Poplar Oxidoreductases Involved In The Oxidative Stress Response : A Crystallographic Snapshot Towards The Understanding Of The Catalytic Mechanisms [RB170. K79 2008 f rb].

Penghasilan species oksigen reaktif (ROS) merupakan akibat daripada persekitaran sentiasa dalam keaadaan aerobik yang tidak boleh dielakkan dan ia boleh disingkirkan oleh beberapa jenis sistem enzim sebaik sahaja dihasilkan. The production of reactive oxygen species (ROS) is an unavoidable consequence of living in an aerobic environment and once produced, it can be removed by several different enzyme systems

Transfer RNA modification and infection – implications for pathogenicity and host responses

Open Access funded by the author(s).Transfer RNA (tRNA) molecules are sumptuously decorated with evolutionary conserved post-transcriptional nucleoside modifications that are essential for structural stability and ensure efficient protein translation. The tRNA modification levels change significantly in response to physiological stresses, altering translation in a number of ways. For instance, tRNA hypomodification leads to translational slowdown, disrupting protein homeostasis and reducing cellular fitness. This highlights the importance of proper tRNA modification as a determinant for maintaining cellular function and viability during stress. Furthermore, the expression of several microbial virulence factors is induced by changes in environmental conditions; a process where tRNA 2-thiolation is unequivocal for pathogenicity. In this review, we discuss the multifaceted implications of tRNA modification for infection by examining the roles of nucleoside modification in tRNA biology. Future development of novel methods and combinatory utilization of existing technologies will bring tRNA modification-mediated regulation of cellular immunity and pathogenicity to the limelight.Peer reviewe

Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome

Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs. Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2*GTP bound with sordarin. The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics. The IRES rearranges from extended to bent to extended conformations. This inchworm-like movement is coupled with ribosomal inter-subunit rotation and 40S head swivel. eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site. Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center. This unlocks 40S domains, facilitating head swivel and biasing IRES translocation via hitherto-elusive intermediates with PKI captured between the A and P sites. The structures suggest missing links in our understanding of tRNA translocation

Structures of Yeast 80S Ribosome-tRNA Complexes in the Rotated and Nonrotated Conformations

SummaryThe structural understanding of eukaryotic translation lags behind that of translation on bacterial ribosomes. Here, we present two subnanometer resolution structures of S. cerevisiae 80S ribosome complexes formed with either one or two tRNAs and bound in response to an mRNA fragment containing the Kozak consensus sequence. The ribosomes adopt two globally different conformations that are related to each other by the rotation of the small subunit. Comparison with bacterial ribosome complexes reveals that the global structures and modes of intersubunit rotation of the yeast ribosome differ significantly from those in the bacterial counterpart, most notably in the regions involving the tRNA, small ribosomal subunit, and conserved helix 69 of the large ribosomal subunit. The structures provide insight into ribosome dynamics implicated in tRNA translocation and help elucidate the role of the Kozak fragment in positioning an open reading frame during translation initiation in eukaryotes

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Etude cristallographique d?oxydoréductases impliquées dans la réponse au stress oxydatif chez le peuplier en vue de la compréhension de leur mécanisme catalytique

