Project description:Hyperlipidemia is a risk factor of arteriosclerosis, stroke, and other coronary heart disease, which has been shown to correlate with single nucleotide polymorphisms of genes essential for lipid metabolism, such as lipoprotein lipase (LPL) and apolipoprotein A5 (APOA5). In this study, the effect of magnolol, the main active component extracted from Magnolia officinalis, on LPL activity was investigated. A dose-dependent up-regulation of LPL activity, possibly through increasing LPL mRNA transcription, was observed in mouse 3T3-L1 pre-adipocytes cultured in the presence of magnolol for 6 days. Subsequently, a transgenic knock-in mice carrying APOA5 c.553G>T variant was established and then fed with corn oil with or without magnolol for four days. The baseline plasma triglyceride levels in transgenic knock-in mice were higher than those in wild-type mice, with the highest increase occurred in homozygous transgenic mice (106 mg/dL vs 51 mg/dL, p<0.01). After the induction of hyperglyceridemia along with the administration of magnolol, the plasma triglyceride level in heterozygous transgenic mice was significantly reduced by half. In summary, magnolol could effectively lower the plasma triglyceride levels in APOA5 c.553G>T variant carrier mice and facilitate the triglyceride metabolism in postprandial hypertriglyceridemia.
Project description:Previously, we reported that BRCA1 inhibits progesterone receptor (PR) activity and blocks progesterone-stimulated gene expression and cell proliferation. In the present manuscript, we studied the mechanism of BRCA1 inhibition of PR activity, using c-Myc as a model progesterone-regulated promoter. Here, we found that BRCA1 has little or no effect on PR ligand-binding affinity. However, BRCA1 overexpression inhibited the R5020-induced recruitment of PR to the c-Myc and mouse mammary tumor virus progesterone response elements (PREs) and blocked R5020-stimulated c-Myc expression, whereas BRCA1 underexpression did the opposite. In EMSAs, BRCA1 overexpression blocked the R5020-induced complex formation between PR and several radiolabeled PRE-containing oligonucleotides, and in vitro-translated BRCA1 blocked the interaction of full-length PR-A or a fragment containing the DNA-binding domain of PR with a radiolabeled PRE oligonucleotide. In further studies, BRCA1 overexpression inhibited the recruitment of coactivators (steroid receptor coactivator 1 and amplified in breast cancer 1) and enhanced the recruitment of a corepressor (histone deacetylase 1) to the c-Myc PRE, whereas BRCA1 knockdown increased the abundance of AIB1 and decreased the abundance of HDAC1 at the c-Myc PRE. These findings suggest that BRCA1 inhibits progestin-stimulated PR activity, in part, by preventing PR from binding to the PRE and by promoting the formation of a corepressor complex rather than a coactivator complex.
Project description:Epidemiological, genetic association, and Mendelian randomization studies have provided strong evidence that lipoprotein (a) [Lp(a)] is an independent causal risk factor for CVD, including myocardial infarction, stroke, peripheral arterial disease, and calcific aortic valve stenosis. Lp(a) levels >50 mg/dl are highly prevalent (20% of the general population) and are overrepresented in patients with CVD and aortic stenosis. These data support the notion that Lp(a) should be a target of therapy for CVD event reduction and to reduce progression of aortic stenosis. However, effective therapies to specifically reduce plasma Lp(a) levels are lacking. Recent animal and human studies have shown that Lp(a) can be specifically targeted with second generation antisense oligonucleotides (ASOs) that inhibit apo(a) mRNA translation. In apo(a) transgenic mice, an apo(a) ASO reduced plasma apo(a)/Lp(a) levels and their associated oxidized phospholipid (OxPL) levels by 86 and 93%, respectively. In cynomolgus monkeys, a second generation apo(a) ASO, ISIS-APO(a)Rx, significantly reduced hepatic apo(a) mRNA expression and plasma Lp(a) levels by >80%. Finally, in a phase I study in normal volunteers, ISIS-APO(a)Rx ASO reduced Lp(a) levels and their associated OxPL levels up to 89 and 93%, respectively, with minimal effects on other lipoproteins. ISIS-APO(a)Rx represents the first specific and potent drug in clinical development to lower Lp(a) levels and may be beneficial in reducing CVD events and progression of calcific aortic valve stenosis.
