Determining the roles of Ist1 inhibition and stimulation of Vps4 ATPase activity

描述(申请人提供)：下调重要的跨膜信号受体，包括表皮生长因子受体(EGFR)，通过调节信号级联的动力学和幅度，对细胞内稳至关重要。在内吞作用之后，激活的信号受体通过内体隔间进行分类。晚期内小体的一个子集通过一种称为多囊泡体(MVB)分选的机制促进受体向腔内小泡(ILV)的运输。进入MVB分选途径的受体在内切溶酶体融合事件后被降解。ILV的形成需要膜出芽，其拓扑结构远离细胞质，这一现象是由运输所需的内体分选复合体(ESCRT-III)和AAA-ATPase Vps4介导的。Vps4 ATPase活性受某些ESCRT-III亚基相互作用的调节，预计会破坏这一过程的突变与神经退行性疾病有关。一些ESCRT-III亚基，包括Vps2和Did2，使用MIT相互作用基序1(MIM1)刺激Vps4 ATPase活性。ESCRT-III的一个亚基Ist1在体外抑制Vps4的活性，尽管这种调节的机制及其在MVB分选中的作用尚不清楚。此外，我们还证明了Ist1也能够刺激Vps4在 在高盐条件下，以MIM1依赖的方式体外培养。这一发现可能与体内将Ist1从抑制Vps4活性转化为刺激Vps4活性的机制有关 在ESCRT-III聚合过程中发生从“封闭”到“开放”状态的构象变化之后。Ist1通过Did2被掺入ESCRT-III聚合物中，这表明与Did2的相互作用代表了Ist1在体内转化为“开放”构象的生理相关模式。本研究培训计划的目标是利用酵母遗传学、细胞生物学和生化方法来(1)阐明Ist1调节Vps4不同模式的分子机制，以及(2)确定Ist1抑制和刺激结构域在体内介导MVB分选中的作用。这项工作的完成将增加对Vps4活性如何由多个ESCRT-III亚基协调导致MVB分选的理解，MVB分选是与神经退行性疾病有关的关键生命过程。这项研究获得的见解将广泛应用于ESCRT-III介导的其他AAA-ATPase调节的潜在机制的研究，如Spastin，这些AAA-ATPase与Ist1相互作用，也与神经退行性疾病有关。

DESCRIPTION (provided by applicant): Down-regulation of important transmembrane signaling receptors, including epidermal growth factor receptor (EGFR), is critical for cellular homeostasis by modulating the kinetics and amplitude of signaling cascades. Following endocytosis, activated signaling receptors are sorted through the endosomal compartment. A subset of late endosomes facilitate receptor trafficking into intralumenal vesicles (ILVs) within the endosomal lumen by a mechanism known as multivesicular body (MVB) sorting. Receptors that enter the MVB sorting pathway are degraded following endo-lysosomal fusion events. ILV formation requires membrane budding with a topology away from the cytoplasm, a phenomenon that is mediated by the endosomal sorting complexes required for transport- III (ESCRT-III), and the AAA-ATPase, Vps4. Vps4 ATPase activity is regulated by interactions with some ESCRT-III subunits, and mutations that are predicted to disrupt this process are implicated in neurodegenerative disease. Some ESCRT-III subunits, including Vps2 and Did2, stimulate Vps4 ATPase activity using MIT-interacting motif 1 (MIM1). One ESCRT-III subunit, Ist1, inhibits Vps4 activity in vitro, although the mechanisms underlying this regulation and its roles in MVB sorting are unclear. In addition, we have demonstrated that Ist1 is also able to stimulate Vps4 activity in vitro in a MIM1- MIT-dependent manner under elevated salt conditions. This finding may be related to an in vivo mechanism that converts Ist1 from an inhibitor to stimulator of Vps4 activity following conformational changes from a "closed" to "open" state that occur during ESCRT-III polymerization. Ist1 is incorporated into ESCRT-III polymers by Did2, suggesting that interactions with Did2 represent the physiologically relevant mode of Ist1 conversion to the "open" conformation in vivo. The goals for this research training program are to utilize yeast genetic, cellular biological, and biochemical approaches to (1) elucidate the molecular mechanisms underlying distinct modes of Vps4 regulation by Ist1 and (2) determine the roles of the Ist1 inhibitory and stimulatory domains in mediating MVB sorting in vivo. Completion of this work will provide an increased understanding of how Vps4 activity is coordinated by multiple ESCRT-III subunits leading to MVB sorting, a critical life process that is implicated in neurodegenerative disease. The insights gained from this research will have broad application to the study potential mechanisms underlying ESCRT-III-mediated regulation of other AAA- ATPases, such as Spastin, which interact with Ist1 and are also implicated in neurodegenerative disease.