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1.
Nat Commun ; 11(1): 4479, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32900999

RESUMO

The giant protein titin is thought to be required for sarcomeric integrity in mature myocytes, but direct evidence for this hypothesis is limited. Here, we describe a mouse model in which Z-disc-anchored TTN is depleted in adult skeletal muscles. Inactivation of TTN causes sarcomere disassembly and Z-disc deformations, force impairment, myocyte de-stiffening, upregulation of TTN-binding mechanosensitive proteins and activation of protein quality-control pathways, concomitant with preferential loss of thick-filament proteins. Interestingly, expression of the myosin-bound Cronos-isoform of TTN, generated from an alternative promoter not affected by the targeting strategy, does not prevent deterioration of sarcomere formation and maintenance. Finally, we demonstrate that loss of Z-disc-anchored TTN recapitulates muscle remodeling in critical illness 'myosinopathy' patients, characterized by TTN-depletion and loss of thick filaments. We conclude that full-length TTN is required to integrate Z-disc and A-band proteins into the mature sarcomere, a function that is lost when TTN expression is pathologically lowered.


Assuntos
Fibras Musculares Esqueléticas/fisiologia , Proteínas Quinases/fisiologia , Sarcômeros/fisiologia , Animais , Fenômenos Biomecânicos , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Força Muscular/fisiologia , Atrofia Muscular/etiologia , Atrofia Muscular/patologia , Atrofia Muscular/fisiopatologia , Doenças Musculares/patologia , Doenças Musculares/fisiopatologia , Miosinas/metabolismo , Proteínas Quinases/deficiência , Proteínas Quinases/genética , Sarcômeros/patologia , Ubiquitinação
2.
PLoS Pathog ; 16(8): e1008761, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32790758

RESUMO

The virus-bacterial synergism implicated in secondary bacterial infections caused by Streptococcus pneumoniae following infection with epidemic or pandemic influenza A virus (IAV) is well documented. However, the molecular mechanisms behind such synergism remain largely ill-defined. In pneumocytes infected with influenza A virus, subsequent infection with S. pneumoniae leads to enhanced pneumococcal intracellular survival. The pneumococcal two-component system SirRH appears essential for such enhanced survival. Through comparative transcriptomic analysis between the ΔsirR and wt strains, a list of 179 differentially expressed genes was defined. Among those, the clpL protein chaperone gene and the psaB Mn+2 transporter gene, which are involved in the stress response, are important in enhancing S. pneumoniae survival in influenza-infected cells. The ΔsirR, ΔclpL and ΔpsaB deletion mutants display increased susceptibility to acidic and oxidative stress and no enhancement of intracellular survival in IAV-infected pneumocyte cells. These results suggest that the SirRH two-component system senses IAV-induced stress conditions and controls adaptive responses that allow survival of S. pneumoniae in IAV-infected pneumocytes.


Assuntos
Proteínas de Bactérias/metabolismo , Coinfecção/mortalidade , Vírus da Influenza A/patogenicidade , Influenza Humana/mortalidade , Pulmão/patologia , Infecções Pneumocócicas/mortalidade , Streptococcus pneumoniae/patogenicidade , Proteínas de Bactérias/genética , Sobrevivência Celular , Coinfecção/epidemiologia , Humanos , Influenza Humana/microbiologia , Influenza Humana/patologia , Influenza Humana/virologia , Pulmão/microbiologia , Pulmão/virologia , Infecções Pneumocócicas/microbiologia , Infecções Pneumocócicas/patologia , Infecções Pneumocócicas/virologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Streptococcus pneumoniae/metabolismo , Estresse Fisiológico , Virulência
3.
Mol Cell ; 79(3): 504-520.e9, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32707033

RESUMO

Protein kinases are essential for signal transduction and control of most cellular processes, including metabolism, membrane transport, motility, and cell cycle. Despite the critical role of kinases in cells and their strong association with diseases, good coverage of their interactions is available for only a fraction of the 535 human kinases. Here, we present a comprehensive mass-spectrometry-based analysis of a human kinase interaction network covering more than 300 kinases. The interaction dataset is a high-quality resource with more than 5,000 previously unreported interactions. We extensively characterized the obtained network and were able to identify previously described, as well as predict new, kinase functional associations, including those of the less well-studied kinases PIM3 and protein O-mannose kinase (POMK). Importantly, the presented interaction map is a valuable resource for assisting biomedical studies. We uncover dozens of kinase-disease associations spanning from genetic disorders to complex diseases, including cancer.


