Deglutarylation of glutaryl-CoA dehydrogenase by deacylating enzyme SIRT5 promotes lysine oxidation in mice

A wide range of protein acyl modifications has been identified on enzymes across various metabolic processes; however, the impact of these modifications remains poorly understood. Protein glutarylation is a recently identified modification that can be nonenzymatically driven by glutaryl-CoA. In mammalian systems, this unique metabolite is only produced in the lysine and tryptophan oxidative pathways. To better understand the biology of protein glutarylation, we studied the relationship between enzymes within the lysine/tryptophan catabolic pathways, protein glutarylation, and regulation by the deglutarylating enzyme sirtuin 5 (SIRT5). Here, we identify glutarylation on the lysine oxidation pathway enzyme glutaryl-CoA dehydrogenase (GCDH) and show increased GCDH glutarylation when glutaryl-CoA production is stimulated by lysine catabolism. Our data reveal that glutarylation of GCDH impacts its function, ultimately decreasing lysine oxidation. We also demonstrate the ability of SIRT5 to deglutarylate GCDH, restoring its enzymatic activity. Finally, metabolomic and bioinformatic analyses indicate an expanded role for SIRT5 in regulating amino acid metabolism. Together, these data support a feedback loop model within the lysine/tryptophan oxidation pathway in which glutaryl-CoA is produced, in turn inhibiting GCDH function via glutaryl modification of GCDH lysine residues and can be relieved by SIRT5 deacylation activity

Role of the Anti-Sigma Factor SpoIIAB in Regulation of σ(G) during Bacillus subtilis Sporulation

RNA polymerase sigma factor σ(F) initiates the prespore-specific program of gene expression during Bacillus subtilis sporulation. σ(F) governs transcription of spoIIIG, encoding the late prespore-specific regulator σ(G). However, transcription of spoIIIG is delayed relative to other genes under the control of σ(F), and after synthesis, σ(G) is initially kept in an inactive form. Activation of σ(G) requires the complete engulfment of the prespore by the mother cell and expression of the spoIIIA and spoIIIJ loci. We screened for random mutations in spoIIIG that bypassed the requirement for spoIIIA for the activation of σ(G). We found a mutation (spoIIIGE156K) that resulted in an amino acid substitution at position 156, which is adjacent to the position of a mutation (E155K) previously shown to prevent interaction of SpoIIAB with σ(G). Comparative modelling techniques and in vivo studies suggested that the spoIIIGE156K mutation interferes with the interaction of SpoIIAB with σ(G). The σ(GE156K) isoform restored σ(G)-directed gene expression to spoIIIA mutant cells. However, expression of sspE-lacZ in the spoIIIA spoIIIGE156K double mutant was delayed relative to completion of the engulfment process and was not confined to the prespore. Rather, β-galactosidase accumulated throughout the entire cell at late times in development. This suggests that the activity of σ(GE156K) is still regulated in the prespore of a spoIIIA mutant, but not by SpoIIAB. In agreement with this suggestion, we also found that expression of spoIIIGE156K from the promoter for the early prespore-specific gene spoIIQ still resulted in sspE-lacZ induction at the normal time during sporulation, coincidently with completion of the engulfment process. In contrast, transcription of spoIIIGE156K, but not of the wild-type spoIIIG gene, from the mother cell-specific spoIID promoter permitted the rapid induction of sspE-lacZ expression. Together, the results suggest that SpoIIAB is either redundant or has no role in the regulation of σ(G) in the prespore

A conserved cysteine residue of Bacillus subtilis SpoIIIJ is important for endospore development.

