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[This corrects the article DOI: 10.1371/journal.pbio.2000374.]. publishersversion published

Even if GAPDH is well characterized from a biochemical and structural point of view, the contribution of its two anion recognition sites to catalysis is still matter of debate. This work presents the crystallization, the strategy for the accumulation of the thioacylenzyme intermediate and for the obtaining of the enzyme-product complex, the resolution and analysis of the corresponding crystallographic structures and the implications in terms of reaction mechanism. The results mainly shed light on the existence of a flip-flop movement of the substrate between the two anion recognition sites during catalysis which is related to the cofactor exchange step. Although structurally related to GAPDHs, the E4PDH is an enzyme fairly less characterized. Many interrogations thus remain on the determinants of cofactor affinity, on the nature of the anion recognition site or on the mechanism of activation of the water molecule that is needed for an efficient hydrolysis step. This dissertation presents the resolution of three crystallographic structures of the E4PDH from E. coli, under the apoenzyme form, in the presence of phosphate or in complex with a cofactor analog. The analysis f these structures allows the characterization of the unique anion recognition site of this enzyme, to propose structural hypotheses to explain the low affinity of this enzyme for its cofactor and also to identify possible candidates for the activation of the nucleophilic water molecule., Si la GAPDH est une enzyme bien caractérisée sur le plan biochimique et structural, la contribution de ses deux sites de reconnaissance anionique lors de la catalyse reste incomprise et nécessitait la détermination des structures de l'intermédiaire thioacylenzyme et du complexe enzyme-produit. Ce manuscrit présente la mise au point des conditions de cristallisation et d'accumulation de l'intermédiaire thioacylenzyme (suivi par microspectrophotométrie) et du complexe enzyme-produit, la résolution et l'analyse des structures cristallographiques correspondantes ainsi que les implications pour le mécanisme catalytique. Les résultats obtenus mettent notamment en évidence un mouvement de "va-et-vient" du substrat au cours de la catalyse entre les deux sites de reconnaissance anionique qui est lié à l'étape d'échange du cofacteur. Bien que structuralement proche des GAPDH, l'E4PDH est une enzyme pour laquelle peu de données sont disponibles. De nombreuses questions restent donc posées concernant notamment les déterminants de l'affinité pour le cofacteur, la nature du site de reconnaissance anionique, ou encore le mécanisme d'activation de la molécule d'eau nécessaire à une étape d'hydrolyse efficace. Sont présentées dans ce manuscrit trois structures cristallographiques de l'E4PDH d'E. coli, sous forme apoenzyme, en présence de phosphate ou en complexe avec un analogue de cofacteur. L'analyse de ces structures a notamment permis de caractériser l'unique site de reconnaissance anionique de l'enzyme, de proposer des hypothèses quant à la faible affinité de l'enzyme pour le cofacteur, et d'identifier un candidat possible pour l'activation de la molécule d'eau hydrolytique.

The crystal structure of the thioacylenzyme intermediate of the phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been solved at 1.8A˚ resolution. Formation of the intermediate was obtained by diffusion of the natural substrate within the crystal of the holoenzyme in the absence of inorganic phosphate. To define the soaking conditions suitable for the isolation and accumulation of the intermediate, a microspectrophotometric characterization of the reaction of GAPDH in single crystals was carried out, following NADH formation at 340 nm. When compared with the structure of the Michaelis complex (Didierjean, C., Corbier, C., Fatih, M., Favier, F., Boschi-Muller, S., Branlant, G., and Aubry, A. (2003) J. Biol. Chem. 278, 12968–12976) the 206–210 loop is shifted and now forms part of the so-called “new Pi” site. The locations of both the O1 atom and the C3-phosphate group of the substrate are also changed. Altogether, the results provide evidence for the flipping of the C3-phosphate group occurring concomitantly or after the redox step.