Assuntos
Redes Reguladoras de Genes , Doenças Genéticas Inatas/genética , Neoplasias/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Biologia Computacional/métodos , Conjuntos de Dados como Assunto , Regulação da Expressão Gênica , Ontologia Genética , Doenças Genéticas Inatas/enzimologia , Doenças Genéticas Inatas/patologia , Humanos , Redes e Vias Metabólicas/genética , Anotação de Sequência Molecular , Distrofias Musculares/enzimologia , Distrofias Musculares/genética , Distrofias Musculares/patologia , Neoplasias/enzimologia , Neoplasias/patologia , Doenças Neurodegenerativas/enzimologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Mapeamento de Interação de Proteínas/métodos , Proteínas Quinases/química , Proteínas Quinases/classificação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais
4.
Life Sci ; 257: 118116, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32702447

RESUMO

Emerging evidence implicates accelerated renal tubular epithelial cell (RTEC) senescence in renal fibrosis progression. Mitophagy protects against kidney injury. However, the mechanistic interplay between cell senescence and mitophagy in RTECs is not clearly defined. The purpose of this study was to evaluate the inhibition of RTEC senescence and renal fibrosis by quercetin and explore the underlying mechanisms. We found that quercetin attenuated RTEC senescence induced by angiotensin II (AngII) in vitro and unilateral ureteral obstruction in vivo. Moreover, we demonstrated that mitochondrial abnormalities such as elevated reactive oxygen species, decreased membrane potential, and fragmentation and accumulation of mitochondrial mass, occurred in AngII-treated RTECs. Quercetin treatment reversed these effects. Furthermore, quercetin enhanced mitophagy in AngII-treated RTECs, which was markedly reduced by treatment with mitophagy-specific inhibitors. Sirtuin-1 (SIRT1) was involved in quercetin-mediated PTEN-induced kinase 1 (PINK1)/Parkin-associated mitophagy activation. Pharmacological antagonism of SIRT1 in AngII-treated RTECs blocked the effects of quercetin on mitophagy and cellular senescence. Finally, quercetin alleviated kidney fibrosis by reducing RTEC senescence via mitophagy. Collectively, the antifibrotic effect of quercetin involved inhibition of RTEC senescence, possibly through activation of SIRT1/PINK1/Parkin-mediated mitophagy. These findings suggest that pharmacological elimination of senescent cells and stimulation of mitophagy represent effective therapeutic strategies to prevent kidney fibrosis.


Assuntos
Antioxidantes/farmacologia , Senescência Celular/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Rim/patologia , Mitofagia/efeitos dos fármacos , Proteínas Quinases/metabolismo , Quercetina/farmacologia , Sirtuína 1/metabolismo , Animais , Antioxidantes/uso terapêutico , Linhagem Celular , Epitélio/efeitos dos fármacos , Fibrose , Citometria de Fluxo , Rim/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Quercetina/uso terapêutico , Ratos
5.
PLoS One ; 15(7): e0235766, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32639993

RESUMO

High-grade serous ovarian carcinoma (HGSOC) remains the deadliest form of epithelial ovarian cancer and despite major efforts little improvement in overall survival has been achieved. Identification of recurring "driver" genetic lesions has the potential to enable design of novel therapies for cancer. Here, we report on a study to find such new therapeutic targets for HGSOC using exome-capture sequencing approach targeting all kinase genes in 127 patient samples. Consistent with previous reports, the most frequently mutated gene was TP53 (97% mutation frequency) followed by BRCA1 (10% mutation frequency). The average mutation frequency of the kinase genes mutated from our panel was 1.5%. Intriguingly, after BRCA1, JAK3 was the most frequently mutated gene (4% mutation frequency). We tested the transforming properties of JAK3 mutants using the Ba/F3 cell-based in vitro functional assay and identified a novel gain-of-function mutation in the kinase domain of JAK3 (p.T1022I). Importantly, p.T1022I JAK3 mutants displayed higher sensitivity to the JAK3-selective inhibitor Tofacitinib compared to controls. For independent validation, we re-sequenced the entire JAK3 coding sequence using tagged amplicon sequencing (TAm-Seq) in 463 HGSOCs resulting in an overall somatic mutation frequency of 1%. TAm-Seq screening of CDK12 in the same population revealed a 7% mutation frequency. Our data confirms that the frequency of mutations in kinase genes in HGSOC is low and provides accurate estimates for the frequency of JAK3 and CDK12 mutations in a large well characterized cohort. Although p.T1022I JAK3 mutations are rare, our functional validation shows that if detected they should be considered as potentially actionable for therapy. The observation of CDK12 mutations in 7% of HGSOC cases provides a strong rationale for routine somatic testing, although more functional and clinical characterization is required to understand which nonsynonymous mutations alterations are associated with homologous recombination deficiency.


