Calpain-1-dependent degradation of troponin I mutants found in familial hypertrophic cardiomyopathy.
The mechanism by which mutations of the cardiac troponin I (cTnI) gene evoke familial hypertrophic cardiomyopathy (fHCM) is unknown. In this investigation the potential effects of three fHCM-related cTnI mutations on Calpain-1-induced cTnI degradation were tested, and a study was made of whether additional conformational changes due to troponin complex formation and protein kinase A-induced phosphorylation affect the intensity of cTnI proteolysis. Purified recombinant wild-type cTnI and three of its fHCM-related missense mutants (R145G, G203S and K206Q), alone or in the troponin complex (i.e. together with troponin C and troponin T), in the non-phosphorylated or protein kinase A-bisphosphorylated forms were proteolyzed in vitro in the presence of Calpain-1 (0.05-2.5 U) at 30 degrees C. Following incubation with Calpain-1 for 0.5, 30, 60 or 120 min, the extent of protein degradation was evaluated through the use of Western immunoblotting and densitometry. The results indicated that both the wild-type and the mutant cTnI molecules were susceptible to Calpain-1. However, the degradation of the cTnI molecules in the troponin complex was less intense than that of the non-complexed forms. Moreover, phosphorylation by protein kinase A conferred effective protection against cTnI proteolysis. The data suggested that mutations in the central inhibitory domain (R145G) and in the C-terminal region (G203S and K206Q) of cTnI do not affect its Calpain-1-mediated degradation, or the phosphorylation-induced protection against proteolysis.
Department of Cardiology, University of Debrecen, MHSC, Debrecen, Hungary.
Judit
J
Barta
Attila
A
Tóth
Kornelia
K
Jaquet
Alexander
A
Redlich
István
I
Edes
Zoltán
Z
Papp
D015153:Blotting, Western; D002154:Calpain / Q000378:metabolism*; D024741:Cardiomyopathy, Hypertrophic, Familial / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003720:Densitometry; D006801:Humans; D009154:Mutation / Q000187:drug effects; D020125:Mutation, Missense; D009206:Myocardium / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors; D019209:Troponin C / Q000378:metabolism; D019210:Troponin I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D020107:Troponin T / Q000378:metabolism
D011994:Recombinant Proteins; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T; D017868:Cyclic AMP-Dependent Protein Kinases; D002154:Calpain