Nitrated products formed on α-synuclein are preferentially incorporated into oligomers but excluded from fibrils: A mechanism for accumulation of neurotoxic species.

The self-assembly of tau into paired helical filaments (PHFs) in neurofibrillary tangles (NFTs) is a significant event in Alzheimer’s disease (AD) pathogenesis. Numerous post-translational modifications enhance or inhibit tau assembly into NFTs. Oxidative stress, which accompanies AD, induces multiple post-translational modifications in proteins, including the formation of dityrosine (DiY) cross-links. Previous studies have revealed that metal-catalysed oxidation (MCO) using Cu2+ and H2O2 leads to the formation of DiY cross-links in two misfolding proteins, Aβ and α-synuclein, associated with AD and Parkinson’s disease respectively. The effect of MCO on tau remains unknown. Here, we examined the effect of MCO and ultra-violet oxidation to study the influence of DiY cross-linking on the self-assembly of the PHF-core tau fragment. We report that DiY cross-linking facilitates tau assembly into tau oligomers that fail to bind thioflavin S, lack β-sheet structure and prevents their elongation into filaments. At a higher concentration, Cu2+ (without H2O2) also facilitates the formation of these tau oligomers. The DiY cross-linked tau oligomers do not cause cell death. Our findings suggest that DiY cross-linking of pre-assembled tau promotes the formation of soluble tau oligomers that show no acute impact on cell viability.

Parkinson's disease is the second most common neurodegenerative disease. It is characterized by aggregation of the protein α-synuclein (α-syn) in Lewy bodies, mitochondrial dysfunction, and increased oxidative stress in the substantia nigra. Oxidative stress leads to several modifications of biomolecules including dityrosine (DiY) crosslinking in proteins, which has recently been detected in α-syn in Lewy bodies from Parkinson's disease patients. Here we report that α-syn is highly susceptible to ultraviolet-induced DiY formation. We investigated DiY formation of α-syn and nine tyrosine-to-alanine mutants and monitored its effect on α-syn fibril formation in vitro. Ultraviolet irradiation of intrinsically disordered α-syn generates DiY-modified monomers and dimers, which inhibit fibril formation of unmodified α-syn by interfering with fibril elongation. The inhibition depends on both the DiY group and its integration into α-syn. When preformed α-syn fibrils are crosslinked by DiY formation, they gain increased resistance to denaturation. DiY-stabilized α-syn fibrils retain their high seeding efficiency even after being exposed to denaturant concentrations that completely depolymerize non-crosslinked seeds. Oxidative stress-associated DiY crosslinking of α-syn therefore entails two opposing effects: (i) inhibition of aggregation by DiY-modified monomers and dimers, and (ii) stabilization of fibrillar aggregates against potential degradation mechanisms, which can lead to promotion of aggregation, especially in the presence of secondary nucleation.

Formation of dityrosine (DT) cross-linkages in proteins is one of the most widely used markers of oxidative stress. Ribonuclease A (RNase A) has 6 Tyr residues and shows a characteristic DT fluorescence peak upon oxidation in addition to major changes in its secondary structure. DT formation can be prevented by using polyphenols (GA, ECG, and EGCG) which are known to have strong antioxidant activity. However, it has been observed that ECG and EGCG initiate protein oligomerization due to protein-polyphenol cross-linkages. To prevent the formation of such cross-linkages we have used β-cyclodextrin (β-CD) to encapsulate the polyphenols and studied its antioxidant properties along with that of free polyphenols. The polyphenol/β-cyclodextrin (β-CD) inclusion complexes not only prevent DT formation but also reduce protein oligomerization. This may be attributed to the fact that the quinone forming rings of ECG and EGCG become encapsulated in the cavity of β-CD and are no longer available for protein cross-linking.

Oxidative stress is a significant source of damage that accumulates during aging and contributes to Alzheimer’s disease (AD) pathogenesis. Oxidation of proteins can give rise to covalent links between adjacent tyrosines known as dityrosine (DiY) cross-linking, amongst other modifications, and this observation suggests that DiY could serve as a biomarker of accumulated oxidative stress over the lifespan. Many studies have focused on understanding the contribution of DiY to AD pathogenesis and have revealed that DiY crosslinks can be found in both Aβ and tau deposits – the two key proteins involved in the formation of amyloid plaques and tau tangles, respectively. However, there is no consensus yet in the field on the impact of DiY on Aβ and tau function, aggregation, and toxicity. Here we review the current understanding of the role of DiY on Aβ and tau gathered over the last 20 years since the first observation, and discuss the effect of this modification for Aβ and tau aggregation, and its potential as a biomarker for AD.

