Michael NILGES, Director
(nilges@pasteur.fr)

Vice Director : Pedro Alzari (alzari@pasteur.fr)
Secretary : Catherine Tran (cathtran@pasteur.fr)
 

The Department mainly studies the structure and properties of molecules of biological interest, particularly as regards their role in human pathology. Bioinformatics, crystallography, NMR, protein biochemistry and pharmaceutical chemistry are associated with chemical synthesis of modified glycoconjugates, peptides and nucleotides and various heterocycles to understand the relationship between protein structure and function and develop new therapeutic strategies.

• 3D structure of proteins and pathological processes

• Molecular mechanisms and pathological processes

• Chemical synthesis of compounds with therapeutic potential

• Technological platforms

• 3D structure of proteins and pathological processes

Crystallography and X-ray diffraction are increasingly effective methods for determining the 3D structure of proteins and studying their function at molecular and atomic level. We have determined the structure of several proteins involved in diseases, such as those of the trans-sialidase and proline-racemase of Trypanosoma cruzi, the Chagas' disease agent and the ectoplasmic region of the AMA1 protein, which plays a key role in host cell invasion by Plasmodium falciparum, the malaria agent. In addition, there is continued focus on Mycobacterium tuberculosis proteins, particularly protein kinases and phosphatases. A protein kinase substrate has been identified and its interaction with the kinase, characterized. The highly complex structure of type 4 “specific site” integrase of Vibrio cholerae associated with DNA may be useful for antibiotic resistance. The structure of the 6-phosphogluconolactonase of Trypanosoma brucei has been solved and polymerase mu has been modeled according to the structure of another enzyme of the polymerase family.

Nuclear Magnetic Resonance can also be used to determine the structure of molecules in solution and their interactions with other partners, proteins, DNA or ligands. In addition to crystallography, this technique can be used to study low-affinity complexes at atomic level. Developed in collaboration with many campus teams, it particularly focuses on bacterial proteins involved in the acquisition of heme, on antigenic determinants recognized by a protective monoclonal antibody in the context of a vaccine against Shigella flexneri, on a DNA repair enzyme expressed by the Toxoplasma gondii parasite responsible for toxoplasmosis and on transcription factors from hyperthermophilic archeobacteria viruses.

The structure of complexes between proteins and ligands or macromolecules can also be studied using modeling, which is used to predict interactions between components. A group of bioinformatics programs has been developed to approach this problem in a new, more effective way.

They are used to study interactions between proteins and their ligands and, in particular, to identify complexes between different M. tuberculosis proteins. Other original calculation methods have been developed. The ARIA program for instance enables automated attribution of NMR spectra, and new algorithms have been developed for large-scale DNA structure prediction and research into potential therapeutic agents using virtual screening. Theoretical research has also been conducted into the use of regular methods for crystallographic structure resolution and protein-solvent interaction modeling.

• Molecular mechanisms and pathological processes

Adenyl cyclase is one of the virulence factors of Bordetella pertussis, the whooping cough agent. Toxin entry into the target cells and its interaction with different cell effectors are used in several biotech applications and particularly in anti-cancer therapy. The characterization of membrane proteins involved in cell division in Escherichia coli is being studied using a “double hybrid” system based on functional complementation of two fragments from the catalytic domain of the cyclase. In addition, a mutant protein of the bacterial envelope, which is incapable of normal folding, is being used as a model for understanding the “quality control” mechanisms of folding in the bacterial periplasmic space.

Sialorphin, an inhibitor of ecto-enkephalinase activity in some metallopeptidases, has been discovered in mammals and particularly in humans through a combination of pharmaceutical, post-genomics and biochemical approaches. Characterized at molecular and functional level, sialorphin is a powerful analgesic and it exercises psychostimulant activity on behavioral responses to sociosexual and environmental stimulation in rats. This molecule can therefore be used as a basis for mimetics of potential therapeutic interest in humans.

