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Hydroxytyrosol (HT), the major phenolic compound in olive oil, is attracting increasing interest for its beneficial properties including a notable antioxidant and anti-inflammatory power. In this study, using a combination of biophysical and cell biology techniques, we have tested the role of HT in the formation of advanced glycation end-products (AGEs). AGEs have a key role in clinical sciences as they have been associated to diabetes, neurodegenerative and cardiovascular diseases. In addition, as the incidence of Alzheimer’s disease (AD) is strongly increased in diabetic patients, AGE formation is supposed to be involved in the development of the pathological hallmarks of AD. Our data show that HT selectively inhibits protein glycation reaction in human insulin, and it is able to counteract the AGE-induced cytotoxicity in human neurotypical cells by acting on SIRT1 level and oxidative stress, as well as on inflammatory response. This study identifies new beneficial properties for HT and suggests it might be a promising molecule in protecting against the AGE-induced toxicity, a key mechanism underlying the development and progression of neurodegenerative disorders.

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.

Glycosaminoglycans are extracellular matrix components related to several biological functions and diseases. Chondroitin sulfate is a sulphated glycosaminoglycan synthesized as part of proteoglycan molecules. They are frequently associated with amyloid deposits and possess an active role in amyloid fibril formation. Recently, a neuroprotective effect of extracellular matrix components against amyloid toxicity and oxidative stress has been reported. Advanced glycation end products (AGEs), the end products of the glycation reaction, have been linked to amyloid-based neurodegenerative disease as associated with oxidative stress and inflammation. In this study we have analyzed the effect of chondroitin sulfate isolated from different species, in comparison with a new biotechnological unsulfated chondroitin, in the amyloid aggregation process of insulin, as well as the ability to prevent the formation of AGEs and related toxicity. The results have showed a determining role of chondroitin sulfate groups in modulating insulin amyloid aggregation. In addition, both sulfated and unsulfated chondroitins have shown protective properties against amyloid and AGEs-induced toxicity. These data are very relevant as a protective effect of these glycosaminoglycans in the AGE-induced toxicity was never observed before. Moreover, considering the issues related to the purity and safety of chondroitin from natural sources, this study suggests a new potential application for the biotechnological chondroitin.

PURPOSE: Excessive consumption of free sugar increases the risk for non-communicable diseases where a proper assessment of this intake is necessary to correctly estimate its association with certain diseases. Urinary sugars have been suggested as objective biomarkers for total and free sugar intake in adults but less is known about this marker in children and adolescents. Therefore, the aim of this exploratory study is to evaluate the relative validity of self-reported intake using urinary sugars in children and adolescents. METHODS: The study was conducted in a convenience subsample of 228 participants aged 5–18 years of the I.Family study that investigates the determinants of food choices, lifestyle and health in European families. Total, free and intrinsic sugar intake (g/day) and sugar density (g/1000 kcal) were assessed using 24-h dietary recalls (24HDRs). Urinary sucrose (USUC) and urinary fructose (UFRU) were measured in morning urine samples and corrected for creatinine excretion (USUC/Cr, UFRU/Cr). Correlation coefficients, the method of triads and linear regression models were used to investigate the relationship between intake of different types of sugar and urinary sugars. RESULTS: The correlation between usual sugar density calculated from multiple 24HDRs and the sum of USUC/Cr and UFRU/Cr (USUC/Cr + UFRU/Cr) was 0.38 (p

Advanced glycation end products (AGEs) are the end products of the glycation reaction and have a great importance in clinical science for their association with oxidative stress and inflammation, which play a major role in most chronic diseases, such as cardiovascular disease, neurodegenerative diseases, and diabetes. Their pathogenic effects are generally induced by the interaction between AGEs and the receptor for advanced glycation end product (RAGE) on the cell surface, which triggers reactive oxygen species production, nuclear factor kB (NF-kB) activation, and inflammation. Pinocembrin, the most abundant flavonoid in propolis, has been recently proven to interfere with RAGE activation in A&beta, &ndash, RAGE-induced toxicity. In the present study, we investigated the ability of pinocembrin to interfere with RAGE signaling pathways activated by AGEs. Interestingly, pinocembrin was able to inhibit oxidative stress and NF-kB activation in cells exposed to AGEs. In addition, it was able to block caspase 3/7 and 9 activation, thus suggesting an active role of this molecule in counteracting AGE&ndash, RAGE-induced toxicity mediated by NF-kB signaling pathways. The ability of pinocembrin to affect the glycation reaction has been also tested. Our data suggest that pinocembrin might be a promising molecule in protecting from AGE-mediated pathogenesis.

