A Model Multi-systems Approach for Understanding the Role of the PIX Pathway in Cardiac Muscle and Cardiomyopathy

理解 PIX 通路在心肌和心肌病中作用的模型多系统方法

基本信息

  • 批准号:
    10532707
  • 负责人:
  • 金额:
    $ 57.56万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2022
  • 资助国家:
    美国
  • 起止时间:
    2022-01-01 至 2025-12-31
  • 项目状态:
    未结题

项目摘要

Myofibrils located at the periphery of the muscle cell are attached to the sarcolemma via costameres, muscle- specific integrin adhesion complexes (IACs), that transmit the force of muscle contraction to the outside of the cell. IACs consist of the transmembrane protein integrin and hundreds of proteins in a complex both in the ECM and especially intracellularly. Although much is known about the composition of IACs and mechanisms that initiate their assembly, we do not know how the composition of an IAC is determined, and what determines where an IAC will form. We exploit the conservation of muscle structure/function and the power of genetics in C. elegans. From a genetic screen, we identified a gene, pix-1, which is required for the formation of IACs at muscle cell boundaries (MCBs), but not the other IACs (M-lines and dense bodies). PIX-1 is the nematode ortholog of human b-PIX, which contains a RhoGEF domain known to be an activator of Rac1 and Cdc42. pix- 1 mutants show decreased whole animal locomotion. As compared to wild type, a pix-1 null mutant, shows 50% reduction in the level of activated (GTP bound) Rac in muscle. In addition to deficiency of PIX-1, overexpression of wild type PIX-1 protein results in decreased locomotion and disrupted MCBs. PIX-1 localizes to all 3 IACs in muscle—M-lines, dense bodies and MCBs. Loss of function mutants in each of the known PIX pathway proteins (PIX, Rac, GIT) result in disrupted MCBs. The protein kinase activity of PAK-1 is essential for its MCB function: a kinase-dead PAK-1 mutant has disrupted MCBs. Although PIX proteins are known to be important in several cell types in mammals and nematodes, our results are the first to demonstrate that a PIX protein is required for assembly of an IAC, and is required in striated muscle. A GAP protein (inactivates Rac) for this pathway has not been reported for any cell type or organism, but we now have two candidate GAPs. Remarkably, we found that heart specific knockout of the mouse ortholog b-PIX results in dilated cardiomyopathy at 8 months of age. Similarly, heart-specific knockdown of the Drosophila ortholog, dPix, also results in cardiomyopathy. We hypothesize that PIX proteins, through their RacGEF activity, direct assembly of IACs by activating PAK kinases to phosphorylate key substrate(s) in muscle. We will leverage the unique advantages of 3 model systems to learn new conserved functions of the PIX pathway in striated and cardiac muscle. Aim 1: Exploiting the power of studying muscle in C. elegans we will: (a) identify the GAP protein(s); (b) determine the localization, function and substrates of PAK kinases, and (c) test the hypothesis that PIX-1 is only required at MCBs because there are additional RacGEF proteins at the other IAC locations. Aim 2: We will determine how b-PIX functions in the mouse heart and how its absence leads to cardiomyopathy. Aim 3: We will take advantage of the power of genetic modifier screens in Drosophila to identify new members of the PIX pathway in muscle, and more specifically, cardiac muscle. There is a need to identify additional cardiomyopathy genes in humans and genes encoding members of the PIX pathway may be new candidates.
位于肌细胞周围的肌原纤维通过肋肌、肌纤维和肌膜附着在肌膜上。 特异性整合素粘附复合物(IACs),将肌肉收缩的力传递到细胞外。 cell. IAC由跨膜蛋白整联蛋白和数百种蛋白质组成, ECM,尤其是细胞内。尽管人们对IAC的组成和机制已经有了很多了解, 我们不知道IAC的组成是如何确定的,以及什么决定了他们的组装, 在那里会形成一个IAC。我们利用肌肉结构/功能的保守性和遗传学的力量, C.优美的从遗传筛选中,我们确定了一个基因,pix-1,这是形成IACs所必需的, 肌细胞边界(MCB),但不是其他IAC(M线和致密体)。PIX-1是线虫 人b-PIX的直系同源物,其含有已知是Rac 1和Cdc 42的激活剂的RhoGEF结构域。pix- 1突变体显示整个动物运动减少。与野生型相比,pix-1无效突变体显示, 肌肉中活化(GTP结合)Rac水平降低50%。除了PIX-1缺乏外, 野生型PIX-1蛋白的过表达导致运动减少和MCB破坏。PIX-1本地化 肌肉-M-线、致密体和MCB中的所有3个IAC。每个已知PIX中的功能缺失突变体 途径蛋白(PIX、Rac、GIT)导致MCB被破坏。PAK-1的蛋白激酶活性对于 其MCB功能:激酶死亡的PAK-1突变体破坏了MCB。虽然PIX蛋白已知是 在哺乳动物和线虫的几种细胞类型中很重要,我们的结果首次证明了PIX 蛋白质是组装IAC所必需的,并且是横纹肌所必需的。GAP蛋白(灭活Rac) 对于任何细胞类型或生物体都没有报道过这种途径,但我们现在有两个候选GAP。 值得注意的是,我们发现小鼠直系同源物b-PIX的心脏特异性敲除导致扩张的 8个月大时的心肌病。类似地,果蝇直系同源基因dPix的心脏特异性敲除, 导致心肌病我们假设PIX蛋白,通过其RacGEF活性, 通过激活PAK激酶使肌肉中的关键底物磷酸化来抑制IAC。我们将利用独特的 3个模型系统的优势,以了解新的保守功能的PIX途径在横纹肌和心脏 肌肉.目的1:挖掘C.我们将:(a)鉴定差距蛋白; (b)确定PAK激酶的定位、功能和底物,以及(c)检验PIX-1是 仅在MCB需要,因为在其他IAC位置存在额外的RacGEF蛋白。目标2:我们 将确定b-PIX如何在小鼠心脏中发挥作用,以及它的缺失如何导致心肌病。目标3: 我们将利用果蝇遗传修饰筛选的力量来识别基因组的新成员。 肌肉中的PIX途径,更具体地说,心肌。有必要确定更多的 人类心肌病基因和编码PIX途径成员的基因可能是新的候选者。

