The fatty acyl composition of phospholipids determines the biophysical character of membranes and impacts the function of membrane proteins. Here, we define a nuclear receptor pathway for the dynamic ...modulation of membrane composition in response to changes in cellular lipid metabolism. Ligand activation of liver X receptors (LXRs) preferentially drives the incorporation of polyunsaturated fatty acids into phospholipids through induction of the remodeling enzyme Lpcat3. Promotion of Lpcat3 activity ameliorates endoplasmic reticulum (ER) stress induced by saturated free fatty acids in vitro or by hepatic lipid accumulation in vivo. Conversely, Lpcat3 knockdown in liver exacerbates ER stress and inflammation. Mechanistically, Lpcat3 modulates inflammation both by regulating inflammatory kinase activation through changes in membrane composition and by affecting substrate availability for inflammatory mediator production. These results outline an endogenous mechanism for the preservation of membrane homeostasis during lipid stress and identify Lpcat3 as an important mediator of LXR effects on metabolism.
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•Induction of Lpcat3 expression by LXRs promotes phospholipid remodeling•LXR-Lpcat3 activation drives unsaturated fatty acid incorporation into phospholipids•Lpcat3 activity in liver modulates lipid-induced ER stress and inflammation•Lpcat3 affects inflammation through regulation of membrane c-Src activity
The sterol regulatory element-binding proteins (SREBP) are key transcriptional regulators of lipid metabolism and cellular growth. It has been proposed that SREBP signaling regulates cellular growth ...through its ability to drive lipid biosynthesis. Unexpectedly, we find that loss of SREBP activity inhibits cancer cell growth and viability by uncoupling fatty acid synthesis from desaturation. Integrated lipid profiling and metabolic flux analysis revealed that cancer cells with attenuated SREBP activity maintain long-chain saturated fatty acid synthesis, while losing fatty acid desaturation capacity. We traced this defect to the uncoupling of fatty acid synthase activity from stearoyl-CoA desaturase 1 (SCD1)-mediated desaturation. This deficiency in desaturation drives an imbalance between the saturated and monounsaturated fatty acid pools resulting in severe lipotoxicity. Importantly, replenishing the monounsaturated fatty acid pool restored growth to SREBP-inhibited cells. These studies highlight the importance of fatty acid desaturation in cancer growth and provide a novel mechanistic explanation for the role of SREBPs in cancer metabolism.
The LXR-regulated E3 ubiquitin ligase IDOL controls LDLR receptor stability independent of SREBP and PCSK9, but its relevance to plasma lipid levels is unknown. Here we demonstrate that the effects ...of the LXR–IDOL axis are both tissue and species specific. In mice, LXR agonist induces Idol transcript levels in peripheral tissues but not in liver, and does not change plasma LDL levels. Accordingly, Idol-deficient mice exhibit elevated LDLR protein levels in peripheral tissues, but not in the liver. By contrast, LXR activation in cynomolgus monkeys induces hepatic IDOL expression, reduces LDLR protein levels, and raises plasma LDL levels. Knockdown of IDOL in monkeys with an antisense oligonucleotide blunts the effect of LXR agonist on LDL levels. These results implicate IDOL as a modulator of plasma lipid levels in primates and support further investigation into IDOL inhibition as a potential strategy for LDL lowering in humans.
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•The activity of the LXR-IDOL-LDLR axis is tissue and species specific•In mice, LXR and IDOL regulate LDLR protein levels in the periphery, but not liver•LXR activation in primates induces hepatic IDOL expression and reduces LDLR protein•Inhibition of IDOL in primates blunts the induction of plasma LDL levels by LXR
The LXR-regulated E3 ubiquitin ligase IDOL controls LDLR stability, but its relevance to plasma lipid levels is unknown. Hong et al. demonstrate that the LXR–IDOL axis regulates hepatic LDLR protein and plasma LDL levels in nonhuman primates, but not in mice.
Pathogenic microorganisms and toxins have evolved a variety of mechanisms to gain access to the host-cell cytosol and thereby exert virulent effects upon the host. One common mechanism of cellular ...entry requires trafficking to an acidified endosome, which promotes translocation across the host membrane. To identify small-molecule inhibitors that block this process, a library of 30,000 small molecules was screened for inhibitors of anthrax lethal toxin. Here we report that 4-bromobenzaldehyde N -(2,6-dimethylphenyl)semicarbazone, the most active compound identified in the screen, inhibits intoxication by lethal toxin and blocks the entry of multiple other acid-dependent bacterial toxins and viruses into mammalian cells. This compound, which we named EGA, also delays lysosomal targeting and degradation of the EGF receptor, indicating that it targets host-membrane trafficking. In contrast, EGA does not block endosomal recycling of transferrin, retrograde trafficking of ricin, phagolysosomal trafficking, or phagosome permeabilization by Franciscella tularensis . Furthermore, EGA does not neutralize acidic organelles, demonstrating that its mechanism of action is distinct from pH-raising agents such as ammonium chloride and bafilomycin A1. EGA is a powerful tool for the study of membrane trafficking and represents a class of host-targeted compounds for therapeutic development to treat infectious disease.
