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Abstract Adipocytes are the primary sites for fatty acid storage, but the synthesis rate of fatty acids is very low. The physiological significance of this phenomenon remains unclear. Here, we show that surplus fatty acid synthesis in adipocytes induces necroptosis and lipodystrophy. Transcriptional activation of FASN elevates fatty acid synthesis, but decreases NADPH level and increases ROS production, which ultimately leads to adipocyte necroptosis. We identify MED20, a subunit of the Mediator complex, as a negative regulator of FASN transcription. Adipocyte-specific male Med20 knockout mice progressively develop lipodystrophy, which is reversed by scavenging ROS. Further, in a murine model of HIV-associated lipodystrophy and a human patient with acquired lipodystrophy, ROS neutralization significantly improves metabolic disorders, indicating a causal role of ROS in disease onset. Our study well explains the low fatty acid synthesis rate in adipocytes, and sheds light on the management of acquired lipodystrophy.

Abstract Triple-negative breast cancer (TNBC) is a subtype of breast cancer with poor outcome and lacks of approved targeted therapy. Overexpression of epidermal growth factor receptor (EGFR) is found in more than 50% TNBC and is suggested as a driving force in progression of TNBC; however, targeting EGFR using antibodies to prevent its dimerization and activation shows no significant benefits for TNBC patients. Here we report that EGFR monomer may activate signal transducer activator of transcription-3 (STAT3) in the absence of transmembrane protein TMEM25, whose expression is frequently decreased in human TNBC. Deficiency of TMEM25 allows EGFR monomer to phosphorylate STAT3 independent of ligand binding, and thus enhances basal STAT3 activation to promote TNBC progression in female mice. Moreover, supplying TMEM25 by adeno-associated virus strongly suppresses STAT3 activation and TNBC progression. Hence, our study reveals a role of monomeric-EGFR/STAT3 signaling pathway in TNBC progression and points out a potential targeted therapy for TNBC.

Obesity has become a global pandemic. WDTC1 is a WD40-containing protein that functions as an anti-obesity factor. WDTC1 inhibits adipogenesis by working as an adaptor of the CUL4-DDB1 E3 ligase complex. It remains unclear about how WDTC1 is regulated. Here, we show that the TRiC/CCT functions as a chaperone to facilitate the protein folding of WDTC1 and proper function in adipogenesis. Through tandem purification, we identified the molecular chaperone TRiC/CCT as WDTC1-interacting proteins. WDTC1 bound the TRiC/CCT through its ADP domain, and the TRiC/CCT recognized WDTC1 through the CCT5 subunit. Disruption of the TRiC/CCT by knocking down CCT1 or CCT5 led to misfolding and lysosomal degradation of WDTC1. Furthermore, the knockdown of CCT1 or CCT5 eliminated the inhibitory effect of WDTC1 on adipogenesis. Our studies uncovered a critical role of the TRiC/CCT in the folding of WDTC1 and expanded our knowledge on the regulation of adipogenesis.

EGFR is aberrantly activated in many cancer types. Here the authors show that small GTPase ARF6 mediates the trafficking of palmitoylated EGFR from Golgi to plasma membrane and the blockade of this sorting system inhibits the growth of EGFR overexpression tumours.

Abstract Glioma is the most common primary tumor in the central nervous system. However, the development of glioma and effective therapeutic strategies remain elusive. Here, we identify GPR17 as a potential target to treat glioma. Data mining with human LGG and GBM samples reveals that GPR17 is negatively correlated with glioma development. Overexpressing GPR17 inhibits glioma cell proliferation and induces apoptosis by raising ROS levels. GPR17-overexpressing glioma cells are less tumorigenic in the brain than in control cells. Mechanistically, GPR17 inhibits the transcription of RNF2, a key component in the PRC1 complex, through cAMP/PKA/NF-κB signaling, leading to reduced histone H2A monoubiquitination. ChIP-Seq and RNA-Seq analyses reveal KLF9 as a direct target of RNF2. KLF9 mediates the functions of GPR17 and RNF2 in glioma cells. Furthermore, activation of GPR17 by its agonist inhibits glioma formation. Our findings have thus identified GPR17 as a key regulator of glioma development and a potential therapeutic target for gliomas.

The mechanistic details of fatty acid uptake into cells remains poorly understood. Here, the authors identify CD36 internalization via cavaeolae and demonstrate dynamic palmitoylationof CD36 is required for endocytic uptake of fatty acids.

