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

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 mu-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 mu-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 Ca2+-dependent proteolytic system, constituted of members of a chaperonin-like protein family and capable of promoting intracellular calpain activation

Modulation of rat brain calpastatin efficiency by post-translational modifications

AbstractCalpains, the thiol proteinases of the calcium-dependent proteolytic system, are regulated by a natural inhibitor, calpastatin, which is present in brain tissue in two forms. Although both calpastatins are highly active on human erythrocyte calpain, only one form shows a high inhibitory efficiency with both rat brain calpain isozymes. The second calpastatin form is almost completely inactive against homologous proteinases and can be converted into an active one by exposure to a phosphoprotein phosphatase, also isolated from rat brain. Phosphorylation of the active calpastatin by protein kinase C and protein kinase A promotes a decrease in its inhibitory efficiency. The interconversion between the two inhibitor forms seems involved in the adjustment of the level of intracellular calpastatin activity on specific cell requirements

Autolysis of human erythrocyte calpain produces two active enzyme forms with different cell localization

AbstractThe 80 kDa human erythrocyte calpain, when exposed to Ca2+, undergoes autoproteolysis that generates a 75 kDa species, with an increase in Ca2+ affinity. It is demonstrated here that this proteolytic modification proceeds through an initial step producing a 78 kDa form which is rapidly converted to the 75 kDa one. In the presence of the calpain inhibitor E-64, the 78 kDa form accumulates and only small amounts of the 75 kDa polypeptide are formed. Following loading of erythrocytes with micromolar concentration of Ca2+, in the presence of the ionophore A23187, the native 80 kDa calpain subunit is extensively translocated and retained at the plasma membrane, this process is accompanied by the appearance of only a small amount of the 75 kDa subunit which is released into the soluble fraction of the cells. Following exposure to μM Ca2+, membrane-bound 80 kDa calpain is converted to the 78 kDa form, this conversion being linearly correlated with the expression of the proteinase activity. Taken together, these results demonstrate that the initial step in calpain activation involves Ca2+-induced translocation to the inner surface of plasma membranes. In the membrane-bound form the native inactive 80 kDa subunit is converted through intramolecular autoproteolysis to a locally active 78 kDa form. Further autoproteolytic intermolecular digestion converts the 78 kDa to the 75 kDa form, no longer being retained by the membrane. This process generates two active forms of calpain, with different intracellular localisations

Calpain digestion and HSP90-based chaperone protection modulate the level of plasma membrane F508del-CFTR

AbstractWe are here showing that peripheral mononuclear blood cells (PBMC) from cystic fibrosis (CF) patients contain almost undetectable amounts of mature 170 kDa CF-transmembrane conductance regulator (CFTR) and a highly represented 100 kDa form. This CFTR protein, resembling the form produced by calpain digestion and present, although in lower amounts, also in normal PBMC, is localized in cytoplasmic internal vesicles. These observations are thus revealing that the calpain-mediated proteolysis is largely increased in cells from CF patients. To characterize the process leading to the accumulation of such split CFTR, FRT cells expressing the F508del-CFTR mutated channel protein and human leukaemic T cell line (JA3), expressing wild type CFTR were used. In in vitro experiments, the sensitivity of the mutated channel to the protease is identical to that of the wild type, whereas in Ca2+-loaded cells F508del-CFTR is more susceptible to digestion. Inhibition of intracellular calpain activity prevents CFTR degradation and leads to a 10-fold increase in the level of F508del-CFTR at the plasma membrane, further indicating the involvement of calpain activity in the maintenance of very low levels of mature channel form. The higher sensitivity to calpain of the mutated 170 kDa CFTR results from a reduced affinity for HSP90 causing a lower degree of protection from calpain digestion. The recovery of HSP90 binding capacity in F508del-CFTR, following digestion, explains the large accumulation of the 100 kDa CFTR form in circulating PBMC from CF patients