Role of Glycation in Amyloid: Effect on the Aggregation Process and Cytotoxicity

Although the aggregation process of amyloidogenic proteins has been widely studied in vitro and many physiological factors have been identified, the molecular mechanisms underlying the formation of aggregates in vivo and under pathological conditions are still poorly understood. Post‐translational modifications are known to affect protein structure and function. Some of these modifications might affect proteins in detrimental ways and lead to their misfolding and accumulation. Reducing sugars play an important role in modifying proteins, forming advanced glycation end‐products (AGEs) in a nonenzymatic process, called glycation. Recently, much attention has been devoted to the role played by glycation in stimulating amyloid aggregation and cellular toxicity. Proteins in amyloid deposits are often found glycated, suggesting a direct correlation between protein glycation and amyloidosis

Characterization of blood redox status of early and mid-late lactating dairy cows

The effect of the stage of lactation on blood redox homeostasis of bovine and buffalo cows was evaluated. The investigation was carried out on early lactating and mid-late lactating cows, reared in a farm located in Campania (southern Italy). Plasma concentration of α-tocopherol and ascorbate, the total antioxidant capacity (TAC), glutathione peroxidase (GPx), and superoxide dismutase activities were higher (P < 0.01) in mid-late lactating cows, thus suggesting a higher consumption of antioxidants during early lactation. Plasma concentration of protein-bound carbonyls (PC) and nitrotyrosine (N-Tyr), and the level of lipid hydroperoxides (LPO) were higher (P < 0.01) in early lactating cows, thus suggesting that lipid peroxidation and peroxynitrite production are crucial in determining oxidative modifications in plasma. TAC was positively correlated with ascorbate concentration (P < 0.03), and negatively correlated with PC concentration (P < 0.002), and ascorbate was negatively correlated with PC (P < 0.03) in mid-late lactating group. These findings demonstrate that circulating ascorbate plays a major role in preventing protein modifications induced by carbonyls, and that ascorbate scavenging effect is impaired during early lactation. We calculated a protein oxidative stress index as the ratio (PC + N-Tyr)/TAC multiplied by 100, and we found that this parameter was higher (P < 0.0001) in early lactating cows. Therefore, it could be useful for assessing the extent of protein oxidative damage in relation to the whole antioxidant status. Further, we suggest that the LPO/GPx ratio multiplied by 100 might be used as lipid oxidative stress index in lactating cows. This index was higher (P < 0.0001) in early lactating cows, and might represent a standard parameter for evaluating the lipid damage depending on a deficiency of the enzymatic antioxidant defence. These parameters are proposed for a possible effective description of physiological changes associated with lactation

Kinetics of amyloid aggregation of mammal apomyoglobins and correlation with their amino acid sequences

AbstractIn protein deposition disorders, a normally soluble protein is deposited as insoluble aggregates, referred to as amyloid. The intrinsic effects of specific mutations on the rates of protein aggregation and amyloid formation of unfolded polypeptide chains can be correlated with changes in hydrophobicity, propensity to convert α-helical to β sheet conformation and charge. In this paper, we report the aggregation rates of buffalo, horse and bovine apomyoglobins. The experimental values were compared with the theoretical ones evaluated considering the amino acid differences among the sequences. Our results show that the mutations which play critical roles in the rate-determining step of apomyoglobin aggregation are those located within the N-terminal region of the molecule

The Role of CyaY in Iron Sulfur Cluster Assembly on the E. coli IscU Scaffold Protein

Progress in understanding the mechanism underlying the enzymatic formation of iron-sulfur clusters is difficult since it involves a complex reaction and a multi-component system. By exploiting different spectroscopies, we characterize the effect on the enzymatic kinetics of cluster formation of CyaY, the bacterial ortholog of frataxin, on cluster formation on the scaffold protein IscU. Frataxin/CyaY is a highly conserved protein implicated in an incurable ataxia in humans. Previous studies had suggested a role of CyaY as an inhibitor of iron sulfur cluster formation. Similar studies on the eukaryotic proteins have however suggested for frataxin a role as an activator. Our studies independently confirm that CyaY slows down the reaction and shed new light onto the mechanism by which CyaY works. We observe that the presence of CyaY does not alter the relative ratio between [2Fe2S]2+ and [4Fe4S]2+ but directly affects enzymatic activity

The role of metal binding in the amyotrophic lateral sclerosis-related aggregation of copper-zinc superoxide dismutase

Protein misfolding and conformational changes are common hallmarks in many neurodegenerative diseases involving formation and deposition of toxic protein aggregates. Although many players are involved in the in vivo protein aggregation, physiological factors such as labile metal ions within the cellular environment are likely to play a key role. In this review, we elucidate the role of metal binding in the aggregation process of copper-zinc superoxide dismutase (SOD1) associated to amyotrophic lateral sclerosis (ALS). SOD1 is an extremely stable Cu-Zn metalloprotein in which metal binding is crucial for folding, enzymatic activity and maintenance of the native conformation. Indeed, demetalation in SOD1 is known to induce misfolding and aggregation in physiological conditions in vitro suggesting that metal binding could play a key role in the pathological aggregation of SOD1. In addition, this study includes recent advances on the role of aberrant metal coordination in promoting SOD1 aggregation, highlighting the influence of metal ion homeostasis in pathologic aggregation processes

Glycation in Demetalated Superoxide Dismutase 1 Prevents Amyloid Aggregation and Produces Cytotoxic Ages Adducts

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

Effector role reversal during evolution: the case of frataxin in Fe-S cluster biosynthesis

Human frataxin (FXN) has been intensively studied since the discovery that the FXN gene is associated with the neurodegenerative disease Friedreich's ataxia. Human FXN is a component of the NFS1-ISD11-ISCU2-FXN (SDUF) core Fe-S assembly complex and activates the cysteine desulfurase and Fe-S cluster biosynthesis reactions. In contrast, the Escherichia coli FXN homologue CyaY inhibits Fe-S cluster biosynthesis. To resolve this discrepancy, enzyme kinetic experiments were performed for the human and E. coli systems in which analogous cysteine desulfurase, Fe-S assembly scaffold, and frataxin components were interchanged. Surprisingly, our results reveal that activation or inhibition by the frataxin homologue is determined by which cysteine desulfurase is present and not by the identity of the frataxin homologue. These data are consistent with a model in which the frataxin-less Fe-S assembly complex exists as a mixture of functional and nonfunctional states, which are stabilized by binding of frataxin homologues. Intriguingly, this appears to be an unusual example in which modifications to an enzyme during evolution inverts or reverses the mode of control imparted by a regulatory molecule

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

Pinocembrin Protects from AGE-Induced Cytotoxicity and Inhibits Non-Enzymatic Glycation in Human Insulin

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 Aβ-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-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