Novel molecular mechanism for extracellular release of proteins implicated in metastatic cancer

项目总结/摘要 一些在细胞内外都有功能的关键蛋白质，通过细胞外基质绕过常规分泌， ER和高尔基体，并通过非常规蛋白质分泌（UPS）途径释放。这些路由 发展到在空间上和时间上控制这些UPS货物的功能和触发释放， 某些刺激，或在常规途径受损时激活。因此，UPS途径通常 由细胞应激触发，例如，在低能量条件下的低氧转移性肿瘤和细胞中。一些起起 货物由伴侣协助，但许多其他货物是独立释放的。他们的释放包括自我- 持续直接穿过细胞膜（I型UPS）或细胞器（III型UPS）。 这里的基本问题是UPS分泌的蛋白质如何进入细胞器，以及它们的基本功能是如何进入细胞器的。 跨膜转运受到调节。确定分子调控机制具有重要意义。 推动新的治疗策略的重要性（例如，UPS调节剂）用于与其 受干扰的细胞分布我们提出了一个新的假说，解释了调节和定向释放 这些关键蛋白质在肿瘤进展中的作用。缺氧引起短暂或持久的细胞酸化。在 在这个模型中，局部酸度和膜曲率之间的相互作用决定了构象状态 以及这些货物的膜结合模式。在III型UPS的背景下，这促进了自维持 蛋白质穿过内体膜的移位和最终的分泌。为了验证这个假设，我们将 确定两个重要的UPS货物蛋白，脑型肌酸的细胞外释放机制 激酶和鞘氨醇激酶亚型1和2。这些蛋白质在各种癌症中的细胞外释放 对转移性细胞的存活有实质性贡献。这种机制是由细胞外 生产其生物活性产品。该提案的主题是潜在的两性蛋白质 能够可逆地与膜相互作用。因此，定义触发构象重排的构象， 这些蛋白质的释放需要鉴定在低能状态下占据的构象状态 低氧转移细胞的生长测试可逆结构重折叠的参与和并入 由于其动态状态和获得高分辨率结构的困难， 膜结合蛋白质状态的信息，特别是膜曲率对蛋白质的影响 结构因此，我们将各种方法结合起来，包括一系列互补和尖端的方法， 方法以及细胞生物学研究。使用类似的方法来研究其他药物的释放机制， 关键的治疗靶点将测试其细胞外释放机制的共性和差异。 最终，我们的研究有可能定义一种非常规的蛋白质分泌途径， 癌细胞和其他病理状况。此外，作为长期目标，我们将弥合我们的基础研究 通过在模式生物中测试我们的关键结论，用转化研究阐明机制的研究。

Project Summary/Abstract Some critical proteins, with functions both inside and outside of cells, circumvent conventional secretion via the ER and Golgi and are released through Unconventional Protein Secretion (UPS) pathways. These routes are evolved either to spatially and temporally control the function and the triggered release of these UPS cargoes by certain stimuli, or to activate upon impairment of the conventional pathway. Hence, UPS pathways are often triggered by cellular stress, e.g., in hypoxic metastatic tumors and cells under low energy conditions. Some UPS cargos are assisted by chaperones, but many others are released independently. Their release involves self- sustained direct crossing of a membrane, either the cell membrane (Type I UPS) or organelles (Type III UPS). The fundamental question here is how UPS secreted proteins enter organelles and how their essential translocation across membranes is regulated. Defining the molecular regulatory mechanisms is of high significance to drive new therapeutic strategies (e.g., UPS modulators) for diseases associated with their perturbed cellular distributions. We propose a novel hypothesis that explains the regulated and directed release of these key proteins in tumor progression. Hypoxia instigates a transient or enduring cellular acidification. In this model, the interplay between the local acidity and membrane curvature determines the conformational states and membrane-binding mode of these cargoes. In the context of a Type III UPS, this promotes self-sustained protein translocation across endosomal membranes and ultimate secretion. To test this hypothesis, we will determine the extracellular release mechanism of two important UPS cargo proteins, the brain-type creatine kinase and sphingosine kinase isoforms 1 and 2. Extracellular release of these proteins in various cancers contributes substantially to the survival of metastatic cells. This mechanism is mediated by extracellular production of their biologically active products. The subjects of this proposal as potential amphitropic proteins are able to reversibly interact with a membrane. Thus, defining the conformational rearrangements triggering the release of these proteins entails identifying the conformational states that are populated under low energy status of the hypoxic metastatic cells. Testing the involvement of reversible structural refolding and incorporation into the membrane is challenging due to their dynamic states and the difficulties of gaining high-resolution structural information of membrane-bound protein states, particularly the effects of membrane curvature on the protein structure. Thus, we have combined approaches encompassing a range of complementary and cutting-edge methods as well as cell biology studies. Using a similar methodology to study the release mechanism of other key therapeutic targets will test commonalities and differences in their extracellular release mechanism. Ultimately, our research has the potential to define an unconventional protein secretion pathway employed by cancer cells and other pathological conditions. In addition, as a long-term goal, we will bridge our basic research studies that elucidate mechanism with translational research by testing our key conclusions in model organisms.