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Synucleinopathies are neurodegenerative diseases that are associated with the misfolding and aggregation of [alpha]-synuclein, including Parkinson's disease, dementia with Lewy bodies and multiple system atrophy.sup.1. Clinically, it is challenging to differentiate Parkinson's disease and multiple system atrophy, especially at the early stages of disease.sup.2. Aggregates of [alpha]-synuclein in distinct synucleinopathies have been proposed to represent different conformational strains of [alpha]-synuclein that can self-propagate and spread from cell to cell.sup.3-6. Protein misfolding cyclic amplification (PMCA) is a technique that has previously been used to detect [alpha]-synuclein aggregates in samples of cerebrospinal fluid with high sensitivity and specificity.sup.7,8. Here we show that the [alpha]-synuclein-PMCA assay can discriminate between samples of cerebrospinal fluid from patients diagnosed with Parkinson's disease and samples from patients with multiple system atrophy, with an overall sensitivity of 95.4%. We used a combination of biochemical, biophysical and biological methods to analyse the product of [alpha]-synuclein-PMCA, and found that the characteristics of the [alpha]-synuclein aggregates in the cerebrospinal fluid could be used to readily distinguish between Parkinson's disease and multiple system atrophy. We also found that the properties of aggregates that were amplified from the cerebrospinal fluid were similar to those of aggregates that were amplified from the brain. These findings suggest that [alpha]-synuclein aggregates that are associated with Parkinson's disease and multiple system atrophy correspond to different conformational strains of [alpha]-synuclein, which can be amplified and detected by [alpha]-synuclein-PMCA. Our results may help to improve our understanding of the mechanism of [alpha]-synuclein misfolding and the structures of the aggregates that are implicated in different synucleinopathies, and may also enable the development of a biochemical assay to discriminate between Parkinson's disease and multiple system atrophy. Protein misfolding cyclic amplification (PMCA) technology can discriminate between patients with Parkinson's disease and patients with multiple system atrophy on the basis of the characteristics of the [alpha]-synuclein aggregates in the cerebrospinal fluid., Author(s): Mohammad Shahnawaz [sup.1] , Abhisek Mukherjee [sup.1] , Sandra Pritzkow [sup.1] , Nicolas Mendez [sup.1] , Prakruti Rabadia [sup.1] , Xiangan Liu [sup.2] , Bo Hu [sup.2] , Ann [...]

Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates multicellular functions through interactions with its receptors on cell surfaces. S1P is enriched and stored in erythrocytes; however, it is not clear whether alterations in S1P are involved in the prevalent and debilitating hemolytic disorder sickle cell disease (SCD). Here, using metabolomic screening, we found that S1P is highly elevated in the blood of mice and humans with SCD. In murine models of SCD, we demonstrated that elevated erythrocyte sphingosine kinase 1 (SPHK1) underlies sickling and disease progression by increasing S1P levels in the blood. Additionally, we observed elevated SPHK1 activity in erythrocytes and increased S1P in blood collected from patients with SCD and demonstrated a direct impact of elevated SPHK1-mediated production of S1P on sickling that was independent of S1P receptor activation in isolated erythrocytes. Together, our findings provide insights into erythrocyte pathophysiology, revealing that a SPHK1-mediated elevation of S1P contributes to sickling and promotes disease progression, and highlight potential therapeutic opportunities for SCD., Introduction Sickle cell disease (SCD) is a devastating and costly genetic disorder associated with high morbidity and mortality. It is the most prevalent autosomal recessive disorder, affecting millions worldwide, with [...]

Nitric oxide (NO) is extremely toxic to Clostridium botulinum, but its molecular targets are unknown. Here, we identify a heine protein sensor (SONO) that displays femtomolar affinity for NO. The crystal structure of the SONO heme domain reveals a previously undescribed fold and a strategically placed tyrosine residue that modulates heme-nitrosyl coordination. Furthermore, the domain architecture of a SONO ortholog cloned from Chlamydomonas reinhardtii indicates that NO signaling through cyclic guanosine monophosphate arose before the origin of multicellular eukaryotes. Our findings have broad implications for understanding bacterial responses to NO, as well as for the activation of mammalian NO-sensitive guanylyl cyclase., Nitric oxide (NO) is a small, short-lived, and highly reactive gaseous molecule. In mammals, NO is biosynthesized from L-arginine by nitric oxide synthases, and it plays a key role in [...]

The authors review the publications on conversion mechanism of arachidonic acid to prostaglandin G (sub)2. The topics of interest include the steps of cyclooxygenation reaction, identification of reaction intermediates and stereoselectivity determinants.

Research has been conducted on the tyrosyl radicals generated via the prostaglandin H synthase-1 reaction with ethyl hydrogen peroxide. The study of these radicals carried out by the use of high-field/high frequency EPR spectroscopy is presented.

Research has been conducted on the ferric endothelial nitric oxide synthase holoenzyme. The low-frequency resonance Raman spectra of this holoenzyme have been investigated in the presence of the L-arginine substrate and the results are reported.

Research has been conducted on the prostaglandin H synthase (PGHS) isoforms 1 and 2 catalyzing the first steps in the prostaglandins' biosynthesis. The characterization of PGHS heme active site and its environment has been carried out via resonance Raman spectroscopy.

The binding kinetics of three different heme ligands to endothelial nitric oxide synthase is described. A P450 reductase domain is attached to an oxygenase domain, where the catalytic site is located.

Research has been conducted on the tyrosyl radicals. The use of low-temperature electron paramagnetic resonance and electron nuclear double resonance in investigating these radicals is discussed.

