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

与转移性癌症有关的蛋白质细胞外释放的新分子机制

基本信息

批准号：
10680461
负责人：
Reza Dastvan
金额：
$ 42.85万
依托单位：
SAINT LOUIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-15 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10680461
关键词：
Acidity Acidosis Atomic Force Microscopy Basic Science Binding Binding Proteins Biochemical Biological Assay Biosensor Brain Cell Survival Cell membrane Cells Cellular Stress Cellular biology Creatine Kinase Cryoelectron Microscopy Crystallography Data Development Disease Disseminated Malignant Neoplasm Electron Spin Resonance Spectroscopy Electrons Enzymes Foundations Goals Golgi Apparatus Human Hypoxia Impairment In Vitro Knowledge Lipids Liposomes MAPK3 gene Malignant Neoplasms Maps Mass Spectrum Analysis Measures Mediating Membrane Membrane Potentials Methodology Methods Mission Modeling Molecular Molecular Chaperones Molecular Conformation Neoplasm Metastasis Organelles Pathologic Pathway interactions Phosphoserine Physiological Process Production Prognosis Protein Conformation Protein Isoforms Protein Kinase Protein Secretion Protein translocation Proteins Proteolysis Protomer Public Health Regulation Research Resolution Role Route Site Solvents Speed Stimulus Structure Testing Translational Research Validation cancer cell colon cancer cell line dimer endosome membrane extracellular flexibility fluorescence imaging innovation membrane model metastatic colorectal model organism mutant novel novel therapeutic intervention protein structure recruit research study restraint sphingosine kinase therapeutic target tumor progression

项目摘要

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.

项目概要/摘要一些关键蛋白质在细胞内外都有功能，通过 ER 和高尔基体通过非常规蛋白质分泌 (UPS) 途径释放。这些路线是进化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，例如在低氧转移性肿瘤和低能量条件下的细胞中。一些UPS 货物由陪同人员协助，但许多其他货物是独立释放的。他们的释放涉及自我持续直接穿过膜，无论是细胞膜（I 型 UPS）还是细胞器（III 型 UPS）。这里的根本问题是 UPS 分泌的蛋白质如何进入细胞器以及它们的必需物质如何跨膜易位受到调节。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 调节剂）治疗与其相关的疾病具有重要意义扰乱细胞分布。我们提出了一个新的假设来解释调节和定向释放这些关键蛋白在肿瘤进展中的作用。缺氧会引发短暂或持久的细胞酸化。在在该模型中，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，这促进了自我维持蛋白质跨内体膜易位和最终分泌。为了检验这个假设，我们将确定两种重要的 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细胞的存活有很大贡献。该机制是由细胞外介导的生产其生物活性产品。该提案的主题是潜在的两亲性蛋白质能够与膜发生可逆的相互作用。因此，定义触发的构象重排这些蛋白质的释放需要识别低能量状态下的构象状态缺氧的转移细胞。测试可逆结构重折叠和掺入的参与由于其动态状态和获得高分辨率结构的困难，膜具有挑战性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，我们结合了涵盖一系列互补和前沿的方法方法以及细胞生物学研究。使用类似的方法研究其他释放机制关键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，作为一个长期目标，我们将加强我们的基础研究通过在模型生物中测试我们的关键结论来阐明转化研究机制的研究。