Molecular Mechanism Underlying The Sequestration By Polyglutamine-expanded Proteins That Leads To Cellular Dysfunction

Protein misfolding and aggregation can lead to neurodegenerative diseases and other major diseases,seriously threatening human health and quality of life.However,the molecular mechanism underlying cellular dysfunction or even death caused by protein aggregation is not fully understood.Polyglutamine(polyQ)disease is a group of neurodegenerative diseases caused by abnormal polyQ expansion.The polyQ sequence that extends over a certain threshold will become an amyloid core and trigger aggregation of the protein.PolyQ disease is caused by a single pathogen that possesses a definite amyloid core.This characteristic makes it the excellent model for studying protein aggregation-related diseases.In this dissertation,we took the protein fragments of polyQ-expanded huntingtin(Htt)and ataxin-7(Atx7)as models to investigate the mechanisms underlying the cytotoxicity induced by protein aggregates in both molecular and cellular aspects.Components such as ubiquitin and ubiquitin adaptors are present in the inclusions of many neurodegenerative diseases,implicating their essential role in the pathological process of diseases triggered by protein aggregation.To elucidate the role of ubiquitin-related proteins in the cytotoxicity induced by the amyloid protein,we investigated the cellular effects and molecular mechanism underlying the sequestration effect of polyQ-expanded Htt on ubiquitin adaptors h HR23 B and UBQLN2.Through the supernatant and pellet fractionation and immunoflorescence staining experiments,we found that polyQ-expanded Htt-N552 could sequester intracellular h HR23 B and UBQLN2 into aggregates through ubiquitin-mediated sequestration,which relies on the UBA(ubquitin-associated domain)domains of ubiquitin adaptors.This sequestration effect could impair the stabilizing effect of h HR23 B on XPC(Xeroderma Pigmentosum group C),leading to the reduction of XPC protein level.This study revealed the mechanism underlying the sequestration effect of aggregated protein on ubiquitin-related proteins.The functional impairment of proteins sequestered by protein aggregates may be responsible for the cytotoxicity and neurodegeneration.As a polyQ protein,the expansion in the polyQ tract of Atx3 leads to the spinal cerebellar ataxia(SCA3).The wide-type Atx3 was found to be present in inclusions,indicating that the wide-type Atx3 may be involved in the development of disease.Thus,we investigated the effects and mechanisms of polyQ-expanded proteins on Atx3.PolyQ-expanded Htt and Atx7 were found to impair the homeostasis of Atx3 and promote degradation of Atx3 mainly through the proteasome pathway.The disruption of Atx3 homeostasis may be attributed to the ubiquitin-mediated sequestration of HSJ1 by the polyQ-expanded proteins.Impairment of Atx3 homeostasis and function may aggravate the cytotoxicity and disease.Therefore,the ubiquitin-mediated sequestration may be important in the cellular dysfunction and pathological change caused by protein aggregates.To further verify the sequestration model and explore its potential application,we introduced a peptide from v IRF1,which can specifically bind USP7,into the polyQ-expanded protein.We expected that this sequence could mediate the sequestration of the tumor target USP7 into aggregates formed by the polyQ fusion protein,thus inhibiting the function of USP7 and up-regulating tumor suppressors,such as P53.We found that sequestration of USP7 by the polyQ fusion protein could down-regulate the protein level of HDM2 but failed to up-regulate P53,which limits its further investigation and application.