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Biogenesis of voltage-gated K+ channels

电压门控 K 通道的生物发生

基本信息

批准号：
10669584
负责人：
Carol J Deutsch
金额：
$ 42.22万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-05-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10669584
关键词：
Address Applications Grants Architecture Attention Autoimmune Diseases Biochemical Biogenesis Biological Biological Assay Biophysics Cells Cessation of life Chemicals Clinical Communication Complex Confined Spaces Coupled Cryoelectron Microscopy Defect Disease Drug Targeting Electronic Health Record Electrophysiology (science)Electrostatics Engineering Etiology Event Failure Feedback Future Generations Genes Genetic Suppression Genome Genotype Human Image Immune Impairment Individual Investigation Laboratories Lead Length Ligand Binding Link Location Lymphocyte Activation Measures Medicine Membrane Modeling Modification Molecular Molecular Chaperones Movement Mutation Nature Nerve Neurons Outcome Pathologic Pathology Peptides Pharmaceutical Preparations Phenotype Potassium Potassium Channel Process Proteins Proteome Regulation Repression Research Ribosomal Proteins Ribosomes Role Severities Source Speed Structure T-Lymphocyte Therapeutic Time Translation Process Translations Variant Vision Voltage-Gated Potassium Channel Work biobank chronic inflammatory disease combat constriction design exome sequencing genetic variant human disease innovation insight lymphocyte proliferation molecular dynamics novel particle pleiotropism programs protein aminoacid sequence protein expression protein folding protein function rare variant reconstruction remediation trafficking transmission process voltage

项目摘要

Our research program aims to understand how a voltage-gated potassium (Kv) channel is made. This is a complicated multi-step process that requires acquisition of local secondary, tertiary, and quaternary structures, either sequentially or as coupled events. How this happens is, for the most part, unknown. Yet the impact of these steps is profound, often with pathological consequences. We focus our attention on the human tetrameric Kv1.3 channel, particularly its early-stage folding, and the determinants that regulate these folding events in the exit tunnel of the ribosome and the ER membrane. Three Aims comprise this grant proposal. The ability of a protein to form helices is a fundamental prerequisite for protein folding and function in all proteomes. However, this process has not been defined in the confined and heterogeneous microenvironment of the ribosome exit tunnel where a protein is first made. Helicity must occur at the right time and place during translation. Failure to meet this requirement impairs peptide targeting, chaperone association, efficient bilayer insertion, and oligomerization. In Aim 1, we specifically ask when, where, how, and why these critical secondary structures arise in the Kv1.3 nascent peptide. We will determine the molecular mechanisms that delay or initiate helix formation and identify the underlying peptide-tunnel interactions that are responsible. To do this, we use biochemical approaches and cryo-EM single particle reconstruction of peptide-ribosome complexes. In Aim 2, we explore an exciting new field of fundamental importance to how proteins are made, namely, how a peptide's sequence generates piconewtons of force that fine tune Kv peptide's rate of elongation and folding efficiency. We use experimental approaches and molecular dynamics simulations to identify the type of peptide-tunnel interactions giving rise to force, the nature of the force, and its consequences as the peptide is elongated. Given that human Kv1.3 is expressed in neuronal and immune cells, and impaired expression produces chronic inflammatory disease and autoimmune disorders, it is compelling to ask whether human disease-linked variants of the KCNA3, the gene that encodes Kv1.3, introduce folding/assembly/trafficking defects. In Aim 3, we address this question using the recently developed “genome- first” approach to determine the clinical consequences of specific KCNA3 rare variants and biophysical determinations of Kv1.3 folding and function to identify the molecular defects. Our overall vision of Kv folding includes complex coupled events between intrapeptide segments, the ribosome exit tunnel, and the ER membrane. We now expand this view by introducing two new concepts for further investigation: 1) repressor/activator activity acts as a molecular switch to govern the time and tunnel location of Kv helix initiation, and 2) cotranslational force generation modulates translation rates and folding. Both concepts represent paradigm shifts that reveal additional levels of regulation for Kv channel folding and may generalize to the biogenesis of other proteins.

我们的研究计划旨在了解电压门控钾 (Kv) 通道是如何形成的。这是一个复杂的多步骤过程，需要获取局部二级、三级和四级结构，顺序或作为耦合事件。在大多数情况下，这是如何发生的尚不清楚。然而影响这些步骤意义深远，往往会带来病态后果。我们将注意力集中在人类四聚体上 Kv1.3通道，特别是其早期折叠，以及调节这些折叠事件的决定因素核糖体和内质网膜的出口通道。本拨款提案包含三个目标。的能力蛋白质形成螺旋是所有蛋白质组中蛋白质折叠和功能的基本先决条件。然而，这一过程尚未在有限且异质的微环境中得到定义。核糖体退出隧道，蛋白质首先在这里产生。螺旋性必须在正确的时间和地点发生翻译。未能满足这一要求会损害肽靶向、伴侣关联、高效双层插入和寡聚化。在目标 1 中，我们特别询问何时、何地、如何以及为何这些关键的问题。二级结构出现在 Kv1.3 新生肽中。我们将确定分子机制延迟或启动螺旋形成并识别潜在的肽-隧道相互作用。为此，我们使用生化方法和肽-核糖体的冷冻电镜单粒子重建复合物。在目标 2 中，我们探索了一个令人兴奋的新领域，这对于蛋白质的制造至关重要，也就是说，肽的序列如何产生皮牛顿的力来微调 Kv 肽的延伸率和折叠效率。我们使用实验方法和分子动力学模拟来识别产生力的肽-隧道相互作用的类型、力的性质及其后果肽被拉长。鉴于人类 Kv1.3 在神经元和免疫细胞中表达，并且受损表达会产生慢性炎症性疾病和自身免疫性疾病，因此我们迫切需要问是否 KCNA3 的人类疾病相关变体（编码 Kv1.3 的基因）引入折叠/组装/运输缺陷。在目标 3 中，我们使用最近开发的“基因组”来解决这个问题第一”方法来确定特定 KCNA3 罕见变异的临床后果和生物物理测定 Kv1.3 折叠和功能以识别分子缺陷。我们对 Kv 折叠的总体愿景包括肽内片段、核糖体出口隧道和 ER 之间的复杂耦合事件膜。我们现在通过引入两个新概念来扩展这一观点以供进一步研究：1）抑制子/激活子活性充当分子开关来控制 Kv 螺旋的时间和隧道位置起始，2) 共平移力的产生调节平移速率和折叠。两个概念代表范式转变，揭示了 Kv 通道折叠的额外调节水平，并可能推广其他蛋白质的生物合成。