A characteristic hallmark of Alzheimer’s Disease (AD) is the pathological aggregation and deposition of tau into paired helical filaments (PHF) in neurofibrillary tangles (NFTs). Oxidative stress is an early event during AD pathogenesis and is associated with tau-mediated AD pathology. Oxidative environments can result in the formation of covalent dityrosine crosslinks that can increase protein stability and insolubility. Dityrosine cross-linking has been shown in Aβ plaques in AD and α-synuclein aggregates in Lewy bodies in ex vivo tissue sections, and this modification may increase the insolubility of these aggregates and their resistance to degradation. Using the PHF-core tau fragment (residues 297 – 391) as a model, we have previously demonstrated that dityrosine formation traps tau assemblies to reduce further elongation. However, it is unknown whether dityrosine crosslinks are found in tau deposits in vivo in AD and its relevance to disease mechanism is unclear. Here, using transmission electron microscope (TEM) double immunogold-labelling, we reveal that neurofibrillary NFTs in AD are heavily decorated with dityrosine crosslinks alongside tau. Single immunogold-labelling TEM and fluorescence spectroscopy revealed the presence of dityrosine on AD brain-derived tau oligomers and fibrils. Using the tau (297–391) PHF-core fragment as a model, we further showed that prefibrillar tau species are more amenable to dityrosine crosslinking than tau fibrils. Dityrosine formation results in heat and SDS stability of oxidised prefibrillar and fibrillar tau assemblies. This finding has implications for understanding the mechanism governing the insolubility and toxicity of tau assemblies in vivo.

Developing effective treatments for neurodegenerative diseases is one of the greatest medical challenges of the 21st century. Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are very common neurological disorders of the elderly (Jankovic et al., 2008). Although many of these clinical entities have been recognized for more than a hundred years, it is only during the past fifteen years that the molecular events that precipitate the diseases have begun to be understood. Mutations in the α-synuclein gene cause early-onset PD, often associated with dementia, and one an α-synuclein mutation segregated with pure DLB (with no Alzheimer's pathology). Neuropathologically these diseases are characterized by the presence of Lewy bodies, intraneuronal inclusions mostly composed of α-synuclein protein fibrils, cementing the notion that this protein has a central role in Lewy body diseases (LBD). Despite the progress that has been made in understanding the underlying disease mechanisms of LBD, there remains an urgent need to develop methods for use in diagnosis. The development of reliable surrogate markers for the presence and abundance of α-synuclein lesions (Lewy bodies) in the brain would naturally facilitate a more streamlined work-up during the early care of PD and DLB patients, and importantly, allow for the biologically guided evaluation of future drug trials aimed at neuroprotection in the synucleinopathies. Recently, we generated new antibodies specific for different forms of α-synuclein such as oligomeric-α-synuclein (o-α-synuclein), phosphorylated-α-synuclein at serine 129 (p-S129-α-synuclein) or total-α-synuclein (t-α-synuclein), to develop highly specific and sensitive ELISA systems to assess the levels of these species in biological fluids. Next, we utilized our assays to explore the potential use of α-synuclein species as biomarkers for PD in cerebrospinal fluid (CSF) samples from a cross-sectional cohort of 46 PD patients and 48 age-matched healthy controls. Thereafter, we validated our results in large longitudinal DATATOP cohort (n = 242) over two-year follow-up period). In our study we also investigated the potential predictive role of CSF Alzheimer's disease (AD) core biomarkers (Aβ42, total tau, p-tau). In our study we also investigated the added predictive value of CSF AD core biomarkers (Aβ42, total tau, p-tau) to CSF α-synuclein species. Interestingly, a strong positive correlation between the changes in CSF t- and o-α-synuclein levels was noted. Such a strong correlation clearly suggests that the increase of CSF t-α-synuclein levels at the early stages of the disease lead to the formation and accumulation of the pathogenic species of o-α-synuclein in the brain that cause neuronal cell death. It has been shown that soluble o-α-synuclein are elevated in brain homogenates from PD and DLB compared to normal brains (Sharon, et al., 2003; Paleologou, et al., 2009). Together with the results from our previous studies, we provide strong evidence for the important role of o-α-synuclein in the pathogenesis of PD and other synucleinopathies (El-Agnaf et al., 2003). Consistent with our recent study (Parnetti, et al., 2014), we also found that the change in CSF Aβ42 levels correlated with the change in UPDRS-mental scores, suggesting that CSF Aβ42 may aid in predicting cognitive decline in PD patients. It's worth mentioning that one of the many important features that distinguished the DATATOP study from other studies is that the untreated PD subjects enrolled in the DATATOP study were at a very early stage of the disease; a stage estimated to be earlier than most of the other clinical trials. At baseline, PD patients presented with minimal disability and were followed until just prior to the initiation of the dopamine replacement therapy, and this is reflected in the low HY2:461–462. Jankovic, J. Parkinson's disease: clinical features and diagnosis. J Neurol Neurosurg Psychiatry 2008;79:368–376, doi:10.1136/jnnp.2007.131045. Kordower, J. H. et al. Disease duration and the integrity of the nigrostriatal system in Parkinson's disease. Brain 2013;136:2419–2431, doi:10.1093/brain/awt192. Paleologou, K. E. et al. Detection of elevated levels of soluble alpha-synuclein oligomers in post-mortem brain extracts from patients with dementia with Lewy bodies. Brain 2009;132, doi:10.1093/brain/awn349. Parnetti, L. et al. Differential role of CSF alpha-synuclein species, tau, and Aβ42 in Parkinson's Disease. Front Aging Neurosci 2014;6:53, doi:10.3389/fnagi.2014.00053. Sharon, R. et al. The formation of highly soluble oligomers of alpha-synuclein is regulated by fatty acids and enhanced in Parkinson's disease. Neuron 2003;37:583–595. Toledo, J. B., Korff, A., Shaw, L. M., Trojanowski, J. Q. & Zhang, J. CSF α-synuclein improves diagnostic and prognostic performance of CSF tau and Aβ in Alzheimer's disease. Acta Neuropathol 2013;126:683–697, doi:10.1007/s00401-013-1148-z.

