Browsing by Author "Herzig, Stephan"

SUMOylation is an essential modification that regulates hundreds of proteins in eukaryotic cells. Owing to its dynamic nature and low steady-state levels, endogenous SUMOylation is challenging to detect. Here, we present a method that allows efficient enrichment and identification of endogenous targets of SUMO1 and the nearly identical SUMO2 and 3 (SUMO 2/3) from vertebrate cells and complex organ tissue. Using monoclonal antibodies for which we mapped the epitope, we enriched SUMOylated proteins by immunoprecipitation and peptide elution. We used this approach in combination with MS to identify SUMOylated proteins, which resulted in the first direct comparison of the endogenous SUMO1- and SUMO2/3-modified proteome in mammalian cells, to our knowledge. This protocol provides an affordable and feasible tool to investigate endogenous SUMOylation in primary cells, tissues and organs, and it will facilitate understanding of SUMO's role in physiology and disease.

Abstract Accumulation of excess nutrients hampers proper liver function and is linked to nonalcoholic fatty liver disease (NAFLD) in obesity. However, the signals responsible for an impaired adaptation of hepatocytes to obesogenic dietary cues remain still largely unknown. Post‐translational modification by the small ubiquitin‐like modifier (SUMO) allows for a dynamic regulation of numerous processes including transcriptional reprogramming. We demonstrate that specific SUMOylation of transcription factor Prox1 represents a nutrient‐sensitive determinant of hepatic fasting metabolism. Prox1 is highly SUMOylated on lysine 556 in the liver of ad libitum and refed mice, while this modification is abolished upon fasting. In the context of diet‐induced obesity, Prox1 SUMOylation becomes less sensitive to fasting cues. The hepatocyte‐selective knock‐in of a SUMOylation‐deficient Prox1 mutant into mice fed a high‐fat/high‐fructose diet leads to a reduction of systemic cholesterol levels, associated with the induction of liver bile acid detoxifying pathways during fasting. The generation of tools to maintain the nutrient‐sensitive SUMO‐switch on Prox1 may thus contribute to the development of “fasting‐based” approaches for the preservation of metabolic health.