Abstract 2305 Decoding the Dark Proteome: A Workflow for Microprotein Identification

Melanoma antigen genes (MAGE) are a group of tumor specific genes that are normally expressed in male germline cells and aberrantly expressed in a wide range of cancers.Cancer patients expressing MAGE-B2, a member of the MAGE family show poor prognosis.Studies highlight MAGE-B2 to have high tumor inducing potential in mouse xenograft studies.The functional basis for MAGE-B2's tumorigenic, pro-proliferative effect is unclear and has been sparsely investigated.We seek to establish a mechanistic basis for MAGE-B2 function and understand the biochemical role of the MAGE family of proteins.To this end we designed recombinant expression constructs of MAGE-B2 with single and tandem affinity tags for generation of heterologous cell lines.We used the cell lines with robust expression of MAGE-B2 to perform unbiased proteomics and generate a global protein interactome for MAGE-B2.Our list of MAGE-B2 interacting proteins is highly enriched in RNA binding proteins, enzymes involved in protein turnover, and proteins mediating complex interactions.First, we used a coupled in vitro transcription and translation assay to enable rapid pairwise screening of interactors to ascertain validity of binding.Second, we performed co-IP experiments with candidate binding partners to triangulate domain specific binding.MAGE-B2 provides non transformed cells with a proproliferative advantage, the mechanistic basis for MAGE-B2's function is unclear.To this end we generated knockout cell lines of MAGE-B2 and various validated and putative binding partners using CRISPR/Cas-9.We checked for successful gene knockouts at the transcript and protein level.Next, we investigated the interactions of MAGE-B2 with the candidate binding partners in the context of their relevant functional complexes to further inform on proliferative effects.