Proteolytic degradation of nitric oxide synthase isoforms by calpain is modulated by the expression levels of HSP90.
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.
Averna
Monica
M
Department of Experimental Medicine (DIMES)-Biochemistry Section and Centre of Excellence for Biomedical Research (CEBR), University of Genoa, Italy.
Stifanese
Roberto
R
De Tullio
Roberta
R
Salamino
Franca
F
Bertuccio
Mara
M
Pontremoli
Sandro
S
Melloni
Edon
E
D000818:Animals; D001011:Aorta / Q000166:cytology; D002135:Calcium-Binding Proteins / Q000378:metabolism / Q000494:pharmacology; D002154:Calpain / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D002417:Cattle; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism / Q000494:pharmacology; D042783:Endothelial Cells / Q000201:enzymology / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology; D004912:Erythrocytes / Q000737:chemistry; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D019169:Jurkat Cells; D008954:Models, Biological; D019001:Nitric Oxide Synthase / Q000302:isolation & purification / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000302:isolation & purification / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000302:isolation & purification / Q000378:metabolism; D011233:Precipitin Tests
D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D018841:HSP90 Heat-Shock Proteins; D007527:Isoenzymes; C033668:calpastatin; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; D002154:Calpain