A Novel Role of Syntaxin 3 as a Transcription Regulator

Syntaxin 3 作为转录调节因子的新作用

基本信息

批准号：
8549216
负责人：
Thomas Weimbs
金额：
$ 18.46万
依托单位：
UNIVERSITY OF CALIFORNIA SANTA BARBARA
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-21 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8549216
关键词：
Affect Alternative Splicing Apical Apoptosis Autosomal Dominant Polycystic Kidney Binding Binding Proteins Biological C-terminal Cell Line Cell Nucleus Cell Polarity Cell membrane Cells Cellular Membrane Chimeric Proteins Cleaved cell Co-Immunoprecipitations Cyst DNA Binding Defect ETV4 gene Ensure Epithelial Epithelial Cells Epithelium Epitopes Event Gene Expression Gene Expression Regulation Genetic Transcription Glutamine Human In Vitro Integral Membrane Protein Kidney Light MDCK cell Malignant Neoplasms Mass Spectrum Analysis Mediating Membrane Membrane Fusion Membrane Protein Traffic Morphogenesis Morphology Neoplasm Metastasis Nuclear Nuclear Import Pathology Pathway interactions Patients Phenotype Play Property Protein Isoforms Proteins RNA Splicing Regulation Renal Tissue Role SNAP receptor Science Series Signal Transduction Site Specimen Testing Tissues Transcriptional Regulation Work cancer cell cancer type carcinogenesis human tissue in vivo knock-down member mouse model nephrogenesis novel research study small hairpin RNA syntaxin syntaxin 3 trafficking transcription factor tumor

项目摘要

DESCRIPTION (provided by applicant): SNARE proteins mediate membrane fusion events in virtually all cellular membrane trafficking pathways. We have discovered an unexpected, novel function of the SNARE protein syntaxin 3 (Stx3). Stx3 normally has a C-terminal trans-membrane anchor and is involved in trafficking to the apical plasma membrane domain of polarized epithelial cells. We found that Stx3 undergoes cleavage at an extremely conserved glutamine residue which removes its trans-membrane domain resulting in a soluble fragment, Stx3(1-225). Furthermore, a novel splice-isoform of Stx3 (Stx3E) lacks the trans-membrane anchor, and is expressed in human kidneys. Both, the cleavage fragment and Stx3E (collectively called "soluble Stx3") bind to the nuclear import factor RanBP5, target to the nucleus and co-activate several transcription factors including ETV4. ETV4 is required for branching morphogenesis in kidney development, and associated with carcinogenesis and tumor metastasis. We found that kidneys from Autosomal Dominant Polycystic Kidney Disease (ADPKD) patients express a small Stx3 fragment - consistent with soluble Stx3 ! We hypothesize that cleavage and transcriptional regulation in the nucleus is a novel function that may be a common feature of syntaxin members of SNARE proteins. This may be a novel signaling mechanism that transduces information from cytoplasmic membrane trafficking events to the nucleus to affect changes in gene expression. If correct, this would introduce a new paradigm of SNARE function. More specifically, we hypothesize that soluble Stx3 plays a role in the regulation of renal epithelial morphogenesis, carcinogenesis and ADPKD. ! To test these hypotheses, we will pursue the following Specific Aims. In Aim 1, we will investigate the biological effects of soluble Stx3 in polarized epithelial cells. This will be done by expressing soluble Stx3 in epithelial cell lines - or knocking down Stx3E expression - and investigating possible effects on cellular parameters including morphology, proliferation, apoptosis and cell polarity. In Aim 2, we will identify the exact cleavage site of Stx3 in vitro and in vivo. We will investigate the expression of soluble Stx3 in epithelial cancers and ADPKD by using human tissue specimens and mouse models of ADPKD. In Aim 3, we will investigate the mechanism of transcriptional activity of soluble Stx3 and its regulation of ETV4. We will test whether soluble Stx3 interacts with the general transcription machinery and whether it regulates ETV4 stability or nuclear localization.

描述（由申请人提供）：SNARE 蛋白介导几乎所有细胞膜运输途径中的膜融合事件。我们发现了 SNARE 蛋白突触蛋白 3 (Stx3) 的一个意想不到的新功能。 Stx3 通常具有 C 端跨膜锚并参与运输至极化上皮细胞的顶端质膜结构域。我们发现 Stx3 在极其保守的谷氨酰胺残基处发生裂解，去除其跨膜结构域，产生可溶性片段 Stx3(1-225)。此外，Stx3 的一种新剪接异构体 (Stx3E) 缺乏跨膜锚，并在人类肾脏中表达。裂解片段和 Stx3E（统称为“可溶性 Stx3”）均与核输入因子 RanBP5 结合，靶向细胞核并共同激活包括 ETV4 在内的多种转录因子。 ETV4是肾脏发育中分支形态发生所必需的，并且与癌发生和肿瘤转移相关。我们发现常染色体显性多囊肾病 (ADPKD) 患者的肾脏表达一个小的 Stx3 片段 - 与可溶性 Stx3 一致！我们假设细胞核中的裂解和转录调节是一种新功能，可能是 SNARE 蛋白突触融合蛋白成员的共同特征。这可能是一种新颖的信号传导机制，可将信息从细胞质膜运输事件转导至细胞核，从而影响基因表达的变化。如果正确的话，这将引入 SNARE 功能的新范例。更具体地说，我们假设可溶性 Stx3 在肾上皮形态发生、癌变和 ADPKD 的调节中发挥作用。！为了检验这些假设，我们将追求以下具体目标。在目标 1 中，我们将研究可溶性 Stx3 在极化上皮细胞中的生物学效应。这将通过在上皮细胞系中表达可溶性 Stx3（或敲低 Stx3E 表达）并研究对细胞参数（包括形态、增殖、凋亡和细胞极性）的可能影响来完成。在目标 2 中，我们将在体外和体内鉴定 Stx3 的确切切割位点。我们将利用人体组织标本和 ADPKD 小鼠模型来研究可溶性 Stx3 在上皮癌和 ADPKD 中的表达。在目标3中，我们将研究可溶性Stx3的转录活性机制及其对ETV4的调控。我们将测试可溶性 Stx3 是否与一般转录机制相互作用，以及它是否调节 ETV4 稳定性或核定位。