Molecular strategies to prevent, inhibit and degrade advanced glycoxidation and advanced lipoxidation end products

The advanced glycoxidation end products (AGEs) and lipoxidation end products (ALEs) contribute to the development of diabetic complications and of other pathologies. The review discusses the possibilities of counteracting the formation and stimulating the degradation of these species by pharmaceuticals and natural compounds. The review discusses inhibitors of ALE and AGE formation, cross-link breakers, ALE/AGE elimination by enzymes and proteolytic systems, receptors for advanced glycation end products (RAGEs) and blockade of the ligand-RAGE axis

Monitoring Alzheimer Amyloid Peptide Aggregation by EPR

Plaques containing the aggregated β-Amyloid (Aβ) peptide in the brain are the main indicators of Alzheimer’s disease. Fibrils, the building blocks of plaques, can also be produced in vitro and consist of a regular arrangement of the peptide. The initial steps of fibril formation are not well understood and could involve smaller aggregates (oligomers) of Aβ. Such oligomers have even been implicated as the toxic agents. Here, a method to study oligomers on the time scale of aggregation is suggested. We have labeled the 40 residue Aβ peptide variant containing an N-terminal cysteine (cys-Aβ) with the MTSL [1-oxyl-2,2,5,5-tetramethyl-Δ-pyrroline-3-methyl] methanethiosulfonate spin label (SL-Aβ). Fibril formation in solutions of pure SL-Aβ and of SL-Aβ mixed with Aβ was shown by Congo-red binding and electron microscopy. Continuous-wave 9 GHz electron paramagnetic resonance reveals three fractions of different spin-label mobility: one attributed to monomeric Aβ, one to a multimer (8–15 monomers), and the last one to larger aggregates or fibrils. The approach, in principle, allows detection of oligomers on the time scale of aggregation

Extrinsic Fluorescent Dyes as Tools for Protein Characterization

Noncovalent, extrinsic fluorescent dyes are applied in various fields of protein analysis, e.g. to characterize folding intermediates, measure surface hydrophobicity, and detect aggregation or fibrillation. The main underlying mechanisms, which explain the fluorescence properties of many extrinsic dyes, are solvent relaxation processes and (twisted) intramolecular charge transfer reactions, which are affected by the environment and by interactions of the dyes with proteins. In recent time, the use of extrinsic fluorescent dyes such as ANS, Bis-ANS, Nile Red, Thioflavin T and others has increased, because of their versatility, sensitivity and suitability for high-throughput screening. The intention of this review is to give an overview of available extrinsic dyes, explain their spectral properties, and show illustrative examples of their various applications in protein characterization

Production of recombinant mink growth hormone in E. coli

Escherichia coli cells expressing mink (Mustela vison) growth hormone were grown in a batch fermentation process. The expression level was estimated to be 27% of the total cellular protein after 3 h of induction with 1 mM isopropyl \u3b2-d-thiogalactoside (IPTG). If the expression of mink growth hormone (mGH) was induced with 0.2 mM IPTG, the concentration of target protein was slightly lower and was found to be 23% at the same time after induction. mGH expressed as inclusion bodies was solubilized in 8 M urea and renatured by dilution protocol at a protein concentration of 1.4-2.1 mg/ml in the presence of glutathione pair in a final concentration of 11.3 mM. [GSH]/[GSSG] ratio equal to 2/1 was used. Two-step purification process comprising of ion-exchange chromatography on Q-Sepharose and hydrophobic chromatography on Phenyl-Sepharose was developed. Some 25-30 mg of highly purified and biologically active mGH was obtained from 4 g of biomass. The method presented in this study allows producing large quantities of mGH and considering initiation of scientific investigation on mGH effect on mink in vivo and availability in fur industry

Origin and pathophysiology of protein carbonylation, nitration and chlorination in age-related brain diseases and aging

Non-enzymatic protein modifications occur inevitably in all living systems. Products of such modifications accumulate during aging of cells and organisms and may contribute to their age-related functional deterioration. This review presents the formation of irreversible protein modifications such as carbonylation, nitration and chlorination, modifications by 4-hydroxynonenal, removal of modified proteins and accumulation of these protein modifications during aging of humans and model organisms, and their enhanced accumulation in age-related brain diseases. © 2018, Gonos et al