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1.
Proc Natl Acad Sci U S A ; 117(15): 8468-8475, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32234780

RESUMO

The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) "killer" domain and neighboring first brace helix of human MLKL with nanomolar affinity. When expressed as genetically encoded reagents in cells, these monobodies potently block necroptotic cell death. However, they did not prevent MLKL recruitment to the "necrosome" and phosphorylation by RIPK3, nor the assembly of MLKL into oligomers, but did block MLKL translocation to membranes where activated MLKL normally disrupts membranes to kill cells. An X-ray crystal structure revealed a monobody-binding site centered on the α4 helix of the MLKL 4HB domain, which mutational analyses showed was crucial for reconstitution of necroptosis signaling. These data implicate the α4 helix of its 4HB domain as a crucial site for recruitment of adaptor proteins that mediate membrane translocation, distinct from known phospholipid binding sites.


Assuntos
Materiais Biomiméticos/farmacologia , Membrana Celular/metabolismo , Domínio de Fibronectina Tipo III , Necrose , Oligopeptídeos/farmacologia , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Cristalografia por Raios X , Humanos , Fosforilação , Conformação Proteica , Proteínas Quinases/química , Multimerização Proteica , Transporte Proteico
2.
Nat Commun ; 11(1): 1361, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170184

RESUMO

Grana are a characteristic feature of higher plants' thylakoid membranes, consisting of stacks of appressed membranes enriched in Photosystem II (PSII) and associated light-harvesting complex II (LHCII) proteins, together forming the PSII-LHCII supercomplex. Grana stacks undergo light-dependent structural changes, mainly by reorganizing the supramolecular structure of PSII-LHCII supercomplexes. LHCII is vital for grana formation, in which also PSII-LHCII supercomplexes are involved. By combining top-down and crosslinking mass spectrometry we uncover the spatial organization of paired PSII-LHCII supercomplexes within thylakoid membranes. The resulting model highlights a basic molecular mechanism whereby plants maintain grana stacking at changing light conditions. This mechanism relies on interactions between stroma-exposed N-terminal loops of LHCII trimers and Lhcb4 subunits facing each other in adjacent membranes. The combination of light-dependent LHCII N-terminal trimming and extensive N-terminal α-acetylation likely affects interactions between pairs of PSII-LHCII supercomplexes across the stromal gap, ultimately mediating membrane folding in grana stacks.


Assuntos
Complexos de Proteínas Captadores de Luz/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Plantas/metabolismo , Proteínas Quinases/metabolismo , Tilacoides/metabolismo , Proteínas de Ligação à Clorofila/metabolismo , Embriófitas , Luz , Complexos de Proteínas Captadores de Luz/química , Espectrometria de Massas/métodos , Modelos Moleculares , Complexo de Proteína do Fotossistema II/química , Proteínas de Plantas/metabolismo , Conformação Proteica , Proteínas Quinases/química , Proteômica
3.
Nature ; 578(7794): 301-305, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32025038

RESUMO

Many biomolecules undergo liquid-liquid phase separation to form liquid-like condensates that mediate diverse cellular functions1,2. Autophagy is able to degrade such condensates using autophagosomes-double-membrane structures that are synthesized de novo at the pre-autophagosomal structure (PAS) in yeast3-5. Whereas Atg proteins that associate with the PAS have been characterized, the physicochemical and functional properties of the PAS remain unclear owing to its small size and fragility. Here we show that the PAS is in fact a liquid-like condensate of Atg proteins. The autophagy-initiating Atg1 complex undergoes phase separation to form liquid droplets in vitro, and point mutations or phosphorylation that inhibit phase separation impair PAS formation in vivo. In vitro experiments show that Atg1-complex droplets can be tethered to membranes via specific protein-protein interactions, explaining the vacuolar membrane localization of the PAS in vivo. We propose that phase separation has a critical, active role in autophagy, whereby it organizes the autophagy machinery at the PAS.


