Structure, function, and disease biology of MICU1/MICU2

MICU1/MICU2的结构、功能和疾病生物学

基本信息

批准号：
9980297
负责人：
Zenon Grabarek
金额：
$ 46.43万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9980297
关键词：
Affinity Alleles Animals Binding Bioenergetics Biology Biophysics Calcium Calcium Binding Calcium Channel Calcium Signaling Calcium-Binding Proteins Cell Line Cells Clinical Communication Complex Cryoelectron Microscopy Data Defect Disease EF Hand Motifs Electrons Electrophysiology (science)Energy Metabolism Engineering Enzymes Escherichia coli Exercise Fatigue Fiber Genetic Genomics Goals Grant Histocytochemistry Histology Homeostasis Homologous Gene Human Human Genome Inherited Ion Channel Kinetics Knock-out Knockout Mice Lead Length Lesion Measures Membrane Potentials Metabolic Mitochondria Mitochondrial Myopathies Modeling Molecular Mus Muscle Muscle Contraction Muscle Fatigue Muscle Weakness Mutation Myopathy NADH Names Neurologic Organelles Oxygen Consumption Patients Performance Peripheral Physiology Plasma Positioning Attribute Process Production Property Proteins Regulation Reporting Resolution Rest Roentgen Rays Role Signal Transduction Skeletal Muscle Strenuous Exercise Structural Models Structure System Technology Testing Tissues Work X-Ray Crystallography common symptom design exercise intolerance genome editing in vivo loss of function mutation metabolic profile metabolomics mouse model mutant nervous system disorder neurotransmission response uptake

项目摘要

ABSTRACT Mitochondria uptake calcium via a calcium activated channel called the uniporter. Calcium uptake allows the organelle's metabolic state to be matched to rapidly changing energy requirements, and, in turn, tune key processes such as neurotransmission and muscle contraction. The uniporter is calcium-activated calcium channel regulated by the calcium binding heterodimer MICU1/MICU2. Mutations in MICU1 have recently been identified as a cause of a new form of a myopathy characterized by fatigue and exercise intolerance without the classical features of mitochondrial myopathy. The precise mechanisms by which MICU1 and MICU2 sense calcium to regulate the uniporter, and how lesions in this heterodimer lead to this highly unusual myopathy are not known. Through this dual PI grant we propose to: (1) Characterize the physicochemical properties of MICU1 and MICU2. The wild type proteins and mutants expressed in E. coli will be characterized with respect to the oligomeric state, structural stability, Ca2+ and Mg2+ binding affinities, and pH sensitivity. (2) Determine the structural basis for the regulation of the uniporter by MICU1 and MICU2. High resolution X-ray structures of MICU2 alone and of the MICU1/2 heterodimer in the apo and Ca2+-bound forms will be determined. Electron cryo-microscopy will be used to determine the structure of a native-like oligomer of MICU1/2 complex. (3) Investigate mitochondrial calcium dynamics in cellular systems. Using genome-editing technology we have engineered a powerful in vivo system of knockout cell lines for studying the effects of engineered and naturally occurring human mutations in MICU1 and MICU2 on the mitochondrial calcium transport kinetics and energetics, and (4) Understand the metabolic and bioenergetic basis of human MICU1 myopathy by investigating the Micu1-/- mouse as a model. We will characterize the muscle histology, mitochondrial bioenergetics, exercise performance and metabolomics, and single fiber contractility to test the hypothesis that loss of MICU1 leads to a myopathy by causing disturbances in energy metabolism. Our aims – spanning the molecular to animal physiology -- will yield a holistic and mechanistic understanding of the regulation of mitochondrial calcium uptake by MICU1 and MICU2 and its contribution to a newly described human myopathy.

抽象的线粒体通过称为单转运蛋白的钙激活通道摄取钙。细胞器的代谢状态与快速变化的能量需求相匹配，进而调节神经传递和肌肉收缩等关键过程是钙激活的钙。由钙结合异二聚体 MICU1/MICU2 调节的通道最近已被证实。被确定为一种新形式的肌病的原因，其特征是疲劳和运动不耐受，但没有线粒体肌病的经典特征 MICU1 和 MICU2 感知的精确机制。钙调节单向转运蛋白，以及这种异二聚体的病变如何导致这种极不寻常的肌病通过这项双重 PI 资助，我们建议：（1）表征其物理化学性质。 MICU1 和 MICU2 在大肠杆菌中表达的野生型蛋白和突变体将得到表征。 (2) 确定 MICU1 和 MICU2 的单向转运蛋白调节的结构基础。将测定单独的MICU2以及apo和Ca2+结合形式的MICU1/2异二聚体。电子冷冻显微镜将用于确定 MICU1/2 的类似天然低聚物的结构 (3) 使用基因组编辑技术研究细胞系统中的线粒体钙动力学。我们设计了一个强大的体内敲除细胞系系统，用于研究工程和 MICU1 和 MICU2 自然发生的人类突变对线粒体钙转运动力学的影响能量学，以及 (4) 了解人类 MICU1 肌病的代谢和生物能量基础研究 Micu1-/- 小鼠作为模型，我们将表征肌肉组织学、线粒体。生物能量学、运动表现和代谢组学以及单纤维收缩力来检验以下假设： MICU1 的缺失会导致能量代谢紊乱，从而导致肌病。分子到动物生理学——将对调节产生整体和机械的理解 MICU1 和 MICU2 线粒体钙摄取及其对新描述的人类的贡献肌病。