项目成果

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GUY Martin BENIAN其他文献

GUY Martin BENIAN的其他文献

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{{ truncateString('GUY Martin BENIAN', 18)}}的其他基金

A Model Multi-systems Approach for Understanding the Role of the PIX Pathway in Cardiac Muscle and Cardiomyopathy
理解 PIX 通路在心肌和心肌病中作用的模型多系统方法
  • 批准号:
    10340546
  • 财政年份:
    2022
  • 资助金额:
    $ 57.56万
  • 项目类别:
ApoE4 and C/EBP: Mutually Regulate Each Other in Alzheimer's Disease
ApoE4 和 C/EBP:在阿尔茨海默病中相互调节
  • 批准号:
    10319519
  • 财政年份:
    2020
  • 资助金额:
    $ 57.56万
  • 项目类别:
ApoE4 and C/EBP: Mutually Regulate Each Other in Alzheimer's Disease
ApoE4 和 C/EBP:在阿尔茨海默病中相互调节
  • 批准号:
    10533321
  • 财政年份:
    2020
  • 资助金额:
    $ 57.56万
  • 项目类别:
The UNC-45 Chaperone as a Modulator of Myosin Biogenesis and Function
UNC-45 伴侣作为肌球蛋白生物发生和功能的调节剂
  • 批准号:
    9267166
  • 财政年份:
    2016
  • 资助金额:
    $ 57.56万
  • 项目类别:
The UNC-45 Chaperone as a Modulator of Myosin Biogenesis and Function
UNC-45 伴侣作为肌球蛋白生物发生和功能的调节剂
  • 批准号:
    9477067
  • 财政年份:
    2016
  • 资助金额:
    $ 57.56万
  • 项目类别:
The UNC-45 Chaperone as a Modulator of Myosin Biogenesis and Function
UNC-45 伴侣作为肌球蛋白生物合成和功能的调节剂
  • 批准号:
    9789043
  • 财政年份:
    2016
  • 资助金额:
    $ 57.56万
  • 项目类别:
Role of UNC-89 (obscurin) in sarcomere assembly and maintenance.
UNC-89(obscurin)在肌节组装和维护中的作用。
  • 批准号:
    8836489
  • 财政年份:
    2014
  • 资助金额:
    $ 57.56万
  • 项目类别:
Role of UNC-89 (obscurin) in sarcomere assembly and maintenance.
UNC-89(obscurin)在肌节组装和维护中的作用。
  • 批准号:
    8632004
  • 财政年份:
    2014
  • 资助金额:
    $ 57.56万
  • 项目类别:
Using C. elegans to study titin and obscurin (UNC-89)
使用线虫研究 titin 和 obscurin (UNC-89)
  • 批准号:
    7847206
  • 财政年份:
    2009
  • 资助金额:
    $ 57.56万
  • 项目类别:
Nematode UNC-98 functions in focal adhensions and nuclei
线虫 UNC-98 在粘着点和细胞核中发挥作用
  • 批准号:
    7483267
  • 财政年份:
    2005
  • 资助金额:
    $ 57.56万
  • 项目类别:
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