Project description:Here, we report that the natural compound pentachloropseudilin (PClP) acts as a reversible and allosteric inhibitor of myosin ATPase and motor activity. IC(50) values are in the range from 1 to 5 ?m for mammalian class-1 myosins and greater than 90 ?m for class-2 and class-5 myosins, and no inhibition was observed with class-6 and class-7 myosins. We show that in mammalian cells, PClP selectively inhibits myosin-1c function. To elucidate the structural basis for PClP-induced allosteric coupling and isoform-specific differences in the inhibitory potency of the compound, we used a multifaceted approach combining direct functional, crystallographic, and in silico modeling studies. Our results indicate that allosteric inhibition by PClP is mediated by the combined effects of global changes in protein dynamics and direct communication between the catalytic and allosteric sites via a cascade of small conformational changes along a conserved communication pathway.
Project description:As part of the avian reproductive effort, large quantities of triglyceride-rich very-low-density lipoprotein (VLDL) particles are transported by receptor-mediated endocytosis into the female germ cells. Although the oocytes are surrounded by a layer of granulosa cells harbouring high levels of active lipoprotein lipase, non-lipolysed VLDL is transported into the yolk. This is because VLDL particles from laying chickens are protected from lipolysis by apolipoprotein (apo)-VLDL-II, a potent dimeric lipoprotein lipase inhibitor [Schneider, Carroll, Severson and Nimpf (1990) J. Lipid Res. 31, 507-513]. To determine whether this protection depends on dimer formation and constitutes a general mechanism to ensure high levels of yolk triglycerides for embryonic utilization in birds, we have now molecularly characterized apo-VLDL-II in the Japanese quail, a frequently used avian species. Quail apo-VLDL-II shows 72% amino acid identity with the chicken protein, with most replacements being in the C-terminal region. Importantly, quail apo-VLDL-II lacks the single cysteine residue present eight residues from the C-terminus of chicken apo-VLDL-II, which is responsible for dimerization of the chicken lipoprotein lipase inhibitor. Nevertheless, monomeric quail and dimeric chicken apo-VLDL-II display, on a molar basis, identical inhibitory effects on lipoprotein lipase, underscoring the biological importance of their function. Furthermore secondary structure prediction of the 3'-untranslated region of the quail message supports a role for loop structures in the strictly oestrogen-dependent production of the lipoprotein lipase inhibitors. Our findings shed new light on the essential role of this small, hormonally regulated, protein in avian reproduction.
Project description:We have investigated the mechanism of action of Aquifex aeolicus IspH [E-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase], together with its inhibition, using a combination of site-directed mutagenesis (K ( M ),V (max)), EPR and (1)H, (2)H, (13)C, (31)P, and (57)Fe-electron-nuclear double resonance (ENDOR) spectroscopy. On addition of HMBPP to an (unreactive) E126A IspH mutant, a reaction intermediate forms that has a very similar EPR spectrum to those seen previously with the HMBPP "parent" molecules, ethylene and allyl alcohol, bound to a nitrogenase FeMo cofactor. The EPR spectrum is broadened on (57)Fe labeling and there is no evidence for the formation of allyl radicals. When combined with ENDOR spectroscopy, the results indicate formation of an organometallic species with HMBPP, a pi/sigma "metallacycle" or eta (2)-alkenyl complex. The complex is poised to interact with H(+) from E126 (and H124) in reduced wt IspH, resulting in loss of water and formation of an eta (1)-allyl complex. After reduction, this forms an eta (3)-allyl pi-complex (i.e. containing an allyl anion) that on protonation (at C2 or C4) results in product formation. We find that alkyne diphosphates (such as propargyl diphosphate) are potent IspH inhibitors and likewise form metallacycle complexes, as evidenced by (1)H, (2)H, and (13)C ENDOR, where hyperfine couplings of approximately 6 MHz for (13)C and 10 MHz for (1)H, are observed. Overall, the results are of broad general interest because they provide new insights into IspH catalysis and inhibition, involving organometallic species, and may be applicable to other Fe(4)S(4)-containing proteins, such as IspG.