Assuntos
Proteína BRCA1/genética , Cistadenocarcinoma Seroso/genética , Janus Quinase 3/genética , Mutação , Neoplasias Ovarianas/genética , Proteínas Quinases/genética , Proteína Supressora de Tumor p53/genética , Proteína BRCA1/metabolismo , Estudos de Casos e Controles , Estudos de Coortes , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Janus Quinase 3/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo
6.
Nat Commun ; 11(1): 3563, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32678104

RESUMO

Rapidly increasing availability of genomic data and ensuing identification of disease associated mutations allows for an unbiased insight into genetic drivers of disease development. However, determination of molecular mechanisms by which individual genomic changes affect biochemical processes remains a major challenge. Here, we develop a multilayered proteomic workflow to explore how genetic lesions modulate the proteome and are translated into molecular phenotypes. Using this workflow we determine how expression of a panel of disease-associated mutations in the Dyrk2 protein kinase alter the composition, topology and activity of this kinase complex as well as the phosphoproteomic state of the cell. The data show that altered protein-protein interactions caused by the mutations are associated with topological changes and affected phosphorylation of known cancer driver proteins, thus linking Dyrk2 mutations with cancer-related biochemical processes. Overall, we discover multiple mutation-specific functionally relevant changes, thus highlighting the extensive plasticity of molecular responses to genetic lesions.


Assuntos
Neoplasias/genética , Neoplasias/patologia , Proteínas Quinases/genética , Proteômica/métodos , Linhagem Celular , Humanos , Espectrometria de Massas , Complexos Multiproteicos , Mutação , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Fenótipo , Fosfoproteínas/metabolismo , Fosforilação , Conformação Proteica , Mapas de Interação de Proteínas , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteoma/metabolismo
7.
PLoS Genet ; 16(7): e1008883, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32609718

RESUMO

Plant steroid hormones brassinosteroids (BRs) regulate plant growth and development at many levels. While negative regulatory factors that inhibit development and are counteracted by BRs exist in the root meristem, these factors have not been characterized. The functions of UPB1 transcription factor in BR-regulated root growth have not been established, although its role in regulating root are well documented. Here, we found that BIN2 interacts with and phosphorylates the UPB1 transcription factor consequently promoting UPB1 stability and transcriptional activity. Genetic analysis revealed that UPB1 deficiency could partially recover the short-root phenotype of BR-deficient mutants. Expression of a mutated UPB1S37AS41A protein lacking a conserved BIN2 phosphorylation sites can rescue shorter root phenotype of bin2-1 mutant. In addition, UPB1 was repressed by BES1 at the transcriptional level. The paclobutrazol-resistant protein family (PRE2/3) interacts with UPB1 and inhibits its transcriptional activity to promote root meristem development, and BIN2-mediated phosphorylation of UPB1 suppresses its interaction with PRE2/3, and subsequently impairing root meristem development. Taken together, our data elucidate a molecular mechanism by which BR promotes root growth via inhibiting BIN2-UPB1 module.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Brassinosteroides/metabolismo , Proteínas Quinases/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas/genética , Meristema/genética , Meristema/crescimento & desenvolvimento , Fosforilação , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Transdução de Sinais/genética
8.
BMC Bioinformatics ; 21(1): 309, 2020 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-32664863

