Prospecting for unannotated enzymes: discovery of a 3',5'-nucleotide bisphosphate phosphatase within the amidohydrolase superfamily.

In bacteria, 3',5'-adenosine bisphosphate (pAp) is generated from 3'-phosphoadenosine 5'-phosphosulfate in the sulfate assimilation pathway, and from coenzyme A by the transfer of the phosphopantetheine group to the acyl-carrier protein. pAp is subsequently hydrolyzed to 5'-AMP and orthophosphate, and this reaction has been shown to be important for superoxide stress tolerance. Herein, we report the discovery of the first instance of an enzyme from the amidohydrolase superfamily that is capable of hydrolyzing pAp. Crystal structures of Cv1693 from Chromobacterium violaceum have been determined to a resolution of 1.9 Å with AMP and orthophosphate bound in the active site. The enzyme has a trinuclear metal center in the active site with three Mn(2+) ions. This enzyme (Cv1693) belongs to the Cluster of Orthologous Groups cog0613 from the polymerase and histidinol phosphatase family of enzymes. The values of kcat and kcat/Km for the hydrolysis of pAp are 22 s(-1) and 1.4 × 10(6) M(-1) s(-1), respectively. The enzyme is promiscuous and is able to hydrolyze other 3',5'-bisphosphonucleotides (pGp, pCp, pUp, and pIp) and 2'-deoxynucleotides with comparable catalytic efficiency. The enzyme is capable of hydrolyzing short oligonucleotides (pdA)5, albeit at rates much lower than that of pAp. Enzymes from two other enzyme families have previously been found to hydrolyze pAp at physiologically significant rates. These enzymes include CysQ from Escherichia coli (cog1218) and YtqI/NrnA from Bacillus subtilis (cog0618). Identification of the functional homologues to the experimentally verified pAp phosphatases from cog0613, cog1218, and cog0618 suggests that there is relatively little overlap of enzymes with this function in sequenced bacterial genomes.

Substrate deconstruction and the nonadditivity of enzyme recognition.

Predicting substrates for enzymes of unknown function is a major postgenomic challenge. Substrate discovery, like inhibitor discovery, is constrained by our ability to explore chemotypes; it would be expanded by orders of magnitude if reactive sites could be probed with fragments rather than fully elaborated substrates, as is done for inhibitor discovery. To explore the feasibility of this approach, substrates of six enzymes from three different superfamilies were deconstructed into 41 overlapping fragments that were tested for activity or binding. Surprisingly, even those fragments containing the key reactive group had little activity, and most fragments did not bind measurably, until they captured most of the substrate features. Removing a single atom from a recognized substrate could often reduce catalytic recognition by 6 log-orders. To explore recognition at atomic resolution, the structures of three fragment complexes of the ß-lactamase substrate cephalothin were determined by X-ray crystallography. Substrate discovery may be difficult to reduce to the fragment level, with implications for function discovery and for the tolerance of enzymes to metabolite promiscuity. Pragmatically, this study supports the development of libraries of fully elaborated metabolites as probes for enzyme function, which currently do not exist.

Discovery of a cyclic phosphodiesterase that catalyzes the sequential hydrolysis of both ester bonds to phosphorus.

The bacterial C-P lyase pathway is responsible for the metabolism of unactivated organophosphonates under conditions of phosphate starvation. The cleavage of the C-P bond within ribose-1-methylphosphonate-5-phosphate to form methane and 5-phospho-ribose-1,2-cyclic phosphate (PRcP) is catalyzed by the radical SAM enzyme PhnJ. In Escherichia coli the cyclic phosphate product is hydrolyzed to ribose-1,5-bisphosphate by PhnP. In this study, we describe the discovery and characterization of an enzyme that can hydrolyze a cyclic phosphodiester directly to a vicinal diol and inorganic phosphate. With PRcP, this enzyme hydrolyzes the phosphate ester at carbon-1 of the ribose moiety to form ribose-2,5-bisphosphate, and then this intermediate is hydrolyzed to ribose-5-phosphate and inorganic phosphate. Ribose-1,5-bisphosphate is neither an intermediate nor a substrate for this enzyme. Orthologues of this enzyme are found in the human pathogens Clostridium difficile and Eggerthella lenta. We propose that this enzyme be called cyclic phosphate dihydrolase (cPDH) and be designated as PhnPP.

Predicting Functional Connectivity From Observed and Latent Structural Connectivity via Eigenvalue Mapping.

