Determining if the Variable Region of Acocostatin is Critical for Apoptotic Induction of HeLa Cells

Apoptosis is a caspase dependent pathway of cell death that can occur when normal cells are released from the extracellular matrix (ECM). Recombinant acocostatin, a member of the PIII-SVMPs disintegrin family, is capable of inducing apoptosis of HeLa cells. Previous crystallographic research suggested that the PIII- SVMP’s variable region, on the cysteine rich domain, might be a site of integrin binding.  Therefore, we hypothesized that amino acids in the variable region of r-acocostatin are responsible for early apoptosis of HeLa cells.  My project involved the production of mutations by site-directed mutagenesis on the sequence coding for the first four amino acids of r-acocostatin’s variable region.  I have produced four consecutive Ala mutants (wild type sequence: CRKE).  The first mutant replaced the Cys for an Ala (ARKE).  The second mutant replaced the Arg for an Ala (AAKE).  The third mutant replaced the Lys for an Ala (AAAE), and the fourth mutant replaced the Glu for an Ala (AAAA). I am in the process of expressing and purifying r-acocostatin’s variable region mutants.  Once the peptides are purified, HeLa cells will be treated with 5 µM of peptide for 24 h.  Apoptosis induction will be tested with a tunnel assay. If the four amino acids are responsible for binding to the integrin receptor, I would expect to detect a reduced amount of cells undergoing apoptosis.  This project was funded by NSF-REU grant DBI 1004350.