Tyrosine residues are sensitive to oxidation and can be converted to hydroperoxides either by superoxide reacting with the Tyr radical or by singlet oxygen. These hydroperoxides rearrange to bicyclic derivatives that are readily reduced to more stable hydroxides. The aromatic character of tyrosine is lost, but the product contains an α-β unsaturated carbonyl group and is, therefore, an electrophile. We have generated hydroxide derivatives of several Tyr-containing peptides and shown using liquid chromatography/mass spectrometry that they undergo Michael addition with GSH. For Tyr-Gly, rate constants of 9.2 and 11.8 m(-1)min(-1) were measured for the two chromatographically distinct isomers. Unusual for GSH addition to an electrophile, the reaction is reversible, with a half-life of many hours for the reverse reaction. These kinetics indicate that with a typical cellular concentration of 5 mm GSH, >95% Tyr-Gly hydroxide would become conjugated with a half-life of ∼15 min. Sperm whale myoglobin forms a hydroperoxide on Tyr-151 in a hydrogen peroxide/superoxide-dependent reaction. We show that its hydroxide derivative reacts with GSH to form a conjugate. Detection of the conjugate required stabilization by reduction; otherwise, the reverse reaction occurred during tryptic digestion and analysis. Our findings represent a novel mechanism for peptide or protein glutathionylation involving a carbon-sulfur cross-link between oxidized Tyr and Cys. As with other electrophiles, the oxidized Tyr should undergo a similar reaction with Cys residues in proteins to give intramolecular or intermolecular protein cross-links. This mechanism could give rise to protein cross-linking in conditions of oxidative stress.

