HIV-1 Tat and morphine interactions dynamically shift striatal monoamine levels and exploratory behaviors over time.

Despite the advent of combination anti-retroviral therapy (cART), nearly half of people infected with HIV treated with cART still exhibit HIV-associated neurocognitive disorders (HAND). HAND can be worsened by co-morbid opioid use disorder. The basal ganglia are particularly vulnerable to HIV-1 and exhibit higher viral loads and more severe pathology, which can be exacerbated by co-exposure to opioids. Evidence suggests that dopaminergic neurotransmission is disrupted by HIV exposure, however, little is known about whether co-exposure to opioids may alter neurotransmitter levels in the striatum and if this in turn influences behavior. Therefore, we assayed motor, anxiety-like, novelty-seeking, exploratory, and social behaviors, and levels of monoamines and their metabolites following 2 weeks and 2 months of Tat and/or morphine exposure in transgenic mice. Morphine decreased dopamine levels, but significantly elevated norepinephrine, the dopamine metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and the serotonin metabolite 5-hydroxyindoleacetic acid, which typically correlated with increased locomotor behavior. The combination of Tat and morphine altered dopamine, DOPAC, and HVA concentrations differently depending on the neurotransmitter/metabolite and duration of exposure but did not affect the numbers of tyrosine hydroxylase-positive neurons in the mesencephalon. Tat exposure increased the latency to interact with novel conspecifics, but not other novel objects, suggesting the viral protein inhibits exploratory behavior initiation in a context-dependent manner. By contrast, and consistent with prior findings that opioid misuse can increase novelty-seeking behavior, morphine exposure increased the time spent exploring a novel environment. Finally, Tat and morphine interacted to affect locomotor activity in a time-dependent manner, while grip strength and rotarod performance were unaffected. Together, our results provide novel insight into the unique effects of HIV-1 Tat and morphine on monoamine neurochemistry that may underlie their divergent effects on motor and exploratory behavior.

Identification of novel F2-isoprostane metabolites by specific UDP-glucuronosyltransferases.

UDP-glucuronosyltransferases (UGTs) catalyze the conjugation of glucuronic acid with endogenous and exogenous lipophilic small molecules to facilitate their inactivation and excretion from the body. This represents approximately 35 % of all phase II metabolic transformations. Fatty acids and their oxidized eicosanoid derivatives can be metabolized by UGTs. F2-isoprostanes (F2-IsoPs) are eicosanoids formed from the free radical oxidation of arachidonic acid. These molecules are potent vasoconstrictors and are widely used as biomarkers of endogenous oxidative damage. An increasing body of evidence demonstrates the efficacy of measuring the ß-oxidation metabolites of F2-IsoPs rather than the unmetabolized F2-IsoPs to quantify oxidative damage in certain settings. Yet, the metabolism of F2-IsoPs is incompletely understood. This study sought to identify and characterize novel phase II metabolites of 15-F2t-IsoP and 5-epi-5-F2t-IsoP, two abundantly produced F2-IsoPs, in human liver microsomes (HLM). Utilizing liquid chromatography-mass spectrometry, we demonstrated that glucuronide conjugates are the major metabolites of these F2-IsoPs in HLM. Further, we showed that these molecules are metabolized by specific UGT isoforms. 15-F2t-IsoP is metabolized by UGT1A3, 1A9, and 2B7, while 5-epi-5-F2t-IsoP is metabolized by UGT1A7, 1A9, and 2B7. We identified, for the first time, the formation of intact glucuronide F2-IsoPs in human urine and showed that F2-IsoP glucuronidation is reduced in people supplemented with eicosapentaenoic and docosahexaenoic acids for 12 weeks. These studies demonstrate that endogenous F2-IsoP levels can be modified by factors other than redox mechanisms.

Transiently increased serotonin has modest or no effects on bone mass accrual in growing female C57BL6/J or growing male and female Lrp5A214V mice.

