The calpain small subunit regulates cell-substrate mechanical interactions during fibroblast migration.

Cell migration involves the dynamic formation and release of cell-substrate adhesions, where the exertion and detection of mechanical forces take place. Members of the calpain family of calcium-dependent proteases are believed to have a central role in these processes, possibly through the regulation of focal adhesion dynamics. The ubiquitous calpains, calpain 1 (mu-calpain) and calpain 2 (m-calpain), are heterodimers consisting of large catalytic subunits encoded by the Capn1 and Capn2 genes, respectively, and the small regulatory subunit encoded by Capn4. We have examined the role of the calpain regulatory small subunit in traction force production and mechanosensing during cell migration. Capn4-deficient or rescued cells were plated on flexible polyacrylamide substrates, for both the detection of traction forces and the application of mechanical stimuli. The total force output of Capn4-deficient cells was approximately 75% lower than that of rescued cells and the forces were more randomly distributed and less dynamic in Capn4-deficient cells than in rescued cells. Furthermore, Capn4-deficient cells were less adhesive than wild-type cells and they also failed to respond to mechanical stimulations by pushing or pulling the flexible substrate, or by engaging dorsal receptors to the extracellular matrix. Surprisingly, fibroblasts deficient in calpain 1 or calpain 2 upon siRNA-mediated knockdown of Capn1 or Capn2, respectively, did not show the same defects in force production or adhesion, although they also failed to respond to mechanical stimulation. Interestingly, stress fibers were aberrant and also contained fewer colocalised vinculin-containing adhesions in Capn4-deficient cells than Capn1- and Capn2-knockdown cells. Together, these results suggest that the calpain small subunit plays an important role in the production of mechanical forces and in mediating mechanosensing during fibroblast migration. Furthermore, the Capn4 gene product might perform functions secondary to, or independent of, its role as a regulatory subunit for calpain 1 and calpain 2.

Genetic disruption of calpain correlates with loss of membrane blebbing and differential expression of RhoGDI-1, cofilin and tropomyosin.

Dynamic regulation of the actin cytoskeleton is important for cell motility, spreading and the formation of membrane surface extensions such as lamellipodia, ruffles and blebs. The ubiquitous calpains contribute to integrin-mediated cytoskeletal remodelling during cell migration and spreading, by cleavage of focal adhesion components and signalling molecules. In the present study, the live-cell morphology of calpain-knockout and wild-type cells was examined by time-lapse fluorescence microscopy, and a role of calpain in mediating the formation of sporadic membrane blebs was established. Membrane blebbing was significantly reduced in calpain-knockout cells, and genetic rescue fully restored the wild-type phenotype in knockout cells. Proteomic comparison of wild-type and knockout cells identified decreased levels of RhoGDI-1 (Rho GDP-dissociation inhibitor) and cofilin 1, and increased levels of tropomyosin in calpain-knockout cells, suggesting a role of calpain in regulating membrane extensions involving these proteins. RhoGDI, cofilin and tropomyosin are known regulators of actin filament dynamics and membrane extensions. The reduced levels of RhoGDI-1 in calpain-knockout cells observed by proteome analysis were confirmed by immunoblotting. Genetic rescue of the calpain-knockout cells enhanced RhoGDI-1-expression 2-fold above that normally present in wild-type cells. These results suggest a regulatory connection between calpain and RhoGDI-1 in promoting formation of membrane blebs.

Conditional disruption of ubiquitous calpains in the mouse.

Ubiquitous mu- and m-calpain proteases are implicated in development and apoptosis. They are heterodimers consisting of 80-kDa catalytic subunits encoded by capn1 and capn2, respectively, and a common 28-kDa regulatory subunit encoded by capn4. The regulatory subunit is required to maintain stability and activity of mu- and m-calpains; thus, genetic disruption of capn4 was predicted to eliminate both calpain activities. Germline disruption of capn4 caused embryonic lethality, hampering the use of those mouse models to explore physiological calpain functions. Here we describe a loxP/cre conditional capn4 targeted mouse model that enables tissue-specific and temporal deletion of calpain activity. Disruption of the floxed capn4 gene using a ubiquitous cytomegalovirus promoter driven Cre recombinase transgene led to midgestation embryonic lethality. Fibroblasts from these embryos lacked detectable regulatory subunit expression, had reduced levels of the mu- and m-calpain catalytic subunits, and had no detectable mu- and m-calpain activities. These defects were corrected with a capn4-encoding lentivirus.

