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Mouse caspase-11 and human caspase-4 and caspase-5 recognize cytosolic lipopolysaccharide (LPS) to induce pyroptosis by cleaving the pore-forming protein GSDMD.sup.1-5. This non-canonical inflammasome defends against Gram-negative bacteria.sup.6,7. Shigella flexneri, which causes bacillary dysentery, lives freely within the host cytosol where these caspases reside. However, the role of caspase-11-mediated pyroptosis in S. flexneri infection is unknown. Here we show that caspase-11 did not protect mice from S. flexneri infection, in contrast to infection with another cytosolic bacterium, Burkholderia thailandensis.sup.8. S. flexneri evaded pyroptosis mediated by caspase-11 or caspase 4 (hereafter referred to as caspase-11/4) using a type III secretion system (T3SS) effector, OspC3. OspC3, but not its paralogues OspC1 and 2, covalently modified caspase-11/4; although it used the NAD.sup.+ donor, this modification was not ADP-ribosylation. Biochemical dissections uncovered an ADP-riboxanation modification on Arg314 and Arg310 in caspase-4 and caspase-11, respectively. The enzymatic activity was shared by OspC1 and 2, whose ankyrin-repeat domains, unlike that of OspC3, could not recognize caspase-11/4. ADP-riboxanation of the arginine blocked autoprocessing of caspase-4/11 as well as their recognition and cleavage of GSDMD. ADP-riboxanation of caspase-11 paralysed pyroptosis-mediated defence in Shigella-infected mice and mutation of ospC3 stimulated caspase-11- and GSDMD-dependent anti-Shigella humoral immunity, generating a vaccine-like protective effect. Our study establishes ADP-riboxanation of arginine as a bacterial virulence mechanism that prevents LPS-induced pyroptosis. This study reports the identification of a new post-translational modification, termed ADP riboxanation, which is mediated by the Shigella effector OspC3 and inactivates the cytosolic LPS sensing pathway of caspase-4 and caspase-11., Author(s): Zilin Li [sup.1] [sup.2] , Wang Liu [sup.1] [sup.2] , Jiaqi Fu [sup.3] , Sen Cheng [sup.2] [sup.3] , Yue Xu [sup.1] [sup.2] , Zhiqiang Wang [sup.2] , Xiaofan [...]

Histone residues play an essential role in the regulation of various biological processes. In the present study, we utilized the H3/H4 histone mutant library to probe the functional aspects of histone residues in amino acid biosynthesis. We found that the histone residue H3R72 plays a crucial role in the regulation of isoleucine biosynthesis. Substitution of the arginine residue (H3R72) of histone H3 to alanine (H3R72A) renders yeast cells unable to grow in minimal medium. Histone mutant H3R72A requires external supplementation of either isoleucine, serine, or threonine for growth in minimal medium. We also observed that the H3R72 residue and leucine amino acid in synthetic complete medium might play a crucial role in determining the intake of isoleucine and threonine in yeast. Furthermore, gene deletion analysis of ILV1 and CHA1 in the H3R72A mutant confirmed that isoleucine is the sole requirement for growth in minimal medium. Altogether, we have identified that histone H3R72 residue may be crucial for yeast growth in minimal medium by regulating isoleucine biosynthesis through the Ilv1 enzyme in the budding yeast Saccharomyces cerevisiae. Key words: yeast, metabolism, histone, amino acids, epigenetics, isoleucine. Les résidus d'acides aminés des histones jouent un rôle essentiel dans la régulation de divers processus biologiques. Dans la présente étude, les auteurs ont utilisé une banque de mutants d'histones H3/H4 pour sonder les aspects fonctionnels des résidus d'acides aminés d'histones dans la biosynthèse des acides aminés. Ils ont découvert que le résidu d'histone H3R72 joue un rôle clé dans la régulation de la biosynthèse de l'isoleucine. La substitution du résidu arginine (H3R72) de l'histone H3 par une alanine (H3R72A) rend les cellules de levure incapables de croître en milieu minimal. Le mutant d'histone H3R72A nécessite un apport externe d'isoleucine, de sérine ou de thréonine pour croître en milieu minimal. Ils ont également observé que le résidu H3R72 et l'acide aminé leucine des milieux complets synthétiques pourraient jouer un rôle clé dans la détermination de l'apport d'isoleucine et de thréonine chez la levure. En outre, l'analyse de la délétion des genes ILV1 et CHA1 dans le mutant H3R72A confirmait que l'isoleucine est la seule exigence pour croître en milieu minimal. En somme, les auteurs ont déterminé que le résidu d'histone H3R72 peut être crucial pour la croissance de la levure en milieu minimal en régulant la biosynthèse de l'isoleucine par l'intermédiaire de l'enzyme Ilv1 chez la levure à bourgeonnement Saccharomyces cerevisiae. [Traduit par la Rédaction] Mots-clés : levure, métabolisme, histone, acides aminés, épigénétique, isoleucine., 1. Introduction The easy gene manipulation and availability of knockout gene libraries in yeast offer an excellent opportunity to understand biochemical pathways (Botstein and Fink 2011; Duina et al. 2014; [...]

