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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 less stable than PRMT6-WT. (A) Western blots for Myc-PRMT6-WT, PRMT6-R35A and PRMT6-KLA, following treatment with CHX (upper panels). Actin was used as a loading control (lower panels). 15 μg of protein from whole cell extract were loaded into each well. (B) Densitometric analysis of Myc-PRMT6-WT and -R35A degradation over time following the addition of CHX. Myc expression was normalized to levels of actin. Each experiment was performed three times with similar results obtained each time; a representative blot is shown.
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Figure 5: PRMT6-R35A is less stable than PRMT6-WT. (A) Western blots for Myc-PRMT6-WT, PRMT6-R35A and PRMT6-KLA, following treatment with CHX (upper panels). Actin was used as a loading control (lower panels). 15 μg of protein from whole cell extract were loaded into each well. (B) Densitometric analysis of Myc-PRMT6-WT and -R35A degradation over time following the addition of CHX. Myc expression was normalized to levels of actin. Each experiment was performed three times with similar results obtained each time; a representative blot is shown.

Mentions: We next measured the stability of the wild-type, KLA, and R35A PRMT6 proteins (Figure 5). Plasmids coding for the various forms of myc-tagged PRMT6 were transfected into HeLa cells, and, at 24 h after transfection, cycloheximide (CHX) was added to culture supernatants. Since CHX inhibits protein synthesis, it can be used to study protein degradation over time in studies in which expression levels of PRMT6 protein are measured by Western-blot. The results show that PRMT6 is very stable (Figure 5A, first panel), but that the mutant R35A protein and the catalytically inactive KLA form of PRMT6 were less stable, with their expression levels decreasing over time. Densitometric quantification of the Western-blot confirmed that the expression levels of both mutant proteins decreased faster than the WT protein following the addition of CHX (Figure 5B). Thus, PRMT6 automethylation is important for its stability.


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 less stable than PRMT6-WT. (A) Western blots for Myc-PRMT6-WT, PRMT6-R35A and PRMT6-KLA, following treatment with CHX (upper panels). Actin was used as a loading control (lower panels). 15 μg of protein from whole cell extract were loaded into each well. (B) Densitometric analysis of Myc-PRMT6-WT and -R35A degradation over time following the addition of CHX. Myc expression was normalized to levels of actin. Each experiment was performed three times with similar results obtained each time; a representative blot is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC3750301&req=5

Figure 5: PRMT6-R35A is less stable than PRMT6-WT. (A) Western blots for Myc-PRMT6-WT, PRMT6-R35A and PRMT6-KLA, following treatment with CHX (upper panels). Actin was used as a loading control (lower panels). 15 μg of protein from whole cell extract were loaded into each well. (B) Densitometric analysis of Myc-PRMT6-WT and -R35A degradation over time following the addition of CHX. Myc expression was normalized to levels of actin. Each experiment was performed three times with similar results obtained each time; a representative blot is shown.
Mentions: We next measured the stability of the wild-type, KLA, and R35A PRMT6 proteins (Figure 5). Plasmids coding for the various forms of myc-tagged PRMT6 were transfected into HeLa cells, and, at 24 h after transfection, cycloheximide (CHX) was added to culture supernatants. Since CHX inhibits protein synthesis, it can be used to study protein degradation over time in studies in which expression levels of PRMT6 protein are measured by Western-blot. The results show that PRMT6 is very stable (Figure 5A, first panel), but that the mutant R35A protein and the catalytically inactive KLA form of PRMT6 were less stable, with their expression levels decreasing over time. Densitometric quantification of the Western-blot confirmed that the expression levels of both mutant proteins decreased faster than the WT protein following the addition of CHX (Figure 5B). Thus, PRMT6 automethylation is important for its stability.

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