Protein glycation is a non-enzymatic, irreversible modification of protein amino groups by reactive carbonyl species leading to the formation of advanced glycation end products (AGEs). Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases related to protein misfolding and amyloid aggregation, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. We have recently shown that glycation of the amyloidogenic W7FW14F apomyoglobin mutant significantly accelerates the amyloid fibrils formation providing evidence that glycation actively participates to the process. In the present study, to test if glycation can be considered also a triggering factor in amyloidosis, we evaluated the ability of different glycation agents to induce amyloid aggregation in the soluble wild-type apomyoglobin. Our results show that glycation covalently modifies apomyoglobin and induces conformational changes that lead to the formation of oligomeric species that are not implicated in amyloid aggregation. Thus, AGEs formation does not trigger amyloid aggregation in the wild-type apomyoglobin but only induce the formation of soluble oligomeric species able to affect cell viability. The molecular bases of cell toxicity induced by AGEs formed upon glycation of wild-type apomyoglobin have been also investigated.

Human insulin is a widely used model protein for the study of amyloid formation as both associated to insulin injection amyloidosis in type II diabetes and highly prone to form amyloid fibrils in vitro. In this study, we aim to gain new structural insights into insulin fibril formation under two different aggregating conditions at neutral and acidic pH, using a combination of fluorescence, circular dichroism, Fourier-transform infrared spectroscopy, and transmission electron miscroscopy. We reveal that fibrils formed at neutral pH are morphologically different from those obtained at lower pH. Moreover, differences in FTIR spectra were also detected. In addition, only insulin fibrils formed at neutral pH showed the characteristic blue-green fluorescence generally associated to amyloid fibrils. So far, the molecular origin of this fluorescence phenomenon has not been clarified and different hypotheses have been proposed. In this respect, our data provide experimental evidence that allow identifying the molecular origin of such intrinsic property.

Curcumin is known for its anti-inflammatory, antioxidant and anticancer activity, as well as for its ability to interfere with amyloid aggregation and non-enzymatic glycation reaction, that makes it an attractive potential drug. However, curcumin therapeutic use is limited because of its low systemic bioavailability and chemical stability as it undergoes rapid hydrolysis in physiological conditions. Recently, much attention has been paid to the biological properties of curcumin degradation products as potential bioactive molecules. Between them, vanillin, a natural vanilla extract, is a stable degradation product of curcumin that could be responsible for mediating its beneficial effects. We have analyzed the effect of vanillin, in comparison with curcumin, in the amyloid aggregation process of insulin as well as its ability to prevent the formation of the advanced glycation end products (AGEs). Employing biophysical, biochemical and cell based assays, we show that vanillin and curcumin similarly affect insulin amyloid aggregation promoting the formation of harmless fibrils. Moreover, vanillin restrains AGE formation and protects from AGE-induced cytotoxicity. Our novel findings not only suggest that the main health benefits observed for curcumin can be ascribed to its degradation product vanillin, but also open new avenues for developing therapeutic applications of curcumin degradation products.

W7FW14F apomyoglobin (W7FW14F ApoMb) amyloid aggregates induce cytotoxicity in SH-SY5Y human neuroblastoma cells through a mechanism not fully elucidated. Amyloid neurotoxicity process involves calcium dyshomeostasis and reactive oxygen species (ROS) production. Another key mediator of the amyloid neurotoxicity is Platelet-Activating Factor (PAF), an inflammatory phospholipid implicated in neurodegenerative diseases. Here, with the aim at evaluating the possible involvement of PAF signaling in the W7FW14F ApoMb-induced cytotoxicity, we show that the presence of CV3899, a PAF receptor (PAF-R) antagonist, prevented the detrimental effect of W7FW14F ApoMb aggregates on SH-SY5Y cell viability. Noticeably, we found that the activation of PAF signaling, following treatment with W7FW14F ApoMb, involves a decreased expression of the PAF acetylhydroase II (PAF-AH II). Interestingly, the reduced PAF-AH II expression was associated with a decreased acetylhydrolase (AH) activity and to an increased sphingosine-transacetylase activity (TA(S)) with production of N-acetylsphingosine (C2-ceramide), a well known mediator of neuronal caspase-dependent apoptosis. These findings suggest that an altered PAF catabolism takes part to the molecular events leading to W7FW14F ApoMb amyloid aggregates-induced cell death.