Project description:Background and purposeCytochrome P450 (CYP, P450) 3A4 is involved in the metabolism of 50% of drugs and its catalytic activity in vivo is not explained only by hepatic expression levels. We previously demonstrated that UDP-glucuronosyltransferase (UGT) 2B7 suppressed CYP3A4 activity through an interaction. In the present study, we target UGT1A9 as another candidate modulator of CYP3A4.Experimental approachWe prepared co-expressed enzymes using the baculovirus-insect cell expression system and compared CYP3A4 activity in the presence and absence of UGT1A9. Wistar rats were treated with dexamethasone and liver microsomes were used to elucidate the role of CYP3A-UGT1A interactions.Key resultsUGT1A9 and UGT2B7 interacted with and suppressed CYP3A4. Kinetic analyses showed that both of the UGTs significantly reduced Vmax of CYP3A4 activity. In addition, C-terminal truncated mutants of UGT1A9 and UGT2B7 still retained the suppressive capacity. Dexamethasone treatment induced hepatic CYP3As and UGT1As at different magnitudes. Turnover of CYP3A was enhanced about twofold by this treatment.Conclusion and implicationsThe changes of kinetic parameters suggested that UGT1A9 suppressed CYP3A4 activity with almost the same mechanism as UGT2B7. The luminal domain of UGTs contains the suppressive interaction site(s), whereas the C-terminal domain may contribute to modulating suppression in a UGT isoform-specific manner. CYP3A-UGT1A interaction seemed to be disturbed by dexamethasone treatment and the suppression was partially cancelled. CYP3A4-UGT interactions would help to better understand the causes of inter/intra-individual differences in CYP3A4 activity.
Project description:Despite the widespread use of the blockade of immune checkpoints, for a significant number of cancer patients, these therapies have proven ineffective, presumably due to the immunosuppressive nature of the tumor microenvironment (TME). Critical drivers of immune escape in the TME include tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), which not only mediate immune suppression, but also facilitate metastatic dissemination and impart resistance to immunotherapies. Thus, strategies that convert them into tumor fighters may offer great therapeutic potential. In this study, we evaluated whether pharmacologic modulation of macrophage phenotype by HDAC inhibitors (HDACi) could produce an anti-tumor effect. We demonstrated that low-dose HDACi trichostatin-A (TSA) markedly reshaped the tumor immune microenvironment by modulating the suppressive activity of infiltrating macrophages and inhibiting the recruitment of MDSCs in various tumors. These actions, in turn, augmented anti-tumor immune responses and further enhanced anti-tumor effects of immunotherapies. HDAC inhibition, however, also upregulated PD-L1, thereby limiting the beneficial therapeutic effects. Indeed, combining low-dose TSA with anti-PD-L1 in this model significantly enhanced the durability of tumor reduction and prolonged survival of tumor-bearing mice, compared with the effect of either treatment alone. These data introduce HDAC inhibition as a potential means to harness the anti-tumor potential of macrophages in cancer therapy.
Project description:Tonically active cholinergic interneurons in the striatum modulate activities of striatal outputs from medium spiny (MS) neurons and significantly influence overall functions of the basal ganglia. Cellular mechanisms of this modulation are not fully understood. Here we show that ambient acetylcholine (ACh) derived from tonically active cholinergic interneurons constitutively upregulates depolarization-induced release of endocannabinoids from MS neurons. The released endocannabinoids cause transient suppression of inhibitory synaptic inputs to MS neurons through acting retrogradely onto presynaptic CB1 cannabinoid receptors. The effects were mediated by postsynaptic M(1) subtype of muscarinic ACh receptors, because the action of a muscarinic agonist to release endocannabinoids and the enhancement of depolarization-induced endocannabinoid release by ambient ACh were both deficient in M1 knock-out mice and were blocked by postsynaptic infusion of guanosine-5'-O-(2-thiodiphosphate). Suppression of spontaneous firings of cholinergic interneurons by inhibiting Ih current reduced the depolarization-induced release of endocannabinoids. Conversely, elevation of ambient ACh concentration by inhibiting choline esterase significantly enhanced the endocannabinoid release. Paired recording from a cholinergic interneuron and an MS neuron revealed that the activity of single cholinergic neuron could influence endocannabinoid-mediated signaling in neighboring MS neurons. These results clearly indicate that striatal endocannabinoid-mediated modulation is under the control of cholinergic interneuron activity. By immunofluorescent and immunoelectron microscopic examinations, we demonstrated that M1 receptor was densely distributed in perikarya and dendrites of dopamine D1 or D2 receptor-positive MS neurons. Thus, we have disclosed a novel mechanism by which the muscarinic system regulates striatal output and may contribute to motor control.
Project description:BMS-284756 (T-3811ME), a novel des-F(6) quinolone, was tested in the supercoiling inhibition and cleavable complex assays against Escherichia coli DNA gyrase, a target of quinolones. The results suggest that BMS-284756 has the same mechanism of action against DNA gyrase as other quinolones and a similar level of potency.