During sporulation in Bacillus subtilis, the onset of activity of the late forespore-specific sigma factor σG coincides with completion of forespore engulfment by the mother cell. At this stage, the forespore becomes a free protoplast, surrounded by the mother cell cytoplasm and separated from it by two membranes that derive from the asymmetric division septum. Continued gene expression in the forespore, isolated from the surrounding medium, relies on the SpoIIIA-SpoIIQ secretion system assembled from proteins synthesised both in the mother cell and in the forespore. The membrane protein insertase SpoIIIJ, of the YidC/Oxa1/Alb3 family, is involved in the assembly of the SpoIIIA-SpoIIQ complex. Here we show that SpoIIIJ exists as a mixture of monomers and dimers stabilised by a disulphide bond. We show that residue Cys134 within transmembrane segment 2 (TM2) of SpoIIIJ is important to stabilise the protein in the dimeric form. Labelling of Cys134 with a Cys-reactive reagent could only be achieved under stringent conditions, suggesting a tight association at least in part through TM2, between monomers in the membrane. Substitution of Cys134 by an Ala results in accumulation of the monomer, and reduces SpoIIIJ function in vivo. Therefore, SpoIIIJ activity in vivo appears to require dimer formation

Efficiency of sporulation^a of strains bearing various <i>spoIIIJ</i> alleles.

a<p>The titre of viable cells and heat-resistant spores was measured 24 h after the onset of sporulation in DSM (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099811#s2" target="_blank">Materials and Methods</a>).</p>b<p>The values are averages ± SD, for three independent experiments.</p

Oligomeric state of SpoIIIJ and SpoIIIJ^C134A.

<p>(A) Blue-Native PAGE of purified SpoIIIJ-His₆. Presumed hexameric (SpoIIIJ₆), dimeric (SpoIIIJ₂) and monomeric (SpoIIIJ) species are indicated. MW markers are shown in kDa. (B) Size exclusion chromatography of SpoIIIJ-His₆ (top) or SpoIIIJ^C134A-His₆ (bottom) in the presence of 0.1% DDM and 500 mM NaCl at pH 7.6 (see the Material and Methods section for details). The vertical black arrows indicate the elution volumes of the size standards and additional peaks. The inserts show the immunoblot analysis of the peaks indicated in the two panels. Purified SpoIIIJ-His₆ (top) or SpoIIIJ^C134A-His₆ (bottom) were included as a migration control (lane P). The areas of the A and B peaks in the two panels were estimated using the ImageJ software (<a href="http://imagej.nih.gov/ij/" target="_blank">http://imagej.nih.gov/ij/</a>) and their ratio indicated.</p

Labelling of YqjG^A50C/C142A-His₆ and SpoIIIJ-His₆ with malPEG.

<p>(A) Topological model of YqjG in the membrane. The numbers refer to the amino acid residues that delimit the transmembrane (TM) segments I to V. The positions of the A50C and C142A mutations are indicated. (B) Strains producing SpoIIIJ-His₆ or YqjG^A50C/C142A-His₆ in a <i>spoIIIJ</i> mutant background were grown in liquid LB, and samples withdrawn. Cells were resuspended in a buffer containing 1 mM TCEP, lysed and membranes isolated. The membranes were resuspended in the presence of 1 mM TCEP and further incubated with or without malPEG in the presence of 2% SDS (lanes 1–2), 1 or 0% SDS (lanes 3 and 4, respectively). Proteins (30 µg) were electrophoretically resolved by SDS-PAGE and immunoblotted with an anti-His₆ antibody for the detection of SpoIIIJ-His₆ and YqjG^A50C/C142A-His₆. (C) Samples from cultures of strains producing SpoIIIJ-His₆ or YqjG^A50C/C142A-His₆ were withdrawn at the onset of the stationary growth phase in LB medium. Samples were treated as in (B), except that no TCEP was added to the French press buffer. Incubation proceeded at the indicated temperatures in the presence of 1 mM TCEP and with or without 1 mM malPEG and SDS, as indicated. In both B and C, grey and black arrows indicate SpoIIIJ-His₆ or YqjG^A50C/C142A-His₆ or labeled (shifted) reaction products, respectively. The position of molecular weight markers (in kDa) is shown.</p

Dimerisation of SpoIIIJ.