A study was conducted to analyze the influence of substituted imidazoles on the heme structure of endothelial nitric oxide synthase. The baculovirus expression system was carried out to prepare recombinant human endothelial nitric oxide synthase while optical spectra were recorded using a Hewlett-Packard diode array spectrophotometer. Results indicated that imidazoles produced differing effects on heme spin and geometry.

Simultaneous spectrophotometric and polarographic measurements of the interaction of prostaglandin H synthase (PGHS) with hyperoxide, fatty acid and oxygen found that tyrosyl radical formation in PGHS requires interactions with only 1 equiv of hyperoxide. Stopped flow studies show that hyperoxide level indirectly affects the rate of Compound II formation. Results support a dioxygenase mechanism for PGHS reaction with fatty acids and oppose a proposed mixed dioxygenase/monooxygenase mechanisms for the reaction with 20:2.

Spectroscopic studies were done to gather evidences to support the hypothesis that a hydroperoxide-induced tyrosyl radical is a key cyclooxygenase intermediate both for the 'basal' isoform of prostaglandin H synthase, PGHS-1 and for the 'inducible' isoform, PGHS-2. Treatment of PGHS-2 with aspirin, a compound known to induce acylation reaction that destroys cyclooxygenase activity, was found not to alter the production of tyrosyl radical that is needed for cyclooxygenase catalysis. Thus, it is suggested that PGHS-2 tyrosyl radical is a potent oxidant for the oxygenase catalysis by PGHS.

The catalytic abilities of Prostaglandin H synthase are better explained by a branched-chain mechanism instead of a tightly coupled mechanism. The two proposed mechanisms are competing explanations. The characteristics observed in experiment, especially the accumulation of micromolar levels of PGG(sub 2), are largely in keeping with predictions of a branched-chain mechanism. However, these observations are opposed to important predictions based upon a tightly coupled mechanism.

Manganese prostaglandin H synthase (PGHS) generates a free radical for the participation in cyclooxygenase catalysis, and the reaction is kinetically competent. The reactivity of Mn-PGHS with Fe-PGHS is compared. Cyclooxygenase specific activity increases with the concentration of Mn-PGHS and reaches a value higher than that of Fe-PGHS. This concentration dependence is due to the low rate of formation of oxidized peroxidase intermediates.

Prostaglandin H synthase was altered using diethyl pyrocarbonate (DEPC) to determine the function of histidine residues in the reaction catalyzed by the enzyme. DEPC modification of the apoenzyme caused a progressive diminution of the enzyme's peroxidase and cyclooxigenase function. Further studies show that the DEPC preferentially modifies histidine residues. This suggests that histidine residues have important functions in the the peroxidase and cyclooxygenase activity of the synthase.

Tyrosyl radicals were characterized and their role in hydroperoxide-dependent regulation of prostaglandin endoperoxide (PGH) synthase were determined. A tyrosyl radical is formed during incubation of PGH synthase 1 with arachidonate or peroxide. No strong evidence for the role of this radical in cyclooxygenase catalysis was found. However, it is proposed that the radical is associated with suicide inactivation of the peroxidase activity of PGH synthase 1.

A study was conducted to examine the effects of three competitive inhibitors (nimesulide, flurbiprofen, and diclofenac) on the proportions and stability of the two radicals in prostaglandin H synthase-2 (PGHS-2) pretreated with peroxide. The modulation of tyrosyl radical stability by cyclooxygenase site ligands offers insight into a mechanism to regulate the reactivity of PGHS tyrosyl radicals with cellular antioxidants.

Electron paramagnetic resonance (EPR) and optical spectroscopy is used for the characterization of cytochrome (cyt) [b.sub.561] on the wild-type and mutant forms of the cytochrome expressed in insect cells. The low-potential heme is assigned to matrix side of the membrane, suggesting that the membrane potential modulates electron transport across the chromaffin granules (CG) membrane.

A new nanotrigger (NT) is designed to synchronize and monitor an enzymatic activity interacting specifically with the conserved NADPH binding site. The analysis has shown that selective targeting of the nanotrigger to NADPH sites might allow controlled initiation of the enzymatic activity of numerous proteins containing an NADPH site.

Hydrogen bonding betweenTyr385 and Tyr348 is shown to be an important structural factor in the interaction of prostaglandin H synthase-2 (PGHS-2) with aspirin. One factor controlling the efficiency of coupling between the PGHS peroxidase and cycloogenase reactions is the rate of Tyr385 radical formation.

The presence of a hydrogen bond to the wide doublet (WD) radical, as well as the distribution in gx values is determined with the help of a high-frequency (HF) EPR and ENDOR study. It is concluded that hydrogen-bounding networks provide pathways for proton-coupled electron transfer in enzymes.

Hydroperoxides induce formation of a tyrosyl radical on Tyr385 in prostaglandin H synthase (PGHS). The Tyr385 radical initiates hydrogen abstraction from arachidonic acid, thereby mechanistically connecting the peroxidase and cyclooxygenase activities.

Research reveals that inactivation of prostaglandin H synthase-1 by peroxide in the presence of cosubstrate leads to a large inactivation of the cyclooxygenase that cause damages to the cyclooxygenase active sites, which acquire a ferryl heme in the peroxidase site and a tyrosyl free radical in the cyclooxygenase site.

Original citation: J Clin Invest. 2014;124(6):2750-2761. doi:10.1172/JCI74604. Citation for this corrigendum: J Clin Invest. 2014;124(7):3274. doi: 10.1172/JCI77393. During the preparation for this manuscript, the authors provided an incorrect citation for [...]

Characterization of a pentadienyl radical intermediate formed during prostaglandin H synthase-2 catalysis is described.