Regulation of ESCRT-III Activity in Yeast

描述(由申请人提供)：运输所需的内体分选复合体(ESCRT)包括一种从酵母到人类保存的细胞机械。ESCRT通过形成腔内小泡(ILV)在晚期内体多泡小体(MVB)的生物发生中发挥作用，ILV直接从内体膜发芽到腔内。ILV通过携带不需要的、受损的或危险的跨膜蛋白，在溶酶体内融合时在溶酶体的水解性内部降解，从而在维持细胞动态平衡方面发挥关键作用。ESCRT-III复合体的功能障碍与神经退行性疾病有关，它的开发对于HIV-1和其他逆转录病毒以及某些细菌病原体的传播至关重要。体外研究表明，ESCRT-III在MVBS的ILV萌发途径中催化膜断裂。本研究的目的是确定在体内控制ESCRT-III活性的调控机制，以发芽酵母酿酒酵母为模型系统。中心假设是ESCRT-III的活性在体内受到控制这种复合体的组装和拆卸的蛋白质的调节。这项研究将研究调节复合体组装和拆卸的蛋白质如何影响ESCRT-III在酵母中的结构和功能。所使用的方法学包括电子显微镜、光学显微镜、蛋白质生物化学和功能分析。这项研究的基本原理是，如果要开发治疗ESCRT-III功能障碍或对抗人类病原体对ESCRT-III的开发的治疗策略，就必须确定ESCRT-III的调节。使用酵母作为模型系统来理解这种调控直接关系到人类的健康，因为ESCRT-III的功能机制是高度保守的。关于预期结果，预计这项研究的完成将揭示调节ESCRT-III活动的核心、广泛保守的机制。这些结果有望产生重要的积极影响，因为它们将从根本上洞察内吞途径中知之甚少的调控步骤，揭示与ESCRT-III相关的疾病预防和治疗的潜在靶点，并启发新的创新方法来了解ESCRT-III的功能机制。

DESCRIPTION (provided by applicant): The endosomal sorting complexes required for transport (ESCRTs) comprise a cellular machinery conserved from yeast to man. ESCRTs function in the biogenesis of late endosomal multivesicular bodies (MVBs) by executing the formation of intralumenal vesicles (ILVs), which bud directly from the endosomal membrane toward the lumen of the compartment. ILVs perform a critical role in maintaining cellular homeostasis by carrying unneeded, damaged, or dangerous transmembrane proteins to be degraded in the hydrolytic interior of the lysosome upon endolysosomal fusion. Dysfunction of the ESCRT-III complex has been linked to neurodegenerative disease, and its exploitation is essential for the spread of HIV-1 and other retroviruses as well as certain bacterial pathogens. In vitro studies have shown that ESCRT-III catalyzes membrane scission in the ILV budding pathway at MVBs. The objective of this research is to determine the regulatory mechanisms that control ESCRT-III activity in vivo, using the budding yeast Saccharomyces cerevisiae as a model system. The central hypothesis is that ESCRT-III activity is regulated in vivo by proteins that control the assembly and disassembly of this complex. This research will study how the structure and function of ESCRT-III is impacted in yeast by proteins that regulate assembly and disassembly of the complex. The methodology to be used includes electron microscopy, light microscopy, protein biochemistry, and functional assays. The rationale for this research is that the regulation of ESCRT-III must be defined if therapeutic strategies are to be developed that will treat ESCRT-III dysfunction or combat exploitations of ESCRT-III by human pathogens. Using yeast as a model system to understand this regulation is directly relevant to human health because the mechanism of ESCRT-III function is highly conserved. With respect to expected outcomes, it is anticipated that completion of this research will reveal the core, broadly conserved mechanisms that regulate ESCRT-III activity. Such results are expected to have an important positive impact because they will yield fundamental insight into a poorly understood regulatory step in the endocytic pathway, reveal potential targets for the prevention and treatment of diseases linked to ESCRT-III, and inspire new and innovative approaches to understand the mechanisms of ESCRT-III function.