Mohsen Nemat-Gorgani

Inhibition of amyloid fibrillation of lysozyme by indole derivatives ) possible mechanism of action

Amyloid aggregation of polypeptides is related to a growing number of pathologic states known as amyloid disorders. There is a great deal of interest in developing small molecule inhibitors of the amyloidogenic processes. In the present article, the inhibitory effects of some indole derivatives on amyloid fibrillation of hen egg white lysozyme (HEWL) are reported. Acidic pH and high temperatures were used to drive HEWL towards amyloid formation. A variety of techniques, ranging from thioflavin T fluorescence and Congo red absorbance assays to far‐UV CD and transmission electron microscopy, were employed to characterize the HEWL fibrillation process. Among the indole derivatives tested, indole 3‐acetic acid, indole 3‐carbinol and tryptophol had the most inhibitory effects on amyloid formation, indole and indole 3‐propionic acid gave some inhibition, and indole aldehyde and tryptophan showed no significant inhibition. Although indoles did not protect the HEWL native state from conformational changes, they were effective in diminishing HEWL amyloid fibril formation, delaying both the nucleation and elongation phases. Disaggregation of previously formed HEWL amyloid fibrils was also enhanced by indole 3‐acetic acid. Various medium conditions, such as the presence of different anions and alcoholic cosolvents, were explored to gain an insight into possible mechanisms. These observations, taken together, suggest that the indole ring is likely to play the main role in inhibition and that the side chain hydroxyl group may contribute positively, in contrast to the side chain carbonyl and intervening methylene groups.

Partial unfolding of carbonic anhydrase provides a method for its immobilization on hydrophobic adsorbents and protects it against irreversible thermoinactivation

Abstract Our previous studies indicated that native carbonic anhydrase does not interact with hydrophobic adsorbents and that it acquires this ability upon denaturation. It was concluded that hydrophobic sites become available and that catalytically active immobilized preparations may be finally obtained. In the present study, alkyl-substituted Sepharose 4B containing octyl, dodecyl or palmityl residues (Sepharose-lipid) were used for adsorptive immobilization of heat-denatured forms of this protein. The enzyme adsorbed on octyl-Sepharose showed the highest k cat / K m(app) and the greatest thermostability as compared to the other two preparations. Moreover, the pH stability and resistance to the effect of KSCN, as a chaotropic salt, was higher for this preparation of the immobilized enzyme. Thermal stability and aggregation studies suggested that the enzyme is protected against irreversible thermoinactivation by interaction with the alkyl residues upon adsorption. Proline prevented both aggregation of the free enzyme at elevated temperatures and its interaction with the hydrophobic support. The higher degree of substitution in octyl-Sepharose appears to provide it with a greater capacity for multipoint interaction. It is suggested that the type of studies presented may provide useful information in connection with formation of intermediate states, in addition to its importance related to protein immobilization by adsorption.

Chemical modification of lysine residues in Bacillus α-amylases : effect on activity and stability

Chemical modification of lysine residues in two bacterial α-amylases, a mesophilic enzyme from Bacillus amyloliquefaciens (BAA) and a thermophilic enzyme from Bacillus licheniformis (BLA) was carried out using citraconic anhydride. 13 ± 1 residues in BAA and 10 ± 1 residues in BLA were found modified under defined experimental conditions. Modification brought about dramatic enhancement of thermal stability of BAA and catalytic activity of BLA. Such alterations were found dependent on the temperature and pH. Results obtained on Tm, the extent of deamidation, changes in the circular dichroism (CD) spectra and kinetic parameters before and after modification are discussed in terms of their contributions to the mechanism of irreversible thermoinactivation and activity enhancement.