RESUMO

BACKGROUND: Despite continued efforts using chemical similarity methods in virtual screening, currently developed approaches suffer from time-consuming multistep procedures and low success rates. We recently developed a machine learning-based chemical binding similarity model considering common structural features from molecules binding to the same, or evolutionarily related targets. The chemical binding similarity measures the resemblance of chemical compounds in terms of binding site similarity to better describe functional similarities that arise from target binding. In this study, we have shown how the chemical binding similarity could be used in virtual screening together with the conventional structure-based methods. RESULTS: The chemical binding similarity, receptor-based pharmacophore, chemical structure similarity, and molecular docking methods were evaluated to identify an effective virtual screening procedure for desired target proteins. When we tested the chemical binding similarity method with test sets of 51 kinases, it outperformed the traditional structural similarity-based methods as well as structure-based methods, such as molecular docking and receptor-based pharmacophore modeling, in terms of finding active compounds. We further validated the results by performing virtual screening (using the chemical binding similarity and receptor-based pharmacophore methods) against a completely blind dataset for mitogen-activated protein kinase kinase 1 (MEK1), ephrin type-B receptor 4 (EPHB4) and wee1-like protein kinase (WEE1). The in vitro kinase binding assay confirmed that 6 out of 13 (46.2%) for MEK1 and 2 out of 12 (16.7%) for EPHB4 were newly identified only by the chemical binding similarity model. CONCLUSIONS: We report that the virtual screening results could further be improved by combining the chemical binding similarity model with 3D-QSAR pharmacophore and molecular docking models. Not only the new inhibitors are identified in this study, but also many of the identified molecules have low structural similarity scores against already reported inhibitors and that show the revelation of novel scaffolds.


Assuntos
Simulação de Acoplamento Molecular , Relação Quantitativa Estrutura-Atividade , Área Sob a Curva , Sítios de Ligação , Humanos , Aprendizado de Máquina , Compostos Orgânicos/química , Compostos Orgânicos/metabolismo , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Ligação Proteica , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Curva ROC
9.
Exp Parasitol ; 216: 107935, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32569599

RESUMO

Toxoplasma gondii is an important pathogen that causes serious public health problems. Currently, therapeutic drugs for toxoplasmosis cause serious side effects, and more effective and novel substances with relatively low toxicity are urgently needed. Ursolic acid (UA) has many properties that can be beneficial to healthcare. In this study, we synthesized eight series of UA derivatives bearing a tetrazole moiety and evaluated their anti-T. gondii activity in vitro using spiramycin as a positive control. Most of the synthesized derivatives exhibited better anti-T. gondii activity in vitro than UA, among which compound 12a exhibited the most potent anti-T. gondii activity. Furthermore, the results of biochemical parameter determination indicated that 12a effectively restored the normal body weight of mice infected with T. gondii, reduced hepatotoxicity, and exerted significant anti-oxidative effects compared with the findings for spiramycin. Additionally, our molecular docking study indicated that the synthesized compounds could act as potential inhibitors of T. gondii calcium-dependent protein kinase 1 (TgCDPK1), with 12a possessing strong affinity for TgCDPK1 via binding to the key amino acids GLU129 and TYR131.


Assuntos
Anti-Infecciosos/farmacologia , Toxoplasma/efeitos dos fármacos , Toxoplasmose Animal/tratamento farmacológico , Toxoplasmose/tratamento farmacológico , Triterpenos/farmacologia , Alanina Transaminase/sangue , Animais , Anti-Infecciosos/química , Anti-Infecciosos/uso terapêutico , Aspartato Aminotransferases/sangue , Coccidiostáticos/química , Coccidiostáticos/farmacologia , Modelos Animais de Doenças , Feminino , Glutationa/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/patologia , Malondialdeído/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Tamanho do Órgão/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases , Distribuição Aleatória , Espiramicina/farmacologia , Baço/efeitos dos fármacos , Baço/patologia , Triterpenos/química , Triterpenos/uso terapêutico
10.
Proc Natl Acad Sci U S A ; 117(25): 14270-14279, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513699

RESUMO

Directional cell migration involves signaling cascades that stimulate actin assembly at the leading edge, and additional pathways must inhibit actin polymerization at the rear. During neuroblast migration in Caenorhabditis elegans, the transmembrane protein MIG-13/Lrp12 acts through the Arp2/3 nucleation-promoting factors WAVE and WASP to guide the anterior migration. Here we show that a tyrosine kinase, SRC-1, directly phosphorylates MIG-13 and promotes its activity on actin assembly at the leading edge. In GFP knockin animals, SRC-1 and MIG-13 distribute along the entire plasma membrane of migrating cells. We reveal that a receptor-like tyrosine phosphatase, PTP-3, maintains the F-actin polarity during neuroblast migration. Recombinant PTP-3 dephosphorylates SRC-1-dependent MIG-13 phosphorylation in vitro. Importantly, the endogenous PTP-3 accumulates at the rear of the migrating neuroblast, and its extracellular domain is essential for directional cell migration. We provide evidence that the asymmetrically localized tyrosine phosphatase PTP-3 spatially restricts MIG-13/Lrp12 receptor activity in migrating cells.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Movimento Celular/fisiologia , Neurônios/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Polaridade Celular/fisiologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Proteínas de Membrana/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Transdução de Sinais
11.
Proc Natl Acad Sci U S A ; 117(25): 14433-14443, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513747

