VLCAD from SIRT3 and SIRT5 knockout mice shows reduced affinity for cardiolipin.

<p>A) The fat blot method was used to evaluate endogenous VLCAD binding to cardiolipin in fasted (20 hr) mouse liver lysates. B) Densitometry was used to quantify binding from panel A. C) Lysate from VLCAD-/- liver was tested as a negative control and shows no detectable signal. D) Western blot was used to confirm that total VLCAD expression is not significantly different between wild-type, SIRT3 KO, and SIRT5 KO mice.</p

The SIRT3/SIRT5 target site K299 is critical for FAD binding and VLCAD activity.

<p>A) Chemical acetylation and succinylation both reduce enzymatic activity of recombinant VLCAD. B) Incubation of acetylated VLCAD with SIRT3 rescues activity, while incubation of succinylated VLCAD with SIRT5 does not (not shown). C) Mutant K298R retains sensitivity to acylation-induced loss of activity,suggesting that K298 does not play a mechanistic role in the reduced activity. D) Likewise, mutant K507R retains sensitivity to acylation-induced loss of activity, suggesting that K507 also does not play a mechanistic role in the reduced activity. E) K299 is highly sensitive to conservative substitution with arginine. K299R lost the yellow color characteristic of FAD and consequently became inactive. All bar graphs depict means and standard deviations of triplicate assays. *P<0.01 versus wild-type or control.</p

SIRT3 and SIRT5 deacylate VLCAD at overlapping sites.

<p>A) Recombinant, unmodified VLCAD (Ctrl) was subjected to chemical succinylation (top) or acetylation (bottom) which was verified by western blotting with anti-succinyllysine (SuK) or anti-acetyllysine (AcK) antibodies. B) Chemically succinylated (Suc) and acetylated (Ac) VLCAD proteins were reacted with SIRT5 and SIRT3, respectively. Changes in succinylation or acetylation were then evaluated by western blotting, with anti-His blotting as loading control. C) Only SIRT3 reacts with chemically acetylated VLCAD as determined by incubating increasing amounts of acetylated VLCAD with SIRT3, SIRT4, or SIRT5 in the presence of radiolabeled NAD+. Shown are the means of duplicate assays. D) Acetylated VLCAD was treated with SIRT3 or inactive mutant SIRT3 (Control). Quantitative mass spectrometry was used to determine the relative abundance of acetylated peptides. Shown are acetylation sites with >2-fold change. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122297#pone.0122297.s001" target="_blank">S1 Dataset</a> for details. E) Succinylated VLCAD was treated with SIRT5 or inactive mutant SIRT5 (Control) and succinylated peptides were quantified by mass spectrometry. Shown are succinylation sites with >2-fold change. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122297#pone.0122297.s002" target="_blank">S2 Dataset</a> for details. D and E both depict the means and standard deviations of quadruplicate assays.</p

SIRT3 and SIRT5 deacylate lysines that localize to the active site and putative membrane binding domain of VLCAD.

<p>K299 (red) hydrogen bonds with neighboring S304 (green), and both are within interacting distance of the essential FAD cofactor (yellow) which is non-covalently bound in the VLCAD active site. B) Amino acid alignment of the region surrounding K299, showing conservation of this residue across diverse species. C) The portion of VLCAD spanning residues 486–518, which includes sirtuin target sites K492 and K507, is disordered in the crystal structure. PsiPred was used to generate a model of the disordered segment which was overlaid upon the structure of a VLCAD monomer. Hydrophobic residues are rendered red, positively charged residues blue, and negatively charged residues green. The active site is indicated as FAD in yellow and acyl-CoA substrate in red. D) Amino acid alignment of the putative membrane-binding amphipathic helix.</p

Identification and characterization of AckA-dependent protein acetylation in <i>Neisseria gonorrhoeae</i>

<div><p><i>Neisseria gonorrhoeae</i>, the causative agent of gonorrhea, has a number of factors known to contribute to pathogenesis; however, a full understanding of these processes and their regulation has proven to be elusive. Post-translational modifications (PTMs) of bacterial proteins are now recognized as one mechanism of protein regulation. In the present study, Western blot analyses, with an anti-acetyl-lysine antibody, indicated that a large number of gonococcal proteins are post-translationally modified. Previous work has shown that N^ε-lysine acetylation can occur non-enzymatically with acetyl-phosphate (AcP) as the acetyl donor. In the current study, an acetate kinase mutant (1291<i>ackA</i>), which accumulates AcP, was generated in <i>N</i>. <i>gonorrhoeae</i>. Broth cultures of <i>N</i>. <i>gonorrhoeae</i> 1291wt and 1291<i>ackA</i> were grown, proteins extracted and digested, and peptides containing acetylated-lysines (K-acetyl) were affinity-enriched from both strains. Mass spectrometric analyses of these samples identified a total of 2686 unique acetylation sites. Label-free relative quantitation of the K-acetyl peptides derived from the <i>ackA</i> and wild-type (wt) strains demonstrated that 109 acetylation sites had an <i>ackA</i>/wt ratio>2 and p-values <0.05 in at least 2/3 of the biological replicates and were designated as “AckA-dependent”. Regulated K-acetyl sites were found in ribosomal proteins, central metabolism proteins, iron acquisition and regulation proteins, pilus assembly and regulation proteins, and a two-component response regulator. Since AckA is part of a metabolic pathway, comparative growth studies of the <i>ackA</i> mutant and wt strains were performed. The mutant showed a growth defect under aerobic conditions, an inability to grow anaerobically, and a defect in biofilm maturation. In conclusion, the current study identified AckA-dependent acetylation sites in <i>N</i>. <i>gonorrhoeae</i> and determined that these sites are found in a diverse group of proteins. This work lays the foundation for future studies focusing on specific acetylation sites that may have relevance in gonococcal pathogenesis and metabolism.</p></div

