Biochimie 122 (2016) 169e187

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Biochimie

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Review An eccentric , CAPN3/p94/calpain-3

Yasuko Ono a, 1, Koichi Ojima a, b, 1, Fumiko Shinkai-Ouchi a, 1, Shoji Hata a, * Hiroyuki Sorimachi a, a Calpain Project, Department of Advanced Science for Biomolecules, Tokyo Metropolitan Institute of Medical Science (IGAKUKEN), 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan b Animal Products Research Division, NARO Institute of Livestock and Grassland Science, Ikenodai 2, Tsukuba, Ibaraki 305-0901, Japan article info abstract

þ Article history: are Ca2 -regulated proteolytic enzymes that are involved in a variety of biological phenomena. Received 9 July 2015 Calpains process substrates by limited proteolysis to modulate various functions in the cell, and Accepted 7 September 2015 are thus called “modulator .” CAPN3, previously called p94 or calpain-3, has unique features Available online 10 September 2015 that are not found in any of the other 14 human calpains, or even in other proteases. For instance, CAPN3 undergoes extremely rapid and exhaustive autodegradation. CAPN3 is also the þ Keywords: first (and so far, the only) intracellular enzyme found to depend on Na for its activation. CAPN3 has both Calpain proteolytic and non-proteolytic functions. It has the interesting distinction of being the only , Autolysis Muscular dystrophy other than a few virus proteases, with the ability to regain protease function after its autolytic dissoci- Molecular evolution ation; this occurs through a process known as intermolecular complementation (iMOC). mutations Structureeactivity relationship causing CAPN3 defects are responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). Substrate Unusual characteristics of CAPN3 have fascinated researchers, but have also hampered conventional biochemical analysis. In this review, we describe significant findings about CAPN3 from its discovery to the present, and suggest promising avenues for future CAPN3 research. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. Introduction ...... 170 2. The discovery of CAPN3 ...... 170 2.1. Discovery of the calpain family ...... 170 2.2. Discovery of a third, organ-specific calpain ...... 171 2.2.1. Early nomenclature ...... 171 2.2.2. CAPN3 definition and orthologs ...... 172

þ Abbreviations: AX, alternative exon; CaM, ; CANP, Ca2 -activated neutral protease (calpain); CAPN, calpain; CAPNS1, calpain small subunit 1; CBSW, calpain-type b-sandwich domain; CTBP1, C-terminal binding protein 1; CysPc, calpain-type core domain; DEF, digestive organ expansion factor; EM, eosinophilic myositis; EoE, eosinophilic esophagitis; iM- and eM-activation, intra- and intermolecular activation; iMOC, intermolecular complementation; iTRAQ, isobaric tag for relative and absolute quantification; IS1 and IS2, insertion sequence 1 and 2; LGMD2A, limb-girdle muscular dystrophy type 2A; mdm, muscular dystrophy with myositis; NS, N-terminal addition sequence; PC1 and PC2, protease core domains 1 and 2; PEF, penta-EF-hand domain; PLEIAD, platform element for inhibition of autolytic degradation of CAPN3; TMD, tibial muscular dystrophy; TTN, connectin/. * Corresponding author. E-mail address: [email protected] (H. Sorimachi). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.biochi.2015.09.010 0300-9084/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 170 Y. Ono et al. / Biochimie 122 (2016) 169e187

2.2.3. Found first in skeletal muscle, and later throughout the body ...... 172 3. Defensive role of CAPN3 against disease ...... 172 3.1. The calpainopathy LGMD2A ...... 173 3.1.1. La Reunion paradox ...... 173 3.1.2. Mouse models ...... 173 3.1.3. Genotype-phenotype correlations ...... 173 3.2. Eosinophilic myositis (EM) ...... 173 3.3. Tumors ...... 173 3.4. Diabetes ...... 174 4. Structure and activity ...... 174 4.1. Structures ...... 174 4.1.1. Tertiary structure ...... 174 4.1.2. Possible homodimerization ...... 174 4.1.3. A variety of splice variants ...... 174 4.2. Activation ...... 174 þ 4.2.1. Rapid, exhaustive, and apparently Ca2 -independent autolytic activity ...... 174 þ 4.2.2. Na -dependency ...... 174 4.2.3. Autolysis and activation ...... 175 4.2.4. Mutations in calpainopathy and CAPN3 activity ...... 176 4.3. Molecular evolution ...... 177 5. Unique features ...... 177 5.1. Re-activation after inactivation ...... 177 5.2. Phosphorylation ...... 178 5.3. Non-proteolytic function ...... 178 5.4. Dynamic translocations in skeletal muscle cells ...... 179 6. Molecules that interact with CAPN3 ...... 180 6.1. Molecules responsible for muscular dystrophies ...... 180 6.1.1. TTN...... 180 6.1.2. Dysferlin and LGMD2B ...... 180 6.1.3. RBBOX1, a Capn3 splicing regulator ...... 181 6.2. Suppressor and activator ...... 181 6.2.1. Pursuit of a CAPN3 suppressor in the cytosol ...... 181 6.2.2. CaM as an activator of CAPN3 ...... 181 6.3. Possible involvement of CAPN3 in cell-cycle regulation ...... 181 6.4. Substrates ...... 181 7. Perspectives and conclusions ...... 182 Acknowledgments ...... 182 References ...... 182

1. Introduction protease; Clan CA, family C02; EC3.4.22.17) was described for the first time by Gordon Guroff [35]. The primary structure of the cal- Proteases are enzymes that hydrolyze peptide bonds, which are pain catalytic subunit was determined in 1984 [37], revealing a otherwise highly stable under physiological conditions. Proteases multi-domain structure consisting of a cysteine protease domain, are themselves , and proteolyze themselves to various ex- now called a Calpain-type cysteine Protease conserved (CysPc) tents; this process is called autolysis. The calpain CAPN3 (previ- domain [38e40],aCalpain-type Beta-SandWich (CBSW) domain, ously called p94, nCL-1, nCANP, and calpain-3) has such strong previously called a C2-domain-like (C2L) domain, and a Penta E-F autolytic activity that it degrades itself within minutes, leaving hand (PEF) [41] domain (Fig. 1). The CysPc domain is composed of researchers wondering whether such a protease can have any two Protease Core subdomains (PC1 and PC2). Until 1989, most physiological function, and how to go about studying such an calpain studies focused on calpain-1 (previously called m-calpain, ephemeral molecule. mCANP, or calpain-I) and calpain-2 (m-calpain, mCANP, or calpain- Despite its rapid autolytic process, CAPN3 has many important II). Thus, these calpains are called “conventional” calpains. The functions, and its idiosyncrasies have drawn a growing number of terms calpain-1 and calpain-2 are now reserved for the active en- enthusiastic researchers. We here review the history of CAPN3 zymes, which are heterodimers consisting of a larger ca. 80-kDa research, and introduce unusual characteristics of CAPN3 from the catalytic subunit, either CAPN1 (previously called mCL or m80K) or perspective of structureefunction relationships. (Although CAPN3 CAPN2 (mCL or m80K), and a smaller ca. 28-kDa regulatory subunit, is involved in postmortem meat quality and tenderization in some CAPNS1 (30K) (Fig. 1). animals [18e31], this review focuses on physiological and patho- The name “calpain,” first used by Takashi Murachi in 1981 [42], physiological functions of CAPN3.) was proposed to be the official name of this protease family in 1990 by Koichi Suzuki [43]. Mammals have more than a dozen calpain (Table 1), half of which are predominantly expressed in 2. The discovery of CAPN3 specific tissues or organs. Calpains with the same domain structure as CAPN1 and CAPN2 are called “classical” calpains, and the rest are 2.1. Discovery of the calpain family “non-classical” (Fig. 1). Genetics studies have revealed that indi- vidual calpains have important and widely divergent physiological In January of 1964, calpain (calcium-dependent -like Y. Ono et al. / Biochimie 122 (2016) 169e187 171

Fig. 1. Schematic showing the structure of the human calpains. The calpains shown in red are predominantly expressed in specific tissues or organs; those in black are more widely expressed. GR, Gly-rich domain; C2, protein kinase C conserved domain 2; MIT, microtubule interacting and transport motif; Zn, zinc-finger motif; SOL, small optic lobes product homology domain; IQ, calmodulin (CaM)-interacting motif.

Table 1 Known calpainopathies and the responsible calpains. CAPNx and Capnx indicate human and mouse genes, respectively.

