Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Md; Leprosy Laboratory, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Md.
J Allergy Clin Immunol. 2018 May;141(5):1844-1853.e2. doi: 10.1016/j.jaci.2017.07.027. Epub 2017 Aug 30.
Sumoylation is a posttranslational reversible modification of cellular proteins through the conjugation of small ubiquitin-related modifier (SUMO) and comprises an important regulator of protein function.
We sought to characterize the molecular mechanism of a novel mutation at the SUMO motif on signal transducer and activator of transcription 1 (STAT1).
STAT1 sequencing and functional characterization were performed in transfection experiments by using immunoblotting and immunoprecipitation in STAT1-deficient cell lines. Transcriptional response and target gene activation were also investigated in PBMCs.
We identified a novel STAT1 mutation (c.2114A>T, p.E705V) within the SUMO motif (IKTE) in a patient with disseminated Rhodococcus species infection, Norwegian scabies, chronic mucocutaneous candidiasis, hypothyroidism, and esophageal squamous cell carcinoma. The mutation is located in the tail segment and is predicted to disrupt STAT1 sumoylation. Immunoprecipitation experiments performed in transfected cells confirmed absent STAT1 sumoylation for E705V, whereas it was present in wild-type (WT) STAT1 cells, as well as the loss-of-function mutants L706S and Y701C. Furthermore, stimulation with IFN-γ led to enhanced STAT1 phosphorylation, enhanced transcriptional activity, and target gene expression in the E705V-transfected compared with WT-transfected cells. Computer modeling of WT and mutant STAT1 molecules showed variations in the accessibility of the phosphorylation site Y701, which corresponded to the loss-of-function and gain-of-function variants.
This is the first report of a mutation in the STAT1 sumoylation motif associated with clinical disease. These data reinforce sumoylation as a key posttranslational regulatory modification of STAT1 and identify a novel mechanism for gain-of-function STAT1 disease in human subjects.
SUMO 化是通过小泛素相关修饰物（SUMO）与细胞蛋白的缀合对其进行的一种翻译后可逆修饰，是蛋白质功能的重要调节因子。
我们旨在研究信号转导和转录激活因子 1（STAT1）SUMO 基序上的新型突变的分子机制。
在 STAT1 缺陷细胞系中通过免疫印迹和免疫沉淀进行 STAT1 测序和功能特征分析，以进行转染实验。还在 PBMC 中研究了转录反应和靶基因激活。
我们在患有播散性罗地西亚菌感染、挪威疥疮、慢性黏膜皮肤念珠菌病、甲状腺功能减退和食管鳞状细胞癌的患者中发现了 STAT1 突变（c.2114A>T，p.E705V），该突变位于 SUMO 基序（IKTE）内。突变位于尾部片段，预计会破坏 STAT1 的 SUMO 化。转染细胞中的免疫沉淀实验证实 E705V 无 STAT1 SUMO 化，而在 WT STAT1 细胞、失活突变体 L706S 和 Y701C 中则存在。此外，与 WT 转染细胞相比，用 IFN-γ 刺激可增强 E705V 转染细胞中的 STAT1 磷酸化、转录活性和靶基因表达。WT 和突变 STAT1 分子的计算机建模显示 Y701 磷酸化位点的可及性存在差异，这与失活和激活功能变体相对应。
这是首次报道与临床疾病相关的 STAT1 SUMO 化基序突变。这些数据强化了 SUMO 化作为 STAT1 的关键翻译后调节修饰，并确定了人类体内激活功能 STAT1 疾病的新机制。
