Understanding the role of protein glycation in the amyloid aggregation process

Protein function and flexibility is directly related to the native distribution of its structural elements and any alteration in protein architecture leads to several abnormalities and accumulation of misfolded proteins. This phenomenon is associated with a range of increasingly common human disorders, including Alzheimer and Parkinson diseases, type II diabetes, and a number of systemic amyloidosis characterized by the accumulation of amyloid aggregates both in the extracellular space of tissues and as intracellular deposits. Post-translational modifications are known to have an active role in the in vivo amyloid aggregation as able to affect protein structure and dynamics. Among them, a key role seems to be played by non-enzymatic glycation, the most unwanted irreversible modification of the protein structure, which strongly affects long-living proteins throughout the body. This study provided an overview of the molecular effects induced by glycation on the amyloid aggregation process of several protein models associated with misfolding diseases. In particular, we analyzed the role of glycation on protein folding, kinetics of amyloid formation, and amyloid cytotoxicity in order to shed light on the role of this post-translational modification in the in vivo amyloid aggregation process

Hydroxytyrosol Prevents Doxorubicin-Induced Oxidative Stress and Apoptosis in Cardiomyocytes

Doxorubicin (Dox) is a highly effective chemotherapeutic agent employed in the handling of hematological and solid tumors. The effective use of Dox in cancer therapy has been seriously limited due to its well-known cardiotoxic side effects, mainly mediated by oxidative damage. Therefore, the identification of an effective and safe antagonist against Dox-induced cardiotoxicity remains a challenge. In this respect, as plant polyphenols have attracted considerable interest due to their antioxidant properties and good safety profile, hydroxytyrosol (HT), the major phenolic compound in olive oil, could be a potential candidate due to its remarkable antioxidant and anticancer powers. In this study, the effect of HT was tested on Dox-induced cardiotoxicity by using a combination of biochemical and cellular biology techniques. Interestingly, HT was able to counteract Dox-induced cytotoxicity in cardiomyocytes by acting on the SOD2 level and the oxidative response, as well as on apoptotic mechanisms mediated by Bcl-2/Bax. At the same time, HT did not to interfere with the antitumorigenic properties of Dox in osteosarcoma cells. This study identifies new, beneficial properties for HT and suggests that it might be a promising molecule for the development of additional therapeutic approaches aimed at preventing anthracycline-related cardiotoxicity and improving long-term outcomes in antineoplastic treatments

Vanillin prevents doxorubicin-induced apoptosis and oxidative stress in rat H9c2 cardiomyocytes

Doxorubicin (doxo) is an effective anticancer compound in several tumor types. However, as a consequence of oxidative stress induction and ROS overproduction, its high cardiotoxicity demands urgent attention. Vanillin possesses antioxidant, antiproliferative, antidepressant and anti-glycating properties. Therefore, we investigated the potential vanillin protective effects against doxo-induced cardiotoxicity in H9c2 cells. Using multiparametric approach, we demonstrated that vanillin restored both cell viability and damage in response to doxo exposure. Contextually, vanillin decreased sub-G1 appearance and caspase-3 and PARP1 activation, reducing the doxo-related apoptosis induction. From a mechanistic point of view, vanillin hindered doxo-induced ROS accumulation and impaired the ERK phosphorylation. Notably, besides the cardioprotective effects, vanillin did not counteract the doxo effectiveness in osteosarcoma cells. Taken together, our results suggest that vanillin ameliorates doxo-induced toxicity in H9c2 cells, opening new avenues for developing alternative therapeutic approaches to prevent the anthracycline-related cardiotoxicity and to improve the long-term outcome of antineoplastic treatment

W-F substitutions in apomyoglobin increase the local flexibility of the N-terminal region causing amyloid aggregation: A H/D exchange study.

Myoglobin is an α-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The simultaneous substitution of the two residues impairs the productive folding of the protein making the polypeptide chain highly prone to aggregate forming amyloid fibrils at physiological pH and room temperature. The role played by tryptophanyl residues in driving the productive folding process was investigated by providing structural details at low resolution of compact intermediate of three mutated apomyoglobins, i.e., W7F, W14F and the amyloid forming mutant W7FW14F. In particular, we followed the hydrogen/deuterium exchange rate of protein segments using proteolysis with pepsin followed by mass spectrometry analysis. The results revealed significant differences in the N-terminal region, consisting in an alteration of the physico-chemical properties of the 7-11 segment for W7F and in an increase of local flexibility of the 12-29 segment for W14F. In the double trypthophanyl substituted mutant, these effects are additive and impair the formation of native-like contacts and favour inter-chain interactions leading to protein aggregation and amyloid formation at physiological pH

Heparin Induces Harmless Fibril Formation in Amyloidogenic W7FW14F Apomyoglobin and Amyloid Aggregation in Wild-Type Protein In Vitro

Glycosaminoglycans (GAGs) are frequently associated with amyloid deposits in most amyloid diseases, and there is evidence to support their active role in amyloid fibril formation. The purpose of this study was to obtain structural insight into GAG-protein interactions and to better elucidate the molecular mechanism underlying the effect of GAGs on the amyloid aggregation process and on the related cytotoxicity. To this aim, using Fourier transform infrared and circular diochroism spectroscopy, electron microscopy and thioflavin fluorescence dye we examined the effect of heparin and other GAGs on the fibrillogenesis and cytotoxicity of aggregates formed by the amyloidogenic W7FW14 apomyoglobin mutant. Although this protein is unrelated to human disease, it is a suitable model for in vitro studies because it forms amyloid-like fibrils under physiological conditions of pH and temperature. Heparin strongly stimulated aggregation into amyloid fibrils, thereby abolishing the lag-phase normally detected following the kinetics of the process, and increasing the yield of fibrils. Moreover, the protein aggregates were harmless when assayed for cytotoxicity in vitro. Neutral or positive compounds did not affect the aggregation rate, and the early aggregates were highly cytotoxic. The surprising result that heparin induced amyloid fibril formation in wild-type apomyoglobin and in the partially folded intermediate state of the mutant, i.e., proteins that normally do not show any tendency to aggregate, suggested that the interaction of heparin with apomyoglobin is highly specific because of the presence, in protein turn regions, of consensus sequences consisting of alternating basic and non-basic residues that are capable of binding heparin molecules. Our data suggest that GAGs play a dual role in amyloidosis, namely, they promote beneficial fibril formation, but they also function as pathological chaperones by inducing amyloid aggregation

Inhibition of aggregate formation as therapeutic target in protein misfolding diseases: effect of tetracycline and trehalose.

Importance of the field: The identification of molecules that inhibit protein deposition or reverse fibril formation could open new strategies for therapeutic intervention in misfolding diseases. Numerous compounds have been shown to inhibit amyloid fibril formation in vitro. Among these compounds, tetracycline and the disaccharide trehalose have been reported to inhibit or reverse amyloid aggregation but their efficiency as potential drugs is controversial. Areas covered in this review: The results obtained using tetracycline and trehalose, reported in the last 15 years, are described and discussed. What the reader will gain: The conclusions have important implications for the development of therapeutic agents for protein deposition diseases. If fibrillar proteins contribute to cell degeneration, then the disassembly of fibrils may reverse or slow down disease progression; however, if the action of therapeutic agents produces intermediates of fibrillation and/or products of fibril disaggregation, then their accumulation could be harmful. Take home message: Care should be taken to ensure that strategies aimed at inhibiting fibril formation do not cause a corresponding increase in the concentration of toxic oligomeric species