CHARACTERIZATION OF THE E6*I PROTEIN THE MAJOR SPLICING PRODUCT OF THE ONCOPROTEIN E6 FROM THE HIGH RISK HUMAN PAPILLOMAVIRUS.

ANGELES HEER, LEONARDO G. ALONSO Y GONZALO PRAT GAY

Buzios, Rio de Janeiro, Brasil

Congreso; VII Congreso Iberoamiericano de Biofisica; 2009

Objectives: The HPV E6 proteins are small oncoproteins of about 150 amino acids in length with two Zn-binding domains, one in the amino terminal and the other in the carboxi terminal domain. Each motif is composed of C-X-X-C-29-C-X-X-C amino acid sequence and is unique among CCCC fingers in which there are usually fewer than 20 amino acids separating the pairs of Cys residues. The most representative E6 spliced mRNA in HPV transformed cells, cervical cancer cell lines and clinical samples, named E6*I, encode for the first 50 amino acid of the E6 protein, including only half of the first Zn binding motif. In this context, we want to characterize E6*I in solution in terms of folding, stability and Zn binding.Methods: We assessed Zn binding stoichometry by tyrosine fluorescence titration, disulfide bridge connectivity by MALDI-TOF spectrometry, conformational properties by circular dichroism and redox properties by HPLC-RP. Results: A synthetic peptide or recombinant E6*I can fold into stable species and bind one mol of Zn per mol of protein. This binding inhibit the oxidation of two of its three cysteins that otherwise oxidize to form an intramolecular disulfide bridge. In addition, the redox state of its cysteines modify the secondary structure of the protein.Conclusion: We found that this E6 spliced RNA could be translated into a short folded protein product. This gnew domainh although is able to bind Zn, is necessary forced to rebuilt its geometry coordination since two of the four cysteines originally present at the full length protein are absent in the spliced form. Redox state of its cysteines can finely tune its conformation and could further regulate its activity expanding its biological function.