Protein N-Terminomics Reveals Major Proteases in Regulating Beige Adipocyte Differentiation

Abstract

In this comprehensive study, we present an innovative analytical platform designed to capture the temporal shifts in both the proteome and protein N-terminome during beige adipocyte differentiation. Employing a refined N-terminomics technique, we achieved a high purity of 97% in isolating protein N-terminal peptides. Our data encompassed 7,171 unique N-terminal peptides, with 3,043 from canonical proteins and 4,129 with neo-N-termini. Strikingly, nearly half (44%) of the proteins revealed distinct temporal trajectories between the global proteome and the N-terminome. This underscores the central role of proteolysis in beige adipocyte differentiation. Experimentally, knockdown of either Pmpcb, Plg, or Cstd in preadipocytes attenuated thermogenesis, manifested by reduced levels of beige adipocyte markers like Cidea, Pgc1a, Ucp1, and Tbx1 and an increase in adipogenic proteins, thereby hampering beige adipocyte maturation. A salient discovery was the non-apoptotic role of caspase 8 protease; inhibiting its proteolytic action amplified Ucp1 expression levels. Collectively, our findings spotlight proteases and their proteolytic by-products as vital regulators in beige adipocyte differentiation.