Serotonin (5HT) is a chemical messenger with biologic activities affecting multiple organs. Within the skeletal system, studies in mice and humans suggest blood 5HT levels affect bone, with elevations impairing and reductions enhancing bone accrual. Other studies, however, have not supported this hypothesis. Recently, administering 5HT to a Piezo1 mutant mouse strain with hyposerotonemia, intestinal dysmotility, and a doubling of femoral trabecular bone mass at 2 months of age, was reported to return the animals' intestinal motility and bone mass to normal. However, whether the 5HT directly affected bone metabolism or indirectly affected metabolism by improving intestinal function was not determined. Therefore, we administered 5HT to mice with normal intestinal function. We randomized female C57BL6/J mice and male and female mice that have increased bone mass due to a missense mutation in the WNT co-receptor LRP5 (Lrp5A214V) to receive 5HT or vehicle via daily IP injection from 4 weeks to 8 weeks of age. We did not observe consistent significant changes for distal femur trabecular, midshaft femur cortical, or vertebral body trabecular bone mass between 5HT treated and vehicle treated mice of either genotype. These data are compatible with other studies that have not observed a correlation between blood 5HT level and bone mass.

Measurement of F2- isoprostanes and isofurans using gas chromatography-mass spectrometry.

F2-Isoprostanes (IsoPs) are isomers of prostaglandin F2α formed from the nonenzymatic free radical-catalyzed peroxidation of arachidonic acid. Since discovery of these molecules by Morrow and Roberts in 1990, F2-IsoPs have been shown to be excellent biomarkers as well as potent mediators of oxidative stress in vivo in humans. Isofurans (IsoFs) are also oxidation products generated from the nonenzymatic oxidation of arachidonic acid. IsoFs are preferentially formed instead of F2-IsoPs in settings of increased oxygen tension. The protocol presented herein is the current methodology that our laboratory uses to quantify F2-IsoPs and IsoFs in biological tissues and fluids using gas chromatography/mass spectrometry (GC/MS). A variety of analytical procedures to measure F2-IsoPs, including other GC/MS methods and liquid chromatography/MS and immunological approaches, are reported in the literature. This method provides a very low limit of quantitation and is suitable for analysis of both F2-IsoPs and IsoFs from a variety of biological sources including urine, plasma, tissues, cerebral spinal fluid, exhaled breath condensate, and amniotic fluid, among others.

Omega-3 fatty acids are oxygenated at the n-7 carbon by the lipoxygenase domain of a fusion protein in the cyanobacterium Acaryochloris marina.

Lipoxygenases (LOX) are found in most organisms that contain polyunsaturated fatty acids, usually existing as individual genes although occasionally encoded as a fusion protein with a catalase-related hemoprotein. Such a fusion protein occurs in the cyanobacterium Acaryochloris marina and herein we report the novel catalytic activity of its LOX domain. The full-length protein and the C-terminal LOX domain were expressed in Escherichia coli, and the catalytic activities characterized by UV, HPLC, GC-MS, and CD. All omega-3 polyunsaturates were oxygenated by the LOX domain at the n-7 position and with R stereospecificity: alpha-linolenic and the most abundant fatty acid in A. marina, stearidonic acid (C18.4omega3), are converted to the corresponding 12R-hydroperoxides, eicosapentaenoic acid to its 14R-hydroperoxide, and docosahexaenoic acid to its 16R-hydroperoxide. Omega-6 polyunsaturates were oxygenated at the n-10 position, forming 9R-hydroperoxy-octadecadienoic acid from linoleic acid and 11R-hydroperoxy-eicosatetraenoic acid from arachidonic acid. The metabolic transformation of stearidonic acid by the full-length fusion protein entails its 12R oxygenation with subsequent conversion by the catalase-related domain to a novel allene epoxide, a likely precursor of cyclopentenone fatty acids or other signaling molecules (Gao et al, J. Biol. Chem. 284:22087-98, 2009). Although omega-3 fatty acids and lipoxygenases are of widespread occurrence, this appears to be the first description of a LOX-catalyzed oxygenation that specifically utilizes the terminal pentadiene of omega-3 fatty acids.

