Stark, L. | bioRxiv Science

bioRxiv2023

Molecular basis of Progressive Familial Intrahepatic Cholestasis 3. A proteomics study.

Background and aimsProgressive familiar intrahepatic cholestasis type 3 (PFIC3) is a severe rare liver disease which affects between 1/50,000 to 1/100,000 children. In physiological conditions, bile is produced by the liver and stored in the gallbladder, then it flows to the small intestine to play its role in fat digestion. To prevent tissue damage, bile acids are kept into phospholipid micelles. Mutations in phosphatidyl choline transporter ABCB4 (MDR3) lead to intrahepatic accumulation of free bile acids that results in liver damage. PFIC3 onset occurs usually at early ages, progress rapidly and the prognosis is poor. Currently, besides the palliative use of ursodeoxycholate, the only available treatment for this disease is liver transplantation, which is really challenging for short-aged patients. MethodsTo gain insight into the pathogenesis of PFIC3 we have performed an integrated proteomics and phosphoproteomics study in human liver samples to then validate the emerging functional hypotheses in a PFIC3 murine model. ResultsWe identified 6,246 protein groups, 324 proteins among them showing differential expression between control and PFIC3. The phosphoproteomic analysis allowed the identification of 5,090 phosphopeptides, from which 215 corresponding to 157 protein groups, were differentially phosphorylated in PFIC3, including MDR3. Regulation of essential cellular processes and structures, such as inflammation, metabolic reprograming, cytoskeleton and extracellular matrix remodeling and cell proliferation were identified as main drivers of the disease. ConclusionOur results provide a strong molecular background that significantly contributes to a better understanding of PFIC3 and provides new concepts that might prove useful in the clinical management of patients. Lay Summary/Key PointsPFIC3 is a rare disease that affect 1/50,000 to 1/100,000 children that present severe symptoms in the first years of life and have scarce therapeutic options. We identified a panel of proteins that recapitulate some of the main cellular processes associated to the progression of liver injury in PFIC3 patients and suggest alternative treatment options.

molecular biology↗

bioRxiv2017

Activation of NF-κB by a novel, CDK4-regulated, nucleolar stress response pathway

p53 as an effector of nucleolar stress is well defined, but p53 independent mechanisms are largely unknown. Like p53, the NF-{kappa}B transcription factor plays a critical role in maintaining cellular homeostasis under stress. Many stresses that stimulate NF-{kappa}B also disrupt nucleoli. However, the link between nucleolar function and activation of the NF-{kappa}B pathway is as yet unknown. Here we demonstrate that siRNA silencing of PolI complex components stimulates NF-{kappa}B signalling. Unlike p53 nucleolar stress response, this effect does not appear to be linked to inhibition of rDNA transcription. We show that specific stress stimuli of NF-{kappa}B induce degradation of a critical component of the PolI complex, TIF-IA. This degradation precedes activation of the NF-{kappa}B pathway and is associated with an atypical nucleolar architecture. It is mimicked by CDK4 inhibition and is dependent upon upstream binding factor (UBF) and p14ARF. We show that blocking stress effects on TIF-IA blocks their ability to activate the NF-{kappa}B pathway. Finally, using ex vivo culture, we show a strong correlation between degradation of TIF-IA and activation of NF-{kappa}B in freshly resected, human colorectal tumours exposed to the chemopreventative agent, aspirin. Together, our study provides compelling evidence for a new, NF-{kappa}B nucleolar stress response pathway that has in vivo relevance and therapeutic implications.

cell biology↗