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1.
Nature ; 577(7789): 266-270, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31827282

RESUMO

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs)1. Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.


Assuntos
Histona Acetiltransferases/metabolismo , Leucemia Mieloide Aguda/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Linhagem Celular Tumoral , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Terciária de Proteína
2.
EMBO J ; 32(5): 713-27, 2013 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-23386062

RESUMO

The intracellular pathogenic bacterium Salmonella enterica serovar typhimurium (Salmonella) relies on acidification of the Salmonella-containing vacuole (SCV) for survival inside host cells. The transport and fusion of membrane-bound compartments in a cell is regulated by small GTPases, including Rac and members of the Rab GTPase family, and their effector proteins. However, the role of these components in survival of intracellular pathogens is not completely understood. Here, we identify Nischarin as a novel dual effector that can interact with members of Rac and Rab GTPase (Rab4, Rab14 and Rab9) families at different endosomal compartments. Nischarin interacts with GTP-bound Rab14 and PI(3)P to direct the maturation of early endosomes to Rab9/CD63-containing late endosomes. Nischarin is recruited to the SCV in a Rab14-dependent manner and enhances acidification of the SCV. Depletion of Nischarin or the Nischarin binding partners--Rac1, Rab14 and Rab9 GTPases--reduced the intracellular growth of Salmonella. Thus, interaction of Nischarin with GTPases may regulate maturation and subsequent acidification of vacuoles produced after phagocytosis of pathogens.


Assuntos
Endossomos/microbiologia , Receptores de Imidazolinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Salmonella typhimurium/crescimento & desenvolvimento , Vacúolos/microbiologia , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Transporte Biológico , Western Blotting , Endossomos/metabolismo , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Receptores de Imidazolinas/genética , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisossomos/metabolismo , Lisossomos/microbiologia , Fosfatos de Fosfatidilinositol/metabolismo , Transporte Proteico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções por Salmonella/microbiologia , Técnicas do Sistema de Duplo-Híbrido , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/genética
3.
J Biol Chem ; 290(43): 25834-46, 2015 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-26342081

RESUMO

The insulin/insulin-like growth factor (IGF)-1 signaling pathway (ISP) plays a fundamental role in long term health in a range of organisms. Protein kinases including Akt and ERK are intimately involved in the ISP. To identify other kinases that may participate in this pathway or intersect with it in a regulatory manner, we performed a whole kinome (779 kinases) siRNA screen for positive or negative regulators of the ISP, using GLUT4 translocation to the cell surface as an output for pathway activity. We identified PFKFB3, a positive regulator of glycolysis that is highly expressed in cancer cells and adipocytes, as a positive ISP regulator. Pharmacological inhibition of PFKFB3 suppressed insulin-stimulated glucose uptake, GLUT4 translocation, and Akt signaling in 3T3-L1 adipocytes. In contrast, overexpression of PFKFB3 in HEK293 cells potentiated insulin-dependent phosphorylation of Akt and Akt substrates. Furthermore, pharmacological modulation of glycolysis in 3T3-L1 adipocytes affected Akt phosphorylation. These data add to an emerging body of evidence that metabolism plays a central role in regulating numerous biological processes including the ISP. Our findings have important implications for diseases such as type 2 diabetes and cancer that are characterized by marked disruption of both metabolism and growth factor signaling.


Assuntos
Glucose/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Insulina/metabolismo , Fosfofrutoquinase-2/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais , Células 3T3-L1 , Animais , Transportador de Glucose Tipo 4/metabolismo , Células HeLa , Humanos , Camundongos , RNA Interferente Pequeno/genética
4.
J Biol Chem ; 287(9): 6128-38, 2012 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-22207758

