RESUMO
Coenzyme A (CoA) is an essential metabolic cofactor used by around 4% of cellular enzymes. Its role is to carry and transfer acetyl and acyl groups to other molecules. Cells can synthesize CoA de novo from vitamin B5 (pantothenate) through five consecutive enzymatic steps. Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the second step of the pathway during which phosphopantothenate reacts with ATP and cysteine to form phosphopantothenoylcysteine. Inborn errors of CoA biosynthesis have been implicated in neurodegeneration with brain iron accumulation (NBIA), a group of rare neurological disorders characterized by accumulation of iron in the basal ganglia and progressive neurodegeneration. Exome sequencing in five individuals from two unrelated families presenting with dilated cardiomyopathy revealed biallelic mutations in PPCS, linking CoA synthesis with a cardiac phenotype. Studies in yeast and fruit flies confirmed the pathogenicity of identified mutations. Biochemical analysis revealed a decrease in CoA levels in fibroblasts of all affected individuals. CoA biosynthesis can occur with pantethine as a source independent from PPCS, suggesting pantethine as targeted treatment for the affected individuals still alive.
Assuntos
Cardiomiopatia Dilatada/enzimologia , Cardiomiopatia Dilatada/genética , Genes Recessivos , Mutação/genética , Peptídeo Sintases/genética , Sequência de Aminoácidos , Animais , Vias Biossintéticas , Cardiomiopatia Dilatada/diagnóstico , Carnitina/análogos & derivados , Carnitina/metabolismo , Pré-Escolar , Coenzima A/biossíntese , Demografia , Drosophila , Estabilidade Enzimática , Feminino , Fibroblastos/metabolismo , Coração/fisiopatologia , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Lactente , Recém-Nascido , Imageamento por Ressonância Magnética , Masculino , Panteteína/administração & dosagem , Panteteína/análogos & derivados , Linhagem , Peptídeo Sintases/sangue , Peptídeo Sintases/química , Peptídeo Sintases/deficiência , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genéticaRESUMO
Designed peptides derived from the islet amyloid polypeptide (IAPP) cross-amyloid interaction surface with Aß (termed interaction surface mimics or ISMs) have been shown to be highly potent inhibitors of Aß amyloid self-assembly. However, the molecular mechanism of their function is not well understood. Using solution-state and solid-state NMR spectroscopy in combination with ensemble-averaged dynamics simulations and other biophysical methods including TEM, fluorescence spectroscopy and microscopy, and DLS, we characterize ISM structural preferences and interactions. We find that the ISM peptide R3-GI is highly dynamic, can adopt a ß-like structure, and oligomerizes into colloid-like assemblies in a process that is reminiscent of liquid-liquid phase separation (LLPS). Our results suggest that such assemblies yield multivalent surfaces for interactions with Aß40. Sequestration of substrates into these colloid-like structures provides a mechanistic basis for ISM function and the design of novel potent anti-amyloid molecules.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Fragmentos de Peptídeos/antagonistas & inibidores , Peptídeos/química , Sequência de Aminoácidos , Peptídeos beta-Amiloides/metabolismo , Microscopia de Fluorescência , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/metabolismo , Peptídeos/metabolismo , Especificidade por SubstratoRESUMO
BACKGROUND/OBJECTIVES: Individuals carrying loss-of-function gene mutations for the adipocyte hormone leptin are morbidly obese, but respond favorably to replacement therapy. Recombinant leptin is however largely ineffective for the vast majority of obese individuals due to leptin resistance. One theory underlying leptin resistance is impaired leptin transport across the blood-brain-barrier (BBB). Here, we aim to gain new insights into the mechanisms of leptin BBB transport, and its role in leptin resistance. METHODS: We developed a novel tool for visualizing leptin transport using infrared fluorescently labeled leptin, combined with tissue clearing and light-sheet fluorescence microscopy. We corroborated these data using western blotting. RESULTS: Using 3D whole brain imaging, we display comparable leptin accumulation in circumventricular organs of lean and obese mice, predominantly in the choroid plexus (CP). Protein quantification revealed comparable leptin levels in microdissected mediobasal hypothalami (MBH) of lean and obese mice (p = 0.99). We further found increased leptin receptor expression in the CP (p = 0.025, p = 0.0002) and a trend toward elevated leptin protein levels in the MBH (p = 0.17, p = 0.078) of obese mice undergoing weight loss interventions by calorie restriction or exendin-4 treatment. CONCLUSIONS: Overall, our findings suggest a crucial role for the CP in controlling the transport of leptin into the cerebrospinal fluid and from there to target areas such as the MBH, potentially mediated via the leptin receptor. Similar leptin levels in circumventricular organs and the MBH of lean and obese mice further suggest intact leptin BBB transport in leptin resistant mice.