Understanding how complex dynamic activity propagates over a static structural network is an overarching question in the field of neuroscience. Previous work has demonstrated that linear graph-theoretic models perform as well as non-linear neural simulations in predicting functional connectivity with the added benefits of low dimensionality and a closed-form solution which make them far less computationally expensive. Here we show a simple model relating the eigenvalues of the structural connectivity and functional networks using the Gamma function, producing a reliable prediction of functional connectivity with a single model parameter. We also investigate the impact of local activity diffusion and long-range interhemispheric connectivity on the structure-function model and show an improvement in functional connectivity prediction when accounting for such latent variables which are often excluded from traditional diffusion tensor imaging (DTI) methods.

Structure, mechanism, and substrate profile for Sco3058: the closest bacterial homologue to human renal dipeptidase .

Human renal dipeptidase, an enzyme associated with glutathione metabolism and the hydrolysis of beta-lactams, is similar in sequence to a cluster of approximately 400 microbial proteins currently annotated as nonspecific dipeptidases within the amidohydrolase superfamily. The closest homologue to the human renal dipeptidase from a fully sequenced microbe is Sco3058 from Streptomyces coelicolor. Dipeptide substrates of Sco3058 were identified by screening a comprehensive series of l-Xaa-l-Xaa, l-Xaa-d-Xaa, and d-Xaa-l-Xaa dipeptide libraries. The substrate specificity profile shows that Sco3058 hydrolyzes a broad range of dipeptides with a marked preference for an l-amino acid at the N-terminus and a d-amino acid at the C-terminus. The best substrate identified was l-Arg-d-Asp (k(cat)/K(m) = 7.6 x 10(5) M(-1) s(-1)). The three-dimensional structure of Sco3058 was determined in the absence and presence of the inhibitors citrate and a phosphinate mimic of l-Ala-d-Asp. The enzyme folds as a (beta/alpha)(8) barrel, and two zinc ions are bound in the active site. Site-directed mutagenesis was used to probe the importance of specific residues that have direct interactions with the substrate analogues in the active site (Asp-22, His-150, Arg-223, and Asp-320). The solvent viscosity and kinetic effects of D(2)O indicate that substrate binding is relatively sticky and that proton transfers do not occurr during the rate-limiting step. A bell-shaped pH-rate profile for k(cat) and k(cat)/K(m) indicated that one group needs to be deprotonated and a second group must be protonated for optimal turnover. Computational docking of high-energy intermediate forms of l/d-Ala-l/d-Ala to the three-dimensional structure of Sco3058 identified the structural determinants for the stereochemical preferences for substrate binding and turnover.

Annotating enzymes of uncertain function: the deacylation of D-amino acids by members of the amidohydrolase superfamily.

The catalytic activities of three members of the amidohydrolase superfamily were discovered using amino acid substrate libraries. Bb3285 from Bordetella bronchiseptica, Gox1177 from Gluconobacter oxidans, and Sco4986 from Streptomyces coelicolor are currently annotated as d-aminoacylases or N-acetyl-d-glutamate deacetylases. These three enzymes are 22-34% identical to one another in amino acid sequence. Substrate libraries containing nearly all combinations of N-formyl-d-Xaa, N-acetyl-d-Xaa, N-succinyl-d-Xaa, and l-Xaa-d-Xaa were used to establish the substrate profiles for these enzymes. It was demonstrated that Bb3285 is restricted to the hydrolysis of N-acyl-substituted derivatives of d-glutamate. The best substrates for this enzyme are N-formyl-d-glutamate (k(cat)/K(m) = 5.8 x 10(6) M(-1) s(-1)), N-acetyl-d-glutamate (k(cat)/K(m) = 5.2 x 10(6) M(-1) s(-1)), and l-methionine-d-glutamate (k(cat)/K(m) = 3.4 x 10(5) M(-1) s(-1)). Gox1177 and Sco4986 preferentially hydrolyze N-acyl-substituted derivatives of hydrophobic d-amino acids. The best substrates for Gox1177 are N-acetyl-d-leucine (k(cat)/K(m) = 3.2 x 10(4) M(-1) s(-1)), N-acetyl-d-tryptophan (k(cat)/K(m) = 4.1 x 10(4) M(-1) s(-1)), and l-tyrosine-d-leucine (k(cat)/K(m) = 1.5 x 10(4) M(-1) s(-1)). A fourth protein, Bb2785 from B. bronchiseptica, did not have d-aminoacylase activity. The best substrates for Sco4986 are N-acetyl-d-phenylalanine and N-acetyl-d-tryptophan. The three-dimensional structures of Bb3285 in the presence of the product acetate or a potent mimic of the tetrahedral intermediate were determined by X-ray diffraction methods. The side chain of the d-glutamate moiety of the inhibitor is ion-paired to Arg-295, while the alpha-carboxylate is ion-paired with Lys-250 and Arg-376. These results have revealed the chemical and structural determinants for substrate specificity in this protein. Bioinformatic analyses of an additional approximately 250 sequences identified as members of this group suggest that there are no simple motifs that allow prediction of substrate specificity for most of these unknowns, highlighting the challenges for computational annotation of some groups of homologous proteins.

