Effect of bacteriophage-encoded chaperonins on amyloid transformation of α-synuclein.

Controversial information about the role of chaperonins in the amyloid transformation of proteins and, in particular, α-synuclein, requires a more detailed study of the observed effects due to the structure and functional state of various chaperonins. In this work, two types of phage chaperonins, the double-ring EL and the single-ring OBP, were shown to stimulate α-synuclein fibrillation in an ATP-dependent manner. Chaperonin morphology does not affect the stimulation of α-synuclein amyloid transformation. However, the ATP-dependent effect of single- and double-ring chaperonins on this process differs, which can lead to different morphology of resulting fibrils. Fibril formation seems to proceed without substrate encapsulation in the internal cavity of chaperonin, because of the structural features of phage chaperonins and their ability to function without co-chaperonins. In the absence of ATP, both chaperonins, on the contrary, completely prevent α-synuclein amyloid transformation, which provides the possibility of their use as anti-amyloid agents, in the form of incomplete molecules or mutants with suppressed ATPase activity.

Regulation by Different Types of Chaperones of Amyloid Transformation of Proteins Involved in the Development of Neurodegenerative Diseases.

The review highlights various aspects of the influence of chaperones on amyloid proteins associated with the development of neurodegenerative diseases and includes studies conducted in our laboratory. Different sections of the article are devoted to the role of chaperones in the pathological transformation of alpha-synuclein and the prion protein. Information about the interaction of the chaperonins GroE and TRiC as well as polymer-based artificial chaperones with amyloidogenic proteins is summarized. Particular attention is paid to the effect of blocking chaperones by misfolded and amyloidogenic proteins. It was noted that the accumulation of functionally inactive chaperones blocked by misfolded proteins might cause the formation of amyloid aggregates and prevent the disassembly of fibrillar structures. Moreover, the blocking of chaperones by various forms of amyloid proteins might lead to pathological changes in the vital activity of cells due to the impaired folding of newly synthesized proteins and their subsequent processing. The final section of the article discusses both the little data on the role of gut microbiota in the propagation of synucleinopathies and prion diseases and the possible involvement of the bacterial chaperone GroE in these processes.

Glycation of glyceraldehyde-3-phosphate dehydrogenase inhibits the binding with α-synuclein and RNA.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) shows great diversity of functions, interaction partners and post-translational modifications. GAPDH undergoes glycation of positively charged residues in diabetic patient's tissues and therefore may change interaction with partners. The influence of GAPDH glycation on interaction with two important partners, α-synuclein and RNA, has been investigated in silico using molecular dynamics simulations and in vitro using surface plasmon resonance measurements. Since positively charged groove including substrate- and NAD+-binding sites is proposed as potential binding site for α-synuclein and RNA, GAPDH was glycated on residues in grooves and randomly distributed over the whole surface. Lysine residues were replaced with negatively charged carboxymethyl lysine as a widespread advanced glycation end product. As results, GAPDH glycation suppressed the interaction with α-synuclein and RNA. Although the modified GAPDH residues participated in binding with α-synuclein, no stable binding site with both glycated forms was observed. Glycation along the whole GAPDH surface completely suppressed interaction with RNA, whereas the alternative possible RNA binding site was identified in case of groove glycation. The findings were supported by direct measurement of the binding affinity. The obtained results clarify effect of glycation on GAPDH interaction with α-synuclein and RNA and elucidate a possible mechanism of interplay between glycation occurred in diabetes and neurodegenerative diseases, which GAPDH and α-synuclein are involved in.

Natural and Synthetic Derivatives of Hydroxycinnamic Acid Modulating the Pathological Transformation of Amyloidogenic Proteins.

This review presents the main properties of hydroxycinnamic acid (HCA) derivatives and their potential application as agents for the prevention and treatment of neurodegenerative diseases. It is partially focused on the successful use of these compounds as inhibitors of amyloidogenic transformation of proteins. Firstly, the prerequisites for the emergence of interest in HCA derivatives, including natural compounds, are described. A separate section is devoted to synthesis and properties of HCA derivatives. Then, the results of molecular modeling of HCA derivatives with prion protein as well as with α-synuclein fibrils are summarized, followed by detailed analysis of the experiments on the effect of natural and synthetic HCA derivatives, as well as structurally similar phenylacetic and benzoic acid derivatives, on the pathological transformation of prion protein and α-synuclein. The ability of HCA derivatives to prevent amyloid transformation of some amyloidogenic proteins, and their presence not only in food products but also as natural metabolites in human blood and tissues, makes them promising for the prevention and treatment of neurodegenerative diseases of amyloid nature.

Alpha-Synuclein Amyloid Aggregation Is Inhibited by Sulfated Aromatic Polymers and Pyridinium Polycation.

