• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Autophagy. Author manuscript; available in PMC Jan 1, 2009.
Published in final edited form as:
Published online Oct 15, 2007.
PMCID: PMC2597496

Parkin-mediated K63-linked polyubiquitination

A signal for targeting misfolded proteins to the aggresome-autophagy pathway


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.

Keywords: Parkinson’s disease, autophagy, aggresome, inclusion body, misfolded proteins, parkin, lysine-63, ubiquitination, HDAC6

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 ...

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.


This work was supported by National Institutes of Health grants NS054597 (J.A.O.) and NS050650 (L.S.C.).


histone deacetylase 6
Huntington’s disease
Parkinson’s disease
ubiquitin-proteasome system
zinc finger ubiquitin-binding domain


1. Ross CA, Poirier MA. Protein aggregation and neurodegenerative disease. Nat Med. 2004;10(Suppl):S10–7. [PubMed]
2. Garcia-Mata R, Gao YS, Sztul E. Hassles with taking out the garbage: aggravating aggresomes. Traffic. 2002;3:388–96. [PubMed]
3. Kopito RR. Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol. 2000;10:524–30. [PubMed]
4. Holmberg CI, Staniszewski KE, Mensah KN, Matouschek A, Morimoto RI. Inefficient degradation of truncated polyglutamine proteins by the proteasome. EMBO J. 2004;23:4307–18. [PMC free article] [PubMed]
5. Kristiansen M, Deriziotis P, Dimcheff DE, Jackson GS, Ovaa H, Naumann H, Clarke AR, van Leeuwen FW, Menendez-Benito V, Dantuma NP, Portis JL, Collinge J, Tabrizi SJ. Disease-associated prion protein oligomers inhibit the 26S proteasome. Mol Cell. 2007;26:175–88. [PubMed]
6. Venkatraman P, Wetzel R, Tanaka M, Nukina N, Goldberg AL. Eukaryotic proteasomes cannot digest polyglutamine sequences and release them during degradation of polyglutamine-containing proteins. Mol Cell. 2004;14:95–104. [PubMed]
7. Fortun J, Dunn WA, Jr., Joy S, Li J, Notterpek L. Emerging role for autophagy in the removal of aggresomes in Schwann cells. J Neurosci. 2003;23:10672–80. [PubMed]
8. Iwata A, Riley BE, Johnston JA, Kopito RR. HDAC6 and microtubules are required for autophagic degradation of aggregated huntingtin. J Biol Chem. 2005;280:40282–92. [PubMed]
9. Taylor JP, Tanaka F, Robitschek J, Sandoval CM, Taye A, Markovic-Plese S, Fischbeck KH. Aggresomes protect cells by enhancing the degradation of toxic polyglutamine-containing protein. Hum Mol Genet. 2003;12:749–57. [PubMed]
10. Iwata A, Christianson JC, Bucci M, Ellerby LM, Nukina N, Forno LS, Kopito RR. Increased susceptibility of cytoplasmic over nuclear polyglutamine aggregates to autophagic degradation. Proc Natl Acad Sci U S A. 2005;102:13135–40. [PMC free article] [PubMed]
11. Moore DJ, West AB, Dawson VL, Dawson TM. Molecular pathophysiology of Parkinson’s disease. Annu Rev Neurosci. 2005;28:57–87. [PubMed]
12. Kitada T, Asakawa S, Hattori N, Matsumine H, Yamamura Y, Minoshima S, Yokochi M, Mizuno Y, Shimizu N. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature. 1998;392:605–8. [PubMed]
13. Hayashi S, Wakabayashi K, Ishikawa A, Nagai H, Saito M, Maruyama M, Takahashi T, Ozawa T, Tsuji S, Takahashi H. An autopsy case of autosomal-recessive juvenile parkinsonism with a homozygous exon 4 deletion in the parkin gene. Mov Disord. 2000;15:884–8. [PubMed]
