Dorothy E. Croall

The calpains: modular designs and functional diversity

SummaryThe calpain family is named for the calcium dependence of the papain-like, thiol protease activity of the well-studied ubiquitous vertebrate enzymes calpain-1 (μ-calpain) and calpain-2 (m-calpain). Proteins showing sequence relatedness to the catalytic core domains of these enzymes are included in this ancient and diverse eukaryotic protein family. Calpains are examples of highly modular organization, with several varieties of amino-terminal or carboxy-terminal modules flanking a conserved core. Acquisition of the penta-EF-hand module involved in calcium binding (and the formation of heterodimers for some calpains) seems to be a relatively late event in calpain evolution. Several alternative mechanisms for binding calcium and associating with membranes/phospholipids are found throughout the family. The gene family is expanded in mammals, trypanosomes and ciliates, with up to 26 members in Tetrahymena, for example; in striking contrast to this, only a single calpain gene is present in many other protozoa and in plants. The many isoforms of calpain and their multiple splice variants complicate the discussion and analysis of the family, and challenge researchers to ascertain the relationships between calpain gene sequences, protein isoforms and their distinct or overlapping functions. In mammals and plants it is clear that a calpain plays an essential role in development. There is increasing evidence that ubiquitous calpains participate in a variety of signal transduction pathways and function in important cellular processes of life and death. In contrast to relatively promiscuous degradative proteases, calpains cleave only a restricted set of protein substrates and use complex substrate-recognition mechanisms, involving primary and secondary structural features of target proteins. The detailed physiological significance of both proteolytically active calpains and those lacking key catalytic residues requires further study.

m-Calpain is required for preimplantation embryonic development in mice

Backgroundμ-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.ResultsTo 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.ConclusionWe 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.

Hepatocellular carcinoma cells resist necrosis during anoxia by preventing phospholipase-mediated calpain activation

Although hepatocellular carcinoma (HCC) cells are more resistant to anoxic injury than normal hepatocytes, the mechanisms responsible for this differential sensitivity remain obscure. Because enhanced calpain protease activity contributes to hepatocyte necrosis, we tested the hypothesis that HCC cells resist anoxia by preventing calpain activation. Cell viability in two rat HCC cell lines (N1S1 and McA‐RH7777 cells) was fourfold greater compared to rat hepatocytes after 4 h of anoxia. Although calpain activity increased twofold in rat hepatocytes during anoxia, no increase in calpain activity occurred in HCC cells. Western and Northern blot analysis revealed greater or equivalent expression of calpains and calpastatin in HCC cells compared to hepatocytes. Because increases in cytosolic free Ca++ (Cai++) and phospholipid degradation products regulate calpains in vitro, we measured Cai++ and phospholipid degradation. Ca++i did not change in any cell types during 60 min of anoxia. In contrast, phospholipid degradation was fourfold greater in hepatocytes compared to HCC cells. Melittin, a phospholipase A2 activator, increased calpain activity and cell necrosis in all cell types; melittin‐induced cell necrosis was ameliorated by a calpain protease inhibitor. In summary, these data demonstrate for the first time 1) calpain activation without a measureable increase in Ca++i, 2) phospholipase‐mediated calpain activation in hepatocytes and HCC cells, and 3) the adaptive mechanism responsible for the resistance of HCC cells to anoxia—an inhibition of phospholipid‐mediated calpain activation. Interruption of phospholipase‐mediated calpain activation may be a therapeutic strategy for preventing anoxic cell injury.

m-Calpain subunits remain associated in the presence of calcium

The 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.

Polyclonal antisera specific for the proenzyme form of each calpain

Each subunit of calpain (EC 3.4.22.17) is proteolytically modified when the enzymes are exposed to calcium. These cleavages appear to be important for regulating the proteolytic activity and calcium-sensitivity of the proteinases. We have synthesized peptides that correspond to the sites of autoproteolytic modification within the catalytic subunit of each calpain. Polyclonal antisera raised against these peptides are highly specific for the unmodified catalytic subunit of each calpain. The antiserum specific for the N-terminal epitope of milli-calpain was used to demonstrate an inverse relationship between the presence of this N-terminal peptide and casein hydrolysis. The antiserum specific for the N-terminal epitope of micro-calpain was used to demonstrate proteolytic modification of the catalytic subunit of mu-calpain in rat erythrocytes treated with ionomycin and calcium.

CLEAVAGE OF CALDESMON AND CALPONIN BY CALPAIN : SUBSTRATE RECOGNITION IS NOT DEPENDENT ON CALMODULIN BINDING DOMAINS

The calmodulin binding proteins, caldesmon and calponin, are cleaved by both major isoforms of calpain in vitro. The patterns of fragments generated by each enzyme are essentially identical for a given substrate. Qualitatively, the cleavage pattern of each substrate is unchanged by the presence or absence of calmodulin suggesting that the interaction between calmodulin and these calmodulin-binding proteins does not alter substrate recognition by calpain. However, calmodulin (at microM concentrations) does have a small, but significant, inhibitory effect directly on calpain as evidenced by slower rates of cleavage of alpha-casein, a protein that does not bind calmodulin. Inhibition is more pronounced with mu-calpain (15-25%) than with m-calpain (6-10%). In order to demonstrate, unequivocally, that substrate recognition does not require an interaction between calpain and a substrates calmodulin-binding domain, recombinant, full-length caldesmon and a mutant lacking the calmodulin binding domain were tested as substrates for calpain in the presence and absence of calmodulin. Calpain produced similar cleavage patterns of the baculovirus expressed caldesmon and the truncated mutant. Competition experiments demonstrated that calpain does not discriminate between the truncated mutant and full length caldesmon. This suggests that substrate recognition by calpain was not altered significantly by the absence of the calmodulin-binding domain. Cleavage of a second calmodulin-binding protein, calponin was also examined. The rate of calponin cleavage was increased in the presence of calmodulin, an observation that is also inconsistent with any requirement for calpain to bind to its calmodulin-binding site. These results demonstrate that calmodulin-binding domains do not provide substrate recognition sites for calpains. It seems likely that the calmodulin-like regions of calpain function to bind calcium and to regulate enzyme conformation as required for activity and that they do not interact directly with most substrates.

Detecting the active conformation of calpain with calpastatin-based reagents.

The specific, calcium-dependent, high affinity interaction between calpain and its endogenous inhibitor calpastatin was exploited to selectively detect the calcium-bound, catalytically competent, conformation of calpain in vitro. Modification of calpastatin domain-1 (Val(114)-Ser(270)) or its N-terminal fragment (Val(114)-Pro(202)), at selected unique cysteine residues with maleimide-AlexaFluor546 did not compromise calpastatin function (inhibition of calpain) or its binding with calpain. Ca(2+)-dependent binding between catalytically dead calpain-2 (Cys(105)Ala) fused with eGFP and these fluorigenic calpastatin peptides generates fluorescent resonance energy transfer (FRET). The FRET signal documents proximity of calpain-2, C-terminally linked fluorophore to specific sites within calpastatin when the proteins form a complex. These results provide important insights into the calcium-dependent interaction between calpain and calpastatin and for holo-calpain-2 in solution experimentally validate some key features of their predicted interactions. These data also provide proof of concept that the calpastatin-based reagents may be useful to selectively detect the active conformation of calpain.