Hyperglycaemia, a characteristic feature of diabetes mellitus, induces endothelial dysfunction and vascular complications by accelerating endothelial cell (EC) senescence and limiting the proliferative potential of these cells. Here we aimed to investigate the effect of stachydrine, a proline betaine present in considerable quantities in juices from fruits of the Citrus genus, on EC under high‐glucose stimulation, and its underlying mechanism. The senescence model of EC was set up by treating cells with high‐glucose (30mM) for different times. Dose‐dependent (0.001–1mM) evaluation of cell viability revealed that stachydrine does not affect cell proliferation with a similar trend up to 72h. Noticeable, stachydrine (0.1mM) significantly attenuated the high‐glucose induced EC growth arrest and senescence. Indeed, co‐treatment with high‐ glucose and stachydrine for 48h kept the percentage of EC in the G0/G1 cell cycle phase near to control values and significantly reduced cell senescence. Western blot analysis and confocal‐laser scanning microscopy revealed that stachydrine also blocked the high‐glucose induced upregulation of p16 INK4A and downregulation of SIRT1 expression and enzyme activity. Taken together, results here presented are the first evidence that stachydrine, a naturally occurring compound abundant in citrus fruit juices, inhibits the deleterious effect of high‐glucose on EC and acts through the modulation of SIRT1 pathway. These results may open new prospective in the identification of stachydrine as an important component of healthier eating patterns in prevention of cardiovascular diseases. J. Cell. Biochem. 114: 2522–2530, 2013.

Apomyoglobin is an excellent example of a monomeric all α-helical globular protein whose folding pathway has been extensively studied and well characterized. Structural perturbation induced by denaturants or high temperature as well as amino acid substitution have been described to induce misfolding and, in some cases, aggregation. In this article, we review the molecular mechanism of the aggregation process through which a misfolded form of a mutated apomyoglobin aggregates at physiological pH and room temperature forming an amyloid fibril. The results are compared with data showing that either amyloid or aggregate formation occurs under particular denaturing conditions or upon cleavage of the residues corresponding to the C-terminal helix of apomyoglobin. The results are discussed in terms of the sequence regions that are more important than others in determining the amyloid aggregation process.

A range of debilitating human diseases is known to be associated with the formation of stable highly organized protein aggregates known as amyloid fibrils. The early prefibrillar aggregates behave as cytotoxic agents and their toxicity appears to result from an intrinsic ability to impair fundamental cellular processes by interacting with cellular membranes, causing oxidative stress and increase in free Ca2+ that lead to apoptotic or necrotic cell death. However, specific signaling pathways that underlie amyloid pathogenicity remain still unclear. This work aimed to clarify cell impairment induced by amyloid aggregated. To this end, we used a combined proteomic and one-dimensional 1H-NMR approach on NIH-3T3 cells exposed to prefibrillar aggregates from the amyloidogenic apomyoglobin mutant W7FW14F. The results indicated that cell exposure to prefibrillar aggregates induces changes of the expression level of proteins and metabolites involved in stress response. The majority of the proteins and metabolites detected are reported to be related to oxidative stress, perturbation of calcium homeostasis, apoptotic and survival pathways, and membrane damage. In conclusion, the combined proteomic and 1H-NMR metabonomic approach, described in this study, contributes to unveil novel proteins and metabolites that could take part to the general framework of the toxicity induced by amyloid aggregates. These findings offer new insights in therapeutic and diagnostic opportunities. J. Cell. Physiol. 228: 1359–1367, 2013. © 2012 Wiley Periodicals, Inc.