The effect of a range of synthetic charged polymers on alpha-synuclein aggregation and amyloid formation was tested. Sulfated aromatic polymers, poly(styrene sulfonate) and poly(anethole sulfonate), have been found to suppress the fibril formation. In this case, small soluble complexes, which do not bind with thioflavin T, have been formed in contrast to the large stick-type fibrils of free alpha-synuclein. Sulfated polysaccharide (dextran sulfate), as well as sulfated vinylic polymer (poly(vinyl sulfate)) and polycarboxylate (poly(methacrylic acid)), enhanced amyloid aggregation. Conversely, pyridinium polycation, poly(N-ethylvinylpyridinium), switched the mechanism of alpha-synuclein aggregation from amyloidogenic to amorphous, which resulted in the formation of large amorphous aggregates that do not bind with thioflavin T. The obtained results are relevant as a model of charged macromolecules influence on amyloidosis development in humans. In addition, these results may be helpful in searching for new approaches for synucleinopathies treatment with the use of natural polymers.

Naturally occurring cinnamic acid derivatives prevent amyloid transformation of alpha-synuclein.

In search of the compounds that interfere with amyloid transformation of alpha-synuclein, 9 natural and synthetic cinnamic acid derivatives were studied. They are structurally similar to a half of curcumin, which has pronounced anti-aggregatory and anti-amyloid effects. We have shown that some of these derivatives prevent ovine prion protein amyloidization. Subsequently, thioflavin T binding assay showed that 3 out of 9 studied compounds effectively prevented amyloid transformation of alpha-synuclein with IC50 of 13, 50 and 251 µM. Molecular modeling approach revealed possible binding sites of the three selected ligands with alpha-synuclein fibrils, while monomeric alpha-synuclein does not bind to the ligands according to experimental results. This led us to believe that compounds may act by changing the structure of primary aggregates, preventing the formation of full-length fibrils. The inhibiting effect of the ligands on aggregation of alpha-synuclein was further confirmed by monitoring aggregation via turbidimetry, susceptibility to proteolytic cleavage, changes in beta-sheet content, and scanning ion-conductance microscopy. Studied derivatives were not cytotoxic, and, moreover, two studied compounds (ferulic and 3,4-dimethoxycinnamic acid) are found in plant sources and are natural metabolites present in human blood, so they can be promising candidate drugs for synucleinopathies, including Parkinson's disease.

Glycation of α-synuclein amplifies the binding with glyceraldehyde-3-phosphate dehydrogenase.

α-Synuclein was recently found to interact with moonlighting glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) involved in neurodegenerative diseases development. In the present work, we have analyzed influence of α-synuclein glycation on this interaction, because the literature data suggest relation between diabetes and Parkinson's disease. According to zeta potential measurement, glycation can shift the charge of α-synuclein to more negative values that was pronounced in case of modification by glyceraldehyde-3-phosphate. We selected carboxymethyl lysine as a typical advanced glycation end product and performed molecular dynamics simulations. The binding was found to be electrostatically driven and was significantly amplified after α-synuclein glycation because of increase the number of acidic residues. Since the main binding site was located in the anion-binding groove, which comprises the active site of GAPDH, enhanced binding of α-synuclein can result in GAPDH inactivation. This hypothesis was proven experimentally. Glycation of α-synuclein resulted in increase of GAPDH inactivation, and this effect was more pronounced in case of modification by glyceraldehyde-3-phosphate. The obtained results can reflect the probable relations between protein glycation and neurodegenerative diseases.

Modification by glyceraldehyde-3-phosphate prevents amyloid transformation of alpha-synuclein.

Numerous investigations point to the relation between diabetes and neurodegenerative disorders. Alpha-synuclein is a protein involved in the development of synucleinopathies including Parkinson's disease. In the present work, alpha-synuclein was for the first time modified by the intermediate product of glycolysis, glyceraldehyde-3-phosphate (GA-3-P). The resulting product was compared with the alpha-synuclein modified by methylglyoxal (MGO). The efficiency of the modification by the aldehydes was evaluated by decrease in free amino group content. The modification products were detected using fluorescence spectroscopy. The effect of modification by two glycating agents on the amyloid transformation of alpha-synuclein was investigated. Transmission electron microscopy analysis of the aggregates produced by the native alpha-synuclein under fibrillation conditions revealed the presence of 355-441-nm fibrils. In the aggregates produced by the modified alpha-synuclein, short fibrils of 65-230 nm or 85-260 nm were detected in the case of the protein treated with MGO and GA-3-P, respectively. Investigation of the aggregates by the fluorescence assay with Thioflavin T and CD spectroscopy showed that, in contrast to native alpha-synuclein, alpha-synuclein treated with GA-3-P does not produce real amyloid structures. Consequently, modification of alpha-synuclein by GA-3-P, the metabolite whose concentration is determined by the activity of glyceraldehyde-3-phosphate dehydrogenase, prevents its amyloid transformation.

