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（~ 100 kDa）GTP酶，其在细胞膜运输、线粒体分裂和其他地方的膜断裂事件中发挥关键作用。发动蛋白同种型的功能在细胞内运输、信号传导、神经元功能等中是中心的，影响许多人类疾病中受影响的过程。最近，动力蛋白的pleckstrin同源（PH）结构域（我们研究的一个焦点）的突变被发现会导致Charcot-Marie-Tooth病，一种遗传性进行性神经病。参与线粒体分裂的动力蛋白家族（人的DRP 1，酵母的Dnmlp）在细胞凋亡中起关键作用，控制细胞凋亡是重要的临床目标。因此，了解这些GTP酶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是重要的。发动蛋白家族成员共有3个共同的结构域：N-末端GTP酶结构域、“中间”结构域和GTP酶效应子结构域（GED）。它们经历由GTP结合和水解调节的组装和拆卸循环。据信，这种循环与精确位置的膜断裂有关，尽管它是如何联系起来的仍然是一个激烈辩论的主题。除了共同的结构域之外，每个发动蛋白家族成员还包含可能将蛋白质靶向其特定位置和/或将其GTdR活性与膜断裂联系起来的独特结构域。发动蛋白具有普列克底物蛋白同源（PH）结构域和脯氨酸/富脯氨酸结构域（PRO）。DRP 1和Dnmlp具有取代PH结构域的不同的“Drp”或“插入B”结构域。我们的重点是了解这些领域的机械作用。尽管动力蛋白的PH结构域结合磷酸肌醇，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，动力蛋白似乎集群Ptdlns（4，5）P2在膜（通过其PH结构域），提供了一个可能的机制，动力蛋白的参与肌动蛋白成核。以这种方式调节脂质分布可能反映了发动蛋白PH结构域（以及其他家族成员中的等效结构域）的“效应器”功能，我们建议对此进行研究。结合细胞和体外生物化学/生物物理方法，我们提出了以下具体目标：1。为了检验发动蛋白PH结构域作为效应物而不是靶向结构域瞬时调节PtdIns（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.验证心磷脂通过与Drp结构域结合在Dnm 1 p/DRP 1功能中发挥关键作用的假设。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.