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

项目摘要/摘要 一些具有细胞内外功能的关键蛋白质，通过 内质网和高尔基体，通过非常规的蛋白质分泌(UPS)途径释放。这些路线是 演变为在空间和时间上控制这些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路径通常是 由细胞应激触发，例如，在低氧转移肿瘤和低能量条件下的细胞中。一些UPS 货物由监护人协助，但许多其他人是独立释放的。他们的释放涉及到自我 细胞膜(I型UPS)或细胞器(III型UPS)的持续直接交叉。 这里的根本问题是UPS分泌的蛋白质是如何进入细胞器的，以及它们的本质是如何 跨膜转运是受调控的。明确分子调控机制具有重要意义 推动新的治疗策略(例如，UPS调节器)对与其相关的疾病的意义 扰乱了细胞的分布。我们提出了一个新的假说来解释调节和定向释放 这些在肿瘤进展中起关键作用的蛋白质。低氧会引起短暂或持久的细胞酸化。在……里面 这个模型，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方式。在III型UPS的背景下，这促进了自我维持 蛋白质跨内体膜转运和最终分泌。为了检验这一假设，我们将 确定两种重要的UPS货运蛋白脑型肌酸的胞外释放机制 激酶和鞘氨醇激酶亚型1和2.这些蛋白在不同癌症中的胞外释放 在很大程度上有助于转移细胞的存活。这一机制是由细胞外介导的 他们的生物活性产品的生产。这项提议的主题是潜在的两亲性蛋白质 能够可逆地与膜相互作用。因此，定义构象重排触发 这些蛋白质的释放需要识别在低能量状态下填充的构象状态 低氧转移细胞。测试可逆结构折叠和整合到 由于其动态状态和获得高分辨率结构的困难，膜是具有挑战性的 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，我们结合了一系列互补和尖端的方法 方法以及细胞生物学研究。用类似的方法研究其他药物的释放机制 关键的治疗靶点将测试它们细胞外释放机制的共性和差异。 最终，我们的研究有可能确定一种非传统的蛋白质分泌途径， 癌细胞和其他病理情况。此外，作为一个长期目标，我们将在基础研究之间架起桥梁 通过在模型生物体中检验我们的关键结论，用翻译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.