A Novel Role of Syntaxin 3 as a Transcription Regulator

Syntaxin 3 作为转录调节因子的新作用

• 批准号: 8385449
• 负责人: Thomas Weimbs
• 金额: $ 21.95万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-21 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8385449
• 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

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. PUBLIC HEALTH RELEVANCE: Our preliminary work has led to the discovery of a novel signaling function of a member of the SNARE membrane fusion proteins, syntaxin 3. This unexpected finding suggests that other syntaxins may have similar signaling properties in addition to their roles in membrane trafficking and would introduce a new paradigm of SNARE function. Our proposed work is highly significant for a wide range of scientific fields in the biomedical sciences and will shed light on potentially significant implications for our understanding of kidney morphogenesis and carcinogenesis.

描述（由申请人提供）：网罗蛋白几乎在所有细胞膜运输途径中介导膜融合事件。我们发现了SNARE蛋白语法3（STX3）的出乎意料的新功能。 STX3通常具有C末端的跨膜锚，并参与运输到极化上皮细胞的顶端质膜结构域。我们发现STX3在极度保守的谷氨酰胺残基上进行裂解，该残基去除其跨膜结构域，从而产生可溶性片段STX3（1-225）。此外，STX3（STX3E）的一种新型的剪接异构体缺乏跨膜锚，并且在人肾脏中表达。裂解片段和STX3E（统称称为“可溶性STX3”）都与核进口因子RANBP5结合，靶向核，并共同激活包括ETV4在内的几种转录因子。 ETV4是在肾脏发育中分支形态发生所必需的，并且与癌变和肿瘤转移有关。我们发现，来自常染色体显性多囊肾脏疾病（ADPKD）患者的肾脏表达了一个小的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蛋白的义敏素成员的共同特征。这可能是一种新型的信号传导机制，可将信息从细胞质膜运输事件传达到细胞核，以影响基因表达的变化。如果正确，这将引入新的SNARE功能范式。更具体地说，我们假设可溶性STX3在调节肾上皮形态发生，致癌作用和ADPKD中起作用。呢为了检验这些假设，我们将追求以下特定目标。在AIM 1中，我们将研究可溶性STX3在极化上皮细胞中的生物学作用。这将通过在上皮细胞系中表达可溶性STX3或敲低STX3E表达 - 并研究对细胞参数的可能影响，包括形态，增殖，凋亡和细胞极性。在AIM 2中，我们将在体外和体内确定STX3的精确切割位点。我们将通过使用ADPKD的人体组织样品和小鼠模型来研究上皮癌和ADPKD中可溶性STX3的表达。在AIM 3中，我们将研究可溶性STX3转录活性的机理及其对ETV4的调节。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。 公共卫生相关性：我们的初步工作导致发现了SNARE膜融合蛋白成员的新信号传导功能，语法3。这一出乎意料的发现表明，其他语法蛋白除了在膜运输中的作用外还具有相似的信号传导特性，并会引入新的圈圈功能。我们提出的工作对于生物医学科学的各种科学领域非常重要，并将阐明对我们对肾脏形态发生和致癌作用的理解的潜在重要意义。