PINK1 Inhibits Local Protein Synthesis to Limit Transmission of Deleterious Mitochondrial DNA Mutations.

We have previously proposed that selective inheritance, the limited transmission of damaging mtDNA mutations from mother to offspring, is based on replication competition in Drosophila melanogaster. This model, which stems from our observation that wild-type mitochondria propagate much more vigorously in the fly ovary than mitochondria carrying fitness-impairing mutations, implies that germ cells recognize the fitness of individual mitochondria and selectively boost the propagation of healthy ones. Here, we demonstrate that the protein kinase PINK1 preferentially accumulates on mitochondria enriched for a deleterious mtDNA mutation. PINK1 phosphorylates Larp to inhibit protein synthesis on the mitochondrial outer membrane. Impaired local translation on defective mitochondria in turn limits the replication of their mtDNA and hence the transmission of deleterious mutations to the offspring. Our work confirms that selective inheritance occurs at the organelle level during Drosophila oogenesis and provides molecular entry points to test this model in other systems.

Acetylation-mediated remodeling of the nucleolus regulates cellular acetyl-CoA responses.

The metabolite acetyl-coenzyme A (acetyl-CoA) serves as an essential element for a wide range of cellular functions including adenosine triphosphate (ATP) production, lipid synthesis, and protein acetylation. Intracellular acetyl-CoA concentrations are associated with nutrient availability, but the mechanisms by which a cell responds to fluctuations in acetyl-CoA levels remain elusive. Here, we generate a cell system to selectively manipulate the nucleo-cytoplasmic levels of acetyl-CoA using clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing and acetate supplementation of the culture media. Using this system and quantitative omics analyses, we demonstrate that acetyl-CoA depletion alters the integrity of the nucleolus, impairing ribosomal RNA synthesis and evoking the ribosomal protein-dependent activation of p53. This nucleolar remodeling appears to be mediated through the class IIa histone deacetylases (HDACs). Our findings highlight acetylation-mediated control of the nucleolus as an important hub linking acetyl-CoA fluctuations to cellular stress responses.

Proteomic, biomechanical and functional analyses define neutrophil heterogeneity in systemic lupus erythematosus.

OBJECTIVES: Low-density granulocytes (LDGs) are a distinct subset of proinflammatory and vasculopathic neutrophils expanded in systemic lupus erythematosus (SLE). Neutrophil trafficking and immune function are intimately linked to cellular biophysical properties. This study used proteomic, biomechanical and functional analyses to further define neutrophil heterogeneity in the context of SLE. METHODS: Proteomic/phosphoproteomic analyses were performed in healthy control (HC) normal density neutrophils (NDNs), SLE NDNs and autologous SLE LDGs. The biophysical properties of these neutrophil subsets were analysed by real-time deformability cytometry and lattice light-sheet microscopy. A two-dimensional endothelial flow system and a three-dimensional microfluidic microvasculature mimetic (MMM) were used to decouple the contributions of cell surface mediators and biophysical properties to neutrophil trafficking, respectively. RESULTS: Proteomic and phosphoproteomic differences were detected between HC and SLE neutrophils and between SLE NDNs and LDGs. Increased abundance of type 1 interferon-regulated proteins and differential phosphorylation of proteins associated with cytoskeletal organisation were identified in SLE LDGs relative to SLE NDNs. The cell surface of SLE LDGs was rougher than in SLE and HC NDNs, suggesting membrane perturbances. While SLE LDGs did not display increased binding to endothelial cells in the two-dimensional assay, they were increasingly retained/trapped in the narrow channels of the lung MMM. CONCLUSIONS: Modulation of the neutrophil proteome and distinct changes in biophysical properties are observed alongside differences in neutrophil trafficking. SLE LDGs may be increasingly retained in microvasculature networks, which has important pathogenic implications in the context of lupus organ damage and small vessel vasculopathy.

Mitochondrial General Control of Amino Acid Synthesis 5 Like 1 Regulates Glutaminolysis, Mammalian Target of Rapamycin Complex 1 Activity, and Murine Liver Regeneration.