Effect of organic solvents on stability and activity of two related alcohol dehydrogenases: a comparative study

A comparative study was performed regarding the catalytic activity and stability of two related enzymes (thermophilic alcohol dehydrogenase from Thermoanaerobacter brockii and its mesophilic counterpart from yeast) in the presence of a number of miscible and immiscible organic solvents. The study was performed in view of the practical usefulness of organic solvents for alcohol dehydrogenases which have been shown to catalyse a variety of industrially-important dehydrogenation reactions. A number of organic solvents of different physicochemical characteristics were used and substantial stabilization was achieved. The non-polar solvents utilized showed the ability to enhance thermal stability of both proteins. Protection against thermal denaturation was especially pronounced by n-dodecane, the solvent with the highest logP used in the present study. Dimethylformamide and dioxane, employed as two miscible organic solvents, showed the ability to cause substrate inhibition and changes in protein conformation as indicated by kinetic and fluorescence studies. A higher resistance of the thermophilic protein to the deleterious effect of pyridine and thermostabilization of the mesophilic enzyme by non-polar solvents are especially emphasized. Combined differences in protein structure and nature of organic solvents are suggested to explain the differences in stability and catalytic activity observed in the present investigation.

Prediction of protein orientation upon immobilization on biological and nonbiological surfaces

We report on a rapid simulation method for predicting protein orientation on a surface based on electrostatic interactions. New methods for predicting protein immobilization are needed because of the increasing use of biosensors and protein microarrays, two technologies that use protein immobilization onto a solid support, and because the orientation of an immobilized protein is important for its function. The proposed simulation model is based on the premise that the protein interacts with the electric field generated by the surface, and this interaction defines the orientation of attachment. Results of this model are in agreement with experimental observations of immobilization of mitochondrial creatine kinase and type I hexokinase on biological membranes. The advantages of our method are that it can be applied to any protein with a known structure; it does not require modeling of the surface at atomic resolution and can be run relatively quickly on readily available computing resources. Finally, we also propose an orientation of membrane-bound cytochrome c, a protein for which the membrane orientation has not been unequivocally determined.

CHEMICAL MODIFICATION OF BACTERIAL ALPHA-AMYLASES: CHANGES IN TERTIARY STRUCTURES AND THE EFFECT OF ADDITIONAL CALCIUM

A comparative study was performed on the effect of calcium on native and chemically modified forms of mesophilic and thermophilic alpha-amylases. Circular dichroism (CD) and irreversible thermoinactivation studies were carried out in the absence and presence of 10 mM calcium. From the CD experiments, changes in the tertiary structure of these enzymes, brought about by modification, were concluded. Furthermore, these changes were found to be influenced by the presence of calcium. Sorbitol was very effective in affording protection against irreversible thermoinactivation of native and modified forms of the enzymes, both in the absence and presence of calcium. Results are discussed in terms of the usefulness of this new approach involving a combination of medium and chemical modification for protein stabilization and enhancement of catalytic potential.

Chemical modification of lysine residues in lysozyme may dramatically influence its amyloid fibrillation.

Studies on the aggregation of mutant proteins have provided new insights into the genetics of amyloid diseases and the role of the net charge of the protein on the rate, extent, and type of aggregate formation. In the present work, hen egg white lysozyme (HEWL) was employed as the model protein. Acetylation and (separately) citraconylation were employed to neutralize the charge on lysine residues. Acetylation of the lysine residues promoted amyloid formation, resulting in more pronounced fibrils and a dramatic decline in the nucleation time. In contrast, citraconylation produced the opposite effect. In both cases, native secondary and tertiary structures appeared to be retained. Studies on the effect of pH on aggregation suggested greater possibilities for amorphous aggregate formation rather than fibrillation at pH values closer to neutrality, in which the protein is known to take up a conformation more similar to its native form. This is in accord with reports in the literature suggesting that formation of amorphous aggregates is more favored under relatively more native conditions. pH 5 provided a critical environment in which a mixture of amorphous and fibrillar structures were observed. Use of Tango and Aggrescan software which describe aggregation tendencies of different parts of a protein structure suggested critical importance of some of the lysine residues in the aggregation process. Results are discussed in terms of the importance of the net charge in control of protein-protein interactions leading to aggregate formation and possible specific roles of lysine residues 96 and 97.

