Ubiquitin is a small
protein of 9kDa inside present in all eukaryotic cells and involved in the
degradation of proteins by covalently binding target proteins which requires
different enzymes, the E1 activating enzyme, the E2 conjugation enzymes, and
the E3 ubiquitin ligase. Deubiquitinating enzymes (DUBs) can reverse the ubiquitination
of substrates thus preventing the degradation of proteins and can be classified
into two main classes, cysteine proteases and metalloproteases.
The first evidence that
ubiquitination plays a role in the induction of autophagy came from studies
that identified p62/SQSTM1, NDP52, and NBR1 as adaptors for poly- and
monoubiquitylated proteins to autophagosomes.
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| Basic outline of the ubiquitination machinery |
As discussed in previous posts, both p62/SQSTM1 and NBR1 bind
cargo via a ubiquitin-associated (UBA) domain and recruit LC3-I via a LC3-I
interacting region (LIR); in a further step, ALFY (autophagy-linked FYVE
domain) then recruits this complex to phosphatidylinositol 3-phosphate
(PtdIns(3)P) and the ATG12-ATG5-ATG16 complex. Deubiqutination of substrates
therefore can prevent the induction of (selective) autophagy and indeed the
expression of the deubiquitinating enzyme USP36 has been demonstrated to
regulate p62/SQSTM1 dependent selective autophagy in Drosophila and human
cells. As the name implies, deubiquitinating enzymes remove Ubiquitin from
poly- and monoubiquitylated proteins and thus antagonise E3 ligases, leading to
the accumulation of proteins which otherwise would be subject to degradation
via the proteasome or selective autophagy. Deubiquitinases (DUBs) often contain
multiple domains, some of which contribute to the recognition of the substrate
and others that facilitate the proteolytic cleavage of the Ubiquitin residues.
In the case of dUSP36, the inactivation of dUSP36 (as well as hUSP36) induces
the formation of nuclear and cytoplasmic aggregates of ubiquitinated proteins,
the latter being degraded by selective autophagy.
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Deubiqutinases inhibit selective autophagy via binding to ubiqutinated substrates toa Uba domain or by binding substrates to a a Ubl domain |
Although the role of USP36
in viral infected cells has not been studied, the role of DUBs in general has
been, although the role of viral DUBs in regulating selective (p62/SQSTM1
dependent) autophagy is not known. In case of bacterial infections however the
situation is different and although this post covers viral DUBs, a short look
at cells infected with Salmonella is warranted.
Salmonella enterica serovar
Typhimurium (S. Typhimurium) SseL 1
In epithelial cells,
replicating Salmonella are localised in clusters close to the MTOC and the
Golgi, leading to the accumulation of mono- and polyubiquitinated proteins. In
contrast to wt strains however, cells infected with a ssaV null mutant (ΔssaV)
strain that lacks a functional SPI-2 T3SS less than 10% of infected cells do
not accumulate ubiquitinated proteins. One of the effectors of SPI-2 T3SS acts
as a deubiquitinase (SseL), while others (SspH1, SspH2 and SlrP) are E3
ligases, suggesting that the formation of the clusters depends on the action of
E3 ligase and a DUB. In HeLa cells infected with a Salmonella strain mutant for
SseL or complemented with an inactive SseLC262A (SseLC/A) mutant (catalytic
inactive mutant), induces the formation of ubiquitinated inclusions which are
positive for p62/SQSTM1 as well as LC3, suggesting that ubiquitinated proteins
are targeted for selective autophagy. In the context of the intracellular
replication of Salmonella, targeting bacteria to the autophagic machinery would
be considered the bacterial equivalent of the degradation of viral components;
the expression of SseL therefore does favour bacterial replication by
stabilising the “bacterial replication centre”.
Viral DUBs: Coronavirus proteases
3CLpro and 3Cpro
Although a number of viral
deubiquitinases have been characterised, non has been shown to inhibit
selective autophagy. Since in various past posts I have discussed the impact of
coronaviral non-structural proteins (nsp) on the induction of the autophagic
pathway in addition to the role the cleavage of p62/SQSTM1 by Coxsackievirus B3
proteases (and the potential impact on selective autophagy), I want to propose
a model in which the coronaviral 3CLpro and 3Cpro
proteases binds and deubiquitinate mono- and polyubiquitinated proteins,
thus inhibiting p62/SQSTM1 dependent autophagy. In the case of
3Cpro/PLPpro,
the N-terminal Ubl domain binds ubiquitinated substrates, thus preventing the
substrate from being recognised by p62/SQSTM1, followed by either proteolytic
cleavage via the cysteine protease activity or -alternatively- by delivering
them to the proteasome in a mechanism resembling the cellular Rad23/ Rhp23
system. In both cases, selective autophagy mediated by p62/SQSTM1 is bypassed
but not necessarily inhibited.
If however, ubiquitinated substrates are not degraded following binding to the viral Ubl domain, these proteins might accumulate either in p62/SQSTM1 positive -akin to in cells expressing the bacterial SseLC262 - or negative structures, the latter accumulating proteins in aggresome like structures. In a similar way, the viral main protease, 3CLpro , may bind substrates and prevent the degradation via selective autophagy or promote the recruitment of LC3-I via sequestering p62/SQSTM1. If the localisation of substrates to the viral proteases is inducing the proteolytic cleavage of these proteins instead of autophagy, also remains to be seen and can not ruled out at present.
