Proteolytic degradation of nitric oxide synthase isoforms by calpain is modulated by the expression levels of HSP90

Abstract

Ca2+ loading of Jurkat and bovine aorta endothelium cells induces the degradation of the neuronal and endothelial nitric oxide synthases that are selectively expressed in these cell lines. For neuronal nitric oxide synthase, this process involves a conservative limited proteolysis without appreciable loss of catalytic activity. By contrast, endothelial nitic oxide synthase digestion proceeds through a parallel loss of protein and catalytic activity. The chaperone heat shock protein 90 (HSP90) is present in a large amount in Jurkat cells and at significantly lower levels in bovine aorta endothelium cells. The differing ratios of HSP90/nitric oxide synthase (NOS) occurring in the two cell types are responsible for the conservative or nonconservative digestion of NOS isozymes. Consistently, we demonstrate that, in the absence of Ca2+, HSP90 forms binary complexes with NOS isozymes or with calpain. When Ca2+ is present, a ternary complex containing the three proteins is produced. In this associated state, HSP90 and NOS forms are almost completely resistant to calpain digestion, probably due to a structural hindrance and a reduction in the catalytic efficiency of the protease. Thus, the recruitment of calpain in the HSP90–NOS complexes reduces the extent of the proteolysis of these two proteins. We have also observed that calpastatin competes with HSP90 for the binding of calpain in reconstructed systems. Digestion of the proteins present in the complexes can occur only when free active calpain is present in the system. This process can be visualized as a novel mechanism involving the association of NOS with HSP90 and the concomitant recruitment of active calpain in ternary complexes in which the proteolysis of both NOS isozymes and HSP90 is significantly reduced.

Functional Role of HSP90 Complexes with Endothelial Nitric-oxide Synthase (eNOS) and Calpain on Nitric Oxide Generation in Endothelial Cells

“…In previous studies (16,17), we have demonstrated that in the presence of Ca 2ϩ , calpain can be recruited in a ternary complex containing eNOS, HSP90, and the protease. In this associated form, HSP90 and NOS become resistant to calpain digestion.…”

“…These observations could be explained on the basis of the well defined property of HSP90 to bind eNOS, forming discrete complexes (11,17,19). Similarly, we have previously observed -ionophore A23187 for 5 min at 37°C in the absence (ϩCa 2ϩ ) or in the presence of 2 mM Mg-ATP, together with 10 mM phosphoenolpyruvate and 2 g of purified pyruvate kinase (ϩCa 2ϩ ϩATP) were lysed, and aliquots (500 g of soluble protein) obtained as described under "Experimental Procedures" were incubated overnight at 4°C with monoclonal anti-HSP90 antibody.…”

“…that in the presence of Ca 2ϩ , the eNOS-HSP90 complex can recruit calpain generating a ternary complex in which both eNOS and HSP90 were resistant to degradation by the endogenous calpain (17). Thus, the formation of such a ternary complex was analyzed by means of the immunoprecipitation of cytosolic HSP90.…”

“…We have also shown that in intact cells and in rat tissues, the level of HSP90 expression is directly correlated with the extent of NOS degradation. Accordingly, in rat aorta, under conditions of calpain activation, endothelial NOS is much more vulnerable to proteolytic degradation than neuronal NOS (17), since HSP90 is more abundant in brain.…”

Calpain and Atherosclerosis

“…Accordingly, calpain systems appear to play key roles in angiogenesis 36,38) , wound closure 39) and maintenance of barrier functions 13,40) in ECs. In addition to motility responses, it has been reported that nitric oxide (NO) production in ECs is modulated through calpain-induced proteolysis of HSP90 42) or calpain-regulated PI3K/AMPK signaling 35) . The physiological significance of these calpain-mediated responses in ECs, however, remains controversial since the observations were mainly derived from cell-based experiments.…”