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

描述（由申请人提供）：重要跨膜信号受体（包括表皮生长因子受体（EGFR））的下调通过调节信号级联的动力学和幅度对细胞稳态至关重要。内吞作用后，激活的信号受体通过内体区室分选。晚期内体的一个子集通过称为多泡体（MVB）分选的机制促进受体运输到内体腔内的腔内囊泡（ILV）中。进入MVB分选途径的受体在内-溶酶体融合事件后降解。ILV形成需要具有远离细胞质的拓扑结构的膜出芽，这是由转运所需的内体分选复合物- III（ESCRT-III）和AAA-ATP酶Vps 4介导的现象。Vps 4 ATP酶活性通过与一些ESCRT-III亚基的相互作用来调节，预测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Vps 2和Did 2，使用MIT相互作用基序1（MIM 1）刺激Vps 4 ATP酶活性。ESCRT-III亚基Ist 1在体外抑制Vps 4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，我们已经证明，Ist 1也能够刺激Vps 4活性， 在体外以MIM 1-MIT依赖性方式在高盐条件下。这一发现可能与将Ist 1从Vps 4活性的抑制剂转化为刺激剂的体内机制有关 随后在ESCRT-III聚合期间发生从“封闭”到“开放”状态的构象变化。Ist 1被纳入ESCRT-III聚合物的Did 2，这表明与Did 2的相互作用代表的生理相关模式的Ist 1转换为“开放”的构象在体内。本研究培训计划的目标是利用酵母遗传学、细胞生物学和生物化学方法来（1）阐明Ist 1对Vps 4不同调控模式的分子机制，（2）确定Ist 1抑制和刺激结构域在体内介导MVB分选中的作用。这项工作的完成将使人们进一步了解Vps 4活性如何通过多个ESCRT-III亚基协调，从而导致MVB分选，这是一个与神经退行性疾病有关的关键生命过程。从这项研究中获得的见解将广泛应用于研究ESCRT-III介导的其他AAA-ATP酶（如Spastin）调节的潜在机制，Spastin与Ist 1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.