Assuntos
Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Leptina/metabolismo , Camundongos Obesos/metabolismo , Obesidade Mórbida/metabolismo , Animais , Transporte Biológico , Barreira Hematoencefálica/diagnóstico por imagem , Western Blotting , Encéfalo/diagnóstico por imagem , Modelos Animais de Doenças , Fluorescência , Células HEK293 , Humanos , Imageamento Tridimensional , Camundongos , Imagem MolecularRESUMO
White adipocytes function as major energy reservoirs in humans by storing substantial amounts of triglycerides, and their dysfunction is associated with metabolic disorders; however, the mechanisms underlying cellular specialization during adipogenesis remain unknown. Here, we generate a spatiotemporal proteomic atlas of human adipogenesis, which elucidates cellular remodelling as well as the spatial reorganization of metabolic pathways to optimize cells for lipid accumulation and highlights the coordinated regulation of protein localization and abundance during adipocyte formation. We identify compartment-specific regulation of protein levels and localization changes of metabolic enzymes to reprogramme branched-chain amino acids and one-carbon metabolism to provide building blocks and reduction equivalents. Additionally, we identify C19orf12 as a differentiation-induced adipocyte lipid droplet protein that interacts with the translocase of the outer membrane complex of lipid droplet-associated mitochondria and regulates adipocyte lipid storage by determining the capacity of mitochondria to metabolize fatty acids. Overall, our study provides a comprehensive resource for understanding human adipogenesis and for future discoveries in the field.
Assuntos
Adipogenia , Proteômica , Humanos , Proteômica/métodos , Metabolismo dos Lipídeos , Mitocôndrias/metabolismo , Gotículas Lipídicas/metabolismo , Proteoma/metabolismo , Adipócitos/metabolismo , Diferenciação CelularRESUMO
Type 2 diabetes (T2D) can be prevented in pre-diabetic individuals with impaired glucose tolerance (IGT). Here, we have used a metabolomics approach to identify candidate biomarkers of pre-diabetes. We quantified 140 metabolites for 4297 fasting serum samples in the population-based Cooperative Health Research in the Region of Augsburg (KORA) cohort. Our study revealed significant metabolic variation in pre-diabetic individuals that are distinct from known diabetes risk indicators, such as glycosylated hemoglobin levels, fasting glucose and insulin. We identified three metabolites (glycine, lysophosphatidylcholine (LPC) (18:2) and acetylcarnitine) that had significantly altered levels in IGT individuals as compared to those with normal glucose tolerance, with P-values ranging from 2.4×10(-4) to 2.1×10(-13). Lower levels of glycine and LPC were found to be predictors not only for IGT but also for T2D, and were independently confirmed in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam cohort. Using metabolite-protein network analysis, we identified seven T2D-related genes that are associated with these three IGT-specific metabolites by multiple interactions with four enzymes. The expression levels of these enzymes correlate with changes in the metabolite concentrations linked to diabetes. Our results may help developing novel strategies to prevent T2D.
Assuntos
Biomarcadores/metabolismo , Metabolômica/métodos , Estado Pré-Diabético/metabolismo , Idoso , Glicemia/metabolismo , Estudos de Casos e Controles , Estudos Transversais , Diabetes Mellitus Tipo 2/metabolismo , Jejum/sangue , Feminino , Alemanha , Teste de Tolerância a Glucose , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Razão de Chances , Estudos Prospectivos , Reprodutibilidade dos Testes , Fatores de RiscoRESUMO
Mitochondrial membrane protein-associated neurodegeneration (MPAN) is a relentlessly progressive neurodegenerative disorder caused by mutations in the C19orf12 gene. C19orf12 has been implicated in playing a role in lipid metabolism, mitochondrial function, and autophagy, however, the precise functions remain unknown. To identify new robust cellular targets for small compound treatments, we evaluated reported mitochondrial function alterations, cellular signaling, and autophagy in a large cohort of MPAN patients and control fibroblasts. We found no consistent alteration of mitochondrial functions or cellular signaling messengers in MPAN fibroblasts. In contrast, we found that autophagy initiation is consistently impaired in MPAN fibroblasts and show that C19orf12 expression correlates with the amount of LC3 puncta, an autophagy marker. Finally, we screened 14 different autophagy modulators to test which can restore this autophagy defect. Amongst these compounds, carbamazepine, ABT-737, LY294002, oridonin, and paroxetine could restore LC3 puncta in the MPAN fibroblasts, identifying them as novel potential therapeutic compounds to treat MPAN. In summary, our study confirms a role for C19orf12 in autophagy, proposes LC3 puncta as a functionally robust and consistent readout for testing compounds, and pinpoints potential therapeutic compounds for MPAN.