Ubiquitous calpains promote caspase-12 and JNK activation during endoplasmic reticulum stress-induced apoptosis.

Ubiquitously expressed mu- and m-calpain proteases are implicated in development and apoptosis. They consist of 80-kDa catalytic subunits encoded by the capn1 and capn2 genes, respectively, and a common 28-kDa regulatory subunit encoded by the capn4 gene. The regulatory subunit is required to maintain the stability and activity of mu- and m-calpains. Accordingly, genetic disruption of capn4 in the mouse eliminated both ubiquitous calpain activities. In embryonic fibroblasts derived from these mice, calpain deficiency correlated with resistance to endoplasmic reticulum (ER) stress-induced apoptosis, and this was directly related to a calpain requirement for activation of both caspase-12 and the ASK1-JNK cascade. This study provides compelling genetic evidence for calpain's role in caspase-12 activation at the ER, and reveals a novel role for the ubiquitous calpains in ER-stress induced apoptosis and JNK activation.

m-Calpain is required for preimplantation embryonic development in mice.

BACKGROUND: Mu-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 (mu-calpain) or Capn2 (m-calpain), and a common regulatory subunit encoded by Capn4. Disruption of the mouse Capn4 gene abolished both mu-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 mu-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 mu-calpain and m-calpain have distinct functions, and that m-calpain is vital for development of the preimplantation murine embryo.

Activation of calpain by Ca2+: roles of the large subunit N-terminal and domain III-IV linker peptides.

The calpains are a family of cysteine proteases with closely related amino acid sequences, but a wide range of Ca(2+) requirements (K(d)). For m-calpain, K(d) is approximately 325microM, for mu-calpain it is approximately 50microM, and for calpain 3 it is not strictly known but may be approximately 0.1microM. On the basis of previous structure determination of m-calpain we postulated that two regions of the calpain large subunits, the N-terminal peptide (residues 1-20) and a domain III-IV linker peptide (residues 514-530 in m-calpain) were important in defining K(d). The mutations Lys10Thr in the N-terminal peptide, and Glu517Pro in the domain linker peptide, reduced K(d) of m-calpain by 30% and 42%, respectively, revealing that these two regions are functionally important. The increased Ca(2+)-sensitivity of these mutants demonstrate that the Lys10-Asp148 salt link and the short beta-sheet interaction involving Glu517 are factors contributing to the high K(d) of m-calpain. Though these two regions are physically remote from the active site and Ca(2+)-binding site, they play significant roles in regulating the response of calpain to Ca(2+). Differences in these interactions in mu-calpain and in calpain 3 are also consistent with their progressively lower K(d) values.

Expression of human, mouse, and rat m-calpains in Escherichia coli and in murine fibroblasts.

The two best known calpains, micro- and m-calpain, are Ca(2+)-dependent cysteine proteases found in all mammalian tissues. They are probably involved in many Ca(2+)-linked signal pathways, although the details are not yet clear. The enzymes are heterodimers of a specific large subunit (micro-80k or m-80k) and a common small subunit (28k). Recombinant calpains have been obtained by co-expression of large and small subunits in Escherichia coli and in Sf9 cells, with variable success. Expression with the 28k subunit is very low, but is much higher with a C-terminal 21k fragment of this subunit. Rat m-calpain (m-80k/21k) is well expressed in E. coli but mouse m-calpain (m-80k/21k) is poorly expressed, even though the amino acid sequences of rat-m-80k and mouse-m-80k are 92% identical. It had also been reported that human m-calpain could be expressed in Sf9 cells but not in E. coli. To investigate these differences, hybrid rat/mouse and rat/human m-calpains were cloned and expressed in E. coli. It was shown that Ile-6 and Pro-127, which are specific to the mouse m-80k sequence, caused poor expression. High expression of human m-calpain in E. coli could be achieved by providing the correct Shine-Dalgarno ribosome binding site. The results provide a simple method to obtain approximately 10mg amounts of human m-calpain and a slightly modified mouse m-calpain. Expression of m-80k-EGFP fusions was also studied, both in E. coli and in mammalian cells, varying both the small subunit and the promoters. m-80k-EGFP alone was not active, but with 21k or 28k subunits was active in both cell types. The EGFP domain was partially cleaved during expression, releasing an active m-80k/21k calpain.