14. Mori H, Kondo T, Yokochi M, Matsumine H, Nakagawa-Hattori Y, Miyake T, Suda K, Mizuno Y. Pathologic and biochemical studies of juvenile parkinsonism linked to chromosome 6q. Neurology. 1998;51:890–2. [PubMed]
15. Takahashi H, Ohama E, Suzuki S, Horikawa Y, Ishikawa A, Morita T, Tsuji S, Ikuta F. Familial juvenile parkinsonism: clinical and pathologic study in a family. Neurology. 1994;44:437–41. [PubMed]
16. Sulzer D. Multiple hit hypotheses for dopamine neuron loss in Parkinson’s disease. Trends Neurosci. 2007 [PubMed]
17. Olzmann JA, Li L, Chudaev MV, Chen J, Perez FA, Palmiter RD, Chin LS. Parkin-mediated K63-linked polyubiquitination targets misfolded DJ-1 to aggresomes via binding to HDAC6. J Cell Biol. 2007;178:1025–38. [PMC free article] [PubMed]
18. Olzmann JA, Brown K, Wilkinson KD, Rees HD, Huai Q, Ke H, Levey AI, Li L, Chin LS. Familial Parkinson’s disease-associated L166P mutation disrupts DJ-1 protein folding and function. J Biol Chem. 2004;279:8506–15. [PubMed]
19. Shendelman S, Jonason A, Martinat C, Leete T, Abeliovich A. DJ-1 is a redox-dependent molecular chaperone that inhibits alpha-synuclein aggregate formation. PLoS Biol. 2004;2:e362. [PMC free article] [PubMed]
20. Kawaguchi Y, Kovacs JJ, McLaurin A, Vance JM, Ito A, Yao TP. The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress. Cell. 2003;115:727–38. [PubMed]
21. Perez FA, Palmiter RD. Parkin-deficient mice are not a robust model of parkinsonism. Proc Natl Acad Sci U S A. 2005;102:2174–9. [PMC free article] [PubMed]
22. Boyault C, Gilquin B, Zhang Y, Rybin V, Garman E, Meyer-Klaucke W, Matthias P, Muller CW, Khochbin S. HDAC6-p97/VCP controlled polyubiquitin chain turnover. EMBO J. 2006;25:3357–66. [PMC free article] [PubMed]
23. Hook SS, Orian A, Cowley SM, Eisenman RN. Histone deacetylase 6 binds polyubiquitin through its zinc finger (PAZ domain) and copurifies with deubiquitinating enzymes. Proc Natl Acad Sci U S A. 2002;99:13425–30. [PMC free article] [PubMed]
24. Seigneurin-Berny D, Verdel A, Curtet S, Lemercier C, Garin J, Rousseaux S, Khochbin S. Identification of components of the murine histone deacetylase 6 complex: link between acetylation and ubiquitination signaling pathways. Mol Cell Biol. 2001;21:8035–44. [PMC free article] [PubMed]
25. Bennett EJ, Shaler TA, Woodman B, Ryu KY, Zaitseva TS, Becker CH, Bates GP, Schulman H, Kopito RR. Global changes to the ubiquitin system in Huntington’s disease. Nature. 2007;448:704–8. [PubMed]
26. Yamamoto A, Lucas JJ, Hen R. Reversal of neuropathology and motor dysfunction in a conditional model of Huntington’s disease. Cell. 2000;101:57–66. [PubMed]
27. Zu T, Duvick LA, Kaytor MD, Berlinger MS, Zoghbi HY, Clark HB, Orr HT. Recovery from polyglutamine-induced neurodegeneration in conditional SCA1 transgenic mice. J Neurosci. 2004;24:8853–61. [PubMed]
PubReader format: click here to try


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • BioAssay
    PubChem BioAssay links
  • Compound
    PubChem Compound links
  • MedGen
    Related information in MedGen
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...