Superoxide dismutase 1 (SOD1) has been implicated with familial amyotrophic lateral sclerosis (fALS) through accumulation of protein amyloid aggregates in motor neurons of patients. Amyloid aggregates and protein inclusions are a common pathological feature of many neurological disorders in which protein aggregation seems to be directly related to neurotoxicity. Although, extensive studies performed on the aggregation process of several amyloidogenic proteins in vitro allowed the identification of many physiological factors involved, the molecular mechanisms underlying the formation of amyloid aggregates in vivo and in pathological conditions are still poorly understood. Post-translational modifications are known to affect protein structure and function and, recently, much attention has been devoted to the role played by non-enzymatic glycation in stimulating amyloid aggregation and cellular toxicity. In particular, glycation seems to have a determining role both in sporadic and familial forms of ALS and SOD1 has been shown to be glycated in vivo The aim of this study was to investigate the role of glycation on the amyloid aggregation process of both wild-type SOD1 and its ALS-related mutant G93A. To this aim, the glycation kinetics of both native and demetalated SOD have been followed using two different glycating agents, i.e., D-ribose and methylglyoxal. The effect of glycation on the structure and the amyloid aggregation propensity of native and ApoSOD has been also investigated using a combination of biophysical and biochemical techniques. In addition, the effect of SOD glycated species on cellular toxicity and reactive oxygen species (ROS) production has been evaluated in different cellular models. The results provided by this study contribute to clarify the role of glycation in amyloid aggregation and suggest a direct implication of glycation in the pathology of fALS.

""Myoglobin is an alpha-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 increases the susceptibility of the polypeptide chain to misfold, causing amyloid aggregation under physiological condition, i.e., neutral pH and room temperature. The role played by tryptophanyl residues in driving the folding process has been investigated by examining three mutated apomyoglobins, i.e., W7F, W14F, and the amyloid-forming mutant W7FW14F, by an integrated approach based on far-ultraviolet (UV) circular dichroism (CD) analysis, fluorescence spectroscopy, and complementary proteolysis. Particular attention has been devoted to examine the conformational and dynamic properties of the equilibrium intermediate formed at pH 4.0, since it represents the early organized structure from which the native fold originates. The results show that the W -> F substitutions at position 7 and 14 differently affect the structural organization of the AGH subdomain of apomyoglobin. The combined effect of the two substitutions in the double mutant impairs the formation of native-like contacts and favors interchain interactions, leading to protein aggregation and amyloid formation.""

Resonance energy transfer between tryptophanyl residues and the apolar fluorescent dye 1-anilino-8-naphthalene sulfonate (ANS) occurs when the fluorophore is bound to native folded sperm whale apomyoglobin. The individual transfer contribution of the two tryptophanyl residues (W7 and W14, both located on the A-helix of the protein) was resolved by measuring the tryptophan–ANS transfer efficiency for the ANS–apomyoglobin complexes formed by wild-type protein and protein mutants containing one or no tryptophanyl residues, i.e. W7F, W14F and W7YW14F. The transfer efficiency of W14 residue was found to be higher than that of W7, thus indicating that W14 acts as the main energy donor in the ANS–apomyoglobin complex. This suggests that the plane containing the anilinonaphthalene ring of the extrinsic fluorophore has a spatial orientation similar to that of W14 and, hence, to the heme group in the holoprotein.

Protein glycation is a non-enzymatic, irreversible modification of protein amino groups by reactive carbonyl species leading to the formation of advanced glycation end products (AGEs). Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases related to protein misfolding and amyloid aggregation, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. We have recently shown that glycation of the amyloidogenic W7FW14F apomyoglobin mutant significantly accelerates the amyloid fibrils formation providing evidence that glycation actively participates to the process. In the present study, to test if glycation can be considered also a triggering factor in amyloidosis, we evaluated the ability of different glycation agents to induce amyloid aggregation in the soluble wild-type apomyoglobin. Our results show that glycation covalently modifies apomyoglobin and induces conformational changes that lead to the formation of oligomeric species that are not implicated in amyloid aggregation. Thus, AGEs formation does not trigger amyloid aggregation in the wild-type apomyoglobin but only induce the formation of soluble oligomeric species able to affect cell viability. The molecular bases of cell toxicity induced by AGEs formed upon glycation of wild-type apomyoglobin have been also investigated.

