Protein and Peptide Letters - Volume 22, Issue 4, 2015

It has long been known that amyloid ß protein (Aß) plays a key role in Alzheimer’s Disease (AD) and in Down Syndrome cognitive decline. Recent findings have shown that soluble forms of Aß (mostly Aß oligomers; Aßo), rather than insoluble forms (fibrils and plaques), are associated with memory impairments in early stages of AD. Since synaptic plasticity and oscillatory network activity are required for memory formation, consolidation and retrieval, numerous attempts have been made to establish whether or not Aßo-induced alterations in synaptic plasticity and oscillatory network activity cause memory impairment. Despite a wealth of uncorrelated experimental evidence, such a relationship remains elusive. Furthermore, the specific cellular mechanisms underlying these disruptions remain to be determined. This review will discuss recent findings about the cellular and network mechanisms involved in Aßo-induced alterations of network oscillations and synaptic plasticity that could be responsible for the learning and memory impairments observed in early AD. Additionally, we will review some of the signal transduction pathways involved in these deleterious effects, which are revealing promising therapeutic targets to ease Aßo-induced brain dysfunction and treat AD.

Numerous small molecules including cytokines primarily associated with immune response have been shown to play a role in normal mesenchymal stem cells (MSC) and tumour cells’ communication. One characteristic that distinguishes MSC from fibroblast and other cells of mesenchymal origin is their pro-tumour migratory behaviour. Recognizing the cytokines as key players of the MSC/tumour cell cross-talk and understanding their intracellular signalling, should lead to a development of more efficient anti-tumour therapies. Those are urgently needed for improving the treatment of patients with glioblastoma multiformae (GBM) that are suffering from most aggressive and incurable type of brain tumours. So far, the “cytokine signalling interference” approach, employing genetically modified MSCs and GBM cells in animal xenograft models pointed to the mechanisms underlying tumour - directed migration and immunomodulatory role of MSCs. There, MSC’s effects on tumour growth were shown to vary substantially, and to depend on the type of the cells and the animal model used. This review is focusing on the cytokines produced by MSCs and their involvement in proliferation, migration, angiogenesis, apoptosis and immune cell infiltration. Recently, targeted therapies have emerged as a promising modality for GBM treatment. New approaches, combining these with MSCs as cellular vectors for modulating cytokines and cytokine receptors’ signalling in GBM may thus prove more efficient at inhibiting glioma progression.

Recent studies have shown that angiotensin-converting enzyme 2 (ACE2)/angiotensin (Ang) -(1-7)/Mas axis activation is able to improve the metabolic profile, enhance glucose tolerance and insulin sensitivity, improve metabolic parameters, and counteract deleterious effects of Ang II. The effects of endogenous ACE 2 activation on the metabolic profile of mice are poorly studied. In this study, 12 weeks old male mice were treated with the ACE 2 activator (diminazene aceturate, DIZE, 1 mg/kg/day, gavage) or saline (control) for 30 days followed by glucose tolerance tests, insulin sensitivity tests, and blood analysis. Epididymal ACE2, ACE, angiotensinogen, acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS) were measured by quantitative RT-PCR. ACE 2 activation treatment lowered body weight (DIZE vs control) (28.69 vs 30.28g, P < 0.001), serum cholesterol (140,0 vs 177.5; P < .05), and serum triglycerides (75,00 vs 165,0; P < .05) as well as epididymal (0.008 vs 0.016; P < .05) and retroperitoneal (0.0024 vs. 0.0068; P < .01) adipose tissue weights. These effects were associated with significantly increased epididymal ACE 2 and decreased ACE and angiotensinogen (AGT) expression. Additionally, DIZE decreased adipogenesis-related gene transcription, such as ACC and FAS mRNA. In conclusion, these results indicate that activation of ACE2 by oral DIZE treatment improves the metabolic profile and reduces fat deposition in mice. These results, along with the reduction of lipogenesis markers open a new perspective for metabolic disorder pharmacotherapy.

The central nervous system (CNS) of Aplysia is a fascinating source to identify and characterize neuropeptides and neurotransmitters because of offering many useful divergent and convergent neuronal aggregates. Here, two neuropeptides were isolated from the extract of CNS of the northwest pacific sea hare, Aplysia kurodai, using HPLC system for fractionation and the anterior byssus retractor muscle (ABRM) of the Mytilis edulis as the bioassay system. Purified peptides, myomodulin A (MMA) and E (MME), were determined by amino acid sequencing and molecular mass analysis. MMA showed a potentiating effect at 100 nM or lower, on the contrary, an inhibitory effect at higher doses from 100 nM on phasic contraction elicited by repetitive electrical stimulation on the ABRM of Mytilus. However, MME only inhibited phasic contraction with all examined concentrations. MME revealed 100 times more potent activity than that of MMA on the relaxing catch-tension of ABRM stimulated by acetylcholine. Both MMA and MME potently stimulated a response on the crop and penial retractor muscle of the African giant snail, Achatina fulica, compared with other known mollusks neuropeptides. These results suggest that MMA and MME may be broadly distributed in CNS of Aplysia to function a neuromodulatory role controlled via excitatory and inhibitory neurons, and may be involved in the digestive and reproductive activity in other mollusk.