Ovarian Hormones Mediate Changes in Adaptive Choice and Motivation in Female Rats.

In female rodents, sexual receptivity is coordinated with cyclic changes in the release of gonadal hormones. Increases in estradiol (E) and progesterone (P) during proestrus and estrus not only induce ovulation but also modulate behaviors that increase the likelihood that the female will find a mate and reproduce. This includes changes in receptive behaviors, such as lordosis, as well as changes in appetitive or proceptive behaviors, including motivation. Interestingly, the direction of these changes in motivation is dependent on the type of reward that is being pursued. While induction of sexual receptivity by E and P increases motivation for access to a male, motivation for a palatable food reward is decreased. These concurrent changes may facilitate adaptive choice across the estrous cycle; females bias their choice for sex when fertilization is most likely to occur, but for food when copulation is unlikely to result in impregnation. In order to test this hypothesis, we developed a novel paradigm to measure the motivated choice between a palatable food reward and access to a male conspecific. Ovariectomized, hormone primed females were trained to operantly respond for both food and sex on a fixed interval (FI) schedule. After training, unprimed and primed females were tested in a chamber that allows them to choose between food and sex while still requiring responding on the FI schedule for reach reward. From this we can not only determine the impact of hormone priming on female choice for food or sex, but also how this is reflected by changes in motivation for each specific reward, as measured by the average number of responses made during each fixed interval. Induction of sexual receptivity by hormone priming biases choice toward sex over food and this change is accompanied by an increase in motivation for sex but a decrease in motivation for food. This work provides evidence in support of a novel framework for understanding how the release of ovarian hormones over the course of the estrous cycle modulates adaptive behavioral choice in females by directly assessing motivation via operant responding when multiple rewards are available.

Oestradiol influences on dopamine release from the nucleus accumbens shell: sex differences and the role of selective oestradiol receptor subtypes.

BACKGROUND AND PURPOSE: Females are more sensitive than males to both the acute and prolonged effects of psychomotor stimulants. In females, this is regulated by oestradiol, which enhances dopamine release in the dorsal striatum. In this study, we tested the acute effect of oestradiol on dopamine release in the nucleus accumbens (NAc) shell after cocaine administration and investigated which oestradiol receptors (ERs) contribute to sex differences in the response to cocaine. EXPERIMENTAL APPROACH: The ability of oestradiol benzoate (EB) to acutely modulate the effect of cocaine on phasic dopamine release in the NAc shell was measured by fast-scan cyclic voltammetry in anaesthetized male and female rats. The roles of ER subtypes, ERα and ERß, was determined with selective agonists. KEY RESULTS: EB acutely enhanced the effect of cocaine on stimulated dopamine release from the NAc shell in females but not in male rats only at levels of stimulation expected to optimally saturate dopamine transporters. Enhanced dopamine release after cocaine administration was also observed in females after selective activation of ERß but not ERα. EB attenuated the effect of cocaine on NAc shell dopamine reuptake in males but not in females. CONCLUSIONS AND IMPLICATIONS: Oestradiol acutely and rapidly regulates dopamine release in females and dopamine reuptake in males. In females, oestradiol rapidly enhances the effect of cocaine on dopamine release, likely via activation of ERß. The effect of oestradiol in males is not seen with selective receptor subtype activation, a topic deserving of further study. LINKED ARTICLES: This article is part of a themed section on The Importance of Sex Differences in Pharmacology Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.21/issuetoc.

An integrated multiplatform bioanalytical strategy for antibody-drug conjugates: a novel case study.