Assuntos
Autofagossomos/química , Autofagossomos/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/química , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fosforilação , Mutação Puntual , Ligação Proteica , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(5): 2693-2703, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31964818

RESUMO

Plants use leucine-rich repeat receptor kinases (LRR-RKs) to sense sequence diverse peptide hormones at the cell surface. A 3.0-Å crystal structure of the LRR-RK GSO1/SGN3 regulating Casparian strip formation in the endodermis reveals a large spiral-shaped ectodomain. The domain provides a binding platform for 21 amino acid CIF peptide ligands, which are tyrosine sulfated by the tyrosylprotein sulfotransferase TPST/SGN2. GSO1/SGN3 harbors a binding pocket for sulfotyrosine and makes extended backbone interactions with CIF2. Quantitative biochemical comparisons reveal that GSO1/SGN3-CIF2 represents one of the strongest receptor-ligand pairs known in plants. Multiple missense mutations are required to block CIF2 binding in vitro and GSO1/SGN3 function in vivo. Using structure-guided sequence analysis we uncover previously uncharacterized CIF peptides conserved among higher plants. Quantitative binding assays with known and novel CIFs suggest that the homologous LRR-RKs GSO1/SGN3 and GSO2 have evolved unique peptide binding properties to control different developmental processes. A quantitative biochemical interaction screen, a CIF peptide antagonist and genetic analyses together implicate SERK proteins as essential coreceptor kinases required for GSO1/SGN3 and GSO2 receptor activation. Our work provides a mechanistic framework for the recognition of sequence-divergent peptide hormones in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Peptídeos/metabolismo , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Arabidopsis/química , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Cinética , Ligantes , Peptídeos/química , Reguladores de Crescimento de Planta/química , Reguladores de Crescimento de Planta/metabolismo , Ligação Proteica , Proteínas Quinases/química , Proteínas Quinases/genética
5.
BMC Genomics ; 21(1): 53, 2020 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-31948407

RESUMO

BACKGROUND: Ca2+ played as a ubiquitous secondary messenger involved in plant growth, development, and responses to various environmental stimuli. Calcium-dependent protein kinases (CDPK) were important Ca2+ sensors, which could directly translate Ca2+ signals into downstream phosphorylation signals. Considering the importance of CDPKs as Ca2+ effectors for regulation of plant stress tolerance and few studies on Brachypodium distachyon were available, it was of interest for us to isolate CDPKs from B. distachyon. RESULTS: A systemic analysis of 30 CDPK family genes in B. distachyon was performed. Results showed that all BdCDPK family members contained conserved catalytic Ser/Thr protein kinase domain, autoinhibitory domain, and EF-hand domain, and a variable N-terminal domain, could be divided into four subgroup (I-IV), based upon sequence homology. Most BdCDPKs had four EF-hands, in which EF2 and EF4 revealed high variability and strong divergence from EF-hand in AtCDPKs. Synteny results indicated that large number of syntenic relationship events existed between rice and B. distachyon, implying their high conservation. Expression profiles indicated that most of BdCDPK genes were involved in phytohormones signal transduction pathways and regulated physiological process in responding to multiple environmental stresses. Moreover, the co-expression network implied that BdCDPKs might be both the activator and the repressor involved in WRKY transcription factors or MAPK cascade genes mediated stress response processes, base on their complex regulatory network. CONCLUSIONS: BdCDPKs might play multiple function in WRKY or MAPK mediated abiotic stresses response and phytohormone signaling transduction in B. distachyon. Our genomics analysis of BdCDPKs could provide fundamental information for further investigation the functions of CDPKs in integrating Ca2+ signalling pathways in response to environments stresses in B. distachyon.


Assuntos
Brachypodium/enzimologia , Proteínas de Plantas/genética , Proteínas Quinases/genética , Brachypodium/genética , Perfilação da Expressão Gênica , Genoma de Planta , Sistema de Sinalização das MAP Quinases/genética , Família Multigênica , Filogenia , Proteínas de Plantas/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Sintenia , Fatores de Transcrição/metabolismo
6.
PLoS One ; 15(1): e0227454, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31914150

