Biological Mechanism of FSGS-1

FSGS-1的生物学机制

基本信息

批准号：
9319727
负责人：
MARTIN R. POLLAK
金额：
$ 41.39万
依托单位：
BETH ISRAEL DEACONESS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-07-21 至 2021-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9319727
关键词：
Actinin Actins Address Adhesions Adriamycin PFS Affinity Albumins Alpha Cell Angiopoietin-2 Animal Model Animals Award Binding Biological Biophysics CRISPR screen CRISPR/Cas technology Calcium Cell Adhesion Cell Survival Cell membrane Cells Characteristics Clustered Regularly Interspaced Short Palindromic Repeats Contracts Cues Cytoskeleton Defect Disease Event Focal Adhesions Focal Segmental Glomerulosclerosis Gene Expression Gene Expression Profile Genes Genetic Transcription Goals Heparin Human In Vitro Induced Mutation Infusion procedures Kidney Kidney Diseases Kidney Failure Liquid substance Measurement Measures Mechanics Mediating Modeling Mus Mutant Strains Mice Mutation Natural experiment Nephrotoxic Organism Pathology Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Property Protamine Sulfate Proteins Proteinuria Recovery Regulation Renal function Role Serine Serum Signal Transduction Site Small Interfering RNA Solid Stress Stretching Surveys Testing Toxic effect Traction Transforming Growth Factor beta Work alpha Actinin biophysical properties cell behavior cell motility disease-causing mutation fluidity functional decline human disease in vivo knockout animal migration mouse model mutant nephrotoxicity podocyte prevent response spatiotemporal tool transcriptome

项目摘要

This project continues to build on our-long-term goal of identifying and understanding genes which when altered cause human kidney disease. Mutations in alpha-actinin-4 (ACTN4) cause a form of kidney disease characterized by progressive decline in function, proteinuria, and focal segmental glomerulosclerosis (FSGS). We have made substantial progress during the current award period in understanding the role of mutation-induced alterations in ACTN4-actin affinity on the biophysical properties of the actin network at in vitro and cellular levels. We demonstrated that genetic alterations in ACTN4 have profound effects on cell behavior including contractility, motility, response to stretch, and gene transcription, and ultimately produce glomerular pathology in the organism. We have generated strong evidence that phosphorylation events can reversibly modulate the ACTN4-actin affinity. These findings suggest a model where the global effects of ACTN4 disease-mutations on strain hardening and network brittleness are detrimental, and where ACTN4 phosphorylation allows for similar, but local and spatiotemporally regulated, changes to the actinin-actin network. Our working hypothesis is that a normally hidden actin-biding site (ABS1) is required for strain-dependent network hardening but at the expense of generating a more brittle cytoskeleton. Mutation or phosphorylation exposes this site. We will further define the role of ACTN4 S159 phosphorylation in regulating the cellular actin cytoskeleton, cellular adhesion and contractility, and lastly, in regulating in vivo podocyte function. We will use these studies as a springboard for identifying the signals, kinases and phosphatases regulating ACTN4 phosphorylation. Finally, we will elucidate the importance of ABS1-mediated strain hardening in cells and in vivo. This next set of studies extend and expand upon the aims of the original proposal, and will advance our understanding of not only ACTN4-mutation induced FSGS but also elucidate the importance of spatiotemporal regulation of the actinin-actin network through phosphorylation events. Specifically, we will: 1. Define the cellular role and regulation of ACTN4 by serine 159 phosphorylation; 2. Define the relationship between ACTN4-mediated, biophysical behavior of cells, alterations to the local microenvironment, and its regulation by phosphorylation; 3. Define the role of regulation of the ABD of ACTN4 by phosphorylation and mutation in the function of the kidney in vivo using new CRISPR-derived animal models we have developed; 4. Extend our understanding of the effects of ACTN4 alterations in regulating gene expression.

该项目继续以我们为了识别和理解基因的长期目标建立改变引起人类肾脏疾病。 α-肌动蛋白4（ACTN4）的突变引起肾脏疾病的形式其功能，蛋白尿和局灶性节段性肾小球硬化的特征是（FSG）。我们在当前奖励期间取得了重大进展，以理解突变引起的ACTN4-肌动蛋白亲和力的改变对肌动蛋白网络的生物物理特性体外和细胞水平。我们证明了ACTN4的遗传改变对细胞具有深远的影响包括收缩力，运动性，对拉伸和基因转录的行为，并最终产生生物体中的肾小球病理。我们已经产生了有力的证据，表明磷酸化事件可以可逆地调节ACTN4-肌动蛋白亲和力。这些发现提出了一个模型，其中的全球影响 ACTN4疾病 - 菌株硬化和网络脆性的疾病 - 有害，而actn4 磷酸化允许类似但局部和时空调节，阳脱素 - 肌动蛋白的变化网络。我们的工作假设是，依赖性依赖性需要一个通常隐藏的肌动蛋白抗原位点（ABS1）网络硬化，但要产生更脆的细胞骨架。突变或磷酸化公开此网站。我们将进一步定义ACTN4 S159磷酸化在调节细胞中的作用肌动蛋白细胞骨架，细胞粘附和收缩力，最后是调节体内足细胞功能。我们将这些研究用作识别调节信号，激酶和磷酸酶的跳板 ACTN4磷酸化。最后，我们将阐明细胞中ABS1介导的应变硬化的重要性和体内。下一组研究扩展并扩展了原始提案的目的，并将促进我们对ATACN4突变引起的FSG的理解，还阐明了通过磷酸化事件对阳历原 - 肌动蛋白网络的时空调节。具体而言，我们将：1。通过丝氨酸159磷酸化定义ACTN4的细胞作用和调节； 2。定义ACTN4介导的细胞生物物理行为之间的关系，对局部的改变微环境及其通过磷酸化调节； 3。定义调节ABD的作用 ACTN4通过新的CRISPR衍生我们开发的动物模型； 4。扩展我们对ACTN4改变影响的理解调节基因表达。