The calpain 1–α‐actinin interaction

Abstract

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...

Reverse Phase Protein array for the quantification and validation of protein biomarkers of beef qualities: The case of meat color from Charolais breed

“…5a). Note that these three proteins strongly participate in Z-disk organization, a compartment which rapidly undergoes proteolysis during muscle ischemia or after a calpain treatment of isolated myofibrils (Raynaud et al, 2003). From these findings concerning structural proteins, we suggest their variation in abundance modifies the degree of light penetrating the structural elements and thereby meat color.…”

“…Additional proteins were found with high discriminative power, and this included α-actinin. This protein is a major component of the Zdisk and strongly associates with actin filaments and structural proteins to stabilize the cytoskeleton (Raynaud et al, 2003). The location of a μcalpain binding site in the C-terminal region of α-actinin situates the protease in the vicinity of TTN (Ohtsuka, Yajima, Maruyama, & Kimura, 1997), a protein described as an α-actinin partner in the Z-line and known as a calpain substrate (Papa et al, 1999).…”

The hyaluronic acid inhibitor 4-methylumbelliferone is an NSMase2 activator—role of Ceramide in MU anti-tumor activity

“…They localize to focal adhesions and cleave focal adhesion-related proteins including integrin receptors, focal adhesion kinase and talin (55, 56). Calpain1 and 2 are two widely characterized isoforms and show increased expression and activity in various tumor cells (52, 53, 57-60). PP2A suppresses the migration and invasion of tumor cells through dephosphorylation of calpain1 and calpain2 (61).…”

Actin Bundles Dynamics and Architecture

“…Titin binding further favors the flexibility of the neck region, allowing ABD domains to acquire the orientation necessary for crosslinking of overlapping parts of actin filaments in the Z-disk (in antiparallel orientation) [ 109 ]. Moreover, phosphorylation of specific residues on human α-actinin-1 (Y12) and α-actinin-4 (Y4, T32, and Y265) [ 122 , 123 ], and limited proteolysis of chicken muscle actinin by calpain-1 and calpain-2 [ 124 , 125 ] also contribute to the regulation of its binding/bundling activity. Thus, α-actinin isoforms are regulated at several different levels, resulting in the functional flexibility needed for fulfilling their numerous cell- and tissue-specific functions.…”