Background

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.

Objective

We sought to characterize the molecular mechanism of a novel mutation at the SUMO motif on signal transducer and activator of transcription 1 (STAT1).

Methods

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.

Results

We identified a novel STAT1 mutation (c.2114A>T, p.E705V) within the SUMO motif (⁷⁰²IKTE⁷⁰⁵) 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.

Conclusion

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.

中文翻译：

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新型信号转导器和转录激活剂 1 突变破坏小泛素相关修饰物缀合，导致功能获得

 背景

Sumoylation 是通过小泛素相关修饰剂 (SUMO) 的缀合对细胞蛋白质进行翻译后可逆修饰，并且是蛋白质功能的重要调节剂。

 客观的

我们试图表征信号转导子和转录激活子 1 (STAT1) 上 SUMO 基序的新突变的分子机制。

 方法

通过在 STAT1 缺陷细胞系中使用免疫印迹和免疫沉淀，在转染实验中进行 STAT1 测序和功能表征。还在 PBMC 中研究了转录反应和靶基因激活。

 结果

我们在一名患有播散性红球菌属感染、挪威疥疮、慢性皮肤粘膜念珠菌病、甲状腺功能减退症和食管鳞状细胞癌的患者中，在 SUMO 基序 ( ⁷⁰² IKTE ⁷⁰⁵ ) 内发现了一种新的STAT1突变 (c.2114A>T, p.E705V)。该突变位于尾部，预计会破坏 STAT1 sumoylation。在转染细胞中进行的免疫沉淀实验证实，E705V 不存在 STAT1 sumoylation，而野生型 (WT) STAT1 细胞以及功能丧失突变体 L706S 和 Y701C 中却存在 STAT1 sumoylation。此外，与 WT 转染细胞相比，IFN-γ 刺激导致 E705V 转染细胞中 STAT1 磷酸化增强、转录活性增强和靶基因表达增强。 WT 和突变 STAT1 分子的计算机模型显示磷酸化位点 Y701 的可及性存在变化，这对应于功能丧失和功能获得变体。

 结论

这是与临床疾病相关的 STAT1 sumoylation 基序突变的首次报道。这些数据强化了 sumoylation 作为 STAT1 的关键翻译后调节修饰，并确定了人类受试者中 STAT1 功能获得性疾病的新机制。