How CHIP ensures health and wellbeing across the life-course

目的：确定蛋白质组学的变化，介导的活动，芯片，以维持“健康和健康”在老化过程中。有令人信服的证据表明，E3-泛素连接酶CHIP在哺乳动物和线虫的整个生命过程中都支持健康衰老和幸福。用于CHIP的失活突变或KO小鼠模型产生快速老化的动物，其也表现出认知下降2，而在人类中，正常CHIP功能的丧失是共济失调伴认知障碍和/或痴呆的原因。CHIP首先被研究为蛋白质质量控制网络的一个组成部分，通过其与Hsp/c70结合的能力，Hsp/c70是分子伴侣机制的核心成员。在这个角色中，CHIP与分子伴侣合作，泛素化错误折叠，突变或聚集的蛋白质，发出降解信号。Ball等人已经将CHIP的非经典作用定义为一种不依赖于伴侣的E3连接酶，其特异性调节一小群天然折叠蛋白，这些蛋白参与生物体健康和衰老的核心过程，包括干扰素信号传导3和胰岛素调节途径1。CHIP的非经典功能在整个生物体水平上支持健康衰老，并在神经元保护中起着至关重要的作用。而CHIP作为伴侣机制的一部分的作用旨在对抗有毒中间体和蛋白质聚集体的发展。因此，CHIP的双重功能使其成为治疗干预的有吸引力的靶标，因为它提供了消除与衰老和认知衰退相关的毒性蛋白质生成中的早期事件同时促进健康衰老的前景。然而，到目前为止，我们还没有一个全面的观点，介导年轻细胞和动物中CHIP依赖的健康衰老过程，以及这些途径如何在整个生命过程中被抑制或激活。拟议的最先进的博士项目将利用基因编辑技术来产生同基因细胞和C。elegans模型，将使用广谱蛋白质组学技术进行分析，包括无标记数据独立质谱法（SWATH-MS）。这一跨学科项目的一个主要组成部分将是生物信息学培训及其在定制参考图书馆开发、蛋白质组数据分析和途径绘图方面的应用。其目的是确定CHIP调节的途径以及它们如何在细胞和整个生物体的生命过程中发生变化。更具体地说，该项目将涉及：（i）生成定制的参考光谱库（使用MS和RNA-seq方法），用于从神经干细胞（NSC）和C. elegans，模型;（ii）使用基因编辑技术生成等基因C. elegans和NSC模型;（iii）由RNA-seq补充的SWATH-MS分析，和（iv）影响健康衰老和/或长寿的WT和突变形式的CHIP的遗传加回。[1]塔沃河Pokrzywa，W.，Kevei，E.，Akyuz，M. E、Balaji，V.，Adrian，S.，Hohfeld，J.，Hoppe，T.（2017）泛素连接酶CHIP通过调节胰岛素受体周转来整合蛋白质稳态和衰老，细胞169，470-482 e413。[2]施角，澳-地H、Rubel，C.，Soss，S. E、桑切斯-霍奇，R.，Zhang，S.，（1991），中国农业科学院，马德里加尔S. C.的方法，Ravi，S.，麦克多诺，H.，佩奇河，巴西-地C.的方法，Chazin，W. J.，帕特森角，毛角，澳-地是的，Willis，M.美国，Luo，H.是的，Li，Y.美国，Stevens，D.一、唐，M。B.，Du，P.，Wang，Y. H、Hu，Z. W.，Xu，Y. M.，和J.C.（2018）在SCAR 16的临床前模型中，CHIP的破坏结构和异常功能介导运动和认知功能的丧失，PLoS Genet 14，e1007664。[3]Narayan，V.，Pion，E.，Landre，V.，Muller，P.，球，K。L.等人（2011）Docking-dependent ubiquitination of the interferon regulatory factor-1 tumor suppressor protein by the ubiquitin ligase CHIP，J Biol Chem 286，607-619.

Purpose: To define the proteomic-changes which mediate the activity of CHIP to maintain 'health and wellness' during aging. There is compelling evidence that the E3-ubiquitin ligase CHIP supports healthy aging and wellbeing through-out the life course in both mammals and nematodes1. Inactivating mutations or KO mouse models for CHIP produce rapidly aging animals which also exhibit cognitive decline2, whereas in human's loss of normal CHIP function is causative for ataxia with cognitive impairment and/or dementia. CHIP was first studied as a component of the protein quality control network through its ability to bind to Hsp/c70, a core member of the molecular chaperone machinery. In this role, CHIP cooperates with the molecular chaperones to ubiquitinate misfolded, mutated or aggregated proteins signalling their degradation. The Ball group and others have defined a non-canonical role for CHIP as a chaperone-independent E3-ligase that specifically regulates a small cohort of native folded proteins involved in processes that are central to organismal health and aging including interferon signalling3 and insulin regulated pathways1.The non-canonical function of CHIP supports healthy aging at the level of the whole organism and plays a vital role in neuronal protection. Whereas the role of CHIP as part of the chaperone machinery aims to combat the development of toxic intermediates and protein aggregates. The dual functions of CHIP therefore make it an attractive target for therapeutic intervention as it offers the prospect of abrogating the early events in toxic protein genesis that is associated with aging and cognitive decline whilst at the same time promoting healthy aging. However, as yet we do not have a comprehensive view of the processes that mediate CHIP-dependent healthy aging in young cells and animals and how these pathways are suppressed or activated through-out the life course.The proposed state-of-the-art PhD project will exploit gene-editing technology to generate isogenic cell and C. elegans models which will be analysed using broad spectrum proteomic-techniques including label free data-independent mass-spectrometry (SWATH-MS). A major component of this cross-discipline project will be training in bioinformatics and its application to the development of bespoke reference libraries, proteomic data analysis and pathway mapping. The aim is to identify CHIP regulated pathways and how they change through-out the life course of cells and whole organisms. More specifically, the project will involve: (i) Generating bespoke reference spectral libraries (using MS and RNA-seq approaches) for peptide identification from neuronal stem cells (NSC) and C. elegans, models; (ii) the use of gene-editing technology to generate isogenic C. elegans and NSC models; (iii) SWATH-MS analysis complemented by RNA-seq, and (iv) genetic add-back of WT and mutant forms of CHIP which affect healthy aging and/or longevity. [1] Tawo, R., Pokrzywa, W., Kevei, E., Akyuz, M. E., Balaji, V., Adrian, S., Hohfeld, J., and Hoppe, T. (2017) The Ubiquitin Ligase CHIP Integrates Proteostasis and Aging by Regulation of Insulin Receptor Turnover, Cell 169, 470-482 e413.[2] Shi, C. H., Rubel, C., Soss, S. E., Sanchez-Hodge, R., Zhang, S., Madrigal, S. C., Ravi, S., McDonough, H., Page, R. C., Chazin, W. J., Patterson, C., Mao, C. Y., Willis, M. S., Luo, H. Y., Li, Y. S., Stevens, D. A., Tang, M. B., Du, P., Wang, Y. H., Hu, Z. W., Xu, Y. M., and Schisler, J. C. (2018) Disrupted structure and aberrant function of CHIP mediates the loss of motor and cognitive function in preclinical models of SCAR16, PLoS Genet 14, e1007664.[3] Narayan, V., Pion, E., Landre, V., Muller, P., and Ball, K. L. (2011) Docking-dependent ubiquitination of the interferon regulatory factor-1 tumor suppressor protein by the ubiquitin ligase CHIP, J Biol Chem 286, 607-619.