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1.
Nat Commun ; 11(1): 1032, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32098961

RESUMO

The AMP-activated protein kinase (AMPK) is a master sensor of the cellular energy status that is crucial for the adaptive response to limited energy availability. AMPK is implicated in the regulation of many cellular processes, including autophagy. However, the precise mechanisms by which AMPK controls these processes and the identities of relevant substrates are not fully understood. Using protein microarrays, we identify Cyclin Y as an AMPK substrate that is phosphorylated at Serine 326 (S326) both in vitro and in cells. Phosphorylation of Cyclin Y at S326 promotes its interaction with the Cyclin-dependent kinase 16 (CDK16), thereby stimulating its catalytic activity. When expressed in cells, Cyclin Y/CDK16 is sufficient to promote autophagy. Moreover, Cyclin Y/CDK16 is necessary for efficient AMPK-dependent activation of autophagy. This functional interaction is mediated by AMPK phosphorylating S326 of Cyclin Y. Collectively, we define Cyclin Y/CDK16 as downstream effector of AMPK for inducing autophagy.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/metabolismo , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Análise Serial de Proteínas , Pironas/farmacologia , Serina/metabolismo , Tiofenos/farmacologia
2.
Proc Natl Acad Sci U S A ; 117(4): 1962-1970, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31932442

RESUMO

Necroptosis is a regulated necrotic cell death pathway, mediated by a supermolecular complex called the necrosome, which contains receptor-interacting protein kinase 1 and 3 (RIPK1, RIPK3) and mixed-lineage kinase domain-like protein (MLKL). Phosphorylation of human RIPK3 at serine 227 (S227) has been shown to be required for downstream MLKL binding and necroptosis progression. Tandem immunoprecipitation of RIPK3 reveals that casein kinase 1 (CK1) family proteins associate with the necrosome upon necroptosis induction, and this interaction depends on the kinase activity of RIPK3. In addition, CK1 proteins colocalize with RIPK3 puncta during necroptosis. Importantly, CK1 proteins directly phosphorylate RIPK3 at S227 in vitro and in vivo. Loss of CK1 proteins abolishes S227 phosphorylation and blocks necroptosis. Furthermore, a RIPK3 mutant with mutations in the CK1 recognition motif fails to be phosphorylated at S227, does not bind or phosphorylate MLKL, and is unable to activate necroptosis. These results strongly suggest that CK1 proteins are necrosome components which are responsible for RIPK3-S227 phosphorylation.


Assuntos
Caseína Quinase Ialfa/metabolismo , Caseína Quinase Idelta/metabolismo , Caseína Quinase Iépsilon/metabolismo , Necroptose , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Serina/metabolismo , Caseína Quinase Ialfa/genética , Caseína Quinase Idelta/genética , Caseína Quinase Iépsilon/genética , Células HeLa , Humanos , Fosforilação , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Serina/genética
3.
Nat Commun ; 11(1): 12, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31896774

RESUMO

Abiotic stresses, including drought and salinity, trigger a complex osmotic-stress and abscisic acid (ABA) signal transduction network. The core ABA signalling components are snf1-related protein kinase2s (SnRK2s), which are activated by ABA-triggered inhibition of type-2C protein-phosphatases (PP2Cs). SnRK2 kinases are also activated by a rapid, largely unknown, ABA-independent osmotic-stress signalling pathway. Here, through a combination of a redundancy-circumventing genetic screen and biochemical analyses, we have identified functionally-redundant MAPKK-kinases (M3Ks) that are necessary for activation of SnRK2 kinases. These M3Ks phosphorylate a specific SnRK2/OST1 site, which is indispensable for ABA-induced reactivation of PP2C-dephosphorylated SnRK2 kinases. ABA-triggered SnRK2 activation, transcription factor phosphorylation and SLAC1 activation require these M3Ks in vitro and in plants. M3K triple knock-out plants show reduced ABA sensitivity and strongly impaired rapid osmotic-stress-induced SnRK2 activation. These findings demonstrate that this M3K clade is required for ABA- and osmotic-stress-activation of SnRK2 kinases, enabling robust ABA and osmotic stress signal transduction.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Pressão Osmótica/fisiologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteínas de Arabidopsis/genética , Feminino , Mutação , Oócitos/metabolismo , Fosforilação , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Serina/metabolismo , Transdução de Sinais , Xenopus laevis
4.
PLoS One ; 15(1): e0226838, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31990908

