L444P ve N370S mutasyonları taşıyan gaucher hastalarında makrootofaji-lizozomal sistem mals

Year 2013, Issue: 1, 59 - 74, 01.01.2013

İncilay (sinici) Lay

Abstract

Gaucher hastalığı, lizozomal bir enzim olan glukoserebrosidazın EC 3.2.1.45 aktivitesinin bozukluğu sonucu ortaya çıkar. Lizozomlara hücre dışından endositoz ve fagositoz ve hücre içinden makrootofaji-lizozomal sistem makromoleküller taşınmaktadır. Bu çalışmada, Gaucher hastalığında biriken glukozilseramid ve glukozilsfingozinin normal otofajik füzyon süreci ve/veya otofajik akışı bozabilme ve klinik fenotipi şiddetlendirme olasılığı araştırıldı. Otofaji indüksiyonu ve otofagozomların otolizozomlara bozuk maturasyonu/füzyonu durumunun, otofajik akış otolizozomlara kargonun taşınması bozukluğundan ayırt edilmesi için, en sık gözlenen L444P ve N370S mutasyonlarını taşıyan Gaucher fibroblastlarında LC3B-II düzeyleri ve lizozomlarda floresan DQ-BSA’nın proteolizi Cellomics KSR kullanılarak izlendi. LC3B-I ve GAPDH’a göre artmış düzeydeki LC3B-II, hastaların fibroblastlarında otofagozomların sayısında artışı ve/veya azalmış lizozoma akış hızını gösterdi. Bu durum hasta fenotipleri ile uyumlu bulundu. Hasta fibroblastlarında DQ-BSA’nın düşük proteolizi, Gaucher hastalığında otofajik akış hızının azaldığını göstermektedir. Azalmış lizozoma akış hızı, L444P mutasyonu taşıyan Tip II, akut nöropatik formda daha belirgindi. Sonuç olarak, L444P ve N370S mutasyonlarında otofajik ve LC3B-II’yi de içeren diğer substratların lizozomlara akış hızı azalmıştır ve bu azalma klinik şiddet ile kabaca uyumludur.

Keywords

Makrootofaji-lizozomal sistem, Gaucher, LC3B-II, DQ-BSA

Macroautophagy-Lysosomal System Mals in Gaucher Patients Carrying L444P and N370S Mutations

Abstract

Gaucher disease is caused by defects in the activity of the lysosomal enzyme, glucocerebrosidase EC 3.2.1.45 . Since lysosomes ultimately are responsible for turning over macromolecules transported to them from both outside endocytosis and phagocytosis and inside the cell macroautophagy-lysosomal system , we explored the possibility that the glucosylceramide and glucosylsphingosine accumulation resulting from Gaucher disease may disrupt the normal autophagic fusion process and/or autophagic flux exacerbating the clinical phenotype. To discriminate between an induction of autophagy and defective maturation/fusion of autophagosomes to autolysosomes, versus autophagic flux turnover of cargo in the autolysosome , LC3B-II levels and proteolysis of internalized fluorescent DQ-BSA in lysosomes with the use of high throughput instrument Cellomics KSR were monitored in Gaucher fibroblasts carrying the most prevelant mutations L444P and N370S. Increased levels of LC3B-II relative to LC3B-I and GAPDH demonstrated an increase in the number of autophagosomes and/or a decrease rate of lysosomal turn-over flux in fibroblasts of patients which were correlated with the phenotypes. Lower turnover of the DQ-BSA in patient fibroblasts indicated that the flux rate was decreased in Gaucher disease. The decrease of lysosomal flux was more significant in the Type II, acute neurophathic form of L444P mutation. We can conclude that L444P and N370S mutations decreased the the rate of turn-over of autophagy and other substrates, including LC3B-II, in the lysosome and that the decreases roughly correlated with the clinical severity.

Keywords

Macroautophagy-lysosomal system, Gaucher, LC3B-II, DQ-BSA

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