The liver X receptors (LXRs) are members of the nuclear receptor superfamily that regulate sterol metabolism and inflammation. We sought to identify previously unknown genes regulated by LXRs in ...macrophages and to determine their contribution to atherogenesis. Here we characterize a novel LXR target gene, the lipopolysaccharide binding protein (LBP) gene. Surprisingly, the ability of LXRs to control LBP expression is cell-type specific, occurring in macrophages but not liver. Treatment of macrophages with oxysterols or loading with modified LDL induces LBP in an LXR-dependent manner, suggesting a potential role for LBP in the cellular response to cholesterol overload. To investigate this further, we performed bone marrow transplant studies. After 18 weeks of Western diet feeding, atherosclerotic lesion burden was assessed revealing markedly smaller lesions in the LBP−/− recipients. Furthermore, loss of bone marrow LBP expression increased apoptosis in atherosclerotic lesions as determined by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Supporting in vitro studies with isolated macrophages showed that LBP expression does not affect cholesterol efflux but promotes the survival of macrophages in the setting of cholesterol loading. The LBP gene is a macrophage-specific LXR target that promotes foam cell survival and atherogenesis.
The development of a multigram synthesis of 3-exo-isopropylbicyclo2.2.1heptan-2-endo-amine hydrochloride (1) (also known as BRD4780 and AGN-192403) is described. The process involves protection of ...the amine as 4-nitrobenzyl carbamate, pNZ, which enables chiral SFC chromatography. The absolute configuration (AC) of the individual enantiomers has been determined by Mosher’s amide method, VCD spectroscopy, and X-ray crystallography. We highlight the VCD approach as a rapid and effective means of AC determination that can be deployed directly on the target compounds.
Nucleoside 5'-triphosphate (dNTP) analogues in which the β,γ-oxygen is mimicked by a CXY group (β,γ-CXY-dNTPs) have provided information about DNA polymerase catalysis and fidelity. Definition of CXY ...stereochemistry is important to elucidate precise binding modes. We previously reported the (
)- and (
)-β,γ-CHX-dGTP diastereomers (X = F, Cl), prepared via P,C-dimorpholinamide CHCl (
,
) and CHF (
,
) bisphosphonates (BPs) equipped with an (
)-mandelic acid as a chiral auxiliary, with final deprotection using H
/Pd. This method also affords the β,γ-CHCl-dTTP (
,
), β,γ-CHF (
,
), and β,γ-CHCl (
,
) dATP diastereomers as documented here, but the reductive deprotection step is not compatible with dCTP or the bromo substituent in β,γ-CHBr-dNTP analogues. To complete assembly of the toolkit, we describe an alternative synthetic strategy featuring ethylbenzylamine or phenylglycine-derived chiral BP synthons incorporating a photolabile protecting group. After acid-catalyzed removal of the (
)-(+)-α-ethylbenzylamine auxiliary, coupling with activated dCMP and photochemical deprotection, the individual diastereomers of β,γ-CHBr- (
,
), β,γ-CHCl- (
, 3
), β,γ-CHF-dCTP (
,
) were obtained. The β,γ-CH(CH
)-dATPs (
,
) were obtained using a methyl (
)-(-)-phenylglycinate auxiliary.
P and
F NMR Δδ values are correlated with CXY stereochemistry and p
values for 13 CXY-bisphosphonic acids and imidodiphosphonic acid are tabulated.
An analogue 2 of Brasilicardin A, 1 (BraA), a potent immunosuppressive and cytotoxic agent, was synthesized in which the natural tricyclic skeleton was replaced with a synthetically more accessible ...substituted tetrahydronaphthalene core. BraA, this analogue (BraL), and cyclosporine A were tested for their ability to inhibit the proliferation of human T cells upon CD3/CD28 activation. Although BraL did not impact T cell activation over the dose range tested, this study shows the inhibitory activity of BraA on human T cells for the first time.
Kinetics studies of dNTP analogues having pyrophosphate-mimicking β,γ-pCXYp leaving groups with variable X and Y substitution reveal striking differences in the chemical transition-state energy for ...DNA polymerase β that depend on all aspects of base-pairing configurations, including whether the incoming dNTP is a purine or pyrimidine and if base-pairings are right (T•A and G•C) or wrong (T•G and G•T). Brønsted plots of the catalytic rate constant (log(k pol)) versus pK a4 for the leaving group exhibit linear free energy relationships (LFERs) with negative slopes ranging from −0.6 to −2.0, consistent with chemical rate-determining transition-states in which the active-site adjusts to charge-stabilization demand during chemistry depending on base-pair configuration. The Brønsted slopes as well as the intercepts differ dramatically and provide the first direct evidence that dNTP base recognition by the enzyme–primer–template complex triggers a conformational change in the catalytic region of the active-site that significantly modifies the rate-determining chemical step.
Drug repurposing has the advantage of identifying potential treatments on a shortened timescale. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high-content screen of 3,713 ...compounds at different stages of clinical development to identify FDA-approved compounds that reduce mucin-1 (MUC1) protein abundance. Elevated MUC1 levels predict the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) and correlate with poor clinical outcomes. Our screen identifies fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo, fostamatinib reduces MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro, SYK inhibition by the active metabolite R406 promotes MUC1 removal from the cell surface. Our work suggests fostamatinib as a repurposing drug candidate for ALI.
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Elevated MUC1 levels predict the development of acute lung injury (ALI)A high-content screen of 3,713 compounds identifies repurposing candidatesR406 removes MUC1 from the apical surface of epithelial cellsFostamatinib treatment reduces MUC1 in a mouse model of lung injury
In a high-content screen, Kost-Alimova et al. identify R406, the active metabolite of fostamatinib, as an FDA-approved candidate repurposing compound for the reduction of MUC1 protein levels in lung epithelium in the setting of acute lung injury.
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