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

  • ️Tue Jan 01 2013

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

Diane N Singhroy et al. Retrovirology. 2013.

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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Figures

Figure 1
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.

Figure 2
Figure 2

The R35 residue is conserved in evolution. Sequence alignment of PRMT6 proteins from various organisms showing conservation of the arginine residue at position 35 (H. sapiens). The consensus sequence was produced using ClustalW2 (

http://www.ebi.ac.uk/Tools/msa/clustalw2/

). R35 is bolded and the underlined text refers to the arginine rich motif.

Figure 3
Figure 3

PRMT6-R35A does not automethylate. Cell-free automethylation assays were performed using the indicated PRMT6 recombinant proteins in the presence of 3H-SAM. Autoradiography (upper) and Coomassie staining (lower) are shown. 3H-SAM was contained in all wells. The first lane contains methylation buffer only, without the presence of PRMT6. The other lanes contain the indicated recombinant proteins. This experiment was performed three times with similar results being obtained each time; a representative result is shown.

Figure 4
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.

Figure 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.

Figure 6
Figure 6

PRMT6 automethylation is necessary for its HIV-1 restriction activity. The indicated forms of PRMT6 plasmid were co-transfected with HIV-1 pNL4.3 proviral DNA into 293T cells. At 48 hours after transfection, cell culture fluids containing virus were collected, quantified by QPCR (A) or by HIV RT activity assay (B), and titrated onto TZM-bl cells (C, D). The amount of transfected PRMT6 expression, i.e. not protein stability, was quantified by Western blots with anti-Myc and anti-actin antibodies followed by densitometric analysis normalized against actin (E). Endogenous levels of PRMT6 in HeLa cells (lane 2), TZM-bl cells (lane 3) and 293T cells (lane 4) were compared to transfected PRMT6 in HeLa cells (lane 1) (F). 15 μg of protein from whole cell extracts were loaded into each well used for analysis in the Western blots in (E) and (F). Actin was used as a loading control. Infectivity was measured by luciferase assay at 48 hours after infection. The control experiment was transfection of pNL4.3 with an empty plasmid (referred to on the figure as pNL4.3 only). Each experiment was performed in triplicate on three separate occasions, with similar results being obtained each time. T-tests were performed for both (A) and (B), showing that only PRMT6-WT was significantly different than the control *: p < 0.05. Two-way ANOVA was performed for (C) and (D), showing that only PRMT6-WT statistically inhibited HIV replication *: p < 0.001.

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