RESUMO

Lipases comprise a large class of hydrolytic enzymes which catalyze the cleavage of the ester bonds in triacylglycerols and find numerous biotechnological applications. Previously, we have cloned the gene coding for a novel esterase PMGL2 from a Siberian permafrost metagenomic DNA library. We have determined the 3D structure of PMGL2 which belongs to the hormone-sensitive lipase (HSL) family and contains a new variant of the active site motif, GCSAG. Similar to many other HSLs, PMGL2 forms dimers in solution and in the crystal. Our results demonstrated that PMGL2 and structurally characterized members of the GTSAG motif subfamily possess a common dimerization interface that significantly differs from that of members of the GDSAG subfamily of known structure. Moreover, PMGL2 had a unique organization of the active site cavity with significantly different topology compared to the other lipolytic enzymes from the HSL family with known structure including the distinct orientation of the active site entrances within the dimer and about four times larger size of the active site cavity. To study the role of the cysteine residue in GCSAG motif of PMGL2, the catalytic properties and structure of its double C173T/C202S mutant were examined and found to be very similar to the wild type protein. The presence of the bound PEG molecule in the active site of the mutant form allowed for precise mapping of the amino acid residues forming the substrate cavity.


Assuntos
Bactérias/enzimologia , Lipase/química , Lipase/metabolismo , Mutação , Pergelissolo/microbiologia , Motivos de Aminoácidos , Bactérias/química , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Lipase/genética , Metagenoma , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Serina/metabolismo , Sibéria , Especificidade por Substrato
5.
Infect Immun ; 88(2)2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31740531

RESUMO

l-Serine is a nonessential amino acid and a key intermediate in several relevant metabolic pathways. In bacteria, the major source of l-serine is the phosphorylated pathway, which comprises three enzymes: d-3-phosphoglycerate dehydrogenase (PGDH; SerA), phosphoserine amino transferase (PSAT; SerC), and l-phosphoserine phosphatase (PSP; SerB). The Brucella abortus genome encodes two PGDHs (SerA-1 and SerA-2), involved in the first step in l-serine biosynthesis, and one PSAT and one PSP, responsible for the second and third steps, respectively. In this study, we demonstrate that the serA1 serA2 double mutant and the serC and serB single mutants are auxotrophic for l-serine. These auxotrophic mutants can be internalized but are unable to replicate in HeLa cells and in J774A.1 macrophage-like cells. Replication defects of auxotrophic mutants can be reverted by cell medium supplementation with l-serine at early times postinfection. In addition, the serB mutant is attenuated in the murine intraperitoneal infection model and has an altered lipid composition, since the lack of l-serine abrogates phosphatidylethanolamine synthesis in this strain. Taken together, these results reveal that limited availability of l-serine within the host cell impairs proliferation of the auxotrophic strains, highlighting the relevance of this biosynthetic pathway in Brucella pathogenicity.


Assuntos
Brucella abortus/crescimento & desenvolvimento , Brucella abortus/metabolismo , Proliferação de Células/fisiologia , Serina/metabolismo , Animais , Vias Biossintéticas/fisiologia , Linhagem Celular Tumoral , Feminino , Células HeLa , Humanos , Macrófagos/metabolismo , Macrófagos/microbiologia , Redes e Vias Metabólicas/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação/fisiologia
6.
Metabolism ; 102: 154000, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31678070

RESUMO

OBJECTIVE: Supplementation with serine attenuates alcoholic fatty liver by regulating homocysteine metabolism and lipogenesis. However, little is known about serine metabolism in fatty liver disease (FLD). We aimed to investigate the changes in serine biosynthetic pathways in humans and animal models of fatty liver and their contribution to the development of FLD. METHODS: High-fat diet (HFD)-induced steatosis and methionine-choline-deficient diet-induced steatohepatitis animal models were employed. Human serum samples were obtained from patients with FLD whose proton density fat fraction was estimated by magnetic resonance imaging. 3-Phosphoglycerate dehydrogenase (Phgdh)-knockout mouse embryonic fibroblasts (MEF) and transgenic mice overexpressing Phgdh (Tg-phgdh) were used to evaluate the role of serine metabolism in the development of FLD. RESULTS: Expression of Phgdh was markedly reduced in the animal models. There were significant negative correlations of the serum serine with the liver fat fraction, serum alanine transaminase, and triglyceride levels among patients with FLD. Increased lipid accumulation and reduced NAD+ and SIRT1 activity were observed in Phgdh-knockout MEF and primary hepatocytes incubated with free fatty acids; these effects were reversed by overexpression of Phgdh. Tg-Phgdh mice showed significantly reduced hepatic triglyceride accumulation compared with wild-type littermates fed a HFD, which was accompanied by increased SIRT1 activity and reduced expression of lipogenic genes and proteins. CONCLUSIONS: Human and experimental data suggest that reduced Phgdh expression and serine levels are closely associated with the development of FLD.


Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/genética , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Serina/metabolismo , Animais , Células Cultivadas , Estudos de Coortes , Dieta Hiperlipídica , Regulação para Baixo , Embrião de Mamíferos , Feminino , Regulação Enzimológica da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Metabolismo dos Lipídeos/genética , Lipogênese/genética , Fígado/química , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/patologia , Serina/análise
7.
Proc Natl Acad Sci U S A ; 117(1): 584-594, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31843895

RESUMO

In this study, we provide critical evidence that STAT2 stability regulation plays an essential role in melanoma cell proliferation and colony growth. We found that the interaction of FBXW7 and STAT2 induced STAT2 destabilization via a ubiquitination-mediated proteasomal degradation pathway. Notably, GSK3ß-mediated STAT2 phosphorylation facilitated STAT2-FBXW7 interactions via the DNA binding domain of STAT2 and domains 1, 2, 6, and 7 of FBXW7 WD40. Importantly, the inverse correlation between protein levels of STAT2 and FBXW7 were observed not only in human melanoma cells but also in a human skin cancer tissue array. The relationship between protein levels of STAT2 and FBXW7, cell proliferation, and colony growth were similarly observed in the melanoma cell lines SK-MEL-2, -5, and -28. Moreover, STAT2 knockdown in melanoma cells suppressed melanoma cell proliferation and colony formation. These data demonstrated that FBXW7-mediated STAT2 stability regulation plays an essential role in melanoma cell proliferation and cancer growth.


Assuntos
Proteína 7 com Repetições F-Box-WD/metabolismo , Melanoma/patologia , Fator de Transcrição STAT2/metabolismo , Neoplasias Cutâneas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação , Estabilidade Proteica , Proteólise , Fator de Transcrição STAT2/química , Fator de Transcrição STAT2/genética , Serina/metabolismo , Transdução de Sinais , Pele/patologia , Treonina/metabolismo , Análise Serial de Tecidos , Ubiquitinação , Repetições WD40
8.
Proc Natl Acad Sci U S A ; 117(1): 328-336, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31843888

RESUMO

The fundamental importance of the 26S proteasome in health and disease suggests that its function must be finely controlled, and yet our knowledge about proteasome regulation remains limited. Posttranslational modifications, especially phosphorylation, of proteasome subunits have been shown to impact proteasome function through different mechanisms, although the vast majority of proteasome phosphorylation events have not been studied. Here, we have characterized 1 of the most frequently detected proteasome phosphosites, namely Ser361 of Rpn1, a base subunit of the 19S regulatory particle. Using a variety of approaches including CRISPR/Cas9-mediated gene editing and quantitative mass spectrometry, we found that loss of Rpn1-S361 phosphorylation reduces proteasome activity, impairs cell proliferation, and causes oxidative stress as well as mitochondrial dysfunction. A screen of the human kinome identified several kinases including PIM1/2/3 that catalyze S361 phosphorylation, while its level is reversibly controlled by the proteasome-resident phosphatase, UBLCP1. Mechanistically, Rpn1-S361 phosphorylation is required for proper assembly of the 26S proteasome, and we have utilized a genetic code expansion system to directly demonstrate that S361-phosphorylated Rpn1 more readily forms a precursor complex with Rpt2, 1 of the first steps of 19S base assembly. These findings have revealed a prevalent and biologically important mechanism governing proteasome formation and function.