The NEMO protein plays a central role in the regulation of the NF-kB signaling pathway by regulating the IKK protein kinase complex. Its biological activity is linked to its ability to form complexes via an oligomerization domain, which is the subject of structural and functional research. Its activity is specifically inhibited by peptides, which prevent the formation of trimeric structures. The effects of a mutation in this domain which is present in a human genetic disease have been characterized in vitro on the recombinant protein carrying this mutation.

Lastly, research into the recombination mechanism in the HIV virus, conducted on using an original system of cells in culture, shows that the secondary structure of the template RNA plays an important role in strand jumps caused by reverse transcriptase.

• Chemical synthesis of compounds with therapeutic potential

Access to synthetic molecules and characterization of their structure and interactions form the subject of several projects that may lead to new therapeutic approaches.

Glycoconjugates

Complex oligosides, which could be used to develop a vaccine response to several major pathogens, have been synthesized, particularly in the case of Shigella flexneri, the bacillary dysentery agent, Vibrio cholerae and the fungus Cryptococcus neoformans. These complex syntheses of saccharide haptens corresponding to previously identified antigens, have in the case of S. flexneri led to glycoconjugates inducing high titers of superior antigens in mice. Glycosylated phosphorylcholine has been used as a building block for preparing several synthetic vaccine compounds for diseases of the respiratory tract, and clinical development of a synthetic vaccine with potential anticancer therapeutic effects is envisaged

Peptides

Chemical synthesis of small proteins is sometimes preferable to their biological production. Expertise in synthesis of long peptides has been used to obtain several small proteins in a very pure state and in particular chemokines, phosphorylated fragments of ß-catenin, a membrane mini-protein (PMP1), antimicrobial peptides and the oligomerization domain of the NEMO protein. In addition, peptides interfering with different subunits of protein phosphatases, created from two canine adenovirus proteins, are currently being studied for their apoptosis inducing factor.

Nucleoside analogs with antibacterial and antiviral properties

The modified nucleoside analogs used in antiviral therapy and generally modified on ribose favor the development of resistance. A series of synthesized analogs with, as a nucleobase, heterocycles capable of being paired with several of the canonical bases, may result in less resistance. Specific kinases ensure phosphorylation of the different monophosphate nucleosides into diphosphate derivatives. They have specific characteristics in the tuberculosis bacillus and are therefore new potential antituberculosis targets. An initial original family of inhibitors acting on bacterial cultures has been identified based on the structure of the M. tuberculosis thymidine monophosphate kinase.

Directed evolution of enzymes

In certain cases, directed evolution can be used to obtain enzymes featuring new or improved functions. Therefore, thermostable DNA-dependent DNA polymerases, featuring thermostable reverse transcriptase catalytic activity, have been selected in vitro using a new method for simultaneously analyzing the catalytic activity of a large number of proteins. This enzyme engineering strategy is also applied to the isolation of glycosyltransferases to facilitate the synthesis of vaccine glycoconjugates.

• Technological platforms

The Department comprises 7 technological platforms (PF), several of which are also affiliated to the Genopole.

The Production of Monoclonal Antibodies and Recombinant Proteins PF can produce cultures of varying sizes, from the micro-fermenter to mass cultures. Monoclonal antibodies are used as diagnosis tools against the plague and cholera, or as research tools in several units on campus. The Macromolecular Crystallization and X-Ray Diffraction Facility PF features two robots for high-speed crystallization which have, in particular, led to the resolution of about fifteen M. tuberculosis protein structures. The Proteomics PF has advanced mass spectrometry equipment, including a spectrometer combined with high-pressure liquid chromatography. The aim of the Molecular Cryomicroscopy PF is to analyze large macromolecular complexes using an ultra-high resolution cryomicroscope. The Protein Microsequencing and Analysis PF has chemical sequencers for obtaining amino acid sequences and ProteinChip Array® (Ciphergen) technology combined with mass spectrometry (SELDI-TOF-MS) for analyzing proteins. The High Throughput Synthesis of Long Oligonucleotides PF produces oligonucleotides for DNA microarrays and modified oligonucleotides. Finally, the Biophysics PF features a combination of technology for research into macromolecules and their interactions, such as surface plasmon resonance (Biacore), microcalorimetry, circular dichroism and analytical ultracentrifugation.