RESUMO

Cholangiocarcinoma (CCA), a malignant tumor originating in the biliary tract, is well known to be associated with adverse clinical outcomes and high mortality rates due to the lack of effective therapy. Evasion of apoptosis is considered a key contributor to therapeutic success and chemotherapy resistance in CCA, highlighting the need for novel therapeutic strategies. In this study, we demonstrated that the induction of necroptosis, a novel regulated form of necrosis, could potentially serve as a novel therapeutic approach for CCA patients. The RNA sequencing data in The Cancer Genome Atlas (TCGA) database were analyzed and revealed that both receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL), two essential mediators of necroptosis, were upregulated in CCA tissues when compared with the levels in normal bile ducts. We demonstrated in a panel of CCA cell lines that RIPK3 was differentially expressed in CCA cell lines, while MLKL was more highly expressed in CCA cell lines than in nontumor cholangiocytes. We therefore showed that treatment with both tumor necrosis factor-α (TNF-α) and Smac mimetic, an inhibitor of apoptosis protein (IAP) antagonist, induced RIPK1/RIPK3/MLKL-dependent necroptosis in CCA cells when caspases were blocked. The necroptotic induction in a panel of CCA cells was correlated with RIPK3 expression. Intriguingly, we demonstrated that Smac mimetic sensitized CCA cells to a low dose of standard chemotherapy, gemcitabine, and induced necroptosis in an RIPK1/RIPK3/MLKL-dependent manner upon caspase inhibition but not in nontumor cholangiocytes. We further demonstrated that Smac mimetic and gemcitabine synergistically induced an increase in TNF-α mRNA levels and that Smac mimetic reversed gemcitabine-induced cell cycle arrest, leading to cell killing. Collectively, our present study demonstrated that TNF-α and gemcitabine induced RIPK1/RIPK3/MLKL-dependent necroptosis upon IAP depletion and caspase inhibition; therefore, our findings have pivotal implications for designing a novel necroptosis-based therapeutic strategy for CCA patients.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Desoxicitidina/análogos & derivados , Necroptose/efeitos dos fármacos , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Triazóis/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Neoplasias do Sistema Biliar/metabolismo , Neoplasias do Sistema Biliar/patologia , Inibidores de Caspase/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Desoxicitidina/farmacologia , Sinergismo Farmacológico , Humanos , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
7.
Nat Struct Mol Biol ; 27(1): 92-104, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31925410

RESUMO

Kinases are involved in disease development and modulation of their activity can be therapeutically beneficial. Drug-resistant mutant kinases are valuable tools in drug discovery efforts, but the prediction of mutants across the kinome is challenging. Here, we generate deep mutational scanning data to identify mutant mammalian kinases that drive resistance to clinically relevant inhibitors. We aggregate these data with subsaturation mutagenesis data and use it to develop, test and validate a framework to prospectively identify residues that mediate kinase activity and drug resistance across the kinome. We validate predicted resistance mutations in CDK4, CDK6, ERK2, EGFR and HER2. Capitalizing on a highly predictable residue, we generate resistance mutations in TBK1, CSNK2A1 and BRAF. Unexpectedly, we uncover a potentially generalizable activation site that mediates drug resistance and confirm its impact in BRAF, EGFR, HER2 and MEK1. We anticipate that the identification of these residues will enable the broad interrogation of the kinome and its inhibitors.


Assuntos
Resistência a Medicamentos , Mutação Puntual , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Descoberta de Drogas , Resistencia a Medicamentos Antineoplásicos , Humanos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteômica
8.
Science ; 367(6476): 431-435, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31974252

RESUMO

The plant embryonic cuticle is a hydrophobic barrier deposited de novo by the embryo during seed development. At germination, it protects the seedling from water loss and is, thus, critical for survival. Embryonic cuticle formation is controlled by a signaling pathway involving the ABNORMAL LEAF SHAPE1 subtilase and the two GASSHO receptor-like kinases. We show that a sulfated peptide, TWISTED SEED1 (TWS1), acts as a GASSHO ligand. Cuticle surveillance depends on the action of the subtilase, which, unlike the TWS1 precursor and the GASSHO receptors, is not produced in the embryo but in the neighboring endosperm. Subtilase-mediated processing of the embryo-derived TWS1 precursor releases the active peptide, triggering GASSHO-dependent cuticle reinforcement in the embryo. Thus, a bidirectional molecular dialogue between embryo and endosperm safeguards cuticle integrity before germination.