Crystal structure of a micro-like calpain reveals a partially activated conformation with low Ca2+ requirement.

The two Ca2+-dependent cysteine proteases, micro- and m-calpain, are involved in various Ca2+-linked signal pathways but differ markedly in their Ca2+ requirements for activation. We have determined the structure of a micro-like calpain, which has 85% micro-calpain sequence (the first 48 and the last 62 residues of the large subunit are those from m-calpain) and a low Ca2+ requirement. This construct was used because micro-calpain itself is too poorly expressed. The structure of micro-like calpain is very similar in overall fold to that of m-calpain as expected, but differs significantly in two aspects. In comparison with m-calpain, the catalytic triad residues in micro-like calpain, His and Cys, are much closer together in the absence of Ca2+, and significant portions of the Ca2+ binding EF-hand motifs are disordered and more flexible. These structural differences imply that Ca2+-free micro-calpain may represent a partially activated structure, requiring lower Ca2+ concentration to trigger its activation.

Calpains mediate p53 activation and neuronal death evoked by DNA damage.

DNA damage is an initiator of neuronal death implicated in neuropathological conditions such as stroke. Previous evidence has shown that apoptotic death of embryonic cortical neurons treated with the DNA damaging agent camptothecin is dependent upon the tumor suppressor p53, an upstream death mediator, and more distal death effectors such as caspases. We show here that the calcium-regulated cysteine proteases, calpains, are activated during DNA damage induced by camptothecin treatment. Moreover, calpain deficiency, calpastatin expression, or pharmacological calpain inhibitors prevent the death of embryonic cortical neurons, indicating the important role of calpain in DNA damage-induced death. Calpain inhibition also significantly reduced and delayed the induction of p53. Consistent with the actions of calpains upstream of p53 and the proximal nature of p53 death signaling, calpain inhibition inhibited cytochrome c release and DEVD-AFC cleavage activity. Taken together, our results indicate that calpains are a key mediator of p53 induction and consequent caspase-dependent neuronal death due to DNA damage.

Glutamate substitutions at a PKA consensus site are consistent with inactivation of calpain by phosphorylation.

Regulation of calpain by phosphorylation has often been suggested, but has proved difficult to detect. Calpains extracted from mammalian tissue are reported to contain 2-4 mol phosphate/mol of enzyme distributed over multiple sites, but phosphate groups are not detectable in the X-ray structures of recombinant calpain. Some serine and threonine residues in the large subunit of rat m-calpain were converted to aspartic or glutamic acid residues, at sites suggested by previous studies, to assess the probable effects of phosphate groups on the enzyme. Expression of the mutant calpains in Escherichia coli, and their heat stabilities, did not differ from those of the wild-type enzyme. m-Calpains with the mutations Ser50Asp, Ser50Glu, Ser67Glu, and Thr70Glu had the same specific activity and Ca(2+) requirement as the wild-type enzyme. In contrast, Ser369Asp-, Ser369Glu-, and Thr370Glu-m-calpain were inactive. This result is consistent with the recent report that phosphorylation at position 369 or 370 in vivo reduced m-calpain activation.

Purification, crystallization and preliminary X-ray analysis of a mu-like calpain.

