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.     

Conformational properties of the aggregation precursor state of HypF-N

The conversion of specific proteins or protein fragments into insoluble, ordered fibrillar aggregates is a fundamental process in protein chemistry, biology, medicine and biotechnology. As this structural conversion seems to be a property shared by many proteins, understanding the mechanism of this process will be of extreme importance. Here we present a structural characterisation of a conformational state populated at low pH by the N-terminal domain of Escherichia coli HypF. Combining different biophysical and biochemical techniques, including near- and far-UV circular dichroism, intrinsic and 8-anilinonaphthalene-1-sulfonate-derived fluorescence, dynamic light scattering and limited proteolysis, we will show that this state is largely unfolded but contains significant secondary structure and hydrophobic clusters. It also appears to be more compact than a random coil-like state but less organised than a molten globule state. Increase of the total ionic strength of the solution induces aggregation of such a pre-molten globule state into amyloid-like protofibrils, as revealed by thioflavin T fluorescence and atomic force microscopy. These results show that a pre-molten globule state can be, among other possible conformational states, one of the precursor states of amyloid formation. In addition, the possibility of triggering aggregation by modulating the ionic strength of the solution provides one a unique opportunity to study both the initial precursor state and the aggregation process.     

The mechanism of inactivation of glucose oxidase from Penicillium amagasakiense under ambient storage conditions

Glucose oxidase (GOx) from Penicillium amagasakiense has a higher specific activity than the more commonly studied Aspergillus niger enzyme, and may therefore be preferred in many medical and industrial applications. The enzyme rapidly inactivates on storage at pH 7.0-7.6 at temperatures between 30 and 40 °C. Results of fluorimetry and circular dichroism spectroscopy indicate that GOx inactivation under these conditions is associated with release of the cofactor FAD and molten globule formation, indicated by major loss of tertiary structure but almost complete retention of secondary structure. Inactivation of GOx at pH < 7 leads to precipitation, but at pH ≥ 7 it leads to non-specific formation of small soluble aggregates detectable by PAGE and size-exclusion chromatography (SEC). Inactivation of P. amagasakiense GOx differs from that of A. niger GOx in displaying complete rather than partial retention of secondary structure and in being promoted rather than prevented by NaCl. The contrasting salt effects may reflect differences in the nature of the interface between subunits in the native dimers and/or the quantity of secondary structure loss upon inactivation.     

Hydrogen peroxide targets the cysteine at the active site and irreversibly inactivates creatine kinase

In our study, we showed that at a relatively low concentration, H₂O₂ can irreversibly inactivate the human brain type of creatine kinase (HBCK) and that HBCK is inactivated in an H₂O₂ concentration-dependent manner. HBCK is completely inactivated when incubated with 2 mM H₂O₂ for 1 h (pH 8.0, 25 °C). Inactivation of HBCK is a two-stage process with a fast stage (k₁ = 0.050 ± 0.002 min⁻¹) and a slow (k₂ = 0.022 ± 0.003 min⁻¹) stage. HBCK inactivation by H₂O₂ was affected by pH and therefore we determined the pH profile of HBCK inactivation by H₂O₂. H₂O₂-induced inactivation could not be recovered by reducing agents such as dl-dithiothreitol, N-acetyl-l-cysteine, and l-glutathione reduced. When HBCK was treated with DTNB, an enzyme substrate that reacts specifically with active site cysteines, the enzyme became resistant to H₂O₂. HBCK binding to Mg²⁺ATP and creatine can also prevent H₂O₂ inactivation. Intrinsic and 1-anilinonaphthalene-8-sulfonate-binding fluorescence data showed no tertiary structure changes after H₂O₂ treatment. The thiol group content of H₂O₂-treated HBCK was reduced by 13% (approximately 1 thiol group per HBCK dimer, theoretically). For further insight, we performed a simulation of HBCK and H₂O₂ docking that suggested the CYS283 residue could interact with H₂O₂. Considering these results and the asymmetrical structure of HBCK, we propose that H₂O₂ specifically targets the active site cysteine of HBCK to inactivate HBCK, but that substrate-bound HBCK is resistant to H₂O₂. Our findings suggest the existence of a previously unknown negative form of regulation of HBCK via reactive oxygen species.     