Assuntos
Endosperma/fisiologia , Germinação , Sementes/fisiologia , Sequência de Aminoácidos , Endosperma/citologia , Endosperma/metabolismo , Ligantes , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Sementes/citologia , Sementes/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Transdução de Sinais , Tabaco/crescimento & desenvolvimento , Tabaco/metabolismo
9.
Blood ; 135(1): 56-70, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31697837

RESUMO

Lineage-defining transcription factors (TFs) are compelling targets for leukemia therapy, yet they are among the most challenging proteins to modulate directly with small molecules. We previously used CRISPR screening to identify a salt-inducible kinase 3 (SIK3) requirement for the growth of acute myeloid leukemia (AML) cell lines that overexpress the lineage TF myocyte enhancer factor (MEF2C). In this context, SIK3 maintains MEF2C function by directly phosphorylating histone deacetylase 4 (HDAC4), a repressive cofactor of MEF2C. In this study, we evaluated whether inhibition of SIK3 with the tool compound YKL-05-099 can suppress MEF2C function and attenuate disease progression in animal models of AML. Genetic targeting of SIK3 or MEF2C selectively suppressed the growth of transformed hematopoietic cells under in vitro and in vivo conditions. Similar phenotypes were obtained when cells were exposed to YKL-05-099, which caused cell-cycle arrest and apoptosis in MEF2C-expressing AML cell lines. An epigenomic analysis revealed that YKL-05-099 rapidly suppressed MEF2C function by altering the phosphorylation state and nuclear localization of HDAC4. Using a gatekeeper allele of SIK3, we found that the antiproliferative effects of YKL-05-099 occurred through on-target inhibition of SIK3 kinase activity. Based on these findings, we treated 2 different mouse models of MLL-AF9 AML with YKL-05-099, which attenuated disease progression in vivo and extended animal survival at well-tolerated doses. These findings validate SIK3 as a therapeutic target in MEF2C-addicted AML and provide a rationale for developing druglike inhibitors of SIK3 for definitive preclinical investigation and for studies in human patients.


Assuntos
Leucemia Mieloide Aguda/prevenção & controle , Fatores de Transcrição MEF2/metabolismo , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Apoptose , Ciclo Celular , Proliferação de Células , Feminino , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Fatores de Transcrição MEF2/genética , Camundongos , Camundongos Endogâmicos C57BL , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
10.
BMB Rep ; 53(1): 47-55, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31818365

RESUMO

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease and has become a major socioeconomic issue in many developed countries. Currently available therapeutic agents for AD provide only symptomatic treatments, mainly because the complete mechanism of the AD pathogenesis is still unclear. Although several different hypotheses have been proposed, mitochondrial dysfunction has gathered interest because of its profound effect on brain bioenergetics and neuronal survival in the pathophysiology of AD. Various therapeutic agents targeting the mitochondrial pathways associated with AD have been developed over the past decade. Although most of these agents are still early in the clinical development process, they are used to restore mitochondrial function, which provides an alternative therapeutic strategy that is likely to slow the progression of the disease. In this mini review, we will survey the AD-related mitochondrial pathways and their small-molecule modulators that have therapeutic potential. We will focus on recently reported examples, and also overview the current challenges and future perspectives of ongoing research. [BMB Reports 2020; 53(1): 47-55].


Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , 17-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 17-Hidroxiesteroide Desidrogenases/metabolismo , 3-Hidroxiacil-CoA Desidrogenases/antagonistas & inibidores , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Animais , Progressão da Doença , Dinaminas/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Neuroesteroides/química , Neuroesteroides/metabolismo , Neuroesteroides/farmacologia , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Receptores de GABA/metabolismo
11.
Plant Sci ; 290: 110317, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31779910

RESUMO

Thaumatin-like protein kinases (TLPKs) are defense related proteins having antimicrobial property. Herein, we identified two TLPKs in the genome of Brachypodium distachyon and Oryza sativa, four in Hordeum vulgare and Sorghum bicolor, and 16 in Triticum aestivum. All the TLPKs were located at only one chromosome in each plant except T. aestivum, where they were located on chromosome 2 and chromosome 3. Paralogous analysis suggested the occurrence of one duplication event (DE) in each B. distachyon and O. sativa, two in H. vulgare while four DEs in T. aestivum genome during the evolution of TLPKs. The majority of TLPKs were intron less, while a few contains one or two introns. The introns were found in each 0, 1 and 2 phase. Protein structure analysis suggested the occurrence of a thaumatin and a kinase domain with a transmembrane (TM) helix in each TLPK. Further, a thaumatin family signature motif "GX[GF]XCXT[GA]DCX(1,2)GX(2,3)C", a "REDDD" motif and 16 cysteine residues were found conserved in the majority of TLPKs. Expression analysis indicated variable expression of TLPKs in various tissues of different cereal crops. They were high expressing in reproductive tissues in B. distachyon, while in leaves in T. aestivum. Modulated expression of TaTLPKs in the presence of fungal pathogen, and heat, drought and salt stress in T. aestivum suggested their roles in stress response. Co-expression analysis showed interaction of TLPKs with various development and stress related genes. The results indicated diverse roles of TLPKs, which can be utilized for the development of eco-friendly pest resistant crops in future.


