PARP power: a structural perspective on PARP1, PARP2, and PARP3 in DNA damage repair and nucleosome remodelling

Poly (ADP-ribose) polymerases (PARP) 1-3 are well-known multi-domain enzymes, catalysing the covalent modification of proteins, DNA, and themselves. They attach mono- or poly-ADP-ribose to targets using NAD+ as a substrate. Poly-ADP-ribosylation (PARylation) is central to the important functions of PARP enzymes in the DNA damage response and nucleosome remodelling. Activation of PARP happens through DNA binding via zinc fingers and/or the WGR domain. Modulation of their activity using PARP inhibitors occupying the NAD+ binding site has proven successful in cancer therapies. For decades, studies set out to elucidate their full-length molecular structure and activation mechanism. In the last five years, significant advances have progressed the structural and functional understanding of PARP1-3, such as understanding allosteric activation via inter-domain contacts, how PARP senses damaged DNA in the crowded nucleus, and the complementary role of histone PARylation factor 1 in modulating the active site of PARP. Here, we review these advances together with the versatility of PARP domains involved in DNA binding, the targets and shape of PARylation and the role of PARPs in nucleosome remodelling

ANÁLISE GENÔMICA DE ACINETOBACTER BAUMANII RESISTENTE AOS CARBAPENÊMICOS ISOLADOS NA REGIÃO AMAZÔNICA: RESULTADOS DA PLATAFORMA GENERATE

Introdução: Acinetobacter baumannii resistentes aos Carbapenêmicos (CRAB) é atualmente um dos principais problemas de saúde pública do mundo, e é considerado pela Organização Mundial da Saúde (OMS) como um patógeno prioritário para pesquisa e desenvolvimento de novos antimicrobianos. Frente a isso, é importante compreender as características genômicas dessas linhagens que estão circulando nos hospitais. No Brasil, ainda existe uma escassez desses dados, principalmente na região norte do país. Diante disso, está sendo criada a plataforma GENERATE que tem como objetivo disponibilizar dados genômicos de bacilos gram-negativos multirresistentes do Brasil. Diante disso o objetivo do trabalho foi analisar as características genômicas de isolados de CRAB isolados no estado de Rondônia incluídos no projeto GENERATE. Metodologia: Nove isolados de CRAB recuperados de hemocultura (n=4) e aspirado traqueal (n=5) de dois hospitais de Porto Velho – RO foram sequenciados utilizando Illumina HiSeq 2500. A montagem e anotação de novo foram realizadas usando os softwares SPAdes e Prokka, respectivamente. O Sequence Type (ST) e análise filogenética foi realizada na plataforma CGE e o resistoma foi obtido no CARD. Resultados: Nossas análises identificaram a presença de cinco STs, sendo eles: ST79 (n=3), ST160 (n=1), ST8554(n=1), ST1 (n=1), e ST2 (n=1). Além disso, dois isolados apresentaram novos STs. Também foi verificado a presença de genes de conferem resistência aos β-lactâmicos (blaTEM-1, blaADC-Like, blaOXA-51-Like, blaOXA-23, blaGES-5), aminoglicosídeos (aac(6’)-ib’, ant(2’’)-Ia, ant(3’’)IIc, aph(3’)-Via, aph(3’’)-Ib, aph(6)Id, aadA, armA), trimetoprima (dfrA1), macrolídeos (mphE, msrE), anfenicóis (florR, catB8), tetraciclinas (tet(B)) e mutações que conferem resistências as quinolonas (gyrA S81L; parC S84L, V104I, D105E). Todos os CRAB possuíam OXA-23, e curiosamente, um isolado também carreava o gene codificador da carbapenemase GES-5, sendo esse até onde sabemos, o segundo relato no mundo. A análise filogenética mostrou que os três isolados ST79 estavam intimamente relacionados, assim como os dois isolados que pertencem a um novo ST. Conclusão: Os dados aqui apresentados revelam uma diversidade de genes que conferem resistência a diversas classes de antimicrobianos. Além disso, identificamos a presença do ST2 que não é muito frequente no Brasil e uma linhagem ST1 co-abrigando blaOXA-23 e blaGES-5. Esses dados reforçam a variabilidade genética de CRAB na Amazôni

Structural analysis of Canavalia maritima and Canavalia gladiata lectins complexed with different dimannosides: New insights into the understanding of the structure-biological activity relationship in legume lectins

Plant lectins, especially those purified from species of the Legummosae family, represent the best studied group of carbohydrate-binding proteins. The legume lectins from Diocleinae subtribe are highly similar proteins that present significant differences in the potency/ efficacy of their biological activities. The structural studies of the interactions between lectins and sugars may clarify the origin of the distinct biological activities observed in this high similar class of proteins. In this way, this work presents a crystallographic study of the ConM and CGL (agglutinins from Canavalia maritima and Canavalia gladiata, respectively) in the following complexes: ConM/ CGL:Man(alpha 1-2)Man(alpha 1-0)Me, ConM/CGL:Man(alpha 1-O)Man(alpha 1-O)Me and ConM/CGL:Man(alpha 1-4)Man(alpha 1-O)Me, which crystallized in different conditions and space group from the native proteins.The structures were solved by molecular replacement, presenting satisfactory values for R-factor and R-factor. Comparisons between ConM, CGL and ConA (Canavalia ensiformis lectin) binding mode with the dimannosides in subject, presented different interactions patterns, which may account for a structural explanation of the distincts biological properties observed in the lectins of Diocleinae subtribe. (C) 2007 Elsevier B.V. All rights reserved

Cryo-EM of NHEJ supercomplexes provides insights into DNA repair.

