Site-directed activation of calpain is promoted by a membrane-associated natural activator protein

Abstract

Human erythrocytes contain a calpain activator protein with a molecular mass of approx. 40 kDa. The activator is present in association with the plasma membrane and promotes expression of calpain activity at a concentration of Ca2+ close to physiological values. The initial step of the activating mechanism involves association of the activator with calpain, followed by autoproteolytic activation of the proteinase in the presence of 1 microM Ca2+, at a rate identical to that induced by 1 mM Ca2+. In a reconstituted system, the activator binds to erythrocyte membranes, but not to phospholipid vesicles, suggesting the participation of an intrinsic membrane protein(s). In its membrane-associated form the activator selectively binds calpain, thus favouring interaction of the proteinase with the inner surface of plasma membranes. These results further confirm the importance of a natural activator protein in promoting intracellular activation of calpain under physiological conditions through a site-directed mechanism, which explains the high specificity of the proteinase for membrane of cytoskeletal proteins.

Cross-talk between calpain and caspase-3/-7 in cisplatin-induced apoptosis of melanoma cells: a major role of calpain inhibition in cell death protection and p53 status

“…Strictly related to the controversial views on biochemistry of calpain activation (reviewed in Molinari and Carafoli, 1997;Kitagaki et al, 2000), a constant debated problem is to reconcile the actual concentration of Ca 2 þ in the cytosol (nanomolar) with the high Ca 2 þ requirement of calpain in vitro; the Ca 2 þ concentrations giving half-maximal calpain activity in vitro, in fact, are 3-50 and 400-800 mM, for m-calpain and m-calpain, respectively (Goll et al, 2002). Although high calcium concentrations are present in the presynaptic terminals of neurons and under specific pathological processes, much lower calcium concentrations should be sufficient to activate calpain in physiological conditions (Salamino et al, 1993;Zhang et al, 1996). Thus, in addition to calcium, several mechanisms have been proposed, including association to specific membrane phospholipids (see for a review, Molinari and Carafoli, 1997), interactions with activating proteins (Melloni et al, 1998(Melloni et al, , 2000, caspase-mediated degradation of the endogenous inhibitor calpastatin (Wang et al, 1998) and, more recently, extracellular signal-related kinase (ERK)-mediated phosphorylation (Glading et al, 2004).…”

Calpain Inhibition Protects against Virus-Induced Apoptotic Myocardial Injury

“…The mechanisms by which reovirus triggers increased cellular calpain activity are not known but may include initiation of calcium fluxes following viral attachment, as demonstrated with rotavirus, a closely related virus (15); upregulation of growth factors which facilitate calpain activation (37,66); or upregulation of endogenous calpain activator proteins which have been characterized for several cell types (49). Calpain may play a physiologic role in the regulation of a variety of cellular transcription factors and cell cycle-regulating factors implicated in apoptosis, including Jun, Fos, p53, cyclin D, and NF-B (3,7,73).…”

Molecular and Functional Properties of a Calpain Activator Protein Specific for μ-Isoforms

“…One unit of calpain activator activity is defined as the amount causing the appearance of one unit of calpain activity in the presence of 2 M Ca 2ϩ (14).…”