BACKGROUND: The bioanalytical strategy for antibody-drug conjugates (ADC) includes numerous measurements integrally designed to provide comprehensive characterization of PK, PD and immunogenicity. This manuscript describes the utilization of reagents specifically tailored to an ADC with a microtubule polymerization inhibitor payload and cathepsin B cleavable linker. METHODS: The PK strategy includes the evaluation of physiological levels of total antibody, active ADC, total ADC, antibody-conjugated payload and unconjugated payload. These data are evaluated in the context of target and antidrug antibody levels to elucidate bioactive ADC. RESULTS & CONCLUSION: Herein, we discuss how this strategy has been applied and present our preliminary observations. Continuously evolving to meet pipeline demands, the integrated bioanalytical data will provide critical insights into the exposure-response relationship.

Comparison of physical and emotional side effects of progesterone or medroxyprogesterone in early postmenopausal women.

OBJECTIVE: To compare the mood and somatic effects during the initial 2 months of medroxyprogesterone acetate (MPA) or progesterone combined with conjugated equine estrogen (CEE) in early postmenopausal women. DESIGN: Twenty-three nondepressed, early postmenopausal women (average age, 52.5 years) completed a 91-day, single-blind pilot study with the following sequence of treatments: 1 week of no substance; 2 weeks of placebo; 2 weeks of progestogen only; 1 week of placebo; and 2 months of "standard hormone replacement therapy cycles," which consisted of (in order) 2 weeks of 0.625 mg CEE, 2 weeks of CEE plus progestogen, 2 weeks of CEE, and 2 weeks of CEE plus progestogen. Ten women who completed the study received MPA (5 mg/day) as their progestogen, and 13 who completed the study received micronized, oil-suspended progesterone (200 mg/day) as their progestogen. All participants made daily assessments of mood using the Profile of Mood States and daily recordings of somatic symptoms. All subjects had plasma follicle-stimulating hormone of greater than 35 IU/L and had not had spontaneous vaginal bleeding for more than 1 year. RESULTS: None of the hormone treatments had a detectable effect on mood. MPA users reported more vaginal bleeding and breast tenderness than progesterone users. CONCLUSIONS: In contrast with the widely held belief among psychiatrists that progesterone depresses mood, neither of the progestogens we used in normal, nondepressed and nonanxious women showed this effect. Absence of an effect on mood was also found when the results of the two progestogens were combined. The lesser side effects of the micronized progesterone-containing regimen suggest that some women may prefer it to an MPA-containing regimen.

Sex differences in the effects of estradiol in the nucleus accumbens and striatum on the response to cocaine: neurochemistry and behavior.

BACKGROUND: Females exhibit more rapid escalation of cocaine use and enhanced cocaine-taking behavior as compared to males. While ovarian hormones likely play a role in this increased vulnerability, research has yet to examine the role of estradiol in affecting the behavioral and neurological response to cocaine in a brain region- and sex-specific way. METHODS: First, we examined stereotypy and locomotor sensitization after repeated cocaine administration (10 mg/kg i.p.) in intact (SHAM) and castrated (CAST) males, and ovariectomized (OVX) females treated with 5 µg estradiol benzoate (EB) or vehicle (OIL). Next, we used in vivo microdialysis to examine the effects of acute EB treatment on cocaine-induced DA in the regions mediating the display of these behaviors (i.e., the dorsolateral striatum, DLS; and the nucleus accumbens, NAc; respectively). RESULTS: We find that EB enhances sensitization of cocaine-induced stereotypy in OVX females after 12 days of cocaine treatment, and after a 10-day withdrawal. Similarly, the OVX/EB females show enhanced locomotor sensitization compared to the other three groups on the same days. Using in vivo microdialysis to assess the neurochemical response, we find that EB rapidly enhances cocaine-induced DA in DLS dialysate of OVX females but not CAST males, and has no effect in NAc of either sex. CONCLUSIONS: With these experiments, we show that there are sex differences in the effects of estradiol to preferentially enhance the response to cocaine in the DLS over the NAc in females, which may contribute to the preferential sensitization of stereotypy in females.

Estradiol, dopamine and motivation.