BACKGROUND AND AIMS: The regenerative capacity of the liver plays a protective role against hepatotoxins and impaired regeneration exacerbates liver dysfunction in nonalcoholic fatty liver disease (NAFLD). Mitochondrial bioenergetic and -synthetic functions are important contributory factors in hepatic regeneration, and the control of mitochondrial protein acetylation is implicated in the mitochondrial susceptibility to liver stressors. Here, we evaluated the role of general control of amino acid synthesis 5 like 1 (GCN5L1), a mediator of mitochondrial metabolism and acetylation, in modulating murine liver regeneration (LR) in response to acute CCl4 -induced hepatotoxicity. APPROACH AND RESULTS: Initial metabolomic screening found that liver GCN5L1 knockout (LKO) mice have augmented glutaminolysis. Absence of GCN5L1 modified enzyme activity of liver-enriched glutaminase enzyme (glutaminase 2; GLS2), and GCN5L1 levels modulated GLS2 oligomerization and acetylation. This metabolic remodeling resulted in the elevation of α-ketoglutarate levels, which are known to activate mammalian target of rapamycin complex 1 (mTORC1). This signaling pathway was induced with increased phosphorylation of S6 kinase in LKO hepatocytes, and inhibition of glutaminolysis reversed aberrant mTORC1 signaling. At the same time, glutaminolysis, activity of GLS2, and activation of mTORC1 signaling were reversed by the genetic reintroduction of the mitochondrial isoform of GCN5L1 into LKO primary hepatocytes. Finally, LKO mice had a more robust regenerative capacity in response to CCl4 hepatoxicity, and this response was blunted by both the mTORC1 inhibitor, rapamycin, and by pharmacological blunting of glutaminolysis. CONCLUSIONS: These data point to a central role of glutaminolysis in modulating the regenerative capacity in the liver. Furthermore, inhibition of mitochondrial GCN5L1 to augment LR may be a useful strategy in disease states linked to hepatotoxicity.

Apolipoprotein E and Periostin Are Potential Biomarkers of Nasal Mucosal Inflammation. A Parallel Approach of In Vitro and In Vivo Secretomes.

No previously suggested biomarkers of nasal mucosal inflammation have been practically applied in clinical fields, and nasal epithelium-derived secreted proteins as biomarkers have not specifically been investigated. The goal of this study was to identify secreted proteins that dynamically change during the differentiation from basal cells to fully differentiated cells and examine whether nasal epithelium-derived proteins can be used as biomarkers of nasal mucosal inflammation, such as chronic rhinosinusitis. To achieve this goal, we analyzed two secretomes using the isobaric tag for relative and absolute quantification technique. From in vitro secretomes, we identified the proteins altered in apical secretions of primary human nasal epithelial cells according to the degree of differentiation; from in vivo secretomes, we identified the increased proteins in nasal lavage fluids obtained from patients 2 weeks after endoscopic sinus surgery for chronic sinusitis. We then used a parallel approach to identify specific biomarkers of nasal mucosal inflammation; first, we selected apolipoprotein E as a nasal epithelial cell-derived biomarker through screening proteins that were upregulated in both in vitro and in vivo secretomes, and verified highly secreted apolipoprotein E in nasal lavage fluids of the patients by Western blotting. Next, we selected periostin as an inflammatory mediator-inducible biomarker from in vivo secretomes, the secretion of which was not induced under in vitro culture conditions. We demonstrated that those two nasal epithelium-derived proteins are possible biomarkers of nasal mucosal inflammation.

The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis.

GCN5L1 interacts with αTAT1 and RanBP2 to regulate hepatic α-tubulin acetylation and lysosome trafficking.

Although GCN5L1 (also known as BLOC1S1) facilitates mitochondrial protein acetylation and controls endosomal-lysosomal trafficking, the mechanisms underpinning these disparate effects are unclear. As microtubule acetylation modulates endosome-lysosome trafficking, we reasoned that exploring the role of GCN5L1 in this biology may enhance our understanding of GCN5L1-mediated protein acetylation. We show that α-tubulin acetylation is reduced in GCN5L1-knockout hepatocytes and restored by GCN5L1 reconstitution. Furthermore, GCN5L1 binds to the α-tubulin acetyltransferase αTAT1, and GCN5L1-mediated α-tubulin acetylation is dependent on αTAT1. Given that cytosolic GCN5L1 has been identified as a component of numerous multiprotein complexes, we explored whether novel interacting partners contribute to this regulation. We identify RanBP2 as a novel interacting partner of GCN5L1 and αTAT1. Genetic silencing of RanBP2 phenocopies GCN5L1 depletion by reducing α-tubulin acetylation, and we find that RanBP2 possesses a tubulin-binding domain, which recruits GCN5L1 to α-tubulin. Finally, we find that genetic depletion of GCN5L1 promotes perinuclear lysosome accumulation and histone deacetylase inhibition partially restores lysosomal positioning. We conclude that the interactions of GCN5L1, RanBP2 and αTAT1 function in concert to control α-tubulin acetylation and may contribute towards the regulation of cellular lysosome positioning. This article has an associated First Person interview with the first author of the paper.

