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Automethylation of protein arginine methyltransferase 6 (PRMT6) regulates its stability and its anti-HIV-1 activity.

Singhroy DN, Mesplède T, Sabbah A, Quashie PK, Falgueyret JP, Wainberg MA - Retrovirology (2013)

Bottom Line: In addition, PRMT6 inhibits HIV-1 replication in cell culture by directly methylating and interfering with the functions of several HIV-1 proteins, i.e. Tat, Rev and nucleocapsid (NC).PRMT6 also displays automethylation capacity but the role of this post-translational modification in its antiretroviral activity remains unknown.These results show that PRMT6 automethylation plays a role in the stability of this protein and that this event is indispensible for its anti-HIV-1 activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: McGill University AIDS Centre, Lady Davis for Medical Research, Jewish General Hospital, 3755 Cote Sainte Catherine, Montreal, QC, H3T 1E2, Canada.

ABSTRACT

Background: Protein arginine methyltransferase 6 (PRMT6) is a nuclear enzyme that methylates arginine residues on histones and transcription factors. In addition, PRMT6 inhibits HIV-1 replication in cell culture by directly methylating and interfering with the functions of several HIV-1 proteins, i.e. Tat, Rev and nucleocapsid (NC). PRMT6 also displays automethylation capacity but the role of this post-translational modification in its antiretroviral activity remains unknown.

Results: Here we report the identification by liquid chromatography-mass spectrometry of R35 within PRMT6 as the target residue for automethylation and have confirmed this by site-directed mutagenesis and in vitro and in vivo methylation assays. We further show that automethylation at position 35 greatly affects PRMT6 stability and is indispensable for its antiretroviral activity, as demonstrated in HIV-1 single-cycle TZM-bl infectivity assays.

Conclusion: These results show that PRMT6 automethylation plays a role in the stability of this protein and that this event is indispensible for its anti-HIV-1 activity.

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PRMT6-R35A is catalytically active. A PRMT6 cell-free methylation assay was performed with HIV-1 Rev protein, a known substrate of PRMT6. The upper frame represents the autoradiograph of Rev methylation by PRMT6. The lower frame represents a Coomassie stained gel of Rev. This experiment was performed three times with similar results obtained each time; a representative result is shown.
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Figure 4: PRMT6-R35A is catalytically active. A PRMT6 cell-free methylation assay was performed with HIV-1 Rev protein, a known substrate of PRMT6. The upper frame represents the autoradiograph of Rev methylation by PRMT6. The lower frame represents a Coomassie stained gel of Rev. This experiment was performed three times with similar results obtained each time; a representative result is shown.

Mentions: To characterize the role of automethylation in the activity of PRMT6, the central residue R35 was mutated to an alanine residue (PRMT6-R35A) and automethylation was measured in vitro in the presence of 3H-SAM (Figure 3). Mutant PRMT6-KLA was used as a negative control. Weak but consistent automethylation was measured for the wild-type recombinant PRMT6 protein but not for either KLA or R35A, suggesting that R35 plays a critical role in automethylation. Therefore our study focused on the role of this residue. In a similar experiment, PRMT6-R35A was shown to be able to methylate the purified HIV-1 Rev protein in vitro, indicating that this mutated derivative protein retains its ability to arginine methylate other proteins but not itself (Figure 4). In the same experiment, PRMT6-KLA was, as expected, completely unable to methylate Rev. Thus, R35 is a PRMT6 primary automethylation site and automethylation may not be essential for the methylation of HIV-1 Rev. We performed similar in vitro methylation experiments using HIV-1 Tat as a substrate and found that PRMT6-R35A maintains methylation activity (data not shown).


Automethylation of protein arginine methyltransferase 6 (PRMT6) regulates its stability and its anti-HIV-1 activity.

Singhroy DN, Mesplède T, Sabbah A, Quashie PK, Falgueyret JP, Wainberg MA - Retrovirology (2013)

PRMT6-R35A is catalytically active. A PRMT6 cell-free methylation assay was performed with HIV-1 Rev protein, a known substrate of PRMT6. The upper frame represents the autoradiograph of Rev methylation by PRMT6. The lower frame represents a Coomassie stained gel of Rev. This experiment was performed three times with similar results obtained each time; a representative result is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750301&req=5

Figure 4: PRMT6-R35A is catalytically active. A PRMT6 cell-free methylation assay was performed with HIV-1 Rev protein, a known substrate of PRMT6. The upper frame represents the autoradiograph of Rev methylation by PRMT6. The lower frame represents a Coomassie stained gel of Rev. This experiment was performed three times with similar results obtained each time; a representative result is shown.
Mentions: To characterize the role of automethylation in the activity of PRMT6, the central residue R35 was mutated to an alanine residue (PRMT6-R35A) and automethylation was measured in vitro in the presence of 3H-SAM (Figure 3). Mutant PRMT6-KLA was used as a negative control. Weak but consistent automethylation was measured for the wild-type recombinant PRMT6 protein but not for either KLA or R35A, suggesting that R35 plays a critical role in automethylation. Therefore our study focused on the role of this residue. In a similar experiment, PRMT6-R35A was shown to be able to methylate the purified HIV-1 Rev protein in vitro, indicating that this mutated derivative protein retains its ability to arginine methylate other proteins but not itself (Figure 4). In the same experiment, PRMT6-KLA was, as expected, completely unable to methylate Rev. Thus, R35 is a PRMT6 primary automethylation site and automethylation may not be essential for the methylation of HIV-1 Rev. We performed similar in vitro methylation experiments using HIV-1 Tat as a substrate and found that PRMT6-R35A maintains methylation activity (data not shown).

Bottom Line: In addition, PRMT6 inhibits HIV-1 replication in cell culture by directly methylating and interfering with the functions of several HIV-1 proteins, i.e. Tat, Rev and nucleocapsid (NC).PRMT6 also displays automethylation capacity but the role of this post-translational modification in its antiretroviral activity remains unknown.These results show that PRMT6 automethylation plays a role in the stability of this protein and that this event is indispensible for its anti-HIV-1 activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: McGill University AIDS Centre, Lady Davis for Medical Research, Jewish General Hospital, 3755 Cote Sainte Catherine, Montreal, QC, H3T 1E2, Canada.

ABSTRACT

Background: Protein arginine methyltransferase 6 (PRMT6) is a nuclear enzyme that methylates arginine residues on histones and transcription factors. In addition, PRMT6 inhibits HIV-1 replication in cell culture by directly methylating and interfering with the functions of several HIV-1 proteins, i.e. Tat, Rev and nucleocapsid (NC). PRMT6 also displays automethylation capacity but the role of this post-translational modification in its antiretroviral activity remains unknown.

Results: Here we report the identification by liquid chromatography-mass spectrometry of R35 within PRMT6 as the target residue for automethylation and have confirmed this by site-directed mutagenesis and in vitro and in vivo methylation assays. We further show that automethylation at position 35 greatly affects PRMT6 stability and is indispensable for its antiretroviral activity, as demonstrated in HIV-1 single-cycle TZM-bl infectivity assays.

Conclusion: These results show that PRMT6 automethylation plays a role in the stability of this protein and that this event is indispensible for its anti-HIV-1 activity.

Show MeSH