Biological Mechanism of FSGS-1

FSGS-1的生物学机制

• 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
• 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

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.

这个项目继续建立在我们的长期目标上，即识别和理解基因， 导致人类肾脏疾病。α-辅肌动蛋白-4（ACTN 4）的突变导致一种肾脏疾病 以功能进行性下降、蛋白尿和局灶节段性肾小球硬化为特征 （FSGS）。我们在当前的奖励期内取得了实质性进展， 突变诱导的ACTN 4-肌动蛋白亲和力的改变对肌动蛋白网络的生物物理性质的影响， 体外和细胞水平。我们证明了ACTN 4的遗传改变对细胞增殖有深远的影响。 行为，包括收缩性、运动性、对拉伸的反应和基因转录，并最终产生 肾小球病理学。我们已经有了强有力的证据表明磷酸化事件可以 可逆地调节ACTN 4-肌动蛋白亲和力。这些发现表明， ACTN 4疾病-应变硬化和网络脆性突变是有害的，并且其中ACTN 4 磷酸化允许类似的，但局部和时空调节，变化的肌动蛋白-肌动蛋白 网络 我们的工作假设是，一个通常隐藏的肌动蛋白结合位点（ABS 1）是应变依赖性 网络硬化，但以产生更脆弱的细胞骨架为代价。突变或磷酸化 暴露了这个网站。我们将进一步明确ACTN 4 S159磷酸化在调节细胞凋亡中的作用。 肌动蛋白细胞骨架、细胞粘附和收缩性，以及最后，调节体内足细胞功能。我们 我将利用这些研究作为跳板，以确定信号，激酶和磷酸酶调节 ACTN 4磷酸化。最后，我们将阐明ABS 1介导的细胞应变硬化的重要性 和体内。下一组研究扩展了原提案的目标，并将 不仅提高了我们对ACTN 4突变诱导的FSGS的理解，而且阐明了 肌动蛋白-肌动蛋白网络通过磷酸化事件的时空调节。 具体来说，我们将：1。通过丝氨酸159磷酸化来定义细胞作用和ACTN 4的调节; 2. 定义ACTN 4介导的细胞生物物理行为、局部免疫功能的改变 微环境及其磷酸化调控; 3.定义ABD的调节作用， 使用新的CRISPR衍生物在体内通过磷酸化和突变在肾脏功能中的作用 我们开发的动物模型; 4.扩展我们对ACTN 4改变在 调节基因表达。