Establishing selective autophagy as novel target for therapeutic interventions in neurodegenerative diseases

背景自噬是一种通过降解蛋白质聚集体和功能失调的细胞器等受损成分来保证细胞蛋白质组和基因组质量的自稳态过程。初始自噬级联的关键是多功能调节蛋白p62。它作为错误折叠的蛋白质货物的适配器，并将它们递送到自噬体进行溶酶体蛋白水解。新出现的数据（包括来自监管人2的数据）表明，负载货物的p62经历了自寡聚化和形成二硫键连接的缀合物，作为其活化机制的一部分。Korolchuk等人还发现了小分子工具化合物，其模拟p62激活的生理机制，并以选择性方式促进自噬。假设和目的我们假设工具化合物允许在分子和细胞水平上对p62激活的机制进行理解。确定这种作用机制将增加我们对p62作为清除致病神经元聚集体的潜在治疗靶点的信心（例如tau和a-突触核蛋白）。这个多学科的项目，由自噬生物学的专业知识监督，（Korolchuk）和结构生物学和药物发现（Yue），以及在药物化学和干细胞生物学领域建立的其他合作，旨在：- 确定p62如何识别其配体- 探索活化过程中p62寡聚体和二硫键连接的缀合物的相互依赖性;- 建立工具化合物能够以p62依赖性方式诱导疾病相关蛋白聚集体的自噬降解的能力。（晶体学）和生物物理（ITC，SPR）方法来确定p62配体的结合模式和决定簇，辅之以诱变研究。p62低聚物和二硫键连接的共轭物的分子基础将通过高端荧光显微镜和冷冻电子显微镜技术进行表征。将在具有野生型和突变型p62构建体的稳定再表达的p62敲除细胞系中研究工具化合物对tau和α-突触核蛋白的自噬降解的影响。潜在影响自噬上调被广泛认为是治疗年龄相关性神经退行性疾病的治疗策略，如帕金森病、阿尔茨海默病和路易体痴呆，其中自噬损伤是疾病的驱动因素。然而，现有的自噬激活剂具有较差的特异性和选择性，并导致多效性效应，这限制了它们的临床潜力。该项目将为靶向p62的概念提供信心，利用其激活机制来产生目前无法治疗的年龄相关疾病所急需的新疗法。这项博士工作的成果将为实现这一目标的下游药物开发计划奠定坚实的基础。

Background Autophagy is a homeostatic process that ensures the quality of cellular proteome and genome, by degrading damaged components such as protein aggregates and dysfunctional cellular organelles. Key to the initial autophagy cascade is the versatile regulator protein p62. It functions as an adapter for misfolded protein cargoes, and delivers them to autophagosomes for lysosomal proteolysis. Emerging data (including from supervisor 2) suggest that cargo-loaded p62 undergoes self-oligomerisation and formation of disulphide-linked conjugates, as part of its activation mechanism. Korolchuk and others have also discovered small molecule tool compounds that mimic the physiological mechanism of p62 activation, and promote autophagy in a selective manner.Hypothesis and aimsWe hypothesise that tool compounds allow for a mechanistic understanding towards the activation of p62 at the molecular and cellular level. Defining such mechanism of action will increase our confidence in p62 as a potential therapeutic target for the clearance of disease-causing neuronal aggregates (e.g. tau and a-synuclein).This multi-disciplinary project, supervised by expertise in the biology of autophagy (Korolchuk) and structural biology and drug discovery (Yue), with additional collaborations established for the fields of medicinal chemistry and stem cell biology, aims to: - determine how p62 recognises its ligands (tool compounds, protein cargoes) at the molecular level;- explore the inter-dependence of p62 oligomers and disulphide-linked conjugates in the activation process;- establish the capacity in which tool compounds can induce autophagic degradation of disease-relevant protein aggregates in a p62-dependent manner.MethodologyThe student will use a panel of protein structural (crystallography) and biophysical (ITC, SPR) approaches to determine the binding modes and determinants of p62 ligands, complemented by mutagenesis studies. The molecular basis of p62 oligomers and disulphide-linked conjugates will be characterised by high-end fluorescence microscopy and cryo-electron microscopy techniques. The effect of tool compounds on the autophagic degradation of tau and a-synuclein will be investigated in p62 knockout cell lines with stable re-expression of wild type and mutant p62 constructs. Where applicable, neuronal models of neurodegenerative diseases can be employed to study aggregate formation and cell viability in physiological context.Potential impact Autophagy up-regulation is widely recognized as a therapeutic strategy for the treatment of age-related neurodegenerative diseases such as Parkinson's Disease and Alzheimer's Disease, and Dementia with Lewy Bodies, in which autophagy impairment is implicated as a disease driver. However, existing autophagy activators have poor specificity and selectivity, and cause pleiotropic effects which limit their clinical potential. The project will serve to provide confidence in the concept of targeting p62, exploiting its activation mechanism for the generation of new therapeutics much needed towards currently untreatable age-related diseases. Outputs for this PhD work will form a solid foundation for downstream drug development programme towards this goal.