RESUMO
Anthracyclines are among the most used and effective anticancer drugs. Their activity has been attributed to DNA double-strand breaks resulting from topoisomerase II poisoning and to eviction of histones from select sites in the genome. Here, we show that the extensively used anthracyclines Doxorubicin, Daunorubicin, and Epirubicin decrease the transcription of nuclear factor kappa B (NF-κB)-dependent gene targets, but not interferon-responsive genes in primary mouse (Mus musculus) macrophages. Using an NMR-based structural approach, we demonstrate that anthracyclines disturb the complexes formed between the NF-κB subunit RelA and its DNA-binding sites. The anthracycline variants Aclarubicin, Doxorubicinone, and the newly developed Dimethyl-doxorubicin, which share anticancer properties with the other anthracyclines but do not induce DNA damage, also suppressed inflammation, thus uncoupling DNA damage from the effects on inflammation. These findings have implications for anticancer therapy and for the development of novel anti-inflammatory drugs with limited side effects for life-threatening conditions such as sepsis.
Assuntos
Antraciclinas , NF-kappa B , Animais , Camundongos , Antraciclinas/farmacologia , Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Dano ao DNA , DNARESUMO
Polyubiquitin chains are flexible multidomain proteins, whose conformational dynamics enable them to regulate multiple biological pathways. Their dynamic is determined by the linkage between ubiquitins and by the number of ubiquitin units. Characterizing polyubiquitin behavior as a function of their length is hampered because of increasing system size and conformational variability. Here, we introduce a new approach to efficiently integrating small-angle x-ray scattering with simulations allowing us to accurately characterize the dynamics of linear di-, tri-, and tetraubiquitin in the free state as well as of diubiquitin in complex with NEMO, a central regulator in the NF-κB pathway. Our results show that the behavior of the diubiquitin subunits is independent of the presence of additional ubiquitin modules and that the dynamics of polyubiquitins with different lengths follow a simple model. Together with experimental data from multiple biophysical techniques, we then rationalize the 2:1 NEMO:polyubiquitin binding.
RESUMO
Celastrol is a natural pentacyclic triterpene extracted from the roots of Tripterygium wilfordi (thunder god vine). Celastrol was reported as a powerful anti-obesity drug with leptin sensitizing properties that decreases food consumption and mediates body weight loss when administered to diet-induced obese mice at 100 µg/kg body weight. The weight lowering properties of celastrol are likely mediated by the CNS, in particular, by the hypothalamus, but the final proof for the accumulation of celastrol in the brain and hypothalamus remains to be established. Here, we aimed to demonstrate that intraperitoneal celastrol administration at 100 µg/kg can rapidly reach the brain and, in particular, the hypothalamus of mice. We developed and validated a sensitive liquid chromatography mass spectrometry method for the quantitative determination of celastrol in murine tissues, namely liver, brain and hypothalamus. Chow-fed lean mice were randomly assigned to the vehicle vs. celastrol groups, injected with saline or 100 µg/kg body weight of celastrol, and sacrificed 30 min or 120 min post injection. Celastrol was extracted from homogenized tissue using ethyl acetate as organic solvent, and quantified using a matrix-matched calibration curve with glycyrrhetinic acid as internal standard. Liver celastrol concentrations were 32.60 ± 8.21 pg/mg and 40.52 ± 15.6 pg/mg, 30 and 120 min after injection, respectively. We found 4.70 ± 0.31 pg/mg celastrol after 30 min, and 16.22 ± 3.33 pg/mg after 120 min in whole brain lysates, and detectable amounts in the hypothalamus. These results corroborate the validity of our methodology, demonstrate the accumulation of celastrol in the brain of mice injected intraperitoneally with a dose of 100 µg/kg, and confirm the CNS as possible site of action for the weight lowering properties of celastrol.