Gene Expression preference Deficiency phenotype References

Capn1 Most cells Platelet dysfunction [1] Capn2 Most cells Embryonic lethal [2,3] CAPN3, Capn3 Skeletal muscle Muscular dystrophy etc. [4e7] CAPN5, Capn5 Most cells Vitreoretinopathy [8,9] Capn6 Embryonic muscle Hypergenesis [10] Capn7 Most cells U.I.a [12] Capn8 Gastrointestinal tract Gastric ulcer [13] Capn9 Gastrointestinal tract Gastric ulcer [13] CAPN10 Most cells Type 2 diabetes [14] Capn11 Testis U.I. [15] Capn12 Hair follicle U.I. [17] Capn13 Most cells N.I. [32] CAPN14 Esophagus Eosinophilic Esophagitis [33] CAPN15/SOLH Most cells N.I. [34] Capn16/Adgb/C6orf103 Testis U.I. [36]

a U.I., under investigation; N.I., no information so far.

functions (Table 1). Although all conditions caused by calpain de- 2.2. Discovery of a third, organ-specific calpain fects called calpainopathies, this review focuses on calpainopathies caused specifically by defects in CAPN3 function, among which 2.2.1. Early nomenclature Limb-Girdle Muscular Dystrophy (LGMD) type 2A (LGMD2A)was Rat cDNA encoding a third calpain, distinct from both CAPN1 the first human disease found to be caused by a calpain deficiency. and CAPN2, was found in 1989. The encoded protein was named

Fig. 2. CAPN3 missense mutations in LGMD2A patients. So far, 229 distinct missense mutations at 189 aars of CAPN3 have been reported in the Leiden Open Variation Database. The secondary structures shown are for inactive calpain-2 (1KFU). 172 Y. Ono et al. / Biochimie 122 (2016) 169e187

Fig. 3. Representative CAPN3 isoforms generated by alternative splicing of CAPN3/Capn3. See Fig. 1 and Table 1 for abbreviations and detailed structures.

“p94” [44]. At the time, we had no information about the subunits vertebrate organisms, some of these calpains may play roles that of a hypothetical active enzyme containing p94 as a catalytic sub- are similar to that of CAPN3. unit, and we wanted to reserve the name “calpain-3” until the nature of the enzyme was elucidated. In retrospect, this may not 2.2.3. Found first in skeletal muscle, and later throughout the body have been the best decision, since the enzyme still has not been CAPN3 was first identified as a calpain present in mammalian completely characterized, even to the present day, due to its skeletal muscle [44]. At the time, Northern blot analysis was not extraordinary instability. The name CAPN3, rather than p94, was sensitive enough to detect CAPN3 mRNA in other tissues we recommended for this molecule at the 2013 FASEB meeting [38,40], examined. However, improvements in sensitivity and detection and our review follows the FASEB recommendation. Even apart revealed that CAPN3 mRNA is present in most tissues. In mammals, from questions of nomenclature, CAPN3 has posed thorny and CAPN3 is expressed far more strongly in skeletal muscles, especially perplexing research issues since its discovery. in the fast (type II) fibers [54], than in other tissues. In the heart, CAPN3 mRNA has been detected but not its protein [55]. During 2.2.2. CAPN3 definition and orthologs human development, CAPN3 appears relatively late compared to After the discovery of CAPN3, related molecules were cloned in other muscle components, such as connectin/titin (TTN) and sar- rapid succession. In 1990, the human gene CAPN3 was determined coglycans (SGCs) [56], and the same is true in mice [57,58]. to be in 15 [45]. Human CAPN3 was cloned in 1995 [4], In chickens, CAPN3 is abundantly expressed in the skeletal followed by the cloning of parts of the rat and mouse Capn3 genes muscles, and considerable amounts are also expressed in the brain, in 1996 [46], of chicken cDNA in 1996 [47], and of bovine and ovine liver, and heart [47,59]. The CAPN3 level decreases seven days after cDNA in 1999 [48]. Since then, nucleotide sequences have been hatching, while the CAPN1 and CAPN2 levels remain constant, collecting in gene databases at an astonishing pace, enabling the suggesting that the function of CAPN3 in chickens differs slightly identification of CAPN3 orthologs in a variety of vertebrates and from that in mammals [60]. The CAPN3 expression patterns also vary making it possible to consider CAPN3 evolution [49,50] (see Figs. 2 somewhat in lower vertebrates [49], indicating that physiological and 4). A clear ortholog, however, has not been found in non- role of CAPN3 may have changed during evolution. vertebrate organisms [39,40,49e51]. Although the calpain Ha- In myocytes, CAPN3 exists everywheredin the cytosol; at the Z- CalpM is found in lobsters, primarily in striated muscle [52,53], line, N2A, and M-line regions; around the sarcoplasmic reticulum this molecule lacks the PEF domain and is thus not an exact CAPN3 (SR); and so on [7,61e66]. CAPN3 carries at least one potential ortholog. nuclear-translocation signal sequence in IS2 [50], although the CAPN3 has three regions that are not found in CAPN1 or CAPN2. nuclear localization of CAPN3 in skeletal muscles has been rarely These regions are at its N-terminus (NS), within the PC2 domain observed in vivo (cf. melanoma cells, Section 3.3). This important (IS1), and between the CBSW and PEF domains (IS2) (Fig. 1). All of issue should be examined further. unique characteristics of CAPN3 originate from IS1 and/or IS2, and these regions define a molecule as CAPN3. The sequence conser- 3. Defensive role of CAPN3 against disease vation of IS1/2, however, is very low among vertebrate CAPN3s (see Fig. 5), rendering a CAPN3 definition by IS1/2 sequences impossible. Calpains are sometimes categorized as disease-causing en- At present, it is reasonable to define a calpain as CAPN3 if it has zymes, and it is true that calpain activity often exacerbates path- more significant similarity to human CAPN3 than to human CAPN1 ological states. However, CAPN3 generally defends the body against or CAPN2. Although this definition excludes all calpains in non- disease, and defects in CAPN3 lead to LGMD2A (for detailed clinical Y. Ono et al. / Biochimie 122 (2016) 169e187 173 symptoms and examples, see Refs. [67e73]) and other diseases. these symptoms [77]. It should also be noted that LGMD2A is completely recessive: heterozygous humans with an entire CAPN3 3.1. The calpainopathy LGMD2A deletion have no symptoms, indicating that half-normal levels of CAPN3 are sufficient to maintain skeletal muscle [85]. 3.1.1. La Reunion paradox In 1995, six years after the discovery of CAPN3, CAPN3 was 3.2. Eosinophilic myositis (EM) identified as the gene responsible for LGMD2A [4,74e76]. This landmark report identified six different CAPN3 mutations in fam- In 2006, CAPN3 mutations were identified in six unrelated EM ilies on La Reunion Island [4]. This was dubbed the “La Reunion patients [90,91], and other cases have since been identified paradox,” because for a rare disease such as LGMD2A, the existence [92e94]. These CAPN3 mutations, which include R49C, R101K, of plural distinct mutations in one gene in a small geographic re- P102S, T184M, G222R, R490W, R769P, W360*, and truncations in gion was considered contradictory [77]. Similar cases have since the PEF domain and in intron 5, have not been linked to specificEM been reported [78e80], and this apparent paradox was later characteristics [91]. Since CAPN3 is also expressed in immune cells, consolidated as a compound heterozygous probability in a the loss of the CAPN3 protein and its function may cause immu- consanguineous population, which is “not so unexpected” [80]. nological defects. CAPN14, which is predominantly expressed in Every pair of individuals and every community has an esophagus mucosa, was recently found to be responsible for “inbreeding coefficient (f)” between 0 and 1, representing the de- eosinophilic esophagitis [33,95] (Table 1). The CAPN14 expression gree of consanguinity (e.g., f ¼ 1 between completely inbred mice, in esophageal epithelial cells is normally upregulated by 0.5 between two clones of an individual, and 0.0625 between two interleukin-13 secreted from immune cells. The induced CAPN14 is cousins). In actuality, the highest f observed in natural populations believed to suppress the immune response, although the mecha- is 0.149, and the f in the La Reunion Island population is presumed nisms are unknown. A decrease in CAP14 induction due to nucle- to be much less than this value. The proportion (P) of affected, otide polymorphisms would create a risk for esophagitis. Since compound-heterozygous individuals to homozygous individuals in CAPN3 and CAPN14 belong to the classical calpains, whose sub- a population with f is given by the following formula [80]: strate specificities are considered similar, CAPN3 may play similar P ¼ (n 1)q(1 f)/[nq(1 f)þf], where n and q are the number and roles in skeletal muscle. However, if CAPN3 mutations perturb the frequency of disease alleles, respectively. Assuming f ¼ 0.07, immune responses, LGMD2A patients should have some immu- n ¼ 6, and q ¼ 0.0012 for La Reunion Island (q is calculated from n nological defects, and this is not the case. Mouse models of CAPN3 and the disease relevance of 4.8 10 5), P is calculated to be 7%, calpainopathy should be re-examined for indications of EM. which is not statistically significant. The values of f and n are probably less and more, respectively, than those presumed above, 3.3. Tumors making P higher (e.g.,iff ¼ 0.04 and n ¼ 12, then q ¼ 0.00058 and P ¼ 13%). In 2004, CAPN3 was found to be highly expressed in melanoma However, this phenomenon does not explain why there are so cell lines [96] and to be downregulated by the anti-melanoma ef- many CAPN3 alleles; the Leiden Open Variation Database, at www. fects of interferon (IFN)-g [97] or -b with mezerein (an antileukemic dmd.nl, lists 493 so far. CAPN3 might be in a mutation-prone locus, reagent) [98]. However, overexpressing CAPN3 does not promote the alleles might have various merits for survival when heterozy- growth in these melanoma cell lines, suggesting that CAPN3 is not gous, and/or some homozygotes may produce relatively benign or causative for melanoma [98]. In fact, CAPN3 is downregulated in late-onset symptoms. However, there is little evidence for any of the metastatic melanoma cell line M14, and the inhibition of con- these possibilities, and the reasons have yet to be clarified. ventional calpains significantly stimulates extra-cellular matrix invasion [99]. It should be noted that these studies examined 3.1.2. Mouse models CAPN3 mRNA only, not the protein. There are several mouse models for CAPN3 calpainopathy. These Two CAPN3 products, hMp84 and hMp78 (see Fig. 3), are include CAPN3 knock-out (CAPN3-KO) mice [5,6,81]. There are also expressed in human melanoma cells through the alternative initi- transgenic (Tg) mice expressing inactive CAPN3:C129S ation exon 1C [100] with and without exon 6, respectively [101]. (CAPN3:C129S-Tg, in which C129, one of the active-site triad aars, is These CAPN3 isoforms were upregulated in M665/2/21 melanoma replaced) [82] or a splicing variant (CAPN3:Dex6-Tg and CAPN3:- cells when treated with apoptosis-inducing cisplatin [101]. Both Dex15-Tg, in which exons 6 and 15 encoding IS1 and a part of IS2, hMp84 and p78 are unstable in melanoma cells, since the full- respectively, are deleted) [83]. CAPN3:C129S knock-in length protein with a presumed autolytic fragment of ca. 62 kDa (CAPN3:C129S-KI) mice express CAPN3:C129S instead of wild-type was only faintly detected [101]. This 62-kDa fragment was detected (WT) CAPN3 [7].Thefirst KO mice showed transmission-ratio in both nuclei and the cytosol. Intriguingly, these variants were distortion, which turned out to be an artifact of the introduced downregulated in malignant melanocytic lesions compared to Pgk1 promoter [81]. All of these mouse models exhibit a myopathy benign nevi in vivo [101]. Conversely, overexpressing hMp84, but phenotype with slight differences in severity, from the most benign not its inactive mutant hMp84:C42S, in A375 and HT-144 mela- to the most severe as follows: CAPN3:C129S-Tg < CAPN3:C129S- noma cells stabilized p53 and upregulated reactive oxygen species KI < CAPN3-KO < CAPN3:Dex15-Tg < CAPN3:Dex6-Tg. These results production, leading to cell death [102]. suggest that CAPN3 and its splicing variants have complex functions In urothelial tumors, CAPN3 was found in a complex with the in muscle. E2F3 transcription factor, which plays a crucial role in carcino- genesis in the human bladder [103]. Accordingly, although CAPN3 3.1.3. Genotype-phenotype correlations is not expressed in normal bovine bladders, CAPN3 has been found Many LGMD2A pathogenic mutations have been reported along in pathological bovine bladders, where its proteolytic activity was with their phenotypes, including 229 missense mutations (Fig. 2) several times higher than that of calpain-2 [103]. [84e87]. However, it is difficult to establish clear genotypeephe- Altogether, CAPN3 plays essential roles in tumor-cell develop- notype correlations because of the high variability of symptoms ment and metastasis. Further studies, however, are required to and severity (see also 4.2.4), even among patients with the same elucidate CAPN3 functions in these events and to determine CAPN3 genotype [84,87e89]. CAPN3 is clearly not the sole factor in whether CAPN3 promotes or suppresses tumor growth, whether 174 Y. Ono et al. / Biochimie 122 (2016) 169e187