RESUMO

During infection, the bacterial pathogen Legionella pneumophila manipulates a variety of host cell signaling pathways, including the Hippo pathway which controls cell proliferation and differentiation in eukaryotes. Our previous studies revealed that L. pneumophila encodes the effector kinase LegK7 which phosphorylates MOB1A, a highly conserved scaffold protein of the Hippo pathway. Here, we show that MOB1A, in addition to being a substrate of LegK7, also functions as an allosteric activator of its kinase activity. A crystallographic analysis of the LegK7-MOB1A complex revealed that the N-terminal half of LegK7 is structurally similar to eukaryotic protein kinases, and that MOB1A directly binds to the LegK7 kinase domain. Substitution of interface residues critical for complex formation abrogated allosteric activation of LegK7 both in vitro and within cells and diminished MOB1A phosphorylation. Importantly, the N-terminal extension (NTE) of MOB1A not only regulated complex formation with LegK7 but also served as a docking site for downstream substrates such as the transcriptional coregulator YAP1. Deletion of the NTE from MOB1A or addition of NTE peptides as binding competitors attenuated YAP1 recruitment to and phosphorylation by LegK7. By providing mechanistic insight into the formation and regulation of the LegK7-MOB1A complex, our study unravels a sophisticated molecular mimicry strategy that is used by L. pneumophila to take control of the host cell Hippo pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Bactérias/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Legionella pneumophila/metabolismo , Proteínas Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Regulação Alostérica , Animais , Proteínas de Bactérias/genética , Proteínas de Ciclo Celular/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Legionella pneumophila/patogenicidade , Doença dos Legionários/microbiologia , Doença dos Legionários/patologia , Macrófagos Alveolares/microbiologia , Macrófagos Alveolares/patologia , Camundongos , Simulação de Dinâmica Molecular , Mimetismo Molecular , Fosforilação , Ligação Proteica , Proteínas Quinases/genética , Células RAW 264.7 , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo
12.
Nat Commun ; 11(1): 3151, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561730

RESUMO

Mixed lineage kinase domain-like (MLKL) is the terminal protein in the pro-inflammatory necroptotic cell death program. RIPK3-mediated phosphorylation is thought to initiate MLKL oligomerization, membrane translocation and membrane disruption, although the precise choreography of events is incompletely understood. Here, we use single-cell imaging approaches to map the chronology of endogenous human MLKL activation during necroptosis. During the effector phase of necroptosis, we observe that phosphorylated MLKL assembles into higher order species on presumed cytoplasmic necrosomes. Subsequently, MLKL co-traffics with tight junction proteins to the cell periphery via Golgi-microtubule-actin-dependent mechanisms. MLKL and tight junction proteins then steadily co-accumulate at the plasma membrane as heterogeneous micron-sized hotspots. Our studies identify MLKL trafficking and plasma membrane accumulation as crucial necroptosis checkpoints. Furthermore, the accumulation of phosphorylated MLKL at intercellular junctions accelerates necroptosis between neighbouring cells, which may be relevant to inflammatory bowel disease and other necroptosis-mediated enteropathies.


Assuntos
Necroptose , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Humanos , Transporte Proteico , Proteínas de Junções Íntimas/metabolismo
13.
Nat Commun ; 11(1): 3150, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561755

RESUMO

MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, MlklD139V, that alters the two-helix 'brace' that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of MlklD139V homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO).


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Sistema Hematopoético/patologia , Necroptose/genética , Proteínas Quinases/genética , Animais , Animais Recém-Nascidos , Doenças Hereditárias Autoinflamatórias , Humanos , Inflamação/genética , Camundongos , Mutação de Sentido Incorreto , Osteomielite/genética , Proteínas Quinases/metabolismo
14.
Nat Commun ; 11(1): 3133, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561764

RESUMO

Proximity proteomics has greatly advanced the analysis of native protein complexes and subcellular structures in culture, but has not been amenable to study development and disease in vivo. Here, we have generated a knock-in mouse with the biotin ligase (BioID) inserted at titin's Z-disc region to identify protein networks that connect the sarcomere to signal transduction and metabolism. Our census of the sarcomeric proteome from neonatal to adult heart and quadriceps reveals how perinatal signaling, protein homeostasis and the shift to adult energy metabolism shape the properties of striated muscle cells. Mapping biotinylation sites to sarcomere structures refines our understanding of myofilament dynamics and supports the hypothesis that myosin filaments penetrate Z-discs to dampen contraction. Extending this proof of concept study to BioID fusion proteins generated with Crispr/CAS9 in animal models recapitulating human pathology will facilitate the future analysis of molecular machines and signaling hubs in physiological, pharmacological, and disease context.