Assuntos
Fármacos Antiobesidade/farmacologia , Encéfalo/efeitos dos fármacos , Fígado/efeitos dos fármacos , Tripterygium/efeitos dos fármacos , Triterpenos/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Dieta HiperlipídicaRESUMO
Celastrol, a plant-derived constituent of traditional Chinese medicine, has been proposed to offer significant potential as an antiobesity drug. However, the molecular mechanism for this activity is unknown. We show that the weight-lowering effects of celastrol are driven by decreased food consumption. Although young Lep ob mice respond with a decrease in food intake and body weight, adult Lep db and Lep ob mice are unresponsive to celastrol, suggesting that functional leptin signaling in adult mice is required to elicit celastrol's catabolic actions. Protein tyrosine phosphatase 1 (PTP1B), a leptin negative-feedback regulator, has been previously reported to be one of celastrol's targets. However, we found that global PTP1B knockout (KO) and wild-type (WT) mice have comparable weight loss and hypophagia when treated with celastrol. Increased levels of uncoupling protein 1 (UCP1) in subcutaneous white and brown adipose tissue suggest celastrol-induced thermogenesis as a further mechanism. However, diet-induced obese UCP1 WT and KO mice have comparable weight loss upon celastrol treatment, and celastrol treatment has no effect on energy expenditure under ambient housing or thermoneutral conditions. Overall, our results suggest that celastrol-induced weight loss is hypophagia driven and age-dependently mediated by functional leptin signaling. Our data encourage reconsideration of therapeutic antiobesity strategies built on leptin sensitization.
Assuntos
Ingestão de Alimentos/efeitos dos fármacos , Obesidade/metabolismo , Extratos Vegetais/farmacologia , Triterpenos/farmacologia , Proteína Desacopladora 1/metabolismo , Redução de Peso/efeitos dos fármacos , Animais , Dieta Hiperlipídica , Metabolismo Energético/efeitos dos fármacos , Camundongos Knockout , Obesidade/genética , Triterpenos Pentacíclicos , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteína Desacopladora 1/genéticaRESUMO
Celastrol is a natural pentacyclic triterpene used in traditional Chinese medicine with significant weight-lowering effects. Celastrol-administered mice at 100 µg/kg decrease food consumption and body weight via a leptin-dependent mechanism, yet its molecular targets in this pathway remain elusive. Here, we demonstrate in vivo that celastrol-induced weight loss is largely mediated by the inhibition of leptin negative regulators protein tyrosine phosphatase (PTP) 1B (PTP1B) and T-cell PTP (TCPTP) in the arcuate nucleus (ARC) of the hypothalamus. We show in vitro that celastrol binds reversibly and inhibits noncompetitively PTP1B and TCPTP. NMR data map the binding site to an allosteric site in the catalytic domain that is in proximity of the active site. By using a panel of PTPs implicated in hypothalamic leptin signaling, we show that celastrol additionally inhibited PTEN and SHP2 but had no activity toward other phosphatases of the PTP family. These results suggest that PTP1B and TCPTP in the ARC are essential for celastrol's weight lowering effects in adult obese mice.
Assuntos
Fármacos Antiobesidade/farmacologia , Obesidade/tratamento farmacológico , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 2/antagonistas & inibidores , Triterpenos/farmacologia , Sítio Alostérico , Animais , Fármacos Antiobesidade/metabolismo , Domínio Catalítico , Dieta Hiperlipídica/efeitos adversos , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Camundongos Transgênicos , Obesidade/etiologia , Triterpenos Pentacíclicos , Proteína Tirosina Fosfatase não Receptora Tipo 1/química , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 2/química , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Relação Estrutura-Atividade , Triterpenos/química , Triterpenos/metabolismo , Redução de Peso/efeitos dos fármacosRESUMO
The NMR structure of the oxidised wild-type cytochrome c3 from Desulfovibrio vulgaris Hildenborough was determined in solution. Using a newly developed methodology, NMR data from the K45Q mutant was then grafted onto data from the wild-type protein to determine the structure in the region of the mutation. The structural origins of the redox-Bohr effect and haem-haem cooperativities are discussed with respect to the redox-related conformational changes observed in solution.
