P. falciparum (N. Aghajari, L. Ballut, S. Violot - master thesis E. Bundhoo)
Approximately 300 million people worldwide are affected by malaria. Malaria is a disease, caused by the protozoan parasite Plasmodium falciparum, which is responsible for more than 1-2 million deaths worldwide annually with a large number of victims being children. It has a massive impact on human health in that it is the world’s second biggest killer after tuberculosis. Despite intense efforts an effective vaccine is still not available. Clinical treatment of P. falciparum malaria has become complicated due to the widespread occurrence of drug resistance, and therefore necessitates the development of new antimalarial drugs. Protein targets being studied include enzymes from the purine metabolism (N. Aghajari with L. Ballut and S. Violot).
Retroviral integrases (P. Gouet - PhD thesis R. Merceron)
Integrase (IN) is the retroviral enzyme responsible for the integration of the DNA copy of the retroviral genome into the host cell DNA. It constitutes a major therapeutic target of the treatment against Human Immunodeficiency Virus (HIV), because it does not have a cellular counterpart. This protein consists of three domains : the N-terminal domain, which binds the viral DNA, the central domain, which contains the catalytic triad (DDE) and the C-terminal domain. The formation of oligomers (dimers and tetramers) is essential for correct IN function. Collaboration has been initiated with the team of C. Ronfort, "Rétrovirus et Intégration Virale", UMR 754 INRA-ENVL-Université Lyon I, to decipher the integration reaction by studying highly stable mutants. The crystal structure of the catalytic domain of an avian integrase, IN ALSV, has been determined to 1.8 Å resolution and has revealed an unexpected dimeric interface (Ballandras et al., 2011).
FIV Capsid protein (C. Guillon - PhD thesis J. Serrière, Master thesis L. Bulens)
Most of the anti-HIV vaccines that are currently being tested in phase II clinical trials include the HIV-1 gag subunit p24 (capsid) and p17 (matrix) proteins. Although it is known that the structure of an antigen influences its immunogenicity, there has been no systematic study of the structure - antigenicity for the capsid and matrix proteins. To this regard, small animal models of HIV infection such as Feline Immunodeficiency Virus (FIV) may help correlating antigenicity and vaccine efficacy. Moreover, an anti-FIV vaccine is of great veterinary interest, as the prevalence of FIV infection can reach 30% in domestic cats in some areas and as FIV is also infecting wild felid species. Furthermore, the FIV CA protein can be a therapeutic target, as demonstrated for HIV. Therefore, our project aims at the determination of the crystal structure of the CA protein of FIV. The structures that will eventually be obtained in this study will be compared with those of HIV. They will also be used to analyze data obtained on specificity, intensity, and antiviral efficacy of the immune responses induced by FIV CA in animal models.
Glycobiology : Probiotics and prebiotics (N. Aghajari, S. Violot, PhD thesis A. Lipski, PhD thesis H. Ben Hlima, PhD thesis M. Dejob)
Infectious diseases are responsible for approximately 40% of death in developing countries and 1% in the industrialized countries. Facing the increase in antibiotic resistant microbial strains, the fact that antibiotic treatment perturbs the intestinal protective flora and thereby predisposes to later infections, the growing number of allergic reactions in general and against medical drugs in particular, and the expanding number of people travelling and thus rapidly propagating bacteria all over the world, the interest for developing health promoting food supplements as probiotics and prebiotics - or inversely to inhibit the growth conditions for pathogens is increasing. In order to gain insights into molecular interactions between proteins from probiotic bacteria and prebiotic carbohydrates, and thereby contribute to the identification of molecular determinants for carbohydrate recognition and to reveal some of the mechanisms underlying the beneficial effects of these sugars and micro-organisms, we perform comparative functional and structural studies of different alpha-glucosidases producing (potentially - for some of them) prebiotics. This type of studies has been conducted for some years now in the laboratory with our work on sucrose isomerases which produce amongst others the prebiotic alpha-D-glucosylpyranosyl-1,6-Dfructofuranose also known as isomaltulose (collaboration with Pr. R. Mattes, University of Stuttgart, Stuttgart Germany) and on L-arabinose isomerases (collaboration with Pr. S. Bejar, Centre de Biotechnologie de Sfax, Sfax Tunisia).
Bacterial type II secretion system (P. Gouet)
This project is performed in collaboration with Vladimir Schevchik, UMR5240 CNRS-UCBL-INSA Lyon and Olivera Francetic, Institut Pasteur Paris. Our aim is to understand how T2SS, a highly dynamic multiprotein machinery, recognizes and translocates folded proteins across the bacterial outer membrane. Funding has been obtained from the ANR in 2010 to (1) elucidate the nature of the secretion signal(s) in the proteins secreted by the T2SS, (2) identify the components of the secretion apparatus involved in recognition of exoproteins and (3) characterize their recognition patterns.