Expression and characterization of alpha-(1,4)-glucan branching enzyme Rv1326c of Mycobacterium tuberculosis H37Rv

Glycogen branching enzyme (GlgB, EC 2.4.1.18) catalyzes the third step of glycogen biosynthesis by the cleavage of an alpha-(1,4)-glucosidic linkage and subsequent transfer of cleaved oligosaccharide to form a new alpha-(1,6)-branch. A single glgB gene Rv1326c is present in Mycobacterium tuberculosis. The predicted amino acid sequence of GlgB of M. tuberculosis has all the conserved regions of alpha-amylase family proteins. The overall amino acid identity to other GlgBs ranges from 48.5 to 99%. The glgB gene of M. tuberculosis was cloned and expressed in Escherichia coli. The recombinant protein was purified to homogeneity using metal affinity and ion exchange chromatography. The recombinant protein is a monomer as evidenced by gel filtration chromatography, is active as an enzyme, and uses amylose as the substrate. Enzyme activity was optimal at pH 7.0, 30 degrees C and divalent cations such as Zn2+ and Cu2+ inhibited activity. CD spectroscopy, proteolytic cleavage and mass spectroscopy analyses revealed that cysteine residues of GlgB form structural disulfide bond(s), which allow the protein to exist in two different redox-dependent conformational states. These conformations have different surface hydrophobicities as evidenced by ANS-fluorescence of oxidized and reduced GlgB. Although the conformational change did not affect the branching enzyme activity, the change in surface hydrophobicity could influence the interaction or dissociation of different cellular proteins with GlgB in response to different physiological states.     

Inhibition of transthyretin amyloid fibril formation by 2,4-dinitrophenol through tetramer stabilization

Transthyretin (TTR), a homotetrameric thyroxine transport protein found in the plasma and cerebrospinal fluid, circulates normally as a innocuous soluble protein. In some individuals, TTR polymerizes to form insoluble amyloid fibrils. TTR amyloid fibril formation and deposition have been associated with several diseases like familial amyloid polyneuropathy and senile systemic amyloidosis. Inhibition of the fibril formation is considered a potential strategy for the therapeutic intervention. The effect of small water-soluble, hydrophobic ligand 2,4-dinitrophenol (2,4-DNP) on TTR amyloid formation has been tested. 2,4-DNP binds to TTR both at acidic and physiological pH, as shown by the quenching of TTR intrinsic fluorescence. Interestingly, 2,4-DNP not only binds to TTR at acidic pH but also inhibits amyloid fibril formation as shown by the light scattering and Congo red-binding assay. Inhibition of fibril formation by 2,4-DNP appears to be through the stabilization of TTR tetramer upon binding to the protein, which includes active site. These findings may have implications for the development of mechanism based small molecular weight compounds as therapeutic agents for the prevention/inhibition of the amyloid diseases.     

4,4'-Dianilino-1,1'-binaphthyl-5,5'-sulfonate, a novel molecule having chaperone-like activity

4,4'-Dianilino-1,1'-binaphthyl-5,5'-sulfonate (bis-ANS) and 1-anilinonaphthalene-8-sulfonate (ANS) are hydrophobic probes that are widely used in protein folding studies, using their capacity to bind to hydrophobic regions of partially unfolded proteins and in turn leading to an increase in fluorescence. Here we reveal a novel chaperone-like activity for bis-ANS, which acted as a highly effective inhibitor for the thermal- or chemical-induced aggregation of alcohol dehydrogenase, insulin or the whole cell extract of Escherichia coli, with ANS showing a much weaker effect. The studies to elucidate the mechanism underlying this activity show that bis-ANS is able to form stable soluble aggregates with the denaturing proteins and dramatically increase its fluorescence intensity upon incubation with aggregation-prone proteins. Moreover, we found that bis-ANS is able to prevent the heat inactivation of citrate synthase. These observations suggest that bis-ANS is able to block the exposed hydrophobic surfaces to suppress protein aggregation, acting in a way similar to what small heat shock proteins (one sub-class of molecular chaperones) do. The data presented here, together with the report that bis-ANS was able to suppress the amyloid formation of the prion peptide [J. Biol. Chem. 279 (2004) 5346], suggest that this molecule may be used as a potential protein stabilizer in addition to its current application as a hydrophobic probe.     

Conformational properties of native sperm whale apomyoglobin in solution.

Apomyoglobin from sperm whale is often used for studies of ligand binding, protein folding, and protein stability. In an effort to describe its conformational properties in solution, homonuclear and heteronuclear (13C and 15N) NMR methods were applied to the protein in its native state. Assignments were confirmed for nuclear Overhauser effects (NOEs) involving side chain and backbone protons in the folded regions of the structure. These NOEs were used to derive distance restraints. The shifts induced by the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS) were inspected in the regions remote from its binding site and served as an indicator of conformational flexibility. 3JalphaH-NH values were obtained to assess dihedral angle averaging and to provide additional restraints. A family of structures was calculated with X-PLOR and an ab initio simulated annealing protocol using holomyoglobin as a template. Where the structure appeared well defined by chemical shift, line width, ANS perturbation, and density of NOEs, the low resolution model of apomyoglobin provides a valid approximation for the structure. The new model offers an improved representation of the folded regions of the protein, which encompass the A, B, E, helices as well as parts of the G and H helices. Regions that are less well defined at this stage of calculations include the CD corner and the end of the H-helix. The EF-F-FG segment remains uncharacterized.