Dopamine production in neurotensin receptor 1 neurons is required for diet-induced obesity and increased day eating on a high-fat diet.

OBJECTIVE: This study aimed to determine a dopaminergic circuit required for diet-induced obesity in mice. METHODS: We created conditional deletion mutants for tyrosine hydroxylase (TH) using neurotensin receptor 1 (Ntsr1) Cre and other Cre drivers and measured feeding and body weight on standard and high-fat diets. We then used an adeno-associated virus to selectively restore TH to the ventral tegmental area (VTA) Ntsr1 neurons in conditional knockout (cKO) mice. RESULTS: Mice with cKO of Th using Vglut2-Cre, Cck-Cre, Calb1-Cre, and Bdnf-Cre were susceptible to obesity on a high-fat diet; however, Ntsr1-Cre Th cKO mice resisted weight gain on a high-fat diet and did not experience an increase in day eating unlike their wild-type littermate controls. Restoration of TH to the VTA Ntsr1 neurons of the Ntsr1-Cre Th cKO mice using an adeno-associated virus resulted in an increase in weight gain and day eating on a high-fat diet. CONCLUSIONS: Ntsr1-Cre Th cKO mice failed to increase day eating on a high-fat diet, offering a possible explanation for their resistance to diet-induced obesity. These results implicate VTA Ntsr1 dopamine neurons as promoting out-of-phase feeding behavior on a high-fat diet that could be an important contributor to diet-induced obesity in humans.

Biochemical and Structural Studies of the Carminomycin 4-O-Methyltransferase DnrK.

Structural and functional studies of the carminomycin 4-O-methyltransferase DnrK are described, with an emphasis on interrogating the acceptor substrate scope of DnrK. Specifically, the evaluation of 100 structurally and functionally diverse natural products and natural product mimetics revealed an array of pharmacophores as productive DnrK substrates. Representative newly identified DnrK substrates from this study included anthracyclines, angucyclines, anthraquinone-fused enediynes, flavonoids, pyranonaphthoquinones, and polyketides. The ligand-bound structure of DnrK bound to a non-native fluorescent hydroxycoumarin acceptor, 4-methylumbelliferone, along with corresponding DnrK kinetic parameters for 4-methylumbelliferone and native acceptor carminomycin are also reported for the first time. The demonstrated unique permissivity of DnrK highlights the potential for DnrK as a new tool in future biocatalytic and/or strain engineering applications. In addition, the comparative bioactivity assessment (cancer cell line cytotoxicity, 4E-BP1 phosphorylation, and axolotl embryo tail regeneration) of a select set of DnrK substrates/products highlights the ability of anthracycline 4-O-methylation to dictate diverse functional outcomes.

Cofactorless oxygenases guide anthraquinone-fused enediyne biosynthesis.

The anthraquinone-fused enediynes (AFEs) combine an anthraquinone moiety and a ten-membered enediyne core capable of generating a cytotoxic diradical species. AFE cyclization is triggered by opening the F-ring epoxide, which is also the site of the most structural diversity. Previous studies of tiancimycin A, a heavily modified AFE, have revealed a cryptic aldehyde blocking installation of the epoxide, and no unassigned oxidases could be predicted within the tnm biosynthetic gene cluster. Here we identify two consecutively acting cofactorless oxygenases derived from methyltransferase and α/ß-hydrolase protein folds, TnmJ and TnmK2, respectively, that are responsible for F-ring tailoring in tiancimycin biosynthesis by comparative genomics. Further biochemical and structural characterizations reveal that the electron-rich AFE anthraquinone moiety assists in catalyzing deformylation, epoxidation and oxidative ring cleavage without exogenous cofactors. These enzymes therefore fill important knowledge gaps for the biosynthesis of this class of molecules and the underappreciated family of cofactorless oxygenases.

Biological Evaluation of the Antibacterial Retinoid CD437 in Cutibacterium acnes Infection.

