Stimulation by quinones of cyanide-resistant respiration in rat liver and heart mitochondria

It was shown that hydrophilic benzo- and naphthoquinones stimulate the cyanide-resistant respiration in liver and muscle mitochondria when succinate or NADH and glutamate or malate are used as oxidation substrates. The substrate-dependent oxygen uptake in the presence of cyanide is initiated by menadione, vicasol, 1.2-naphthoquinone, coenzyme Q0 and duroquinone. Rotenone and antimycin A do not inhibit the cyanide-resistant respiration. Oxidation of glutamate and malate in the course of CN-resistant respiration is inhibited by ortho- and bathophenanthroline and p-chloromercurybenzoate, whereas succinate oxidation by tenoyltrifluoroacetone, carboxin and pentachlorophenol. Superoxide dismutase, Cu2+ and catalase inhibit the CN-resistant respiration in the presence of quinones. Addition of catalase to the experimental cell causes O2 release.     

Phosphorylation modulates the alpha-helical structure and polymerization of a peptide from the third tau microtubule-binding repeat

Paired helical filaments (PHFs) isolated from patients with Alzheimer's disease (AD) mainly consist of the microtubule-associated protein tau in a hyperphosphorylated form. It has been found that PHFs are the first example of pathological protein aggregation associated with formation of alpha-helices [Biochemistry (2002) 41, 7150-5]. In an effort to investigate the interplay between phosphorylation and the putative role of short regions of alpha-helix in the polymerization of tau, we have focused on the region of tau encompassing residues 317 to 335. This region is able to form protein fibrils in vitro and has two serines that are often found phosphorylated in PHFs. Using trifluoroethanol as an indicator of the alpha-helix, we find that the stability of the alpha-helix conformation is enhanced by phosphorylation. Circular dichroism data show that the phosphorylated peptide in water presents a content in alpha-helix similar to the unphosphorylated peptide at 40% of trifluoroethanol. Phosphorylation also stimulates the effect of juglone in promoting the in vitro polymerization. Furthermore, Fourier transformed infrared spectroscopy of samples of phosphorylated peptide polymerized with juglone renders a spectrum with maxima at approximately 1665 and approximately 1675 cm(-1), which are suggestive of a mixture of turns and alpha-helix conformations. Our results provide a direct mechanistic connection between phosphorylation and polymerization in tau. The connection between phosphorylation and polymerization appears to involve formation of alpha-helix structure.     

Tau protein kinase I converts normal tau protein into A68-like component of paired helical filaments.

From bovine brain microtubules we purified tau protein kinase I (TPKI, Mr 45,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and tau protein kinase II (TPKII) whose activity was attributed to a 30-kDa protein on SDS-PAGE by affinity-labeling using an ATP analog. Both kinases were activated by tubulin. TPKII, but not TPKI, phosphorylated tau fragment peptides previously used for detection of a Ser/ThrPro kinase activity. Therefore, TPKII was considered to be the Ser/ThrPro kinase. TPKI was more effective than TPKII for producing the decrease of tau-1 immunoreactivity and mobility shift of tau on SDS-PAGE. Moreover, TPKI, but not TPKII nor other well-known protein kinases, generated an epitope present on paired helical filaments. These findings suggested that tau phosphorylated by TPKI resembled A-68, a component of paired helical filaments.     

Antibody used to identify penicillin-binding protein 2' in methicillin-resistant strains of Staphylococcus aureus (MRSA)

We found that tau, one of the major microtubule-associated proteins, is a good substrate for protein kinase C. The phosphorylation occurred mainly on serine residues and the sites phosphorylated by protein kinase C were largely different from those phosphorylated by cAMP-dependent protein kinase as analyzed by phosphopeptide mapping. The protein kinase C-mediated phosphorylation of tau reduced its abilities to promote tubulin polymerization and to cross-link actin filaments. The reduction in its abilities was in proportion to the number of phosphates incorporated into tau.     

Reversible beta-pleated sheet formation of a phosphorylated synthetic tau peptide.

