Association of Calpastatin with Inactive Calpain

Abstract

It is generally accepted that the Ca 2؉ -dependent interaction of calpain with calpastatin is the most relevant mechanism involved in the regulation of Ca 2؉ -induced proteolysis. We now report that a calpain-calpastatin association can occur also in the absence of Ca 2؉ or at very low Ca 2؉ concentrations, reflecting the physiological conditions under which calpain retains its inactive conformational state. The calpastatin binding region is localized in the non-inhibitory L-domain containing the amino acid sequences encoded by exons 4 -7. This calpastatin region recognizes a calpain sequence located near the end of the DIIdomain. Interaction of calpain with calpastatins lacking these sequences becomes strictly Ca 2؉ -dependent because, under these conditions, the transition to an active state of the protease is an obligatory requirement. The occurrence of the molecular association between Ca 2؉ -free calpain and various recombinant calpastatin forms has been demonstrated by the following experimental results. Addition of calpastatin protected calpain from trypsin digestion. Calpain was coprecipitated when calpastatin was immunoprecipitated. The calpastatin molecular size increased following exposure to calpain. The two proteins comigrated in zymogram analysis. Furthermore, calpain-calpastatin interaction was perturbed by protein kinase C phosphorylation occurring at sites located at the exons involved in the association. At a functional level, calpain-calpastatin interaction at a physiological concentration of Ca 2؉ represents a novel mechanism for the control of the amount of the active form of the protease potentially generated in response to an intracellular Ca 2؉ influx.Several reports have indicated that calpain can acquire different conformational states depending on the presence of low or high [Ca 2ϩ ] (1-9). Binding of Ca 2ϩ to sites present in both the large and small calpain subunits induces a conformational switch, which ultimately results in the rearrangement of the two catalytic subdomains into a functionally active catalytic site (3)(4)(5)(6)(7)(8).This conformational modification is the limiting step in the calpain activation process and may be followed by an autoproteolytic degradation that removes the N-terminal region of domains I and V, stabilizing a low Ca 2ϩ -requiring form of the protease. The active enzyme form is regulated by the endogenous proteinaceous inhibitor calpastatin (10 -12), which has a peculiar molecular structure consisting of one N-terminal region (L-domain) and four repetitive inhibitory units (13-21), each containing three highly conserved regions called A, B, and C (22, 23). Sequence B contains the calpain consensus sequence; sequences A and C do not seem to be required for expression of the inhibitory activity, as indicated by experiments showing that a 27-residue peptide derived from sequence B can interact and inhibit calpain (23-25). Although not required for inhibition, sequences A and C can exert other functions such as interaction with domains IV and VI in the p...

Adaptive Modifications in the Calpain/Calpastatin System in Brain Cells after Persistent Alteration in Ca2+ Homeostasis

“…Aliquots of the crude extracts (corresponding to 50 g of protein) were diluted in 60 l of 0.1 M TrisHCl, pH 6.8, containing 20% glycerol, 10 mM 2-mercaptoethanol, and 10 mM EDTA and loaded on a 10% polyacrylamide gel containing 1 mg/ml casein. Although the step required to measure calpain activity after the digestion of casein was not performed, this protein substrate was still added to the gel, in accordance with the original procedure (11). Moreover, the presence of casein during electrophoresis did not modify the mobility of both calpain and calpastatin as well as their association capacity.…”

“…Calpain was identified with the polyclonal antibody anti--calpain antibody (Sigma). Calpastatin was identified by using mAb 35.23 (11). The immunoreactive material was identified by ECL detection system (43) and detected by the Bio-Rad Chemi Doc XRS apparatus.…”

“…Briefly, aliquots of the fresh tissues (90 -300 mg) collected from control and transgenic mice as described above were resuspended in 5 volumes (w/v) of ice-cold buffer A containing 2 mM EDTA, minced in tissue grinder, and lysed by sonication. Zymogram analysis was carried out as described (11,47,48) with the following modifications. Aliquots of the crude extracts (corresponding to 50 g of protein) were diluted in 60 l of 0.1 M TrisHCl, pH 6.8, containing 20% glycerol, 10 mM 2-mercaptoethanol, and 10 mM EDTA and loaded on a 10% polyacrylamide gel containing 1 mg/ml casein.…”

“…Thus, the increase in intracellular calpain-mediated fluorescence is not due to an increase in calpain level but is indicative of the formation of the calpaincalpastatin complex after elevation in [Ca 2ϩ ] i . The redistribution of calpastatin in the cell-soluble fraction and the formation of the enzyme-inhibitor complex is an essential step for the control of calpain translocation at its sites of activation, but at the same time it exposes calpastatin to degradation by calpain being the inhibitor protein also a substrate of the protease (7,8,11,23,24). Calpastatin diffusion and formation of the protease- inhibitor complex can, thus, be reasonably considered as an indication of a dysregulation of intracellular Ca 2ϩ homeostasis.…”

Variation in the Yak Calpastatin Gene (Cast)

“…It plays an important role in the calpain/calpastatin system [12]. Given the crucial role that exon 6 may play in the calpastatin/calpain system, it is therefore seems likely that the variation revealed here in exon 6 of yak CAST could have both structural and functional effects on the calpastatin system and potentially affect the tenderisation of yak meat.…”