A Novel Role of Syntaxin 3 as a Transcription Regulator

描述(由申请人提供)：SNARE蛋白在几乎所有细胞膜运输途径中介导膜融合事件。我们发现了SNARE蛋白合成素3(Stx3)一个意想不到的新功能。Stx3通常有一个C末端跨膜锚定，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在一个极其保守的谷氨酰胺残基上发生切割，该残基去除了它的跨膜结构域，产生了一个可溶片段Stx3(1-225)。此外，一种新的Stx3剪接异构体(Stx3E)缺乏跨膜锚定，并在人肾脏中表达。切割片段和Stx3E(统称为可溶性Stx3)都与核进口因子RanBP5结合，靶向细胞核，并共同激活包括ETV4在内的几个转录因子。ETV4是肾脏发育中分支形态发生所必需的，并与肿瘤的发生和转移有关。我们发现，常染色体显性遗传性多囊肾病(ADPKD)患者的肾脏表达一个小的Stx3片段--与可溶性Stx3一致！我们推测，核内的切割和转录调节是一种新的功能，可能是SNARE蛋白的合成素成员的共同特征。这可能是一种新的信号机制，将细胞膜转运事件中的信息传递到细胞核，从而影响基因表达的变化。如果这是正确的，这将引入一种新的陷阱功能范式。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的调控中发挥作用。好了！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作用。这将通过在上皮细胞系中表达可溶性Stx3-或下调Stx3E的表达-并研究对细胞参数的可能影响，包括形态、增殖、凋亡和细胞极性来实现。在目标2中，我们将在体外和体内确定Stx3的确切切割位置。我们将利用人组织标本和小鼠ADPKD模型来研究可溶性Stx3在上皮性肿瘤和ADPKD中的表达。在目标3中，我们将研究可溶性Stx3转录活性的机制及其对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的稳定性或核定位。 与公共卫生相关：我们的初步工作发现了SNARE膜融合蛋白的一种新的信号功能--Synaxin 3。这一意想不到的发现表明，除了在膜运输中发挥作用外，其他Synaxins可能具有类似的信号特性，并将引入一种新的SNARE功能范式。我们提出的工作对于生物医学科学中广泛的科学领域具有非常重要的意义，并将阐明对我们理解肾脏形态发生和癌症发生的潜在重要影响。

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.