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Insulin is a key hormone regulating glucose homeostasis, intimately associated with glycemia and is exposed to glycation by glucose, reducing sugars and other highly reactive carbonyls, particularly in diabetes. Glycation of insulin has been reported to differentially affect protein structure, stability and aggregation depending on the glycating agent and experimental conditions. Under reducing conditions glycation produces higher insulin oligomerization thus accelerating amyloid formation whereas, in non-reducing conditions, glycation inhibits amyloid formation. To better detail the effect of glycation on insulin malfunction and toxicity, we investigated the effect of another glycating agent, the D -ribose. Recently, ribosylation has received great interest due to its role in protein glycation and its consequential effects such as protein aggregation, oxidative stress and cell death. Moreover, unusual high concentration of D -ribose has been detected in the urine of type II diabetics. Our results show that, using ribose, as glycating agent, the insulin conformation is preserved and does not evolve in amyloid aggregates because of the block of the α-helix to β-sheet transition, which initiates the aggregation process, maintaining the protein in a soluble state. At the same time, ribose-glycated insulin strongly affects the cell viability, starting a death pathway consisting in the activation of caspases 9 and 3/7, intracellular ROS production and activation of the transcription factor NF-kB.

Proteins and polypeptides containing a high proportion of β-sheets have been recently reported to exhibit, in their amyloid aggregated states, an intrinsic fluorescence in the blue-green range of wavelength where the aromatic residues do not emit. Lately, growing attention has been devoted to the identification of a specific, structure-related fluorophore for the detection of amyloid aggregates due to their implications in the physio-pathology of neurodegenerative diseases and other aggregation-related diseases. Indeed, the appearance of blue-green fluorescence could be used as an alternative method for the investigation and the detection of the aggregation state without using external probes. Several hypotheses have been suggested to explain the molecular bases of this rather unusual intrinsic emission. In particular, it has been related to an expansion of the electronic delocalization of π-electrons of peptide bonds through the backbone-to-backbone hydrogen bonds connecting the β-sheets. Alternatively, the formation of the intrinsic chromophore has been associated to chemical modifications of the aromatic residues or arising from dipolar coupling between excited states of aromatic amino acids densely packed in the fibril structures. More recently, it has been proposed that the blue-green amyloid fluorophore does not require neither the presence of aromatic residues nor multiple bond conjugation. In this study, we critically review the above hypotheses with particular attention to the electronic transitions responsible for the appearance of blue-green fluorescence in amyloid fibrils.

Amyloidosis is a protein folding disorder in which normally soluble proteins are deposited extracellularly as insoluble fibrils, impairing tissue structure and function. Charged polyelectrolytes such as glycosaminoglycans (GAGs) are frequently found associated with the proteinaceous deposits in tissues of patients affected by amyloid diseases. Experimental evidence indicate that they can play an active role in favoring amyloid fibril formation and stabilization. Binding of GAGs to amyloid fibrils occurs mainly through electrostatic interactions involving the negative polyelectrolyte charges and positively charged side chains residues of aggregating protein. Similarly to catalyst for reactions, GAGs favor aggregation, nucleation and amyloid fibril formation functioning as a structural templates for the self-assembly of highly cytotoxic oligomeric precursors, rich in β-sheets, into harmless amyloid fibrils. Moreover, the GAGs amyloid promoting activity can be facilitated through specific interactions via consensus binding sites between amyloid polypeptide and GAGs molecules. We review the effect of GAGs on amyloid deposition as well as proteins not strictly related to diseases. In addition, we consider the potential of the GAGs therapy in amyloidosis.

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.

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.

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.