<p>(A) SpoIIIJ occurs as a monomer and a dimer. The dimer is thought to be stabilised by a disulphide bond involving residue Cys134. However, this residue is not essential for dimer formation. (B) A disulphide bond between cysteine residues (red circles), along with other interactions promotes the formation and/or maintenance of the dimeric form of SpoIIIJ. TM segments 1, 2 and 3 are depicted as green, blue, and yellow circles, respectively.</p

A LysM domain intervenes in sequential protein-protein and protein-peptidoglycan interactions important for spore coat assembly in Bacillus subtilis

At a late stage in spore development in Bacillus subtilis, the mother cell directs synthesis of a layer of peptidoglycan known as the cortex between the two forespore membranes, as well as the assembly of a protective protein coat at the surface of the forespore outer membrane. SafA, the key determinant of inner coat assembly, is first recruited to the surface of the developing spore and then encases the spore under the control of the morphogenetic protein SpoVID. SafA has a LysM peptidoglycan-binding domain, SafALysM, and localizes to the cortex-coat interface in mature spores. SafALysM is followed by a region, A, required for an interaction with SpoVID and encasement. We now show that residues D10 and N30 in SafALysM, while involved in the interaction with peptidoglycan, are also required for the interaction with SpoVID and encasement. We further show that single alanine substitutions on residues S11, L12, and I39 of SafALysM that strongly impair binding to purified cortex peptidoglycan affect a later stage in the localization of SafA that is also dependent on the activity of SpoVE, a transglycosylase required for cortex formation. The assembly of SafA thus involves sequential protein-protein and protein-peptidoglycan interactions, mediated by the LysM domain, which are required first for encasement then for the final localization of the protein in mature spores.IMPORTANCE Bacillus subtilis spores are encased in a multiprotein coat that surrounds an underlying peptidoglycan layer, the cortex. How the connection between the two layers is enforced is not well established. Here, we elucidate the role of the peptidoglycan-binding LysM domain, present in two proteins, SafA and SpoVID, that govern the localization of additional proteins to the coat. We found that SafALysM is a protein-protein interaction module during the early stages of coat assembly and a cortex-binding module at late stages in morphogenesis, with the cortex-binding function promoting a tight connection between the cortex and the coat. In contrast, SpoVIDLysM functions only as a protein-protein interaction domain that targets SpoVID to the spore surface at the onset of coat assembly.</p

Epidemiological and virological characteristics of influenza B: results of the Global Influenza B Study

INTRODUCTION: Literature on influenza focuses on influenza A, despite influenza B having a large public health impact. The Global Influenza B Study aims to collect information on global epidemiology and burden of disease of influenza B since 2000. METHODS: Twenty-six countries in the Southern (n = 5) and Northern (n = 7) hemispheres and intertropical belt (n = 14) provided virological and epidemiological data. We calculated the proportion of influenza cases due to type B and Victoria and Yamagata lineages in each country and season; tested the correlation between proportion of influenza B and maximum weekly influenza-like illness (ILI) rate during the same season; determined the frequency of vaccine mismatches; and described the age distribution of cases by virus type. RESULTS: The database included 935 673 influenza cases (2000-2013). Overall median proportion of influenza B was 22·6%, with no statistically significant differences across seasons. During seasons where influenza B was dominant or co-circulated (>20% of total detections), Victoria and Yamagata lineages predominated during 64% and 36% of seasons, respectively, and a vaccine mismatch was observed in ≈25% of seasons. Proportion of influenza B was inversely correlated with maximum ILI rate in the same season in the Northern and (with borderline significance) Southern hemispheres. Patients infected with influenza B were usually younger (5-17 years) than patients infected with influenza A. CONCLUSION: Influenza B is a common disease with some epidemiological differences from influenza A. This should be considered when optimizing control/prevention strategies in different regions and reducing the global burden of disease due to influenza