Assuntos
Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Ensaios Enzimáticos , Técnicas de Introdução de Genes , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/metabolismo , Proteínas Nucleares/genética , Estresse Oxidativo , Fosfoproteínas Fosfatases/genética , Fosforilação/fisiologia , Complexo de Endopeptidases do Proteassoma/genética , Subunidades Proteicas/genética , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/metabolismo , Serina/metabolismo , Transativadores/genética , Transativadores/metabolismo
9.
Mol Pharmacol ; 97(1): 9-22, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31707355

RESUMO

Folate-dependent one-carbon (C1) metabolism is compartmentalized in the mitochondria and cytosol and is a source of critical metabolites for proliferating tumors. Mitochondrial C1 metabolism including serine hydroxymethyltransferase 2 (SHMT2) generates glycine for de novo purine nucleotide and glutathione biosynthesis and is an important source of NADPH, ATP, and formate, which affords C1 units as 10-formyl-tetrahydrofolate and 5,10-methylene-tetrahydrofolate for nucleotide biosynthesis in the cytosol. We previously discovered novel first-in-class multitargeted pyrrolo[3,2-d]pyrimidine inhibitors of SHMT2 and de novo purine biosynthesis at glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase with potent in vitro and in vivo antitumor efficacy toward pancreatic adenocarcinoma cells. In this report, we extend our findings to an expanded panel of pancreatic cancer models. We used our lead analog AGF347 [(4-(4-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)butyl)-2-fluorobenzoyl)-l-glutamic acid] to characterize pharmacodynamic determinants of antitumor efficacy for this series and demonstrated plasma membrane transport into the cytosol, uptake from cytosol into mitochondria, and metabolism to AGF347 polyglutamates in both cytosol and mitochondria. Antitumor effects of AGF347 downstream of SHMT2 and purine biosynthesis included suppression of mammalian target of rapamycin signaling, and glutathione depletion with increased levels of reactive oxygen species. Our results provide important insights into the cellular pharmacology of novel pyrrolo[3,2-d]pyrimidine inhibitors as antitumor compounds and establish AGF347 as a unique agent for potential clinical application for pancreatic cancer, as well as other malignancies. SIGNIFICANCE STATEMENT: This study establishes the antitumor efficacies of novel inhibitors of serine hydroxymethyltransferase 2 and of cytosolic targets toward a panel of clinically relevant pancreatic cancer cells and demonstrates the important roles of plasma membrane transport, mitochondrial accumulation, and metabolism to polyglutamates of the lead compound AGF347 to drug activity. We also establish that loss of serine catabolism and purine biosynthesis resulting from AGF347 treatment impacts mammalian target of rapamycin signaling, glutathione pools, and reactive oxygen species, contributing to antitumor efficacy.


Assuntos
Antineoplásicos/farmacologia , Citosol/efeitos dos fármacos , Glicina Hidroximetiltransferase/antagonistas & inibidores , Mitocôndrias/efeitos dos fármacos , Pirimidinas/farmacologia , Pirróis/farmacologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citosol/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Técnicas de Inativação de Genes , Glutationa/biossíntese , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/metabolismo , Humanos , Leucovorina/análogos & derivados , Leucovorina/metabolismo , Mitocôndrias/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Nucleotídeos de Purina/biossíntese , Pirimidinas/química , Pirimidinas/uso terapêutico , Pirróis/química , Pirróis/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Serina/metabolismo , Tetra-Hidrofolatos/metabolismo
10.
Nat Chem Biol ; 16(2): 122-125, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31873221

RESUMO

We have identified a molecular interaction between the reversibly oxidized form of protein tyrosine phosphatase 1B (PTP1B) and 14-3-3ζ that regulates PTP1B activity. Destabilizing the transient interaction between 14-3-3ζ and PTP1B prevented PTP1B inactivation by reactive oxygen species and decreased epidermal growth factor receptor phosphorylation. Our data suggest that destabilizing the interaction between 14-3-3ζ and the reversibly oxidized and inactive form of PTP1B may establish a path to PTP1B activation in cells.


Assuntos
Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteínas 14-3-3/metabolismo , Biotinilação , Ativação Enzimática , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Oxirredução , Fosforilação , Mapas de Interação de Proteínas , Proteína Tirosina Fosfatase não Receptora Tipo 1/química , Espécies Reativas de Oxigênio/metabolismo , Serina/metabolismo , Tirosina/metabolismo
11.
Molecules ; 24(19)2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31581474

RESUMO

Imatinib is an effective anticancer drug for the treatment of leukemia. Interestingly, when an FDA-approved drug library was tested for agents that block peroxisome proliferator-activated receptor γ (PPARγ) phosphorylation at Ser245 to evaluate possibilities of antidiabetic drug repositioning, imatinib was determined as a PPARγ antagonist ligand. However, it is not well understood how imatinib binds to PPARγ or would improve insulin sensitivity without classical agonism. Here, we report the crystal structure of the PPARγ R288A mutant in complex with imatinib. Imatinib bound to Arm2 and Arm3 regions in the ligand-binding domain (LBD) of PPARγ, of which the Arm3 region is closely related to the inhibition of PPARγ phosphorylation at Ser245. The binding of imatinib in LBD induced a stable conformation of helix H2' and the Ω loop compared with the ligand-free state. In contrast, imatinib does not interact with Tyr473 on PPARγ helix H12, which is important for the classical agonism associated with side effects. Our study provides new structural insights into the PPARγ regulation by imatinib and may contribute to the development of new antidiabetic drugs targeting PPARγ while minimizing known side effects.