DNA damage can lead to autophagy. Here the authors reveal a molecular mechanism for ATR/Chk1 signaling-induced autophagy in response to DNA damage, through an ATR/Chk1/RhoB-mediated lysosomal recruitment of TSC complex and subsequent mTORC1 inhibition.

Summary: The CNS plays a pivotal role in energy homeostasis, but whether oligodendrocytes are involved has been largely unexplored. Here, we show that signaling through GPR17, a G-protein-coupled receptor predominantly expressed in the oligodendrocyte lineage, regulates food intake by modulating hypothalamic neuronal activities. GPR17-null mice and mice with an oligodendrocyte-specific knockout of GPR17 have lean phenotypes on a high-fat diet, suggesting that GPR17 regulates body weight by way of oligodendrocytes. Downregulation of GPR17 results in activation of cAMP-protein kinase A (PKA) signaling in oligodendrocytes and upregulated expression of pyruvate dehydrogenase kinase 1 (PDK1), which promotes lactate production. Elevation of lactate activates AKT and STAT3 signaling in the hypothalamic neurons, leading to increased expression of Pomc and suppression of Agrp. Our findings uncover a critical role of oligodendrocytes in metabolic homeostasis, where GPR17 modulates the production of lactate, which, in turn, acts as a metabolic signal to regulate neuronal activity. : Ou et. al. show that Gpr17 in oligodendrocytes contributes to whole-body metabolic regulation. Gpr17-deficient mice exhibit decreased body weight on long-term high-fat feeding by reducing food intake. Loss of Gpr17 increases oligodendrocytic lactate production, which results in the lean phenotype of the KO animals. Keywords: oligodendroyctes, GPR17, lactate, metabolic homeostasis

Protein palmitoylation is a post-translational lipid modification of proteins. Accumulating evidence reveals that palmitoylation functions as a sorting signal to direct proteins to destinations; however, the sorting mechanism remains largely unknown. Here, we show that ARF6 plays a general role in targeting palmitoylated proteins from the Golgi to the plasma membrane (PM). Through shRNA screening, we identified ARF6 as the key small GTPase in targeting CD36, a palmitoylated protein, from the Golgi to the PM. We found that the N-terminal myristoylation of ARF6 is required for its binding with palmitoylated CD36, and the GTP-bound form of ARF6 facilitates the delivery of CD36 to the PM. Analysis of stable isotope labeling by amino acids in cell culture revealed that ARF6 might facilitate the sorting of 359 of the 531 palmitoylated PM proteins, indicating a general role of ARF6. Our study has thus identified a sorting mechanism for targeting palmitoylated proteins from the Golgi to the PM. [ABSTRACT FROM AUTHOR]

Nuclear receptor Nur77 participates in multiple metabolic regulations and plays paradoxical roles in tumorigeneses. Herein, we demonstrated that the knockout of Nur77 stimulated mammary tumor development in two mouse models, which would be reversed by a specific reexpression of Nur77 in mammary tissues. Mechanistically, Nur77 interacted and recruited corepressors, the SWI/SNF complex, to the promoters of CD36 and FABP4 to suppress their transcriptions, which hampered the fatty acid uptake, leading to the inhibition of cell proliferation. Peroxisome proliferator-activated receptor-γ (PPARγ) played an antagonistic role in this process through binding to Nur77 to facilitate ubiquitin ligase Trim13-mediated ubiquitination and degradation of Nur77. Cocrystallographic and functional analysis revealed that Csn-B, a Nur77-targeting compound, promoted the formation of Nur77 homodimer to prevent PPARγ binding by steric hindrance, thereby strengthening the Nur77's inhibitory role in breast cancer. Therefore, our study reveals a regulatory function of Nur77 in breast cancer via impeding fatty acid uptake. [ABSTRACT FROM AUTHOR]

Skeletal muscle absorbs long-chain fatty acids (LCFAs) that are either oxidized in mitochondria or temporarily stored as triglycerides in lipid droplets (LDs). So far, it is still not fully understood how lipid uptake and storage are regulated in muscle and whether these are important for whole-body lipid homeostasis. Here we show that the small GTPase Rab8a regulates lipid uptake and storage in skeletal muscle. Muscle-specific Rab8a deletion caused hyperlipidemia and exacerbated hepatosteatosis induced by a high-fat diet. Mechanistically, Rab8a deficiency decreased LCFA entry into skeletal muscle and inhibited LD fusion in muscle cells. Consequently, blood lipid levels were elevated and stimulated hepatic mammalian target of rapamycin, which enhanced hepatosteatosis by upregulating hepatic lipogenesis and cholesterol biosynthesis. Our results demonstrate the significance of lipid uptake and storage in muscle in regulating whole-body lipid homeostasis, and they shed light on the roles of skeletal muscle in the pathogenesis of hyperlipidemia and hepatosteatosis. [ABSTRACT FROM AUTHOR]

