ABSTRACT
Highly substituted pyridine scaffolds are found in many biologically active natural products and therapeutics. Accordingly, numerous complementary de novo approaches to obtain differentially substituted pyridines have been disclosed. This article delineates the evolution of the synthetic strategies designed to assemble the demanding tetrasubstituted pyridine core present in the limonoid alkaloids isolated from Xylocarpus granatum, including xylogranatopyridine B, granatumine A and related congeners. In addition, NMR calculations suggested structural misassignment of several limonoid alkaloids, and predicted their C3-epimers as the correct structures, which was further validated unequivocally through chemical synthesis. The materials produced in this study were evaluated for cytotoxicity, anti-oxidant effects, anti-inflammatory action, PTP1B and Nlrp3 inflammasome inhibition, which led to compelling anti-inflammatory activity and anti-oxidant effects being discovered.
ABSTRACT
OBJECTIVE: To determine the role of fatty acid oxidation on the cellular, molecular, and physiologic response of brown adipose tissue to disparate paradigms of chronic thermogenic stimulation. METHODS: Mice with an adipose-specific loss of Carnitine Palmitoyltransferase 2 (Cpt2A-/-), that lack mitochondrial long chain fatty acid ß-oxidation, were subjected to environmental and pharmacologic interventions known to promote thermogenic programming in adipose tissue. RESULTS: Chronic administration of ß3-adrenergic (CL-316243) or thyroid hormone (GC-1) agonists induced a loss of BAT morphology and UCP1 expression in Cpt2A-/- mice. Fatty acid oxidation was also required for the browning of white adipose tissue (WAT) and the induction of UCP1 in WAT. In contrast, chronic cold (15 °C) stimulation induced UCP1 and thermogenic programming in both control and Cpt2A-/- adipose tissue albeit to a lesser extent in Cpt2A-/- mice. However, thermoneutral housing also induced the loss of UCP1 and BAT morphology in Cpt2A-/- mice. Therefore, adipose fatty acid oxidation is required for both the acute agonist-induced activation of BAT and the maintenance of quiescent BAT. Consistent with this data, Cpt2A-/- BAT exhibited increased macrophage infiltration, inflammation and fibrosis irrespective of BAT activation. Finally, obese Cpt2A-/- mice housed at thermoneutrality exhibited a loss of interscapular BAT and were refractory to ß3-adrenergic-induced energy expenditure and weight loss. CONCLUSION: Mitochondrial long chain fatty acid ß-oxidation is critical for the maintenance of the brown adipocyte phenotype both during times of activation and quiescence.
Subject(s)
Adipose Tissue, Brown/metabolism , Fatty Acids/metabolism , Obesity/metabolism , Thermogenesis , Animals , Carnitine O-Palmitoyltransferase/genetics , Carnitine O-Palmitoyltransferase/metabolism , Macrophages/metabolism , Male , Mice , Oxidation-Reduction , Uncoupling Protein 1/genetics , Uncoupling Protein 1/metabolismABSTRACT
Fatty acid oxidation in macrophages has been suggested to play a causative role in high-fat diet-induced metabolic dysfunction, particularly in the etiology of adipose-driven insulin resistance. To understand the contribution of macrophage fatty acid oxidation directly to metabolic dysfunction in high-fat diet-induced obesity, we generated mice with a myeloid-specific knockout of carnitine palmitoyltransferase II (CPT2 MÏ-KO), an obligate step in mitochondrial long-chain fatty acid oxidation. While fatty acid oxidation was clearly induced upon IL-4 stimulation, fatty acid oxidation-deficient CPT2 MÏ-KO bone marrow-derived macrophages displayed canonical markers of M2 polarization following IL-4 stimulation in vitro. In addition, loss of macrophage fatty acid oxidation in vivo did not alter the progression of high-fat diet-induced obesity, inflammation, macrophage polarization, oxidative stress, or glucose intolerance. These data suggest that although IL-4-stimulated alternatively activated macrophages upregulate fatty acid oxidation, fatty acid oxidation is dispensable for macrophage polarization and high-fat diet-induced metabolic dysfunction. Macrophage fatty acid oxidation likely plays a correlative, rather than causative, role in systemic metabolic dysfunction.