CAPN3 involvement depends on the cell type, and whether other lacking exons 6, 15, and/or 16) [58]. Since the IS1/2 regions are calpains are also involved. responsible for many of unique characteristics of p94, these splice variants' properties are rather different from those of p94. Lp82 is 3.4. Diabetes stable (unlike p94), and it functions much like the conventional calpains. Adding IS1, but not NS or IS2, makes Lp82 as unstable as The relationship between CAPN3 expression and diabetes has p94 [123,126]. Intriguingly, the expression patterns of CAPN3 var- been examined using fat sand rats (Psammomys obesus) as an ani- iants in the lens and retina differ among mammalian species [127], mal model of obesity [104]. In these rats, the amount of CAPN3 in and functional exon 1B only exists in rodents [127,128]. skeletal muscles is negatively correlated with fat mass and the In skeletal muscle, the ratio of CAPN3 variants to p94 decreases concentration of circulating glucose, and positively correlated with as development proceeds [57], eventually dropping to less than 1%, carbohydrate oxidation and energy expenditure. On the other hand, whereas the proportion of variants in the heart, brain, lung, and fasting and refeeding do not affect CAPN3 expression in healthy retina, and other tissues remains at 10e100% [129]. Cattle have a human skeletal muscles [105]. At present, these are the only reports short CAPN3 isoform that encodes the N-terminal 12.9-kDa peptide examining CAPN3 and diabetes, and this area deserves further [130]. Taken together with the disease phenotype of CAPN3:Dex6- study to evaluate whether CAPN3 is involved in diabetes-related and Dex15-Tg mice, CAPN3 splicing variations and expression biological phenomena. changes are likely to play important roles in skeletal-muscle development. 4. Structure and activity The CAPN3/Capn3 genes have the initiation exons 1C (AX2), 12B (AX3), and 14B (AX4) in addition to those functioning in skeletal 4.1. Structures muscle (exon 1) and the lens and retina (1B). Exon 1C is driven by a rather non-specific promoter, whereas 12B and 14B are predomi- 4.1.1. Tertiary structure nantly expressed in the testis and melanoma, respectively [100]. Three-D structure of CAPN3 has not been solved entirely. The variant hUp84, which uses exon 1C, is abundant in immune However, its PEF domain is highly similar to that in other PEF tissues and in some lymphocytes, and active CAPN3 with exon 1 is þ proteins [106]. Unlike other calpains, CAPN3 PEF binds four Ca2 also present in these tissues [131,132]. The presence of these vari- ions, to EF1, 2, 3, and 5. In other calpains that have been analyzed, ants in peripheral blood cells can be used to diagnose LGMD2A þ EF5 is used to form dimers [107], not to bind Ca2 . Since CAPN3 and [133]. CAPN2 are highly similar (47% identity) except for NS/IS1/IS2, 3D CAPN3 modeling was performed without these regions [108]. 4.2. Activation Based on the aar positions of most of the missense mutations that þ produce calpainopathies, these mutations are likely to perturb 4.2.1. Rapid, exhaustive, and apparently Ca2 -independent inter-domain interactions and interfere with the proper CAPN3 autolytic activity conformation [108]. The most striking feature of CAPN3 is its (1) extremely rapid, (2) þ exhaustive, and (3) apparently Ca2 -independent autodegradation 4.1.2. Possible homodimerization activity [65]. This characteristic autolysis was observed in CAPN3 PEF proteins often dimerize with other PEF proteins, whether generated in in vitro translation systems [65,134], in a baculovirus- identical or distinct. For example, the CAPN1, CAPN2, CAPN13, Sf9 expression system [135], by expression in cultured cells (except CAPNS1 [109e112]; grancalcin [113,114], ALG2 [115], and sorcin for skeletal-muscle primary cultured cells) [65], and in endogenous [116] PEFs form homodimers, and the CAPN2-CAPNS1 [117e120] or CAPN3 in skeletal-muscle cell extracts [65,135]. CAPN9-CAPNS1 [112] PEFs form heterodimers. Accordingly, CAPN3 The extreme rapidity of CAPN3 autolysis depends on IS1 and IS2 PEFs dimerize with each other [106,112,121]. This finding implies [65]. The exhaustiveness probably involves other protease(s), as well that full-length CAPN3 may also homodimerize, and the estimated as ability of CAPN3 to undergo interMOlecular Complementation resulting 3D structure places the two active sites at opposite ends (iMOC) (see Section 5.1). Finally, this autolysis, which appears to be þ [106]. independent from Ca2 since it is not inhibited by excess EDTA, is þ However, interaction between full-length CAPN3 molecules has actually activated by Na [136] (see Section 4.2.2). These fascinating not yet been reported. Since the dimerization of PEF proteins is properties are unique among known calpains. þ affected by solution conditions, especially the Ca2 concentration, On the other hand, in the presence of physiological intracellular þ þ full-length CAPN3 may homodimerize only under specific condi- Na concentrations ([Na ]i), CAPN3 is activated by physiological 2þ tions. Nevertheless, the native quaternary structures of CAPN3 have [Ca ]i; these levels are much lower than those known to activate yet to be investigated, including interactions of CAPN3 with other other classical calpains, including CAPN3:Dex6/15/16 (p94D) proteins, described below. [137e139]. Conventional calpains require salt bridges between the PC2 and CBSW domains and a linker between the CBSW and PEF 4.1.3. A variety of splice variants domains for their activation and stability [117,118]. Mutations dis- þ Unlike CAPN1 and CAPN2, CAPN3 produces many splicing vari- rupting these interactions lower the [Ca2 ] required to activate ants (Fig. 3, Table 2). The first variant, discovered in 1998, was Lp82, calpain-2 [140,141]. In CAPN3, the aars corresponding to these þ which is predominantly expressed in the rodent lens [122], positions are different, which may explain low Ca2 requirement of þ particularly in the cytoplasm of the cortical and nuclear fibers CAPN3 for activation. In addition, stability and Ca2 sensitivity of [123]. Lp82 uses exon 1B (also called AX1), which adds an N-ter- calpain-2 are also regulated by N region of CAPN2, which interacts minus that is distinct from that of adult skeletal-muscle type full- with the PEF domain [141e143], and this region is markedly length CAPN3 (p94) and that lacks exons 6, 15, and 16. As a different in CAPN3. This difference in N regions may also contribute þ result, the NS, IS1, and IS2 regions are almost entirely missing from to lower Kd of CAPN3 for Ca2 [141]. Lp82. þ Other splicing variations have since been reported, including 4.2.2. Na -dependency þ Lp85 (Lp82 þ IS3) in the lens [124], Rt88 (Lp82 þ IS1) in the retina CAPN3 is activated by Na in vitro, although the physiological þ þ [125], and several isoforms expressed in embryonic muscles (p94 relevance of this regulation is still elusive. The [Na ] and [Ca2 ] Y. Ono et al. / Biochimie 122 (2016) 169e187 175