Assuntos
Hipoglicemiantes/farmacologia , Mesilato de Imatinib/farmacologia , PPAR gama/química , PPAR gama/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Reposicionamento de Medicamentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hipoglicemiantes/química , Mesilato de Imatinib/química , Modelos Moleculares , Mutação , PPAR gama/genética , Fosforilação/efeitos dos fármacos , Domínios Proteicos , Estrutura Secundária de Proteína , Serina/metabolismo
12.
J Agric Food Chem ; 67(46): 12953-12961, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31638789

RESUMO

Most common sphingolipids are comprised of "typical" sphingoid bases (sphinganine, sphingosine, and structurally related compounds) and are produced via the condensation of l-serine with a fatty acyl-CoA by serine palmitoyltransferase. Some organisms, including mammals, also produce "atypical" sphingoid bases that lack a 1-hydroxyl group as a result of the utilization of l-alanine or glycine instead of l-serine, resulting in the formation of 1-deoxy- or 1-desoxymethylsphingoid bases, respectively. Elevated production of "atypical" sphingolipids has been associated with human disease, but 1-deoxysphingoid bases have also been found to have potential as anticancer compounds, hence, the importance of knowing more about the occurrence of these compounds in food. Most of the "typical" and "atypical" sphingoid bases are found as the N-acyl metabolites (e.g., ceramides and 1-deoxyceramides) in mammals, but this has not been uniformly assessed in previous studies nor determined in consumed food. Therefore, we developed a method for the quantitative analysis of "typical" and "atypical" sphingoid bases and their N-acyl derivatives by reverse-phase liquid chromatography coupled to electrospray ionization tandem mass spectrometry. On the basis of these analyses, there was considerable variability in the amounts and molecular subspecies of atypical sphingoid bases and their N-acyl metabolites found in different edible sources. These findings demonstrate that a broader assessment of the types of sphingolipids in foods is needed because some diets might contain sufficient amounts of atypical as well as typical sphingolipids that could have beneficial or possibly deleterious effects on human health.


Assuntos
Acil Coenzima A/química , Esfingolipídeos/química , Acil Coenzima A/metabolismo , Serina/química , Serina/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Esfingolipídeos/metabolismo
13.
N Engl J Med ; 381(15): 1422-1433, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31509666

RESUMO

BACKGROUND: Identifying mechanisms of diseases with complex inheritance patterns, such as macular telangiectasia type 2, is challenging. A link between macular telangiectasia type 2 and altered serine metabolism has been established previously. METHODS: Through exome sequence analysis of a patient with macular telangiectasia type 2 and his family members, we identified a variant in SPTLC1 encoding a subunit of serine palmitoyltransferase (SPT). Because mutations affecting SPT are known to cause hereditary sensory and autonomic neuropathy type 1 (HSAN1), we examined 10 additional persons with HSAN1 for ophthalmologic disease. We assayed serum amino acid and sphingoid base levels, including levels of deoxysphingolipids, in patients who had macular telangiectasia type 2 but did not have HSAN1 or pathogenic variants affecting SPT. We characterized mice with low serine levels and tested the effects of deoxysphingolipids on human retinal organoids. RESULTS: Two variants known to cause HSAN1 were identified as causal for macular telangiectasia type 2: of 11 patients with HSAN1, 9 also had macular telangiectasia type 2. Circulating deoxysphingolipid levels were 84.2% higher among 125 patients with macular telangiectasia type 2 who did not have pathogenic variants affecting SPT than among 94 unaffected controls. Deoxysphingolipid levels were negatively correlated with serine levels, which were 20.6% lower than among controls. Reduction of serine levels in mice led to increases in levels of retinal deoxysphingolipids and compromised visual function. Deoxysphingolipids caused photoreceptor-cell death in retinal organoids, but not in the presence of regulators of lipid metabolism. CONCLUSIONS: Elevated levels of atypical deoxysphingolipids, caused by variant SPTLC1 or SPTLC2 or by low serine levels, were risk factors for macular telangiectasia type 2, as well as for peripheral neuropathy. (Funded by the Lowy Medical Research Institute and others.).