Serine416 of human tau protein is believed to be phosphorylated in Alzheimer neurofibrillary tangles. We synthesized a fragment of tau, consisting of amino acids 408-421 in both non-phosphorylated and serine416-phosphorylated forms. Circular dichroism in a trifluoroethanol-water mixture indicated a beta-turn----beta-pleated sheet conformational transition upon phosphorylation. The beta-structure formation is intermolecular and can be inhibited by addition of Ca2+ ions or a phosphorylated tripeptide, but not with its non-phosphorylated analog. The presence of the phosphorylated tau peptide did not facilitate the formation of beta-pleated sheets of a phosphorylated neurofilament fragment. Multivalent cations induced a conformational transition of this phosphorylated neurofilament peptide, but the effect was less specific than the transition induced in the tau fragment, and it could also be reversed with the competing phosphorylated tripeptide.     

Tubulin, actin, and tau protein interactions and the study of their macromolecular assemblies

The intracellular polymerization of cytoskeletal proteins into their supramolecular assemblies raises many questions regarding the regulatory patterns that control this process. Binding experiments using the ELISA solid phase system, together with protein assembly assays and electron microscopical studies provided clues on the protein-protein associations in the polymerization of tubulin and actin networks. In vitro reconstitution experiments of these cytoskeletal filaments using purified tau, tubulin, and actin proteins were carried out. Tau protein association with tubulin immobilized in a solid phase support system was inhibited by actin monomer, and a higher inhibition was attained in the presence of preassembled actin filaments. Conversely, tubulin and assembled microtubules strongly inhibited tau interaction with actin in the solid phase system. Actin filaments decreased the extent of in vitro tau-induced tubulin assembly. Studies on the morphological aspects of microtubules and actin filaments coexisting in vitro, revealed the association between both cytoskeletal filaments, and in some cases, the presence of fine filamentous structures bridging these polymers. Immunogold studies showed the association of tau along polymerized microtubules and actin filaments, even though a preferential localization of labeled tau with microtubules was revealed. The studies provide further evidence for the involvement of tau protein in modulating the interactions of microtubules and actin polymers in the organization of the cytsokeletal network.     

The pathology of the neuronal cytoskeleton in Alzheimer's disease.

The two characteristic neuropathological lesions of Alzheimer's disease are the neurofibrillary tangles and the senile plaques. Neurofibrillary tangles are made of abnormal filaments (PHF) accumulating in neurons and mainly composed of a modified form of the microtubule-associated protein tau (PHF-tau). Senile plaques are composed of a cluster of dystrophic neurites surrounding an extracellular deposit of amyloid fibers made of a 42 amino-acid peptide (beta-amyloid peptide). The abnormal filaments contain the complete sequences of the different tau isoforms. The PHF-tau proteins can be distinguished from the normal tau proteins by the presence of several phosphorylated sites. One of these sites is phosphorylated by a calcium-calmodulin-dependent kinase. The relationship between PHF-tau and the cytoskeletal pathology in Alzheimer's disease is further discussed.     

In vitro tau fibrillization: mapping protein regions

We have investigated the propensity to form fibrillar aggregates of a variety of fragments and variants of the tau protein under the influence of a tau fibrillization inducer: coenzyme Q(0). To better identify fibrillization hotspots, we compare the polymerization propensity of tau fragments containing the sequence of putative hotspots with that of tau variants with that same sequence deleted. We also investigate the effects of biologically occurring modifications such as phosphorylation and deamidation. We found that residues 305 to 335 are essential for in vitro tau fibrillization. Residues 306 to 311 facilitate in vitro assembly, but are not sufficient to mimic the in vivo fibrillization of tau. Furthermore, the propensity of the 306-311 sequence to form fibrils is highly decreased by chemical modifications of tyrosine 310 that are commonly found in vivo.     