Assuntos
Grupo dos Citocromos c/química , Grupo dos Citocromos c/genética , Desulfovibrio vulgaris/química , Desulfovibrio vulgaris/genética , Compostos Férricos/química , Heme/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Oxigênio/administração & dosagem , Oxigênio/metabolismo , Mutação Puntual , Conformação Proteica , Estrutura Secundária de Proteína , SoluçõesRESUMO
hnRNP K and hnRNP E1/E2 are RNA-binding proteins comprised of three hnRNP K-homology (KH) domains. These proteins are involved in the translational control and stabilization of mRNAs in erythroid cells. hnRNP E1 and hnRNP K regulate the translation of reticulocyte 15-lipoxygenase (r15-LOX) mRNA. Both proteins bind specifically to the differentiation control element (DICE) in the 3' untranslated region (3'UTR) of the r15-LOX mRNA. It has been shown that hnRNP K is a substrate of the tyrosine kinase c-Src and that tyrosine phosphorylation by c-Src inhibits the binding of hnRNP K to the DICE. Here, we investigate which of the three KH domains of hnRNP E1 and hnRNP K mediate the DICE interaction. Using RNA-binding assays, we demonstrate DICE-binding of the KH domains 1 and 3 of hnRNP E1, and KH domain 3 of hnRNP K. Furthermore, with RNA-binding assays, NMR experiments and in vitro translation studies, we show that tyrosine 458 in KH domain 3 of hnRNP K is important for the DICE interaction and we provide evidence that it is a target of c-Src.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/química , Proteínas Tirosina Quinases/química , Tirosina/química , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Araquidonato 15-Lipoxigenase/química , Proteína Tirosina Quinase CSK , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Quinases da Família srcRESUMO
The heterogeneous nuclear ribonucleoprotein (hnRNP) K is implicated in multiple functions in the regulation of gene expression and acts as a hub at the intersection of signaling pathways and processes involving nucleic acids. Central to its function is its ability to bind both ssDNA and ssRNA via its KH (hnRNP K homology) domains. We determined crystal structures of hnRNP K KH3 domain complexed with 15-mer and 6-mer (CTC(4)) ssDNAs at 2.4 and 1.8 A resolution, respectively, and show that the KH3 domain binds specifically to both TCCC and CCCC sequences. In parallel, we used NMR to compare the binding affinity and mode of interaction of the KH3 domain with several ssRNA ligands and CTC(4) ssDNA. Based on a structure alignment of the KH3-CTC(4) complex with known structures of other KH domains in complex with ssRNA, we discuss recognition of tetranucleotide sequences by KH domains.
Assuntos
Cristalografia por Raios X/métodos , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/química , Sequência de Aminoácidos , Citosina/química , DNA/química , DNA de Cadeia Simples/química , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Ácidos Nucleicos/química , Peptídeos/química , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , RNA/química , Ribonucleoproteínas/química , EspectrofotometriaRESUMO
Trafficking of the G protein-coupled receptor (GPCR) Smoothened (Smo) to the primary cilium (PC) is a potential target to inhibit oncogenic Hh pathway activation in a large number of tumors. One drawback is the appearance of Smo mutations that resist drug treatment, which is a common reason for cancer treatment failure. Here, we undertook a high content screen with compounds in preclinical or clinical development and identified ten small molecules that prevent constitutive active mutant SmoM2 transport into PC for subsequent Hh pathway activation. Eight of the ten small molecules act through direct interference with the G protein-coupled receptor associated sorting protein 2 (Gprasp2)-SmoM2 ciliary targeting complex, whereas one antagonist of ionotropic receptors prevents intracellular trafficking of Smo to the PC. Together, these findings identify several compounds with the potential to treat drug-resistant SmoM2-driven cancer forms, but also reveal off-target effects of established drugs in the clinics.
Assuntos
Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Oncogenes , Receptor Smoothened/metabolismo , Animais , Camundongos , Neoplasias Pancreáticas/metabolismo , Transporte Proteico , Transdução de SinaisRESUMO
The seven-transmembrane receptor Smoothened (Smo) activates all Hedgehog (Hh) signaling by translocation into the primary cilia (PC), but how this is regulated is not well understood. Here we show that Pitchfork (Pifo) and the G protein-coupled receptor associated sorting protein 2 (Gprasp2) are essential components of an Hh induced ciliary targeting complex able to regulate Smo translocation to the PC. Depletion of Pifo or Gprasp2 leads to failure of Smo translocation to the PC and lack of Hh target gene activation. Together, our results identify a novel protein complex that is regulated by Hh signaling and required for Smo ciliary trafficking and Hh pathway activation.
