Mechanisms and Integration of Signal Pathway: A Role for Calpains?

In order to survive cells must sense and respond to changes in their environment. Environmental cues trigger a variety of events within cells. The concentration and movements of calcium ions are essential regulators of many of these cellular responses. Proper control of intracellular calcium is essential because at thigh levels calcium can lead to cell damage or death. Calcium accomplishes it effects through binding to specific proteins such as calmodulin and calpain. Calmodulin, named for its ability to bind calcium and to modulate the activity of other cellular components, is an important mediator of calcium signals and its mechanism of action is relatively well understood. The calpains are proteolytic enzymes that are regulated by calcium binding. Proteolytic enzymes modify cellular proteins such that the target protein is destroyed or is altered in its function. Calpains can modify those proteins, called pumps or channels, that directly allow calcium ion movement and therefore calpains may be involved directly in helping cells to regulate calcium signals. Through its action on other key regulator proteins and enzymes, calpains might also help to integrate cellular responses that involve calcium with other events that do not. Although the activity of calpain is controlled by calcium its action may be prevented by another protein (calpastatin) that specifically inhibits these enzymes. Although calpastatin does not bind calcium directly it binds to calpain only when calpain has bound calcium. Thus the interactions between these two proteins is important in regulating calpain function. By comparison with calmodulin, there is little known about how calcium regulates calpain structure, function or its interactions with other proteins. Thus the immediate goals for this proposal are to understand more about the biochemical mechanisms that regulate calpain. This project aims to answer two major questions: 1) how does calcium binding to calpain regulate the enzymatic activity? 2) can a calpastatin insensitive calpain be generated by exploiting what is known about the interaction between calpain and calpastatin? An enzyme with this property will be useful, in future studies, for determining the role of calpains in cells. These goals will be accomplished through techniques of protein chemistry and expression of recombinant DNA molecules to generate modified enzymes or enzyme fragments for study. This knowledge will provide information on the role of calpain in critical cellular processes. %%% Calcium is important in the regulation of many essential intracellular processes. Many of the effecPts of calcium occur after it binds to specific proteins. One of these proteins is calpain. Calpains are proteolytic enzymes that are regulated by calcium binding, and in turn modify other cellular proteins in ways which often alter their function. Little is known of the mechanism by which calcium modifies calpain, and this project will study the biochemical mechanisms that regulate calpain and the role of calcium in these processes

The calpains: modular designs and functional diversity

The eukaryotic calpains are a family of calcium-dependent papain-like proteases and their non-enzymatic relatives whose varied physiological functions are beginning to be fully explored

MUTATION OF AN IQ-MOTIF: EVIDENCE FOR AN EXOSITE IN CALPAIN-2?

These studies aimed to test the hypothesis that calcium regulates calpain activity by a mechanism analogous to that used by Ca2+- calmodulin (CaM) to regulate its target enzymes. Site directed mutagenesis of a putative IQ motif, IQ413xxxR417 generated catalytic subunits modified at both, or each, of the key residues of the motif to yield ISxxxQ, ISxxxR, and IQxxxQ. Each of the mutant catalytic subunits was co-expressed with a truncated (21k) small subunit in E. coli. The heterodimeric enzymes were purified by standard methods suggesting each was properly folded. Electrophoresis under native conditions, in the absence of calcium, showed no differences between wt and enzyme mutated at R417Q. Each of these enzymes was also stable to incubation at 45 ºC. In contrast enzymes containing Q413S were resolved into multiple bands on native gels and were not stable at 45 ºC. Two calcium dependent activities were assessed: casein hydrolysis and autoproteolysis. Each of the mutants retained significant autolytic activity but lost much of their caseinolytic activity. We propose two interpretations of the results based in part on the calcium-free structures determined for calpain-2 (Hosfield et al , 1999 EMBO J. 18, 6880 and Strobl et al 2000, PNAS 97, 588). The ability of the enzymes to autoproteolyze suggests that the conformational change required for alignment of the catalytic residues is unimpaired in the mutants. Impaired casein hydrolysis may result from 1) inability to release product after cleavage or 2) failure to form a required substrate binding exosite. The latter concept is consistent with many previously known calpain attributes. Exosites provide a mechanism for achieving high substrate specificity amongst enzymes that share conserved active sites, such as those of the thrombin family and perhaps the calpain family. Confirmation and definition of such exosites will be important as they could provide targets for design of isoform selective inhibitors