Table 2 Representative mammalian CAPN3/Capn3 transcripts.

required for autolysis are 100 and 0.1 mM, respectively, in the samples extracted using saline may need to be performed again þ þ absence of the other ion. When both ions are present, however, using buffers lacking Na . It is important to exclude Na from any 2þ CAPN3 is activated additively by both ions; e.g., when [Ca ]i is 0.1, buffer when diagnosing LGMD2A [144]. þ 1, or 10 mM, [Na ]i of 15, 8, or 2 mM half-maximally activates CAPN3, respectively [136]. In other words, CAPN3 is activated by ca. 4.2.3. Autolysis and activation þ 2þ physiological [Na ]i and [Ca ]i. CAPN3 is the first (and so far, the The activation of conventional calpains occurs almost concom- þ only) Na -dependent intracellular enzyme. itantly with their N-terminal autolysis [145e147], which is reported þ Mutations in the potential Ca2 -Binding Site(s) in PC1 (CBS-1), to precede caseinolytic activity of calpain [148] and to parallel the þ but not PC2 (CBS-2), compromise Na -dependency of CAPN3, calpains' autodegradation and inactivation [149].Bydefinition, þ indicating that CAPN3 CBS-1 may have evolved to bind Na . De- calpain activity must exist during autolysis, but further “activation” þ leting both IS1 and IS2 completely abolishes Na -dependent acti- may be required for calpain to proteolyze substrates. This point can þ vation of CAPN3 without changing its Ca2 -dependent activity be clarified by using more specificdefinitions of activation. þ [136]. Thus, Na -dependent activity of CAPN3 requires cooperation The first step of calpain activation is its intraMolecular (iM)- þ þ among CBS-1, IS1, and IS2. activation upon binding Ca2 (or, in the case of CAPN3, Na also). þ Potential substrates of Na -activated CAPN3 include tropomy- This iM-activation enables calpains to hydrolyze peptide bonds; osin, LIM-domain-binding protein 3, and a-actinin-3, all of which however, steric hindrance, with the N region for example, prevents are in the Z-line [136]. CAPN3 localizes to the Z-line by binding a- calpains from proteolyzing substrates. A second step of calpain þ actinin-2 (ACTN2, sarcomeric-a-actinin) [57], indicating that Na activation is that of extraMolecular (eM)-activation, which allows dependency of CAPN3 may play a role in its functions in the Z-line. calpains to contact substrates. Whether autolysis is required for the Autolytic activity of CAPN3 is suppressed in skeletal muscle cells eM-activation of conventional calpains is still controversial. by its binding to TTN or PLEIAD (see Section 6.2.1). However, Due to strong autolytic activity of CAPN3, the full-length WT immediately after homogenizing tissues, CAPN3 rapidly autode- protein can hardly be manipulated in vitro. Therefore, most þ grades in solutions containing Na , such as buffered saline. This biochemical CAPN3 studies have used mutants or partial CAPN3 was not recognized until recently, and early studies of tissue proteins. A study using only the CAPN3 CysPc showed that CAPN3 176 Y. Ono et al. / Biochimie 122 (2016) 169e187

þ þ iM-activation is provoked by ca. 0.1 mM Ca2 but not by Na , and true in vivo (e.g., inactive CAPN3:V354G is lost in vivo [87]). This that its eM-activation requires IS1 nicking [150e152]. IS1 is pro- suggests the existence of one or more CAPN3-mutant degrading teolyzed intramolecularly; in the presence of IS1, even small-Mr proteases that are present in skeletal muscles but not in non- calpain inhibitors like E64 cannot prevent CAPN3 autolysis. These muscle cells. This is also the case with some mutations outside of findings explain several of unique properties of full-length CAPN3, CysPc. D607 in IS2 is conserved from fish to humans (see Fig. 7), and þ but not its rapid autolysis or dependency on Na ; these charac- a D607A mutation attenuates the CAPN3 activity [160]. Further- þ teristics involve the IS2 region. Ca2 -dependence of full-length more, screening by p94-trapping, which uses a yeast two-hybrid CAPN3 has also been demonstrated in a baculovirus-expression system to detect activity-attenuated CAPN3 mutants, identified system [153] and in skeletal-muscle skinned fibers [154]. I603T, F604 L/S, V605A, and D607N [160]. Notably, a neighboring The autolytic sites in IS1 are located after Y274, N296, and Y322 mutation, S606L, causes LGMD2A [161]. Taken together, the short, [155]; CAPN3:Y274A loses both its autolytic and proteolytic activ- evolutionarily conserved stretch around D607 in IS2 is essential for ities [156]. These findings support the idea that IS1 proteolysis is proteolytic activity of CAPN3. required for full-length CAPN3 eM-activation. Moreover, autolysis in IS1 does not immediately cause the dissociation of CAPN3 frag- 4.2.4.2. Case (2): mutations that disable eM-activation. Some IS1 fi ments; it is the subsequent proteolysis in IS2 that nally inactivates mutations, including Y274A, cause defective IS1 nicking, which CAPN3 [126,157]. The TTN N2A region suppresses IS2 proteolysis disables eM-activation [156]. There is an a-helix in the middle of and the subsequent dissociation of CAPN3 fragments. IS1 that is disrupted by L286P [152]. Interestingly, the CysPc-only In some case, two CAPN3 fragments that are cleaved only in IS1 CAPN3:L286P is spontaneously activated during the purification dissociate, thereby inactivating CAPN3. These fragments can process. This type of CAPN3 mutant may be iM- and eM-activated fi become active again by the CAPN3-speci c mechanism called iMOC simultaneously, resulting in self-destruction, as described in þ 2þ (see Section 5.1). Since physiological [Na ]i and [Ca ]i are enough case (3). for CAPN3 activation, CAPN3 is probably already iM-activated in the cell, requiring only IS1 nicking for its eM-activation (Fig. 4). 4.2.4.3. Case (3): mutations that accelerate autolysis. If rapid auto- lytic activity of CAPN3 is accelerated even further, the CAPN3 pro- 4.2.4. Mutations in calpainopathy and CAPN3 activity tein is lost before it can accomplish its function. This is the case with Although mutations that inactivate CAPN3 cause calpainopathy, some mutations in the PEF domain, including S744G and R769Q not all calpainopathy-mutant CAPN3s lose their proteolytic activity [159]. Indeed, compared to WT, autolysis is accelerated in calpain-2 fi [158]. There are ve defective CAPN3 states: (1) completely inactive with mutations corresponding to those causing LGMD2A: T344M (loss of iM-activation ability), (2), inactive as to substrate proteol- (T417M of CAPN3), D346E (D419E), D362K (E435K), R417W ysis by loss of eM-activation ability, (3) inactive due to overly rapid (R490W), and G423R (G496R) [162]. autolytic activity or (4) improper substrate recognition, and (5) Studies using CAPN3 KO mice with transgenic expression of defective in non-proteolytic functions but with proteolytic CAPN3 or its mutants showed that R448H and D705G proteolyzed activity (5). muscle extracts faster than WT CAPN3, probably due to their inability to bind TTN [163]. This experimental system allows any 4.2.4.1. Case (1): inactive mutations. Mutations at evolutionarily CAPN3 mutant to be expressed in vivo in place of WT CAPN3. conserved aars in the CysPc domain are highly likely to inactivate Moreover, in skeletal muscle extracts, CAPN3 showed slowed CAPN3. This inactivation stabilizes CAPN3 when ectopically autolytic activity, and this feature might be useful for a CAPN3 expressed in cultured cells [159]. However, this is not necessarily autolytic assay.