The apomyoglobin mutant W7FW14F forms amyloid-like fibrils at physiological pH. We examined the kinetics of fibrillogenesis using three techniques: the time dependence of the fluorescence emission of thioflavin T and 1-anilino-8-naphthalenesulfonate, circular dichroism measurements, and electron microscopy. We found that in the early stage of fibril formation, non-native apomyoglobin molecules containing beta-structure elements aggregate to form a nucleus. Subsequently, more molecules aggregate around the nucleus, thereby resulting in fibril elongation. We evaluated by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) the cytotoxicity of these aggregates at the early stage of fibril elongation versus mature fibrils and the wild-type protein. Similar to other amyloid-forming proteins, cell toxicity was not due to insoluble mature fibrils but rather to early pre-fibrillar aggregates. Propidium iodide uptake showed that cell toxicity is the result of altered membrane permeability. Phalloidin staining showed that membrane damage is not associated to an altered cell shape caused by changes in the cytoskeleton.

The solvent accessibilities to the tryptophanyl microenvironments of wild type sperm whale apomyoglobin (apoMb) and two mutants (W7F and W14F) containing a single tryptophan are measured by fluorescence quenching studies. The results are compared to those relative to horse apoMb. In the wild type sperm whale protein, no difference is noticed in the solvent accessibility of the two indole residues, as documented by the values of the Stern-Volmer constants. By contrast, the two tryptophan residues of horse apoMb are exposed to the solvent in a different way, thus indicating that some local conformational differences exist between the two homologous proteins in solution. The single W 3 F substitution at either position 7 or 14 determines local conformational changes that increase the accessibility of the remaining indole residue but do not affect the overall architecture of the protein molecule. © 2003 Wiley Periodicals, Inc. Biopolymers (Biospectros- copy) 70: 649 - 654, 2003

Myoglobin is an alpha-helical globular protein that contains two highly conserved tryptophan residues located at positions 7 and 14 in the N-terminal region of the protein. Replacement of both indole residues with phenylalanine residues, i.e. W7F/W14F, results in the expression of an unstable, not correctly folded protein that does not bind the prosthetic group. Here we report data (Congo red and thioflavine T binding assay, birefringence, and electron microscopy) showing that the double Trp/Phe replacements render apomyoglobin molecules highly susceptible to aggregation and amyloid-like fibril formation under physiological conditions in which most of the wild-type protein is in the native state. In refolding experiments, like the wild-type protein, the W7F/W14F apomyoglobin mutant formed a soluble, partially folded helical state between pH 2.0 and pH 4.0. A pH increase from 4.0 to 7.0 restored the native structure only in the case of the wild-type protein and determined aggregation of W7F/W14F. The circular dichroism spectrum recorded immediately after neutralization showed that the polypeptide consists mainly of beta-structures. In conclusion, under physiological pH conditions, some mutations that affect folding may cause protein aggregation and the formation of amyloid-like fibrils.

W7FW14F apomyoglobin (W7FW14F ApoMb) amyloid aggregates induce cytotoxicity in SH-SY5Y human neuroblastoma cells through a mechanism not fully elucidated. Amyloid neurotoxicity process involves calcium dyshomeostasis and reactive oxygen species (ROS) production. Another key mediator of the amyloid neurotoxicity is Platelet-Activating Factor (PAF), an inflammatory phospholipid implicated in neurodegenerative diseases. Here, with the aim at evaluating the possible involvement of PAF signaling in the W7FW14F ApoMb-induced cytotoxicity, we show that the presence of CV3899, a PAF receptor (PAF-R) antagonist, prevented the detrimental effect of W7FW14F ApoMb aggregates on SH-SY5Y cell viability. Noticeably, we found that the activation of PAF signaling, following treatment with W7FW14F ApoMb, involves a decreased expression of the PAF acetylhydroase II (PAF-AH II). Interestingly, the reduced PAF-AH II expression was associated with a decreased acetylhydrolase (AH) activity and to an increased sphingosine-transacetylase activity (TA(S)) with production of N-acetylsphingosine (C2-ceramide), a well known mediator of neuronal caspase-dependent apoptosis. These findings suggest that an altered PAF catabolism takes part to the molecular events leading to W7FW14F ApoMb amyloid aggregates-induced cell death.

