Michel Azoulay

New synthetic glycolipids for targeted gene transfer: synthesis, formulation in lipoplexes and specific interaction with lectin.

Nonviral gene delivery systems are a promising approach for gene therapy applications, despite their low in vivo gene transfer efficiency. One approach to enhance this efficiency is to incorporate targeting elements into cationic lipid/DNA complexes (lipoplexes). Ligand-containing lipoplexes have to retain their efficiency while exposing accessible ligand on their surface. Physicochemical properties (particle size, surface charge, and efficacy of DNA complexation) of the lipoplexes largely determine their gene transfer efficiency. We synthesized glycolipids with various galactosylated head ligand and incorporated them into lipoplexes. We showed that incorporation of up to 33% mol of glycolipid did not change the physicochemical properties of lipoplexes. Some of our glycolipids yielded lipoplexes whose galactosyl heads were well exposed on the surface as demonstrated by a strong interaction with Ricinus communis agglutinin. Glycolipid-containing lipoplexes gave an efficient gene transfer on hepatocytes, although no ligand-targeted transfection could be observed.

SNAP‐tag Based Proteomics Approach for the Study of the Retrograde Route

Proteomics is a powerful technique for protein identification at large scales. A number of proteomics approaches have been developed to study the steady state composition of intracellular compartments. Here, we report a novel vectorial proteomics strategy to identify plasma membrane proteins that undergo retrograde transport to the trans‐Golgi network (TGN). This strategy is based on the covalent modification of the plasma membrane proteome with a membrane impermeable benzylguanine derivative. Benzylguanine‐tagged plasma membrane proteins that are subsequently targeted to the retrograde route are covalently captured by a TGN‐localized SNAP‐tagged fusion protein, which allows for their identification. The approach was validated step‐by‐step using a well explored retrograde cargo protein, the B‐subunit of Shiga toxin. It was then extended to the proteomics format. Among other hits we found one of the historically first identified cargo proteins that undergo retrograde transport, which further validated our approach. Most of the other hits were kinases, receptors or transporters. In conclusion, we have pioneered a vectorial proteomics approach that complements traditional methods for the study of retrograde protein trafficking. This approach is of generic nature and could in principle be extended to other endocytic pathways.

SYNTHESIS OF NEW GALACTOSYL AND LACTOSYL CARBAMATE-CONTAINING GLYCOLIPIDS

An efficient synthesis of mono-, di- and tetrasaccharides linked to a lipid has been developed. Galactose or lactose was covalently coupled to a glycyldioctadecylamide, via well-defined chemical steps, in both an α and β anomeric configuration. The multiantennary galactosyl ligands were obtained using 1,3-diamino-2-propanol as a scaffold.

2-Nitro and 4-nitro-quinone-methides are not irreversible inhibitors of bovine β-glucuronidase

4-Benzylamino-(and 4-chloromethyl)-2-nitro-beta-D-glucuronides (4, 10) and their 2-substituted-4-nitro regioisomers (7, 13) were prepared by glycosidation of the 3-nitro-4-hydroxy- and the 2-hydroxy-5-nitro-benzylic alcohol, respectively, with a glucuronyl donor. Carbonate activation followed by reaction with benzylamine or methanesulfonyl chloride afforded, after complete deprotection, the target molecules 4, 7, 10 and 13. These compounds have been synthesized to determine whether these molecules are (or not) glucuronidase inhibitors. After incubation with bovine liver beta-glucuronidase, none of the cleavage products (the titled quinone-methides) showed to be irreversible inhibitors of this enzyme.

Chemistry-based protein modification strategy for endocytic pathway analysis

Background information. The integrated analysis of intracellular trafficking pathways is one of the current challenges in the field of cell biology, and functional proteomics has become a powerful technique for the large‐scale identification of proteins or lipids and the elucidation of biological processes in their natural contexts. For this, new dynamic strategies must be devised to trace proteins that follow a specific pathway such that their initial and final destinations can be detected by automated means.

p-NITROPHENYL β-D-GLUCOPYRANOSIDURONIC ANALOGS AS POTENTIAL SUBSTRATES FOR β-GLUCURONIDASE

Abstract Three p-nitrophenyl-β-D-glycosides including gluco-hex-4-enopyranosiduronic, glucofuranosiduronic and xylofuranosiduronic moieties have been prepared. None of them appeared to be a better substrate for β-glucuronidase from bovine origin than usual p-nitrophenyl-β-D-glucuronide.