þ 2þ Fig. 4. A scheme of activity regulation of CAPN3. Translated full-length CAPN3 (FL-CAPN3) is iM-activated by physiological [Na ]i and [Ca ]i (1), and autolyzes in IS1, producing nicked (but not dissociated) FL-CAPN3 (nFL-CAPN3) (2). Since nFL-CAPN3 is eM-activated and metastable, it functions as a protease (3). Then, nFL-CAPN3 is inactivated as its components (N31K and C58K) dissociate from each other (4), and/or degradative reaction predominates through autolysis in IS2 and other sites with the aid of other protease(s) (40). N31K and C58K reconstitute the activity by iMOC1 and/or iMOC2 (5), which accelerates autolysis of FL-CAPN3 (6). Reconstituted CAPN3 by iMOC1 (iMOC-CAPN3) localizes and functions distinctly from FL/nFL-CAPN3 under alternative regulation (7), and/or is ultimately inactivated by further autolysis (70). Y. Ono et al. / Biochimie 122 (2016) 169e187 177

4.2.4.4. Case (4): mutations that disrupt substrate recognition. teleost CAPN3; the IS1 in tetrapod CAPN3 may be missing or In conventional calpains, CBSW and PEF are important for substrate composed differently in teleosts (Fig. 6). Thus, teleost Capn3s may recognition. CBSW interacts with substrate aars at the P3 and represent a vivid example of sub- and/or neo-functionalization P5eP10 positions [119,120,164], and PEF is thought to assist in during gene evolution, in which CAPN3A and CAPN3B partitioned recognizing CaM-binding proteins as substrates [165]. Therefore, the functions of a single ancestral CAPN3 and/or acquired new mutations may disrupt the conformation of these domains, functions for one or both. compromising proper substrate recognition of CAPN3. For example, Another significant feature of CAPN3 gene evolution is that þ R490W and R572Q show reduced, Ca2 -dependent autolysis but CAPN3 has the most conserved orthologs among vertebrate clas- can barely proteolyze calpastatin or CAPN3:C129S, which are good sical calpainsdmore than either CAPN1 or CAPN2 [49] (Table 3). substrates for WT CAPN3 [159]. This is astonishing, considering that Capn2 disruption causes em- bryonic lethality [2,3], while Capn3 results in muscular dystrophy 4.2.4.5. Case (5): mutations that interfere with non-proteolytic [5,6]. This finding suggests that vertebrates are essentially insepa- functions. The phenotype of CAPN3:C129S-KI mice is between rable from their skeletal muscles, which constitute and maintain that of WT and CAPN3-KO mice [7,64], leading to the recognition of the whole body shape, and that CAPN3 is essentially involved in two independent CAPN3 functions: proteolytic and non- these functions. Alternatively, ancestral CAPN3 might have had proteolytic. C129S has no proteolytic activity, but its non- functions so pivotal to cell survivaldthe regulation of cell cycles, for proteolytic function is intact. Some mutations in regions that are instance (see Section 6.3)das to warrant the conservation of not directly involved in proteolytic function (cases 1e4) may CAPN3 among vertebrates independently of its expression pattern. compromise only the non-proteolytic function, leaving the pro- Further research will hopefully uncover novel CAPN3 functions. teolytic function intact. This may be the case with A702V, which was found in a homozygous patient that had normal CAPN3 protein 5. Unique features levels and activity [158]. Although extreme instability and rapid autodegradation of 4.3. Molecular evolution CAPN3 is one of its most startling features, it has several other unusual characteristics. Although the physiological significance of As mentioned above, clear CAPN3 orthologs have been found some of these properties has yet to be established, they certainly only in vertebrates (Fig. 5). Notably, teleosts (bony fish) have two make CAPN3 an enigmatic yet attractive research subject. paralogous genes as orthologs for most mammalian genes, including calpains, due to genome duplication in the teleost 5.1. Re-activation after inactivation ancestor, and evolutionary CAPN3 traits have been glimpsed in these fish [166]. Interestingly, the two CAPN3 zebrafish genes show The amazing rapidity of CAPN3 autodegradation is thought to distinct expression patterns: during early development, Capn3a regulate its activity. A recent study demonstrated that de novo iMOC expression begins in the lens and expands into the brain between between the two autolytic fragments of CAPN3 reconstitutes an the eyes, while Capn3b expression is enriched in the digestive or- active core protease domain [168] (see Fig. 4). Unlike the sustained gans and in the head region [167]. interaction that occurs between autolytic fragments in the course Capn3a and Capn3b also have contrasting structural features. A of autolysis, iMOC enables the mutual compensation of two possible nuclear localization signal is detected in the IS2 region of different CAPN3 mutant molecules if the combination is appro- CAPN3B but not CAPN3A. On the other hand, the Capn3a genomic priate. It is currently hypothesized that iMOC may rescue a fraction environment mimics that of human CAPN3, being located at the 30 of CAPN3 protease function in some cases of LGMD2A caused by region of GANC [100] (Fig. 5). Such trends are conserved among distinct missense mutations in both CAPN3 alleles, and this process Capn3s in other teleosts. So far, it has been difficult to identify IS1 in may alleviate symptoms [169].

Presence of IS1 / IS2 Location of GANC gene CAPN3(3A) CAPN3B Human, Mouse, IS1 IS2 ganc CAPN3 Tetrapod Chicken, Turtle

Lobe-finned fish Frog IS1 IS2 N/A

Coelacanth IS1 IS2 N/A Jawed fish Spotted gar IS1 IS2 ganc Capn3a

TGD Medaka*, Platyfish, Nile tilapia IS1 IS2 IS1 IS2 Ray-finned fish

Vertebrate Stickleback, Pufferfish, Teleost Fugu, Atlantic cod**, IS1 IS2 IS1 IS2 Amazon molly, Zebrafish*** ganc Capn3a

Cave fish IS1 IS2 IS1 IS2

Jawless fish Lamprey IS1 IS2

Fig. 5. Summary of CAPN3 orthologs. CAPN3s from different branches are categorized according to their molecular structure and chromosomal location; the topology of the phylogenetic tree is arbitrary. Gray indicates that the annotated sequence does not align perfectly with human CAPN3. *, ***: no exon sequences have been found that correspond to exons 15 and 16, respectively, in humans. **No canonical nuclear localization signal has been found, and the GANC gene is not identified in Ensembl (www.ensembl.org). N/A, the genes encoding GANC and CAPN3 are found in different . TGD, teleost genome duplication. For sequence alignment, see Ref. [11]. 178 Y. Ono et al. / Biochimie 122 (2016) 169e187