Membrane Integrity and Amyloid Cytotoxicity: A Model Study Involving Mitochondria and Lysozyme Fibrillation Products

Recent findings implicate that fibrillation products, the protein aggregates formed during the various steps leading to formation of mature fibrils, induce neurotoxicity predominantly in their intermediate oligomeric state. This has been shown to occur by increasing membrane permeability, eventually leading to cell death. Despite accumulating reports describing mechanisms of membrane permeabilization by oligomers in model membranes, studies directly targeted at characterizing the events occurring in biological membranes are rare. In the present report, we describe interaction of the original native structure, prefibrils and fibrils of hen egg white lysozyme (HEWL) with mitochondrial membranes, as an in vitro biological model, with the aim of gaining insight into possible mechanism of cytotoxicity at the membrane level. These structures were first characterized using a range of techniques, including fluorescence, size-exclusion chromatography, dynamic light scattering, transmission electron microscopy, dot blot analysis and circular dichroism. HEWL oligomers were found to be flexible/hydrophobic structures with the capacity to interact with mitochondrial membranes. Possible permeabilization of mitochondria was explored utilizing sensitive fluorometric and luminometric assays. Results presented demonstrate release of mitochondrial enzymes upon exposure to HEWL oligomers, but not native enzyme monomer or mature fibrils, in a concentration-dependent manner. Release of cytochrome c was also observed, as reported earlier, and membrane stabilization promoted by addition of calcium prevented release. Moreover, the oligomer-membrane interaction was influenced by high concentrations of NaCl and spermine. The observed release of proteins from mitochondria is suggested to occur by a nonspecific perturbation mechanism.

Large Proteins Have a Great Tendency to Aggregate but a Low Propensity to Form Amyloid Fibrils

The assembly of soluble proteins into ordered fibrillar aggregates with cross-β structure is an essential event of many human diseases. The polypeptides undergoing aggregation are generally small in size. To explore if the small size is a primary determinant for the formation of amyloids under pathological conditions we have created two databases of proteins, forming amyloid-related and non-amyloid deposits in human diseases, respectively. The size distributions of the two protein populations are well separated, with the systems forming non-amyloid deposits appearing significantly larger. We have then investigated the propensity of the 486-residue hexokinase-B from Saccharomyces cerevisiae (YHKB) to form amyloid-like fibrils in vitro. This size is intermediate between the size distributions of amyloid and non-amyloid forming proteins. Aggregation was induced under conditions known to be most effective for amyloid formation by normally globular proteins: (i) low pH with salts, (ii) pH 5.5 with trifluoroethanol. In both situations YHKB aggregated very rapidly into species with significant β-sheet structure, as detected using circular dichroism and X-ray diffraction, but a weak Thioflavin T and Congo red binding. Moreover, atomic force microscopy indicated a morphology distinct from typical amyloid fibrils. Both types of aggregates were cytotoxic to human neuroblastoma cells, as indicated by the MTT assay. This analysis indicates that large proteins have a high tendency to form toxic aggregates, but low propensity to form regular amyloid in vivo and that such a behavior is intrinsically determined by the size of the protein, as suggested by the in vitro analysis of our sample protein.

Sugars protect native and apo yeast alcohol dehydrogenase against irreversible thermoinactivation

Abstract In the present study, irreversible thermoinactivation of holo- and apo-yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) and protection by sugars (mannitol, sorbitol, sucrose and trehalose) were investigated at 50°C and pH 7.8. The apo-protein was obtained by removing the structural zinc with the catalytic zinc remaining on the enzyme. Thiol group oxidation, aggregation and deamidation were examined. Hypochlorous acid and cupric chloride were used in relation to thiol group oxidation. Zn 2+ mobilization was measured spectrophotometrically using the metallochromic indicator 4-(2-pyridylazo)resorcinol (PAR). The presence of sugars provided significant protection against all the three deleterious processes. It is concluded that use of sugars may provide an effective approach for stabilization of holo- and apo-YADH via alteration of protein microenvironment.