REDOX PROTEIN IMPORT TO EUKARYOTIC CELLS BY THE CELL PENETRATING PEPTIDE TAT

BALABAN CECILIA L.; MONTANER ANELEY; SOLADANO ANABEL; BANCHIO CLAUDIA; CECCARELLI EDUARDO A.

Mar del Plata

Congreso; LI Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2015

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

Cell penetrating peptides (CPPs) are short peptides (<30 aa) that cross cellular membranes with low cytotoxicity carrying drugs, proteins, nucleic acids or nanoparticles. Tat peptide, corresponding to HIV-1 Tat protein, has been studied for years for that purpose.Leptospira Interrogans Heme-oxygenase (LepHO) and Ferredoxin-NADP+ reductase (LepFNR) collaboratively catalyze heme degradation. HOs and FNRs have reported cell protection in oxidative challenging conditions.We analyzed the translocation to eukaryotic cells of recombinant proteins containing TAT CPP fused to two bacterial redox enzymes in search of a biological tool to alleviate oxidative stress damage. LepHO and LepFNR fusion proteins containing an N-terminal His-TAT extension were expressed, purified, and heme turnover determined to confirm that CPP extension does not block electron transport between the enzymes. His-TAT-LepHO and His-TAT-LepFNR were imported to SHSY5Y human neuroblastoma cells in a concentration dependent manner up to 4 μM. After 15 min both proteins were detected inside the cells and accumulate up to 1 h. Furthermore, both fusion proteins were efficiently delivered together at the same rate as if they were delivered alone.Cells treated with both fusion proteins were challenged to survive an insult. The obtained results open the possibility of using this system as a tool for tissue preservation