Three oxidoreductases (glutathione peroxidase, GPX; thioredoxin, Trx and glutaredoxin, Grx) from Populus trichocarpa × deltoides (poplar tree) were characterized using X-ray crystallography approach. GPXs are a group of enzymes that regulate the levels of oxygen species in cells, and protect them against oxidative damage. In this study, I have determined the crystal structures of the reduced and oxidized form of poplar GPX5 (PtGPX5). Comparison of both redox structures indicates that a drastic conformational change is necessary to bring the two distant cysteine residues together to form an intramolecular disulfide bond. Trxs regulate various protein partners through the thiol-disulfide(s) reduction. The aim of this study is thus to precisely describe the catalytic mechanism of a new isoform of Trx, PtTrxh4, since it has been demonstrated recently to be reduced by Grx. PtTrxh4 contains three cysteines; one localized in an N-terminal extension (Cys4) and two in the usual Trx active site (WC1GPC2). Two crystal structures of PtTrxh4 solved in this study, wild-type and C61S mutant, allow us to propose a four-step disulfide cascade catalytic mechanism in accordance with enzymatic studies. Grxs are highly conserved redox-proteins that utilize electrons from GSH particularly to catalyze thiol-disulfide exchange reactions. Here, I present the structure of glutathionylated PtGrxS12, the first structure of plant Grx of subclass 1 with an atypical 28WCSYS32 active site. Protein structures solved here shed lights to our understanding of the redox mechanism in plant and to the enzyme-substrate interactions.La structure de trois oxydoréductases (la glutathion peroxydase (Gpx), la thiorédoxine (Trx) et la glutarédoxine (Grx)) de Populus trichocarpa × deltoides (le peuplier) a été caractérisée par diffraction des rayons X. Les Gpxs forment un groupe d'enzymes qui régulent la concentration des espèces réactives de l'oxygène (ROS) dans les cellules, et qui les protègent des effets d'un stress oxydant. Contrairement à leurs homologues d'origine animale, les Gpxs végétales ne dépendent pas du glutathion (GSH) mais des Trx pour leur fonctionnement. Dans cette étude, j'ai résolu les structures des formes réduite et oxydée de la Gpx5 de peuplier et montré que des changements conformationnels drastiques sont nécessaires pour passer d'une forme à l'autre. Les Trxs régulent diverses protéines cibles par la réduction de leur pont disulfure. Mon objectif était de comprendre le mécanisme catalytique d'une nouvelle isoforme, la PtTrxh4, dont la capacité à accepter des électrons de la Grx a été récemment démontrée. Cette PtTrxh4 contient trois cystéines, la première localisée dans une extension en position N-terminale (Cys4) et deux situées dans le site actif classique (WC1GPC2) de la Trx. Les résolutions des structures de l'enzyme sauvage et du mutant C4S m'ont permis de proposer un mécanisme catalytique en quatre étapes en accord avec les études enzymatiques. Les Grxs sont des protéines qui utilisent des électrons du GSH en particulier pour catalyser des réactions d'échange de thiol-disulfure. Ici, je présente la structure de la PtGrxS12 (en complexe avec le GSH), la première structure de la Grx végétale de sous-classe 1 ayant un site actif de motif atypique 28WCSYS32

Etude cristallographique d oxydoréductases impliquées dans la réponse au stress oxydatif chez le peuplier en vue de la compréhension de leur mécanisme catalytique

La structure de trois oxydoréductases (la glutathion peroxydase (Gpx), la thiorédoxine (Trx) et la glutarédoxine (Grx)) de Populus trichocarpa . deltoides (le peuplier) a été caractérisée par diffraction des rayons X. Les Gpxs forment un groupe d enzymes qui régulent la concentration des espèces réactives de l'oxygène (ROS) dans les cellules, et qui les protègent des effets d un stress oxydant. Contrairement à leurs homologues d origine animale, les Gpxs végétales ne dépendent pas du glutathion (GSH) mais des Trx pour leur fonctionnement. Dans cette étude, j'ai résolu les structures des formes réduite et oxydée de la Gpx5 de peuplier et montré que des changements conformationnels drastiques sont nécessaires pour passer d une forme à l autre. Les Trxs régulent diverses protéines cibles par la réduction de leur pont disulfure. Mon objectif était de comprendre le mécanisme catalytique d une nouvelle isoforme, la PtTrxh4, dont la capacité à accepter des électrons de la Grx a été récemment démontrée. Cette PtTrxh4 contient trois cystéines, la première localisée dans une extension en position N-terminale (Cys4) et deux situées dans le site actif classique (WC1GPC2) de la Trx. Les résolutions des structures de l enzyme sauvage et du mutant C4S m ont permis de proposer un mécanisme catalytique en quatre étapes en accord avec les études enzymatiques. Les Grxs sont des protéines qui utilisent des électrons du GSH en particulier pour catalyser des réactions d'échange de thiol-disulfure. Ici, je présente la structure de la PtGrxS12 (en complexe avec le GSH), la première structure de la Grx végétale de sous-classe 1 ayant un site actif de motif atypique 28WCSYS32.Three oxidoreductases (glutathione peroxidase, GPX; thioredoxin, Trx and glutaredoxin, Grx) from Populus trichocarpa . deltoides (poplar tree) were characterized using X-ray crystallography approach. GPXs are a group of enzymes that regulate the levels of oxygen species in cells, and protect them against oxidative damage. In this study, I have determined the crystal structures of the reduced and oxidized form of poplar GPX5 (PtGPX5). Comparison of both redox structures indicates that a drastic conformational change is necessary to bring the two distant cysteine residues together to form an intramolecular disulfide bond. Trxs regulate various protein partners through the thiol-disulfide(s) reduction. The aim of this study is thus to precisely describe the catalytic mechanism of a new isoform of Trx, PtTrxh4, since it has been demonstrated recently to be reduced by Grx. PtTrxh4 contains three cysteines; one localized in an N-terminal extension (Cys4) and two in the usual Trx active site (WC1GPC2). Two crystal structures of PtTrxh4 solved in this study, wild-type and C61S mutant, allow us to propose a four-step disulfide cascade catalytic mechanism in accordance with enzymatic studies. Grxs are highly conserved redox-proteins that utilize electrons from GSH particularly to catalyze thiol-disulfide exchange reactions. Here, I present the structure of glutathionylated PtGrxS12, the first structure of plant Grx of subclass 1 with an atypical 28WCSYS32 active site. Protein structures solved here shed lights to our understanding of the redox mechanism in plant and to the enzyme-substrate interactions.NANCY1-Bib. numérique (543959902) / SudocSudocFranceF