Acne vulgaris is a complex skin disease involving infection by Cutibacterium acnes, inflammation, and hyperkeratinization. We evaluated the activity of the retinoid 6-[3-(adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) and 16 other retinoid analogs as potential anti-C. acnes compounds and found that CD437 displayed the highest antimicrobial activity with an MIC against C. acnes (ATCC 6919 and HM-513) of 1 µg/mL. CD437 demonstrated an MBC of 2 µg/mL compared to up to 64 µg/mL for the retinoid adapalene and up to 16 µg/mL for tetracycline, which are commonly used clinically to treat acne. Membrane permeability assays demonstrated that exposure of C. acnes ATCC 6919 to CD437 damaged the integrity of C. acnes ATCC 6919 bacterial membranes, and this finding was confirmed with scanning electron microscopy. Additionally, CD437 downregulated the expression of C. acnes ATCC 6919 virulence factors, including the genes encoding Christie-Atkins-Munch-Petersen factor 1 (CAMP1), CAMP2, glycerol-ester hydrolase B (GehB), sialidase B, and neuraminidase. In a mouse skin infection model of C. acnes ATCC 6919, topical treatment with CD437 ameliorated skin lesions and reduced the bacterial burden in situ (P < 0.001). In human NHEK primary cells, CD437 reduced the transcriptional levels of the coding genes for inflammatory cytokines (interleukin-1α, ~10-fold; interleukin-6, ~20-fold; interleukin-8, ~30-fold; and tumor necrosis factor-alpha, ~6-fold) and downregulated the transcriptional levels of KRT10 (~10-fold), FLG (~4-fold), and TGM1 (~2-fold), indicating that CD437 can diminish inflammation and hyperkeratinization. In summary, CD437 deserves further attention for its dual function as a potential acne therapeutic that potentially acts on both the pathogen and the host.

Application of a Biocatalytic Strategy for the Preparation of Tiancimycin-Based Antibody-Drug Conjugates Revealing Key Insights into Structure-Activity Relationships.

Antibody-drug conjugates (ADCs) are cancer chemotherapeutics that utilize a monoclonal antibody (mAb)-based delivery system, a cytotoxic payload, and a chemical linker. ADC payloads must be strategically functionalized to allow linker attachment without perturbing the potency required for ADC efficacy. We previously developed a biocatalytic system for the precise functionalization of tiancimycin (TNM)-based payloads. The TNMs are anthraquinone-fused enediynes (AFEs) and have yet to be translated into the clinic. Herein, we report the translation of biocatalytically functionalized TNMs into ADCs in combination with the dual-variable domain (DVD)-mAb platform. The DVD enables both site-specific conjugation and a plug-and-play modularity for antigen-targeting specificity. We evaluated three linker chemistries in terms of TNM-based ADC potency and antigen selectivity, demonstrating a trade-off between potency and selectivity. This represents the first application of AFE-based payloads to DVDs for ADC development, a workflow that is generalizable to further advance AFE-based ADCs for multiple cancer types.

Thiocysteine lyases as polyketide synthase domains installing hydropersulfide into natural products and a hydropersulfide methyltransferase.

Nature forms S-S bonds by oxidizing two sulfhydryl groups, and no enzyme installing an intact hydropersulfide (-SSH) group into a natural product has been identified to date. The leinamycin (LNM) family of natural products features intact S-S bonds, and previously we reported an SH domain (LnmJ-SH) within the LNM hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line as a cysteine lyase that plays a role in sulfur incorporation. Here we report the characterization of an S-adenosyl methionine (SAM)-dependent hydropersulfide methyltransferase (GnmP) for guangnanmycin (GNM) biosynthesis, discovery of hydropersulfides as the nascent products of the GNM and LNM hybrid NRPS-PKS assembly lines, and revelation of three SH domains (GnmT-SH, LnmJ-SH, and WsmR-SH) within the GNM, LNM, and weishanmycin (WSM) hybrid NRPS-PKS assembly lines as thiocysteine lyases. Based on these findings, we propose a biosynthetic model for the LNM family of natural products, featuring thiocysteine lyases as PKS domains that directly install a -SSH group into the GNM, LNM, or WSM polyketide scaffold. Genome mining reveals that SH domains are widespread in Nature, extending beyond the LNM family of natural products. The SH domains could also be leveraged as biocatalysts to install an -SSH group into other biologically relevant scaffolds.