Downregulated proteins in <i>N. gonorrhoeae</i> biofilms listed by functional roles^a: Biofilm/Planktonic ratios expressed as log₂ values, 2-fold decrease (log₂ values ≤−1.000) measured in at least one Extract, and p-values <0.05.

a<p>Functional role assignments (both main and JCVI sub-roles) for the <i>N. gonorrhoeae</i> proteins were downloaded from the JCVI-CMR website.</p>b<p>Average log₂ values for biofilm/planktonic protein ratios.</p>c<p>(N) represents the number of measurements (biological replicates) averaged.</p>d<p>Protein with more than one functional role assignment provided (Main role/JCVI sub-role): NGO0639, Energy metabolism/Glycolysis/gluconeogenesis.</p>e<p>Protein functional role category tentatively assigned in the present study by orthology to an <i>N. meningitidis</i> protein (see Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038303#pone.0038303.s010" target="_blank">Table S6</a> for details).</p

Plot of the functional classifications of all upregulated and downregulated proteins in the biofilm compared to the planktonic growth form.

<p>All of the tabulated proteins met a 1.5-fold cutoff threshold for differential expression, with quantitation p-values <0.05. Functional role assignments for the <i>N. gonorrhoeae</i> proteins were downloaded from the JCVI-CMR website (<a href="http://cmr.jcvi.org/tigr-scripts/CMR/CmrHomePage.cgi" target="_blank">http://cmr.jcvi.org/tigr-scripts/CMR/CmrHomePage.cgi</a>). In a few cases where functional role assignments were not available, the <i>N. gonorrhoeae</i> proteins were given tentative assignments in the present study after conducting BLAST and KEGG orthology searches (see Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038303#pone.0038303.s009" target="_blank">Tables S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038303#pone.0038303.s010" target="_blank">S6</a> for details).</p

Mass spectrometric workflow.

<p>(A) Three biological replicates of <i>N</i>. <i>gonorrhoeae</i> strains 1291wt and 1291<i>ackA</i> were grown overnight in broth cultures. Cells were harvested, washed, and a protein lysate was generated and digested with trypsin. Affinity enrichment for acetyl-lysine (K-acetyl)-containing peptides was performed using a polyclonal anti-acetyl-Lys antibody. Enriched K-acetyl peptides were analyzed by high-resolution label-free LC-MS/MS and quantified using MS1 quantitation. (B) An example of a peptide analyzed by MS1 quantitation using Skyline. MS1 ion chromatograms of the precursor ions for M, M+1, and M+2 for the acetylated peptide IKLD<u><b>K*</b></u>LVSEGFER, at K-329, from the zinc-binding alcohol dehydrogenase protein for 1291wt and 1291<i>ackA</i> are shown. MS1 quantitation was performed by extracting and measuring the peak areas from the most intense isotopic precursors and subsequently comparing the wt and <i>ackA</i> mutant peptides.</p

Upregulated proteins in <i>N. gonorrhoeae</i> biofilms listed by functional roles^a: Biofilm/Planktonic ratios expressed as log₂ values, 2-fold increase (log₂ values ≥1.000) measured in at least one Extract, and p-values <0.05.

a<p>Functional role assignments (both main and JCVI sub-roles) for the <i>N. gonorrhoeae</i> proteins were downloaded from the JCVI-CMR website.</p>b<p>Average log₂ values for biofilm/planktonic protein ratios.</p>c<p>(N) represents the number of measurements (biological replicates) averaged.</p>d<p>Proteins with more than one functional role assignment provided (Main role/JCVI sub-role): NGO0094, Cellular processes/DNA transformation, Cellular processes/Pathogenesis; NGO1812, Energy metabolism/Fermentation; NGO0718, Energy metabolism/Sugars.</p>e<p>Protein functional role category tentatively assigned in the present study by orthology to an <i>N. meningitidis</i> protein (see Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038303#pone.0038303.s009" target="_blank">Table S5</a> for details).</p

1D SDS-PAGE gels of mixed protein extracts (biofilm + planktonic) from one representative biological replicate.

<p>The four gels show (A) molecular weight markers, with molecular weights given in KDa, (B) the Extract 1 protein mixture, 40 µg loaded, (C) the Extract 2 protein mixture, 20 µg loaded, and (D) the Extract 3 protein mixture, 40 µg loaded. Bands were excised from the gel lanes as indicated by the numbered red circles.</p