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Title: Investigating the protein targets of the neuroprotective E3 ligase, CHIP
Author: Dias, Catarina
ISNI:       0000 0004 8508 576X
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2019
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Identifying early defects in protein homeostasis ("proteostasis") in neurodegeneration could shed light into disease-promoting events and provide promising therapeutic targets for disease modification. A key player in the regulation of proteostasis is the dual function chaperone and E3 ligase C-terminus of Hsc-70 interacting protein (CHIP). Its role in neurodegenerative diseases, including dementia with Lewy bodies, and the severe progeria and proteotoxic phenotype seen in CHIP KO mice support its neuroprotective effects, but the underpinning molecular mechanisms remain largely unknown and understudied. This project aimed to identify the protein targets of CHIP in a neuronal cell model of disease. I knocked out CHIP expression using CRISPR/Cas9 technology from inducedpluripotent stem cells (iPSC) derived from a synucleinopathy patient with dementia and differentiated them into cortical neurons. A label-free quantitative mass spectrometry to analyse CHIP-dependent changes was conducted and the proteome defined. This comparative proteomic analysis revealed that of all the proteins identified and significantly changed between CHIP KO and WT lines, only 35 proteins could be (directly or indirectly) regulated by CHIP. This supports the emerging hypothesis that CHIP can be chaperone-independent docking-dependent E3 ligase, having tight specificity for substrates, in this model of synucleinopathy with mild proteotoxic stress. Annexin and other calcium-binding proteins, in particular Annexin A2 and its interacting protein S100-A11, were the most over-represented proteins in the CHIP KO cortical neurons. From this comprehensive target discovery investigation, I have validated Annexin A2 increases in CHIP KO cells and have detected an endogenous Annexin A2:CHIP interaction, both in vitro and in situ, in different CHIP cell models. Moreover, CHIP-dependent ubiquitination of Annexin A2 was also identified, both in vitro and in situ. Single-chain antibodies against CHIP have been engineered to modulate its activity. By collapsing the higher molecular weight structures of CHIP, CHIP-dependent ubiquitination of Annexin A2, but not other substrates, is enhanced. Annexins are a conserved family of calcium-regulated phospholipid-binding proteins that are required for membrane repair and maintenance of membrane homeostasis. Given these functions, several types of membrane damage assays were conducted and suggested that CHIP KO cells are more sensitive to damage, despite retaining the ability to repair. This impairment in membrane resilience was also seen in CHIP KO cells expressing a E3 ligase-dead CHIP mutant, but the phenotype was partly rescued in the cell lines expressing a chaperone-dead CHIP mutant or wild-type CHIP. Compromised Annexin A2:S100A11 interactions (important for the repair complex) and a different lipidomic profile between CHIP WT and KO cells could contribute to this phenotype. Although there are reports of annexins being overexpressed in some neurodegenerative diseases, there have been no follow-up studies deciphering the molecular mechanisms of annexins within neurons. I have identified Annexin A2 as a substrate for CHIP and revealed other novel calcium-regulated membrane-binding CHIP targets. Thus, CHIP is likely to play a role in regulating membrane protein homeostasis and maintaining membrane integrity, which may help to explain the neuroprotective actions of CHIP. This is of relevance within the emerging field of impaired membrane integrity in the context of neurodegeneration.
Supervisor: Ball, Kathryn ; Kunath, Tilo Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: CHIP ; Annexin A2 ; membrane damage ; CHIP KO cells ; S100-A11 proteins ; membrane integrity ; neurodegeneration