The structure and peroxidase activity of a 33-kDa catalase-related protein from Mycobacterium avium ssp. paratuberculosis.

True catalases are tyrosine-liganded, usually tetrameric, hemoproteins with subunit sizes of approximately 55-84 kDa. Recently characterized hemoproteins with a catalase-related structure, yet lacking in catalatic activity, include the 40-43 kDa allene oxide synthases of marine invertebrates and cyanobacteria. Herein, we describe the 1.8 A X-ray crystal structure of a 33 kDa subunit hemoprotein from Mycobacterium avium ssp. paratuberculosis (annotated as MAP-2744c), that retains the core elements of the catalase fold and exhibits an organic peroxide-dependent peroxidase activity. MAP-2744c exhibits negligible catalatic activity, weak peroxidatic activity using hydrogen peroxide (20/s) and strong peroxidase activity (approximately 300/s) using organic hydroperoxides as co-substrate. Key amino acid differences significantly impact prosthetic group conformation and placement and confer a distinct activity to this prototypical member of a group of conserved bacterial "minicatalases". Its structural features and the result of the enzyme assays support a role for MAP-2744c and its close homologues in mitigating challenge by a variety of reactive oxygen species.

Evidence for an ionic intermediate in the transformation of fatty acid hydroperoxide by a catalase-related allene oxide synthase from the Cyanobacterium Acaryochloris marina.

Allene oxides are reactive epoxides biosynthesized from fatty acid hydroperoxides by specialized cytochrome P450s or by catalase-related hemoproteins. Here we cloned, expressed, and characterized a gene encoding a lipoxygenase-catalase/peroxidase fusion protein from Acaryochloris marina. We identified novel allene oxide synthase (AOS) activity and a by-product that provides evidence of the reaction mechanism. The fatty acids 18.4omega3 and 18.3omega3 are oxygenated to the 12R-hydroperoxide by the lipoxygenase domain and converted to the corresponding 12R,13-epoxy allene oxide by the catalase-related domain. Linoleic acid is oxygenated to its 9R-hydroperoxide and then, surprisingly, converted approximately 70% to an epoxyalcohol identified spectroscopically and by chemical synthesis as 9R,10S-epoxy-13S-hydroxyoctadeca-11E-enoic acid and only approximately 30% to the 9R,10-epoxy allene oxide. Experiments using oxygen-18-labeled 9R-hydroperoxide substrate and enzyme incubations conducted in H2(18)O indicated that approximately 72% of the oxygen in the epoxyalcohol 13S-hydroxyl arises from water, a finding that points to an ionic intermediate (epoxy allylic carbocation) during catalysis. AOS and epoxyalcohol synthase activities are mechanistically related, with a reacting intermediate undergoing a net hydrogen abstraction or hydroxylation, respectively. The existence of epoxy allylic carbocations in fatty acid transformations is widely implicated although for AOS reactions, without direct experimental support. Our findings place together in strong association the reactions of allene oxide synthesis and an ionic reaction intermediate in the AOS-catalyzed transformation.

Role of the conserved distal heme asparagine of coral allene oxide synthase (Asn137) and human catalase (Asn148): mutations affect the rate but not the essential chemistry of the enzymatic transformations.

A catalase-related allene oxide synthase (cAOS) and true catalases that metabolize hydrogen peroxide have similar structure around the heme. One of the distal heme residues considered to help control catalysis is a highly conserved asparagine. Here we addressed the role of this residue in metabolism of the natural substrate 8R-hydroperoxyeicosatetraenoic acid by cAOS and in H(2)O(2) breakdown by catalase. In cAOS, the mutations N137A, N137Q, N137S, N137D, and N137H drastically reduced the rate of reaction (to 0.8-4% of wild-type), yet the mutants all formed the allene oxide as product. This is remarkable because there are many potential heme-catalyzed transformations of fatty acid hydroperoxides and special enzymatic control must be required. In human catalase, the N148A, N148S, or N148D mutations only reduced rates to approximately 20% of wild-type. The distal heme Asn is not essential in either catalase or cAOS. Its conservation throughout evolution may relate to a role in optimizing catalysis.

