaved as positions. these positions are and from their to the cleaved such that is the residue position to the cleaved bond. the on the where residue is known as the residues revealed some at the and positions T. Kikuchi T. Fukui I. Murachi T. J. Biochem. (Tokyo). 1986; 99: 173-179Crossref PubMed Scopus (33) Google Scholar, T. Kikuchi T. Murachi T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The high and of limited the to residues that reflected in and this a in the to a sequence motif from cleavage sites in proteins and revealed sequence other than that from substrates J. Res. PubMed Scopus Google Scholar, T. K. K. J. Biochem. (Tokyo). PubMed Scopus Google Scholar, D. K. B. PubMed Scopus (11) Google Scholar, K. Akanuma H. K. Kawashima S. J. Biochem. (Tokyo). PubMed Scopus Google Scholar, S. J. C. P. K. Biol. Chem. 1986; PubMed Scopus Google Scholar). This that such as peptide or are in cleavage site A of known calpain cleavage sites in substrates has revealed for residues at positions to P. P. A. A. A. F. P. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). because such the of the to these to small or peptidomimetic substrates and inhibitors is because the data compiled from substrates, the sequences of the cleavage sites may have a in directing the be involved in the recognition. is that substrates of calpain may have to a the peptide to to the In this optimal sequence data not be from a from a of other to the of cysteine proteases, calpains significant at primed positions P. P. A. A. A. F. P. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. Biochem. 2003; PubMed Scopus Google Scholar). designed to primed are to other substrates that unprimed positions. A of residue preference to be for primed positions. for the determination of substrate specificity for and of a large of Curr. Opin. Chem. Biol. 2003; 7: PubMed Scopus Google Scholar). are to of sequence are to cleavage to the and of the digestion for of a methodology was to for the determination of the cleavage specificity of proteases using a limited of peptide This first described by Turk et al. Nat. Biotechnol. 2001; PubMed Scopus Google a of of digestion of peptide library by sequencing to determine the of residue sequencing to a specific position in the The first primed residue specificity and this is in the design of a second, partially degenerate library, used to unprimed residue Nat. Biotechnol. 2001; PubMed Scopus Google Scholar, 2004; PubMed Scopus Google Scholar). We have and improved upon this methodology to determine the complete cleavage specificity profile as well as the optimal cleavage sequence for the core of of the calpain family a core that can be into and or and P.L. J. PubMed Scopus Google Scholar, S. C. M. R. H. K. A. H. H. Suzuki K. W. S. A. PubMed Scopus Google Scholar). core a site that upon the into an active T. P.L. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). of the proteolytic core of the μ-calpain isoform as an in a with the calpain has a specificity for cleavage of substrates and a response to active site-directed inhibitors T. D. P.L. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). this has a the in the of calcium and a for the specificity of The in this that μI-II a preference for hydrophobic amino acids at unprimed is with a novel residue specificity for primed positions is one that is not from of substrate cleavage shows a preference for or at at and amino acids at The optimal (MER) sequence from primed specificity was by to cleavage to a specific a partially degenerate peptide. The of the optimal cleavage sequence in an fluorescent substrate in a turnover of the substrate with compounds other known calpain cleavage from the of peptide. The used resonance energy substrates in the of the Discovery at The as and for as a in was from from other from The core of was from as previously described T. D. P.L. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google used in the determination of cleavage specificity to an to all amino acids to and to K. Biotechnol. 2002; Google in a fluorescent resonance energy peptide high of peptide in to a to of with of the degenerate library by the was at in using peptide library and of by μI-II at in and using of the The digestion was by the of of the library or of the the peptide library or the calcium was a specific by to the of with of in was to of and to for at at was in a using an of and an of of the at degenerate library was by μI-II as described of digestion to where digestion to the at the for the digestion for μI-II a was by the of and to of the at partially degenerate library to a was partially by μI-II as described A was with and a previously in and for peptide using as described This was a of to for Edman The the from all then by and the to any The of was then to with of in to of that with the peptide of was at the or the for The resulting from using of For the library, the resulting from a small of was by sequencing an and the from the sequencing of the was for the The selectivity of a residue at any one position is as the of the of the residue at the position to the of the residue in the of any The was determined by amino for the library or from the sequencing of an of library for the The not for because of amino and sequencing by was at the Discovery on a For a of and was with of a digestion The shown are a of with signals of of by on a The was and in or and at The of these substrate by the substrates in and using the of as an of their in and at The was by the of a calcium to a calcium of using enzyme of and or The in a to and the of for the substrates and for in was in the of to for the determined for from the shown where and are the substrate and to of the and and are the substrate and of the The a of substrate that been to an The was determined by of the of the to the of of the