AMBRA1 interplay with cullin E3 ubiquitin ligases regulates autophagy dynamics.

Autophagy maintains cellular homeostasis by degrading harmful or unnecessary intracellular components. How the autophagy response is induced rapidly and transiently remains largely unknown. We report that the E3 ubiquitin ligases Cullin-5 and Cullin-4 regulate the onset and termination of autophagy, respectively, by dynamically interacting with AMBRA1, a regulator of autophagy. Under normal conditions, Cullin-4 binding to AMBRA1 limits its protein abundance. Autophagy stimuli promote AMBRA1 stabilization by causing ULK1-dependent Cullin-4 release. Notably, Cullin-4/AMBRA1 dissociation is transient, and the re-established interaction triggers AMBRA1 degradation, terminating the autophagy response. Moreover, Cullin-4 inhibits the interaction between AMBRA1 and another Cullin E3 ligase. Indeed, upon Cullin-4 dissociation, AMBRA1 binds and inhibits Cullin-5, thus promoting the accumulation of the mTOR inhibitor DEPTOR. Through DEPTOR stabilization, AMBRA1 establishes a feedback loop that ensures the rapid onset of autophagy by enhancing mTOR inactivation. Our findings show that Cullin-mediated degradation of autophagy regulators temporally controls the autophagy response.

Liver protein profiling in chronic hepatitis C: identification of potential predictive markers for interferon therapy outcome.

The current anti-hepatitis C virus (HCV) therapy, based on pegylated-interferon alpha and ribavirin, has limited success rate and is accompanied by several side effects. The aim of this study was to identify protein profiles in pretreatment liver biopsies of HCV patients correlating with the outcome of antiviral therapy. Cytosolic or membrane/organelle-enriched protein extracts from liver biopsies of eight HCV patients were analyzed by two-dimensional fluorescence difference gel electrophoresis and mass spectrometry. Overall, this analysis identified 21 proteins whose expression levels correlate with therapy response. These factors are involved in interferon-mediated antiviral activity, stress response, and energy metabolism. Moreover, we found that post-translational modifications of dihydroxyacetone kinase were also associated with therapy outcome. Differential expression of the five best performing markers (STAT1, Mx1, DD4, DAK, and PD-ECGF) was confirmed by immunoblotting assays in an independent group of HCV patients. Finally, we showed that a prediction model based on the expression levels of these markers classifies responder and nonresponder patients with an accuracy of 85.7%. These results provide evidence that the analysis of pretreatment liver protein profiles is valuable for discriminating between responder and nonresponder HCV patients, and may contribute to reduce the number of nonresponder patients exposed to therapy-associated risks.

Autophagy protects cells from HCV-induced defects in lipid metabolism.

BACKGROUND & AIMS: Autophagy is a lysosome-mediated catabolic process that mediates degradation and recycling of all major components of eukaryotic cells. Different stresses, including viral and bacterial infection, induce autophagy, which can promote cell survival by removing the stress inducer or by attenuating its dangerous effects. High levels of autophagy occur during infection of cells with hepatitis C virus (HCV), but the clinical relevance of this process is not clear. METHODS: Levels of autophagy were analyzed in liver biopsy samples from 22 patients with HCV infection using microtubule-associated protein-1 light chain 3 immunoblotting; associations with histological and metabolic parameters were evaluated by Pearson correlation analysis. We investigated the role of HCV-induced autophagy in lipid degradation in cells infected with the virus or replicons, and analyzed autophagosome contents by confocal microscopy and by measuring lipid levels after inhibition of autophagy by Beclin 1 knockdown or lysosome inhibitors. RESULTS: In liver biopsy samples from patients with HCV, there was an inverse correlation between microvesicular steatosis and level of autophagy (r = -0.617; P = .002). HCV selectively induced autophagy of lipids in virus-infected and replicon cells. In each system, autophagosomes frequently colocalized with lipid deposits, mainly formed by unesterified cholesterol. Inhibition of the autophagic process in these cells significantly increased the induction of cholesterol accumulation by HCV. CONCLUSIONS: Autophagy counteracts the alterations in lipid metabolism induced by HCV. Disruption of the autophagic process might contribute to development of steatosis in patients with HCV.

Proteomic analysis reveals a major role for contact inhibition in the terminal differentiation of hepatocytes.

BACKGROUND & AIMS: Hepatocytes are considered an exception of the paradigmatic inverse correlation between cell proliferation and terminal differentiation. In fact, hepatic vital functions are guaranteed by proliferating parenchymal cells during liver regeneration. However, a fine molecular characterization of the relationship between proliferation and differentiation in hepatocytes has been hampered by the lack of reliable in vivo or in vitro models. METHODS: The hepatocyte terminal differentiation program was characterized in the immortalized, untransformed and differentiated hepatocytic cell line MMH, using several techniques. Particularly, two-dimensional difference gel electrophoresis combined to tandem mass spectrometry proteomic approach was used. Cell cycle and cell adhesion properties of MMH have been altered using either myc-overexpression and MEK1/2 inhibition or a constitutive active beta-catenin mutant, respectively. RESULTS: The hepatocyte terminal differentiation program is stimulated by the exit from the cell cycle induced by cell-cell contact. Comparative proteomic analysis of proliferating versus quiescent hepatocytes validated the importance of contact inhibition, identifying 68 differently expressed gene products, representing 49 unique proteins. Notably, enzymes involved in important liver functions such as detoxification processes, lipid metabolism, iron and vitamin A storage and secretion, anti-inflammatory response and exocytosis were found significantly up-regulated in quiescent hepatocytes. Finally, we found that: (i) cell cycle arrest induced by MEK1/2 inhibition is not sufficient to induce hepatic product expression; (ii) constitutive activation of beta-catenin counteracts the contact inhibition-induced terminal differentiation. CONCLUSION: The hepatocyte terminal differentiation program requires a quiescent state maintained by cell-cell contact through the E-cadherin/beta-catenin pathway, rather than the inhibition of proliferation.

Analysis of the periplasmic proteome of Pseudomonas aeruginosa, a metabolically versatile opportunistic pathogen.

The Gram-negative bacterium Pseudomonas aeruginosa is a main cause of infection in hospitalized, burned, immunocompromised, and cystic fibrosis patients. Many processes essential for P. aeruginosa pathogenesis, e.g., nutrient uptake, antibiotic resistance, and virulence, take place in the cell envelope and depend on components residing in the periplasmic space. Recent high-throughput studies focused on P. aeruginosa membrane compartments. However, the composition and dynamics of its periplasm remain largely uncharacterized. Here, we report a detailed description of the periplasmic proteome of the wild-type P. aeruginosa strain PAO1 by 2-DE and MALDI-TOF/TOF analysis. Three extraction methods were compared at proteome level in order to achieve the most reliable and comprehensive periplasmic protein map. A total of 495 spots representing 395 different proteins were identified. Most of the high intensity spots corresponded to periplasmic proteins, while cytoplasmic contaminants were mainly detected among faint spots. The majority of the identified periplasmic proteins is involved in transport, cell-envelope integrity, and protein folding control. Notably, more than 30% still has an unpredicted function. This work provides the first overview of the P. aeruginosa periplasm and offers the basis for future studies on periplasmic proteome changes occurring during P. aeruginosa adaptation to different environments and/or antibiotic treatments.