MARCKS-related protein regulates cytoskeletal organization at cell-cell and cell-substrate contacts in epithelial cells.

Treatment of epithelial cells with interferon-γ and TNF-α (IFN/TNF) results in increased paracellular permeability. To identify relevant proteins mediating barrier disruption, we performed proximity-dependent biotinylation (BioID) of occludin and found that tagging of MARCKS-related protein (MRP; also known as MARCKSL1) increased ∼20-fold following IFN/TNF administration. GFP-MRP was focused at the lateral cell membrane and its overexpression potentiated the physiological response of the tight junction barrier to cytokines. However, deletion of MRP did not abrogate the cytokine responses, suggesting that MRP is not required in the occludin-dependent IFN/TNF response. Instead, our results reveal a key role for MRP in epithelial cells in control of multiple actin-based structures, likely by regulation of integrin signaling. Changes in focal adhesion organization and basal actin stress fibers in MRP-knockout (KO) cells were reminiscent of those seen in FAK-KO cells. In addition, we found alterations in cell-cell interactions in MRP-KO cells associated with increased junctional tension, suggesting that MRP may play a role in focal adhesion-adherens junction cross talk. Together, our results are consistent with a key role for MRP in cytoskeletal organization of cell contacts in epithelial cells.

The protein acetylase GCN5L1 modulates hepatic fatty acid oxidation activity via acetylation of the mitochondrial ß-oxidation enzyme HADHA.

Sirtuin 3 (SIRT3) deacetylates and activates several mitochondrial fatty acid oxidation enzymes in the liver. Here, we investigated whether the protein acetylase GCN5 general control of amino acid synthesis 5-like 1 (GCN5L1), previously shown to oppose SIRT3 activity, is involved in the regulation of hepatic fatty acid oxidation. We show that GCN5L1 abundance is significantly up-regulated in response to an acute high-fat diet (HFD). Transgenic GCN5L1 overexpression in the mouse liver increased protein acetylation levels, and proteomic detection of specific lysine residues identified numerous sites that are co-regulated by GCN5L1 and SIRT3. We analyzed several fatty acid oxidation proteins identified by the proteomic screen and found that hyperacetylation of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit α (HADHA) correlates with increased GCN5L1 levels. Stable GCN5L1 knockdown in HepG2 cells reduced HADHA acetylation and increased activities of fatty acid oxidation enzymes. Mice with a liver-specific deletion of GCN5L1 were protected from hepatic lipid accumulation following a chronic HFD and did not exhibit hyperacetylation of HADHA compared with WT controls. Finally, we found that GCN5L1-knockout mice lack HADHA that is hyperacetylated at three specific lysine residues (Lys-350, Lys-383, and Lys-406) and that acetylation at these sites is significantly associated with increased HADHA activity. We conclude that GCN5L1-mediated regulation of mitochondrial protein acetylation plays a role in hepatic metabolic homeostasis.

Rapid Identification of New Delhi Metallo-ß-Lactamase (NDM) Using Tryptic Peptides and LC-MS/MS.

There is significant interest in the development of mass spectrometry (MS) methods for antimicrobial resistance protein detection, given the ability of these methods to confirm protein expression. In this work, we studied the performance of a liquid chromatography, tandem MS multiple-reaction monitoring (LC-MS/MS MRM) method for the direct detection of the New Delhi metallo-ß-lactamase (NDM) carbapenemase in clinical isolates. Using a genoproteomic approach, we selected three unique peptides (SLGNLGDADTEHYAASAR, AFGAAFPK, and ASMIVMSHSAPDSR) specific to NDM that were efficiently ionized and spectrally well-defined. These three peptides were used to build an assay with turnaround time of 90 min. In a blind set, the assay detected 21/24 blaNDM-containing isolates and 76/76 isolates with negative results, corresponding to a sensitivity value of 87.5% (95% confidence interval [CI], 67.6% to 97.3%) and a specificity value of 100% (95% CI, 95.3% to 100%). One of the missed identifications was determined by protein fractionation to be due to low (∼0.1 fm/µg) NDM protein expression (below the assay limit of detection). Parallel disk diffusion susceptibility testing demonstrated this isolate to be meropenem susceptible, consistent with low NDM expression. Total proteomic analysis of the other two missed identifications did not detect NDM peptides but detected other proteins expressed from the blaNDM-containing plasmids, confirming that the plasmids were not lost. The measurement of relative NDM concentrations over the entire isolate test set demonstrated variability spanning 4 orders of magnitude, further confirming the remarkable range that may be seen in levels of NDM expression. This report highlights the sensitivity of LC-MS/MS to variations in NDM protein expression, with implications for how this technology may be used.