Protein arginine methyltransferase 5 (PRMT5) catalyzes symmetric dimethylation (SDM) of arginine, a posttranslational modification involved in oncogenesis and embryonic development. However, the role and mechanisms by which PRMT5 modulates Th cell polarization and autoimmune disease have not yet been elucidated. Here, we found that PRMT5 promoted SREBP1 SDM and the induction of cholesterol biosynthetic pathway enzymes that produce retinoid-related orphan receptor (ROR) agonists that activate [ROR[gamma]t. Specific loss of PRMT5 in the [CD4.sup.+] Th cell compartment suppressed Th17 differentiation and protected mice from developing experimental autoimmune encephalomyelitis (EAE). We also found that PRMT5 controlled thymic and peripheral homeostasis in the [CD4.sup.+] Th cell life cycle and invariant NK (iNK) T cell development and [CD8.sup.+] T cell maintenance. This work demonstrates that PRMT5 expression in recently activated T cells is necessary for the cholesterol biosynthesis metabolic gene expression program that generates [ROR[gamma]t agonistic activity and promotes Th17 differentiation and EAE. These results point to Th PRMT5 and its downstream cholesterol biosynthesis pathway as promising therapeutic targets in Th17-mediated diseases., Introduction Arginine methylation is a posttranslational modification of histones and other proteins that regulates important physiological processes, including stem cell biology, differentiation, and development. Conversely, arginine methylation dysregulation occurs in [...]

Increased oxidative stress and disturbance in nitric oxide bioavailability lead to endothelial dysfunction and cardiovascular complication in renal disease. Gentamicin (GM), a commonly used antibiotic, exhibits a toxic effect on renal proximal tubules. Prevention of its nephrotoxicity is important. Therefore, we investigated whether heme oxygenase 1HO-1) induction influenced kidney and vascular function in GM-administered rats. GM (100 mg x [kg.sup.-1] x [day.sup.-1]; i.p.) was given to rats alone or together with hemin (20 mg x [kg.sup.-1] on alternate days; i.p.) for 14 days. Plasma and kidney L-arginine, asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) as well as kidney 4-hydroxynonenal (HNE) levels and myeloperoxidase (MPO) activity were measured. Histopathological examinations of kidney and relaxation and contraction responses of aorta were also examined. GM increased serum SDMA, urea nitrogen (BUN), and creatinine levels and caused histopathological alterations in the kidney. GM elevated HO-1 protein and mRNA expressions, 4-HNE level, and MPO activity and decreased antioxidant enzyme activities and L-arginine levels in the kidney. Decreased relaxation and contraction were detected in the aorta. Hemin restored renal oxidative stress and inflammatory changes together with vascular dysfunction, but did not affect SDMA, BUN, or creatinine levels. We conclude that HO-1 induction may be effective in improving renal oxidative stress, inflammation, and vascular dysfunction mediated by GM. Key words: gentamicin, vascular reactivity, heme oxygenase 1, dimethylarginines, oxidative stress. L'augmentation du stress oxydatif et les perturbations dans la biodisponibilite de l'oxyde nitrique menent a un dysfonctionnement endothelial et a des complications cardiovasculaires en insuffisance renale. La gentamicine (GM), un antibiotique courant, exerce des effets toxiques sur les tubules proximaux des reins: il est important de prevenir sa nephrotoxicite. Par consequent, nous avons evalue si l'induction de l'heme oxygenase 1 HO-1) influencait les fonctions renales et vasculaires chez des rats ayant recu de la GM. Nous avons administre de la GM (100 mgx [kg.sup.- 1] x [jour.sup.-1]; par voie i.p.) a des rats seule ou en association avec de l'hemine (20 mg x [kg.sup.-1] un jour sur deux; par voie i.p.) pendant 14 jours. Nous avons mesure les taux plasmatiques et renaux de L-arginine, de dimethylarginines asymetriques (ADMA) et symetriques (SDMA), ainsi que les taux renaux de 4-hydroxynonenal (HNE) et l'activite de la myeloperoxidase (MPO). Nous avons aussi effectue des etudes histopathologiques des reins et etudie les reactions de relaxation et de contraction aortiques. La GM entrainait une augmentation des taux seriques de SDMA, d'azote ureique (BUN) et de creatinine, ainsi que des modifications histopathologiques dans les reins. La GM entrainait une augmentation de l'expression de la proteine et de l'ARNm pour l'HO-1, des taux de 4-HNE et de l'activite de la MPO, ainsi qu'une diminution de l'activite des enzymes antioxydants et des taux de L-arginine dans les reins. Nous avons decele une diminution de la relaxation et de la contraction dans l'aorte. L'hemine a entraine un retablissement des modifications de stress oxydatif et d'inflammation dans les reins avec un dysfonctionnement vasculaire, mais n'a eu aucun effet sur les taux de SDMA, de BUN, ni de creatinine. On en arrive a la conclusion que l'induction de l'HO-1 pourrait avoir une efficacite pour attenuer le stress oxydatif, l'inflammation et les dysfonctionnements vasculaires medies par la GM. [Traduit par la Redaction] Mots-des: gentamicine, reactivite vasculaire, heme oxygenase 1, dimethylarginines, stress oxydatif., Introduction Gentamicin (GM) is an aminoglycoside antibiotic utilized to treat gram-negative bacterial infections. GM's major adverse effect, which limits its clinical use, is its toxic effect on the kidneys (Quiros [...]

