内质网
分泌物
蛋白酶体
细胞生物学
高尔基体
转染
溶酶体
内质网相关蛋白降解
突变体
HEK 293细胞
细胞内
化学
ADAMTS13号
分泌途径
蛋白质降解
分子生物学
未折叠蛋白反应
生物
生物化学
免疫学
酶
基因
血栓性血小板减少性紫癜
血小板
作者
Mary Underwood,Flora Peyvandi,Isabella Garagiola,Samuel J. Machin,Ian Mackie
标识
DOI:10.1016/j.thromres.2016.09.014
摘要
Introduction Over 150 mutations have been identified in the ADAMTS13 gene in patients with congenital thrombotic thrombocytopenic purpura (TTP). The majority of these (86%), lead to reduced (<50%) secretion of mutant recombinant ADAMTS13. The mechanism by which this occurs has not been investigated in vitro. Two novel ADAMTS13 mutations (p.I143T and p.Y570C) identified in two congenital adolescence onset TTP patients were studied, to investigate their effects on ADAMTS13 secretion and subcellular localisation. Materials and Methods HEK293T cells were transiently transfected with wild type or mutant ADAMTS13 cDNA. Immunofluorescence and confocal microscopy were used to study localisation within the endoplasmic reticulum (ER) and Golgi. The cell proteasome and lysosomes were inhibited in cells stably expressing ADAMTS13 to investigate degradation of ADAMTS13 by either organelle. Results Both mutations severely impaired secretion and both mutants localised within the ER and Golgi. Proteasome inhibition led to the intracellular accumulation of both mutants, suggesting proteasome degradation. Lysosome inhibition on the other hand did not lead to increased intracellular accumulation of the mutants. Conclusions Proteasome degradation of these ADAMTS13 mutants contributed to their reduced secretion.
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