Identification of the OXA-48 Carbapenemase Family by Use of Tryptic Peptides and Liquid Chromatography-Tandem Mass Spectrometry.

Phenotypic detection of the OXA-48-type class D ß-lactamases in Enterobacteriaceae is challenging. We describe a rapid (less than 90 min) assay for the identification of OXA-48 family carbapenemases in subcultured bacterial isolates based on a genoproteomic approach. Following in silico trypsin digestion to ascertain theoretical core peptides common to the OXA-48 family, liquid chromatography-tandem mass spectrometry (LC-MS/MS) data-dependent acquisition was used to identify candidate peptide markers. Two peptides were selected based on performance characteristics: ANQAFLPASTFK, a core peptide common to all 12 OXA-48 family ß-lactamase members, and YSVVPVYQEFAR, a highly specific peptide common to 11 of 12 OXA-48 family proteins providing the basis for an LC-MS/MS multiple reaction monitoring assay. An accuracy assessment was performed that included 98 isolates, 26 of which were OXA-48 positive. Two additional specificity assessments were performed including a mixture of isolates positive for OXA-48, KPC, NDM, VIM, and IMP carbapenemases. A combination of expert rules and expert judgment was applied by blinded operators to identify positive isolates. All isolates containing an OXA-48 family carbapenemase across all three test sets were correctly identified with no false positives, demonstrating 100% sensitivity (95% confidence interval [CI], 91.2% to 100%) and 100% specificity (95% CI, 96.2% to 100%) for the assay. These findings provide a framework for an LC-MS/MS-based method for the direct detection of OXA-48 family carbapenemases from cultured isolates that may have utility in predicting carbapenem resistance and tracking hospital outbreaks of OXA-48-carrying organisms.

Strand-Specific Dual RNA Sequencing of Bronchial Epithelial Cells Infected with Influenza A/H3N2 Viruses Reveals Splicing of Gene Segment 6 and Novel Host-Virus Interactions.

Host-influenza virus interplay at the transcript level has been extensively characterized in epithelial cells. Yet, there are no studies that simultaneously characterize human host and influenza A virus (IAV) genomes. We infected human bronchial epithelial BEAS-2B cells with two seasonal IAV/H3N2 strains, Brisbane/10/07 and Perth/16/09 (reference strains for past vaccine seasons) and the well-characterized laboratory strain Udorn/307/72. Strand-specific RNA sequencing (RNA-seq) of the infected BEAS-2B cells allowed for simultaneous analysis of host and viral transcriptomes, in addition to pathogen genomes, to reveal changes in mRNA expression and alternative splicing (AS). In general, patterns of global and immune gene expression induced by the three IAVs were mostly shared. However, AS of host transcripts and small nuclear RNAs differed between the seasonal and laboratory strains. Analysis of viral transcriptomes showed deletions of the polymerase components (defective interfering-like RNAs) within the genome. Surprisingly, we found that the neuraminidase gene undergoes AS and that the splicing event differs between seasonal and laboratory strains. Our findings reveal novel elements of the host-virus interaction and highlight the importance of RNA-seq in identifying molecular changes at the genome level that may contribute to shaping RNA-based innate immunity.IMPORTANCE The use of massively parallel RNA sequencing (RNA-seq) has revealed insights into human and pathogen genomes and their evolution. Dual RNA-seq allows simultaneous dissection of host and pathogen genomes and strand-specific RNA-seq provides information about the polarity of the RNA. This is important in the case of negative-strand RNA viruses like influenza virus, which generate positive (complementary and mRNA) and negative-strand RNAs (genome) that differ in their potential to trigger innate immunity. Here, we characterize interactions between human bronchial epithelial cells and three influenza A/H3N2 strains using strand-specific dual RNA-seq. We focused on this subtype because of its epidemiological importance in causing significant morbidity and mortality during influenza epidemics. We report novel elements that differ between seasonal and laboratory strains highlighting the complexity of the host-virus interplay at the RNA level.