The X-ray structure of m-calpain in the absence of Ca(2+) has been described, but it has not been possible to obtain sufficient mu-calpain for structure determination. Comparison of the two structures is of interest in attempting to understand their different Ca(2+) requirements. Here, the crystallization in the absence of Ca(2+) of an inactive mutant hybrid calpain (MW approximately 100 kDa), which contains 85% of the rat mu-calpain sequence and is well expressed in Escherichia coli, is described. The properties of this calpain in its active form and particularly its Ca(2+) requirement are close to those expected for wild-type mu-calpain. Clusters of plate-shaped crystals were obtained by vapour diffusion with polyethylene glycol (M(r) approximately 6000) as precipitating agent in the presence of detergent. The crystals diffract to a resolution of 2.7 A at a synchrotron source. The space group is P2(1), with unit-cell parameters a = 72.7, b = 184.6, c = 86.3 A, beta = 100.7 degrees. There are two molecules in the asymmetric unit, corresponding to a solvent content of 57.1%.

Calpain is required for MMP-2 and u-PA expression in SV40 large T-antigen-immortalized cells.

The absence of both mu- and m-calpain activity, caused by disruption of the capn4 gene in mice, retarded migration, and disrupted the cytoskeleton, both in primary capn4(-/-) embryonic fibroblasts (mEF) and in capn4(-/-) mEF immortalized with SV40 large T-antigen (TAg). These results are thought to reflect the role of calpain in integrin signaling to the cytoskeleton. The integrins are also involved, together with matrix metalloproteinases (MMP) and plasminogen activators (PA), in cellular invasion. This study therefore aimed to establish whether links exist between the calpain, MMP, and PA systems, using both primary and TAg-immortalized capn4(+/+) and capn4(-/-) embryonic fibroblasts. Both Matrigel invasion, and expression of MMP-2 and u-PA activities, correlated with calpain expression in TAg-containing cells, but not in primary cells. MMP-2 mRNA synthesis also correlated with calpain expression in the presence of TAg, but u-PA mRNA synthesis was not so correlated. The results suggest that calpain acquires new regulatory roles in the presence of TAg. Calpain is also required for v-Src-mediated transformation. It appears that calpain may have previously unsuspected roles in oncogenic transformation.

Origins of the difference in Ca2+ requirement for activation of mu- and m-calpain.

The mu- and m-calpains are closely related Ca(2+)-dependent cysteine proteases having different in vitro Ca(2+) requirements ( K (d)), of approx. 25 and 325 microM respectively. The two isoforms are heterodimers of slightly different large (80 kDa) subunits and an identical small (28 kDa) subunit, so that the difference in K (d) values must reside in the large subunits. As assayed here, these K (d) values relate to the Ca(2+) required for the first phase of calpain activation and do not reflect the lower Ca(2+) then required by fully activated calpain. On the basis of sequence comparison and the X-ray structure of m-calpain, many m-type residues in the C-terminal EF-hand-containing domain IV were converted into the corresponding mu-type residues, but these mutations did not produce the expected decrease in K (d). In a series of hybrid (mu/m) large-subunit calpains, the K (d) values decreased progressively towards that of mu-calpain as the proportion of mu-type sequence increased from 0 to 90%. K (d) values cannot therefore be ascribed to one or a few specific intramolecular interactions, but reflect the global response of the whole molecule to Ca(2+) binding. Nonetheless, 25% of the difference in K (d) values between mu- and m-calpain can be ascribed to the N-terminal peptide of the large subunit, whereas the C-terminal EF-hand-containing domain IV accounts for 65% of the difference.

A Ca(2+) switch aligns the active site of calpain.

Ca(2+) signaling by calpains leads to controlled proteolysis during processes ranging from cytoskeleton remodeling in mammals to sex determination in nematodes. Deregulated Ca(2+) levels result in aberrant proteolysis by calpains, which contributes to tissue damage in heart and brain ischemias as well as neurodegeneration in Alzheimer's disease. Here we show that activation of the protease core of mu calpain requires cooperative binding of two Ca(2+) atoms at two non-EF-hand sites revealed in the 2.1 A crystal structure. Conservation of the Ca(2+) binding residues defines an ancestral general mechanism of activation for most calpain isoforms, including some that lack EF-hand domains. The protease region is not affected by the endogenous inhibitor, calpastatin, and may contribute to calpain-mediated pathologies when the core is released by autoproteolysis.