👤 Kristoffer Rigbolt

We recently showed that the functional capacity for ureagenesis is deficient in non-alcoholic fatty liver disease (NAFLD) patients. The aim of this study was to assess expression of urea cycle-related genes to elucidate a possible gene regulatory basis to the functional problem. Liver mRNA expression analyses within the gene pathway governing hepatic nitrogen conversion were performed in 20 non-diabetic, biopsy-proven NAFLD patients (8 simple steatosis; 12 non-alcoholic steatohepatitis [NASH]) and 12 obese and 14 lean healthy individuals. Sixteen NAFLD patients were included for gene expression validation. Relationship between gene expressions and functional capacity for ureagenesis was described. Gene expression of most urea cycle-related enzymes were downregulated in NAFLD vs both control groups; markedly so for the urea cycle flux-generating carbamoyl phosphate synthetase (CPS1) (~3.5-fold, P < .0001). In NASH, CPS1 downregulation paralleled the deficit in ureagenesis (P = .03). Additionally, expression of several genes involved in amino acid uptake and degradation, and the glucagon receptor gene, were downregulated in NAFLD. Conversely, glutamine synthetase (GS) expression increased >1.5-fold (P ≤ .03), inversely related to CPS1 expression (P = .004). NAFLD downregulated the expression of urea cycle-related genes. Downregulation of urea cycle flux-generating CPS1 correlated with the loss of functional capacity for ureagenesis in NASH. On gene level, these changes coincided with an increase in the major ammonia scavenging enzyme GS. The effects seemed related to a fatty liver as such rather than NASH or obesity. The findings support gene regulatory mechanisms involved in the deficient ureagenesis of NAFLD, but it remains unexplained how hepatocyte fat accumulation exerts these effects. Show less

Protein ubiquitination is a dynamic reversible post-translational modification that plays a key role in the regulation of numerous cellular processes including signal transduction, endocytosis, cell cycle control, DNA repair and gene transcription. The conjugation of the small protein ubiquitin or chains of ubiquitin molecules of various types and lengths to targeted proteins is known to alter proteins' lifespan, localization and function and to modulate protein interactions. Despite its central importance in various aspects of cellular life and function there are only a limited number of reports investigating ubiquitination on a proteomic scale, mainly due to the inherited complexity and heterogeneity of ubiquitination. We describe here a quantitative proteomics strategy based on the specificity of ubiquitin binding domains (UBDs) and Stable Isotope Labeling by Amino acids in Cell culture (SILAC) for selectively decoding ubiquitination-driven processes involved in the regulation of cellular signaling networks. We applied this approach to characterize the temporal dynamics of ubiquitination events accompanying epidermal growth factor receptor (EGFR) signal transduction. We used recombinant UBDs derived from endocytic adaptor proteins for specific enrichment of ubiquitinated complexes from the EGFR network and subsequent quantitative analyses by high accuracy mass spectrometry. We show that the strategy is suitable for profiling the dynamics of ubiquitination occurring on individual proteins as well as ubiquitination-dependent events in signaling pathways. In addition to a detailed seven time-point profile of EGFR ubiquitination over 30 minutes of ligand stimulation, our data determined prominent involvement of Lysine-63 ubiquitin branching in EGF signaling. Furthermore, we found two centrosomal proteins, PCM1 and Azi1, to form a multi-protein complex with the ubiquitin E3 ligases MIB1 and WWP2 downstream of the EGFR, thereby revealing possible ubiquitination cross-talk between EGF signaling and centrosomal-dependent rearrangements of the microtubules. This is a general strategy that can be utilized to study the dynamics of other cellular systems and post-translational modifications. Show less