Uloga odnosa kalpaina 3 i titina u etiopatogenezi pojasne mišićne distrofije tip 2A (LGMD2A) [The role of Calpain 3 and Titin relationship in the Ethiopatogenesis of limb girdle muscular dystrophy type 2A (LGMD2A)]

Milić, Astrid (2007) Uloga odnosa kalpaina 3 i titina u etiopatogenezi pojasne mišićne distrofije tip 2A (LGMD2A) [The role of Calpain 3 and Titin relationship in the Ethiopatogenesis of limb girdle muscular dystrophy type 2A (LGMD2A)]. PhD thesis, Sveučilište u Zagrebu.

Preview

PDF Download (10MB) | Preview

Abstract

This PhD thesis was directed towards a better understanding of calpain 3 function, especially possible involvement of calpain 3 and titin relationship in the ethiopathogenesis of LGMD2A. In order to improve presently available LGMD2A diagnostic methods, as well as those used in calpain 3 fundamental research, the first part of the thesis was focussed on the development of an in vitro calpain 3 activity assay in which the expression, the autocatalytic and proteolytic properties of calpain 3 are tested at once. This activity assay was validated on 34 human biopsies with moleculary diagnosed LGMD2A, revealing that 30% of LGMD2A biopsies have a normal calpain 3 activity. This finding suggests that additional calpain 3 properties could be responsible for the appearance of LGMD2A phenotype. The work aimed at better understanding of the relationship between calpain 3 and titin resulted in the identification of two C-terminal titin fragments, p45 and p15, that are products of in vivo cleavage by calpain 3. Titin fragments were not observed in the absence of calpain 3 (LGMD2A), as well as in the presence of mutations in the last exon of the gene coding for titin (LGMD2J). This inadequate processing of titin represents a common characteristic of LGMD2A and LGMD2J and may be, at least partially, part of their pathophysiological mechanisms. The search for calpain 3 substrates resulted in the identification of MURF1, MURF2alt and PSMC3 as calpain 3 in vitro substrates. Implication of these proteins in the process of protein degradation suggests a possible involvement of calpain 3, and therefore LGMD2A phenotype, in this process.

Abstract in Croatian

Disertacija je bila usmjerena prema boljem razumijevanju funkcije kalpaina 3, posebice uloge odnosa kalpaina 3 i titina u etiopatogenezi pojasne mišićne distrofije tip 2A (LGMD2A). U cilju poboljšanja trenutno dostupnih LGMD2A dijagnostičkih metoda, kao i onih koji se koriste za osnovna znanstvena istraživanja kalpaina 3, prvi dio disertacije bio je posvećen razvitku i optimizaciji enzimskog testa kojim se istovremeno uz izražaj analizira i autolitička i proteolitička aktivnost kalpaina 3 u in vitro uvjetima. Primjena enzimskog testa na 34 uzorka mišića molekularno potvrđenih bolesnika s LGMD2A pokazala je da u 30% LGMD2A biopsija kalpain 3 ima normalnu enzimsku aktivnost. Ovaj rezultat upućuje na zaključak da bi, osim proteolitičke aktivnosti kalpaina 3, i neka njegova druga svojstva mogla biti odgovorna za nastanak LGMD2A. Istraživanje usmjereno prema boljem razumijevanju odnosa kalpaina 3 i titina pokazalo je da u C-terminalnom dijelu titina postoje dva mjesta podložna cijepanju kalpainom 3. Rezultirajući proteinski fragmenti, p45 i p15, odsutni su ukoliko nema izražaja kalpaina 3 (LGMD2A), ali i ukoliko su prisutne mutacije u Mex6 egzonu gena za titin (LGMD2J). Stoga se može zaključiti da je, unatoč različitom genskom uzroku, izostanak cijepanja titina zajednička karakterisitka LGMD2A i LGMD2J, te bi mogao, barem djelomično, biti odgovoran za nastanak ove dvije distrofije. Ispitivanje različitih proteina kao potencijalnih supstrata kalpaina 3 pokazalo je da su MURF1, MURF2alt i PSMC3 cijepani kalpainom 3 u in vitro uvjetima. Uključenost sva tri supstrata u proces proteinske razgradnje upućuje na moguću povezanost kalpaina 3, odnosno nastanka LGMD2A fenotipa, sa tim procesom.

Actions (login required)

View Item

Downloads