Protein turnover is a tightly controlled process that is crucial for the removal of aberrant polypeptides and for cellular signalling. Whereas ubiquitin marks eukaryotic proteins for proteasomal degradation, a general tagging system for the equivalent bacterial Clp proteases is not known. Here we describe the targeting mechanism of the ClpCClpP proteolytic complex from Bacillus subtilis. Quantitative affinity proteomics using a ClpP-trapping mutant show that proteins phosphorylated on arginine residues are selectively targeted to ClpCClpP. In vitro reconstitution experiments demonstrate that arginine phosphorylation by the McsB kinase is required and sufficient for the degradation of substrate proteins. The docking site for phosphoarginine is located in the amino-terminal domain of the ClpC ATPase, as resolved at high resolution in a co-crystal structure. Together, our data demonstrate that phosphoarginine functions as a bona fide degradation tag for the ClpCClpP protease. This system, which is widely distributed across Gram-positive bacteria, is functionally analogous to the eukaryotic ubiquitinproteasome system., Author(s): Dbora Broch Trentini [1]; Marcin Jzef Suskiewicz [1]; Alexander Heuck [1]; Robert Kurzbauer [1]; Luiza Deszcz [1]; Karl Mechtler [1, 2]; Tim Clausen (corresponding author) [1] Proteins destined for [...]

The mechanistic Target of Rapamycin Complex 1 (mTORCl) is a major regulator of eukaryotic growth that coordinates anabolic and catabolic cellular processes with inputs such as growth factors and nutrients, including amino acids (1-3). In mammals arginine is particularly important, promoting diverse physiological effects such as immune cell activation, insulin secretion, and muscle growth, largely mediated through activation of mTORCl (refs 4-7). Arginine activates mTORCl upstream of the Rag family of GTPases (8), through either the lysosomal amino acid transporter SLC38A9 or the GATOR2-interacting Cellular Arginine Sensor for mTORCl (CASTOR1) (9-12). However, the mechanism by which the mTORC1 pathway detects and transmits this arginine signal has been elusive. Here, we present the 1.8 Å crystal structure of arginine-bound CASTOR1. Homodimeric cAsTOR1 binds arginine at the interface of two Aspartate kinase, Chorismate mutase, TyrA (ACT) domains, enabling allosteric control of the adjacent GATOR2-binding site to trigger dissociation from GATOR2 and downstream activation of mTORC1. Our data reveal that CASTOR1 shares substantial structural homology with the lysine-binding regulatory domain of prokaryotic aspartate kinases, suggesting that the mTORC1 pathway exploited an ancient, amino-acid-dependent allosteric mechanism to acquire arginine sensitivity. Together, these results establish a structural basis for arginine sensing by the mTORC1 pathway and provide insights into the evolution of a mammalian nutrient sensor., To understand the molecular mechanisms through which CASTOR1 detects the presence of arginine and signals it to mTORC1, we determined the crystal structure of arginine-bound CASTOR1 to 1.8 Å resolution [...]

