Inhibition of selective autophagy by members of the herpesvirus ubiquitin-deconjugase family
Summary (2 min read)
Introduction
- Autophagy is a protein degradation and recycling machinery that regulates cellular homeostasis and participates in the host defense against infection by capturing and destroying invading microorganisms (1, 2).
- The strategies by which pathogens capture autophagy are beginning to be elucidated at the molecular level, which provides new insight on pathogenesis and highlights potential targets for therapeutic intervention (19).
- The authors found that similar to BPLF1, the catalytic domains of HCMV-UL48, KSHV-ORF64 and HSV1-UL36 bind to and deubiquitinate SQSTM1/p62 but the efficiency and ubiquitin chain .
Immunoblotting and immunoprecipitation
- It is The copyright holder for this preprintthis version posted March 30, 2021.
- For co-immunoprecipitation, the cell lysates were incubated for 4 h with anti-FLAG agarose affinity gel (Sigma, A-2220).
- Precipitated complexes were washed with lysis buffer and eluted with the FLAG peptide (Sigma, F4799) at a concentration of 400 g/ml.
- Equal amounts of proteins were fractionated in a polyacrylamide Bis-Tris 4– 12% gradient gels (Invitrogen, NP0321PK2).
Filter-trap assay
- Cells were harvested 48 h after transfection, resuspended in PBS (Biowest, X0515-500) containing protease inhibitor cocktail and samples were snap frozen and stored at -20°C.
- Lysates were sonicated with a QSonica Q125 sonicator with settings 20% amplitude, pulsating 1s on/1s off total time of 30 sec.
- After sonication protein amounts were measured with Bio-Rad .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
- It is The copyright holder for this preprintthis version posted March 30, 2021.
- Images were analyzed with the ImageJ software.
Immunofluorescence and confocal microscopy
- HeLa cells were grown to semi-confluence on glass cover slips in Dulbecco’s modified Eagle’s medium (Sigma, D6429) containing 10% fetal calf serum and 10 g/ml ciprofloxacin and transfected with the indicated plasmids using the JetPEI or Lipofectamine 2000 kit as recommended by the manufacturers.
- The cells were labeled in 3% BSA-PBS using rabbit anti-LC3, mouse antiSQSTM1/p62 and goat anti-FLAG or mouse anti-FLAG antibodies followed by the appropriate Alexa Fluor 488, 555, 594 or 647 conjugated secondary antibodies.
- Image analysis .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
- It is The copyright holder for this preprintthis version posted March 30, 2021.
- The size of SQSTM1/p62 structures and colocalization of LC3 and SQSTM1/p62 labels was analyzed by creating a mask based on thresholding the SQSTM1/p62 fluorescence.
Results
- The herpesvirus DUBs interact with SQSTM1/p62 and regulate its ubiquitination.
- It is The copyright holder for this preprintthis version posted March 30, 2021.
- To investigate whether HCMVUL48, KSHV-ORF64 and HSV1-UL36 share with BPLF1 the capacity to prevent the colocalization of SQSTM1/p62 with LC3, HeLa cells transfected with plasmids expressing the viral enzymes were co-stained with antibodies specific for LC3 and SQSTM1/p62.
- .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
- The authors have previously shown that overexpression of a SQSTM1/p62 mutant where the Lys and Glu residues are substituted with Arg and Ala, respectively (SQSTM1/p62-E409A, K420R) can override the capacity of BPLF1 to inhibit selective autophagy, suggesting that deubiquitination of SQSTM1/p62 Lys420 may be critical for the effect of the viral enzyme (20).
Discussion
- While compelling evidence points to a key role of selective autophagy in the clearance of both the incoming viruses and newly synthesized viral proteins (19), different viruses have evolved distinct strategies for counteracting this cellular defense to promote their replication and spread.
- CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
- It is The copyright holder for this preprintthis version posted March 30, 2021.
- A detailed mapping of the interaction site(s) of SQSTM1/p62 with the herpesvirus homologs would be required to clarify this issue.