The gonadal hormone estradiol modulates mesolimbic dopamine systems in the female rat. This modulatory effect is thought to be responsible for the observed effects of estradiol on motivated behaviors. Dopamine acting in the nucleus accumbens is thought to be important for the attribution of incentive motivational properties to cues that predict reward delivery, while dopamine in the striatum is associated with the expression of repetitive or stereotyped behaviors. Elevated concentrations of estradiol are associated with increased motivation for sex or cues associated with access to a mate, while simultaneously attenuating motivation for food. This shift in motivational salience is important for adaptive choice behavior in the natural environment. Additionally, estradiol's adaptive effects on motivation can be maladaptive when increasing motivation for non-natural reinforcers, such as drugs of abuse. Here we discuss the effect of estradiol on mesotelencephalic dopamine transmission and subsequent effects on motivated behaviors.

Male rats that differ in novelty exploration demonstrate distinct patterns of sexual behavior.

High- versus low-novelty exploration predicts a variety of behavioral differences. For example, rats selectively bred for high-novelty exploration (bred-high responders, bHR) exhibit exaggerated aggression, impulsivity, and proclivity to addictive behaviors compared with low-novelty reactive rats (bred-low responders, bLRs), which are characterized by a high anxiety/depressive-like phenotype. Since bHR/bLR rats exhibit differences in dopaminergic circuitry and differential response to rewarding stimuli (i.e., psychostimulants, food), the present study examined whether they also differ in another key hedonic behavior-sex. Thus, adult bHR/bLR males were given five 30-min opportunities to engage in sexual activity with a receptive female. Sexual behavior and motivation were examined and compared between the groups. The bHR/bLR phenotype affected both sexual motivation and behavior, with bLR males demonstrating reduced motivation for sex compared with bHR males (i.e., fewer animals copulated, longer latency to engage in sex). The bHR males required more intromissions at a faster pace per ejaculation than did bLR males. Thus, neurobiological differences that affect motivation for drugs of abuse, aggression, and impulsivity in rats also affect sexual motivation and performance.

Quantitative assessment of female sexual motivation in the rat: Hormonal control of motivation.

While a good deal of information has been garnered in the last few decades regarding the neural and hormonal control of female sexual behavior, literature elucidating these mechanisms with respect to female sexual motivation has been scarce. We believe that one reason for this is the lack of a standardized paradigm that will quantify female sexual motivation while allowing for sexual interaction to occur. Here we describe a two-chambered apparatus that utilizes operant responding (nose poking) to quantify female sexual motivation. During the test, the female exhibits nose pokes to gain access to a sexually active male, with whom she is allowed to mate. Therefore, this apparatus allows for examination of sexual behavior as well as quantification of sexual motivation by assessing the number of nose pokes the female will exhibit within a fixed interval to gain access to the male. We report that hormone priming significantly increases sexual motivation in the female as indicated by the number of nose pokes she will exhibit to gain access to the male. Additionally, hormone primed females enter the male compartment after a shorter period and spend more time in direct contact with the male compared to when they are not hormone primed. In contrast, when females are not hormone primed they spend more time in view, but out of reach, of the male. This paradigm will help to advance the study of female sexual motivation, providing a method for quantifiable assessment of female sexual motivation while allowing for sexual activity to occur.

Effects of a selectively bred novelty-seeking phenotype on the motivation to take cocaine in male and female rats.

BACKGROUND: Gender and enhanced novelty reactivity can predispose certain individuals to drug abuse. Previous research in male and female rats selectively bred for high or low locomotor reactivity to novelty found that bred High Responders (bHRs) acquire cocaine self-administration more rapidly than bred Low Responders (bLRs) and that bHR females in particular self-administered more cocaine than the other groups. The experiments presented here aimed to determine whether an individual's sex and behavioral phenotype interact to affect motivation to take cocaine. METHODS: We examined motivation for taking cocaine in two experiments using a range of doses on a progressive ratio (PR) schedule of responding in bHR or bLR males and females. Additionally, we included a measure of continuing to respond in the absence of reinforcement, a feature of addiction that has been recently incorporated into tests of animal models on the basis of the criteria for substance use disorder in the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition. Statistical analyses were performed using PASW Statistics 18.0 software. Data were analyzed using repeated-measures analysis of variance followed by a Bonferroni correction post hoc test when applicable. RESULTS: We found sex differences as well as effects of novelty reactivity on the motivation to self-administer cocaine. Specifically, females demonstrated higher breaking points on the PR schedule compared with males, regardless of phenotype, and bHR males and females exhibited higher motivation than bLR animals at a number of the doses studied. CONCLUSIONS: An individual's sex continues to be a predisposing factor with respect to drug abuse liability and can be compounded by additional individual differences such as reactivity to novelty.