We have previously determined the crystal structure of pectate lyase, PelI, from the phytopathogen E. chysantemi to 1.45 Å resolution (Crézé et al., 2008), which is translocated by the T2SS machinery across the outer membrane. Our objective is now to identify the short structural element(s) and individual residues that act as species-specific secretion signal.
Human 5’-nucleotidase II (N. Aghajari)
Nucleoside analogues are widely used as antiviral and anticancer agents. These compounds mimic physiological metabolites and interfere with key steps during nucleic acids biosynthesis and/or in cancer cell proliferation, respectively. The development of novel cytotoxic agents is currently limited by the appearance of resistance mechanisms mainly consisting in altered nucleotide metabolism. Among, the five cytosolic human nucléotidases involved in the regulation of cellular nucleotide pools, the over expression of 5’-nucleotidase II (cN-II) has been shown to be a predictive and non-favorable factor for patient survival when treated by cytarabine (a well-know nucleoside analogue used to treat leukaemia). Thus, the involvement of cN-II as a new resistance mechanism to cytotoxic nucleoside analogues is suspected. The main objective of our proposal is to design and to study inhibitors of this enzyme in order to potentiate the efficiency of nucleoside analogues currently used in clinic. The rational design of cN-II inhibitors, such as allosteric effectors and substrate analogues, i.e. purine mononucleotides, will be performed using molecular modeling, x-ray crystallography and a novel NMR-fragment based drug-design approach. Collaboration with Pr. C. Dumontet (CHU & University Lyon 1), Dr. S. Peyrottes & Pr. C. Périgaud (CNRS, Montpellier), Dr. L. Chaloin & Dr. C. Lionne (CNRS, Montpellier) and Dr. I. Krimm & Pr. J.M. Lancelin (University Lyon 1).
Web Server ESPript/ENDscript (P. Gouet, X. Robert)
We created the web server ESPript/ENDscript to facilitate the generation of article quality figures containing 1D/2D/3D structure information. ENDscript uses programs such as BLAST, ClustalW and PHYLODENDRON to work on protein sequences and such as DSSP, CNS and MOLSCRIPT to work on protein coordinates. It enables the creation, from a single Protein Data Bank identifier, of a multiple sequence alignment figure adorned with secondary structure elements of each sequence of known 3D structure. Similar 3D structures are superimposed in turn with the program PROFIT and a final figure is drawn with BOBSCRIPT, which shows sequence and structure conservation along the C-alpha trace of the query. ESPript and ENDscript are available at http://espript.ibcp.fr and http://endscript.ibcp.fr.
Infectious diseases/cancer : BmrA (N. Aghajari - Post-doc M. Rhimi)
BmrA (Bacillus Multidrug Resistance ATP) is a prokaryotic ABC half-transporter of 65 kDa leading to a chemoresistance phenotype. It is functional as a homodimer, for which each monomer is constituted of a cytosolic nucleotide-binding domain, NBD, and a transmembrane domain, TMD. The TMD is responsible for the binding and the transport of several structurally-unrelated compounds, by a mechanism that remains to be elucidated, whereas the NBD allows the reset of the transport cycle by hydrolysis of ATP. In order to contribute to the understanding of the molecular mechanism of drug transport and the multi-specificity for drugs of this family of ABC MDR transporters, structural studies of different constructions are conducted in collaboration with P. Falson & A. Di-Pietro (IBCP-BMSSI) and J.M. Jault (IBS, UMR 5090, Grenoble).
BMP-1/tolloid like proteinases (N. Aghajari)
We study regulatory mechanisms of BMP-1/tolloid like proteinases (BTPs also known as procollagen C-proteinases) which are enzymes implicated in a number of key events during tissue morphogenesis and tissue repair. The research project proposed aims at a better understanding of the mechanism by which specific enhancer proteins regulate BTP activity, by solving the 3D structure of the complex of the substrate:enhancer complex. Collaboration with D. Hulmes and C. Moali (IBCP-DyHTIT).
FIV Matrix protein (C. Guillon, P. Gouet - PhD Thesis J. Serrière)
As for the CA protein, the matrix protein MA, encoded by the gag gene of the Feline Immunodeficiency Virus (FIV), is an interesting target for a veterinary vaccine, but also as a model retroviral MA protein. This project aims at the determination of the structure of the FIV MA protein. We will compare structural data of FIV MA that will eventually be obtained with those of other retroviral MA proteins. The structure of FIV MA will also be correlated with specificity, intensity and efficacy of the immune responses induced by this protein in animal models to explore the structure/function/antigenicity relationship of the FIV MA protein and its specificity compared to other retroviral infections. Collaboration with the team of B. Verrier (IBCP-DyTHIT).
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