Development of Therapeutic Vaccines and Studies of Human DNA Topoisomerase I
During the past years, my laboratory has focused on two projects: 1) using linear array epitope (LAE) approach to develop therapeutic vaccine, 2) studying the posttranslational modification of DNA topoisomerase I.
This laboratory is particularly excited about the future of therapeutic vaccines. Our goal is to focus on the development of therapeutic vaccines against major human diseases, such as cancer, aging-related and infectious diseases. With the advance of molecular biology, new methodologies have been developed for powerful vaccines. My laboratory has been responsible for pioneering the linear array epitope (LAE) technology. This powerful technology, which combines the use of receptor- mediated uptake and efficient antigen presentation, allows the development of antibodies against any antigen including self-antigen, and therefore opens the door for novel therapeutic applications. Using this technology, we have successfully developed an anti-GnRH vaccine and are in the process of developing other molecular vaccines such as LAE vaccine against HER2 for HER2 overexpressed breast cancer therapy and LAE vaccine against A_43 for Alzheimer¡¦s disease prevention.
The second project is to study the posttranslational regulation of DNA topoisomerase I (TOP1). Human TOP1 has recently been identified as a SUMO1 target in vivo. In response to TOP1-mediated DNA damage induced by camptothecin, multiple SUMO1 molecules are conjugated to the N-terminal domain of a single TOP1 molecule. We have made an effort to establish an in vitro system for studying SUMO1 conjugation to TOP1 using purified SAE1/2, UBC9, SUMO1 and TOP1 peptides. Consistent with results from in vivo studies, multiple SUMO1 molecules were found to be conjugated to the N-terminal domain of a single TOP1 molecule. Based on our present data, we propose the mechanism of SUMO1 polysumoylation as diagrammatically shown. The full-length SUMO1 peptide initially undergoes maturation through cleavage by a SUMO protease (such as Ulp protease), which yields SUMO1 with the exposed C-terminal glycine residue. The SAE2 subunit of the SAE1/SAE2 heterodimer complex (E1) forms a thioester bond with the C-terminal glycine of SUMO1 in the presence of ATP (green arrow). The SAE1/SAE2 complex then transfers SUMO1 from the SAE2 subunit to Ubc9 (purple arrow). This Ubc9-SUMO1 conjugate can further accept another SUMO1 transferred from another Ubc9-SUMO1 to form a Ubc9-polySUMO1 conjugate (light blue arrow). In the presence of substrate, this SUMO1 modifier or the whole block of the polymeric SUMO1 chain can be transferred from Ubc9-SUMO1 or Ubc9-polySUMO1 conjugates to the substrate protein, respectively (deep blue arrow). Hence, the substrate protein can be mono-sumoylated or polysumoylated. Alternatively, Ubc9-polySUMO1 may repeatedly transfer a single SUMO1 molecule to substrate protein until a polymeric SUMO1 chain is formed on substrate protein. In summary, our study offer new insight into hTOP1 polysumoylation in response to TOP1-mediated DNA damage, and may have general implications in protein polysumoylation. We are currently carrying out more detailed analysis in vitro to elucidate the molecular mechanism of polysumoylation.