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 -calpain isoform by a peptide library-based approach using the proteolytic core of -calpain (I-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 I-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 transferbased substrate with optimal cleavage motifs on the primed and non-primed sides (PLFAER). The -calpain core shows a far greater turnover rate for our substrate than for those based on the cleavage site of ␣-spectrin or the proteolytic sequence consensus compiled from substrate alignments.Calpains (clan CA, family C2 in the MEROPS data base (1)), a family of calcium-activated intracellular proteases, are found in animals, plants, and possibly bacteria (2). They convert intracellular calcium signals (3) into a proteolytic signal by catalyzing the limited cleavage of target proteins (4, 5). Among the known cellular substrates of calpain are numerous cytoskeletal proteins, as well as some receptors and integral membrane proteins like the Na ϩ /Ca 2ϩ exchanger, NCX-3 (6). 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 (7,8). 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 (9); autoproteolytic inactivation; or simply the dissipation of local high calcium levels. Unr...

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The progressive decline in kidney function and concomitant loss of renal 1alpha-hydroxylase (CYP27B1) in chronic kidney disease (CKD) are associated with a gradual loss of circulating 25-hydroxyvitamin D(3) (25(OH)D(3)) and 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)). However, only the decrease in 1alpha,25(OH)(2)D(3) can be explained by the decline of CYP27B1, suggesting that insufficiency of both metabolites may reflect their accelerated degradation by the key catabolic enzyme 24-hydroxylase (CYP24). To determine whether CYP24 is involved in causing vitamin D insufficiency and/or resistance to vitamin D therapy in CKD, we determined the regulation of CYP24 and CYP27B1 in normal rats and rats treated with adenine to induce CKD. As expected, CYP24 decreased whereas CYP27B1 increased when normal animals were rendered vitamin D deficient. Unexpectedly, renal CYP24 mRNA and protein expression were markedly elevated, irrespective of the vitamin D status of the rats. A significant decrease in serum 1alpha,25(OH)(2)D(3) levels was found in uremic rats; however, we did not find a coincident decline in CYP27B1. Analysis in human kidney biopsies confirmed the association of elevated CYP24 with kidney disease. Thus, our findings suggest that dysregulation of CYP24 may be a significant mechanism contributing to vitamin D insufficiency and resistance to vitamin D therapy in CKD.

Calpains are intracellular calcium-activated cysteine proteases whose unregulated proteolysis following the loss of calcium homeostasis can lead to acute degeneration during ischemic episodes and trauma, as well as Alzheimer's disease and cataract formation. The determination of the crystal structure of the proteolytic core of mu-calpain (muI-II) in a calcium-bound active conformation has made structure-guided design of active site inhibitors feasible. We present here high-resolution crystal structures of rat muI-II complexed with two reversible calpain-specific inhibitors employing cyclic hemiacetal (SNJ-1715) and alpha-ketoamide (SNJ-1945) chemistries that reveal new details about the interactions of inhibitors with this enzyme. The SNJ-1715 complex confirms that the free aldehyde is the reactive species of the cornea-permeable cyclic hemiacetal. The alpha-ketoamide warhead of SNJ-1945 binds with the hydroxyl group of the tetrahedral adduct pointing toward the catalytic histidine rather than the oxyanion hole. The muI-II-SNJ-1945 complex shows residue Glu261 displaced from the S1' site by the inhibitor, resulting in an extended "open" conformation of the domain II gating loop and an unobstructed S1' site. This conformation offers an additional template for structure-based drug design extending to the primed subsites. An important role for the highly conserved Glu261 is proposed.