Assuntos
Produtos Agrícolas/genética , Proteínas de Plantas/genética , Proteínas Quinases/genética , Transcrição Genética , Sequência de Aminoácidos , Produtos Agrícolas/metabolismo , Perfilação da Expressão Gênica , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Alinhamento de Sequência
12.
Gene ; 723: 144134, 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31589960

RESUMO

Viral kinases are known to undergo autophosphorylation and also phosphorylate viral and host substrates. Viral kinases have been implicated in various diseases and are also known to acquire host kinases for mimicking cellular functions and exhibit virulence. Although substantial analyses have been reported in the literature on diversity of viral kinases, there is a gap in the understanding of sequence and structural similarity among kinases from different classes of viruses. In this study, we performed a comprehensive analysis of protein kinases encoded in viral genomes. Homology search methods have been used to identify kinases from 104,282 viral genomic datasets. Serine/threonine and tyrosine kinases are identified only in 390 viral genomes. Out of seven viral classes that are based on nature of genetic material, only viruses having double-stranded DNA and single-stranded RNA retroviruses are found to encode kinases. The 716 identified protein kinases are classified into 63 subfamilies based on their sequence similarity within each cluster, and sequence signatures have been identified for each subfamily. 11 clusters are well represented with at least 10 members in each of these clusters. Kinases from dsDNA viruses, Phycodnaviridae which infect green algae and Herpesvirales that infect vertebrates including human, form a major group. From our analysis, it has been observed that the protein kinases in viruses belonging to same taxonomic lineages form discrete clusters and the kinases encoded in alphaherpesvirus form host-specific clusters. A comprehensive sequence and structure-based analysis enabled us to identify the conserved residues or motifs in kinase catalytic domain regions across all viral kinases. Conserved sequence regions that are specific to a particular viral kinase cluster and the kinases that show close similarity to eukaryotic kinases were identified by using sequence and three-dimensional structural regions of eukaryotic kinases as reference. The regions specific to each viral kinase cluster can be used as signatures in the future in classifying uncharacterized viral kinases. We note that kinases from giant viruses Marseilleviridae have close similarity to viral oncogenes in the functional regions and in putative substrate binding regions indicating their possible role in cancer.


Assuntos
Proteínas Quinases/química , Proteínas Quinases/genética , Vírus/classificação , Domínio Catalítico , Biologia Computacional/métodos , Bases de Dados de Proteínas , Variação Genética , Fosforilação , Filogenia , Proteínas Quinases/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Fatores de Virulência/química , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Vírus/enzimologia , Vírus/patogenicidade
13.
Org Biomol Chem ; 18(3): 379-390, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31844862

RESUMO

The accessory gene regulator (agr) quorum-sensing system is arguably the most important regulator of staphylococcus virulence and has been the focus of tremendous interest in the development of effective therapies for pathogenic bacterial infections. With regards to chemotherapeutic based strategies, the significant proportion of currently reported agr-system modulating molecules are mimics of the native ArgC substrate, which is a thioester-based macrocyclic peptide know as the auto-inducing peptide. Over the past two decades, more than two-hundred synthetic analogues have been reported. This review traces the development of the synthetic strategies employed to synthesise these analogues with a particular focus on macrocyclisation. At present these synthetic approaches can be clustered into five broad categories (1) solution-phase cyclisation, (2) immobilised carbodiimide assisted cyclisation, (3) concomitant on-resin cleavage and macrocyclisation, (4) Boc-compatible chemoselective thioesterification, and (5) Fmoc-compatible chemoselective thioesterification. The advantages and limitation provided by each of the approaches are compared and contrasted with a view towards potential reaction scale-up.


