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; GONZALO PRAT GAY

Brasil

Congreso; VII congreso Iberoamericano de Biofisica; 2009

Objectives: The HPV E6 proteins are small oncoproteins of about 150 amino acids in lengthwith two Zn‐binding domains, one in the amino terminal and the other in the carboxi terminaldomain. Each motif is composed of C‐X‐X‐C‐29‐C‐X‐X‐C amino acid sequence and is uniqueamong CCCC fingers in which there are usually fewer than 20 amino acids separating the pairsof Cys residues.The most representative E6 spliced mRNA in HPV transformed cells, cervical cancer cell linesand 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 Znbinding.Methods: We assessed Zn binding stoichometry by tyrosine fluorescence titration, disulfidebridge connectivity by MALDI‐TOF spectrometry, conformational properties by circulardichroism and redox properties by HPLC‐RP.Results: A synthetic peptide or recombinant E6*I can fold into stable species and bind one molof Zn per mol of protein. This binding inhibit the oxidation of two of its three cysteins thatotherwise oxidize to form an intramolecular disulfide bridge. In addition, the redox state of itscysteines modify the secondary structure of the protein.Conclusion: We found that this E6 spliced RNA could be translated into a short folded proteinproduct. This “new domain” although is able to bind Zn, is necessary forced to rebuilt itsgeometry coordination since two of the four cysteines originally present at the full lengthprotein are absent in the spliced form. Redox state of its cysteines can finely tune itsconformation and could further regulate its activity expanding its biological function.