Previous experiments in suitable animal models and in mild hypercholesterolemic individuals have shown that the consumption of lupin proteins may be useful for controlling total and low-density lipoprotein (LDL) cholesterol levels. With the objective of providing evidence that peptides deriving from the hydrolysis of lupin proteins may be responsible of the observed activities and for investigating the mechanism of action, HepG2 cells were treated with lupin peptides obtained by either pepsin (P) or trypsin (T) hydrolysis, and molecular and functional investigations were performed on the LDL receptor/SREBP2 pathway. For the first time, this paper provides experimental evidence that lupin peptides are able to interfere with the HMGCoAR activity, up-regulating the LDL receptor (136 and 84% vs the control for P and T peptides, respectively, at 1 mg/mL) and SREBP2 proteins (148 and 73% vs the control for P and T peptides, respectively, at 1 mg/mL) via the activation of PI3K/Akt/GSK3β pathways and increasing the LDL uptake at HepG2 cell line (40 and 50% vs the control for P and T peptides, respectively, at 1 mg/mL). These results may be useful in explaining the activities observed in vivo in animals and humans treated with lupin protein.

Many cellular processes are regulated by the coordination of several post-translational modifications that allow a very fine modulation of substrates. Recently it has been reported that there is a relationship between sumoylation and ubiquitination. Here we propose that the nucleolus is the key organelle in which SUMO-1 conjugates accumulate in response to proteasome inhibition. We demonstrated that, upon proteasome inhibition, the SUMO-1 nuclear dot localization is redirected to nucleolar structures. To better understand this process we investigated, by quantitative proteomics, the effect of proteasome activity on endogenous nucleolar SUMO-1 targets. 193 potential SUMO-1 substrates were identified, and interestingly in several purified SUMO-1 conjugates ubiquitin chains were found to be present, confirming the coordination of these two modifications. 23 SUMO-1 targets were confirmed by an in vitro sumoylation reaction performed on nuclear substrates. They belong to protein families such as small nuclear ribonucleoproteins, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, histones, RNA-binding proteins, and transcription factor regulators. Among these, histone H1, histone H3, and p160 Myb-binding protein 1A were further characterized as novel SUMO-1 substrates. The analysis of the nature of the SUMO-1 targets identified in this study strongly indicates that sumoylation, acting in coordination with the ubiquitin-proteasome system, regulates the maintenance of nucleolar integrity.