Differential assembly of human tau isoforms in the presence of arachidonic acid

Six tau isoforms arise from the alternative splicing of a single gene in humans. Insoluble, filamentous deposits of tau protein occur in a number of neurodegenerative diseases, and in some of these diseases, the deposition of polymers enriched in certain tau isoforms has been documented. Because of these findings, we have undertaken studies on the efficacy of fatty acid-induced polymerization of the individual tau isoforms found in the adult human CNS. The polymerization of each tau isoform in the presence of two concentrations of arachidonic acid indicated that isoforms lacking N-terminal exons e2 and e3 formed small, globular oligomers that did not go on to elongate into straight (SF) or paired helical (PHF) filaments under our buffer conditions. The polymerization of all isoforms containing e2 or e2 and e3 occurred readily at a high arachidonic acid concentration. Conversely, at a lower arachidonic acid concentration, only tau isoforms containing four microtubule binding repeats assembled well. Under all buffer conditions employed, filaments formed from three of the isoforms containing e2 and e3 resembled SFs in morphology but began to form PHF-like structures following extended incubation at 37 degrees C. These results indicate that polymerization of the intact tau molecule may be facilitated by e2 and e3. Moreover, tau isoforms containing three versus four microtubule binding repeats display different assembly properties depending on the solvent conditions employed.     

In vitro assembly of tau protein: mapping the regions involved in filament formation

Unraveling the mechanism of self-assembly of the protein tau into paired helical filaments (PHFs) is a crucial step toward the understanding of Alzheimer's and other neuropathological diseases at the molecular level. In an effort to map the role of different regions of tau in the mechanism of self-assembly, we have studied the polymerization ability of different tau fragments using an in vitro assay. Our results indicate that the N-terminal domain interferes with tau's ability to polymerize in vitro. The effect seems to be size dependent. Particularly, an isoform of tau from the peripheral nervous system, which has a much larger N-terminal domain, was found unable to form filaments in our in vitro assay. This finding can explain why in Alzheimer's patients PHFs only accumulate in the neurons from the central nervous system. We also report that a short segment of tau located in the third microtubule binding repeat (residues 317 to 335, peptide 1/2R) is probably the minimal segment of that region able to grow into filaments in vitro and in the presence of heparin. In contrast with whole peptide 1/2R, peptides corresponding to either the N-terminal or C-terminal halves of this segment were unable to form filaments. Finally, our polymerization studies of peptides from the C-terminal domain reveal a short sequence spanning residues 391 to 407 that grows into filaments in vitro. This tau segment forms filaments regardless of whether is incubated with heparin. Moreover, such filaments differ in diameter and morphology, suggesting a different mechanism of self-assembly.     

Ubiquitination and abnormal phosphorylation of paired helical filaments in Alzheimer's disease

The most characteristic cellular change in Alzheimer's disease is the accumulation of aberrant filaments, the paired helical filaments (PHF), in the affected neurons. There is growing evidence from a number of laboratories that dementia correlates better with the accumulation of PHF than of the extracellular amyloid, the second major lesion of Alzheimer's disease. PHF are both morphologically and biochemically unlike any of the normal neurofibrils. The major polypeptides in isolated PHF are microtubule-associated protein tau. Tau in PHF is phosphorylated differently from tau in microtubules. This abnormal phosphorylation of tau in PHF occurs at several sites. The accumulation of abnormally phosphorylated tau in the affected neurons in Alzheimer's disease brain precedes both the formation and the ubiquitination of the neurofibrillary tangles. In Alzheimer's disease brain, tubulin is assembly competent, but the in vitro assembly of microtubules is not observed. In vitro, the phosphate groups in PHF are less accessible than those of tau to alkaline phosphatase. The in vitro dephosphorylated PHF polypeptides stimulate microtubule assembly from bovine tubulin. It is hypothesized that a defect in the protein phosphorylation/dephosphorylation system is one of the earliest events in the cytoskeletal pathology in Alzheimer's disease. Production of nonfunctional tau by its phosphorylation and its polymerization into PHF most probably contributes to a microtubule assembly defect, and consequently, to a compromise in both axoplasmic flow and neuronal function. Index Entries: Alzheimer's disease; mechanisms of neuronal degeneration; neurofibrillary changes; paired helical filaments: biochemistry; microtubule-associated protein tau; abnormal phosphorylation; ubiquitination; microtubule assembly; axoplasmic flow; protein phosphorylation/dephosphorylation.     