Non-homologous end joining (NHEJ) is one of two critical mechanisms utilized in humans to repair DNA double-strand breaks (DSBs). Unrepaired or incorrect repair of DSBs can lead to apoptosis or cancer. NHEJ involves several proteins, including the Ku70/80 heterodimer, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), X-ray cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), and ligase IV. These core proteins bind DSBs and ligate the damaged DNA ends. However, details of the structural assembly of these proteins remain unclear. Here, we present cryo-EM structures of NHEJ supercomplexes that are composed of these core proteins and DNA, revealing the detailed structural architecture of this assembly. We describe monomeric and dimeric forms of this supercomplex and also propose the existence of alternate dimeric forms of long-range synaptic complexes. Finally, we show that mutational disruption of several structural features within these NHEJ complexes negatively affects DNA repair.Wellcome Trust for a Programme Grant (O93167/Z/10/Z; 2011–2016) and Investigator Award (200814/Z/16/Z

“Special K” Drug on Adolescent Rats: Oxidative Damage and Neurobehavioral Impairments

Ketamine is used in clinical practice as an anesthetic that pharmacologically modulates neurotransmission in postsynaptic receptors, such as NMDA receptors. However, widespread recreational use of ketamine in “party drug” worldwide since the 1990s quickly spread to the Asian orient region. Thus, this study aimed at investigating the behavioral and oxidative effects after immediate withdrawal of intermittent administration of ketamine in adolescent female rats. For this, twenty female Wistar rats were randomly divided into two groups: control and ketamine group (n=10/group). Animals received ketamine (10 mg/kg/day) or saline intraperitoneally for three consecutive days. Three hours after the last administration, animals were submitted to open field, elevated plus-maze, forced swim tests, and inhibitory avoidance paradigm. Twenty-four hours after behavioral tests, the blood and hippocampus were collected for the biochemical analyses. Superoxide dismutase, catalase, nitrite, and lipid peroxidation (LPO) were measured in the blood samples. Nitrite and LPO were measured in the hippocampus. The present findings demonstrate that the early hours of ketamine withdrawal induced oxidative biochemistry unbalance in the blood samples, with elevated levels of nitrite and LPO. In addition, we showed for the first time that ketamine withdrawal induced depressive- and anxiety-like profile, as well as short-term memory impairment in adolescent rodents. The neurobehavioral deficits were accompanied by the hippocampal nitrite and LPO-elevated levels

Crystallization and preliminary X-ray diffraction analysis of the lectin from Dioclea rostrata Benth seeds

D. rostrata lectin was crystallized by hanging-drop vapor diffusion. The crystal belongs to the orthorhombic space group I222 and diffracted to 1.87 Å resolution

Structural and functional basis of inositol hexaphosphate stimulation of NHEJ through stabilization of Ku-XLF interaction.

Acknowledgements: JBC thank the I2BC platforms for Interactions of Macromolecules (PIM) and for Crystallization supported by French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-0005. We thank the HTX platform at EMBL, Grenoble. We thank Magali Aumont-Nicaise and Adrian Velazquez-Campoy for their help with ITC data. AG was supported by a CIFRE (ANRT) fellowship. JBC was supported by ANR-20-CE11-0026, ANR-17-CE12-0020 and ANR-21-CE12-0019-01. We also thank the staff from PROXIMA-1, PROXIMA-2a and SWING beamlines for their help in data collection and synchrotron SOLEIL (Saint-Aubin, France) for provision of synchrotron radiation facilities. SJ, MJM, and ER thank Mauro Modesti for providing SNAP tag Ku protein. PC, SB and PF were supported by the Ligue Contre le Cancer as Equipe Labellisée 2018 and ANR-20-CE11-0026. PC is a scientist from INSERM. We acknowledge the TRI imaging facility, member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04). We would like to thank the Imaging Core Facility TRI-IPBS, in particular Antonio Peixoto and Eve Pitot for technical assistance. A.C., A.K. and T.L.B. thank the Wellcome Trust for support of this research through the award of an Investigator Award (200814/Z/16/Z; 2016–2022).Funder: Ligue Contre le Cancer; DOI: https://doi.org/10.13039/501100004099The classical Non-Homologous End Joining (c-NHEJ) pathway is the predominant process in mammals for repairing endogenous, accidental or programmed DNA Double-Strand Breaks. c-NHEJ is regulated by several accessory factors, post-translational modifications, endogenous chemical agents and metabolites. The metabolite inositol-hexaphosphate (IP6) stimulates c-NHEJ by interacting with the Ku70-Ku80 heterodimer (Ku). We report cryo-EM structures of apo- and DNA-bound Ku in complex with IP6, at 3.5 Å and 2.74 Å resolutions respectively, and an X-ray crystallography structure of a Ku in complex with DNA and IP6 at 3.7 Å. The Ku-IP6 interaction is mediated predominantly via salt bridges at the interface of the Ku70 and Ku80 subunits. This interaction is distant from the DNA, DNA-PKcs, APLF and PAXX binding sites and in close proximity to XLF binding site. Biophysical experiments show that IP6 binding increases the thermal stability of Ku by 2°C in a DNA-dependent manner, stabilizes Ku on DNA and enhances XLF affinity for Ku. In cells, selected mutagenesis of the IP6 binding pocket reduces both Ku accrual at damaged sites and XLF enrolment in the NHEJ complex, which translate into a lower end-joining efficiency. Thus, this study defines the molecular bases of the IP6 metabolite stimulatory effect on the c-NHEJ repair activity