Rapid detection of colistin resistance protein MCR-1 by LC-MS/MS.

BACKGROUND: Colistin (polymyxin E) and polymixin B are important bactericidal antibiotics used in the treatment of serious infections caused by multi-drug resistant Gram-negative organisms. Transferrable plasmid-mediated colistin resistance, conferred by the product of the mcr-1 gene, has emerged as a global healthcare threat. Consequently, the rapid detection of the MCR-1 protein in clinical bacterial isolates has become increasingly important. We used a genoproteomic approach to identify unique peptides of the MCR-1 protein that could be detected rapidly by liquid chromatography tandem mass spectrometry (LC-MS/MS). METHODS: MCR-1 tryptic peptides that were efficiently ionized and readily detectable were characterized in a set of mcr-1-containing isolates with triple quadrupole LC-MS. Three optimal peptides were selected for the development of a rapid multiple reaction monitoring LC-MS/MS assay for the MCR-1 protein. To investigate the feasibility of rapid detection of the MCR-1 protein in bacterial isolates using this assay, a blinded 99-sample test set was built that included three additional mcr-1-containing clinical isolates tested in triplicate (9 samples) and 90 negative control isolates. RESULTS: All of the mcr-1-containing isolates in the test set were accurately identified with no false positive detections by three independent, blinded operators, yielding an overall performance of 100% sensitivity and specificity for multiple operators. Among the three peptides tested in this study, the best performing was DTFPQLAK. The isolate-to-result time for the assay as implemented is less than 90 min. CONCLUSIONS: This work demonstrates the feasibility of rapid detection of the MCR-1 protein in bacterial isolates by LC-MS/MS.

The NAD-dependent deacetylase SIRT2 is required for programmed necrosis.

Although initially viewed as unregulated, increasing evidence suggests that cellular necrosis often proceeds through a specific molecular program. In particular, death ligands such as tumour necrosis factor (TNF)-α activate necrosis by stimulating the formation of a complex containing receptor-interacting protein 1 (RIP1) and receptor-interacting protein 3 (RIP3). Relatively little is known regarding how this complex formation is regulated. Here, we show that the NAD-dependent deacetylase SIRT2 binds constitutively to RIP3 and that deletion or knockdown of SIRT2 prevents formation of the RIP1-RIP3 complex in mice. Furthermore, genetic or pharmacological inhibition of SIRT2 blocks cellular necrosis induced by TNF-α. We further demonstrate that RIP1 is a critical target of SIRT2-dependent deacetylation. Using gain- and loss-of-function mutants, we demonstrate that acetylation of RIP1 lysine 530 modulates RIP1-RIP3 complex formation and TNF-α-stimulated necrosis. In the setting of ischaemia-reperfusion injury, RIP1 is deacetylated in a SIRT2-dependent fashion. Furthermore, the hearts of Sirt2(-/-) mice, or wild-type mice treated with a specific pharmacological inhibitor of SIRT2, show marked protection from ischaemic injury. Taken together, these results implicate SIRT2 as an important regulator of programmed necrosis and indicate that inhibitors of this deacetylase may constitute a novel approach to protect against necrotic injuries, including ischaemic stroke and myocardial infarction.

Oncogenesis driven by the Ras/Raf pathway requires the SUMO E2 ligase Ubc9.

The small GTPase KRAS is frequently mutated in human cancer and currently there are no targeted therapies for KRAS mutant tumors. Here, we show that the small ubiquitin-like modifier (SUMO) pathway is required for KRAS-driven transformation. RNAi depletion of the SUMO E2 ligase Ubc9 suppresses 3D growth of KRAS mutant colorectal cancer cells in vitro and attenuates tumor growth in vivo. In KRAS mutant cells, a subset of proteins exhibit elevated levels of SUMOylation. Among these proteins, KAP1, CHD1, and EIF3L collectively support anchorage-independent growth, and the SUMOylation of KAP1 is necessary for its activity in this context. Thus, the SUMO pathway critically contributes to the transformed phenotype of KRAS mutant cells and Ubc9 presents a potential target for the treatment of KRAS mutant colorectal cancer.

