Phosphoinositide signaling in autophagy

项目摘要 磷酸肌醇（PIP）是真核生物膜的次要组分，但却是细胞膜的主要调节剂。 功能协调发展的这七种PIP几乎参与了细胞生理学的各个方面。一种细胞 由PIP调节的过程是自噬，这是一种对广泛的细胞功能和组织至关重要的过程。 发展，并在许多人类疾病中失调。发现于晚期核内体和溶酶体，PI（3，5）P2 是自噬体成熟所必需的，PI（3，5）P2生物合成的失调与 几种神经系统疾病。然而，PI（3，5）P2 调节自噬的机制知之甚少。PIP信号通常由脂质-蛋白质相互作用介导。我们 在上一个资助周期的努力下，已经开发出了一种检测脂质相互作用的单分子测定法 与哺乳动物全细胞裂解物中的蛋白质，使用它，我们发现了广泛的PIP相互作用 在人普列克底物蛋白同源（PH）结构域蛋白的大家族中。XPLN，具有双重活动 作为RhoA鸟嘌呤核苷酸交换因子（GEF）和哺乳动物靶点的内源性抑制剂， 雷帕霉素复合物2（mTORC 2），已成为一种新的PI（3，5）P2相互作用蛋白，我们也有 发现XPLN在体内调节自噬。在XPLN是效应器的工作假设的指导下， PI（3，5）P2的，并在介导PIP信号在自噬的调节中起着核心作用，我们提出， 研究将破译XPLN-PIP相互作用的生化基础，以及它们如何控制XPLN活性， 功能还将研究蛋白激酶C对XPLN磷酸化的作用。我们会问， 生物化学机制是哺乳动物细胞中自噬调节的基础。最后，生理 新机制的相关性将在损伤诱导骨骼肌的小鼠模型中进行探索 再生，这需要自噬。我们在脂质信号传导方面的专业知识，强大的初步数据， 生物化学、生物物理、细胞生物学和动物模型方法的独特组合将确保 成功的结果可能对生物化学和功能的理解产生重大影响， PIP信号传导和自噬的调节。

PROJECT SUMMARY Phosphoinositides (PIPs) are minor components of the eukaryotic membrane but major regulators of cellular functions. The seven PIPs are critically involved in nearly every aspect of cell physiology. One of the cellular processes regulated by PIPs is autophagy, a process essential for a broad range of cellular functions and tissue development, and dysregulated in many human diseases. Found on late endosomes and lysosomes, PI(3,5)P2 is necessary for autophagosome maturation, and dysregulation of PI(3,5)P2 biogenesis has been linked to several neurological disorders through defective autophagy. However, the mechanism by which PI(3,5)P2 regulates autophagy is poorly understood. PIP signaling is often mediated by lipid-protein interactions. Our efforts in the last grant cycle have led to the development of a single-molecule assay that detects lipid interaction with proteins in mammalian whole-cell lysates, using which we have discovered widespread PIP interactions within the large family of human pleckstrin homology (PH) domain-containing proteins. XPLN, with dual activities as a RhoA guanine nucleotide exchange factor (GEF) and an endogenous inhibitor of mammalian target of rapamycin complex 2 (mTORC2), has emerged as a novel PI(3,5)P2-interacting protein, and we have also discovered that XPLN regulates autophagy in vivo. Guided by the working hypothesis that XPLN is an effector of PI(3,5)P2 and plays a central role in mediating PIP signaling in the regulation of autophagy, our proposed studies will decipher the biochemical basis of XPLN-PIP interactions and how they control XPLN activity and function. The role of XPLN phosphorylation by protein kinase C will also be investigated. We will ask how those biochemical mechanisms underlie the regulation of autophagy in mammalian cells. Finally, physiological relevance of the new mechanisms will be probed in a mouse model of injury-induced skeletal muscle regeneration, for which autophagy is required. Our expertise in lipid signaling, strong preliminary data, and a unique combination of biochemical, biophysical, cell biology, and animal model approaches will ensure a successful outcome that is likely to have significant impact on the biochemical and functional understanding of PIP signaling and regulation of autophagy.