Cytoplasmic localization of SIRT2 is dependent on constitutive nuclear export.

<p>(A) HeLa cells were transfected with GFP-SIRT2 and analyzed by immunofluorescence for SIRT2 localization. (B) Cells from (A) were scored for their distribution as cytoplasmic or pancellular. (C) HeLa cells were transfected with GFP-SIRT2 and incubated with or without leptomycin B (LMB). (D) Cells from (C) were scored for localization of GFP-SIRT2 in the nucleus. (E) HeLa cells were transfected with GFP-SIRT2 followed by treatment with LMB. At indicated times, cells were fixed and visualized for nuclear localization.</p

Overexpression of SIRT2 leads increases mutlinucleation.

<p>HeLa cells were transfected with GFP, and wild-type or H187Y GFP-SIRT2 and 48 hours after transfection, cells were scored for the presence of multiple nuclei. Percentages represent average of counting ∼200 cells in three independent transfections with error bars representing standard deviation.</p

Enrichment of the three proteins on the midbody is shown in the merge images.

<p>(A) HeLa cells were stained with antisera for SIRT2 (green) and Aurora B (blue) and acetylated tubulin (red), and analyzed by confocal microscopy. Enrichment of the three proteins on the midbody is shown in the merged images. (B) 293T cells were transfected with Myc-Aurora A with or without SIRT2-FLAG. Cellular lysates were immunoprecipitated with anti-FLAG and probed by western blotting with antisera specific for FLAG and Myc. 10% of protein input was analyzed by western blotting with antisera for FLAG or Myc.</p

Mutational analysis of SIRT2 NES sequence.

<p>(A) HeLa cells were transfected with GFP-SIRT2 wild-type or single and double point mutants of the proposed NES sequence. (B) Subcellular distribution results of all single and double mutants of SIRT2 NES analyzed in (A). (C) 293T cells were transfected with GFP-SIRT2 single and double point mutants and lysates were separated by SDS–PAGE and visualized by western blotting with an antiserum specific for GFP. (D) HeLa cells were transfected with SIRT2-FLAG and fibrillarin-GFP, treated with or without LMB for 2 hrs and subsequently stained for FLAG and visualize by confocal microscopy.</p

Resting primary CD4⁺ T cells support both productive and latent HIV infection.

<p>(A) Diagram of the HIV Duo-Fluo I virus, in which eGFP has replaced the <i>nef</i> gene, and a whole transcription unit—consisting of an EF1α promoter driving the expression of an mCherry fluorescent marker—has been inserted downstream. Upon infection with the HIV Duo-Fluo I virus, cells that express GFP alone or GFP and mCherry are considered productively infected; cells that express only mCherry are considered latently infected; cells that lack expression of either fluorescent marker are considered uninfected. (B) Expression of the activation markers CD69 and CD25 in resting primary CD4^<b>+</b> T cells either left untreated or stimulated with CCL19, IL-7, or αCD3/αCD28 activating beads for 72 h. Mean Fluorescence Intensity (MFI) for CD25 expression is also shown. (C) Infection profiles of untreated or stimulated primary CD4^<b>+</b> T cells 6 days after infection via flow cytometry. Untreated resting CD4^<b>+</b> T cells were infected with either HIV Duo-Fluo I virus alone or the Vpx-containing HIV Duo-Fluo I virus. Stimulated cells were infected with HIV Duo-Fluo I alone. Productive infection (GFP+ and GFP/mCherry double-positive) and latent infection (mCherry+) were analyzed by flow cytometry. Data shown are from a single donor but are representative of three separate donors. (D) Quantified values of latent infection and productive infection from panel C. Data represents the average of three donors. (E) Ratios of latent infection to productive infection were calculated using data from panel D. Data represent the average of three donors. (F) Quantified values for reactivation of pre-integration latent virus and post-integration provirus calculated from the isolated uninfected populations (GFP/mCherry double-negative) of untreated and stimulated primary CD4^<b>+</b> T cells via flow cytometry (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004955#ppat.1004955.s004" target="_blank">S4 Fig</a>). Six days after infection, uninfected cells were isolated via fluorescence-activated cell sorting (FACS) and were either left unstimulated or stimulated with αCD3/αCD28 activating beads alone or αCD3/αCD28 activating beads in the presence of raltegravir for 48 h. Reactivatable pre-integration latent virus was calculated by subtracting the amount of productive infection from cells treated with αCD3/αCD28 activating beads alone and cells treated with αCD3/αCD28 activating beads in the presence of raltegravir. Reactivatable post-integration latent provirus was calculated by subtracting the amount of productive infection from unstimulated cells and cells treated with αCD3/αCD28 activating beads in the presence of raltegravir. Data represent the average of three donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, non-significant.</p

Primary CD4⁺ T cells transitioning from an activated state back to a resting state are more likely to become latently infected.

