Parkin-mediated ubiquitination of mutant glucocerebrosidase leads to competition with its substrates PARIS and ARTS

Abstract

BackgroundParkinson’s disease (PD) is a movement neurodegenerative disorder characterized by death of dopaminergic neurons in the substantia nigra pars compacta of the brain that leads to movement impairments including bradykinesia, resting tremor, postural instability and rigidity. Mutations in several genes have been associated with familial PD, such as parkin, pink, DJ-1, LRKK2 and α-synuclein. Lately, mutations in the GBA gene were recognized as a major cause for the development of PD.Mutations in the GBA gene, which encodes for lysosomal β-glucocerebrosidase (GCase), lead to Gaucher disease (GD), an autosomal recessive sphingolipidosis characterized by accumulation of glucosylceramide, mainly in monocyte-derived cells. It is a heterogeneous disease, with Type 1 patients that do not present any primary neurological signs, and Type 2 or Type 3 patients who suffer from a neurological disease. The propensity of type 1 GD patients and carriers of GD mutations to develop PD is significantly higher than that of the non-GD population.We have shown in the past that parkin and mutant GCase, expressed in heterologous systems, interact with each other, and that normal but not mutant parkin mediates K48-dependent proteasomal degradation of mutant GCase variants.MethodsWe tested possible competition between mutant GCase and PARIS or ARTS on the E3 ubiquitin ligase parkin, using coimmunoprecipitation assays and quantitative real-time PCR.ResultsWe show that endogenous mutant GCase variants associate with parkin and undergo parkin-dependent degradation. Mutant GCase competes with the known parkin substrates PARIS and ARTS, whose accumulation leads to apoptosis. Dopaminergic cells expressing mutant GCase are more susceptible to apoptotic stimuli than dopaminergic cells expressing normal GCase, present increased cleavage of caspase 3 and caspase 9 levels and undergo cell death.ConclusionsOur results imply that presence of mutant GCase leads to accumulation of parkin substrates like PARIS and ARTS, which may cause apoptotic death of cells.

Mitochondrial dysfunction and mitophagy defect triggered by heterozygous GBA mutations

“…This effect may be attributed to the ER retention of mutant GBA protein, resulting in an unfolded protein response [67,68]. For example, N370S and L444P GBA mutant proteins, but not WT GBA, interact with PRKN/PARK2 within the ER [69], preventing the binding of PRKN/PARK2 to other substrates [70]. …”

Mutant glucocerebrosidase in Gaucher disease recruits Hsp27 to the Hsp90 chaperone complex for proteasomal degradation

“…The fact that Hsp27 recruitment was significantly diminished in nonfunctional Hsp90 mutants confirmed that proper Hsp90 biologic function was integral to recognition and degradation of mutant GCase. GCase mutants are heavily ubiquitinated and degraded (7,19), a process that depends upon involvement of the Hsp90 complex (13). The mutant-specific recruitment of Hsp27 to the Hsp90 chaperone complex suggested that Hsp27 was related to the ubiquitination and degradation of mutant GCase.…”

Ambroxol-induced rescue of defective glucocerebrosidase is associated with increased LIMP-2 and saposin C levels in GBA1 mutant Parkinson's disease cells

“…Moreover, Ballabio's group has shown that mTOR, one of GSK3 downstream target kinases, interacts with and phosphorylates TFEB on the lysosomal membrane, thereby preventing its translocation to the nucleus, further indicating a connection between these pathways (Settembre et al, 2012). In parallel, parkin, a PD-related E3 ubiquitin ligase whose expression is regulated also by ER stress (Bouman et al, 2011), is involved in mitophagy (Calì et al, 2013) and in the ubiquitination and proteasomal degradation of several substrates, including mutant GCase (Ron et al, 2010;Bendikov-Bar et al, 2014).…”