Assuntos
Neuropatias Hereditárias Sensoriais e Autônomas/genética , Mutação , Telangiectasia Retiniana/genética , Serina C-Palmitoiltransferase/genética , Serina/metabolismo , Esfingolipídeos/metabolismo , Adulto , Idoso , Animais , Análise Mutacional de DNA , Modelos Animais de Doenças , Exoma/genética , Feminino , Neuropatias Hereditárias Sensoriais e Autônomas/complicações , Neuropatias Hereditárias Sensoriais e Autônomas/metabolismo , Humanos , Metabolismo dos Lipídeos , Macula Lutea/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Linhagem , Telangiectasia Retiniana/complicações , Telangiectasia Retiniana/metabolismo , Fatores de Risco , Serina/sangue , Esfingosina/análogos & derivados , Esfingosina/análise , Adulto Jovem
14.
BioDrugs ; 33(5): 539-553, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31392631

RESUMO

MYC is a master transcriptional regulator that controls almost all cellular processes. Over the last several decades, researchers have strived to define the context-dependent transcriptional gene programs that are controlled by MYC, as well as the mechanisms that regulate MYC function, in an effort to better understand the contribution of this oncoprotein to cancer progression. There are a wealth of data indicating that deregulation of MYC activity occurs in a large number of cancers and significantly contributes to disease progression, metastatic potential, and therapeutic resistance. Although the therapeutic targeting of MYC in cancer is highly desirable, there remain substantial structural and functional challenges that have impeded direct MYC-targeted drug development and efficacy. While efforts to drug the 'undruggable' may seem futile given these challenges and considering the broad reach of MYC, significant strides have been made to identify points of regulation that can be exploited for therapeutic purposes. These include targeting the deregulation of MYC transcription in cancer through small-molecule inhibitors that induce epigenetic silencing or that regulate the G-quadruplex structures within the MYC promoter. Alternatively, compounds that disrupt the DNA-binding activities of MYC have been the long-standing focus of many research groups, since this method would prevent downstream MYC oncogenic activities regardless of upstream alterations. Finally, proteins involved in the post-translational regulation of MYC have been identified as important surrogate targets to reduce MYC activity downstream of aberrant cell stimulatory signals. Given the complex regulation of the MYC signaling pathway, a combination of these approaches may provide the most durable response, but this has yet to be shown. Here, we provide a comprehensive overview of the different therapeutic strategies being employed to target oncogenic MYC function, with a focus on post-translational mechanisms.


Assuntos
Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Genes myc , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Animais , Humanos , Morfolinas/farmacologia , Neoplasias/genética , Fosforilação , Multimerização Proteica/efeitos dos fármacos , Proteína Fosfatase 2/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Piranos/farmacologia , Serina/metabolismo
15.
J Ind Microbiol Biotechnol ; 46(12): 1685-1695, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31420796

RESUMO

Glutathione is a bioactive tripeptide composed of glycine, L-cysteine, and L-glutamate, and has been widely used in pharmaceutical, food, and healthy products. The current metabolic studies of glutathione were mainly focused on the native producing strains with precursor amino acid supplementation. In the present work, Corynebacterium glutamicum, a workhorse for industrial production of a series of amino acids, was engineered to produce glutathione. First, the introduction of glutathione synthetase gene gshF from Streptococcus agalactiae fulfilled the ability of glutathione production in C. glutamicum and revealed that L-cysteine was the limiting factor. Then, considering the inherent capability of L-glutamate synthesis and the availability of external addition of low-cost glycine, L-cysteine biosynthesis was enhanced using a varieties of pathway engineering methods, such as disrupting the degradation pathways of L-cysteine and L-serine, and removing the repressor responsible for sulfur metabolism. Finally, the simultaneously introduction of gshF and enhancement of cysteine formation enabled C. glutamicum strain to produce glutathione greatly. Without external addition of L-cysteine and L-glutamate, 756 mg/L glutathione was produced. This is first time to demonstrate the potential of the glutathione non-producing strain C. glutamicum for glutathione production and provide a novel strategy to construct glutathione-producing strains.