Ligand-dependent tau filament formation: implications for Alzheimer's disease progression.

The mechanism through which arachidonic acid induces the polymerization of tau protein into filaments under reducing conditions was characterized through a combination of fluorescence spectroscopy and electron microscopy. Results show that polymerization follows a ligand-mediated mechanism, where binding of arachidonic acid is an obligate step preceding tau-tau interaction. Homopolymerization begins with rapid (on the order of seconds) nucleation, followed by a slower elongation phase (on the order of hours). Although essentially all synthetic filaments have straight morphology at early time points, they interact with thioflavin-S and monoclonal antibody Alz50 much like authentic paired helical filaments, suggesting that the conformation of tau protein is similar in the two filament forms. Over a period of days, synthetic straight filaments gradually adopt paired helical morphology. These results define a novel pathway of tau filament formation under reducing conditions, where oxidation may contribute to final paired helical morphology, but is not a necessary prerequisite for efficient nucleation or elongation of tau filaments.     

Role of cysteine-291 and cysteine-322 in the polymerization of human tau into Alzheimer-like filaments.

Filamentous tau pathology is central to a large number of dementing disorders, including Alzheimer's disease in which polymerized tau is hyperphosphorylated. Previous studies on heparin-dependent tau polymerization, using recombinant tau isoforms lacking Cys-291, suggest that tau dimerization via Cys-322 is critical for initiation of assembly of soluble tau into filaments. We report heparin-dependent in vitro polymerization of human recombinant tau (1-383 isoform), containing both Cys-291 and Cys-322, into paired helical filaments as characterized by electron microscopy. Tau polymerization, under physiological tau concentrations in the presence of dithiothreitol (DTT), was followed by a Thioflavine S fluorescence assay. To understand the molecular basis for heparin-induced tau polymerization, we expressed and purified C291A, C322A, and C291A/C322A tau mutants. The DTT requirement for tau polymerization was abolished using either the C291A or C322A tau mutant and polymerization was not observed with the C291A/C322A tau double mutant. Analysis by sodium dodecyl sulfate gel electrophoresis showed that, unlike wild type tau, a significant amount of the C291A mutant and the C322A mutant is present as a disulfide bonded dimer. Taken together these results suggest that, in isoforms containing both Cys-291 and Cys-322, a dimeric tau with an intermolecular disulfide bond through either Cys-291 or Cys-322 is presumably acting as a seed for initiation of tau polymerization.     

Brain Levels of Microtubule-Associated Protein τ Are Elevated in Alzheimer''s Disease: A Radioimmuno-Slot-Blot Assay for Nanograms of the Protein

The microtubule-associated protein tau, which stimulates the assembly of alpha-beta tubulin heterodimers into microtubules, is abnormally phosphorylated in Alzheimer's disease (AD) brain and is the major component of paired helical filaments. In the present study, the levels of tau and abnormally phosphorylated tau were determined in brain homogenates of AD and age-matched control cases. A radioimmuno-slot-blot assay was developed, using a primary monoclonal antibody, Tau-1, and a secondary antibody, antimouse 125I-immunoglobulin G. To assay the abnormally phosphorylated tau, the blots were treated with alkaline phosphatase before immunolabeling. The levels of total tau were about eightfold higher in AD (7.3 +/- 2.7 ng/micrograms of protein) than in control cases (0.9 +/- 0.2 ng/micrograms), and this increase was in the form of the abnormally phosphorylated protein. These studies indicate that the abnormal phosphorylation--not a decrease in the level of tau--is a likely cause of neurofibrillary degeneration in AD.     

