Homodimerization of calpain 3 penta-EF-hand domain

Ravulapalli, R.; Díaz, Beatriz García; Campbell, Robert L.; Davies, Peter L.

doi:10.1042/bj20041821

Cited by 39 publications

(44 citation statements)

References 48 publications

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“…uncontrolled) autolysis, at least in the conditions here where the free [Ca 2ϩ ] is tightly controlled at a low level; this is consistent with a previous report describing the successful isolation and purification in its full-length form of recombinant calpain-3 expressed in baculovirus-infected insect cells (18). Although calpain-3 can form homodimers in some circumstances (37,38), there is as yet no evidence that this does occur in muscle fibers. Unless the calpain-3 is normally bound to titin in such a homodimeric form, it seems probable that the calpain-3 was present as monomers in the wash solution in the experiments here, because it had diffused out only very slowly and was diluted to a comparatively very low concentration in the wash solution (volume ϳ1000-fold greater than fiber volume).…”

Section: Discussionsupporting

confidence: 76%

Endogenous Calpain-3 Activation Is Primarily Governed by Small Increases in Resting Cytoplasmic [Ca2+] and Is Not Dependent on Stretch

Murphy

Lamb

2009

Journal of Biological Chemistry

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Proteolytically active calpain-3/p94 is clearly vital for normal muscle function, since its absence leads to limb girdle muscular dystrophy 2A, but its function and regulatory control are poorly understood. Here we use single muscle fibers, individually skinned by microdissection, to investigate the diffusibility and autolytic activation of calpain-3 in situ. Virtually all calpain-3 present in mature muscle fibers is tightly bound in the vicinity of the titin N2A line and triad junctions and remains so irrespective of fiber stretching or raised [Ca 2؉ ]. Most calpain-3 is evidently bound within the contractile filament lattice, because (i) its slow diffusional loss is slowed further by locking myosin and actin into rigor and (ii) detergent dispersion of membranes causes rapid washout of most ryanodine receptors and sarcoplasmic reticulum Ca 2؉ pumps with little accompanying washout of calpain-3. Calpain-3 autolyzes (becoming proteolytically active) in a tightly calcium-dependent manner. It remains in its nonactivated full-length form if [Ca 2؉ ] is maintained at <50 nM, the normal resting level, even with brief increases to 2-20 M during repeated tetanic contractions, but it becomes active (though still bound) if [Ca 2؉ ] is kept slightly elevated at 200 nM (ϳ20% autolysis in 1 h). Calpain-3 did not spontaneously autolyze even when free in solution with 200 nM Ca 2؉ for up to 60 min. These findings explain why calpain-3 remains quiescent with normal exercise but is activated following eccentric (stretching) contractions, when resting [Ca 2؉ ] is elevated, and how a protease such as calpain-3 can be very Ca 2؉ -sensitive yet highly specific in its actions.

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Section: Discussionsupporting

confidence: 76%

Endogenous Calpain-3 Activation Is Primarily Governed by Small Increases in Resting Cytoplasmic [Ca2+] and Is Not Dependent on Stretch

Murphy

Lamb

2009

Journal of Biological Chemistry

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“…These domains can harbor one or more binding sites, known as exosites (148), that play important roles as mediators for a great diversity of interactions, most of them involving protein binding. Such is the case of the penta-EF-hands in the members of the calpain-calpastatin system (149) or the caspase recruitment domain (CARD) domains present in caspases that are responsible for protease oligomerization and in the latter case also for recruitment to the apoptosome and activation (150). Similar roles have been also described for the interaction of the complement CUB and the epidermal growth factor modules in C1s and C1r complement proteins in the C1 complex assembly (151).…”

