Limits...
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.

Show MeSH
Mass spectrometric analysis of PRMT6 arginine methylated residues. Following in vitro and in vivo methylation, methylated arginine residues in recombinant PRMT6 were mapped by LC-MS/MS. (A) Percent coverage obtained for PRMT6 WT (−/+ SAM) and the PRMT6 KLA mutant. Recombinant PRMT6 was digested with Trypsin in an ammonium bicarbonate buffer and peptides were separated onto a C18 column and sequenced by LC-MS (Methods). To evaluate trace amounts of dimethylated arginine in mutated protein, a 10 fold concentrated peptide solution was injected. Percentages coverage for PRMT6, PRMT6 + SAM and the PRMT6 mutant were 44%, 57%, and 52%, respectively (highlighted in green). Dimethylated arginine was identified only with PRMT6 WT +/− SAM (R29; R35; R37). Dimethylated residues are represented with an (*) above the residue. (B) To evaluate the percentage of dimethylated arginine on PRMT6, ions corresponding to EAALERPR (m/z 471.26) and EAALER*PR (m/z 485.27) were extracted. Dimethylation was only observed in the WT protein and in the WT protein + SAM (not shown). There was no detectable signal at m/z 485.27 in the PRMT6 KLA mutant even when used at 10-fold the WT protein concentration. Areas under the curve (AUC) were studied for both peptides from the PRMT6 WT protein; assuming no differences in ionization efficiency, the methylated protein apparently represents 10 to 20% of total protein. MS/MS spectra observed for the methylated (C) EAALERPR and non-methylated (D) peptide of wild type PRMT6. Observed ions are indicated in bold. (E)In vivo methylation assays were performed in HeLa cells with transfected myc-tagged PRMT6. Samples were digested and processed as described for the in vitro methylation assays. The percent coverage for PRMT6-WT was 63% (highlighted in green). Dimethylated arginines were identified for residues R29, R35, R38, R39 and R82, and are represented with an (*). Amino acids mutated in PRMT6-KLA are indicated by enlarged letters.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Mass spectrometric analysis of PRMT6 arginine methylated residues. Following in vitro and in vivo methylation, methylated arginine residues in recombinant PRMT6 were mapped by LC-MS/MS. (A) Percent coverage obtained for PRMT6 WT (−/+ SAM) and the PRMT6 KLA mutant. Recombinant PRMT6 was digested with Trypsin in an ammonium bicarbonate buffer and peptides were separated onto a C18 column and sequenced by LC-MS (Methods). To evaluate trace amounts of dimethylated arginine in mutated protein, a 10 fold concentrated peptide solution was injected. Percentages coverage for PRMT6, PRMT6 + SAM and the PRMT6 mutant were 44%, 57%, and 52%, respectively (highlighted in green). Dimethylated arginine was identified only with PRMT6 WT +/− SAM (R29; R35; R37). Dimethylated residues are represented with an (*) above the residue. (B) To evaluate the percentage of dimethylated arginine on PRMT6, ions corresponding to EAALERPR (m/z 471.26) and EAALER*PR (m/z 485.27) were extracted. Dimethylation was only observed in the WT protein and in the WT protein + SAM (not shown). There was no detectable signal at m/z 485.27 in the PRMT6 KLA mutant even when used at 10-fold the WT protein concentration. Areas under the curve (AUC) were studied for both peptides from the PRMT6 WT protein; assuming no differences in ionization efficiency, the methylated protein apparently represents 10 to 20% of total protein. MS/MS spectra observed for the methylated (C) EAALERPR and non-methylated (D) peptide of wild type PRMT6. Observed ions are indicated in bold. (E)In vivo methylation assays were performed in HeLa cells with transfected myc-tagged PRMT6. Samples were digested and processed as described for the in vitro methylation assays. The percent coverage for PRMT6-WT was 63% (highlighted in green). Dimethylated arginines were identified for residues R29, R35, R38, R39 and R82, and are represented with an (*). Amino acids mutated in PRMT6-KLA are indicated by enlarged letters.

Mentions: PRMT6 was previously shown to be automethylated [17]. Now, we wished to identify the arginine residues that are involved and, therefore, used purified recombinant PRMT6 in an in vitro methylation assay in the presence and absence of the methyl group donor S-adenosyl-methionine (SAM) with catalytically inactive recombinant PRMT6 V86K/D88A (PRMT6-KLA) serving as a control. Post-translational methylation was determined by mass spectrometry and showed that R29, R35 and R37 in the N-terminal region of the protein were methylated in the PRMT6 wild-type protein with and without SAM but not in the KLA inactive mutant (Figure 1A). Extracting ions from the peptides that contain R35 from both proteins confirmed the absence of methylation on this residue within the KLA mutant (Figure 1B). The observation that PRMT6-WT was methylated in the absence of added substrate suggested that most of the active protein was automethylated during bacterial expression and also provided an explanation for the low levels of automethylation observed in previous studies [17]. We also confirmed that R35 is methylated in vivo by conducting an in vivo methylation assay (Figure 1E).