Assuntos
Carbono-Nitrogênio Ligases/genética , Proteínas de Escherichia coli/genética , Proteínas Quinases/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Proteínas Repressoras/genética , Sarcômeros/metabolismo , Animais , Animais Recém-Nascidos , Biotinilação/genética , Feminino , Técnicas de Introdução de Genes , Masculino , Redes e Vias Metabólicas , Camundongos Transgênicos , Modelos Animais , Miocárdio/citologia , Miocárdio/metabolismo , Estudo de Prova de Conceito , Mapas de Interação de Proteínas/fisiologia , Proteínas Quinases/genética , Proteostase/fisiologia , Músculo Quadríceps/citologia , Músculo Quadríceps/metabolismo , Sarcômeros/genética , Transdução de Sinais/fisiologia , Relação Estrutura-Atividade
15.
Mol Cell Biol ; 40(17)2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32541066

RESUMO

Rad3 is the orthologue of ATR and the sensor kinase of the DNA replication checkpoint in Schizosaccharomyces pombe Under replication stress, it initiates checkpoint signaling at the forks necessary for maintaining genome stability and cell survival. To better understand the checkpoint initiation process, we have carried out a genetic screen in fission yeast by random mutation of the genome, looking for mutants defective in response to the replication stress induced by hydroxyurea. In addition to the previously reported mutant with a C-to-Y change at position 307 encoded by tel2 (tel2-C307Y mutant) (Y.-J. Xu, S. Khan, A. C. Didier, M. Wozniak, et al., Mol Cell Biol 39:e00175-19, 2019, https://doi.org/10.1128/MCB.00175-19), this screen has identified six mutations in rqh1 encoding a RecQ DNA helicase. Surprisingly, these rqh1 mutations, except for a start codon mutation, are all in the helicase domain, indicating that the helicase activity of Rqh1 plays an important role in the replication checkpoint. In support of this notion, integration of two helicase-inactive mutations or deletion of rqh1 generated a similar Rad3 signaling defect, and heterologous expression of human RECQ1, BLM, and RECQ4 restored the Rad3 signaling and partially rescued a rqh1 helicase mutant. Therefore, the replication checkpoint function of Rqh1 is highly conserved, and mutations in the helicase domain of these human enzymes may cause the checkpoint defect and contribute to the cancer predisposition syndromes.


Assuntos
Quinase do Ponto de Checagem 2/metabolismo , DNA Helicases/metabolismo , Replicação do DNA , DNA Fúngico/biossíntese , Proteínas de Schizosaccharomyces pombe/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , DNA Helicases/genética , DNA Fúngico/genética , DNA Fúngico/metabolismo , Instabilidade Genômica , Hidroxiureia/farmacologia , Proteínas Quinases/metabolismo , RecQ Helicases/genética , RecQ Helicases/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Transdução de Sinais/efeitos dos fármacos
16.
PLoS Genet ; 16(6): e1008840, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32579556

RESUMO

The S. cerevisiae ISR1 gene encodes a putative kinase with no ascribed function. Here, we show that Isr1 acts as a negative regulator of the highly-conserved hexosamine biosynthesis pathway (HBP), which converts glucose into uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), the carbohydrate precursor to protein glycosylation, GPI-anchor formation, and chitin biosynthesis. Overexpression of ISR1 is lethal and, at lower levels, causes sensitivity to tunicamycin and resistance to calcofluor white, implying impaired protein glycosylation and reduced chitin deposition. Gfa1 is the first enzyme in the HBP and is conserved from bacteria and yeast to humans. The lethality caused by ISR1 overexpression is rescued by co-overexpression of GFA1 or exogenous glucosamine, which bypasses GFA1's essential function. Gfa1 is phosphorylated in an Isr1-dependent fashion and mutation of Isr1-dependent sites ameliorates the lethality associated with ISR1 overexpression. Isr1 contains a phosphodegron that is phosphorylated by Pho85 and subsequently ubiquitinated by the SCF-Cdc4 complex, largely confining Isr1 protein levels to the time of bud emergence. Mutation of this phosphodegron stabilizes Isr1 and recapitulates the overexpression phenotypes. As Pho85 is a cell cycle and nutrient responsive kinase, this tight regulation of Isr1 may serve to dynamically regulate flux through the HBP and modulate how the cell's energy resources are converted into structural carbohydrates in response to changing cellular needs.


