VIRUSES & PARASITES: surface mapping of Env glycoprotein from HIV and role of hexose-6-phosphate mutarotase from Trypanosoma brucei in oxidative stress.

Protein studies on HIV Most efforts in HIV vaccine development have been based on the use of structural proteins Env, and most recently of regulatory protein Tat. The rational for this approach is based on these facts: i) the presence of anti-Tat antibodies seems to protect infected individuals from progressing to AIDS ii) biologically active Tat is immunomodulatory. In addition Tat is released by infected cells and binds to the surface of both HIV-1 infected and surrounding uninfected cells instructing these to become more prone to infection. There are evidences for a specific interaction between Tat and Env, which enhances virus attachment and entry into cells. The molecular complex seems to be generated upon interaction of the cysteine rich and basic region of Tat and the gp120 V3 loop. AIMS: studies on the Tat and Env interaction by surface mapping and measurement of binding induced conformational changes by the assay of amino acids chemical reactivity changes. METHODS: chemical modification of cys and lys residues, proteolysis, HPLC, mass spectrometry. MAIN RESULTS: 4 disulfide bridges of 10 present in GP120 are accessible to reducing agents under mild conditions. These disulfides are found both in the monomeric GP120 and in trimeric GP140, and the binding of TAT does not modify the labelling pattern. Labelling of lys residues evidences 18 corresponding peptides in both GP120 and GP140, with 2 additional lys labelled in GP140, and the binding of TAT shields a single lys residue. CONCLUSIONS: Conformations of monomeric and trimeric ENV are very similar, the binding of TAT does not induce significant conformational changes. Protein studies on T. brucei T. brucei is a protozoan parasite responsible for African trypanosomiasis. Antigenic variation or intracellular parasitic life make prospects for vaccine development bleak. Only few drugs are available, which are however inadequate because of toxicity, inefficacy and resistance. However in recent years research has identified enzymes, other proteins and peculiar organelles as potential drug targets. Glycolysis, which in trypanosomatids takes place in specialized organelles called glycosomes, is considered a potential target because trypanosomes are completely dependent on it when living in the mammalian bloodstream. The pentose-phosphate pathway (PPP) is also very important since provides both NADPH for biosyntheses, detoxifications and protection against oxidative stress, and ribose for nucleotide biosynthesis. Glucose occurs in 2 anomeric forms: a-D-glucose and b-D-glucose. a- and b-anomers spontaneously interconvert but quite likely intercoversion is slow under intracellular conditions. Proteomic studies have identified a hexose-6-phosphate mutarotase (H6PM) in T. brucei. In all available exokinase crystal-structures containing glucose, the a-anomer was found. PGI is specific for a-G6P while G6PDH for b-G6P. It seems likely that H6PM plays an important role to provide the PPP with its substrate. AIMS: To well characterize the enzyme and assess its importance for the parasite in dealing with oxidative stress. METHODS: protein purification from recombinant bacteria, over-expressing the enzyme with a hystidine tail and kinetic studies. Knock-down of gene expression by RNA interference. Vitality analysis of RNAi bloodstream trypanosomes in presence or absence of H2O2. MAIN RESULTS: Knockdown shows a small effect on growth of bloodstreamform T. brucei under normal conditions, but dramatic effect when the cells are subjected to oxidative stress. The increased sensitivity to oxidative stress is similar to that in G6PDH-depleted cells. CONCLUSIONS: H6PM is important for oxidative defence, probably by accelerating anomeric conversion of G6P so its flux through PPP increases.