Parkin-mediated K63-linked polyubiquitination

Abstract

Pathological inclusions containing misfolded proteins are a prominent feature common to many age-related neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. In cultured cells, when the production of misfolded proteins exceeds the capacity of the chaperone refolding system and the ubiquitin-proteasome degradation pathway, misfolded proteins are actively transported along microtubules to pericentriolar inclusions called aggresomes. The aggresomes sequester potentially toxic misfolded proteins and facilitate their clearance by autophagy. The molecular mechanism(s) that targets misfolded proteins to the aggresome-autophagy pathway is mostly unknown. Our recent work identifies parkin-mediated K63-linked polyubiquitination as a signal that couples misfolded proteins to the dynein motor complex via the adaptor protein histone deacetylase 6 and thereby promotes sequestration of misfolded proteins into aggresomes and subsequent clearance by autophagy. Our findings provide insight into the mechanisms underlying aggresome formation and suggest that parkin and K63-linked polyubiquitination may play a role in the autophagic clearance of misfolded proteins.

A common feature of neurodegenerative diseases is the abnormal accumulation of misfolded proteins, leading to the assembly of toxic oligomers and aggregates.¹ In cultured cells, misfolded proteins are generally handled by very efficient protein quality control systems, which include a host of molecular chaperones and the ubiquitin-proteasome system (UPS).^2,3 However, when these systems are impaired or overwhelmed, misfolded and aggregated proteins are actively sequestered into aggresomes, a specialized type of intracellular inclusion body formed at the centrosome by dynein-mediated retrograde transport.^2,3 Studies indicate that aggregated proteins are inherently resistant to degradation by the proteasome.^4-6 As substrates for autophagy, aggresomes facilitate the clearance of degradation-resistant aggregates and act as another cellular defense mechanism to reduce misfolded protein-induced cytotoxicity.^7-10 The machinery and mechanisms underlying the recognition and targeting of misfolded and aggregated proteins to aggresomes remain poorly understood.

Parkinson’s disease (PD) is a debilitating neurodegenerative disease characterized by the relatively selective loss of nigral dopa-minergic neurons and the presence of intraneuronal cytoplasmic inclusions called Lewy bodies.¹¹ Mutations in the gene encoding the E3 ubiquitin-protein ligase parkin cause an autosomal recessive, early onset form of Parkinson’s disease (PD) that is unique in its lack of the hallmark inclusion bodies.^12-15 It has been hypothesized that parkin function might be required for the formation of Lewy bodies.¹⁶ In a recent study, we examined the role of parkin in the cellular management of misfolded proteins.¹⁷ The PD-linked L166P mutant DJ-1 was chosen as a model substrate because we and others have previously shown that it is a misfolded protein that is efficiently degraded by the ubiquitin-proteasome system under normal conditions.^17-19 The results from our recent study indicate that, under the conditions in which proteasome function is impaired, parkin cooperates with the heterodimeric E2 ubiquitin-conjugating enzyme UbcH13/Uev1a to selectively mediate K63-linked polyubiquitination of the misfolded L166P mutant DJ-1, but not the correctly folded wild-type DJ-1 (Fig. 1, step).¹⁷ K63-linked polyubiquitination of misfolded DJ-1 had no effect on its proteasomal degradation and instead facilitated binding to histone deacetylase 6 (HDAC6) (Fig. 1, step 2),¹⁷ a dynein adaptor protein that simultaneously binds ubiquitinated proteins via a zinc finger ubiquitin-binding domain (ZnF-UBP) and the dynein motor via a distinct dynein binding domain.²⁰ Indeed parkin expression promoted retrograde transport of misfolded DJ-1 into perinuclear aggresomes and its redistribution into a detergent-insoluble pool (Fig. 1, step 3).¹⁷ Moreover, mouse embryonic fibroblasts from parkin-deficient mice²¹ exhibited a pronounced deficit in the ability to target misfolded DJ-1 to aggresomes.¹⁷ By using ubiquitin mutants that are unable to form K63-linked polyubiquitin chains, we found that inhibition of K63-linked polyubiquitination impaired recruitment of misfolded DJ-1 to aggresomes and instead resulted in the accumulation of misfolded DJ-1 in small aggregates distributed throughout the cytoplasm of the cell.¹⁷ Previous in vitro binding studies have shown that HDAC6 binds both monoubiquitin and polyubiquitin chains.^22-24 However, our studies suggest that HDAC6 interacts preferentially with K63-linked polyubiquitin chains in vivo, suggesting that K63-linked polyubiquitination acts as a specific signal for regulating dynein-mediated retrograde transport in cells.¹⁷