IS1 Human 251 IMKKAIERGSLMGCSIDDGTNMTYGTSPSGLNMGELIARMVRNMDNSLLQDSDLDPRGSDER---PTRTIIPVQYETRMACGLVRGHA 335 Spotted gar (a) 242 IMSKALERGSLMGCSIDDTMGITPGV-PAPNALGTLIDRMISQTEADKSKPGEPPTGCTLTF---STHSLVPVRFETRTVTGLVKGHA 325 Medaka** (a) 232 TMKKALERGSLMGCSIDDGFCFRSGTVALKSKSHQQITRCKNDDKQRLFKAMDCGPYIRQSK---YLQSLVPARFETRTTTGLVKGHA 316 Platyfish* (a) 232 IMKKALERGSLMGCSIDVRDWLS------YFDYI---RSVVCVLLICFLIFSLPLFLVSSCSLLNSLVPARFETRTVTGLVKGHA 307 Nile tilapia** (a) 232 IMKKALERGSLMGCSIDTLTSSPLLTSPKDLLYVNFS---HQVSSKSNLSPLNFHNLFF------NLQSLVPARFETRTVTGLVKGHA 310 Cave fish* (b) 246 IMKKALNRGSLMGCSIDVSVFNLYNFFPALNLYRPLLR--LVNHKLRRESTSDLTSPHLKQLYRHLPWSLVPSASETKTETGLVRGHA 331 Lamprey (a) 242 IMVKATSRRSLMGCSIDDGMCWRCGV-PAPDSIGLV----RSNMSPPILRHIQLTPIGIEDAK--KRKIAMLTHAESRTPMGLVRNHA 322

Fig. 6. Sequences found in the IS1 region of CAPN3 from species other than tetrapods. The arrow indicates one of the active-site aars, H334, in humans. In tetrapod CAPN3s, IS1 is encoded, in-frame, by a single exon. *, **: the first (white arrowhead) and the last (black arrowhead) aars, respectively, are not in-frame. For full sequence alignment, see Ref. [11].

Protease compatibility for iMOC has been reported only for a CAPN3-Tg mice show almost no phenotype [83,173], suggesting few viruses [170], and in no other proteases so far. Uncovering the that excess CAPN3 may be absorbed by myofibrils via increased molecular mechanisms and physiological relevance of CAPN3 iMOC phosphorylation. Intriguingly, S629 is well conserved among and its physiological relevance should be an interesting avenue of eutheria, with the exception of a subset of small rodents and research. bats, in which S629 is replaced by G. Large eutheria may take advantage of P-CAPN3, stocking an abundance in skeletal mus- cles to protect and maintain the muscles and their functions. 5.2. Phosphorylation Further studies are needed to reveal P-CAPN3 role in normal as well as pathogenic (e.g., LGMD2A) and physiologically stressed Two phosphorylated aars have been found in human CAPN3, at (e.g., exercised) muscles. S629 and S636 in IS2 (Fig. 7); the former is the major site [171]. The autolytic CAPN3 activity is slightly attenuated by the defective phosphorylation of S629 but not S636, suggesting that the autolysis 5.3. Non-proteolytic function is regulated in part through S629 phosphorylation [171]. Phos- phorylated CAPN3 (P-CAPN3) is enriched in myofibrils rather than Non-proteolytic functions of CAPN3 are independent of its in the cytoplasm [163,171]. As CAPN3 directly binds TTN and ACTN2 proteolytic function. This idea emerged from data comparing [57,62], it is possible that CAPN3 is selectively phosphorylated in CAPN3-KO and -KI mice with WT mice [64]. CAPN3 binding to myofibrils, and this property may be related to TTN-based me- Aldolase A (ALDOA) is essential to localize ALDOA to the triads, chano-transduction signaling. where the complex interacts with the (RyR) in Phosphorylation may also increase affinity of CAPN3 for the SR [174]. Indeed, IS2 region of CAPN3 interacts with RyR, sarco/ þ myofibrils, causing CAPN3 to accumulate in myofibrils (Fig. 8). endoplasmic reticulum Ca2 -ATPase, -1 (CSQ1), and We found that the amounts of both CAPN3 and P-CAPN3 are triadine, locating proximally to the SR [64]. Notably, CAPN3 does reduced in LGMD2A muscle tissues [171,172].Incontrast,WT not proteolyze these molecules in vitro. Furthermore, myotubes and

Table 3 Sequence conservation of CAPN3 vs. CAPN1 among vertebrates. The upper and lower values indicate the identity and similarity of aa sequences, respectively. Y. Ono et al. / Biochimie 122 (2016) 169e187 179

601 610 620 630 638 Human PII FVSDRANSNK ELGVDQESEEGKGKTSPDKQKQSPQ Guinea pig PII FVSDRANSNK ELGVDHESEEGKDKASPDKQEKSPQ Mouse PII FVSDRANSNK ELGVDQEAEEGKDKAGPEKRGETPQ Rat PII FVSDRANSNK ELGVDQEAEEGKDKTGPDKQGESPQ Bat PII FVSDRANSNK ELSVDQEAEEGKDKIGPDKPEKPPQ Opossum PII FVSDRANSNK ELGVDHESQEGKDK--PEKPTQPTS Chicken PII FVSDRANSNK ELTTDE--DAGKDGEKTHVDEKK-- Green anoles PII FVSDRANKNK DPKGGE--GHKKDKEKTITDKKKKA Frog PII FVSDRSNSNK ELTVDGATDEDQKKLNADQKDKTSV Medaka PIV FVSDRARTNK EIEHEGI-----RGEKKKKPKRKPL Spotted gar PII FVSDRANANK EIEPESYVHEEEEKKDKMKPAPASA Platyfish PIV FVSDRAHANK EIEHDGI-----QGEKRKKPKKQLQ Coelacanth PII FVSDRANHNK EKTDNEKPGKEEPASKPETKKKPAP Lamprey PIV FVSDR--TKK GQQQPESPDESEPEDGNEESNKLAE

Fig. 7. Conserved sequences of IS2 region found in CAPN3. The sequences correspond to 601e638 aars of human CAPN3. The arrows indicate two phosphorylation sites found in human (see Section 5.2). For full sequence alignment, see Ref. [11].

(A) (B) CAPN3 kinase phosphatase phosphatase

P CAPN3 CAPN3 P CAPN3 cytosol or

P P myofibrils CAPN3 CAPN3 kinase CAPN3

Fig. 8. Possible mechanisms for anchoring P-CAPN3 to the myofibrils. These mechanisms postulate the existence of an as-yet unidentified CAPN3-phosphorylating kinase, either in the cytosol (A) or myofibril-bound (B). In (A), P-CAPN3 is recruited to the myofibrils by an increased affinity for myofibrils. In (B), recruited CAPN3 is phosphorylated by myofibril- bound kinase, which may or may not increase the affinity of the molecule for the myofibrils. In either case, dissociated P-CAPN3 is rapidly dephosphorylated by phosphatases in the cytosol, increasing the accumulation of P-CAPN3 in the myofibril fraction.