Section: Exosite-mediated Interactionsmentioning

confidence: 88%

Metadegradomics

Doucet

Butler

Rodrı́guez

et al. 2008

Molecular & Cellular Proteomics

138

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Post-translational modifications enable extra layers of control of the proteome, and perhaps the most important is proteolysis, a major irreversible modification affecting every protein. The intersection of the protease web with a proteome sculpts that proteome, dynamically modifying its state and function. Protease expression is distorted in cancer, so perturbing signaling pathways and the secretome of the tumor and reactive stromal cells. Indeed many cancer biomarkers are stable proteolytic fragments. It is crucial to determine which proteases contribute to the pathology versus their roles in homeostasis and in mitigating cancer. Thus the full substrate repertoire of a protease, termed the substrate degradome, must be deciphered to define protease function and to identify drug targets. Degradomics has been used to identify many substrates of matrix metalloproteinases that are important proteases in cancer. Here we review recent degradomics technologies that allow for the broadly applicable identification and quantification of proteases (the protease degradome) and their activity state, substrates, and interactors. Quantitative proteomics using stable isotope labeling, such as ICAT, isobaric tags for relative and absolute quantification (iTRAQ), and stable isotope labeling by amino acids in cell culture (SILAC), can reveal protease substrates by taking advantage of the natural compartmentalization of membrane proteins that are shed into the extracellular space. Identifying the actual cleavage sites in a complex proteome relies on positional proteomics and utilizes selection strategies to enrich for proteasegenerated neo-N termini of proteins. In so doing, important functional information is generated. Finally protease substrates and interactors can be identified by interactomics based on affinity purification of protease complexes using exosite scanning and inactive catalytic domain capture strategies followed by mass spectrometry analysis. At the global level, the N terminome analysis of whole communities of proteases in tissues and organs in vivo provides a full scale understanding of the protease web and the web-sculpted proteome, so defining metadegradomics. Molecular & Cellular Proteomics 7: 1925-1951, 2008.

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“…Evidence for this association came initially from the absence of small subunit in calpain-3 preparations (12) and from the apparent molecular weight of the enzyme (~180,000 Da) being twice that expected for the monomer (94 000 Da) when autolysis was suppressed by mutation of the catalytic Cys to Ser (13). Subsequently, we showed direct evidence of homodimerization of the calpain-3 PEF domain (14,15) and have subsequently solved the crystal structure of the Ca 2+ -bound PEF homodimer (16). Other structural differences from the large subunit of calpain-1 and -2 are the presence of two insertion sequences (IS) that are not seen in any of the other calpain isoforms.…”

mentioning

confidence: 80%

Structures of human calpain-3 protease core with and without bound inhibitor reveal mechanisms of calpain activation

Campbell

Davies

2018

Journal of Biological Chemistry

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Limb-girdle muscular dystrophy type 2a arises from mutations in the Ca 2+ -activated intracellular cysteine protease calpain-3. This calpain isoform is abundant in skeletal muscle and differs from the main isoforms, calpain-1 and -2, in being a homodimer and having two short insertion sequences. The first of these, IS1, interrupts the protease core and must be cleaved for activation and substrate binding. Here, to learn how calpain-3 can be regulated and inhibited, we determined the structures of the calpain-3 protease core with IS1 present or proteolytically excised. To prevent intramolecular IS1 autoproteolysis, we converted the active-site Cys to Ala. Small-angle X-ray scattering (SAXS) analysis of the C129A mutant suggested that IS1 is disordered and mobile enough to occupy several locations. Surprisingly, this was also true for the apo version of this mutant. We therefore concluded that IS1 might have a binding partner in the sarcomere and is unstructured in its absence. After autoproteolytic IS1 removal from the active Cys-129 calpain-3 protease core, we could solve its crystal structures with and without the cysteine protease inhibitors E-64 and leupeptin covalently bound to the active-site cysteine. In each structure, the active state of the protease core was assembled by the cooperative binding of two Ca 2+ ions to the equivalent sites used in calpain-1 and -2.

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Homodimerization of calpain 3 penta-EF-hand domain

Cited by 39 publications

References 48 publications

Endogenous Calpain-3 Activation Is Primarily Governed by Small Increases in Resting Cytoplasmic [Ca2+] and Is Not Dependent on Stretch

Endogenous Calpain-3 Activation Is Primarily Governed by Small Increases in Resting Cytoplasmic [Ca2+] and Is Not Dependent on Stretch

Metadegradomics

Structures of human calpain-3 protease core with and without bound inhibitor reveal mechanisms of calpain activation

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