In vitro polymerization of tau protein monitored by laser light scattering: method and application to the study of FTDP-17 mutants

Tau polymerization into the filaments that compose neurofibrillary tangles is seminal to the development of many neurodegenerative diseases. It is therefore important to understand the mechanisms involved in this process. However, a consensus method for monitoring tau polymerization in vitro has been lacking. Here we demonstrate that illuminating tau polymerization reactions with laser light and measuring the increased scattering at 90 degrees to the incident beam with a digital camera results in data that closely approximate the mass of tau polymer formation in vitro. The validity of the technique was demonstrated over a range of tau concentrations and through multiple angle scattering measurements. In addition, laser light scattering data closely correlated with quantitative electron microscopy measurements of the mass of tau filaments. Laser light scattering was then used to measure the efficiency with which the mutant tau proteins found in frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17) form filamentous structures. Several of these mutant proteins display enhanced polymerization in the presence of arachidonic acid, suggesting a direct role for these mutations in tau the filament formation that characterizes FTDP-17.     

Quinone reductase 2 substrate specificity and inhibition pharmacology

Quinone reductase 2 is a mammalian cytosolic FAD-dependent enzyme, the activity of which is not supported by conventional nicotinamide nucleotides. An endobiotic substrate has never been reported for this enzyme nor a set of molecular tools, such as inhibitors. In the present work, we used the recombinant human enzyme, expressed in CHO cells for the systematic screening of both co-substrates and substrates. The co-substrates survey showed that the natural occurring compound, N-ribosylnicotinamide, was a poor co-substrate. The synthetic N-benzylnicotinamide is a better one compared to any other compounds tested. We found that tetrahydrofolic acid acted as a co-substrate for the reduction of menadione catalysed by quinone reductase 2, although with poor potency (Km approximately 2 mM). Among a series of commercially available quinones, a single one was found to be substrate of quinone reductase 2, in the presence of N-benzyldihydronicotinamide: coenzyme Q0. Finally, we tested a series of 197 flavonoids as potential inhibitors. We found apigenin, genistein or kaempferol as good inhibitor of quinone reductase 2 activity with IC50 in the 100 nM range. These compounds, co-substrate, substrate and inhibitors will permit to better know this enzyme, the role of which is still poorly understood.     

Structural and functional implications of tau hyperphosphorylation: information from phosphorylation-mimicking mutated tau proteins

Abnormal tau-immunoreactive filaments are a hallmark of tauopathies, including Alzheimer's disease (AD). A higher phosphorylation ("hyperphosphorylation") state of tau protein may represent a critical event. To determine the potential role of tau hyperphosphorylation in these disorders, mutated tau proteins were produced where serine/threonine residues known to be highly phosphorylated in tau filaments isolated from AD patients were substituted for glutamate to simulate a paired helical filament (PHF)-like tau hyperphosphorylation. We demonstrate that, like hyperphosphorylation, glutamate substitutions induce compact structure elements and SDS-resistant conformational domains in tau protein. Hyperphosphorylation-mimicking glutamate-mutated tau proteins display a complete functional loss in its ability to promote microtubule nucleation which can partially be overcome by addition of the osmolyte trimethylamine N-oxide (TMAO), which is similar to phosphorylated tau. In addition, glutamate-mutated tau proteins fail to interact with the dominant brain protein phosphatase 2A isoform ABalphaC, and exhibit a reduced ability to assemble into filaments. Interestingly, wild-type tau and phosphorylation-mimicking tau similarly bind to microtubules when added alone, but the mutated tau is almost completely displaced from the microtubule surface by equimolar concentrations of wild-type tau. The data indicate that glutamate-mutated tau proteins provide a useful model for analyzing the functional consequences of tau hyperphosphorylation. They suggest that several mechanisms contribute to the abnormal tau accumulation observed during tauopathies, in particular a selective displacement of hyperphosphorylated tau from microtubules, a functional loss in promoting microtubule nucleation, and a failure to interact with phosphatases.     

The biological significance of non-enzymatic reaction of menadione with plasma thiols: enhancement of menadione-induced cytotoxicity to platelets by the presence of blood plasma.

