Acyl-CoA-binding Protein Is a Potent m-Calpain Activator

Abstract

Acyl-CoA-binding protein, a 20-kDa homodimer that exerts many physiological functions, promotes activation of the classic calpain forms, most markedly that of the m-isozyme. This protein factor was purified from rat skeletal muscle and was also expressed in Escherichia coli. Both native and recombinant acyl-CoA-binding proteins show the same molecular properties and an identical capacity to decrease the [Ca 2؉ ] required for m-calpain activity. The binding of long-chain acyl-CoAs to acyl-CoA-binding protein does not modify the activating effect on calpains. Acyl-CoA-binding protein seems to be involved in the m-calpain regulation process, whereas the previously identified UK114 activator is a specific modulator of -calpain. Acyl-CoA-binding protein is proposed as a new component of the Ca 2؉ -dependent proteolytic system. A comparative analysis among levels of classic calpains and their activator proteins is also reported.How the calcium-dependent proteolytic system is activated is an intriguing question in terms of understanding its general properties, physiological functions, and involvement in the occurrence of tissue damage (1-4). Calpains, the enzymatic components of the system, are primarily regulated by calcium ions. The binding of Ca 2ϩ to calmodulin-like domains of the proteinases induces a conformational change that constitutes the initial limiting step of the overall sequential activation process (5). In this new conformation calpains undergo limited autoproteolysis that irreversibly removes the molecular constraints responsible for stabilizing the native inactive form (1-4). In their autoproteolyzed active forms, calpains acquire the highest affinity for calpastatin, which becomes the only negative modulator of calpain activity (6). In rat brain we have recently identified a protein factor, called UK114, which specifically interacts with -calpains and accelerates their activation steps, increasing the affinity for calcium ions (7). The reduced time of calpain activation is consistent with the proposed involvement of the Ca 2ϩ -dependent proteolytic system in specific signal transduction pathways, resulting, for instance, in granule secretion in various cell types (8) or in the conversion of protein kinase C into an autoproteolyzed form produced during the differentiation of murine erythroleukemia cells (9).The rat brain -calpain activator shows a strict specificity for all -calpain isoforms, whereas it has no effect on the classic m-calpains (10), the activation of which requires a calcium concentration at least two orders of magnitude higher than that required by -calpain. Despite reports indicating that free calcium ions can be accumulated in specific cell regions at high micromolar concentrations (11), the calcium requirement for m-calpain activation can apparently be satisfied only during tissue necrosis, a situation far removed from physiological conditions (12). We now report that a highly conserved protein, called acylCoA-binding protein (ACBP) 1 (13-15), shows a potent m-calpain-activating pr...

N Terminus of Calpain 1 Is a Mitochondrial Targeting Sequence

“…Purified -calpain requires 3-50 M Ca 2ϩ for halfmaximal activity (10). Acyl-CoA binding protein, a calpain activator protein, is also present in the mitochondrial IMS and may lower the Ca 2ϩ activation requirements of -calpain (65,66). This suggests the possibility that mitochondrial -calpain may be activated by transient increases in Ca 2ϩ in the IMS (for a recent review, see Ref.…”

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

“…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). In the present study, with the exception of late apoptotic detached cells, we do not detect any change in cytosolic and releasable reticular calcium throughout the time of cisplatin treatment, where calpain activity is early increased over control cells.…”

The Calpain System Is Involved in the Constitutive Regulation of β-Catenin Signaling Functions

“…Rat skeletal muscle m-calpain activity was monitored after incubating for 3 min with His 6 -tagged recombinant proteins Gas2, Gas2DN, and Calpastatin in a calpain assay buffer containing 50 mM sodium borate buffer, pH 7.5, 50 M CaCl 2 , and rat skeletal muscle m-calpain. Activity was measured 10 min after the addition of 1 mM calcium and 2 mg/ml denatured globin as previously described (15). Gas2 and Calpastatin, known calpain inhibitors, were used as control of calpain activity in the same assay.…”

“…For the construction of His 6 -tagged Gas2DN and Calpastatin fusion proteins, specific oligonucleotides upstream and downstream containing, respectively, NcoI and BamHI or NcoI and XhoI sites were used to generate polymerase chain reaction fragments of Gas2DN or Calpastatin that were cloned in pETM11 vector. One unit of calpain activity was defined as the amount of the enzyme causing the production of 1 mol acid-soluble NH 2 revealed with fluorescamine and using acid-denatured globin as substrate (15). Rat skeletal muscle m-calpain activity was monitored after incubating for 3 min with His 6 -tagged recombinant proteins Gas2, Gas2DN, and Calpastatin in a calpain assay buffer containing 50 mM sodium borate buffer, pH 7.5, 50 M CaCl 2 , and rat skeletal muscle m-calpain.…”