m-Calpain is required for preimplantation embryonic development in mice

BACKGROUND: μ-calpain and m-calpain are ubiquitously expressed proteases implicated in cellular migration, cell cycle progression, degenerative processes and cell death. These heterodimeric enzymes are composed of distinct catalytic subunits, encoded by Capn1 (μ-calpain) or Capn2 (m-calpain), and a common regulatory subunit encoded by Capn4. Disruption of the mouse Capn4 gene abolished both μ-calpain and m-calpain activity, and resulted in embryonic lethality, thereby suggesting essential roles for one or both of these enzymes during mammalian embryogenesis. Disruption of the Capn1 gene produced viable, fertile mice implying that either m-calpain could compensate for the loss of μ-calpain, or that the loss of m-calpain was responsible for death of Capn4(-/- )mice. RESULTS: To distinguish between the alternatives described above, we deleted an essential coding region in the mouse Capn2 gene in embryonic stems cells and transmitted this mutant allele through the mouse germline. Breeding of heterozygous animals failed to produce homozygous mutant live offspring or implanted embryos. A nested PCR genotyping protocol was established, and homozygous preimplantation mutant embryos were detected at the morula but not at the blastocyts stage. CONCLUSION: We conclude that homozygous disruption of the Capn2 gene results in pre-implantation embryonic lethality between the morula and blastocyst stage. This establishes that μ-calpain and m-calpain have distinct functions, and that m-calpain is vital for development of the preimplantation murine embryo

A biochemical approach to define the interactome for calpain2 in endothelial cells

Current repositories for protein-protein interactions and high throughput screening methods focus on individual gene products and do not consider the significance of calcium induced conformational changes. These limitations suggest the need for alternative strategies to better define the calpain2 interactome. Affinity capture coupled with LC-MS/MS and proteomic analysis of the recovered proteins provides a powerful approach to identify protein-protein interactions for the heterodimeric calpain2. CAPN2 (rat) was modified to be catalytically incompetent (C105A) and fused with a C-terminal 15 residue peptide optimized for biotinylation by the biotin protein ligase, BirA. The resulting CAPN2*, heterodimerized with truncated CAPNS1, was purified from E. coli, and biotinylated in vitro. Biotinylated calpain2* served as ‘bait’ for streptavidin affinity capture of calpain2 and its interacting proteins from lysates of bovine aortic (BAEC) and human umbilical vein (HUVEC) endothelial cells (ECs). Protein-calpain2 complexes were formed in the presence of calcium to allow EGTA elution of interacting proteins and LC-MS/MS analysis in the absence of an abundance of bait peptides. Capture of the well characterized calpain inhibitor protein calpastatin (CAST), and a known substrate, vimentin provide proof of concept and validates the conformational integrity of the bait calpain2*. Significant overlap between datasets (two from BAEC and one HUVEC) is also encouraging. Of numerous other proteins including several annexins, ANXA1 was confirmed as a substrate for calpain2. Findings are expected to contribute to continuing efforts in the field to better characterize calpain2’s selection of substrates and may reveal other important clues to calpain’s localization and regulation

m-Calpain subunits remain associated in the presence of calcium

AbstractThe hypothesis that calpain subunits dissociate in the presence of Ca2+ has been tested by methods which avoid interference by Ca2+-induced aggregation and large subunit autolysis. Inactive Cys105Ser-m-calpain, bound either to Ni-NTA-agarose or to immobilized casein, after incubation with Ca2+, could be recovered in high yield as a heterodimer. Natural bovine m-calpain, after irreversible inhibition with Z-LLY-CHN2, also bound to immobilized casein and was eluted as a heterodimer. The Ca2+ requirements of calpain containing a small subunit with EF-hand mutations were higher, both before and after autolysis, than those of wild-type calpain. In mixtures of wild-type and mutant enzymes, subunit exchange did not occur in the presence of Ca2+. The results demonstrate that the subunits in both natural and recombinant m-calpain, in the given experimental conditions, remain associated in the presence of Ca2+ both before and after autolysis

Crystallization and X-ray crystallographic analysis of m-calpain, a Ca²⁺-dependent protease

The absolute requirement of Ca2+ for proteolytic activity is a feature unique to the calpains, a family of heterodimeric cysteine proteases. Conditions are described which give rise to diffraction-quality crystals of m-calpain in two crystal forms, P1 and P21. Data have been collected from native crystals of m-calpain in both P1 and P21 forms, to 2.6 and 2.15 Å, respectively. Selenomethionine-containing crystals have been grown in both forms, and anomalous data from the P21 selenomethionine enzyme provided the location of 17 of the 19 Se atoms in the protein.</jats:p