these a for of a substrate was The to convert in to in of cleaved substrate determined by the hydrolysis of a known of substrate to to using a of The turnover rate was by the rate of substrate hydrolysis at substrate by the enzyme at the specificity profile at primed an degenerate peptide library was and to Edman to the of residues found at primed position This was and using a with a specificity at primed positions. of the library with the residues in the first of sequencing not with the known specificity of the J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the library was by the of using with amino those of the from to form a fluorescent S. S. P. W. W. M. PubMed Scopus Google Scholar). The calcium of this by the of in the of not in that cleavage from the of μI-II and not a E. Because residues at a of one residue for degenerate the of the signal from the complete digestion of the peptide library by with the signal from digestion by μI-II an of by μI-II the first for an from the digestion to was to sequencing The sequencing a for and in the first sequencing a preference for these residues at position of a peptide The position shows a preference for the residues and cleavage in the first may partially the for and in the shows a in selectivity from the first to the This a preference for at position The sequencing a selectivity for with that the residue at may with the with The and preference for a residue for all from a of residues to those hydrophobic not specificity for positions than at the specificity profile for unprimed the consensus motif for the first primed was used to cleavage in a library to the to the unprimed residues that cleavage to this this cleavage in hydrolysis that are residues the of sequencing of these to unprimed positions in the substrate and the of the and that the are in an a or a motif to the positions was in the This motif the purpose of cleavage from to the because residues have a such that the peptide is not The original methodology for sequence specificity at unprimed positions used a library to for the of the by the of and on Nat. Biotechnol. 2001; PubMed Scopus Google Scholar, 2004; PubMed Scopus Google Scholar). Because the may be or the an on to digestion by this in the of the from the was This in signal upon sequencing of the from the not This of the methodology was modified to the by the peptide a to a peptide dendrimer K. Biotechnol. 2002; Google Scholar). In this approach, the are of than the to the for their by that the have to the the of the an was the and the peptide of the library in high to to the digestion of the library, a second, that these to degenerate The importance of an in this methodology is by the profile from a digestion the are to a processing of these to A of that cleavage is to the peptide bond. the peptide to the resulting from hydrolysis at the of the library was shown by to be to the other and and of cleavage at this to cleavage the other positions sequencing of an to of the library the residues and for second, and sequencing not the from digestion of the library an at to the of degenerate The sequencing for these a for hydrophobic residues at the unprimed positions A preference for is in the and sequencing to positions and on these is a selectivity for the residues and at and residues and at The second, and sequencing a at positions and for and as well as and The that the preference for amino acids at is not because not be to Edman as by the selectivity at was to from the cleaved to for the unprimed positions to the primed positions to A of the residues for position is in of the residue preference for position in a peptide in a of the the optimal cleavage sequence from the library-based approach and to with previously calpain substrates in of cleavage a of substrates based on J. PubMed Scopus Google Scholar) such that an to an and to a are by a sequence of these in the of by and a in of not that for of the can be sensitive substrates used at are limited in their for the determination of at high This is to the is in compounds by the of with The an in the with substrate and can therefore be as of the not into to the fluorescent substrates in our at We therefore our of the substrates to the determination of μI-II turnover rate at one enzyme one substrate using substrates at The sequence for the substrate the cleavage motif was designed to cleavage was and at because and the peptide in an was at because the is to and substrate cleavage by this optimized substrate was found to be than than an substrate the of the consensus sequences described by et al. P. P. A. A. A. F. P. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google and than than the substrate a cleavage site of a calpain substrate S. M. Biochem. 2003; PubMed Scopus Google Scholar) at a of The amino substrate was cleaved at an rate than the substrates, than at T. Kikuchi T. Murachi T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of as an substrate is partially by the of the of with the used in the substrates not substrates a in turnover rate by μI-II with a to at shows a to at The cleavage specificity of calpains to be by the sequence of other the such as the for an or possibly a specific to be in the cleavage sites 36: PubMed Scopus Google Scholar, K. Akanuma H. K. Kawashima S. J. Biochem. (Tokyo). PubMed Scopus Google Scholar). the cleavage is to an a that is of the hydrolysis of small substrates, calpains will at positions where the sequences flanking the the The broad specificity of calpains for the hydrolysis of is reflected in the of all residues to at substrate position the of at A and as well as in the of cleavage of the degenerate library by μI-II that digestion of the library by a of an with μI-II the of digestion an as though a of cleavage to the digestion The unprimed specificity from our approach well with using small inhibitors and is with the by and T. Kikuchi T. Fukui I. Murachi T. J. Biochem. (Tokyo). 