Abstract 1038 Isolation and identification of cyanobacterial secondary metabolites responsible for increased Ubiquitin transfer

Ubiquitination is a post-translational modification that is fundamental to most biological processes in Eukaryotes. Ubiquitin conjugation is controlled by an enzymatic cascade that comprises of the E1 (Ubiquitin-activating), E2 (Ubiquitin-conjugating), and E3 (Ubiquitin ligases) enzymes. Ubiquitination controls many cellular processes, such as DNA repair and apoptosis, but its main role is targeting proteins to the proteasome for degradation and this function is critical for normal cellular functions. Therefore, dysregulation of this process can lead to many diseases, like cancer, developmental, and neurodegenerative disease. Accordingly, discovery of small molecules that can modulate this pathway would enhance our understanding of this important pathway and could represent new therapeutic approaches for a variety of diseases. Since prokaryotes lack ubiquitin conjugation machinery, we sought out to identify new molecules that could regulate the ubiquitin pathway. We screened crude fractionated libraries of compounds sourced from the marine cyanobacteria Rivularia spp. using in vitro ubiquitination assays. We observed that a middle range polarity fraction of the Rivularia spp. that would increase both substrate and auto-ubiquitination activities for several different E3s. This included UHRF1, a RING E3 ubiquitin ligase that controls DNA methylation, and IAP2, a protein that controls apoptosis. Additionally, we also observed an increase in E2∼Ub formed by E1 indicating that the compound was impacting steps upstream of the E3. Following our experiments, further fractionation of one of the active fractions was performed to simplify the complexity of the mixture. Additionally, we could identify the active fraction after additional chromatography of the crude fraction. We are currently trying to identify the compound's structure using NMR and mass spectrometry along with determining what precise steps by which the small molecule increases ubiquitination activity.