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

Abstract

A natural calpain activator protein has been isolated from bovine brain and characterized in its properties and molecular structure. The protein is a homodimer with a molecular mass of about 30 kDa and results in being almost identical to UK114 goat liver protein. Significant similarities with mouse HR12 protein were also observed, whereas a lower degree of similarity was found with a family of heat-responsive proteins named YJGF and YABJ from Haemophilus influenzae and Bacillus subtilis, respectively. The brain activator expresses a strict specificity for the -calpain isoform, being completely ineffective on the m-calpain form. As expected, also UK114 was found to possess calpain-activating properties, indistinguishable from those of bovine brain activator. A protein showing the same calpain-activating activity has been also isolated from human red cells, indicating that this factor is widely expressed. All these activators are efficient on -calpain independently from the source of the proteinase.The high degree of specificity of the calpain activator for a single calpain isoform may be relevant for the understanding of sophisticated intracellular mechanisms underlying intracellular proteolysis. These data are indicating the existence of a new component of the Ca 2؉ -dependent proteolytic system, constituted of members of a chaperonin-like protein family and capable of promoting intracellular calpain activation.Calpains are a family of dimeric proteinases all characterized by an absolute dependence on Ca 2ϩ (1-7). In the absence of this metal ion, calpains are stabilized in an inactive conformational state, by inter-and intramolecular constraints (8, 9). Binding of Ca 2ϩ to the proteinase molecules produces both dissociation of the heterodimers (10) and conformational changes of the 80-kDa catalytic subunits, triggering the enzyme activation that is completed by an autoproteolytic event (11,12). The concentrations of Ca 2ϩ inducing the conformational changes required for the activation of both -and mcalpain are at least one order of magnitude higher than the actual concentrations of this metal ion in cells. Experiments designed to identify possible mechanisms effective in reducing the calcium requirement of calpains have demonstrated that association of the proteinase to phospholipid vesicles (12) or to nuclei (13) are effective in increasing its affinity for Ca 2ϩ . A more relevant physiological significance is represented by a calpain activator protein recently identified in human red blood cells (14) and in rat skeletal muscle (15). This protein factor, which is significantly effective in reducing the requirement of the proteinase for calcium ions, binds Ca 2ϩ with high affinity (10) and associates to the particulate fraction of the cells; in fact, it is recovered in the soluble fraction only when cell lysis is performed by a medium containing metal chelators.Furthermore, the activator-Ca 2ϩ complex interacts with calpain and thereby induces those conformational changes required to trigger the activation pro...

Regulating cell migration: calpains make the cut

“…Similarly, regulation of protein-protein interactions changes the calcium requirements of calpains (Melloni et al, 2000a;Melloni et al, 2000b;Melloni et al, 1998;Melloni et al, 2000c;Michetti et al, 1991;Salamino et al, 1993), but their roles in activation are not clear. Finally, calpains are regulated by their best-known interacting partner, the endogenous calpain inhibitor calpastatin (Wendt et al, 2004).…”

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

“…1 unit of calpain activator activity is defined as the amount that elicits 1 unit of calpain activity in the presence of 5 M Ca 2ϩ (7). Purification of Calpain Activators from Rat Skeletal Muscle-Samples of rat skeletal muscle (100 g) were minced and homogenized in a Waring blender by means of 3 bursts (30 s each) in 10 volumes of ice-cold 0.25 M sucrose containing 1 mM EDTA, 0.5 mM 2-mercaptoeth-* This work was supported by grants from the Italian National Research Council (CNR), "Target Project Biotechnology" and "Biotechnology Project" and from the Italian Ministry of University and Scientific and Technilogical Research (MURST), National Interest Projects (PRIN 1997(PRIN -1998.…”

“…The disrupted cell suspension was centrifuged at 20,000 ϫ g for 15 min, and the supernatant was collected. The purification of calpain activators was then carried out as described previously for calpain activator purification from rat brain (7). Briefly, the pH value of the supernatant was adjusted to 5.8, and the sample was heated to 90°C for 3 min and centrifuged.…”

“…The primers for PCR were selected externally to the translated sequence of mouse brain cDNA for ACBP (accession number X61431). The sense primer was 5Ј-CTTGATTGCTCCTGCTTCTG-3Ј (nucleotides [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]; the antisense primer was 5Ј-TATGTCCTCACAGGTCACACG-3Ј (complementary to nucleotides 353-373). PCR was carried out by using Amplitaq DNA polymerase and by performing 30 cycles of amplification as described previously (24).…”

“…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).…”