- Conceivably, the failure of UL36 to inhibit the formation of Lys63 linked chains on Lys7 could explain the retained formation of large SQSTM1/p62 aggregates that colocalize with LC3 in UL36 expressing cells, but differences in the ubiquitination status of this residue are not sufficient to explain the concomitant failure to inhibit selective autophagy.
Data availability
- All data that support the findings of this study are contained within the manuscript and are available on reasonable request.
- It is The copyright holder for this preprintthis version posted March 30, 2021.
Figure legends
- Figure 1. EBV-BPLF1, HSV-1-UL36, HCMV-UL48 and KSHV-ORF64 interact with SQSTM1/p62 but differentially affect its ubiquitination.
- It is The copyright holder for this preprintthis version posted March 30, 2021.
- HeLa cells were co-transfected with plasmids expressing aggregation-prone HTTQ109-GFP and increasing amounts of the viral DUBs.
- Blots from one representative experiment out of two are shown.
- In addition, intramolecular blockade of the UBA by Lys63-linked chains may redirect SQSTM1/p62 to other functions.
Did you find this useful? Give us your feedback
References
3,676 citations
3,398 citations
2,708 citations
2,379 citations
1,918 citations
Related Papers (5)
Frequently Asked Questions (11)
Q2. What is the role of UL36 in the autophagic degradation of cargo?
SQSTM1/p62 functions in the autophagic degradation of ubiquitinated cargo by physically linking the cargo to the autophagic machinery via binding to autophagosome-anchored LC3 (32).
Q3. What is the role of the ubiquitin chain in the interaction with the cargo?
The interaction with the cargo is mediated by the C-terminal ubiquitin associated domain (UBA) and is dependent on the ubiquitination of critical residues, which prevents SQSTM1/p62 homodimerization and the consequent occlusion of the cargo-binding site (25).
Q4. What is the role of autophagy in cellular homeostasis?
Autophagy is a protein degradation and recycling machinery that regulates cellular homeostasis and participates in the host defense against infection by capturing and destroying invading microorganisms (1, 2).
Q5. What is the role of Lys420 in the activity of viral enzymes?
The capacity of the SQSTM1/p62-E409A, K420R mutant to rescue the inhibitory effects of BPLF1, UL48, and ORF64 suggests that deubiquitination of Lys420 is critical for the activity of viral enzymes.
Q6. What is the ubiquitinating enzyme in the UL36 molecule?
The deubiquitinating enzymes encoded in the N-terminus of HSV1-UL36, HCMV-UL48, EBV-BPLF1 and KSHV-ORF64 bind to and deubiquitinate SQSTM1/p62.
Q7. Who has granted bioRxiv a license to display the preprint in perpetuity?
Plasmid encoding codon optimized 3xFLAG-tagged HSV-UL36 (amino acid.CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
Q8. What type of ubiquitin chains was not investigated?
Several ubiquitin ligases, including the Keap1/Cullin3 ligase (35), and under ubiquitin-stress conditions the UBE2D2/UBE2D3 conjugating enzymes (25), were shown to polyubiquitinate the Lys420 residue, but the type of ubiquitin chains was not investigated.
Q9. What is the role of Lys63 in the regulation of autophagy?
elimination of all types of chains may be required to stabilize the formation of homodimers that prevent cargo binding and inhibit autophagy; whereas persistent Lys63-linked ubiquitination appears to allow autophagy.
Q10. How many plasmids were encoding the viral DUBs?
HeLa cells were co-transfected with fixed amounts of plasmids encoding the viral DUBs and HTT109-GFP and increasing amounts of plasmids encoding the indicated SQSTM1/p62 mutants.
Q11. What is the effect of the mutant on the HTTQ109-GFP aggregates?
The weaker inhibitory activity of KSHV-ORF64 required high amount of transfected plasmid to achieve detectable levels of HTTQ109-GFP aggregates, which did not allow testing the effect of a large excess of the SQSTM1/p62-E409A, K420R mutant.