8700 Mono(ADP-Ribosyl)ation of Ribosomal Proteins Promotes Differentiation During Adipogenesis

Abstract Disclosure: X. Tan: None. S. Challa: None. C.V. Camacho: None. T.S. Nandu: None. M.S. Stokes: None. W.L. Kraus: Other; Self; W.L.K. is a founder and stockholder for Ribon Therapeutics, Inc; a founder, member of the SAB, member of the BOD, and a stockholder for and ARase Therapeutics, Inc. He is also coholder of U.S. Patent. ADP-ribosylation, a crucial post-translational modification, involves the covalent attachment of ADP-ribose units to amino acids on proteins through ADP-ribosyl transferase (ART) enzymes, utilizing NAD+ synthesized by nicotinamide mononucleotide adenylyl transferases (NMNATs). We previously showed that NMNAT-2, a cytoplasmic NAD+ synthase, increases during adipogenesis, leading to reduced nuclear NAD+ levels through compartmentalized NMN depletion and enhanced differentiation of preadipocytes. However, the implications of increased cytoplasmic NAD+ synthesis by NMNAT-2 in adipocytes remain unknown. Our current findings demonstrate elevated ribosome MARylation during 3T3-L1 preadipocyte differentiation, driven by NMNAT-2 induction. Inhibiting NAD+ biosynthesis by knockdown of NMNAT-2 or FK866 results in reduced ribosome MARylation, with PARP16 identified as the primary ART mediating MARylation. Knockdown of NMNAT-2 or PARP16 impaired global protein synthesis and preadipocyte differentiation. Treatment with the PARP16 inhibitor DB008 hindered ribosomal MARylation and preadipocyte differentiation. Immunofluorescence and subcellular fractionation analysis demonstrated that MARylated ribosomes are mostly associated with the endoplasmic reticulum. Using TMT mass spectrometry, we identified numerous high-confidence ADP-ribosylation sites on glutamate and aspartate residues in both undifferentiated and differentiated cells. We examined five proteins with specific sites of MARylation that are consistently more modified in the differentiated state. Among these, ADP-ribosylation of RPSA significantly promote preadipocyte differentiation; mutation of the site inhibits this effect. Ongoing investigations involve studying the impact of Parp16 knockout on high fat diet-induced obesity in mice to further determine the biological role of ribosome protein MARylation during adipogenesis. This work is supported by a grant from the NIH/NIDDK (R01 DK069710) and the DOD OCRP (OC200311). Presentation: 6/1/2024