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)

Mass spectrometric analysis of PRMT6 arginine methylated residues. Following in vitro and in vivo methylation, methylated arginine residues in recombinant PRMT6 were mapped by LC-MS/MS. (A) Percent coverage obtained for PRMT6 WT (−/+ SAM) and the PRMT6 KLA mutant. Recombinant PRMT6 was digested with Trypsin in an ammonium bicarbonate buffer and peptides were separated onto a C18 column and sequenced by LC-MS (Methods). To evaluate trace amounts of dimethylated arginine in mutated protein, a 10 fold concentrated peptide solution was injected. Percentages coverage for PRMT6, PRMT6 + SAM and the PRMT6 mutant were 44%, 57%, and 52%, respectively (highlighted in green). Dimethylated arginine was identified only with PRMT6 WT +/− SAM (R29; R35; R37). Dimethylated residues are represented with an (*) above the residue. (B) To evaluate the percentage of dimethylated arginine on PRMT6, ions corresponding to EAALERPR (m/z 471.26) and EAALER*PR (m/z 485.27) were extracted. Dimethylation was only observed in the WT protein and in the WT protein + SAM (not shown). There was no detectable signal at m/z 485.27 in the PRMT6 KLA mutant even when used at 10-fold the WT protein concentration. Areas under the curve (AUC) were studied for both peptides from the PRMT6 WT protein; assuming no differences in ionization efficiency, the methylated protein apparently represents 10 to 20% of total protein. MS/MS spectra observed for the methylated (C) EAALERPR and non-methylated (D) peptide of wild type PRMT6. Observed ions are indicated in bold. (E)In vivo methylation assays were performed in HeLa cells with transfected myc-tagged PRMT6. Samples were digested and processed as described for the in vitro methylation assays. The percent coverage for PRMT6-WT was 63% (highlighted in green). Dimethylated arginines were identified for residues R29, R35, R38, R39 and R82, and are represented with an (*). Amino acids mutated in PRMT6-KLA are indicated by enlarged letters.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Mass spectrometric analysis of PRMT6 arginine methylated residues. Following in vitro and in vivo methylation, methylated arginine residues in recombinant PRMT6 were mapped by LC-MS/MS. (A) Percent coverage obtained for PRMT6 WT (−/+ SAM) and the PRMT6 KLA mutant. Recombinant PRMT6 was digested with Trypsin in an ammonium bicarbonate buffer and peptides were separated onto a C18 column and sequenced by LC-MS (Methods). To evaluate trace amounts of dimethylated arginine in mutated protein, a 10 fold concentrated peptide solution was injected. Percentages coverage for PRMT6, PRMT6 + SAM and the PRMT6 mutant were 44%, 57%, and 52%, respectively (highlighted in green). Dimethylated arginine was identified only with PRMT6 WT +/− SAM (R29; R35; R37). Dimethylated residues are represented with an (*) above the residue. (B) To evaluate the percentage of dimethylated arginine on PRMT6, ions corresponding to EAALERPR (m/z 471.26) and EAALER*PR (m/z 485.27) were extracted. Dimethylation was only observed in the WT protein and in the WT protein + SAM (not shown). There was no detectable signal at m/z 485.27 in the PRMT6 KLA mutant even when used at 10-fold the WT protein concentration. Areas under the curve (AUC) were studied for both peptides from the PRMT6 WT protein; assuming no differences in ionization efficiency, the methylated protein apparently represents 10 to 20% of total protein. MS/MS spectra observed for the methylated (C) EAALERPR and non-methylated (D) peptide of wild type PRMT6. Observed ions are indicated in bold. (E)In vivo methylation assays were performed in HeLa cells with transfected myc-tagged PRMT6. Samples were digested and processed as described for the in vitro methylation assays. The percent coverage for PRMT6-WT was 63% (highlighted in green). Dimethylated arginines were identified for residues R29, R35, R38, R39 and R82, and are represented with an (*). Amino acids mutated in PRMT6-KLA are indicated by enlarged letters.
Mentions: PRMT6 was previously shown to be automethylated [17]. Now, we wished to identify the arginine residues that are involved and, therefore, used purified recombinant PRMT6 in an in vitro methylation assay in the presence and absence of the methyl group donor S-adenosyl-methionine (SAM) with catalytically inactive recombinant PRMT6 V86K/D88A (PRMT6-KLA) serving as a control. Post-translational methylation was determined by mass spectrometry and showed that R29, R35 and R37 in the N-terminal region of the protein were methylated in the PRMT6 wild-type protein with and without SAM but not in the KLA inactive mutant (Figure 1A). Extracting ions from the peptides that contain R35 from both proteins confirmed the absence of methylation on this residue within the KLA mutant (Figure 1B). The observation that PRMT6-WT was methylated in the absence of added substrate suggested that most of the active protein was automethylated during bacterial expression and also provided an explanation for the low levels of automethylation observed in previous studies [17]. We also confirmed that R35 is methylated in vivo by conducting an in vivo methylation assay (Figure 1E).

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