Amyloids are a class of insoluble proteinaceous substances generally composed of linear un-branched fibrils that are formed from misfolded proteins. Conformational diseases such as Alzheimer’s disease, transmissible spongiform encephalopathies, and familial amyloidosis are associated with the presence of amyloid aggregates in the affected tissues. The majority of the cases are sporadic, suggesting that several factors must contribute to the onset and progression of these disorders. Among them, in the past ten years, non enzymatic glycation of proteins has been reported to stimulate protein aggregation and amyloid deposition. In this review, we analyze the most recent advances in this field suggesting that the effects induced by glycation may not be generalized as strongly depending on the protein structure. Indeed, being a post-translational modification, glycation could differentially affects the aggregation process in promoting, accelerating and/or stabilizing on-pathway and off-pathway species.

Mammalian myoglobins contain two tryptophanyl residues at the invariant positions 7 (A-5) and 14 (A-12) in the N-terminal region (A helix) of the protein molecule. The crucial role of tryptophanyl residues has been investigated by site-directed mutagenesis and molecular dynamics simulation. The apomyoglobin mutants with a double W!F substitution were found to be not correctly folded and therefore not expressed as holoprotein. The introduction of a tyrosyl residue at position 7, that is, W7YW14F, resulted in the expression of a correctly folded myoglobin. Not correctly folded apomyoglobins were found with the following mutants: W7FW14Y, W7EW14F, W7FW14E, W7KW14F, W7FW14K. Moreover, in all these cases, very low levels of expression were observed. The acid-induced denaturation curves of wild-type and folded mutant W7YW14F, obtained following the fluorescence variation of the extrinsic fluorophore 1-anilino-8-naphthalenesulfonate, revealed that the stability of the native state of mutant apoprotein is decreased, thus indicating that the replacement W!Y in position 7 is able to restore a correct folding but not the same stability. Molecular dynamics simulation indicated that both tryptophans are involved in forming favorable, specific tertiary interactions in the native apomyoglobin structure. The lack of some of these interactions caused by tryptophanyl replacement affects the overall protein structure and may provide an explanation for the observed stability decrease. In the case of the double W!F substitution, the simulated structure shows conclusively the domain formed by helices A, G and H to be not correctly folded. This effect is attenuated if at least one of the two residues is conserved or a tyrosyl residue replaces W7.

Amyloidosis is the accumulation of insoluble proteinaceous aggregates in vivo and is implicated in many neurodegenerative diseases, including Alzheimer's, Huntington's, and Parkinson's diseases. This article briefly reviews the current knowledge of amyloid aggregate toxicity and inflammatory signaling in the nervous system. In particular, we focus our attention on the role of platelet-activating factor (PAF) as mediator of amyloid cytotoxicity.

Hyperglycaemia, a characteristic feature of diabetes mellitus, induces endothelial dysfunction and vascular complications by accelerating endothelial cell (EC) senescence and limiting the proliferative potential of these cells. Here we aimed to investigate the effect of stachydrine, a proline betaine present in considerable quantities in juices from fruits of the Citrus genus, on EC under high-glucose stimulation, and its underlying mechanism. The senescence model of EC was set up by treating cells with high-glucose (30 mM) for different times. Dose-dependent (0.001-1 mM) evaluation of cell viability revealed that stachydrine does not affect cell proliferation with a similar trend up to 72 h. Noticeable, stachydrine (0.1 mM) significantly attenuated the high-glucose induced EC growth arrest and senescence. Indeed, co-treatment with high-glucose and stachydrine for 48 h kept the percentage of EC in the G0 /G1 cell cycle phase near to control values and significantly reduced cell senescence. Western blot analysis and confocal-laser scanning microscopy revealed that stachydrine also blocked the high-glucose induced upregulation of p16(INK4A) and downregulation of SIRT1 expression and enzyme activity. Taken together, results here presented are the first evidence that stachydrine, a naturally occurring compound abundant in citrus fruit juices, inhibits the deleterious effect of high-glucose on EC and acts through the modulation of SIRT1 pathway. These results may open new prospective in the identification of stachydrine as an important component of healthier eating patterns in prevention of cardiovascular diseases.