Ghrelin, an octanoylated peptide hormone produced in the stomach. rises dramatically in mouse plasma during chronic severe calorie deprivation, an event that is essential to maintain life. The mechanism for this increase is not understood. Here, we study the control of ghrelin secretion in tissue culture cells derived from mice bearing ghrelinomas induced by a tissue-specific SV40 T-antigen transgene. We found thatthe ghrelin-secreting cells express high levels of mRNA encoding β1-adrenergic receptors. Addition of norepinephrine or epinephrine to the culture medium stimulated ghrelin secretion, and this effect was blocked by atenolol, a selective β1-adrenergic antagonist. When WT mice were treated with reserpine to deplete adrenergic neurotransmitters from sympathetic neurons, the fasting-induced increase in plasma ghrelin was blocked. Inhibition was also seen following atenolol administration. We conclude that ghrelin secretion during fasting is induced by adrenergic agents released by sympathetic neurons and acting directly on β1 receptors on the ghrelin- secreting cells of the stomach. [ABSTRACT FROM AUTHOR]

Ghrelin O-acyltransferase (GOAT) attaches octanoate to proghrelin, which is processed to ghrelin, an octanoylated peptide hormone that stimulates release of growth hormone (GH) from pituitary cells. Elimination of the gene encoding ghrelin or its receptor produces only mild phenotypes in mice. Thus, the essential function of ghrelin is obscure. Here, we eliminate the Goat gene in mice, thereby eliminating all octanoylated ghrelin from blood. On normal or high fat diets, Goar-/- mice grew and maintained the same weights as wild-type (WI) littermates. When subjected to 60% calorie restriction, WT and Goat-/- mice both lost 30% of body weight and 75% of body fat within 4 days. In both lines, fasting blood glucose initially declined equally. After 4 days, glucose stabilized in WI mice at 58-76 mg/dL. In Goat-/- mice, glucose continued to decline, reaching 12-36 mg/dL on day 7. At this point, WT mice showed normal physical activity, whereas Goat-/- mice were moribund. GH rose progressively in calorie-restricted WT mice and less in Goat-/- mice. Infusion of either ghrelin or GH normalized blood glucose in Goat-/- mice and prevented death. Thus, an essential function of ghrelin in mice is elevation of GH levels during severe calorie restriction, thereby preserving blood glucose and preventing death. [ABSTRACT FROM AUTHOR]

The discovery of ghrelin O-acyltransferase (GOAT) opens the way to the design of drugs that block the attachment of an octanoyl group to the appetite-stimulating peptide hormone ghrelin, potentially preventing obesity. Here, we develop a biochemical assay that uses membranes from insect cells infected with baculovirus encoding mouse GOAT. The GOAT-containing membranes transferred the [3H]octanoyl group from [3H]octanoyl CoA to recombinant proghrelin in vitro. Transfer depended on the serine at residue 3 of proghrelin. which is the known site of acylation. GOAT also transferred [3Hloctanoyl to a pentapeptide containing only the N-terminal five amino acids of proghrelin. GOAT activity could be inhibited by an octanoylated ghrelin pentapeptide, and its potency was enhanced 45-fold when the octanoylated serine-3 was replaced by octanoylated diaminopropionic acid. The data suggest that GOAT is subjected to end-product inhibition and this inhibition is better achieved with substrates having the octanoyl group attached through an amide linkage rather than the corresponding ester. These insights may facilitate the future design of useful inhibitors of GOAT. [ABSTRACT FROM AUTHOR]

Muscle creatine kinase (CK) is a crucial enzyme in energy metabolism, and it exists in two forms, the reduced form (R-CK) and the oxidized form (O-CK). In contrast with R-CK, O-CK contained an intrachain disulfide bond in each subunit. Here we explored the properties of O-CK and its regulatory role on muscle CK. The intrachain disulfide bond in O-CK was demonstrated to be formed between Cys74 and Cys146 by site-directed mutagenesis. Biophysical analysis indicated that O-CK showed decreased catalytic activity and that it might be structurally unstable. Further assays through guanidine hydrochloride denaturation and proteolysis by trypsin and protease K revealed that the tertiary structure of O-CK was more easily disturbed than that of R-CK. Surprisingly, O-CK, unlike R-CK, cannot interact with the M-line protein myomesin through biosensor assay, indicating that O-CK might have no role in muscle contraction. Through in vitro ubiquitination assay, CK was demonstrated to be a specific substrate of muscle ring finger protein 1 (MURF-1). O-CK can be rapidly ubiquitinated by MURF-1, while R-CK can hardly be ubiquitinated, implying that CK might be degraded by the ATP-ubiquitin-proteasome pathway through the generation of O-CK. The results above were further confirmed by molecular modeling of the structure of O-CK. Therefore, it can be concluded that the generation of O-CK was a negative regulation of R-CK and that O-CK might play essential roles in the molecular turnover of MM-CK. [ABSTRACT FROM AUTHOR]