Assuntos
Corynebacterium glutamicum/metabolismo , Glutationa/biossíntese , Corynebacterium glutamicum/genética , Cisteína/metabolismo , Ácido Glutâmico/metabolismo , Glicina/metabolismo , Engenharia Metabólica/métodos , Redes e Vias Metabólicas , Serina/metabolismo
16.
Cancer Sci ; 110(10): 3110-3121, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31385398

RESUMO

Hepatocellular carcinoma (HCC) is one of the most common malignancies and the fourth leading cause of cancer-related death worldwide. Our previous study showed that EYA4 functioned by suppressing growth of HCC tumor cells, but its molecular mechanism is still not elucidated. Based on the results of gene microassay, EYA4 was inversely correlated with MYCBP and was verified in human HCC tissues by immunohistochemistry and western blot. Overexpressed and KO EYA4 in human HCC cell lines confirmed the negative correlation between EYA4 and MYCBP by qRT-PCR and western blot. Transfected siRNA of MYCBP in EYA4 overexpressed cells and overexpressed MYCBP in EYA4 KO cells could efficiently rescue the proliferation and G2/M arrest effects of EYA4 on HCC cells. Mechanistically, armed with serine/threonine-specific protein phosphatase activity, EYA4 reduced nuclear translocation of ß-catenin by dephosphorylating ß-catenin at Ser552, thereby suppressing the transcription of MYCBP which was induced by ß-catenin/LEF1 binding to the promoter of MYCBP. Clinically, HCC patients with highly expressed EYA4 and poorly expressed MYCBP had significantly longer disease-free survival and overall survival than HCC patients with poorly expressed EYA4 and highly expressed MYCBP. In conclusion, EYA4 suppressed HCC tumor cell growth by repressing MYCBP by dephosphorylating ß-catenin S552. EYA4 combined with MYCBP could be potential prognostic biomarkers in HCC.


Assuntos
Carcinoma Hepatocelular/metabolismo , Proteínas de Ligação a DNA/genética , Neoplasias Hepáticas/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , beta Catenina/metabolismo , Adulto , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Masculino , Pessoa de Meia-Idade , Fosforilação , Prognóstico , Serina/metabolismo , Análise de Sobrevida , Fatores de Transcrição/metabolismo , Transcrição Genética , beta Catenina/química
17.
Elife ; 82019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31385803

RESUMO

The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of the C-terminal domain (CTD) of the largest subunit (RPB1) of RNA polymerase II and is essential for the transition from transcription initiation to elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activity in vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and hinders understanding of the transition from transcription initiation to elongation. Through in vitro reconstruction of CTD phosphorylation, mass spectrometry analysis, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of P-TEFb and alters its specificity from Ser5 to Ser2. The loss of Tyr1 phosphorylation causes an accumulation of RNA polymerase II in the promoter region as detected by ChIP-seq. We demonstrate the ability of Tyr1 phosphorylation to generate a heterogeneous CTD modification landscape that expands the CTD's coding potential. These findings provide direct experimental evidence for a combinatorial CTD phosphorylation code wherein previously installed modifications direct the identity and abundance of subsequent coding events by influencing the behavior of downstream enzymes.


Assuntos
Fator B de Elongação Transcricional Positiva/metabolismo , Processamento de Proteína Pós-Traducional , RNA Polimerase II/metabolismo , Serina/metabolismo , Tirosina/metabolismo , Humanos , Fosforilação , Transcrição Genética
18.
Nutrients ; 11(8)2019 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-31426549

RESUMO

The biological effects of insulin signaling are regulated by the phosphorylation of insulin receptor substrate 1 (IRS1) at serine (Ser) residues. In the brain, phosphorylation of IRS1 at specific Ser sites increases in patients with Alzheimer's disease (AD) and its animal models. However, whether the activation of Ser sites on neural IRS1 is related to any type of memory decline remains unclear. Here, we show the modifications of IRS1 through its phosphorylation at etiology-specific Ser sites in various animal models of memory decline, such as diabetic, aged, and amyloid precursor protein (APP) knock-in NL-G-F (APPKINL-G-F) mice. Substantial phosphorylation of IRS1 at specific Ser sites occurs in type 2 diabetes- or age-related memory deficits independently of amyloid-ß (Aß). Furthermore, we present the first evidence that, in APPKINL-G-F mice showing Aß42 elevation, the increased phosphorylation of IRS1 at multiple Ser sites occurs without memory impairment. Our findings suggest that the phosphorylation of IRS1 at specific Ser sites is a potential marker of Aß-unrelated memory deficits caused by type 2 diabetes and aging; however, in Aß-related memory decline, the modifications of IRS1 may be a marker of early detection of Aß42 elevation prior to the onset of memory decline in AD.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Insulina/metabolismo , Transtornos da Memória/metabolismo , Memória , Envelhecimento , Doença de Alzheimer/complicações , Animais , Encéfalo/metabolismo , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Masculino , Transtornos da Memória/etiologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação , Serina/metabolismo , Transdução de Sinais
19.
Basic Res Cardiol ; 114(5): 40, 2019 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-31463533