Targeted disruption of PDE3B, but not PDE3A, protects murine heart from ischemia/reperfusion injury.

Although inhibition of cyclic nucleotide phosphodiesterase type 3 (PDE3) has been reported to protect rodent heart against ischemia/reperfusion (I/R) injury, neither the specific PDE3 isoform involved nor the underlying mechanisms have been identified. Targeted disruption of PDE3 subfamily B (PDE3B), but not of PDE3 subfamily A (PDE3A), protected mouse heart from I/R injury in vivo and in vitro, with reduced infarct size and improved cardiac function. The cardioprotective effect in PDE3B(-/-) heart was reversed by blocking cAMP-dependent PKA and by paxilline, an inhibitor of mitochondrial calcium-activated K channels, the opening of which is potentiated by cAMP/PKA signaling. Compared with WT mitochondria, PDE3B(-/-) mitochondria were enriched in antiapoptotic Bcl-2, produced less reactive oxygen species, and more frequently contacted transverse tubules where PDE3B was localized with caveolin-3. Moreover, a PDE3B(-/-) mitochondrial fraction containing connexin-43 and caveolin-3 was more resistant to Ca(2+)-induced opening of the mitochondrial permeability transition pore. Proteomics analyses indicated that PDE3B(-/-) heart mitochondria fractions were enriched in buoyant ischemia-induced caveolin-3-enriched fractions (ICEFs) containing cardioprotective proteins. Accumulation of proteins into ICEFs was PKA dependent and was achieved by ischemic preconditioning or treatment of WT heart with the PDE3 inhibitor cilostamide. Taken together, these findings indicate that PDE3B deletion confers cardioprotective effects because of cAMP/PKA-induced preconditioning, which is associated with the accumulation of proteins with cardioprotective function in ICEFs. To our knowledge, our study is the first to define a role for PDE3B in cardioprotection against I/R injury and suggests PDE3B as a target for cardiovascular therapies.

Structural Insights into Substrate Recognition and Catalysis in Outer Membrane Protein B (OmpB) by Protein-lysine Methyltransferases from Rickettsia.

Rickettsia belong to a family of Gram-negative obligate intracellular infectious bacteria that are the causative agents of typhus and spotted fever. Outer membrane protein B (OmpB) occurs in all rickettsial species, serves as a protective envelope, mediates host cell adhesion and invasion, and is a major immunodominant antigen. OmpBs from virulent strains contain multiple trimethylated lysine residues, whereas the avirulent strain contains mainly monomethyllysine. Two protein-lysine methyltransferases (PKMTs) that catalyze methylation of recombinant OmpB at multiple sites with varying sequences have been identified and overexpressed. PKMT1 catalyzes predominantly monomethylation, whereas PKMT2 catalyzes mainly trimethylation. Rickettsial PKMT1 and PKMT2 are unusual in that their primary substrate appears to be limited to OmpB, and both are capable of methylating multiple lysyl residues with broad sequence specificity. Here we report the crystal structures of PKMT1 from Rickettsia prowazekii and PKMT2 from Rickettsia typhi, both the apo form and in complex with its cofactor S-adenosylmethionine or S-adenosylhomocysteine. The structure of PKMT1 in complex with S-adenosylhomocysteine is solved to a resolution of 1.9 Å. Both enzymes are dimeric with each monomer containing an S-adenosylmethionine binding domain with a core Rossmann fold, a dimerization domain, a middle domain, a C-terminal domain, and a centrally located open cavity. Based on the crystal structures, residues involved in catalysis, cofactor binding, and substrate interactions were examined using site-directed mutagenesis followed by steady state kinetic analysis to ascertain their catalytic functions in solution. Together, our data reveal new structural and mechanistic insights into how rickettsial methyltransferases catalyze OmpB methylation.

Lecithin:Cholesterol Acyltransferase Activation by Sulfhydryl-Reactive Small Molecules: Role of Cysteine-31.