1986; 99: 173-179Crossref PubMed Scopus (33) Google Scholar) that inhibitors an with a residue preference at in the of and that small substrates are cleaved with at and at T. Kikuchi T. Murachi T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). peptidomimetic calpain inhibitors that unprimed to the such as calpain inhibitors that our with large hydrophobic residues inhibitor inhibitor inhibitor or inhibitor at inhibitors and or inhibitors and at and inhibitors and at on the peptidomimetic inhibitors are therefore to optimal with to the of amino acids by the unprimed of the cleavage specificity using substrates is from the consensus motif from the of known cleavage sites of substrates P. P. A. A. A. F. P. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). the cleaved sequences of substrates there is a large of residues at and small residues at and a paucity of or residues at positions or P. P. A. A. A. F. P. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). A for the at unprimed for positions to the cleaved is that the primed sequence the selectivity at unprimed positions. a is that the sequence the cleavage site of a substrate not need to be to an optimized cleavage sequence because may or transient substrate to an to cleavage to a specific bond. This be of the sequence the cleavage site is optimized for cleavage by calpain 36: PubMed Scopus Google Scholar). A this is where a and in a is cleaved by active this or of the specific residues in the as the of and the of the cleavage be by the the the be an of the of specific substrates to cleavage by calpain by the of cleavage the or site of to this at position where and are peptide substrates and the consensus from substrate sequences P. P. A. A. A. F. P. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google that this position may an in the site of cleavage in with this of the residue at position of the hydrophobic residue at position of the cleavage site of α-spectrin and to a their to calpain 36: PubMed Scopus Google Scholar, J. A. Nat. Biol. 2004; PubMed Scopus Google Scholar). A preference for is found at positions to of have revealed that substrates to the active site in an T. Chem. Rev. 2005; PubMed Scopus Google Scholar). Because peptide we that the at these positions is to this than to sequence by the This may be of importance in calpains because of the of a active site T. D. P.L. J. Mol. Biol. 2004; PubMed Scopus Google Scholar) that substrates have to to the active The selectivity at positions to the cleaved may be partially as an from the of a proteolytic that is of than the the of the residue preference using at positions and with that of using small substrates and inhibitors using calpain T. Kikuchi T. Fukui I. Murachi T. J. Biochem. (Tokyo). 1986; 99: 173-179Crossref PubMed Scopus (33) Google Scholar, T. Kikuchi T. Murachi T. J. Biol. Chem. Full Text PDF PubMed Google Scholar) that the in the those to the active are not from those in the substrates have been to be substrates for calpains in with T. Kikuchi T. Murachi T. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. T. Kikuchi T. Murachi T. Biochem. Biophys. PubMed Scopus Google Scholar) and This is with the calpain substrate a turnover rate than the substrate with the turnover this large is to the of primed site in the designed to at the and positions T. Kikuchi T. Murachi T. J. Biol. Chem. Full Text PDF PubMed Google our substrates, to proteins and primed and unprimed positions the enzyme of the Because substrates unprimed positions by they be to be substrates with By an optimal cleavage sequence for positions our the of other P. P. A. A. A. F. P. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. Biochem. 2003; PubMed Scopus Google Scholar) that the primed of calpains are involved in peptide recognition. The importance of primed residues is by the substrates the optimal sequence and the consensus sequence from known cleavage sites substrates, and a in their turnover rate at substrate strongly the of residues in the of cleavage by The of μI-II some to the residue the unprimed of the a the and are with the specificity at positions to and A binding at the shown to the of in the T. D. P.L. J. Mol. Biol. 2004; PubMed Scopus Google the of other such as and as well as the of The found at the can residues such as and The on the primed of the are with a preference for and residues on this of the cleaved In with our residue is one of the the active site T. D. P.L. J. Mol. Biol. 2004; PubMed Scopus Google be in a position to with a at In addition, the is in to the of that with or at In the calpain family ubiquitous and that have sequence and a other some of and the T. 2001; PubMed Scopus Google Scholar). The library-based approach to cleavage specificity used a to in the specificity of the such be in the design of substrates and In we have improved upon the peptide library-based approach described by Turk and 2004; PubMed Scopus Google Scholar) for the cleavage specificity of proteases at positions by the degenerate library a peptide dendrimer to the of and This approach was used to the cleavage sequence of μ-calpain at positions flanking the bond. This sequence was in the design of a substrate that is sensitive to hydrolysis by μ-calpain than the consensus sequence from the P. P. A. A. A. F. P. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) and a sequence from an substrate This sequence from sequences in substrate cleavage sites at all residue positions for that calpain substrates in the have sequences the cleavage sites that have been designed to be as a to substrate We and L. for for and of the Discovery at for and the and the at the for for amino and

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