Transcriptomic Profiling Suggests That Promysalin Alters the Metabolic Flux, Motility, and Iron Regulation in Pseudomonas putida KT2440.

Promysalin, a secondary metabolite produced by P. putida RW10S1, is a narrow-spectrum antibiotic that targets P. aeruginosa over other Pseudomonas spp. P. putida KT2440, a nonproducing strain, displays increased swarming motility and decreased pyoverdine production in the presence of exogenous promysalin. Herein, proteomic and transcriptomic experiments were used to provide insight about how promysalin elicits responses in PPKT2440 and rationalize its species selectivity. RNA-sequencing results suggest that promysalin affects PPKT2440 by (1) increasing swarming in a flagella-independent manner; (2) causing cells to behave as if they were experiencing an iron-deficient environment, and (3) shifting metabolism away from glucose conversion to pyruvate via the Entner-Doudoroff pathway. These findings highlight nature's ability to develop small molecules with specific targets, resulting in exquisite selectivity.

Sex differences in circadian food anticipatory activity are not altered by individual manipulations of sex hormones or sex chromosome copy number in mice.

Recent studies in mice have demonstrated a sexual dimorphism in circadian entrainment to scheduled feeding. On a time restricted diet, males tend to develop food anticipatory activity (FAA) sooner than females and with a higher amplitude of activity. The underlying cause of this sex difference remains unknown. One study suggests that sex hormones, both androgens and estrogens, modulate food anticipatory activity in mice. Here we present results suggesting that the sex difference in FAA is unrelated to gonadal sex hormones. While a sex difference between males and females in FAA on a timed, calorie restricted diet was observed there were no differences between intact and gonadectomized mice in the onset or magnitude of FAA. To test other sources of the sex difference in circadian entrainment to scheduled feeding, we used sex chromosome copy number mutants, but there was no difference in FAA when comparing XX, XY-, XY-;Sry Tg, and XX;Sry Tg mice, demonstrating that gene dosage of sex chromosomes does not mediate the sex difference in FAA. Next, we masculinized female mice by treating them with 17-beta estradiol during the neonatal period; yet again, we saw no difference in FAA between control and masculinized females. Finally, we observed that there was no longer a sex difference in FAA for older mice, suggesting that the sex difference in FAA is age-dependent. Thus, our study demonstrates that singular manipulations of gonadal hormones, sex chromosomes, or developmental patterning are not able to explain the difference in FAA between young male and female mice.

Diverted Total Synthesis of Promysalin Analogs Demonstrates That an Iron-Binding Motif Is Responsible for Its Narrow-Spectrum Antibacterial Activity.

Promysalin is a species-specific Pseudomonad metabolite with unique bioactivity. To better understand the mode of action of this natural product, we synthesized 16 analogs utilizing diverted total synthesis (DTS). Our analog studies revealed that the bioactivity of promysalin is sensitive to changes within its hydrogen bond network whereby alteration has drastic biological consequences. The DTS library not only yielded three analogs that retained potency but also provided insights that resulted in the identification of a previously unknown ability of promysalin to bind iron. These findings coupled with previous observations hint at a complex multifaceted role of the natural product within the rhizosphere.

Daily scheduled high fat meals moderately entrain behavioral anticipatory activity, body temperature, and hypothalamic c-Fos activation.