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 the12-29 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.

Neurodegenerative diseases are associated with misfolding and deposition of specific proteins, either intra or extracellularly in the nervous system. Advanced glycation end products (AGEs) originate from different molecular species that become glycated after exposure to sugars. Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. To this concern, in the present study we have investigated the effect of glycation on the aggregation pathway of the amyloidogenic W7FW14F apomyoglobin. Although this protein has not been related to any amyloid disease, it represents a good model to resemble proteins that intrinsically evolve toward the formation of amyloid aggregates in physiological conditions. We show that D-ribose, but not D-glucose, rapidly induces the W7FW14F apomyoglobin to generate AGEs in a time-dependent manner and protein ribosylation is likely to involve lysine residues on the polypeptide chain. Ribosylation of the W7FW14F apomyoglobin strongly affects its aggregation kinetics producing amyloid fibrils within few days. Cytotoxicity of the glycated aggregates has also been tested using a cell viability assay. We propose that ribosylation in the W7FW14F apomyoglobin induces the formation of a cross-link that strongly reduces the flexibility of the H helix and/or induce a conformational change that favor fibril formation. These results open new perspectives for AGEs biological role as they can be considered not only a triggering factor in amyloidosis but also a player in later stages of the aggregation process.

The pressure dependence of the flexibility of the 8-anilino-1-naphthalene sulfonate (ANS)-apomyoglobin complex was investigated in the range between atmospheric pressure and 2.4 kbar by frequency domain fluorometry. We examined two structural states: native and acidic compact. The conformational dynamics of the ANS-apomyoglobin complex were deduced by studying the emission decay of ANS, which can form a noncovalent complex with the apoprotein in both the native and the acidic compact forms. Because the free fluorophore has a very short lifetime (less than 75 ps), its contribution can be separated from the long-lived emission. The latter arises from ANS molecules bound to the protein and provides information on the structural and dynamic characteristics of the macromolecule. The fluorescence emission decay of the ANS-apomyoglobin complex at neutral pH has a broad fluorescence lifetime distribution (width at half-maximum = 4.1 ns). The small changes in the fluorescence distribution parameters that occur with changes in pressure indicate that the ANS-apomyoglobin complex at neutral pH holds its compactness even at 2.4 kbar. A small contraction of molecular volume has been detected at low pressure, followed by a slight swelling with an increase in flexibility at higher pressures. The heterogeneity of ANS fluorescence in the acidic compact state of apomyoglobin is even greater than that in the native form (distribution width = 10 ns); moreover, the acidic compact state appears more expanded and accessible to solvent molecules than the native state, as suggested by the distribution center, which is 11 ns for the former and 19 ns for the latter. The lifetime distribution center remains constant with increasing pressure, which suggests that no other binding site is formed at high pressure.

Initiation factor eIF2 binds GTP and promotes the binding of methionyl-tRNA to ribosomes. Biochemical and sequence evidence suggests that the GTP might bind to either the beta- or gamma-subunit of eIF2. Mutations were made in the NKXD consensus elements found in both subunits and individual mutant forms were overexpressed in transiently transfected COS-1 cells. The effect on the translational efficiency of a reporter mRNA for dihydrofolate reductase was monitored. Mutations in the gamma-subunit cause severe repression of protein synthesis, whereas those in the beta-subunit are only mildly inhibitory. The results support the view that GTP binds exclusively to the gamma-subunit.

Prion proteins are known as the main agents of transmissible spongiform encephalopathies affecting humans as well as animals. A recombinant ovine prion protein was found to be in vitro able to act as an effective substrate for a microbial isoform of transglutaminase, an enzyme catalyzing the formation of isopeptide bonds inside the proteins. We proved that transglutaminase modifies the structure of the prion protein by leading to the formation of three intra-molecular crosslinks and that the crosslinked protein form is more competent in amyloid formation compared to the unmodified one. In addition, the crosslinked prion protein was shown also to be more resistant to proteinase K digestion. Our findings suggest a possible use of transglutaminase in stabilizing the prion protein three-dimensional structure in order to investigate the molecular basis of the conversion of the protein into its pathological form.