All during fish postmortem evolution, structural muscle proteins are targets for various proteases. During the prerigor period (24 hours at 4 degrees C for sea bass), cytoskeletal proteins are affected by the first proteolytic events. These cleavages disrupt connections between myofibrils and the extracellular matrix, induce segmentation of myofibril cores, and modify the rheological properties of tissue. Dystrophin, a cytoskeletal actin-binding protein, is a relevant in situ marker for muscular proteolysis in the prerigor period. The immunodetection of dystrophin allowed the monitoring of early proteolysis during fish storage. Using antidystrophin antibodies directed toward the carboxy-terminal region, a highly sensitive domain exposed to calpain activity, we showed that proteolysis kinetics are strongly influenced by the muscular lipid content. In particular, comparison between low-fat diets (11.3% lipid) and high-fat diets (30% lipid), used during sea bass farming (90 days), revealed a faster proteolysis rate during the first 8 hours of storage at 0 degrees C with the high-fat diet. The origin of this faster proteolysis is discussed on the basis of a possible activation or translocation of calpains related to lipid accumulation in muscle fibers and cytoskeleton alterations.

CapZ is a widely distributed and highly conserved, heterodimeric protein, that nucleates actin polymerization and binds to the barbed ends of actin filaments, preventing the addition or loss of actin monomers. CapZ interaction with actin filaments was shown to be of high affinity and decreased in the presence of PIP2. CapZ was located in nascent Z-lines during skeletal muscle myofibrillogenesis before the striated appearance of thin filaments in sarcomers. In this study, the stabilization and the anchorage of thin filaments were explored through identification of CapZ partners in the Z-line. Fish (sea bass) striated white muscle and its related Z-line proteins were selected since they correspond to the simplest Z-line organization. We report here the interaction between purified CapZ and alpha-actinin, a major component of Z filaments and polar links in Z-discs. Affinity of CapZ for alpha-actinin, estimated by fluorescence and immunochemical assays, is in the microM range. This association was found to be independent of actin and shown to be weakened in the presence of phosphoinositides. Binding contacts on the alpha-actinin molecule lie in the 55 kDa repetitive domain. A model including CapZ/alpha-actinin/titin/actin interactions is proposed considering Luther's 3D Z-line reconstruction.

Calpain 1 behaviour toward cytoskeletal targets was investigated using two a-actinin isoforms from smooth and skeletal muscles. These two isoforms which are, respectively, sensitive and resistant to calpain cleavage, interact with the protease when using in vitro binding assays. The stability of the complexes in EGTA ¼ 0.5 ± 0.1 lM] was improved in the presence of 1 mM calcium ionsLocation of the binding structures shows that the C-terminal domain of a-actinin and each calpain subunit, 28 and 80 kDa, participates in the interaction. In particular, the autolysed calpain form (76/18) affords a similar binding compared to the 80/28 intact enzyme, with an identified binding site in the catalytic subunit, located in the C-terminal region of the chain (domain III-IV). The in vivo colocalization of calpain 1 and a-actinin was shown to be likely in the presence of calcium, when permeabilized muscle fibres were supplemented by exogenous calpain 1 and the presence of calpain 1 in Z-line cores was shown by gold-labelled antibodies. The demonstration of such a colocalization was brought by coimmunoprecipitation experiments of calpain 1 and a-actinin from C2.7 myogenic cells. We propose that calpain 1 interacts in a resting state with cytoskeletal targets, and that this binding is strengthened in pathological conditions, such as ischaemia and dystrophies, associated with high calcium concentrations.Keywords: calpain; cytoskeleton; alpha-actinin; muscle; calcium.Calpain 1 (Calp1) and calpain 2 (Calp2) are intracellular Ca 2+ -dependent thiol endoproteases [1], expressed throughout the animal kingdom, and recently reported in the plant kingdom [2]. These two proteases are particularly implicated [1] in the selective proteolysis of factors involved in the cell cycle, in myocyte fusion, during apoptosis in association with caspases or in the cleavage of membrane-cytoskeleton complexes during cell motility phases [3]. Many of the substrates are transcription and signalling factors with intracellular presence of less than 2 h [4] or cytoskeletal proteins with long half-lives, generally specialized in the cross-link or the membrane anchorage of fibrillar components [5]. The hypothesis according to which calpains would be released from complexes with calpastatin (its natural inhibitor) to join membrane phospholipids where protease activation is achieved was proposed [6], but the origin of recognition of specific substrates by calpains [7,8] remains unclear.A statistical analysis of the presence of PEST sequences in the target, critical for calpain recognition [9,10], gives valuable scores with short half-life proteins, but is not appropriate in the case of several cytoskeletal actin-binding proteins [1]. For example, filamin, dystrophin and talin are known to be cleaved in vivo by calpains. It should be noted that the accessibility of the calmodulin (CaM)-binding domain in PEST sequences is an important factor to consider [11], as demonstrated for IjBa, a CaM and calpain-binding protein [12]. Moreover, we have shown recently in mus...