14834). Received by CS. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Fundacao para a Ciencia e Tecnologia https://www.fct.pt/index.phtml.en (grant number SFRH/BPD/41416/2007; SFRH/BPD/64702/2009; SFRH/BPD/109347/2015; SFRH/BD/61495/2009; Ciencia, 2007; SAU-NEU/105215/2008;). Received by RMO, HVM, PG, TFP, TFO. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Dorothea Schlozer Program (University of Gottingen). Received by EMS. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. JPND: Denmark, Innofond; Germany, BMBF; Sweden, SRC; Switzerland, SNSF; United Kingdom, Medical Research Council www.jpnd.eu. Received by TFO. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Alexander-von-Humboldt Research Fellowship for Postdoctoral Researchers https.//www.humboldt-foundation.de/web/humboldt-fellowship-postdoc.html. Received by LF. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. DFG Center for Nanoscale Microscopy and Molecular Physiology of the Brain http://www.cnmpb.de/index.php?lang=en_EN. Received by TFO and MZ. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. EU Joint Programme - Neurodegenerative Disease Research (JPND) project http://www.neurodegenerationresearch.eu (aSynProtec). Received by TFO. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways. Sirtuin 2 was previously shown to modulate proteotoxicity associated with age-associated neurodegenerative disorders such as Alzheimer and Parkinson disease (PD). However, the precise molecular mechanisms involved remain unclear. Here, we provide mechanistic insight into the interplay between sirtuin 2 and α-synuclein, the major component of the pathognomonic protein inclusions in PD and other synucleinopathies. We found that α-synuclein is acetylated on lysines 6 and 10 and that these residues are deacetylated by sirtuin 2. Genetic manipulation of sirtuin 2 levels in vitro and in vivo modulates the levels of α-synuclein acetylation, its aggregation, and autophagy. Strikingly, mutants blocking acetylation exacerbate α-synuclein toxicity in vivo, in the substantia nigra of rats. Our study identifies α-synuclein acetylation as a key regulatory mechanism governing α-synuclein aggregation and toxicity, demonstrating the potential therapeutic value of sirtuin 2 inhibition in synucleinopathies. © 2017 de Oliveira et al. publishersversion published

Sirt2 is a target for the treatment of neurological, metabolic, and age-related diseases including cancer. Here we report a series of Sirt2 inhibitors based on the 1,2,4-oxadiazole scaffold. These compounds are potent Sirt2 inhibitors active at single-digit μM level by using the Sirt2 substrate α-tubulin-acetylLys40 peptide and inactive up to 100 μM against Sirt1, -3, and -5 (deacetylase and desuccinylase activities). Their mechanism of inhibition is uncompetitive toward both the peptide substrate and NAD(+), and the crystal structure of a 1,2,4-oxadiazole analog in complex with Sirt2 and ADP-ribose reveals its orientation in a still unexplored subcavity useful for further inhibitor development. Tested in leukemia cell lines, 35 and 39 induced apoptosis and/or showed antiproliferative effects at 10 or 25 μM after 48 h. Western blot analyses confirmed the involvement of Sirt2 inhibition for their effects in NB4 and in U937 cells. Our results provide novel Sirt2 inhibitors with a compact scaffold and structural insights for further inhibitor improvement.

Modulators of sirtuins are considered promising therapeutic targets for the treatment of cancer, cardiovascular, metabolic, inflammatory, and neurodegenerative diseases. Here we prepared new 1,4-dihydropyridines (DHPs) bearing changes at the C2/C6, C3/C5, C4, or N1 position. Tested with the SIRTainty procedure, some of them displayed increased SIRT1 activation with respect to the prototype 3a, high NO release in HaCat cells, and ameliorated skin repair in a mouse model of wound healing. In C2C12 myoblasts, two of them improved mitochondrial density and functions. All the effects were reverted by coadministration of compound C (9), an AMPK inhibitor, or of EX-527 (10), a SIRT1 inhibitor, highlighting the involvement of the SIRT1/AMPK pathway in the action of DHPs. Finally, tested in a panel of cancer cells, the water-soluble form of 3a, compound 8, displayed antiproliferative effects in the range of 8-35 μM and increased H4K16 deacetylation, suggesting a possible role for SIRT1 activators in cancer therapy.