The formation of amyloid fibrils are thought to contribute to pathogenesis of many amyloids associated human diseases. Here the impact of curcumin on amyloid formation of human serum albumin (HSA) was studied. Incubation of HSA at 68°C under physiologic pH led to amyloid fibril formation. Thioflavin T (ThT) fluorescence was used for determination of amyloid fibril formation. Atomic force microscopy experiments indicated different fibril structure of HSA incubated with or without curcumin. The monitoring of the changes in reactive oxygen species (ROS) levels upon incubation of curcumin with HSA showed a significant decrease in ROS levels. Similar experiments were also carried out in the presence of aflatoxin M1 (AFM1) and lead (Pb) ions. Our results indicated that AFM1 and Pb ions promote the fibrillation of HSA and accelerate ROS production, which were inhibited in the presence of curcumin. Thus, curcumin mitigates protein fibrillation activity and diminishes ROS generation.

α-Synuclein forms amyloid deposits in the dopaminergic neurons; a process that is believed to contribute to the Parkinson’s disease. An emerging theme in amyloid research is the hypothesis that the toxic species produced during amyloid formation share common physic-chemical features and exert their effects by common modes. This prompted the idea that molecules able to inhibit a protein aggregation process may cross-react with other amyloidogenic proteins, interfering in their fibrils formation. We investigate the ability of analogues of the heptapeptide H-Arg-Lys-Val-MePhe-Tyr-Thr-Trp- OH2, an inhibitor of Aβ-peptide aggregation, to cross-react with α-synuclein interfering with its fibril formation. The influence of the MePhe topography on the interaction with α-synuclein has also been evaluated, replacing the MePhe residue with either Phe or the conformationally restricted Tic residues. Peptides interact with good affinity with the

Ofloxacin (OFX) and moxifloxacin (MOX) are the most promising second line drugs for tuberculosis treatment. Although the primary mechanism of action of OFX and MOX is gyrase inhibition, other possible mechanisms cannot be ruled out. Being the functional moiety of cell, the proteins act as primary targets for developing drugs, diagnostics and therapeutics. In this study we have investigated the proteomic changes of Mycobacterium tuberculosis isolates induced by OFX and MOX by applying comparative proteomic approaches based on two-dinensional gel electrophoresis (2DE) along with matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI TOF/TOF-MS) and bioinformatic tools. The findings are likely to provide new understanding of OFX and MOX mechanisms that might be helpful in exploring new diagnostics and drug targets. Our study explored eleven proteins (Rv2889c, Rv2623, Rv0952, Rv1827, Rv1932, Rv0054, Rv1080c, Rv3418c, Rv3914, Rv1636 and Rv0009) that were overexpressed in the presence of drugs. Among them, Rv2623, Rv1827 and Rv1636 were identified as proteins with unknown function. InterProScan and molecular docking revealed that the conserved domain of hypothetical proteins interact with OFX and MOX which indicate a probable inhibition/modulation of the functioning of these proteins by both drugs, which might be overexpressed to overcome this effect.

Standard trypsin digestion protocol of proteins followed by MALDI-MS analysis has been realized as an important tool for the identification and characterization of proteins. In this article, we proposed the elimination of the step of ‘staining/de-staining of gel pieces’ in in-gel digestion protocol in order to improve the efficiency of trypsin digestion. Coomassie dye is known to interfere with digestion of proteins by trypsin and the procedure of staining-de-staining could result in loss of photoaffinity probe, post translational modifications and catalytic activities of enzymes. Further, we studied parameters like hydrophobicity and isoelectric point, and attempted to quantitatively relate it to the efficiency of trypsin digestion. We suggest that properties of proteins should be considered and trypsin digestion protocol should be appropriately modified as per sequence and other information.

This report describes the purification of an aspartic protease (salpichroin) from ripe fruits of Salpichroa origanifolia (Solanaceae) starting with precipitation using organic solvents and anionexchange chromatography with 32.1% recovery and 13.4-fold purification. SDS-PAGE and zymograms of this enzyme showed a single band corresponding to an apparent molecular mass of approximately 32 kDa. The biochemical and kinetic characterization of the pure enzyme showed an acidic behavior with an optimal pH value around 3.0–4.5 with hemoglobin and 5.5–6.0 with casein. Salpichroin activity was inhibited by pepstatin but not by phenylmethylsulfonyl fluoride, E-64, EDTA or 1,10-phenanthroline, thus suggesting an aspartic protease behavior. Salpichroin hydrolyzed natural substrates, such as casein and hemoglobin, with high specific activity. Kinetic studies conducted with the synthetic peptide H-Pro- Thr-Glu-Phe-p-(NO2)-Phe-Arg-Leu-OH showed lower affinity (Km 494 µM) than other representative aspartic proteases. By investigating the cleavage of oxidized insulin β-chain to establish the hydrolytic specificity of salpichroin, we found six cleavage sites on the substrate of peptide bonds similar to those of chymosin. MALDI-TOF/TOF-MS of the tryptic ingel digest of salpichroin showed that the isolated protease shared homologous sequences with other plant proteases of the A1 aspartic protease family. This is the first report concerning the isolation and biochemical characterization of an aspartic protease isolated from Salpichroa origanifolia fruits.