Rapid Proteasomal Degradation of Mutant Proteins Is the Primary Mechanism Leading to Tumorigenesis in Patients With Missense AIP Mutations.
Rapid Proteasomal Degradation of Mutant Proteins Is the Primary Mechanism Leading to Tumorigenesis in Patients With Missense AIP Mutations.
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DOI：
10.1210/jc.2016-1307
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发表时间：
2016-08
期刊：
影响因子：
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通讯作者：
Korbonits M
中科院分区：
文献类型：
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作者：
Hernández-Ramírez LC;Martucci F;Morgan RM;Trivellin G;Tilley D;Ramos-Guajardo N;Iacovazzo D;D'Acquisto F;Prodromou C;Korbonits M
芳香烃受体相互作用蛋白(AIP)基因突变在垂体腺瘤中的致病作用尚不完全清楚。我们已经确定了错义AIPmuts功能丧失的主要机制。这项研究试图分析野生型和错义AIP变异体的蛋白质周转机制/速度，将蛋白质半衰期与临床参数相关联。对AIPmut阳性患者的资料进行了半衰期和蛋白质-蛋白质相互作用实验和横断面分析。数据来自我们的垂体腺瘤患者队列和文献报道的病例。用放线菌素追踪法和蛋白酶体抑制法分析了两个细胞系和HEK293细胞中高表达的15个AIP变异体内源性AIP的蛋白质周转。谷胱甘肽-S-转移酶下拉和定量质谱分析鉴定了与AIP降解有关的蛋白质；免疫共沉淀和基因敲除证实了结果。收集相关临床资料。野生型和突变型AIP蛋白的半衰期及其与临床参数的相关性。HEK293和淋巴母细胞的内源性AIP半衰期相似(43.5和32.7h)。AIP变异体分为稳定蛋白(中位数77.7h；四分位区间60.7~92.9h)、半衰期短(中位数27h；IQR 21.6~28.7h)和极短半衰期(中位数7.7h；IQR 5.6~10.5h)；蛋白酶体抑制挽救了突变蛋白的快速降解。实验半衰期与肢端肥大症/巨人症的诊断年龄显著相关(r=0.411；P=0.002)。含有Fbxo3的Skp1-CUL1-F-box蛋白复合体被鉴定为识别AIP的E3泛素连接酶。AIP是一种稳定的蛋白质，由Skp1-CUL1-F-box蛋白复合体驱动泛素化。蛋白酶体降解增强是AIPmuts的一种新的致病机制，与表型有直接关系。我们确定，导致蛋白质半衰期缩短的蛋白质不稳定是AIP基因突变的一种新的致病机制，对垂体腺瘤患者具有临床意义。
The pathogenic effect of mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene (AIPmuts) in pituitary adenomas is incompletely understood. We have identified the primary mechanism of loss of function for missense AIPmuts. This study sought to analyze the mechanism/speed of protein turnover of wild-type and missense AIP variants, correlating protein half-life with clinical parameters. Half-life and protein–protein interaction experiments and cross-sectional analysis of AIPmut positive patients' data were performed in a clinical academic research institution. Data were obtained from our cohort of pituitary adenoma patients and literature-reported cases. Protein turnover of endogenous AIP in two cell lines and fifteen AIP variants overexpressed in HEK293 cells was analyzed via cycloheximide chase and proteasome inhibition. Glutathione-S-transferase pull-down and quantitative mass spectrometry identified proteins involved in AIP degradation; results were confirmed by coimmunoprecipitation and gene knockdown. Relevant clinical data was collected. Half-life of wild-type and mutant AIP proteins and its correlation with clinical parameters. Endogenous AIP half-life was similar in HEK293 and lymphoblastoid cells (43.5 and 32.7 h). AIP variants were divided into stable proteins (median, 77.7 h; interquartile range [IQR], 60.7–92.9 h), and those with short (median, 27 h; IQR, 21.6–28.7 h) or very short (median, 7.7 h; IQR, 5.6–10.5 h) half-life; proteasomal inhibition rescued the rapid degradation of mutant proteins. The experimental half-life significantly correlated with age at diagnosis of acromegaly/gigantism (r = 0.411; P = .002). The FBXO3-containing SKP1–CUL1–F-box protein complex was identified as the E3 ubiquitin-ligase recognizing AIP. AIP is a stable protein, driven to ubiquitination by the SKP1–CUL1–F-box protein complex. Enhanced proteasomal degradation is a novel pathogenic mechanism for AIPmuts, with direct implications for the phenotype. We determined that protein instability, leading to shortened protein half-life, is a novel pathogenic mechanism for mutations in the AIP gene with clinical significance for pituitary adenoma patients.