Figure 1.

A model of parkin function in the clearance of misfolded proteins by the aggresome-autophagy pathway. Under conditions of proteasomal impairment, parkin coordinates the E2 enzyme UbcH13/Uev1a to mediate K63-linked polyubiquitination of misfolded proteins (step 1). This K63-linked polyubiquitin chain promotes binding to HDAC6 (step 2) and thereby links the ubiquitinated proteins to the dynein motor complex for transport to the aggresome (step 3). Autophagic membrane and machinery are recruited to the aggresome (step 4). The autophagosome then fuses with lysosomes to form an autophagolysosome and thus allows the degradation of misfolded and aggregated proteins by lysosomal hydrolases (step 5).

Our data are consistent with a role for autophagy in the clearance of aggresomes.^7-9 We found that the L166P mutant DJ-1 aggresomes stained with the classical marker of autophagy monodansylcadaverine and were tightly ringed by lysosomes, suggesting that aggresomes are an intermediate structure in a pathway destined to bring about eventual degradation by autophagy (Fig. 1).¹⁷ Although autophagy is generally considered to be a non-specific bulk degradation process, one possibility is that parkin, by promoting the delivery of misfolded proteins to centrosomally localized aggresomes, may facilitate the selective clearance of misfolded proteins by autophagy (Fig. 1, steps 4 and 5). Indeed recent studies provide evidence that autophagy-related (Atg) proteins and lysosomes are recruited to aggresomes via retrograde microtubule transport, and perhaps concentration of aggregated proteins and autophagy components provides a measure of selectivity.^{8, 10} However, whether aggresomes play a role in selective autophagic clearance of misfolded proteins remains a controversial issue that has not been definitively addressed.

Our studies have yielded new insights into the molecular mechanisms underlying aggresome formation in cells and may have important implications regarding the formation of pathological inclusion bodies. Interestingly, Bennett et al. recently showed that K63-linked polyubiquitin chains accumulate in cultured cells expressing a huntingtin fragment containing an expanded polyglutamine repeat, in the brains from multiple Huntington’s disease (HD) mouse models, and in brains of HD patients, suggesting that K63-linked polyubiquitination may be involved in the pathogenesis of HD.²⁵ In addition, polyglutamine-containing inclusion bodies formed in cell culture and in animal models can be cleared if the production of misfolded proteins is halted.^8-10,26,27 Thus emerging data implicates autophagy in the clearance of aggresomes and pathological inclusion bodies. However, the mechanism underlying inclusion body formation and the precise role of K63-linked polyubiquitination in disease is unclear.

As with most studies, our findings raise significant questions, including: what is the role of parkin and K63-linked polyubiquitination in autophagy and disease? Do other E3 enzymes play similar roles in the cell? What are the mechanisms that regulate parkin recruitment of different E2 enzymes and conjugation of different types of ubiquitin chains? Are pathological inclusion bodies formed by similar mechanisms as aggresomes? Future studies of parkin, HDAC6, and K63-linked polyubiquitination may advance our understanding of the mechanisms underlying the clearance of misfolded proteins by the aggresome-autophagy pathway and could provide novel targets for therapeutic intervention in neurodegenerative diseases.

Abbreviations

HDAC6

histone deacetylase 6

HD

Huntington’s disease

PD

Parkinson’s disease

UPS

ubiquitin-proteasome system

ZnF-UBP

zinc finger ubiquitin-binding domain