Assuntos
Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Hexosaminas/biossíntese , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Metabolismo Energético , Glucose/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Mutação , Fosforilação , Proteínas Quinases/genética , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Proteínas de Saccharomyces cerevisiae/genética , Uridina Difosfato N-Acetilglicosamina/metabolismo
17.
Life Sci ; 256: 117923, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32522567

RESUMO

AIMS: Liver kinase B1 (LKB1) deficiency is associated with reduced expression of programmed death ligand 1 (PD-L1) and inferior clinical outcomes of PD-1/PD-L1 blockade in non-small cell lung cancer (NSCLC). This study aimed to investigate the mechanism by which LKB1 regulates PD-L1 expression and its role in programmed death 1 (PD-1) blockade therapy in NSCLC. MAIN METHODS: The impact of LKB1 on PD-L1 was assessed by western blot, qRT-PCR and immunohistochemistry in NSCLC. Activators/inhibitors of AMPK and NRF2 were applied to explore the mechanisms underlying the regulation of PD-L1 by LKB1. Efficiency of combined application of metformin and PD-1 blockade was evaluated in immunocompetent C57BL/6 mice. KEY FINDINGS: A remarkable positive correlation between LKB1 and PD-L1 expression was demonstrated in NSCLC tissues. Knockdown of LKB1 decreased PD-L1 in TC-1 cells, whereas overexpression of LKB1 increased PD-L1 in A549 cells. We further characterized that AMPK mediated the upregulation of PD-L1 by LKB1. Inhibition of AMPK or NRF2 markedly reduced PD-L1 in LKB1-intact NSCLC cells. In contrast, activation of AMPK or NRF2 reversed PD-L1 expression in LKB1-deficient NSCLC cells. Combined administration of metformin and anti-PD-1 antibody efficiently inhibited the growth of LKB1-intact tumors, whereas no obvious suppression was observed in LKB1-deficient tumors. SIGNIFICANCE: These findings demonstrated that LKB1 upregulates PD-L1 expression in NSCLC by activating the AMPK and KEAP1/NRF2 signaling. Activation of LKB1-AMPK with metformin improves the therapeutic effect of PD-1 blockade in NSCLC with wild-type LKB1.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Metformina/farmacologia , Receptor de Morte Celular Programada 1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células A549 , Animais , Anticorpos Monoclonais Humanizados/metabolismo , Antineoplásicos/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Metformina/metabolismo , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Transdução de Sinais , Ativação Transcricional , Regulação para Cima
18.
Gene ; 756: 144917, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32590104

RESUMO

The self-incompatibility system of Corylus is a sporophytic type that is phenotypically similar to that of Brassica. While the molecular mechanism of sporophytic self-incompatibility (SSI) has been investigated extensively in Brassica (Brassicaceae), little is known about the corresponding mechanism in Corylus (Betulaceae). Here, we discuss the SSI mechanism with respect to S-locus receptor kinase (SRK) gene homologs. To obtain two SRK candidate unigenes, we compared all of the unigenes in a transcriptional protein database from our previous study with BnSRK-1 (AB270767) using BLASTX with a cutoff e-value of 10-5. We then cloned the full-length cDNA of ChaSRK1 and ChaSRK2 genes from Ping'ou hybrid hazelnut (Corylus heterophylla × Corylus avellana) using RACE techniques. Bioinformatics approaches were used to analyze the cDNA sequences, protein sequences, and domains of the encoded proteins. The full-length ChaSRK1 cDNA was 2883 base pairs (bp) with a coding sequence (CDS) of 2,547 bp encoding 849 amino acid residues. The full-length ChaSRK2 cDNA was 2,693 bp, with a CDS of 2,433 bp encoding 811 amino acids. The ChaSRK1/2 proteins contained an S-domain (extracellular domain), a transmembrane domain, a Ser/Thr protein kinase active site (kinase domain), and DUF3660 and/or DUF3403 domains. The lengths of 18 partial SRK homologs ranged from 1347 to 1451 bp, and they contained the same structural domains as ChaSRK1 and ChaSRK2. Phylogenetic analysis revealed that all SRK homologs could be divided into two categories that were similar to the classification of SRKs in Brassica. The expression patterns of ChaSRK1 and ChaSRK2 differed: ChaSRK2 was predominantly expressed in mature stigmatic styles, while ChaSRK1 was expressed in other tissues with the highest in the root tips of Corylus. Using dual-color fluorescence in situ hybridization, ChaSRK1/2 expression was found to be localized in papillar cells. Collectively, these results revealed that SRKs from Corylus had similar characteristics to SRKs from Brassica. We therefore speculated that the SSI mechanism in Corylus might be more similar to the Brassica mechanism than to other SSI types.