In the context of viral replication, this system might be used to recruit viral proteins to replication enters whilst preventing them from being degraded. Alternatively -or additionally?-, the recruitment of p62/SQSTM1 by this non-canonical system might explain why Atg5 is not required for the replication of the murine CoV, MHV. The expression of the viral 3CLpro and 3Cpro proteases however might not only prevent selective autophagy but also the formation of EDEMosomes in viral infected cells and thus the ERAD pathway.
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| CoV PLPpro might sequester and cleave substrates via the Ubl domain bypassing p62/SQSTM1 |
If however, ubiquitinated substrates are not degraded following binding to the viral Ubl domain, these proteins might accumulate either in p62/SQSTM1 positive -akin to in cells expressing the bacterial SseLC262 - or negative structures, the latter accumulating proteins in aggresome like structures. In a similar way, the viral main protease, 3CLpro , may bind substrates and prevent the degradation via selective autophagy or promote the recruitment of LC3-I via sequestering p62/SQSTM1. If the localisation of substrates to the viral proteases is inducing the proteolytic cleavage of these proteins instead of autophagy, also remains to be seen and can not ruled out at present.
In the context of viral replication, this system might be used to recruit viral proteins to replication enters whilst preventing them from being degraded. Alternatively -or additionally?-, the recruitment of p62/SQSTM1 by this non-canonical system might explain why Atg5 is not required for the replication of the murine CoV, MHV. The expression of the viral 3CLpro and 3Cpro proteases however might not only prevent selective autophagy but also the formation of EDEMosomes in viral infected cells and thus the ERAD pathway.
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| Sequestering of p62/SQSTM1 by CoV PLPpro might inhibit the formation of the EDEMosome and promote LC3-i recruitment |
Central to this question is if p62/SQSTM1 is required for the replication of CoV. A simple way to investigate this would be to infect p62 -/- MEF with MHV. Further questions to be answered are of course related to the localisation of p62/SQSTM1 in cells expressing the viral proteases both in the absence and presence of the orf1a polyprotein as well as nsp-3/-4/-6, in addition to the questions relating to the accumulation of mono- and polyubiquitinated proteins. To extent this beyond CoV, one has to look at the arteriviral equivalents of the coronaviral proteins as well as other viral DUBs such as HAUSP.
As always, who is up to
the challenge?
Further reading
Taillebourg E, Gregoire I, Viargues P, Jacomin AC, Thevenon D, Faure M, & Fauvarque MO (2012). The deubiquitinating enzyme USP36 controls selective autophagy activation by ubiquitinated proteins. Autophagy, 8 (5), 767-79 PMID: 22622177
Birmingham CL, & Brumell JH (2006). Autophagy recognizes intracellular Salmonella enterica serovar Typhimurium in damaged vacuoles. Autophagy, 2 (3), 156-8 PMID: 16874057
Mesquita FS, Thomas M, Sachse M, Santos AJ, Figueira R, & Holden DW (2012). The Salmonella deubiquitinase SseL inhibits selective autophagy of cytosolic aggregates. PLoS pathogens, 8 (6) PMID: 22719249
González CM, Wang L, & Damania B (2009). Kaposi's sarcoma-associated herpesvirus encodes a viral deubiquitinase. Journal of virology, 83 (19), 10224-33 PMID: 19640989
van Kasteren PB, Bailey-Elkin BA, James TW, Ninaber DK, Beugeling C, Khajehpour M, Snijder EJ, Mark BL, & Kikkert M (2013). Deubiquitinase function of arterivirus papain-like protease 2 suppresses the innate immune response in infected host cells. Proceedings of the National Academy of Sciences of the United States of America, 110 (9) PMID: 23401522
Wu X, Zhang M, & Sun SC (2011). Mutual regulation between deubiquitinase CYLD and retroviral oncoprotein Tax. Cell & bioscience, 1 PMID: 21824392
Kanjanahaluethai A, Chen Z, Jukneliene D, & Baker SC (2007). Membrane topology of murine coronavirus replicase nonstructural protein 3. Virology, 361 (2), 391-401 PMID: 17222884
Ratia K, Saikatendu KS, Santarsiero BD, Barretto N, Baker SC, Stevens RC, & Mesecar AD (2006). Severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme. Proceedings of the National Academy of Sciences of the United States of America, 103 (15), 5717-22 PMID: 16581910
Báez-Santos YM, Mielech AM, Deng X, Baker S, & Mesecar AD (2014). Catalytic Function and Substrate Specificity of the PLpro Domain of nsp3 from the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Journal of virology PMID: 25142582
Clementz MA, Chen Z, Banach BS, Wang Y, Sun L, Ratia K, Baez-Santos YM, Wang J, Takayama J, Ghosh AK, Li K, Mesecar AD, & Baker SC (2010). Deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases. Journal of virology, 84 (9), 4619-29 PMID: 20181693
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