Two fused proteins of dimeric arginine kinase (AK) from sea cucumber and dimeric creatine kinase (CK) from rabbit muscle, named AK-CK and CK-AK, were obtained through the expression of fused AK and CK genes. Both AK-CK and CK-AK had about 50% AK activity and about 2-fold K m values for arginine of native AK, as well as about 50% CK activity and about 2-fold K m values for creatine of native CK. This indicated that both AK and CK moieties are fully active in the two fused proteins. The structures of AK, CK, AK-CK, and CK-AK were compared by collecting data of far-UV circular dichroism, intrinsic fluorescence, 1-anilinonaphthalene-8-sulfonate binding fluorescence, and size-exclusion chromatography. The results indicated that dimeric AK and CK differed in the maximum emission wavelength, the exposure extent of hydrophobic surfaces, and molecular size, though they have a close evolutionary relationship. The structure and thermodynamic stability of AK, CK, AK-CK, and CK-AK were compared by guanidine hydrochloride (GdnHCl) titration. Dimeric AK was more dependent on the cooperation of two subunits than CK according to the analysis of residual AK or CK activity with GdnHCl concentration increase. Additionally, AK and CK had different denaturation curves induced by GdnHCl, but almost the same thermodynamic stability. The two fused proteins, AK-CK and CK-AK, had similar secondary structure, tertiary structure, molecular size, structure, and thermodynamic stability, which indicated that the expression order of AK and CK genes might have little effect on the characteristics of the fused proteins and might further verify the close relationship of dimeric AK and CK. [ABSTRACT FROM AUTHOR]

DREB1/C-repeat binding factor (CBF) is a plant-specific family of transcription factors and plays a crucial role in freeze tolerance. In the present work, two groups of drought-responsive element binding factor (DREB)-like genes were isolated from Brassica napus, named Group I and Group II. The two groups of genes were both induced by low temperature, but the expression of Group I preceded that of Group II. The Group I DREBs could specifically bind with the DRE cis-acting element and activate the expression of downstream genes, but Group II factors were trans-inactive although they still had the ability to bind with DRE, which was confirmed by electrophoretic mobility shift assay. Fluorescence quenching assays indicated that the DRE binding ability of the two groups was similar. Co-expression of Group II could depress the trans-activation activity of Group I DREB in a concentration-dependent manner. These results strongly suggested that the trans-active Group I DREBs were expressed at the early stage of cold stress to open the DRE-mediated signaling pathway in cold stress, whereas the trans-inactive Group II DREBs were expressed at the later stage to close the signal pathway in a competitive manner. The results herein provide a new insight into the regulation mechanisms of the DRE-mediated signaling pathway in response to cold stress. [ABSTRACT FROM AUTHOR]

As a depressant of the central nervous system, the clinical effect of sodium barbital has been extensively studied. Here we report on sodium barbital as an inhibitor of rabbit-muscle creatine kinase (CK), which plays a significant role in energy homeostasis in the muscles. Although sodium barbital gradually inhibits the activity of CK with increased concentration, the inhibition effect can be completely reversed by dilution, indicating that the inactivation process is reversible. Detailed kinetics analysis, according to a previously presented theory, indicates that sodium barbital functions as a non complexing inhibitor, and its inhibition effect on CK is a slow reversible inactivation. In this study, a kinetic model of the substrate reaction is presented, and the microscopic rate constants for the reaction of sodium barbital with the free enzyme and the enzyme–substrate complexes are determined. Kinetic analysis reveals that sodium barbital might compete with both creatine and ATP, but mainly with creatine, to inhibit the activity of CK. The results suggest that CK might be a target for sodium barbital in vivo. [ABSTRACT FROM AUTHOR]