Mechanism of flavin reduction in the alkanesulfonate monooxygenase system.

The alkanesulfonate monooxygenase system from Escherichia coli is involved in scavenging sulfur from alkanesulfonates under sulfur starvation. An FMN reductase (SsuE) catalyzes the reduction of FMN by NADPH, and the reduced flavin is transferred to the monooxygenase (SsuD). Rapid reaction kinetic analyses were performed to define the microscopic steps involved in SsuE catalyzed flavin reduction. Results from single-wavelength analyses at 450 and 550 nm showed that reduction of FMN occurs in three distinct phases. Following a possible rapid equilibrium binding of FMN and NADPH to SsuE (MC-1) that occurs before the first detectable step, an initial fast phase (241 s(-1)) corresponds to the interaction of NADPH with FMN (CT-1). The second phase is a slow conversion (11 s(-1)) to form a charge-transfer complex of reduced FMNH(2) with NADP(+) (CT-2), and represents electron transfer from the pyridine nucleotide to the flavin. The third step (19 s(-1)) is the decay of the charge-transfer complex to SsuE with bound products (MC-2) or product release from the CT-2 complex. Results from isotope studies with [(4R)-(2)H]NADPH demonstrates a rate-limiting step in electron transfer from NADPH to FMN, and may imply a partial rate-limiting step from CT-2 to MC-2 or the direct release of products from CT-2. While the utilization of flavin as a substrate by the alkanesulfonate monooxygenase system is novel, the mechanism for flavin reduction follows an analogous reaction path as standard flavoproteins.

Altered mechanism of the alkanesulfonate FMN reductase with the monooxygenase enzyme.

The two-component alkanesulfonate monooxygenase system from Escherichia coli is comprised of an FMN reductase (SsuE) and a monooxygenase enzyme (SsuD) that together catalyze the oxidation of alkanesulfonate to the corresponding aldehyde and sulfite products. To determine the effects of protein interactions on catalysis, the steady-state kinetic parameters for SsuE were determined in single-enzyme assays and in the presence of the monooxygenase enzyme and alkanesulfonate substrate. In single-enzyme kinetic assays, SsuE followed an ordered sequential mechanism, with NADPH as the first substrate to bind and NADP+ as the last product to dissociate. However, in the presence of SsuD and octanesulfonate the kinetic mechanism of SsuE is altered to a rapid equilibrium ordered mechanism, and the Km value for FMN is increased 10-fold. These results suggest that both the SsuD enzyme and alkanesulfonate substrate are required to ensure that the FMN reductase reaction proceeds to form the ternary complex with the subsequent generation of reduced flavin transfer.

Crystallization and preliminary X-ray crystallographic studies of the alkanesulfonate FMN reductase from Escherichia coli.

The alkanesulfonate FMN reductase (SsuE) from Escherichia coli catalyzes the reduction of FMN by NADPH to provide reduced flavin for the monooxygenase (SsuD) enzyme. The vapor-diffusion technique yielded single crystals that grow as hexagonal rods and diffract to 2.9 A resolution using synchrotron X-ray radiation. The protein crystallizes in the primitive hexagonal space group P622. The SsuE protein lacks any cysteine or methionine residues owing to the role of the SsuE enzyme in the acquisition of sulfur during sulfate starvation. Therefore, substitution of two leucine residues (Leu114 and Leu165) to methionine was performed to obtain selenomethionine-containing SsuE for MAD phasing. The selenomethionine derivative of SsuE has been expressed and purified and crystals of the protein have been obtained with and without bound FMN. These preliminary studies should lead to the structure solution of SsuE. It is anticipated that this new protein structure will provide detailed structural information on specific active-site regions of the protein and insight into the mechanism of flavin reduction and transfer of reduced flavin.