The type III effector EspF coordinates membrane trafficking by the spatiotemporal activation of two eukaryotic signaling pathways

Bacterial toxins and effector proteins hijack eukaryotic enzymes that are spatially localized and display rapid signaling kinetics. However, the molecular mechanisms by which virulence factors engage highly dynamic substrates in the host cell environment are poorly understood. Here, we demonstrate that the enteropathogenic Escherichia coli (EPEC) type III effector protein EspF nucleates a multiprotein signaling complex composed of eukaryotic sorting nexin 9 (SNX9) and neuronal Wiskott-Aldrich syndrome protein (N-WASP). We demonstrate that a specific and high affinity association between EspF and SNX9 induces membrane remodeling in host cells. These membrane-remodeling events are directly coupled to N-WASP/Arp2/3–mediated actin nucleation. In addition to providing a biochemical mechanism of EspF function, we find that EspF dynamically localizes to membrane-trafficking organelles in a spatiotemporal pattern that correlates with SNX9 and N-WASP activity in living cells. Thus, our findings suggest that the EspF-dependent assembly of SNX9 and N-WASP represents a novel form of signaling mimicry used to promote EPEC pathogenesis and gastrointestinal disease

SUCLA2 mutations cause global protein succinylation contributing to the pathomechanism of a hereditary mitochondrial disease

Mitochondrial acyl-coenzyme A species are emerging as important sources of protein modification and damage. Succinyl-CoA ligase (SCL) deficiency causes a mitochondrial encephalomyopathy of unknown pathomechanism. Here, we show that succinyl-CoA accumulates in cells derived from patients with recessive mutations in the tricarboxylic acid cycle (TCA) gene succinyl-CoA ligase subunit-beta (SUCLA2), causing global protein hyper-succinylation. Using mass spectrometry, we quantify nearly 1,000 protein succinylation sites on 366 proteins from patient-derived fibroblasts and myotubes. Interestingly, hyper-succinylated proteins are distributed across cellular compartments, and many are known targets of the (NAD(+))-dependent desuccinylase SIRT5. To test the contribution of hyper-succinylation to disease progression, we develop a zebrafish model of the SCL deficiency and find that SIRT5 gain-of-function reduces global protein succinylation and improves survival. Thus, increased succinyl-CoA levels contribute to the pathology of SCL deficiency through post-translational modifications. The pathomechanism of succinyl-CoA ligase (SCL) deficiency, a hereditary mitochondrial disease, is not fully understood. Here, the authors show that increased succinyl-CoA levels contribute to SCL pathology by causing global protein hyper-succinylation.Peer reviewe

Temporal dynamics of liver mitochondrial protein acetylation and succinylation and metabolites due to high fat diet and/or excess glucose or fructose.

Dietary macronutrient composition alters metabolism through several mechanisms, including post-translational modification (PTM) of proteins. To connect diet and molecular changes, here we performed short- and long-term feeding of mice with standard chow diet (SCD) and high-fat diet (HFD), with or without glucose or fructose supplementation, and quantified liver metabolites, 861 proteins, and 1,815 protein level-corrected mitochondrial acetylation and succinylation sites. Nearly half the acylation sites were altered by at least one diet; nutrient-specific changes in protein acylation sometimes encompass entire pathways. Although acetyl-CoA is an intermediate in both sugar and fat metabolism, acetyl-CoA had a dichotomous fate depending on its source; chronic feeding of dietary sugars induced protein hyperacetylation, whereas the same duration of HFD did not. Instead, HFD resulted in citrate accumulation, anaplerotic metabolism of amino acids, and protein hypo-succinylation. Together, our results demonstrate novel connections between dietary macronutrients, protein post-translational modifications, and regulation of fuel selection in liver

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 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

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

Multiplexed, Scheduled, High-Resolution Parallel Reaction Monitoring on a Full Scan QqTOF Instrument with Integrated Data-Dependent and Targeted Mass Spectrometric Workflows

Recent advances in commercial mass spectrometers with higher resolving power and faster scanning capabilities have expanded their functionality beyond traditional data-dependent acquisition (DDA) to targeted proteomics with higher precision and multiplexing. Using an orthogonal quadrupole time-of flight (QqTOF) LC-MS system, we investigated the feasibility of implementing large-scale targeted quantitative assays using scheduled, high resolution multiple reaction monitoring (sMRM-HR), also referred to as parallel reaction monitoring (sPRM). We assessed the selectivity and reproducibility of PRM, also referred to as parallel reaction monitoring, by measuring standard peptide concentration curves and system suitability assays. By evaluating up to 500 peptides in a single assay, the robustness and accuracy of PRM assays were compared to traditional SRM workflows on triple quadrupole instruments. The high resolution and high mass accuracy of the full scan MS/MS spectra resulted in sufficient selectivity to monitor 6–10 MS/MS fragment ions per target precursor, providing flexibility in postacquisition assay refinement and optimization. The general applicability of the sPRM workflow was assessed in complex biological samples by first targeting 532 peptide precursor ions in a yeast lysate, and then 466 peptide precursors from a previously generated candidate list of differentially expressed proteins in whole cell lysates from <i>E. coli</i>. Lastly, we found that sPRM assays could be rapidly and efficiently developed in Skyline from DDA libraries when acquired on the same QqTOF platform, greatly facilitating their successful implementation. These results establish a robust sPRM workflow on a QqTOF platform to rapidly transition from discovery analysis to highly multiplexed, targeted peptide quantitation