Protein disulfide isomerase (PDI) functions as an isomerase to catalyze thiol:disulfide exchange, as a chaperone to assist protein folding, and as a subunit of prolyl-4-hydroxylase and microsomal triglyceride transfer protein. At a lower concentration of 0.2 µM, PDI facilitated the aggregation of unfolded rabbit muscle creatine kinase (CK) and exhibited anti-chaperone activity, which was shown to be mainly due to the hydrophobic interactions between PDI and CK and was independent of the cross-linking of disulfide bonds. At concentrations above 1 µM, PDI acted as a protector against aggregation but an inhibitor of reactivation during CK refolding. The inhibition effect of PDI on CK reactivation was fur. ther characterized as due to the formation of PDI-CK complexes through intermolecular disulfide bonds, a process involving Cys-36 and Cys-295 of PDI. Two disulfide-linked complexes containing both PDI and CK were obtained, and the large, soluble aggregates around 400 kDa were composed of 1 molecule of tetrameric PDI and 2 molecules of inactive intermediate dimeric CK, whereas the smaller one, around 200 kDa, was formed by 1 dimeric PDI and 1 dimeric CK. To our knowledge this is the first study revealing that PDI could switch its conformation from dimer to tetramer in its functions as a foldase. According to the observations in this research and our previous study of the folding pathways of CK, a working model was proposed for the molecular mechanism of CK refolding catalyzed by PDI. [ABSTRACT FROM AUTHOR]

Many metabolic diseases are caused by disorders of lipid homeostasis. CIDEC, a lipid droplet (LD)-associated protein, plays a critical role in controlling LD fusion and lipid storage. However, regulators of CIDEC remain largely unknown. Here, we established a homogeneous time-resolved fluorescence (HTRF)-based high-throughput screening method and identified LPXN as a positive regulatory candidate for CIDEC. LPXN and Hic-5, the members of the Paxillin family, are focal adhesion adaptor proteins that contribute to the recruitment of specific kinases and phosphatases, cofactors, and structural proteins, participating in the transduction of extracellular signals into intracellular responses. Our data showed that Hic-5 and LPXN significantly increased the protein level of CIDEC and enhanced CIDEC stability not through triacylglycerol synthesis and FAK signaling pathways. Hic-5 and LPXN reduced the ubiquitination of CIDEC and inhibited its proteasome degradation pathway. Furthermore, Hic-5 and LPXN enlarged LDs and promoted lipid storage in adipocytes. Therefore, we identified Hic-5 and LPXN as novel regulators of CIDEC. Our current findings also suggest intervention with Hic-5 and LPXN might ameliorate ectopic fat storage by enhancing the lipid storage capacity of white adipose tissues. [ABSTRACT FROM AUTHOR]

The article offers information on monoubiquitination of p120-catenin essential for transforming growth factor beta (TGF) induced epithelial-mesenchymal transition and tumor metastasis. Topics include report on the E3 ubiquitin ligase Smurf1 that targets p120-catenin, a core component of adherens junction (AJ) complex, for monoubiquitination during transforming growth factor beta induced EMT, thereby leading to AJ dissociation.

Dehydration-responsive element-binding proteins (DREBs) and ethylene-responsive element (ERE) binding factors are two major subfamilies of the AP2/ethylene-responsive element-binding protein family and play crucial roles in the regulation of abiotic- and biotic-stress responses, respectively. In the present work, we have reported a previously identified DREB-like factor, TINY, that was involved and both abiotic- and biotic-stress signaling pathways. TINY was capable of binding to both DRE and ERE with similar affinity and could activate the expression of reporter genes driven by either of these two elements in tobacco cells. The 15th amino acid in the APETALA2 (AP2)/ethylene- responsive element-binding factor domain was demonstrated to be essential for its specific binding to ERE, whereas the 14th and 19th amino acids were responsible for the binding to DRE. The expression of TINY was greatly activated by drought, cold, ethylene, and slightly by methyl jasmonate. Additionally, overexpression of TINY in A rabidopsis resulted in elevated expressions of both the DRE- and the ERE-containing genes. Moreover, the expression of DRE-regulated genes, such as COR6.6 and ERD10, was up-regulated upon ethylene treatment, and the expression of ERE-regulated genes, such as HLS1, was also increased by cold stress, when the expression of TINY was being induced. These results strongly suggested that TINY might play a role in the cross-talk between abiotic- and biotic-stress-responsive gene expressions by connecting the DRE- and ERE-mediated signaling pathways. The results herein might promote the understanding of the mechanisms of specific DNA recognition and gene expression regulation by DREBs. [ABSTRACT FROM AUTHOR]