Objective: The pathogenic mechanism of UCHL1 in PD remains unclear. In this study, we aimed to investigate the role of UCHL1 in the pathogenesis of PD.

Background: Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease. The ubiquitin carboxy-terminal hydrolase L1 (UCHL1), a member of deubiquitinating enzymes, is primarily abundant in neurons. Down-regulation and dysfunction of UCHL1 have been detected in the brains of PD patients. In addition, UCHL1 gene mutants are linked to PD.

Methods: To study the mechanism of UCHL1 in the pathogenesis of PD, we used MPP+ to reproduce a cellular model of PD in SH-SY5Y cells. The effects of UCHL1 knockdown and overexpression in MPP+-induced cellular model of PD were determined. Immunoprecipitation and mass spectrometric analysis (IP-MS) were used to screen UCHL1 interacting proteins. Among these proteins, we focused on Mortalin. The interaction between UCHL1 and Mortalin was confirmed by Co-immunoprecipitation (Co-IP) in vivo and vitro using mice brain and HEK293T cells lysates. Co-IP was also applied to detect whether UCHL1 affects Mortalin ubiquitination. We used MG132 to block proteasome-dependent degradation and tested whether UCHL1 would influence Mortalin protein level in SH-SY5Y cells. UCHL1-siRNA transfected SH-SY5Y cells were fractionated into cytoplasmic and mitochondrial fractions, followed by western blotting to detect Mortalin localization change.

Results: UCHL1 knockdown enhanced MPP+-induced cytotoxicity in SH-SY5Y cells, as reflected by decreased cell viability, increased apoptosis protein level and declined mitochondrial membrane potential, while UCHL1 overexpression could rescue MPP+-induced cytotoxicity in SH-SY5Y cells. In total, 234 UCHL1 interacting proteins were screened through IP-MS. According to the findings, Mortalin was chosen as a target protein due to its association with PD reported previously. Mortalin co-immunoprecipitated with Mortalin in vivo and vitro was from rat brains and HEK293T cells lysates respectively. Furthermore, UCHL1 ubiquitinated Mortalin as ubiquitin ligase. Instead of affecting the protein level of Mortalin, UCHL1-mediated ubiquitination facilitated the localization of Mortalin to mitochondria.

Conclusions: The present data suggested that UCHL1 could mediate Mortalin to mitochondrial localization by ubiquitinating Mortalin as an ubiquitin ligase.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/ubiquitin-carboxyl-terminal-hydrolase-1-uchl1-mediated-ubiquitination-attributed-to-localization-of-mortalin-to-mitochondria/