TULP1 Missense Mutations Induces the Endoplasmic Reticulum Unfolded Protein Response Stress Complex (ER-UPR)
TULP1 Missense Mutations Induces the Endoplasmic Reticulum Unfolded Protein Response Stress Complex (ER-UPR)
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DOI：
10.1007/978-3-319-17121-0_30
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发表时间：
2016-01-01
期刊：
影响因子：
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通讯作者：
Hagstrom, Stephanie A.
中科院分区：
文献类型：
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作者：
Lobo, Glenn P.;Ebke, Lindsey A.;Hagstrom, Stephanie A.
TULP1基因突变与早发性视网膜色素变性(RP)有关；然而，与TULP1突变的有害影响相关的分子机制尚不清楚。一些研究表明，基因突变导致的错误折叠蛋白可以在内质网(ER)内积聚，导致未折叠蛋白反应(UPR)复合体的激活，继而导致细胞凋亡。我们假设TULP1突变产生错误折叠的蛋白质产物，这些蛋白质产物在内质网中积累，并通过UPR诱导细胞凋亡。为了验证我们的假设，我们首先对TULP1错义突变(I459K、R420P和F491L)进行了三次电子分析，这些突变预测了错误折叠的蛋白质产物。随后，将突变型TULP1-GFP和野生型(Wt)TULP1-GFP瞬时导入hTERT-RPE-1细胞。用ER追踪器和共聚焦显微镜对细胞进行染色，结果显示wt-TULP1主要定位于细胞质和质膜。相反，三种突变型TULP1蛋白均显示与内质网共定位的细胞质斑点染色。此外，表达突变TULP1蛋白的细胞的蛋白质印迹分析显示，ER-UPR复合体的下游靶点被诱导，包括Bip/GPR-78、磷酸化PERK(Thr980)和CHOP。我们的体外分析表明，突变的TULP1蛋白被错误折叠并积聚在内质网中，导致诱导UPR应激反应复合体。
Mutations in the TULP1 gene are associated with early-onset retinitis pigmentosa (RP); however, the molecular mechanisms related to the deleterious effects of TULP1 mutations remains unknown. Several studies have shown that misfolded proteins secondary to genetic mutations can accumulate within the endoplasmic reticulum (ER), causing activation of the unfolded protein response (UPR) complex followed by cellular apoptosis. We hypothesize that TULP1 mutations produce misfolded protein products that accumulate in the ER and induce cellular apoptosis via the UPR. To test our hypothesis, we first performed three in-silico analyses of TULP1 missense mutations (I459K, R420P and F491L), which predicted misfolded protein products. Subsequently, the three mutant TULP1-GFP constructs and wildtype (wt) TULP1-GFP were transiently transfected into hTERT-RPE-1 cells. Staining of cells using ER tracker followed by confocal microscopy showed wt-TULP1 localized predominantly to the cytoplasm and plasma membrane. In contrast, all three mutant TULP1 proteins revealed cytoplasmic punctate staining which colocalized with the ER. Furthermore, western blot analysis of cells expressing mutant TULP1 proteins revealed induction of downstream targets of the ER-UPR complex, including BiP/GPR-78, phosphorylated-PERK (Thr980) and CHOP. Our in-vitro analyses suggest that mutant TULP1 proteins are misfolded and accumulate within the ER leading to induction of the UPR stress response complex.