How CHIP ensures health and wellbeing across the life-course

目的：确定在衰老过程中调节芯片活性以维持健康和健康的蛋白质组学变化。有令人信服的证据表明，E3-泛素连接酶芯片在哺乳动物和线虫的整个生命过程中都支持健康的衰老和福祉1。CHIP的突变失活或KO小鼠模型会产生快速老化的动物，这些动物也表现出认知能力下降2，而在人类中，正常CHIP功能的丧失是导致共济失调并伴有认知障碍和/或痴呆的原因。CHIP最初是作为蛋白质质量控制网络的一个组成部分而被研究的，因为它能够与分子伴侣机制的核心成员HSP/C70结合。在这一角色中，CHIP与分子伴侣合作，泛化错误折叠、突变或聚集的蛋白质，发出它们降解的信号。鲍尔等人将CHIP的非规范作用定义为一种不依赖于伴侣的E3-连接酶，它特异性地调节一小部分天然折叠蛋白，参与对机体健康和衰老至关重要的过程，包括干扰素信号和胰岛素调节途径1。CHIP的非规范功能在整个有机体水平上支持健康衰老，并在神经元保护中发挥重要作用。而芯片作为伴侣机制的一部分，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一个有吸引力的靶点，因为它提供了一种前景，即在消除与衰老和认知衰退相关的有毒蛋白质发生的早期事件的同时，促进健康衰老。然而，到目前为止，我们还没有一个全面的观点来介导年轻细胞和动物中依赖芯片的健康衰老的过程，以及这些途径在整个生命过程中是如何被抑制或激活的。拟议的最先进的PHD项目将利用基因编辑技术来产生等基因细胞和线虫模型，并将使用包括无标记数据独立质谱仪(SWATH-MS)在内的广谱蛋白质组技术来分析这些模型。这一跨学科项目的一个主要组成部分将是生物信息学方面的培训及其在开发定制参考库、蛋白质组数据分析和路径绘制方面的应用。其目的是识别芯片调控的通路，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：(1)建立定制的参考光谱库(使用MS和RNA-SEQ方法)，用于从神经元干细胞和线虫中鉴定多肽；(2)利用基因编辑技术生成等基因线虫和线虫模型；(3)辅以RNA-SEQ的条带-MS分析；以及(4)影响健康衰老和/或寿命的WT和突变形式的芯片的遗传添加。[1]Tawo，R.，Pokrzywa，W.，Kevin，E.，Akyuz，M.E.，Balaji，V.，禤浩焯，S.，Hohfeld，J.和Hoppe，T.(2017年)泛素连接酶芯片通过调节胰岛素受体周转整合蛋白质稳定和衰老，细胞169,470-482e413。[2]施正宏，鲁贝尔，C.，索斯，S.E.，桑切斯-霍奇，R.，张，S.，Madrigal，S.C.，Ravi，S.，McDonough，H.，Page，R.C.，Chazin，W.J.，Patterson，C.，毛春云，Willis，M.S.，Roo，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)临床前SCAR16，PLoS Genet 14，e1007664.[3]Narayan，V.，Pion，E.，Landre.V.，Muller，P.和Ball，K.L.(2011)泛素连接酶芯片对接依赖的干扰素调节因子-1肿瘤抑制蛋白泛素化，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.