Assuntos
Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Cílios/genética , Cílios/metabolismo , Proteínas Hedgehog/genética , Proteínas de Homeodomínio/genética , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Transporte Proteico/fisiologia , Receptores Acoplados a Proteínas G/genética , Receptor SmoothenedRESUMO
The IκB kinase (IKK) complex acts as the gatekeeper of canonical NF-κB signaling, thereby regulating immunity, inflammation and cancer. It consists of the catalytic subunits IKKα and IKKß and the regulatory subunit NEMO/IKKγ. Here, we show that the ubiquitin binding domain (UBAN) in NEMO is essential for IKK/NF-κB activation in response to TNFα, but not IL-1ß stimulation. By screening a natural compound library we identified an anthraquinone derivative that acts as an inhibitor of NEMO-ubiquitin binding (iNUB). Using biochemical and NMR experiments we demonstrate that iNUB binds to NEMOUBAN and competes for interaction with methionine-1-linked linear ubiquitin chains. iNUB inhibited NF-κB activation upon UBAN-dependent TNFα and TCR/CD28, but not UBAN-independent IL-1ß stimulation. Moreover, iNUB was selectively killing lymphoma cells that are addicted to chronic B-cell receptor triggered IKK/NF-κB activation. Thus, iNUB disrupts the NEMO-ubiquitin protein-protein interaction interface and thereby inhibits physiological and pathological NF-κB signaling.
Assuntos
Antraquinonas/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ubiquitina/metabolismo , Animais , Avaliação Pré-Clínica de Medicamentos , Células HeLa , Humanos , Interleucina-1beta/fisiologia , Camundongos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Fator de Necrose Tumoral alfa/fisiologia , UbiquitinaçãoRESUMO
Kinase inhibition is considered to be an important therapeutic target for LRRK2 mediated Parkinson's disease (PD). Many LRRK2 kinase inhibitors have been reported but have yet to be optimized in order to qualify as drug candidates for the treatment of the disease. In order to start a structure-function analysis of such inhibitors, we mutated the active site of Dictyostelium Roco4 kinase to resemble LRRK2. Here, we show saturation transfer difference (STD) NMR and the first cocrystal structures of two potent in vitro inhibitors, LRRK2-IN-1 and compound 19, with mutated Roco4. Our data demonstrate that this system can serve as an excellent tool for the structural characterization and optimization of LRRK2 inhibitors using X-ray crystallography and NMR spectroscopy.
Assuntos
Dictyostelium/enzimologia , Proteínas Serina-Treonina Quinases/química , Proteínas de Protozoários/química , Cristalografia por Raios X , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Simulação de Acoplamento Molecular , Morfolinas/química , Mutação Puntual , Ligação Proteica , Conformação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Pirimidinas/químicaRESUMO
OBJECTIVE: Metformin is used as a first-line oral treatment for type 2 diabetes (T2D). However, the underlying mechanism is not fully understood. Here, we aimed to comprehensively investigate the pleiotropic effects of metformin. RESEARCH DESIGN AND METHODS: We analyzed both metabolomic and genomic data of the population-based KORA cohort. To evaluate the effect of metformin treatment on metabolite concentrations, we quantified 131 metabolites in fasting serum samples and used multivariable linear regression models in three independent cross-sectional studies (n = 151 patients with T2D treated with metformin [mt-T2D]). Additionally, we used linear mixed-effect models to study the longitudinal KORA samples (n = 912) and performed mediation analyses to investigate the effects of metformin intake on blood lipid profiles. We combined genotyping data with the identified metformin-associated metabolites in KORA individuals (n = 1,809) and explored the underlying pathways. RESULTS: We found significantly lower (P < 5.0E-06) concentrations of three metabolites (acyl-alkyl phosphatidylcholines [PCs]) when comparing mt-T2D with four control groups who were not using glucose-lowering oral medication. These findings were controlled for conventional risk factors of T2D and replicated in two independent studies. Furthermore, we observed that the levels of these metabolites decreased significantly in patients after they started metformin treatment during 7 years' follow-up. The reduction of these metabolites was also associated with a lowered blood level of LDL cholesterol (LDL-C). Variations of these three metabolites were significantly associated with 17 genes (including FADS1 and FADS2) and controlled by AMPK, a metformin target. CONCLUSIONS: Our results indicate that metformin intake activates AMPK and consequently suppresses FADS, which leads to reduced levels of the three acyl-alkyl PCs and LDL-C. Our findings suggest potential beneficial effects of metformin in the prevention of cardiovascular disease.