Assuntos
Corylus/enzimologia , Corylus/fisiologia , Proteínas de Plantas/genética , Proteínas Quinases/genética , Autoincompatibilidade em Angiospermas , Sequência de Bases , Brassica/fisiologia , Clonagem Molecular , Corylus/genética , Filogenia , Alinhamento de Sequência , Transcriptoma
19.
Nat Commun ; 11(1): 3060, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561735

RESUMO

The MLKL pseudokinase is the terminal effector in the necroptosis cell death pathway. Phosphorylation by its upstream regulator, RIPK3, triggers MLKL's conversion from a dormant cytoplasmic protein into oligomers that translocate to, and permeabilize, the plasma membrane to kill cells. The precise mechanisms underlying these processes are incompletely understood, and were proposed to differ between mouse and human cells. Here, we examine the divergence of activation mechanisms among nine vertebrate MLKL orthologues, revealing remarkable specificity of mouse and human RIPK3 for MLKL orthologues. Pig MLKL can restore necroptotic signaling in human cells; while horse and pig, but not rat, MLKL can reconstitute the mouse pathway. This selectivity can be rationalized from the distinct conformations observed in the crystal structures of horse and rat MLKL pseudokinase domains. These studies identify important differences in necroptotic signaling between species, and suggest that, more broadly, divergent regulatory mechanisms may exist among orthologous pseudoenzymes.


Assuntos
Proteínas Quinases/química , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Animais , Galinhas , Cristalografia por Raios X , Citoplasma/enzimologia , Células HEK293 , Cavalos , Humanos , Camundongos , Necroptose , Necrose/metabolismo , Fosforilação , Conformação Proteica , Ratos , Transdução de Sinais , Smegmamorpha , Suínos , Células U937 , Xenopus
20.
Nat Commun ; 11(1): 3167, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576831

RESUMO

Legumes tightly regulate nodule number to balance the cost of supporting symbiotic rhizobia with the benefits of nitrogen fixation. C-terminally Encoded Peptides (CEPs) and CLAVATA3-like (CLE) peptides positively and negatively regulate nodulation, respectively, through independent systemic pathways, but how these regulations are coordinated remains unknown. Here, we show that rhizobia, Nod Factors, and cytokinins induce a symbiosis-specific CEP gene, MtCEP7, which positively regulates rhizobial infection. Via grafting and split root studies, we reveal that MtCEP7 increases nodule number systemically through the MtCRA2 receptor. MtCEP7 and MtCLE13 expression in rhizobia-inoculated roots rely on the MtCRE1 cytokinin receptor and on the MtNIN transcription factor. MtNIN binds and transactivates MtCEP7 and MtCLE13, and a NIN Binding Site (NBS) identified within the proximal MtCEP7 promoter is required for its symbiotic activation. Overall, these results demonstrate that a cytokinin-MtCRE1-MtNIN regulatory module coordinates the expression of two antagonistic, symbiosis-related, peptide hormones from different families to fine-tune nodule number.


Assuntos
Peptídeos/química , Nodulação/fisiologia , Rhizobium/metabolismo , Fatores de Transcrição/metabolismo , Citocininas/metabolismo , Epiderme , Regulação da Expressão Gênica de Plantas , Lotus/metabolismo , Medicago truncatula , Peptídeos/genética , Proteínas de Plantas , Nodulação/genética , Raízes de Plantas/metabolismo , Regiões Promotoras Genéticas , Proteínas Quinases , Sinais Direcionadores de Proteínas/genética , Nódulos Radiculares de Plantas , Sinorhizobium meliloti/metabolismo , Simbiose
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