Mechanisms of dynamin family GTPases

描述（由申请人提供）：该提案侧重于动力蛋白家族大（~100kDa） gtp酶，该酶在细胞膜运输，线粒体裂变和其他地方的膜断裂事件中起关键作用。动力蛋白异构体的功能在细胞内运输、信号传导、神经元功能等方面起着中心作用，影响着许多人类疾病的过程。最近，在动力蛋白的pleckstrin同源（PH）结构域的突变（我们的研究重点之一）被发现导致腓骨肌萎缩症，一种遗传性进行性神经病变。参与线粒体分裂的动力蛋白亲属（人类DRP1，酵母Dnmlp）在细胞凋亡中起关键作用，其控制是重要的临床目标。因此，了解这些gtpase的机制对于开发控制细胞凋亡、癌症、神经元变性和许多其他疾病的方法非常重要。Dynamin家族成员共有3个共同结构域：n端GTPase结构域、中间结构域和GTPase-effector结构域（GED）。它们经历由GTP结合和水解调控的组装和拆卸循环。人们相信，这种循环与膜在精确位置的断裂有关，尽管它是如何联系起来的仍然是一个激烈争论的话题。除了共同结构域外，每个动力蛋白家族成员都包含独特的结构域，这些结构域可能将蛋白质靶向到其特定位置和/或将其GTPase活性与膜断裂联系起来。Dynamin具有pleckstrin同源结构域（PH）和脯氨酸/精氨酸富结构域（PRO）。DRP1和Dnmlp具有不同的“Drp”或“插入B”结构域，取代PH结构域。我们的重点是了解这些领域的机制作用。虽然动力蛋白的PH结构域与磷酸肌苷结合，这对其功能很重要，但我们的初步数据表明，它不像其他PH结构域那样作为膜靶向模块。相反，动力蛋白似乎在膜上聚集Ptdlns(4,5)P2（通过其PH结构域），这为动力蛋白参与肌动蛋白成核提供了可能的机制。以这种方式调节脂质分布可能反映了动力蛋白PH结构域的“效应”功能（可能在其他家族成员中也有类似的结构域），我们建议对此进行研究。结合细胞和体外生化/生物物理方法，我们提出以下具体目标：为了验证动力蛋白PH结构域作为效应器而不是靶向性结构域瞬时调节Ptdlns(4,5)P2密度的假设。我们还将研究动力蛋白以Ptdlns(4,5) p2依赖的方式促进肌动蛋白成核的机制。2. 为了验证心磷脂通过结合Drp结构域在Dnm1p/DRP1功能中起关键作用的假设。我们还将研究心磷脂识别的结构基础。总之，这些研究有望为这些重要的大型gtp酶的作用机制提供有价值的见解。

DESCRIPTION (provided by applicant): This proposal focuses on the dynamin-family large (~100kDa) GTPases that play critical roles in membrane scission events during cellular membrane trafficking, mitochondrial fission, and elsewhere. The functions of dynamin isoforms are central in intracellular trafficking, signaling, neuronal function, etc, impacting processes affected in many human diseases. Most recently, mutations in the pleckstrin homology (PH) domain of dynamin (one focus of our studies) were found to cause Charcot-Marie-Tooth disease, a hereditary progressive neuropathy. The dynamin relatives involved in mitochondrial fission (DRP1 in human, Dnmlp in yeast) play a key role in apoptosis, control of which is an important clinical goal. Thus, understanding the mechanisms of these GTPases is important for developing approaches to control apoptosis, cancer, neuronal degeneration, and many other diseases. Dynamin family members share 3 common domains: an N-terminal GTPase domain, a 'middle' domain, and a GTPase-effector domain (GED). They undergo cycles of assembly and disassembly regulated by GTP binding and hydrolysis. It is believed that this cycling is linked to the scission of membranes at precise locations, although how it is linked remains a subject of intense debate. In addition to the common domains, each dynamin family member contains unique domains likely to target the protein to its specific location and/or to link its GTPase activity to membrane scission. Dynamin has a pleckstrin homology (PH) domain and a proline/arginine-rich domain (PRO). DRP1 and Dnmlp have a distinct 'Drp' or 'insert B' domain that replaces the PH domain. Our focus is to understand the mechanistic role of these domains. Although the PH domain of dynamin binds phosphoinositides, and this is important for its function, our preliminary data suggest that it does not act as a membrane targeting module like other PH domains. Instead, dynamin appears to cluster Ptdlns(4,5)P2 in membranes (through its PH domain), providing a possible mechanism for dynamin's involvement in actin nucleation. Modulating lipid distribution in this way may reflect an 'effector' function for the PH domain of dynamin (and perhaps equivalent domains in other family members), which we propose to investigate. Combining cellular and in vitro biochemical/biophysical approaches, we propose the following specific aims: 1. To test the hypothesis that the dynamin PH domain functions as an effector, rather than a targeting, domain - transiently modulating Ptdlns(4,5)P2 density. We will also investigate the mechanisms through which dynamin promotes actin nucleation in a Ptdlns(4,5)P2-dependent manner. 2. To test the hypothesis that cardiolipin plays a critical role in Dnm1p/DRP1 function by binding to the Drp domain. We will also investigate the structural basis for cardiolipin recognition. Together, these studies promise to provide valuable insight into the mechanisms of action of these important large GTPases.