RESUMO

Akt plays a major role in insulin regulation of metabolism in muscle, fat, and liver. Here, we show that in 3T3-L1 adipocytes, Akt operates optimally over a limited dynamic range. This indicates that Akt is a highly sensitive amplification step in the pathway. With robust insulin stimulation, substantial changes in Akt phosphorylation using either pharmacologic or genetic manipulations had relatively little effect on Akt activity. By integrating these data we observed that half-maximal Akt activity was achieved at a threshold level of Akt phosphorylation corresponding to 5-22% of its full dynamic range. This behavior was also associated with lack of concordance or demultiplexing in the behavior of downstream components. Most notably, FoxO1 phosphorylation was more sensitive to insulin and did not exhibit a change in its rate of phosphorylation between 1 and 100 nm insulin compared with other substrates (AS160, TSC2, GSK3). Similar differences were observed between various insulin-regulated pathways such as GLUT4 translocation and protein synthesis. These data indicate that Akt itself is a major amplification switch in the insulin signaling pathway and that features of the pathway enable the insulin signal to be split or demultiplexed into discrete outputs. This has important implications for the role of this pathway in disease.


Assuntos
Adipócitos/enzimologia , Resistência à Insulina/fisiologia , Insulina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Antibióticos Antineoplásicos/farmacologia , Simulação por Computador , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Transportador de Glucose Tipo 4/metabolismo , Hipoglicemiantes/metabolismo , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina/metabolismo , Camundongos , Dinâmica não Linear , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo
5.
Acta Crystallogr D Struct Biol ; 76(Pt 9): 889-898, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32876064

RESUMO

Cancer is one of the leading causes of mortality in humans, and recent work has focused on the area of immuno-oncology, in which the immune system is used to specifically target cancerous cells. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is an emerging therapeutic target in human cancers owing to its role in degrading cyclic GMP-AMP (cGAMP), an agonist of the stimulator of interferon genes (STING). The available structures of ENPP1 are of the mouse enzyme, and no structures are available with anything other than native nucleotides. Here, the first X-ray crystal structures of the human ENPP1 enzyme in an apo form, with bound nucleotides and with two known inhibitors are presented. The availability of these structures and a robust crystallization system will allow the development of structure-based drug-design campaigns against this attractive cancer therapeutic target.


Assuntos
Inibidores Enzimáticos , Proteínas de Membrana/agonistas , Neoplasias/enzimologia , Diester Fosfórico Hidrolases , Pirofosfatases , Inibidores Enzimáticos/química , Humanos , Diester Fosfórico Hidrolases/química , Ligação Proteica , Conformação Proteica , Pirofosfatases/química
6.
NPJ Syst Biol Appl ; 1: 15010, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-28725461

RESUMO

OBJECTIVE: Insulin resistance (IR) is one of the earliest predictors of type 2 diabetes. However, diagnosis of IR is limited. High fat fed mouse models provide key insights into IR. We hypothesized that early features of IR are associated with persistent changes in gene expression (GE) and endeavored to (a) develop novel methods for improving signal:noise in analysis of human GE using mouse models; (b) identify a GE motif that accurately diagnoses IR in humans; and (c) identify novel biology associated with IR in humans. METHODS: We integrated human muscle GE data with longitudinal mouse GE data and developed an unbiased three-level cross-species analysis platform (single gene, gene set, and networks) to generate a gene expression motif (GEM) indicative of IR. A logistic regression classification model validated GEM in three independent human data sets (n=115). RESULTS: This GEM of 93 genes substantially improved diagnosis of IR compared with routine clinical measures across multiple independent data sets. Individuals misclassified by GEM possessed other metabolic features raising the possibility that they represent a separate metabolic subclass. The GEM was enriched in pathways previously implicated in insulin action and revealed novel associations between ß-catenin and Jak1 and IR. Functional analyses using small molecule inhibitors showed an important role for these proteins in insulin action. CONCLUSIONS: This study shows that systems approaches for identifying molecular signatures provides a powerful way to stratify individuals into discrete metabolic groups. Moreover, we speculate that the ß-catenin pathway may represent a novel biomarker for IR in humans that warrant future investigation.

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