[Na.sup.+]/[H.sup.+] exchanger isoform 1 (NHE1) is the principal plasma membrane [Na.sup.+]/[H.sup.+] exchanger of mammalian cells and functions by exchanging one intracellular proton for one extracellular sodium ion. Critical transmembrane segments of [Na.sup.+]/[H.sup.+] exchangers have discontinuous transmembrane helices, which result in a dipole within the membrane. Amino acid R425 has been suggested to play an important role in neutralizing one such helix dipole. To investigate this hypothesis, R425 was mutated to alanine, glutamine, histidine, or lysine and the mutant NHE1 proteins were expressed and characterized in NHE1-deficient cells. The R425A and R425E mutants exhibited complete loss of expression of mature, fully glycosylated NHE1, reduced expression overall, and greatly reduced cell surface targeting. The cell surface targeting, expression, and activity of the R425H and R425K mutant proteins were also impaired, though residual NHE1 activity remained. When reduced targeting and expression were accounted for, the R425H and R425K mutant proteins had activity similar to that of the wild-type protein. The results suggest that R425 is critical for NHE1 expression, targeting, and activity and that replacement with another basic residue can rescue activity. The findings are consistent with a role for R425 in both neutralizing a helix dipole and maintaining NHE1 structure and function. Key words: [Na.sup.+]/[H.sup.+] exchanger, pH regulation, transmembrane segment, helix dipole. L'isoforme 1 de l'echangeur [Na.sup.+]/[H.sup.+] (NHE1) est le principal echangeur [Na.sup.+]/[H.sup.+] de la membrane plasmique des cellules de mammiferes et elle fonctionne en echangeant un proton intercellulaire pour un sodium extracellulaire. Des segments transmembranaires cruciaux des echangeurs [Na.sup.+]/[H.sup.+] comportent des helices transmembranaires discontinues, ce qui resulte en la formation d'un dipole a l'interieur de la membrane. On a suggere que l'acide amine R425 joue un role important dans la neutralisation d'un tel dipole de l'helice. Afin verifier cette hypothese, le R425 a ete mute en alanine, glutamine, histidine ou lysine, et les mutants de NHE1 ont ete exprimes et caracterises dans des cellules depourvues de NHE-1. Les mutants R425A et R425E presentaient une perte complete d'expression de NHE1 pleinement glycosyle, une reduction globale reduite et un ciblage a la surface cellulaire grandement reduit. Le ciblage a la surface cellulaire, l'expression et l'activite des proteines mutantes R425H et R425K etaient aussi affectes mais une activite NHE1 residuelle demeurait. Apres correction sur le plan de la reduction du ciblage et de l'expression, les proteines mutantes R425H et R425 K presentaient une activite similaire a celle de la proteine sauvage. Les resultats suggerent que le R425 est crucial a l'expression, au ciblage et a l'activite de NHE1, et que son remplacement par un autre residu basique peut preserver l'activite. Ces donnees sont en coherence avec le role du R425 dans la neutralisation du dipole de l'helice et le maintien de la structure et de la fonction de NHE1. [Traduit par la Redaction] Mots-cles: echangeur [Na.sup.+]/[H.sup.+], regulation du pH, segment transmembranaire, dipole de l'helice., Introduction The [Na.sup.+]/[H.sup.+] exchanger is a pH regulatory plasma membrane protein present in all mammalian cells. The NHE1 isoform of the protein is an integral membrane protein. It has a [...]

Larval attachment and metamorphosis, also collectively known as settlement, plays an important role in the life history of marine invertebrates with biphasic life cycle. Bugula neritina can settle on any substrate without substrate selection. This complex transformation usually involves an active selection process, while the larval decision to settle is regulated by both exogenous and endogenous factors. This study focuses on the involvement of arginine kinase (AK), an important enzyme that regulates energy metabolism in marine invertebrates, during larval settlement of the bryozoan Bugula neritina. We reveal that AK was highly expressed in the swimming larvae of B. neritina, while its expression was down-regulated post-attachment. When treated with AK inhibitors, the larval settlement rate was significantly decreased. In addition, the immunostaining results indicated that AK was mainly localized to the neuro-muscular cord in swimming larvae. Overall, our results suggest the involvement of AK in regulating larval settlement of B. neritina., Author(s): Yu Zhang [sup.1] [sup.2] , Xiao-Xue Yang [sup.2] , Yue-Him Wong [sup.2] [sup.3] , Pei-Yuan Qian [sup.2] Author Affiliations: (Aff1) 0000 0001 0472 9649, grid.263488.3, Shenzhen Key Laboratory of [...]