Fasting-sensitive SUMO-switch on Prox1 controls hepatic cholesterol metabolism.

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.

Identification and analysis of endogenous SUMO1 and SUMO2/3 targets in mammalian cells and tissues using monoclonal antibodies.

SUMOylation is a protein modification that regulates the function of hundreds of proteins. Detecting endogenous SUMOylation is challenging: most small ubiquitin-related modifier (SUMO) targets are low in abundance, and only a fraction of a protein's cellular pool is typically SUMOylated. Here we present a step-by-step protocol for the enrichment of endogenous SUMO targets from mammalian cells and tissues (specifically, mouse liver), based on the use of monoclonal antibodies that are available to the scientific community. The protocol comprises (i) production of antibodies and affinity matrix, (ii) denaturing cell lysis, and (iii) SUMO immunoprecipitation followed by peptide elution. Production of affinity matrix and cell lysis requires ∼1 d. The immunoprecipitation with peptide elution can be performed in 2 d. As SUMO proteins are conserved, this protocol should also be applicable to other organisms, including many vertebrates and Drosophila melanogaster.

Detecting endogenous SUMO targets in mammalian cells and tissues.

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.

SCFFbxw5 mediates transient degradation of actin remodeller Eps8 to allow proper mitotic progression.

Eps8, a bi-functional actin cytoskeleton remodeller, is a positive regulator of cell proliferation and motility. Here, we describe an unrecognized mechanism regulating Eps8 that is required for proper mitotic progression: whereas Eps8 is stable in G1 and S phase, its half-life drops sharply in G2. This requires G2-specific proteasomal degradation mediated by the ubiquitin E3 ligase SCF(Fbxw5). Consistent with a short window of degradation, Eps8 disappears from the cell cortex early in mitosis, but reappears at the midzone of dividing cells. Failure to reduce Eps8 levels in G2 prolongs its localization at the cell cortex and markedly delays cell rounding and prometaphase duration. However, during late stages of mitosis and cytokinesis, Eps8 capping activity is required to prevent membrane blebbing and cell-shape deformations. Our findings identify SCF(Fbxw5)-driven fluctuation of Eps8 levels as an important mechanism that contributes to cell-shape changes during entry into-and exit from-mitosis.