High glucose induces an early and transient cytoprotective autophagy in retinal Müller cells.

PURPOSE: We investigated the autophagic response of rat Müller rMC-1 cells during a short-term high glucose challenge. METHODS: rMC-1 cells were maintained in 5 mM glucose (LG) or exposed to 25 mM glucose (HG). Western blot analysis was used to evaluate the expression levels of markers of autophagy (LC3-II, p62) and glial activation (AQP4), as well as the activation of TRAF2/JNK, ERK and AKT pathways. Autophagic flux assessment was performed using the autophagy inhibitor chloroquine. ROS levels were measured by flow cytometry using dichlorofluorescein diacetate. ERK involvement in autophagy induction was addressed using the ERK inhibitor FR180204. The effect of autophagy inhibition on cell viability was evaluated by SRB assay. RESULTS: Activation of autophagy was observed in the first 2-6 h of HG exposure. This early autophagic response was transient, not accompanied by an increase in AQP4 or in the phospho-activation of JNK, a key mediator of cellular response to oxidative stress, and required ERK activity. Cells exposed to HG had a lower viability upon autophagy inhibition by chloroquine, as compared to those maintained in LG. CONCLUSION: A short-term HG challenge triggers in rMC-1 cells a process improving the ability to cope with stressful conditions, which involves ERK and an early and transient autophagy activation.

The proteasome as a druggable target with multiple therapeutic potentialities: Cutting and non-cutting edges.

Ubiquitin Proteasome System (UPS) is an adaptable and finely tuned system that sustains proteostasis network under a large variety of physiopathological conditions. Its dysregulation is often associated with the onset and progression of human diseases; hence, UPS modulation has emerged as a promising new avenue for the development of treatments of several relevant pathologies, such as cancer and neurodegeneration. The clinical interest in proteasome inhibition has considerably increased after the FDA approval in 2003 of bortezomib for relapsed/refractory multiple myeloma, which is now used in the front-line setting. Thereafter, two other proteasome inhibitors (carfilzomib and ixazomib), designed to overcome resistance to bortezomib, have been approved for treatment-experienced patients, and a variety of novel inhibitors are currently under preclinical and clinical investigation not only for haematological malignancies but also for solid tumours. However, since UPS collapse leads to toxic misfolded proteins accumulation, proteasome is attracting even more interest as a target for the care of neurodegenerative diseases, which are sustained by UPS impairment. Thus, conceptually, proteasome activation represents an innovative and largely unexplored target for drug development. According to a multidisciplinary approach, spanning from chemistry, biochemistry, molecular biology to pharmacology, this review will summarize the most recent available literature regarding different aspects of proteasome biology, focusing on structure, function and regulation of proteasome in physiological and pathological processes, mostly cancer and neurodegenerative diseases, connecting biochemical features and clinical studies of proteasome targeting drugs.

[Contribution of ISPESL (National Institute for Occupational Safety and Prevention) for protecting workers from exposure to avian influenza]. / Il contributo dell'ISPESL per la protezione di lavoratori esposti a virus dell'influenza aviaria.

Influenza virus A/H5N1 occurs mainly in birds, in which is highly contagious and deadly, and does not usually infect people. Most of the cases occurred in humans resulted from people having direct or close contact with H5N1 infected poultry or contaminated surfaces. The circulation of influenza viruses in birds, humans and other hosts represents a public and animal health threat, with important economic consequences. Controlling avian influenza in poultry, in particular with biosecurity measures, is the primary method to reduce human risk from infection. Enhanced surveillance both in poultry and in wild birds proved effective for the early detection of the infection. Worldwide most countries developed strategic plans, guidelines and recommendations for effective disease prevention and control. Moreover documents were specifically prepared to keep specific categories of workers adequately informed on how to avoid or minimize exposure to the viruses. In accordance with the Italian Decree 626/94, recently amended by the Decree 81/08, regarding the protection of workers from risks related to exposure to biological agents at work, the Department of Occupational Medicine of ISPESL prepared one booklet directed to people working with poultry and, together with Corpo Nazionale Vigili del Fuoco, two booklets addressed to fire brigade who could be at various levels involved in outbreak disease control and eradication activities. In fact information and training are essential aspects of a global preventive and protective strategy.

Ubiquitin binds the amyloid ß peptide and interferes with its clearance pathways.

Several lines of evidence point to a compromised proteostasis associated with a reduction of the Ubiquitin Proteasome System (UPS) activity in patients affected by Alzheimer's Disease (AD) and suggest that the amyloid ß peptide (Aß) is an important player in the game. Inspired also by many reports, underlining the presence of ubiquitin (Ub) in the amyloid plaques of AD brains, here we set out to test whether Ub may bind the Aß peptide and have any effect on its clearance pathways. By using an integrated array of MALDI-TOF/UPLC-HRMS, fluorescence, NMR, SPR, Microscale Thermophoresis (MST) and molecular dynamics studies, we consistently demonstrated that Aß40 binds Ub with a 1 : 1 stoichiometry and K d in the high micromolar range. In particular, we show that the N-terminal domain of the Aß peptide (through residues D1, E3 and R5) interacts with the C-terminal tail of Ub (involving residues K63 and E64), inducing the central region of Aß (14HQKLVFFAEDVGSNK28) to adopt a mixed α-helix/ß-turn structure. ELISA assays, carried out in neuroblastoma cell lysates, suggest that Aß competitively binds Ub also in the presence of the entire pool of cytosolic Ub binding proteins. Ub-bound Aß has a lower tendency to aggregate into amyloid-like fibrils and is more slowly degraded by the Insulin Degrading Enzyme (IDE). Finally, we observe that the water soluble fragment Aß1-16 significantly inhibits Ub chain growth reactions. These results evidence how the non-covalent interaction between Aß peptides and Ub may have relevant effects on the regulation of the upstream events of the UPS and pave the way to future in vivo studies addressing the role played by Aß peptide in the malfunction of proteome maintenance occurring in AD.

Effect of cisplatin on proteasome activity.

Cisplatin is a widely used chemotherapy drug which exerts cytotoxic activity by affecting both nuclear and cytosolic pathways. Herewith, we report, for the first time, that cisplatin inhibits proteasome activity in vitro. Cisplatin induces a dose dependent inhibition of the three enzymatic activities of proteasome (i.e., the chymotrypsin-like activity, the trypsin-like activity and the caspase-like activity). Moreover, cisplatin administration to neuroblastoma cells brings about a fast loss of proteasome particle activity, which is followed by a de novo synthesis of proteasome. Lastly, we report that the simultaneous administration of lactacystin and cisplatin enhances the cytotoxicity of cisplatin alone. The overall bulk of data opens to an intriguing scenario, concerning the biological effects of cisplatin in the control of cellular life, which goes beyond the well established genotoxic effect.