Binding of alpha-synuclein to partially oxidized glyceraldehyde-3-phosphate dehydrogenase induces subsequent inactivation of the enzyme.

According to literature data, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) co-localizes with alpha-synuclein in Lewy bodies in Parkinson's disease, which suggests the involvement of this protein in the development of synucleinopathies. The goal of the present work was to investigate the direct interaction between alpha-synuclein and GAPDH and to evaluate possible influence of this interaction on the catalytic properties of GAPDH. Molecular dynamic simulations predicted the binding of alpha-synuclein to the positively charged groove comprising NAD+-binding pocket of GAPDH. The formation of the complex between alpha-synuclein and GAPDH in vitro was confirmed by different experimental approaches. The binding of alpha-synuclein to GAPDH with partially oxidized active site cysteines resulted in the subsequent inactivation of the enzyme, decreased its thermostability and increased its propensity for aggregation. At the same time, the formation of the complex between GAPDH and monomeric alpha-synuclein prevented amyloid transformation of alpha-synuclein. This work presents the first evidence for the fact that the initial oxidation of GAPDH induces the binding of alpha-synuclein to the enzyme, leading to further inactivation of GAPDH and, as a consequence, inhibition of glycolysis. The described mechanism may contribute to the metabolic disorders that are characteristic for synucleinopathies.

Glyceraldehyde-3-phosphate dehydrogenase: Aggregation mechanisms and impact on amyloid neurodegenerative diseases.

The review analyses data on specific features of aggregation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and possible role of this enzyme in the development of neurodegenerative diseases. Different post-translational modifications of the enzyme are considered: oxidation, nitrosylation, and S-glutathionylation of the active site sulfhydryl groups, as well as phosphorylation, glycation and homocysteinylation of other amino acid residues. Modification of the sulfhydryl groups of the enzyme inhibits the enzymatic activity of GAPDH, resulting in slowdown of glycolysis, and may lead to the dissociation of the cofactor NAD from the active site of the enzyme. The resulting apo-GAPDH (without NAD) is less stable and prone to dissociation, denaturation, and subsequent aggregation. These processes could play a crucial role in the translocation of GAPDH subunits from the cytoplasm into the nucleus, which is linked to the induction of apoptosis. Phosphorylation and glycation of GAPDH are presumably involved in the regulation of protein-protein interactions and intracellular localization of the enzyme. Besides, glycation by dicarbonyl compounds and aldehydes may directly inhibit glycolysis. Homocysteinylation of GAPDH may stabilize aggregates of the enzyme by additional disulfide bonding. All types of post-translational modifications affect aggregation of GAPDH. A special attention is given to the role of chaperones in the amyloidogenic transformation of proteins and to confirmation of the hypothesis on blocking of the chaperones by misfolded protein forms. The denatured GAPDH forms were shown to interact directly with amyloidogenic proteins (alpha-synuclein and amyloid-beta peptide) and to play a crucial role in blocking of chaperone system.

Sperm-specific glyceraldehyde-3-phosphate dehydrogenase is stabilized by additional proline residues and an interdomain salt bridge.

Sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) exhibits enhanced stability compared to the somatic isoenzyme (GAPD). A comparative analysis of the structures of these isoenzymes revealed characteristic features, which could be important for the stability of GAPDS: six specific proline residues and three buried salt bridges. To evaluate the impact of these structural elements into the stability of this isoenzyme, we obtained two series of mutant GAPDS: 1) six mutants each containing a substitution of one of the specific prolines by alanine, and 2) three mutants each containing a mutation breaking one of the salt bridges. Stability of the mutants was evaluated by differential scanning calorimetry and by their resistance towards guanidine hydrochloride (GdnHCl). The most effect on thermostability was observed for the mutants P326A and P164A: the Tm values of the heat-absorption curves decreased by 6.0 and 3.3°C compared to the wild type protein, respectively. The resistance towards GdnHCl was affected most by the mutation D311N breaking the salt bridge between the catalytic and NAD(+)-binding domains: the inactivation rate constant in the presence of GdnHCl increased six-fold, and the value of GdnHCl concentration corresponding to the protein half-denaturation decreased from 1.83 to 1.35M. Besides, the mutation D311N enhanced the enzymatic activity of the protein two-fold. The results suggest that the residues P164 (ß-turn), P326 (first position of α-helix), and the interdomain salt bridge D311-H124 are significant for the enhanced stability of GAPDS. The salt bridge D311-H124 enhances stability of the active site of GAPDS at the expense of the catalytic activity.