Determining the mechanism of mitochondrial outer membrane fusion

确定线粒体外膜融合的机制

基本信息

批准号：
9701515
负责人：
Suzanne C Hoppins
金额：
$ 12.77万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-02-01 至 2022-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9701515
关键词：
Address Alleles Alzheimer&apos s Disease Apoptosis Apoptotic Binding Biochemical Biochemical Reaction Biological Assay C-terminal Catalytic Domain Cell Cycle Progression Cell Death Cell physiology Cells Characteristics Chimeric Proteins Complex Couples Dependence Disease Dissection Dynamin Environment Evaluation Event Family Foundations GTP Binding Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Hydrolysis Immune response Impairment In Vitro Kinetics Length Lipids Liposomes Malignant Neoplasms Mediating Membrane Membrane Fusion Methods Missense Mutation Mitochondria Molecular Molecular Conformation Molecular Machines Monitor Movement Mutation Mutation Analysis N-terminal Nucleotides Outer Mitochondrial Membrane Point Mutation Process Production Property Proteins Public Health Recombinants Role Structure System Work experimental study falls insight member mitochondrial dysfunction mutant proteoliposomes reconstitution self assembly support network

项目摘要

Project Summary Mitochondria are required for a myriad of cellular functions. The mitochondrial network in cells is established and maintained by the coordinate activities of movement, fusion and division. Mitochondrial fusion in particular is beneficial as it increases cellular energy and protects against cell death. The molecular machines that mediate mitochondrial outer and inner membrane fusion are members of the dynamin superfamily. As such, they are large self-assembling GTPases that harness the energy from nucleotide hydrolysis to remodel membranes. How these proteins couple self-assembly and the catalytic cycle to membrane tethering and lipid mixing is not known. The focus of this work is to examine the molecular mechanism of mitochondrial outer membrane fusion. Although Mitofusin1 (Mfn1) and Mitofusin2 (Mfn2) are both required for mitochondrial outer membrane fusion, they are functionally distinct. We discovered that mitochondrial fusion is most efficient when Mfn1 and Mfn2 are on opposite membranes. This suggests that they have unique molecular characteristics. To address this, we will compare and contrast Mfn1 and Mfn2 throughout our analyses and determine their unique biochemical properties. Our evaluation of the GTPase domain will reveal the mechanism of nucleotide binding, hydrolysis and release. We will reconstitute Mitofusin-dependent membrane tethering. This will allow us to determine the role of the catalytic cycle in membrane tethering and the role of lipid composition on Mitofusin tethering activity. Using disease associated missense mutations, we will identify unique features of the GTPase domain. We will identify the molecular determinants of Mitofusin complex assembly. We will combine cellular studies with powerful biochemical analyses, including reconstitution of lipid and content mixing with proteoliposomes. These studies will provide new insight into the unique mechanism of mitochondrial outer membrane fusion.

项目摘要线粒体是无数细胞功能所必需的。建立了细胞中的线粒体网络并由运动，融合和分裂的协调活动维护。线粒体融合特别是有益于增加细胞能量并防止细胞死亡。分子机介导线粒体外膜和内膜融合是Dynamin超家族的成员。作为这样，它们是大型的自组装GTP酶，可利用核苷酸水解到重塑的能量膜。这些蛋白质如何自组装以及催化周期与膜束缚和脂质混合尚不清楚。这项工作的重点是检查线粒体外部的分子机制膜融合。虽然线粒体外部需要Mitofusin1（MFN1）和Mitofusin2（MFN2）膜融合，它们在功能上是不同的。我们发现线粒体融合是最有效的当MFN1和MFN2在相对的膜上时。这表明它们具有独特的分子特征。为了解决这个问题，我们将在分析过程中比较和对比MFN1和MFN2，确定其独特的生化特性。我们对GTPase域的评估将揭示核苷酸结合，水解和释放的机理。我们将重构依赖丝曲表示膜束缚。这将使我们能够确定催化周期在膜束缚中的作用和脂质组成在丝线链蛋白链接活性中的作用。使用疾病相关的错义突变，我们将确定GTPase域的独特功能。我们将识别丝线表示的分子决定因素复杂的组件。我们将将细胞研究与强大的生化分析相结合，包括脂质和含量混合与蛋白质脂质体的重构。这些研究将为您提供新的见解线粒体外膜融合的独特机制。