Assuntos
Antibacterianos/síntese química , Peptídeos Cíclicos/síntese química , Peptidomiméticos/síntese química , Percepção de Quorum/efeitos dos fármacos , Staphylococcus/efeitos dos fármacos , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Ciclização , Peptídeos Cíclicos/farmacologia , Peptidomiméticos/farmacologia , Proteínas Quinases/química , Staphylococcus/química
14.
Nat Struct Mol Biol ; 26(12): 1089-1093, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31792449

RESUMO

We report the 3.45-Å resolution cryo-EM structure of human SMG1-SMG8-SMG9, a phosphatidylinositol-3-kinase (PI(3)K)-related protein kinase (PIKK) complex central to messenger RNA surveillance. Structural and MS analyses reveal the presence of inositol hexaphosphate (InsP6) in the SMG1 kinase. We show that the InsP6-binding site is conserved in mammalian target of rapamycin (mTOR) and potentially other PIKK members, and that it is required for optimal in vitro phosphorylation of both SMG1 and mTOR substrates.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ácido Fítico/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Modelos Moleculares , Ácido Fítico/química , Ligação Proteica , Conformação Proteica , Proteínas Quinases/química , Proteínas Quinases/ultraestrutura , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/ultraestrutura , Estabilidade de RNA
15.
BMC Bioinformatics ; 20(1): 617, 2019 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-31783725

RESUMO

BACKGROUND: The kinase pocket structural information is important for drug discovery targeting cancer or other diseases. Although some kinase sequence, structure or drug databases have been developed, the databases cannot be directly used in the kinase drug study. Therefore, a comprehensive database of human kinase protein pockets is urgently needed to be developed. RESULTS: Here, we have developed HKPocket, a comprehensive Human Kinase Pocket database. This database provides sequence, structure, hydrophilic-hydrophobic, critical interactions, and druggability information including 1717 pockets from 255 kinases. We further divided these pockets into 91 pocket clusters using structural and position features in each kinase group. The pocket structural information would be useful for preliminary drug screening. Then, the potential drugs can be further selected and optimized by analyzing the sequence conservation, critical interactions, and hydrophobicity of identified drug pockets. HKPocket also provides online visualization and pse files of all identified pockets. CONCLUSION: The HKPocket database would be helpful for drug screening and optimization. Besides, drugs targeting the non-catalytic pockets would cause fewer side effects. HKPocket is available at http://zhaoserver.com.cn/HKPocket/HKPocket.html.


Assuntos
Bases de Dados de Proteínas , Desenho de Fármacos , Proteínas Quinases/metabolismo , Software , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Ligantes , Conformação Proteica , Proteínas Quinases/química
16.
mBio ; 10(6)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848291

RESUMO

Clostridium perfringens type F strains cause gastrointestinal disease when they produce a pore-forming toxin named C. perfringens enterotoxin (CPE). In human enterocyte-like Caco-2 cells, low CPE concentrations cause caspase-3-dependent apoptosis, while high CPE concentrations cause necrosis. Since necrosis or apoptosis sometimes involves receptor-interacting serine/threonine-protein kinase-1 or 3 (RIP1 or RIP3), this study examined whether those kinases are important for CPE-induced apoptosis or necrosis. Highly specific RIP1 or RIP3 inhibitors reduced both CPE-induced apoptosis and necrosis in Caco-2 cells. Those findings suggested that the form of necrosis induced by treating Caco-2 cells with high CPE concentrations involves necroptosis, which was confirmed when high, but not low, CPE concentrations were shown to induce oligomerization of mixed-lineage kinase domain-like pseudokinase (MLKL), a key late step in necroptosis. Furthermore, an MLKL oligomerization inhibitor reduced cell death caused by high, but not low, CPE concentrations. Supporting RIP1 and RIP3 involvement in CPE-induced necroptosis, inhibitors of those kinases also reduced MLKL oligomerization during treatment with high CPE concentrations. Calpain inhibitors similarly blocked MLKL oligomerization induced by high CPE concentrations, implicating calpain activation as a key intermediate in initiating CPE-induced necroptosis. In two other CPE-sensitive cell lines, i.e., Vero cells and human enterocyte-like T84 cells, low CPE concentrations also caused primarily apoptosis/late apoptosis, while high CPE concentrations mainly induced necroptosis. Collectively, these results establish that high, but not low, CPE concentrations cause necroptosis and suggest that RIP1, RIP3, MLKL, or calpain inhibitors can be explored as potential therapeutics against CPE effects in vivo IMPORTANCE C. perfringens type F strains are a common cause of food poisoning and antibiotic-associated diarrhea. Type F strain virulence requires production of C. perfringens enterotoxin (CPE). In Caco-2 cells, high CPE concentrations cause necrosis while low enterotoxin concentrations induce apoptosis. The current study determined that receptor-interacting serine/threonine-protein kinases 1 and 3 are involved in both CPE-induced apoptosis and necrosis in Caco-2 cells, while mixed-lineage kinase domain-like pseudokinase (MLKL) oligomerization is involved in CPE-induced necrosis, thereby indicating that this form of CPE-induced cell death involves necroptosis. High CPE concentrations also caused necroptosis in T84 and Vero cells. Calpain activation was identified as a key intermediate for CPE-induced necroptosis. These results suggest inhibitors of RIP1, RIP3, MLKL oligomerization, or calpain are useful therapeutics against CPE-mediated diseases.