When fed in restricted amounts, rodents show robust activity in the hours preceding expected meal delivery. This process, termed food anticipatory activity (FAA), is independent of the light-entrained clock, the suprachiasmatic nucleus, yet beyond this basic observation there is little agreement on the neuronal underpinnings of FAA. One complication in studying FAA using a calorie restriction model is that much of the brain is activated in response to this strong hunger signal. Thus, daily timed access to palatable meals in the presence of continuous access to standard chow has been employed as a model to study FAA in rats. In order to exploit the extensive genetic resources available in the murine system we extended this model to mice, which will anticipate rodent high fat diet but not chocolate or other sweet daily meals (Hsu, Patton, Mistlberger, and Steele; 2010, PLoS ONE e12903). In this study we test additional fatty meals, including peanut butter and cheese, both of which induced modest FAA. Measurement of core body temperature revealed a moderate preprandial increase in temperature in mice fed high fat diet but entrainment due to handling complicated interpretation of these results. Finally, we examined activation patterns of neurons by immunostaining for the immediate early gene c-Fos and observed a modest amount of entrainment of gene expression in the hypothalamus of mice fed a daily fatty palatable meal.

Cholinergic modulation of locomotion and striatal dopamine release is mediated by alpha6alpha4* nicotinic acetylcholine receptors.

Dopamine (DA) release in striatum is governed by firing rates of midbrain DA neurons, striatal cholinergic tone, and nicotinic ACh receptors (nAChRs) on DA presynaptic terminals. DA neurons selectively express alpha6* nAChRs, which show high ACh and nicotine sensitivity. To help identify nAChR subtypes that control DA transmission, we studied transgenic mice expressing hypersensitive alpha6(L9'S)* receptors. alpha6(L9'S) mice are hyperactive, travel greater distance, exhibit increased ambulatory behaviors such as walking, turning, and rearing, and show decreased pausing, hanging, drinking, and grooming. These effects were mediated by alpha6alpha4* pentamers, as alpha6(L9'S) mice lacking alpha4 subunits displayed essentially normal behavior. In alpha6(L9'S) mice, receptor numbers are normal, but loss of alpha4 subunits leads to fewer and less sensitive alpha6* receptors. Gain-of-function nicotine-stimulated DA release from striatal synaptosomes requires alpha4 subunits, implicating alpha6alpha4beta2* nAChRs in alpha6(L9'S) mouse behaviors. In brain slices, we applied electrochemical measurements to study control of DA release by alpha6(L9'S) nAChRs. Burst stimulation of DA fibers elicited increased DA release relative to single action potentials selectively in alpha6(L9'S), but not WT or alpha4KO/alpha6(L9'S), mice. Thus, increased nAChR activity, like decreased activity, leads to enhanced extracellular DA release during phasic firing. Bursts may directly enhance DA release from alpha6(L9'S) presynaptic terminals, as there was no difference in striatal DA receptor numbers or DA transporter levels or function in vitro. These results implicate alpha6alpha4beta2* nAChRs in cholinergic control of DA transmission, and strongly suggest that these receptors are candidate drug targets for disorders involving the DA system.

Loss of Hsp70 exacerbates pathogenesis but not levels of fibrillar aggregates in a mouse model of Huntington's disease.

Endogenous protein quality control machinery has long been suspected of influencing the onset and progression of neurodegenerative diseases characterized by accumulation of misfolded proteins. Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expansion of a polyglutamine (polyQ) tract in the protein huntingtin (htt), which leads to its aggregation and accumulation in inclusion bodies. Here, we demonstrate in a mouse model of HD that deletion of the molecular chaperones Hsp70.1 and Hsp70.3 significantly exacerbated numerous physical, behavioral and neuropathological outcome measures, including survival, body weight, tremor, limb clasping and open field activities. Deletion of Hsp70.1 and Hsp70.3 significantly increased the size of inclusion bodies formed by mutant htt exon 1, but surprisingly did not affect the levels of fibrillar aggregates. Moreover, the lack of Hsp70s significantly decreased levels of the calcium regulated protein c-Fos, a marker for neuronal activity. In contrast, deletion of Hsp70s did not accelerate disease in a mouse model of infectious prion-mediated neurodegeneration, ruling out the possibility that the Hsp70.1/70.3 mice are nonspecifically sensitized to all protein misfolding disorders. Thus, endogenous Hsp70s are a critical component of the cellular defense against the toxic effects of misfolded htt protein in neurons, but buffer toxicity by mechanisms independent of the deposition of fibrillar aggregates.

