[Autophagy 5:6, 862-863; 16 August 2009]; ©2009 Landes Bioscience
Autophagic Punctum
A novel link between autophagy and the ubiquitin-proteasome system Viktor I. Korolchuk,† Fiona M. Menzies† and David C. Rubinsztein* Department of Medical Genetics; University of Cambridge; Cambridge Institute for Medical Research; Addenbrooke’s Hospital; Cambridge, UK †These
authors contributed equally to this work.
Key words: autophagy, ubiquitin proteasome system, p62, SQSTM1, p97
Autophagy and the ubiquitin-proteasome system (UPS) are the major routes for intracellular protein degradation. These two pathways were previously thought to be largely distinct. Here we summarize our recent work that demonstrates that longterm autophagy inhibition slows the clearance of short-lived UPS-specific substrates, like p53. This is caused by the accumulation of p62 after autophagy inhibition. These data suggest that the ramifications of a block in autophagy may be much wider than what was previously thought. Rather than simply decreasing clearance of autophagic substrates, while UPS flux is undisturbed, the cell will have to contend with a decrease in clearance by both major routes. Macroautophagy (hereafter autophagy) and the UPS are normally viewed as two essentially independent cellular incinerators. Indeed, differences in their mechanics, their substrates (with autophagic clients often being bulkier and having longer halflives) and the time frames for each pathway (autophagy is a slower process), as well as the reductionist approach routinely adopted by contemporary biology, have allowed the persistence of this belief. Nevertheless, some cross-talk mechanisms have been suggested. For example, it has been proposed that UPS inhibition stimulates autophagic activity. We have characterized an alternative cross-talk mechanism where inhibition of autophagy ultimately leads to the impaired degradation of UPS clients. Inhibition of autophagy by RNA interference (RNAi) or chemical inhibitors results in slower degradation of bona fide UPS substrates, like the exogenous proteasome reporter, UbG76V-GFP, and endogenous p53. As *Correspondence to: David C. Rubinsztein; University of Cambridge; Cambridge Institute for Medical Research; Wellcome Trust/MRC Building; Addenbrooke’s Hospital; Hills Road Cambridge, Cambs CB2 2XY UK; Tel.: 44.1223.762608; Fax: 44.1223.331206; Email:
[email protected] Submitted: 04/03/09; Revised: 04/24/09; Accepted: 04/24/09 Previously published online as an Autophagy E-publication: http://www.landesbioscience.com/journals/autophagy/article/8840 Punctum to: Korolchuk VI, Mansilla A, Menzies FM, Rubinsztein DC. Autophagy inhibition compromises degradation of ubiquitin-proteasome pathway substrates. Mol Cell 2009; 33:517–27; PMID: 19250912; DOI: 10.1016/j.molcel.2009.01.021.
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autophagy inhibition leads to the accumulation of ubiquitinated proteins, but does not decrease the proteolytic activities of proteasomes isolated from such treated cells, we hypothesize that autophagy inhibition is delaying the delivery of ubiquitinated substrates to the proteasome. Generally, proteasomal substrates are tagged with ubiquitin chains that serve as recognition motifs enabling them to be targeted for delivery to the proteasome. This incompletely understood targeting process involves proteins that contain ubiquitin-binding domains. It is known from previous studies that a Ubiquitin Associated (UBA) domain-containing protein, p62 (also called SQSTM1/A170), is an autophagy substrate that accumulates in autophagy-deficient cells. Therefore, we hypothesize that elevated levels of p62 following autophagy inhibition might sequester ubiquitinated short-lived substrates, thereby delaying their degradation. Consistent with this hypothesis, knockdown of p62 rescues the levels of UPS substrates in autophagy-deficient cells. In addition, overexpression of p62 alone is sufficient to produce the inhibitory effect on the UPS in otherwise normal cells. Importantly, this effect is also seen in autophagy-incompetent cells, demonstrating that overexpression of p62 is acting via the UPS, rather than implicating a scenario where these proteins may be substrates for both autophagy and the proteasome. This effect of p62 was partially dependent on its UBA domain. We observed that p62 not only binds to ubiquitinated proteins but also competes for binding to ubiquitinated proteins with another ubiquitin-binding protein, called p97. Interestingly, one of the known functions of p97 is to facilitate the delivery of ubiquitinated substrates to the proteasome. Thus, elevated levels of p62 may potentially act by denying shuttling proteins like p97 access to ubiquitinated UPS substrates. This hypothesis is consistent with data showing that overexpression of p97 reduces the binding of p62 to ubiquitinated proteins and partially rescues the UPS-inhibition mediated by p62 overexpression. From an experimental point of view, these findings will make it harder to characterize a protein as an autophagy substrate based on its accumulation following a longer-term block of autophagy. From a cellular point of view, it is interesting to consider what the physiological relevance of this novel autophagy-UPS link may be. Could there be an advantage to the cell in inhibiting proteasomal clearance in conditions where autophagic flux is reduced? We have demonstrated that prolonged inhibition of autophagy leads
Autophagy
2009; Vol. 5 Issue 6
A novel link between autophagy and the ubiquitin-proteasome system
to an accumulation of short-lived UPS substrates. Among these substrates may be proteins that are central to the control of cell homeostasis. For example, the level of p53, an important regulator of both cell cycle and cell death, is increased by approximately twofold. From this, one could infer that a range of signalling events may occur in cells with blocked autophagy. In the case of p53, these increased levels may predispose the cells to apoptosis, and there may be other processes altered by substrates we have not yet characterized. It is possible that the link between these two pathways allows the cell to sense its autophagic status and react accordingly. While much further work has to be carried out to investigate the possibility of these types of cellular control processes, the clear cross-talk between autophagy and the UPS opens new directions for the study of autophagy and its cellular effects. Furthermore, these mechanisms could be relevant in diseases where there may be prolonged autophagy inhibition, as this may account for abnormal pathological processes that are dependent on the cellular levels of short-lived regulatory proteins, many of which are UPS clients.
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