muscle fibers from CAPN3-KO mice exhibit reduced responses to contraction, in which a number of molecules cooperate þ caffeine (RyR agonist) and to Ca2 release from the SR, respectively, [183e185]. compared to WT responses [174,175]. In addition, disrupting CAPN3 CAPN3 also locates to the Z-line [57,62]. Intriguingly, CAPN3 þ does not affect Ca2 uptake into the SR [174]. alters its position depending on sarcomere length: as the sarcomere In CAPN3:C129S-KI mice, the RyR and ALDOA localization and extends, CAPN3 becomes less abundant at the M-lines and more þ Ca2 influx/efflux are unaffected [64]. These observations indicate abundant at the N2A regions [57]. This is probably because the that CAPN3 has a non-proteolytic functiondthat is, a structural extension of the TTN I-band exposes CAPN3-binding sites in the þ property that is responsible for regulating SR Ca2 release. This dual N2A regions, while this area is hidden in contracted sarcomeres by character of CAPN3 may account for the wide range of LGMD2A TTN tangled IGs. Furthermore, the CAPN3 accumulation at N2A symptoms, at least in part. regions is facilitated by protease activity of CAPN3 [7,57]. In other words, inactive CAPN3 cannot rapidly access the TTN N2A regions in extended sarcomeres, and this inability may be a contributing 5.4. Dynamic translocations in skeletal muscle cells factor in LGMD2A. As a scaffold to form a protein complex for mechanical signal TTN, which was identified as the first known CAPN3 binding transduction [182], the TTN N2A region binds muscle ankyrin partner in 1995 [62,176], is an essential regulator of CAPN3. TTN is repeat domain protein 2 (ANKRD2, also called MARP2 or ARPP) the largest single polypeptide, spanning half a sarcomeredthat is, [186]. ANKRD2 participates in stress-response pathways and is the distance between the Z- and M-lines, which correspond to N upregulated after exercise [187,188].Insufficient ANKRD2 and C termini of TTN. TTN functions as a molecular spring, retaining recruitment is observed in exercised CAPN3:C129S-KI mice, the thick filaments at the sarcomere center through an elastic indicating that protease activity of CAPN3 is necessary for this property of its I-band region, which is composed of a series of mechanosensory transduction [7]. For this physical stress immunoglobulin (IG) domains [177e179]. response, CAPN3 protease activity is required during exercise CAPN3 binds to N2A and M-line regions of TTN [62,180], rather than after. In addition, CAPN3 activation has been detected where CAPN3 is proposed to play a role in TTN-based mecha- in muscles up to six days after exercise [7,189e191], suggesting nosensory transduction pathways. In stretched sarcomeres, that protease activity of CAPN3 has different roles. In other CAPN3 accumulates at the N2A region, where a protein complex words, while CAPN3 activation during and immediately after is formed in response to mechanical stimulation. The TTN I-band exercise triggers mechanosensory transduction, CAPN3 autolysis region extends, spring-like, as sarcomeres stretch [181,182],and after exercise suggests functions such as sarcomere remodeling thus serves as an ideal “scale” to sense sarcomere extension/ 180 Y. Ono et al. / Biochimie 122 (2016) 169e187

[192]. Monitoring CAPN3 activity poses technical challenges; which expressed mutant and WT TTN at a ratio of ca. 1:1, showed a CAPN3 autolysis in skeletal muscle cells in situ cannot be skeletal-muscle dystrophic phenotype from 9 months of age, detected by immunoblotting. Thus, innovative techniques are whereas the heart was normal. HO mice exhibited a more severe required for probing CAPN3 activity in situ to clarify its functions phenotype with dilated cardiomyopathy. Surprisingly, even though during and after exercise. FINmaj changed only four aars, the mutant TTN lost its last five C- terminal IGs (ca. 84-kDa around M6 to M10, Fig. 9) [16]. The FINmaj mutation causes a pathological cleavage of TTN in 6. Molecules that interact with CAPN3 M10 and at somewhere around M6 (Fig. 9). FINmaj and some other mutations are located in the middle of the M10 b-strand or a-helix 6.1. Molecules responsible for muscular dystrophies [16,206], disrupting an otherwise rather rigid IG conformation [207] and rendering M10 susceptible to cleavage. This cleavage is 6.1.1. TTN carried out by both CAPN3 and calpain-2 [16], which are reported to 6.1.1.1. Muscular dystrophy with myositis (mdm). As mentioned share several other substrate cleavage sites [186,208]. Although this above, TTN serves as a large-scale CAPN3 reservoir. TTN N2A region cleavage has not been confirmed in vivo, the loss of the 84-kDa has at least three CAPN3-binding sites [186], and binding at these extreme C-terminal region is probably caused by further cleav- sites suppresses rapid autolysis of CAPN3 [157]. A severe muscular ages of TTN around M6 (Fig. 9), carried out by CAPN3, calpain-2, and dystrophy phenotype in mdm mice was first described in 1985 possibly other protease(s). Although these cleavages were not [193,194]. Ttn became a causative candidate in 1993 [195], and the reduced in CAPN3 KO muscle, the other protease(s) involved in Ttn small deletion (83 aars) responsible for the mdm phenotype was these pathological cleavages in vivo have yet to be identified. identified in 2002 [194]. TTN M10 binds the 800-kDa IG-repetitive protein, obscurin The relationship between CAPN3 and mdm is still elusive. A (OBSCN), and its smaller paralog, obscurin-like 1 (OBSL1, secondary reduction of CAPN3 in mdm mice was observed in some 130e230 kDa) [209e212]. These proteins, together with the cases [196,197] but not in another [198]; this discrepancy may be myosin-interacting M-line proteins called myomesins (MYOM1-3), due to differences in the developmental stage of the mdm mice or form an elastic web in the M-line [213,214]. Thus, pathological the sample-preparation methods. The phenotype of mdm mice was cleavage in M10 very likely disrupts the integrity of the M-line unaffected by Capn3 KO, but was exacerbated by transgenic CAPN3 structure, including the dissociation of OBSCN/OBSL1 from TTN expression [197]. These findings suggest that CAPN3 may be per- [211,212], which would make the TTN M-line susceptible to pro- turbed by the mdm mutation, which is not a primary cause of the teolysis. Myospryn (also called CMYA5) binds to both CAPN3 and mdm phenotype. the C-terminal region of TTN [215]. Myospryn appears to be pro- teolyzed by co-expressed CAPN3 in cultured cells, and its binding to 6.1.1.2. Tibial muscular dystrophy (TMD) and M-line TTN proteolysis. CAPN3 slightly suppresses CAPN3 autolysis [215]. Thus, this mole- TMD is an autosomal dominant late-onset distal myopathy that was cule may also be involved in the M-line instability caused by M10 identified in 1993 [199]. Since it was mapped to chromosome 2q31, proteolysis. TTN became a candidate causative gene, as with mdm [196,200]. Finally, several mutations in last two exons of TTN (Mex5 and 6), in 6.1.2. Dysferlin and LGMD2B which a few aars in the TTN C-terminal region were changed, Dysferlin (DYSF) is a multi C2-containing protein involved in identified as being responsible for TMD, for LGMD2J (when ho- membrane repair. This protein is responsible for LGMD2B, Miyoshi mozygous), for hereditary myopathy with early respiratory failure, myopathy, and distal myopathy [216]. Secondary CAPN3 reduction and for early-onset myopathy with fatal cardiomyopathy has been reported in LGMD2B, as with TMD, suggesting a link be- [201e204]. tween CAPN3 and DYSF [217]. Biochemical studies show that The first identified mutation (FINmaj) changed EVTW to VKEK CAPN3 directly interacts with DYSF [218] and with the DYSF- (Fig. 9) [201]. Hetero- (HE) and homozygotes (HO) of the FINmaj interacting protein AHNAK (also called desmoyokin) [219]. knock-in mouse model showed increased embryonic lethality. AHNAK is proteolyzed by CAPN3, suggesting that CAPN3 regulates Once born, however, these mice appeared to grow normally the DYSF complex [219], although CAPN3 is not directly involved in and have a lifespan similar to that of WT mice [205]. HE mice, membrane repair [220,221]. Since LGMD2A and 2B account for the