Parkinson’s disease (PD) is the second most common neurological disease and belongs to a group of neurodegenerative disorders called synucleinopathies in which pathological aggregates of N-terminally acetylated α-synuclein (NAcα-Syn) accumulate in various regions of the brain. In PD, these NAcα-Syn aggregates have been found to contain covalent dityrosine crosslinks, which can occur either intermolecularly or intramolecularly. Cerebral metal imbalance is also a hallmark of PD, warranting investigations into the effects of brain biometals on NAcα-Syn. NAcα-Syn is an intrinsically disordered protein, and metal-mediated conformational modifications of this structurally dynamic protein have been demonstrated to influence its propensity for dityrosine formation. In this study, a library of tyrosine-to-phenylalanine (Y-to-F) NAcα-Syn constructs were designed in order to elucidate the nature and the precise residues involved in dityrosine crosslinking of Fe-bound NAcα-Syn. The structural capacity of each mutant to form dityrosine crosslinks was assessed using Photo-Induced Cross-Linking of Unmodified Proteins (PICUP), demonstrating that coordination of either FeIII or FeII to NAcα-Syn inhibits dityrosine crosslinking among the C-terminal residues. We further demonstrate that Y39 is the main contributor to dityrosine formation of Fe-bound NAcα-Syn, while Y125 is the main residue involved in dityrosine crosslinks in unmetalated NAcα-Syn. Our results confirm that iron coordination has a global effect on NAcα-Syn structure and reactivity.

P 072 - Oxidative modifications of α- and β- caseins induced by AAPH-derived peroxyl radicals: Role of tryptophan and tyrosine residues

Idiopathic pulmonary fibrosis (IPF) is a noninflammatory progressive lung disease. Oxidative damage is a hallmark of IPF, but the sources and consequences of oxidant generation in the lungs are unclear. In this study, we addressed the link between the H2O2-generating enzyme NADPH oxidase 4 (NOX4) and di-tyrosine (DT), an oxidative post-translational modification in IPF lungs. We performed immunohistochemical staining for DT and NOX4 in pulmonary tissue from patients with IPF and controls using validated antibodies. In the healthy lung, DT showed little or no staining and NOX4 was mostly present in normal vascular endothelium. On the other hand, both markers were detected in several cell types in the IPF patients, including vascular smooth muscle cells and epithelium (bronchial cells and epithelial cells type II). The link between NOX4 and DT was addressed in human fibroblasts deficient for NOX4 activity (mutation in the CYBA gene). Induction of NOX4 by Transforming growth factor beta 1 (TGFβ1) in fibroblasts led to moderate DT staining after the addition of a heme-containing peroxidase in control cells but not in the fibroblasts deficient for NOX4 activity. Our data indicate that DT is a histological marker of IPF and that NOX4 can generate a sufficient amount of H2O2 for DT formation in vitro.

Enkephalins are bioactive pentapeptides (Tyr-Gly-Gly-Phe-Leu (Leu-enk) and Tyr-Gly-Gly-Phe-Met (Met-enk)) produced while an organism is under mental and/or physical stress. In the course of their biological action they are exposed to reactive oxygen and nitrogen species. We have reinvestigated the reactions of (•)OH radicals toward these peptides in order to elucidate the oxidation mechanisms and the final products. Nanosecond pulse radiolysis was used to obtain the spectra of the reaction intermediates and their kinetics. Additional insight into details of the oxidation mechanism was gained by identification of main final products by means of UV-vis spectrophotometry, HPLC coupled with fluorescence spectroscopy, and mass spectrometry. The key processes are different in both peptides. In Leu-enk, the first step is an (•)OH radical addition to the aromatic rings of Tyr and Phe residues that leads to hydroxylated residues, dihydroxyphenylalanine (DOPA) from Tyr and tyrosine isomers from Phe, respectively. In Met-enk, these processes are less important, an additional target being the sulfur atom of the methionine residue. Depending on pH either an OH-adduct (hydroxysulfuranyl radical) or a sulfur radical cation undergo intramolecular electron transfer with Tyr residue resulting in a repair of Met and oxidation of Tyr to tyrosyl radicals and a final formation of dityrosine. At low pH, the OH-adducts to Tyr residue are precursors of tyrosyl radicals and dityrosine. Thus, the final products coming from oxidation of the Tyr residue depend strongly on the neighboring residues and the pH.