RESUMO

Connexin 43 (Cx43)-associated gap junctions form electrical and mechanical conduits between adjacent ventricular cardiomyocytes, ensuring coordinate electrical excitation and synchronic contraction for each heartbeat. Cx43 dephosphorylation is a characteristic of ischemia, arrhythmia, and a failing and aging myocardium, but the exact phosphosite(s) triggering myocardial apoptosis and electrical disturbance and its underlying mechanisms are unclear. We previously found that Cx43-serine 282 phosphorylation (pS282) can regulate cardiomyocyte survival and electrical stability. Here, we investigated the hypothesis that S282 dephosphorylation occurs in and contributes to ischemia/reperfusion (I/R)-induced cardiac injury. We found enhanced Cx43-pS262 and Cx43-pS368 but decreased Cx43-pS282 in rat hearts subjected to I/R (30 min/2 h). I/R rats had ventricular arrhythmias and myocardial apoptosis with activation of the p38 mitogen-activated protein kinase (p38)/factor-associated suicide (Fas)/Fas-associating protein with a novel death domain (FADD) pathway. Similarly, S282 dephosphorylation, abnormal Ca2+ transients, cell apoptosis and p38/Fas/FADD activation also occurred in neonatal rat ventricular myocytes exposed to anoxia/reoxygenation (12/6 h). To confirm the causative role of S282 dephosphorylation in cardiac injury, rat hearts were intramyocardially injected with a virus carrying the S282 mutant substituted with alanine (S282A), thus causing arrhythmias and reducing cardiac output and myocardial apoptosis with p38/Fas/FADD pathway activation. Moreover, Cx43-S282A+/- mice displayed arrhythmias and impaired cardiac output with global myocardial apoptosis. Our findings revealed that Cx43 dephosphorylation at S282 triggers arrhythmias and, at least partly, contributes to cardiomyocyte death upon I/R by activating the p38/Fas/FADD pathway, providing a novel molecular mechanism and potential target for protecting against cardiac I/R injury.


Assuntos
Arritmias Cardíacas/metabolismo , Conexina 43/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miócitos Cardíacos/patologia , Animais , Apoptose/fisiologia , Arritmias Cardíacas/fisiopatologia , Masculino , Traumatismo por Reperfusão Miocárdica/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Serina/metabolismo
20.
Mol Cells ; 42(8): 604-616, 2019 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-31446747

RESUMO

Phosphoserine phosphatase (PSPH) is one of the key enzymes of the L-serine synthesis pathway. PSPH is reported to affect the progression and survival of several cancers in an L-serine synthesis-independent manner, but the mechanism remains elusive. We demonstrate that PSPH promotes lung cancer progression through a noncanonical L-serine-independent pathway. PSPH was significantly associated with the prognosis of lung cancer patients and regulated the invasion and colony formation of lung cancer cells. Interestingly, L-serine had no effect on the altered invasion and colony formation by PSPH. Upon measuring the phosphatase activity of PSPH on a serine-phosphorylated peptide, we found that PSPH dephosphorylated phospho-serine in peptide sequences. To identify the target proteins of PSPH, we analyzed the protein phosphorylation profile and the PSPH-interacting protein profile using proteomic analyses and found one putative target protein, IRS-1. Immunoprecipitation and immunoblot assays validated a specific interaction between PSPH and IRS1 and the dephosphorylation of phospho-IRS-1 by PSPH in lung cancer cells. We suggest that the specific interaction and dephosphorylation activity of PSPH have novel therapeutic potential for lung cancer treatment, while the metabolic activity of PSPH, as a therapeutic target, is controversial.


Assuntos
Progressão da Doença , Proteínas Substratos do Receptor de Insulina/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Monoéster Fosfórico Hidrolases/metabolismo , Serina/metabolismo , Células A549 , Animais , Humanos , Camundongos , Invasividade Neoplásica , Fosforilação , Transdução de Sinais , Análise de Sobrevida , Ensaio Tumoral de Célula-Tronco
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