Small methyltransferase RlmH assembles a composite active site to methylate a ribosomal pseudouridine

Eubacterial ribosomal large-subunit methyltransferase H (RlmH) methylates 23S ribosomal RNA pseudouridine 1915 (Psi1915), which lies near the ribosomal decoding center. The smallest member of the SPOUT superfamily of methyltransferases, RlmH lacks the RNA recognition domain found in larger methyltransferases. The catalytic mechanism of RlmH enzyme is unknown. Here, we describe the structures of RlmH bound to S-adenosyl-methionine (SAM) and the methyltransferase inhibitor sinefungin. Our structural and biochemical studies reveal catalytically essential residues in the dimer-mediated asymmetrical active site. One monomer provides the SAM-binding site, whereas the conserved C-terminal tail of the second monomer provides residues essential for catalysis. Our findings elucidate the mechanism by which a small protein dimer assembles a functionally asymmetric architecture

Taura syndrome virus IRES initiates translation by binding its tRNA-mRNA-like structural element in the ribosomal decoding center

In cap-dependent translation initiation, the open reading frame (ORF) of mRNA is established by the placement of the AUG start codon and initiator tRNA in the ribosomal peptidyl (P) site. Internal ribosome entry sites (IRESs) promote translation of mRNAs in a cap-independent manner. We report two structures of the ribosome-bound Taura syndrome virus (TSV) IRES belonging to the family of Dicistroviridae intergenic IRESs. Intersubunit rotational states differ in these structures, suggesting that ribosome dynamics play a role in IRES translocation. Pseudoknot I of the IRES occupies the ribosomal decoding center at the aminoacyl (A) site in a manner resembling that of the tRNA anticodon-mRNA codon. The structures reveal that the TSV IRES initiates translation by a previously unseen mechanism, which is conceptually distinct from initiator tRNA-dependent mechanisms. Specifically, the ORF of the IRES-driven mRNA is established by the placement of the preceding tRNA-mRNA-like structure in the A site, whereas the 40S P site remains unoccupied during this initial step

Redox based anti-oxidant systems in plants: Biochemical and structural analyses

International audienceWe provide in this paper a comparative biochemical and structural analysis of the major thiol oxidoreductases (thioredoxin and glutaredoxin) of photosynthetic organisms in relation with their reductases and with target proteins, especially those involved either in the detoxication of peroxides such as hydrogen peroxide (thiol-peroxidases) or in the repair of oxidized methionines in proteins (methionine sulfoxide reductases). Particular emphasis will be given to the catalytic and regeneration mechanisms used by these enzymes. In addition, the protein–protein interactions of these systems will be discussed, leading to an integrated view of the functioning of these systems in various plant sub-cellular compartments. © 2007 Elsevier B.V. All rights reserved