The major long term goal of this proposal is to understand the redox regulatory mechanisms which control helix-loop-helix (bHLH) protein activity in mammalian cells. It has been shown that the activity of members of the E protein family of bHLH proteins is regulated by the formation of an intermolecular disulfide bond which is absolutely required for stable homodimer formation, DNA binding, and transcriptional activation. Homodimers are stable in lymphoid cells but are unstable in non-lymphoid cells due to the presence of an activity which can reduce this disulfide bond. Therefore, homodimer formation probably contributes to changes in gene expression during B cell differentiation.
The specific aims of this proposal are: 1) to further explore the disulfide model of E protein regulation by determining if a peptide link between E protein monomers can bypass the requirement for the intermolecular disulfide bond; to determine if a member of the E protein family (E2-2) which lacks the conserved cysteine required for disulfide formation can act as a homodimer if forced to form a crosslink. 2) to purify and clone the protein(s) associated with the activity required for the reduction of the intermolecular disulfide bond. Protein purification will be accomplished by standard biochemical means using a gel mobility shift assay to follow activity through purification.
These aims serve to further our understanding of transcriptional control mechanisms in this case of lymphoid cell differentiation by the E protein family of HLH proteins.
| Markus, M; Benezra, R (1999) Two isoforms of protein disulfide isomerase alter the dimerization status of E2A proteins by a redox mechanism. J Biol Chem 274:1040-9 |