To test the hypothesis that the non-enzymatic reaction of quinones with thiols in plasma can generate reactive oxygens (ROS), thereby leading to potentiated cellular toxicity, we have studied the effect of a representative quinone compound, menadione, on plasma isolated from rats. The experimental results are as follows: (1) menadione generated ROS via non-enzymatic reaction with protein thiols in plasma; (2) the presence of plasma increased menadione-induced cytotoxicity to platelets; (3) pretreatment of plasma with a thiol-depleting agent significantly suppressed menadione-induced ROS and cytotoxicity. These results suggest that the non-enzymatic reaction of menadione with plasma thiols could be an important process in quinone-induced cellular toxicity.     

Pseudophosphorylation of tau protein alters its ability for self-aggregation

Filamentous tau protein deposits are a pathological hallmark of a group of neurodegenerative disorders (tauopathies). Tau protein in these aggregates is highly phosphorylated at different phosphorylation sites. Although tau filaments can be formed by heparin-induced aggregation of unphosphorylated recombinant tau, it is not known how tau phosphorylation modulates aggregation behaviour. Analysis of the effect of tau phosphorylation at defined single or multiple sites is hampered by the low specificity of protein kinases and the highly dynamic turnover of phosphorylation in vivo. To overcome this problem we employed site-directed mutagenesis to convert serine and threonine to aspartic acid or glutamic acid, which introduce a negative charge and conformational change that mimic phosphorylation. We tested 14 different mutated tau proteins for their propensity for self-aggregation and formation of tau filaments. Tau aggregation was monitored with thioflavin S fluorescence in the presence of different inducers such as heparin, Al3+, Fe2+ and Fe3+. We found that mutations in the N-terminal portion up to amino acid 208 mainly suppress tau aggregation, whereas mutations in the C-terminal region mainly lead to an enhanced aggregation. Mutations in the middle portion of tau showed a mixed picture of suppression and enhancement of aggregation. A single amino acid change Ser422Glu has aggregation-favouring properties with all four inducers.     

Direct interaction between yeast NADH-ubiquinone oxidoreductase, succinate-ubiquinone oxidoreductase, and ubiquinol-cytochrome c oxidoreductase in the reduction of exogenous quinones

The reduction of the following exogenous quinones by succinate and NADH was studied in mitochondria isolated from both wild type and ubiquinone (Q)-deficient strains of yeast: ubiquinone-0 (Q0), ubiquinone-1 (Q1), ubiquinone-2 (Q2), and its decyl analogue 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB), duroquinone (DQ), menadione (MQ), vitamin K1 (2-methyl-3-phytyl-1,4-naphthoquinone), the plastoquinone analogue 2,3,6-trimethyl-1,4-benzoquinone (PQOc1), plastoquinone-2 (PQ2), and its decyl analogue (2,3-dimethyl-6-decyl-1,4-benzoquinone). Reduction of the small quinones DQ, Q0, Q1, and PQOc1 by NADH occurred in both wild type and Q-deficient mitochondria in a reaction inhibited more than 50% by myxothiazol and less than 20% by antimycin. The reduction of these small quinones by succinate also occurred in wild type mitochondria in a reaction inhibited more than 50% by antimycin but did not occur in Q-deficient mitochondria suggesting that endogenous Q6 is involved in their reduction. In addition, the inhibitory effects of antimycin and myxothiazol, specific inhibitors of the cytochrome b-c1 complex, on the reduction of these small quinones suggest the involvement of this complex in the electron transfer reaction. By contrast, the reduction of Q2 and DB by succinate was insensitive to inhibitors and by NADH was 20-30% inhibited by myxothiazol suggesting that these analogues are directly reduced by the primary dehydrogenases. The dependence of the sensitivity to the inhibitors on the substrate used suggests that succinate-ubiquinone oxidoreductase interacts specifically with center i (the antimycin-sensitive site) and NADH ubiquinone oxidoreductase preferentially with center o (the myxothiazol-sensitive site) of the cytochrome b-c1 complex. The NADH dehydrogenase involved in the myxothiazol-sensitive quinone reduction faces the matrix side of the inner membrane suggesting that center o may be localized within the membrane at a similar depth as center i.