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识别和理解基因的长期目标上，当 基因改变会导致人类肾脏疾病。α-肌动蛋白-4(ACTN4)基因突变导致一种肾脏疾病 以功能进行性下降、蛋白尿和局灶性节段性肾小球硬化为特征 (FSGS)。在当前的获奖期内，我们在理解 ACTN4-肌动蛋白亲和力突变对肌动蛋白网络生物物理性质的影响 体外和细胞水平。我们证明了ACTN4的基因改变对细胞有深远的影响 行为包括收缩、运动、对拉伸的反应和基因转录，并最终产生 生物体中的肾小球病理。我们已经产生了强有力的证据表明，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，在这个模型中， ACTN4病-菌株硬化和网络脆性的突变是有害的，其中ACTN4 磷酸化可以使肌动蛋白发生类似的、但局部的和时空调节的变化。 网络。 我们的工作假设是，通常隐藏的肌动蛋白结合位点(Abs1)对于菌株依赖是必需的。 网络硬化，但代价是产生更脆弱的细胞骨架。突变或磷酸化 暴露了这个网站。我们将进一步确定ACTN4 S159磷酸化在调节细胞内 肌动蛋白、细胞骨架、细胞黏附和收缩，最后，在体内调节足细胞功能。我们 将利用这些研究作为一个跳板来识别信号、激酶和磷酸酶调节 ACTN4磷酸化。最后，我们将阐明abs1介导的细胞应变硬化的重要性。 在活体内。下一组研究将在最初提案目标的基础上不断扩展，并将 加深我们对ACTN4突变诱导的FSGS的理解，并阐明ACTN4突变在FSGS中的重要性 肌动蛋白-肌动蛋白网络通过磷酸化事件的时空调节。 具体地说，我们将：1.通过丝氨酸159的磷酸化来确定ACTN4的细胞作用和调控； 明确ACTN4介导的细胞生物物理行为与局部改变之间的关系 微环境及其磷酸化调节；3.明确ABD的调节作用 ACTN4通过磷酸化和突变对体内肾脏功能的影响使用新的CRISPR来源 我们开发的动物模型；4.扩大我们对ACTN4改变在 调控基因表达。