Introduction: Bioprosthetic heart valves (BHVs), fabricated from glutaraldehyde-fixed bovine pericardium or porcine aortic valves, have been widely used in valve replacement surgery. However, BHVs fail over time due to structural degeneration. Despite frequent observations of calcification-associated BHV failure, more than 25% of BHV structural failures occur without calcification. Previously, we reported that oxidative stress may have a pivotal role in noncalcific BHV failure. Moreover, oxidation-induced formation of dityrosine (DT) crosslinks via hydroxyl- and tyrosyl-radical mediated pathways was only detected in clinical BHV explants. Yet the mechanism of DT formation and its role in structural degeneration of clinical BHV is incompletely understood. Hypothesis: Oxidative damage to BHV is due to a specific spatial localization of DT crosslink formation mediated by myeloperoxidase (MPO) and/or iron (Fe) deposition in clinical BHV explants Method & Results: BHVs from 7 human subjects were collected and subjected to mass spectrometry to measure DT in paraffin sections of BHV. 4 different regions of each BHV leaflet (free edge, sewing cushion, commissure, and middle region) were sampled as well as entire cross-sections of each cusp. DT was only detected in the mid–cusp (137.37±49.96 μmol/mol) and was not significantly different from DT quantitated in the cross-sections (191.29±65.98 μmol/mol). DT was not detected in other regions, implying a specific spatial localization of oxidative stress in BHV. MPO, a neutrophil derived enzyme, is responsible for reactive oxygen species (ROS) generation. Immunofluoresence microscopy demonstrated significant presence of MPO in the BHV, implying that MPO may be responsible for the DT formation in BHV. Fe deposition due to intracuspal hematomas and/or Fe-association with calcific deposits can also be responsible for ROS and DT formation. Fe was shown to be present in histology sections in failed BHV by Prussian Blue staining, indicating that Fe deposits may also in part be responsible for the observed DT formation. Conclusion: These results demonstrated a mid-leaflet localization of DT formation in clinically failed BHV explants. This may in part be mediated by MPO and/or Fe-deposits in clinical BHV.

Protein phosphorylation is the most frequent post-translational modification (PTM) that plays important regulatory roles in a wide range of biological processes. Phosphorylation mainly occurs on serine (Ser), threonine (Thr), and tyrosine (Tyr) residues, with the phosphorylated Tyr sites accounting for ~1–2% of all phosphorylated residues. Tyr phosphorylation was initially believed to be less common in plants compared to animals; however, recent investigation indicates otherwise. Although they lack typical protein Tyr kinases, plants possess many dual-specificity protein kinases that were implicated in diverse cellular processes by phosphorylating Ser, Thr, and Tyr residues. Analyses of sequenced plant genomes also identified protein Tyr phosphatases and dual-specificity protein phosphatases. Recent studies have revealed important regulatory roles of Tyr phosphorylation in many different aspects of plant growth and development and plant interactions with the environment. This short review summarizes studies that implicated the Tyr phosphorylation in biosynthesis and signaling of plant hormones.

Previous studies demonstrated that alpha-synuclein (alpha-syn) fibrillization is inhibited by dopamine, and studies to understand the molecular basis of this process were conducted (Conway, K. A., Rochet, J. C., Bieganski, R. M., and Lansbury, P. T., Jr. (2001) Science 294, 1346-1349). Dopamine inhibition of alpha-syn fibrillization generated exclusively spherical oligomers that depended on dopamine autoxidation but not alpha-syn oxidation, because mutagenesis of Met, His, and Tyr residues in alpha-syn did not abrogate this inhibition. However, truncation of alpha-syn at residue 125 restored the ability of alpha-syn to fibrillize in the presence of dopamine. Mutagenesis and competition studies with specific synthetic peptides identified alpha-syn residues 125-129 (i.e. YEMPS) as an important region in the dopamine-induced inhibition of alpha-syn fibrillization. Significantly, the dopamine oxidation product dopaminochrome was identified as a specific inhibitor of alpha-syn fibrillization. Dopaminochrome promotes the formation of spherical oligomers by inducing conformational changes, as these oligomers regained the ability to fibrillize by simple denaturation/renaturation. Taken together, these data indicate that dopamine inhibits alpha-syn fibrillization by inducing structural changes in alpha-syn that can occur through the interaction of dopaminochrome with the 125YEMPS129 motif of alpha-syn. These results suggest that the dopamine autoxidation can prevent alpha-syn fibrillization in dopaminergic neurons through a novel mechanism. Thus, decreased dopamine levels in substantia nigra neurons might promote alpha-syn aggregation in Parkinson's disease.

Inactivation of rabbit muscle glycogen phosphorylase b by peroxynitrite revisited: Does the nitration of Tyr 613 in the allosteric inhibition site control enzymatic function?