<p>(A) Schematic of experimental procedure. Primary CD4^<b>+</b> T cells were isolated from uninfected donor blood and stimulated with αCD3/αCD28 activating beads in the presence of IL-2 for 72 h and were then allowed to return to a resting state over 20 days in the presence of IL-2. Cells were infected at peak activation (day 4) and every 5 days thereafter as they returned to a resting state. (B) Expression of activation markers CD69 and CD25 as the cells transition from an activated state to a resting state. Flow cytometry was performed 72 h post activation and every 5 days after the activation beads were removed. Data shown are from a single donor, but representative of three separate donors. Percentage of live cells is calculated from the live gate (forward vs side scatter plots) in the FACS analysis and represents the average of three donors. (C) Quantified values of the cells’ activation status from panel B. Data represents the average of three donors. (D) Infection profiles of primary CD4^<b>+</b> T cells as they transition back to a resting state. Cells were spinoculated with HIV Duo-Fluo I 72 h after activation and every 5 days after the activation beads were removed. Infection was analyzed by flow cytometry 72 h post-infection. Data shown are from a single donor, but representative of three separate donors. (E) Quantified values of latent infection and productive infection from panel D. Data represents the average of three donors. (F) Ratios of latent infection to productive infection were calculated using data from panel E. Data represents the average of three donors. *, P < 0.05; **, P < 0.01; ns, non-significant.</p

Primary CD4⁺ T cells and total lymphoid cell populations isolated from peripheral blood and tonsillar and splenic tissues are more likely to become latently infected.

<p>(A) Expression of activation markers CD69 and CD25 on CD4^<b>+</b> T cells and total lymphoid cell populations either left untreated or stimulated with αCD3/αCD28 activating beads for 72 h. Data shown are from a single donor, but are representative of three separate donors. (B) Infection profiles of CD4^<b>+</b> T cells and total lymphoid cell populations from panel A. Cells were infected with HIV Duo-Fluo I and analyzed for productive and latent infection 72 h after infection. Data shown are from a single donor, but are representative of three separate donors. (C) Quantification of latent infection and productive infection from panel B. Data represent the average of three donors. (D) Ratios of latent infection to productive infection were calculated using data from panel C. Data represent the average of three donors. *, P < 0.05; ***, P < 0.001; ns, nonsignificant.</p

The BAF complex represses basal transcription at the HIV promoter.

<p>(A) Table of subunit composition of the two distinct SWI/SNF complexes, BAF and PBAF, in mammals. (B) GFP expression was monitored by flow cytometry at indicated times after siRNA transfection to measure HIV promoter activity. Results are presented as MFI for cells treated with a control siRNA or siRNAs specific for SWI/SNF subunits. (C) Same experiments as shown in (B) for clone D, with clone E, another Jurkat cell line containing an integrated LTR-GFP virus. Error bars represent the SEM of five independent experiments. * <i>p</i><0.05. (D) Jurkat cells containing an integrated LTR-GFP virus (clone D) were transfected with control siRNA or siRNAs targeting various SWI/SNF complex subunits as indicated. Western blot analysis shows expression of each SWI/SNF subunit after its specific depletion 0, 2, 4, 6, 7, 8, 10, and 14 d after siRNA transfection with each specific antibody and β-actin loading control as indicated.</p

PBAF, recruited by K50K51 acetylated Tat, is a co-factor for Tat activation of the HIV promoter.

<p>(A) J-Lat A2 cells containing an integrated LTR-Tat-FLAG-GFP were stimulated with PMA to induce expression of Tat-FLAG. Tat was immunoprecipitated from untreated or PMA-stimulated cell lysates and its associated proteins were examined by SDS-PAGE and Western blotting with antibodies against the BAF- or PBAF-specific subunits BAF250a and BAF180, and protein kinase D-1 and 14-3-3 as controls. (B) Tat co-immunoprecipitation with BAF180 is modulated by Tat acetylation. Tat (wild-type or K50R/L51R) was immunoprecipitated using anti-FLAG antibody and analyzed by Western blotting using antibody specific for BAF180. Tat acetylation levels were assessed using an anti-acetyl lysine antibody. All proteins were expressed at similar levels under the different experimental conditions as shown by the Inputs. (C) 1G5 Jurkat cells containing integrated LTR-Luciferase (LTR-Luc) were nucleofected with siRNAs against BAF180, BAF250, or with a control siRNA pool. Expression of BAF180, BAF250, and β-actin was analyzed by Western blotting after depletion of either BAF180 or BAF250. (D) Transactivation of the HIV promoter by Tat is reduced in the absence of BAF180. 48 h after siRNA depletion of BAF180 or BAF250, cells were re-transfected with either a control or Tat-expression vector (CMV-driven), and luciferase assay performed after 24 h. Error bars represent the SEM of three independent experiments. * <i>p</i><0.05. (D).</p

Mitochondrial Acetylome Analysis in a Mouse Model of Alcohol-Induced Liver Injury Utilizing SIRT3 Knockout Mice

Mitochondrial protein hyperacetylation is a known consequence of sustained ethanol consumption and has been proposed to play a role in the pathogenesis of alcoholic liver disease (ALD). The mechanisms underlying this altered acetylome, however, remain unknown. The mitochondrial deacetylase sirtuin 3 (SIRT3) is reported to be the major regulator of mitochondrial protein deacetylation and remains a central focus for studies on protein acetylation. To investigate the mechanisms underlying ethanol-induced mitochondrial acetylation, we employed a model for ALD in both wild-type (WT) and SIRT3 knockout (KO) mice using a proteomics and bioinformatics approach. Here, WT and SIRT3 KO groups were compared in a mouse model of chronic ethanol consumption, revealing pathways relevant to ALD, including lipid and fatty acid metabolism, antioxidant response, amino acid biosynthesis and the electron-transport chain, each displaying proteins with altered acetylation. Interestingly, protein hyperacetylation resulting from ethanol consumption and SIRT3 ablation suggests ethanol-induced hyperacetylation targets numerous biological processes within the mitochondria, the majority of which are known to be acetylated through SIRT3-dependent mechanisms. These findings reveal overall increases in 91 mitochondrial targets for protein acetylation, identifying numerous critical metabolic and antioxidant pathways associated with ALD, suggesting an important role for mitochondrial protein acetylation in the pathogenesis of ALD