Fig. 9. TTN C-terminal mutations found in TMD, and cleavage sites by calpains. A structure based on the sequence of N2A-type TTN (NP_596869; 33,423 aars, ca. 3.7 MDa, 1e1.5 mm). Red arrows indicate the C termini of sites cleaved by both CAPN3 and the conventional calpains; black arrows indicate those cleaved by calpain-2. The dotted arrow indicates a site that is probably cleaved by endogenous conventional calpains; the cleavage is suppressed by deleting mdm. The 44-kDa proteolytic fragment cleaved at the C-terminus of E33,017 is physiologically detected under a static condition [16]. Blue vertical lines indicate TMD mutations. Y. Ono et al. / Biochimie 122 (2016) 169e187 181 majority of LGMDs, the relationships among these molecules Accordingly, knocking down CAPN3 in HepG2 cells increases the should be examined further. proportion of G1-arrested cells. This increase in G1-arrested cells is conserved in zebrafish, 6.1.3. RBBOX1, a Capn3 splicing regulator which has two CAPN3 genes, Capn3a and Capn3b, as described Facioscapulohumeral muscular dystrophy (FSHD) is an auto- above. CAPN3B, but not CAPN3A, is involved in downregulation of somal dominant disorder characterized by facial and shoulder- p53 in a manner depending on active site C120 of CAPN3B [167], girdle muscle weakness [222]. A deletion at the 4q35 region has although which CAPN3 isoform(s) are involved is unknown at been found in FSHD patients. This deletion upregulates several present. We also do not know whether these observations are genes in this region, including FRG1 (FSHD region gene 1), which exceptional and specific to restricted conditions, or if CAPN3 pro- encodes a possible RNA-processing protein [223]. Mice over- teolytic activity is generally involved regulating p53 or cell cycles. expressing FRG1 have an FSHD-like phenotype [224] with a So far, systemic defects related to dysregulated cell cycles have not downregulation of RBBOX1, an alternative splicing regulator; been reported in calpainopathy patients or Capn3 KO mice. How- knocking down RBBOX1 decreases full-length CAPN3 and increases ever, CAPN3 variants lacking IS1/2, such as hUp84 and hMp84, are the Dex6 CAPN3 variants [225]. As mentioned above, CAPN3:Dex6- very likely used for these functions, which can be taken over by Tg mice have a severe phenotype, indicating that the deficiencies other classical calpains when CAPN3 is defective. In other words, found in FSHD can likely be ascribed, at least in part, to aberrant cell-cycle control may be one of the most important functions for CAPN3 splicing. Of note, some FSHD-like patients also carry CAPN3 classical calpains, at least in vertebrates. mutations [226]. The transcription of rRNA and construction of ribosomes take place in the nucleolus. Thus, nucleolar localization of CAPN3 im- 6.2. Suppressor and activator plies that it has functions related to RNA metabolism. During our PLEIAD screening, we identified several RNA-binding proteins 6.2.1. Pursuit of a CAPN3 suppressor in the cytosol involved in stress granules ([208] and unpublished results). Interaction proteomics is a powerful, highly sensitive method Together with the fact that translational machinery components for capturing protein networks. Using CAPN3 as a bait, this meth- are potential CAPN3 substrates (see Section 6.4), the recruitment of odology identified a PLatform Element for Inhibition of Autolytic CAPN3 to the nucleolus by DEF may be physiologically relevant. In Degradation of CAPN3 (PLEIAD) [208]; this molecule was previ- addition, the conventional calpains proteolyze heterogeneous nu- ously known as C5orf25 and as SIMC1, a poly-SUMO interacting clear ribonucleoprotein (RNP) K [235] and SMN protein, both of protein [227]. Protein co-expression experiments demonstrated which are involved in small nuclear RNP biogenesis [236,237]. One that PLEIAD moderates both protease activity and autolysis of of the PEF proteins, ALG-2, is associated with the P-body, another CAPN3, as in the case of N2A fragment of TTN. Cellular localization location where RNA metabolism takes place [238]. These points of PLEIAD is dominantly cytosolic, with occasional sarcomeric I- merit further investigation. band localization. Therefore, PLEIAD is regarded as a candidate No clear CAPN3 orthologs have been identified in non- regulator of CAPN3 residing outside of the sarcomere structure. vertebrates. In these organisms, CAPN3 functions may be carried C-terminal region of PLEIAD, which suppresses CAPN3 activity, out by other calpains. For example, Saccharomyces cerevisiae has a is well conserved in vertebrates. As with CAPN3, no homologous single calpain gene, RIM13 (previously called CPL1) [239,240], sequence for PLEIAD has yet been identified in invertebrates. which may act with Utp25, a yeast DEF ortholog that mediates Intriguingly, N-terminal region of PLEIAD also interacts with the rRNA processing. In this context, the lack of any calpain in Schizo- transcriptional regulator, C-Terminal Binding Protein 1 (CTBP1), saccharomycetes [39] is mysterious, since it has Utp25 orthologs. which is a good substrate for calpains including CAPN3 [208]. Alternatively, the calpains' p53-degrading function may have Although this finding implies that PLEIAD is a scaffold for prote- been acquired during the evolution from invertebrates to verte- olysis by CAPN3, further studies of physiological functions of brates. The N-terminal 100e200 aar regions of DEF/Utp25 are not PLEIAD are necessary to confirm this. conserved between vertebrates and invertebrates, whereas human and zebrafish share 53% identical aars in these sequences. In any 6.2.2. CaM as an activator of CAPN3 case, the involvement of CAPN3 and other calpains in the cell cycle Recently, CaM was shown to promote CAPN3 activation [228]. should be investigated in the near future. Crude muscle extracts of CAPN3-KO mice that Tg-overexpressed CAPN3 WT or C129S were used for CAPN3 assays as described 6.4. Substrates above. In this assay, ectopically added CaM facilitated the autolysis of CAPN3 as well as the proteolysis of TTN by CAPN3 [228], sug- CAPN3 WT, but not C129S, has strong autolytic and proteolytic gesting CaM as the first positive regulator of CAPN3 to be activity when expressed in cultured cells, without any additional discovered. activation process [65]. Thus, it is easy to test whether CAPN3 proteolyzes particular proteins in cultured cells. These types of 6.3. Possible involvement of CAPN3 in cell-cycle regulation studies have identified various in vitro substrates, including ALDOA [174], ANKRD23/CARP/MARP1 [241], C/EBPa [242], filamin C In MCF7 cells (derived from human breast cancer) and HepG2 [156,243],IkB [244], ezrin, talin, vinexin [156], conventional cal- cells (derived from human hepatoblastoma), the degradation of pains, calpastatin [139], b-oxidation enzyme (VLCAD) [245], TTN p53 in the nucleus is mediated by CAPN3 and Digestive organ N2A, M- and Z-line regions [16,156,186], PIAS [246], RyR [247], Expansion Factor (DEF, also called DIEXF and C1orf107) [167]. The AHNAK [219], spectrin [159,248], MyoD [249], and CTBP1 [208]. p53 molecule is a well-known regulator of a variety of cellular Proteomic studies have identified additional substrate candi- functions; its turnover is mediated mainly by MDM1 via the dates, including eukaryotic translation initiation and elongation ubiquitin (Ub)-proteasome system, and also by Ub-independent factors [134], glyceraldehyde-3-phosphate dehydrogenase [134,250], proteasome degradation and by calpains [229e232]. DEF is a PDZ and LIM domain 1 (PDLIM1) [251], troponin T, and CSQ1 [136]. nucleolar protein involved in rRNA processing and p53 regulation Mostofthesecandidateshaveyettobeverified for physiological [233,234]. CAPN3 directly binds to DEF and negatively regulates proteolysis by CAPN3. However, several molecules identified in in- p53, downregulating p53-mediated gene expression [167]. dependent studies and involved in the same biological context are 182 Y. Ono et al. / Biochimie 122 (2016) 169e187 likely to have physiological significance, including myofibril com- Acknowledgments ponents and molecules involved in protein translation and glycolysis. There are too few known cleavage site sequences to analyze We thank all of the colleagues who worked with us on our substrate specificity of CAPN3 at present. Considering that CAPN1, CAPN3 research at the Tokyo Metropolitan Institute of Medical CAPN2, and CAPN3 are highly similar, specificity of CAPN3 should Science and the University of Tokyo, and we thank Dr. Leslie be similar to those of calpain-1 and -2, which are almost identical Miglietta and Dr. Grace Gray for critical reading of the manuscript. [164,252e254]. Using several CAPN3 cleavable fragment se- This work was supported in part by JSPS.KAKENHI 26670166 (to quences, a possible CAPN3-recognizing motif in the vicinity of FSO), 25440059 (to YO), 26450172, 23780152 (to SH), 23500477 (to cleavage sites was proposed [246]. However, since the cleavage KO), 15H02389, and 23247021 (to HS), the Open Partnership Joint sites of these fragments have not been identified, the distances Projects of JSPS Bilateral Joint Research Projects (to HS), a Takeda between these motifs and the cleavage sites are unclear. There is no Science Foundation Research Grant (to HS), the Council for Science, such motif around the cleavage sites identified in calpastatin [139], Technology, and Innovation (CSTI) Cross-ministerial Strategic CTBP1 [208], PDLIM1 [251], or TTN N2A [186]. Innovation Promotion Program (SIP), “Technologies for creating The substrate specificity of conventional calpains is neither next-generation agriculture, forestry and fisheries” (funding explicit nor ambiguous. Although a clear rule has not been found, agency: Bio-Oriented Technology Research Advancement Institu- cleavage sites can be predicted fairly successfully [164,255e257]. tion, NARO) (to HS), a research grant of The Naito Foundation (to Cleavage studies unexpectedly revealed that calpains proteolyze HS), a Toray Science and Technology Grant (to YO), a Kato Memorial many sites in vitro if the peptides are unstructured. In other words, Bioscience Foundation Research grant (to SH), and a SUNTORY the limited proteolytic action of calpains in vivo is mostly defined by Research grant (to SH). the native structures of the substrates. The same can be expected for CAPN3, and researchers must be conscious of 3D structures References when evaluating substrate proteolysis of CAPN3 and physiological significance. [1] M. Azam, S.S. Andrabi, K.E. Sahr, L. Kamath, A. Kuliopulos, A.H. Chishti, Disruption of the mouse mu-calpain gene reveals an essential role in platelet e 7. 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