Type 2 diabetes is characterized by progressive β cell dysfunction, with lipotoxicity playing a possible pathogenetic role. Palmitate is often used to examine the direct effects of lipotoxicity and it may cause mitochondrial alterations by activating protein acetylation. However, it is unknown whether palmitate influences protein acetylation in β cells. We investigated lysine acetylation in mitochondrial proteins from INS-1E β cells (INS-1E) and in proteins from human pancreatic islets (HPI) after 24 h palmitate exposure. First, we confirmed that palmitate damages β cells and demonstrated that chemical inhibition of deacetylation also impairs INS-1E function and survival. Then, by 2-D gel electrophoresis, Western Blot and Liquid Chromatography-Mass Spectrometry we evaluated the effects of palmitate on protein acetylation. In mitochondrial preparations from palmitate-treated INS-1E, 32 acetylated spots were detected, with 13 proteins resulting over-acetylated. In HPI, 136 acetylated proteins were found, of which 11 were over-acetylated upon culture with palmitate. Interestingly, three proteins, glutamate dehydrogenase, mitochondrial superoxide dismutase, and SREBP-1, were over-acetylated in both INS-1E and HPI. Therefore, prolonged exposure to palmitate induces changes in β cell protein lysine acetylation and this modification could play a role in causing β cell damage. Dysregulated acetylation may be a target to counteract palmitate-induced β cell lipotoxicity.

The molecular mechanisms of interaction between G(s) and the A(2A) adenosine receptor were investigated using synthetic peptides corresponding to various segments of the Galpha(s) carboxyl terminus. Synthetic peptides were tested for their ability to modulate binding of a selective radiolabeled agonist, [(3)H]2-[4-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxam idoade nosine ([(3)H]CGS21680), to A(2A) adenosine receptors in rat striatal membranes. The Galpha(s) peptides stimulated specific binding both in the presence and absence of 100 microM guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS). Three peptides, Galpha(s)(378-394)C(379)A, Galpha(s)(376-394)C(379)A, and Galpha(s)(374-394)C(379)A, were the most effective. In the presence of GTPgammaS, peptide Galpha(s)(374-394)C(379)A increased specific binding in a dose-dependent fashion. However, the peptide did not stabilize the high-affinity state of the A(2A) adenosine receptor for [(3)H]CGS21680. Binding assays with a radiolabeled selective antagonist, [(3)H]5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4, 3-e]-1,2,4-triazolo[1,5-c]pyrimidine ([(3)H]SCH58261), showed that the addition of the Galpha(s) peptide modified the slope of the 5'-N-ethylcarboxamidoadenosine (NECA) competition curve, suggesting modulation of receptor affinity states. In the presence of GTPgammaS, the displacement curve was right-shifted, whereas the addition of Galpha(s)(374-394)C(379)A caused a partial left-shift. Both curves were fitted by one-site models. This same Galpha(s) peptide was also able to disrupt G(s)-coupled signal transduction as indicated by inhibition of the A(2A) receptor-stimulated adenylyl cyclase activity without affecting either basal or forskolin-stimulated enzymatic activity in the same membrane preparations. Shorter peptides from Galpha(s) and Galpha(i1/2) carboxyl termini were not effective. NMR spectroscopy showed the strong propensity of peptide Galpha(s)(374-394)C(379)A to assume a compact carboxyl-terminal alpha-helical conformation in solution. Overall, our results point out the conformation requirement of Galpha(s) carboxyl-terminal peptides to modulate agonist binding to rat A(2A) adenosine receptors and disrupt signal transduction.

Chronic fatigue syndrome (CFS) is a debilitating and complex disorder characterized by unexplained fatigue not improved by rest. An area of investigation is the likely connection of CFS with defective mitochondrial function. In a previous work, we investigated the proteomic salivary profile in a couple of monozygotic twins discordant for CFS. Following this work, we analyzed mitochondrial proteins in the same couple of twins. Nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-MS) was used to study the mitochondria extracted from platelets of the twins. Subsequently, we selected three proteins that were validated using western blot analysis in a big cohort of subjects (n=45 CFS; n=45 healthy), using whole saliva (WS). The selected proteins were as follows: aconitate hydratase (ACON), ATP synthase subunit beta (ATPB) and malate dehydrogenase (MDHM). Results for ATPB and ACON confirmed their upregulation in CFS. However, the MDHM alteration was not confirmed. Thereafter, seeing the great variability of clinical features of CFS patients, we decided to analyze the expression of our proteins after splitting patients according to clinical parameters. For each marker, the values were actually higher in the group of patients who had clinical features similar to the ill twin. In conclusion, these results suggest that our potential markers could be one of the criteria to be taken into account for helping in diagnosis. Furthermore, the identification of biomarkers present in particular subgroups of CFS patients may help in shedding light upon the complex entity of CFS. Moreover, it could help in developing tailored treatments.

Matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) of intact proteins is mostly performed using time-of-flight (TOF) based mass spectrometers, operated in linear mode. Linear MALDI-TOF systems provide limited mass resolving power and mass accuracy, which complicates assigning identities to the peaks in the MSI datasets. In this work we report ultra-high mass resolution MALDI-MSI based on 15T Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for the analysis of intact proteins directly from non-embedded and OCT-embedded mouse and human (control and type 2 diabetes) pancreas so that small endocrine compartments (islets of Langerhans) may be analyzed in control and pathological tissues. Sample preparation methods, in terms of increased sensitivity while limiting lateral diffusion of analytes, have been investigated. By combining protein localization, high mass accuracy, and the clearly resolved isotope patterns we were able to assign protein identities with additional confidence, including proteins of similar average mass and with interspersed isotopomers. These capabilities allowed us to ascertain the presence of many protein adducts that, with a low resolving power instrument, could be misinterpreted as distinct protein ions

Neurodegenerative diseases represent a heterogeneous group of disorders that share common features like abnormal protein aggregation, perturbed Ca2+ homeostasis, excitotoxicity, impairment of mitochondrial functions, apoptosis, inflammation, and oxidative stress. Despite recent advances in the research of biomarkers, early diagnosis, and pharmacotherapy, there are no treatments that can halt the progression of these age-associated neurodegenerative diseases. Numerous epidemiological studies indicate that long-term intake of a Mediterranean diet, characterized by a high consumption of extra virgin olive oil, correlates with better cognition in aged populations. Olive oil phenolic compounds have been demonstrated to have different biological activities like antioxidant, antithrombotic, and anti-inflammatory activities. Oleocanthal, a phenolic component of extra virgin olive oil, is getting more and more scientific attention due to its interesting biological activities. The aim of this research was to characterize the neuroprotective effects of oleocanthal against H2O2-induced oxidative stress in neuron-like SH-SY5Y cells. Moreover, protein expression profiling, combined with pathways analyses, was used to investigate the molecular events related to the protective effects. Oleocanthal was demonstrated to counteract oxidative stress, increasing cell viability, reducing reactive oxygen species (ROS) production, and increasing reduced glutathione (GSH) intracellular level. Proteomic analysis revealed that oleocanthal significantly modulates 19 proteins in the presence of H2O2. In particular, oleocanthal up-regulated proteins related to the proteasome, the chaperone heat shock protein 90, the glycolytic enzyme pyruvate kinase, and the antioxidant enzyme peroxiredoxin 1. Moreover, oleocanthal protection seems to be mediated by Akt activation. These data offer new insights into the molecular mechanisms behind oleocanthal protection against oxidative stress.