Lecithin:cholesterol acyltransferase (LCAT) catalyzes plasma cholesteryl ester formation and is defective in familial lecithin:cholesterol acyltransferase deficiency (FLD), an autosomal recessive disorder characterized by low high-density lipoprotein, anemia, and renal disease. This study aimed to investigate the mechanism by which compound A [3-(5-(ethylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile], a small heterocyclic amine, activates LCAT. The effect of compound A on LCAT was tested in human plasma and with recombinant LCAT. Mass spectrometry and nuclear magnetic resonance were used to determine compound A adduct formation with LCAT. Molecular modeling was performed to gain insight into the effects of compound A on LCAT structure and activity. Compound A increased LCAT activity in a subset (three of nine) of LCAT mutations to levels comparable to FLD heterozygotes. The site-directed mutation LCAT-Cys31Gly prevented activation by compound A. Substitution of Cys31 with charged residues (Glu, Arg, and Lys) decreased LCAT activity, whereas bulky hydrophobic groups (Trp, Leu, Phe, and Met) increased activity up to 3-fold (P < 0.005). Mass spectrometry of a tryptic digestion of LCAT incubated with compound A revealed a +103.017 m/z adduct on Cys31, consistent with the addition of a single hydrophobic cyanopyrazine ring. Molecular modeling identified potential interactions of compound A near Cys31 and structural changes correlating with enhanced activity. Functional groups important for LCAT activation by compound A were identified by testing compound A derivatives. Finally, sulfhydryl-reactive ß-lactams were developed as a new class of LCAT activators. In conclusion, compound A activates LCAT, including some FLD mutations, by forming a hydrophobic adduct with Cys31, thus providing a mechanistic rationale for the design of future LCAT activators.

A Genoproteomic Approach to Detect Peptide Markers of Bacterial Respiratory Pathogens.

BACKGROUND: Rapid identification of respiratory pathogens may facilitate targeted antimicrobial therapy. Direct identification of bacteria in bronchoalveolar lavage (BAL) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is confounded by interfering substances. We describe a method to identify unique peptide markers of 5 gram-negative bacteria by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for direct pathogen identification in BAL. METHODS: In silico translation and digestion were performed on 14-25 whole genomes representing strains of Acinetobacter baumannii, Moraxella catarrhalis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Klebsiella pneumoniae. Peptides constituting theoretical core peptidomes in each were identified. Rapid tryptic digestion was performed; peptides were analyzed by LC-MS/MS and compared with the theoretical core peptidomes. High-confidence core peptides (false discovery rate <1%) were identified and analyzed with the lowest common ancestor search to yield potential species-specific peptide markers. The species specificity of each peptide was verified with protein BLAST. Further, 1 or 2 pathogens were serially diluted into pooled inflamed BAL, and a targeted LC-MS/MS assay was used to detect 25 peptides simultaneously. RESULTS: Five unique peptides with the highest abundance for each pathogen distinguished these pathogens with varied detection sensitivities. Peptide markers for A. baumannii and P. aeruginosa, when spiked simultaneously into inflamed BAL, were detected with as few as 3.6 (0.2) × 103 and 2.2 (0.6) × 103 colony-forming units, respectively, by targeted LC-MS/MS. CONCLUSIONS: This proof-of-concept study shows the feasibility of identifying unique peptides in BAL for 5 gram-negative bacterial pathogens, and it may provide a novel approach for rapid direct identification of bacterial pathogens in BAL.

Peptide affinity analysis of proteins that bind to an unstructured region containing the transactivating domain of the osmoprotective transcription factor NFAT5.

NFAT5 is a transcription factor originally identified because it is activated by hypertonicity and that activation increases expression of genes that protect against the adverse effects of the hypertonicity. However, its targets also include genes not obviously related to tonicity. The transactivating domain of NFAT5 is contained in its COOH-terminal region, which is predicted to be unstructured. Unstructured regions are common in transcription factors particularly in transactivating domains where they can bind co-regulatory proteins essential to their function. To identify potential binding partners of NFAT5 from either cytoplasmic or nuclear HEK293 cell extracts, we used peptide affinity chromatography followed by mass spectrometry. Peptide aptamer-baits consisted of overlapping 20 amino acid peptides within the predicted COOH-terminal unstructured region of NFAT5. We identify a total of 351 unique protein preys that associate with at least one COOH-terminal peptide bait from NFAT5 in either cytoplasmic or nuclear extracts from cells incubated at various tonicities (NaCl varied). In addition to finding many proteins already known to associate with NFAT5, we found many new ones whose function suggest novel aspects of NFAT5 regulation, interaction, and function. Relatively few of the proteins pulled down by peptide baits from NFAT5 are generally involved in transcription, and most, therefore, are likely to be specifically related to the regulation of NFAT5 or its function. The novel associated proteins are involved with cancer, effects of hypertonicity on chromatin, development, splicing of mRNA, transcription, and vesicle trafficking.