Ivana Sirangelo

Fibrillogenesis and cytotoxic activity of the amyloid-forming apomyoglobin mutant W7FW14F

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 β-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.

Apomyoglobin folding intermediates characterized by the hydrophobic fluorescent probe 8-anilino-1-naphthalene sulfonate (ANS)

Folding apomyoglobin intermediates were investigated by optical techniques including steady-state fluorescence, frequency domain fluorometry, and absorption spectroscopy. The investigated chromophores were the aromatic residues, i.e., tyrosyl and tryptophanyl residues, and the extrinsic probe (8-anilino-1-naphthalenesulfonate, ANS) which is particularly useful for studying partly structured forms appearing in the early stage of protein folding. The emission decay of the extrinsic probe as well as resonance energy transfer from tryptophanyl residues to ANS permitted to identify and characterize partly folded forms obtained under different experimental conditions. The results indicate that the intermediates so far detected (I-1 and I-2 states) are distinct structural states. The differences concern the solvent accessibility to the aromatic side chains and the conformational dynamics of the protein region forming the binding site for the extrinsic fluorophore.

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.

The Effect of Glycosaminoglycans (GAGs) on Amyloid Aggregation and Toxicity

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.

Tetracycline inhibits W7FW14F apomyoglobin fibril extension and keeps the amyloid protein in a pre-fibrillar, highly cytotoxic state

A significant number of fatal diseases are classified as protein deposition disorders, in which a normally soluble protein is deposited in an insoluble amyloid form. It has been reported that tetracycline exhibits anti‐amyloidogenic activity by inhibiting aggregate formation and disaggregating preformed fibrils. In this work, we examined the effect induced by the presence of tetracycline on the fibrillogenesis and cytotoxicity of the amyloid‐forming apomyoglobin mutant W7FW14F. Like other amyloid‐forming proteins, early prefibrillar aggregates formed by this protein are highly cytotoxic, whereas insoluble mature fibrils are not. The effect induced by tetracycline on the fibrillation process has been examined by atomic force microscopy, light scattering, DPH staining, and thioflavin T fluorescence. The cytotoxicity of the amyloid aggregates was estimated by measuring cell viability using MTT assay. The results show that tetracycline acts as anti‐aggregating agent, which inhibits the fibril elongation process but not the early aggregation steps leading to the formation of soluble oligomeric aggregates. Thus, this inhibition keeps the W7FW14F mutant in a prefibrillar, highly cytotoxic state. In this respect, a careful usage of tetracycline as fibril inhibitor is indicated.

Heme binding inhibits the fibrillization of amyloidogenic apomyoglobin and determines lack of aggregate cytotoxicity

Myoglobin is an α‐helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N‐terminal region. The double W/F replacement renders apomyoglobin highly susceptible to aggregation and amyloid‐like fibril formation under physiological conditions. In this work we analyze the early stage of W7FW14F apomyoglobin aggregation following the time dependence of the process by far‐UV CD, Fourier‐transform infrared (FTIR) spectroscopy, and heme‐binding properties. The results show that the aggregation of W7FW14F apomyoglobin starts from a native‐like globin state able to bind the prosthetic group with spectroscopic properties similar to those observed for wild‐type apoprotein. Nevertheless, it rapidly aggregates, forming amyloid fibrils. However, when the prosthetic group is added before the beginning of aggregation, amyloid fibrillization is inhibited, although the aggregation process is not prevented. Moreover, the apomyoglobin aggregates formed in these conditions are not cytotoxic differently from what is observed for all amyloidogenic proteins. These results open new insights into the relationship between the structure adopted by the protein into the aggregates and their ability to trigger the impairment of cell viability.

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.

Glycation Accelerates Fibrillization of the Amyloidogenic W7FW14F Apomyoglobin

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.

Differential effects of glycation on protein aggregation and amyloid formation

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 Alzheimers 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 10 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.

Stachydrine ameliorates high‐glucose induced endothelial cell senescence and SIRT1 downregulation

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 p16INK4A 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.