Direct and selective elimination of specific prions and amyloids by 4,5-dianilinophthalimide and analogs.

Mechanisms to safely eliminate amyloids and preamyloid oligomers associated with many devastating diseases are urgently needed. Biophysical principles dictate that small molecules are unlikely to perturb large intermolecular protein-protein interfaces, let alone extraordinarily stable amyloid interfaces. Yet 4,5-dianilinophthalimide (DAPH-1) reverses Abeta42 amyloidogenesis and neurotoxicity, which is associated with Alzheimer's disease. Here, we show that DAPH-1 and select derivatives are ineffective against several amyloidogenic proteins, including tau, alpha-synuclein, Ure2, and PrP, but antagonize the yeast prion protein, Sup35, in vitro and in vivo. This allowed us to exploit several powerful new tools created for studying the conformational transitions of Sup35 and decipher the mechanisms by which DAPH-1 and related compounds antagonize the prion state. During fibrillization, inhibitory DAPHs alter the folding of Sup35's amyloidogenic core, preventing amyloidogenic oligomerization and specific recognition events that nucleate prion assembly. Select DAPHs also are capable of attacking preformed amyloids. They remodel Sup35 prion-specific intermolecular interfaces to create morphologically altered aggregates with diminished infectivity and self-templating activity. Our studies provide mechanistic insights and reinvigorate hopes for small-molecule therapies that specifically disrupt intermolecular amyloid contacts.

Diminishing apoptosis by deletion of Bax or overexpression of Bcl-2 does not protect against infectious prion toxicity in vivo.

B-cell lymphoma protein 2 (Bcl-2) and Bcl-2-associated X protein (Bax), key antiapoptotic and proapoptotic proteins, respectively, have important roles in acute and chronic models of neurologic disease. Several studies have implicated Bax and Bcl-2 in mediating neurotoxicity in prion diseases. To determine whether diminishing apoptotic cell death is protective in an infectious prion disease model we inoculated mice that either were null for proapoptotic Bax or overexpressed antiapoptotic Bcl-2. Interestingly, genetic manipulation of apoptosis did not lessen the clinical severity of disease. Moreover, some disease parameters, such as behavioral alterations and death, occurred slightly earlier in mice that are null for Bax or overexpress Bcl-2. These results suggest that Bax and Bcl-2 mediated apoptotic pathways are not the major contributing factor to the clinical or pathological features of infectious prion disease.

Prion protein is expressed on long-term repopulating hematopoietic stem cells and is important for their self-renewal.

Although the wild-type prion protein (PrP) is abundant and widely expressed in various types of tissues and cells, its physiological function(s) remain unknown, and PrP knockout mice do not exhibit overt and undisputed phenotypes. Here we showed that PrP is expressed on the surface of several bone marrow cell populations successively enriched in long-term (LT) hematopoietic stem cells (HSCs) using flow cytometry analysis. Affinity purification of the PrP-positive and -negative fractions from these populations, followed by competitive bone marrow reconstitution assays, shows that all LT HSCs express PrP. HSCs from PrP-null bone marrow exhibited impaired self-renewal in serial transplantation of lethally irradiated mouse recipients both in the presence and absence of competitors. When treated with a cell cycle-specific myelotoxic agent, the animals reconstituted with PrP-null HSCs exhibit increased sensitivity to hematopoietic cell depletion. Ectopic expression of PrP in PrP-null bone marrow cells by retroviral infection rescued the defective hematopoietic engraftment during serial transplantation. Therefore, PrP is a marker for HSCs and supports their self-renewal.

Increase in activity during calorie restriction requires Sirt1.

Sir2 (silent information regulator 2) is a nicotinamide adenine dinucleotide-dependent deacetylase required for longevity due to calorie restriction in yeast and Drosophila. In mammals, calorie restriction induces a complex pattern of physiological and behavioral changes. Here we report that the mammalian Sir2 ortholog, Sirt1, is required for the induction of a phenotype by calorie restriction in mice.