Subject(s)
Fatty Acids/immunology , Interleukin-4/immunology , Macrophage Activation/immunology , Macrophages/immunology , Metabolic Diseases/immunology , Obesity/immunology , Animals , Cells, Cultured , Diet, High-Fat , Male , Metabolic Diseases/pathology , Mice , Mice, Knockout , Mice, Transgenic , Oxidation-ReductionABSTRACT
Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) plays an important role in driving immortalization of EBV-infected B cells through regulating the expression of many viral and cellular genes. We report a structural study of the tumor suppressor BS69/ZMYND11 C-terminal region, comprised of tandem coiled-coil-MYND domains (BS69CC-MYND), in complex with an EBNA2 peptide containing a PXLXP motif. The coiled-coil domain of BS69 self-associates to bring two separate MYND domains in close proximity, thereby enhancing the BS69 MYND-EBNA2 interaction. ITC analysis of BS69CC-MYND with a C-terminal fragment of EBNA2 further suggests that the BS69CC-MYND homodimer synergistically binds to the two EBNA2 PXLXP motifs that are respectively located in the conserved regions CR7 and CR8. Furthermore, we showed that EBNA2 interacts with BS69 and down-regulates its expression at both mRNA and protein levels in EBV-infected B cells. Ectopic BS69CC-MYND is recruited to viral target promoters through interactions with EBNA2, inhibits EBNA2-mediated transcription activation, and impairs proliferation of lymphoblastoid cell lines (LCLs). Substitution of critical residues in the MYND domain impairs the BS69-EBNA2 interaction and abolishes the BS69 inhibition of the EBNA2-mediated transactivation and LCL proliferation. This study identifies the BS69 C-terminal domains as an inhibitor of EBNA2, which may have important implications in development of novel therapeutic strategies against EBV infection.
Subject(s)
Carrier Proteins/metabolism , Epstein-Barr Virus Infections/virology , Herpesvirus 4, Human/physiology , Viral Proteins/antagonists & inhibitors , Amino Acid Sequence , Animals , B-Lymphocytes/virology , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cell Cycle Proteins , Cell Line , Co-Repressor Proteins , Crystallography , DNA-Binding Proteins , Epstein-Barr Virus Nuclear Antigens/genetics , Epstein-Barr Virus Nuclear Antigens/metabolism , Humans , Mice , Molecular Sequence Data , Promoter Regions, Genetic/genetics , Protein Binding , Protein Interaction Mapping , Protein Structure, Tertiary , Sequence Alignment , Tandem Repeat Sequences , Transcriptional Activation , Viral Proteins/genetics , Viral Proteins/metabolismSubject(s)
Carnitine O-Palmitoyltransferase/genetics , Fatty Acids/metabolism , Macrophages/metabolism , Oxygen Consumption/physiology , Animals , Arginase/drug effects , Arginase/metabolism , Carnitine O-Palmitoyltransferase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Epoxy Compounds/pharmacology , Glycolysis , Intercellular Signaling Peptides and Proteins/metabolism , Interleukin-4/pharmacology , Lectins, C-Type/drug effects , Lectins, C-Type/metabolism , Macrophages/drug effects , Macrophages/immunology , Mice , Mice, Knockout , Oxidation-Reduction/drug effects , Oxygen Consumption/drug effectsABSTRACT
Gene-specific activation by enhancers involves their communication with the basal RNA polymerase II transcription machinery at the core promoter. Core promoters are diverse and may contain a variety of sequence elements such as the TATA box, the Initiator (INR), and the downstream promoter element (DPE) recognized, respectively, by the TATA-binding protein (TBP) and TBP-associated factors of the TFIID complex. Core promoter elements contribute to the gene selectivity of enhancers, and INR/DPE-specific enhancers and activators have been identified. Here, we identify a TATA box-selective activating sequence upstream of the human ß-actin (ACTB) gene that mediates serum response factor (SRF)-induced transcription from TATA-dependent but not INR-dependent promoters and requires the TATA-binding/bending activity of TBP, which is otherwise dispensable for transcription from a TATA-less promoter. The SRF-dependent ACTB sequence is stereospecific on TATA promoters but activates in an orientation-independent manner a composite TATA/INR-containing promoter. More generally, we show that SRF-regulated genes of the actin/cytoskeleton/contractile family tend to have a TATA box. These results suggest distinct TATA-dependent and INR-dependent mechanisms of TFIID-mediated transcription in mammalian cells that are compatible with only certain stereospecific combinations of activators, and that a TBP-TATA binding mechanism is important for SRF activation of the actin/cytoskeleton-related gene family.