Assuntos
Infecções por Clostridium/metabolismo , Infecções por Clostridium/microbiologia , Clostridium perfringens/fisiologia , Enterotoxinas/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Apoptose , Células CACO-2 , Chlorocebus aethiops , Interações Hospedeiro-Patógeno , Humanos , Modelos Biológicos , Proteínas Quinases/química , Multimerização Proteica , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Células Vero
17.
PLoS One ; 14(11): e0225091, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31714948

RESUMO

CBL-interacting protein kinases (CIPKs) have been shown to regulate a variety of environmental stress-related signalling pathways in plants. Foxtail millet (Setaria italica (L.) P. Beauv) is known worldwide as a relatively stress-tolerant C4 crop species. Although the foxtail millet genome sequence has been released, little is known about the functions of CIPKs in foxtail millet. Therefore, a systematic genome-wide analysis of CIPK genes in foxtail millet was performed. In total, 35 CIPK members were identified in foxtail millet and divided into four subgroups (I to IV) on the basis of their phylogenetic relationships. Phylogenetic and gene structure analyses clearly divided all SiCIPKs into intron-poor and intron-rich clades. Cis-element analysis subsequently indicated that these SiCIPKs may be involved in responses to abiotic stimuli, hormones, and light signalling during plant growth and development, and stress-induced expression profile analysis revealed that all the SiCIPKs are involved in various stress signalling pathways. These results suggest that the CIPK genes in foxtail millet exhibit the basic characteristics of CIPK family members and play important roles in response to abiotic stresses. The results of this study will contribute to future functional characterization of abiotic stress responses mediated by CIPKs in foxtail millet.


Assuntos
Ácido Abscísico/farmacologia , Proteínas Quinases/genética , Setaria (Planta)/enzimologia , Estresse Fisiológico , Motivos de Aminoácidos , Cromossomos de Plantas/genética , Sequência Conservada , Evolução Molecular , Éxons/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Íntrons/genética , Família Multigênica , Filogenia , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Setaria (Planta)/efeitos dos fármacos , Setaria (Planta)/genética , Setaria (Planta)/fisiologia , Regulação para Cima/efeitos dos fármacos
18.
ACS Chem Biol ; 14(12): 2585-2594, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31638770

RESUMO

Drug optimization is guided by biophysical methods with increasing popularity. In the context of lead structure modifications, the introduction of methyl groups is a simple but potentially powerful approach. Hence, it is crucial to systematically investigate the influence of ligand methylation on biophysical characteristics such as thermodynamics. Here, we investigate the influence of ligand methylation in different positions and combinations on the drug-kinase interaction. Binding modes and complex structures were analyzed using protein crystallography. Thermodynamic signatures were measured via isothermal titration calorimetry (ITC). An extensive computational analysis supported the understanding of the underlying mechanisms. We found that not only position but also stereochemistry of the methyl group has an influence on binding potency as well as the thermodynamic signature of ligand binding to the protein. Strikingly, the combination of single methyl groups does not lead to additive effects. In our case, the merger of two methyl groups in one ligand leads to an entirely new alternative ligand binding mode in the protein ligand complex. Moreover, the combination of the two methyl groups also resulted in a nonadditive thermodynamic profile of ligand binding. Molecular dynamics (MD) simulations revealed distinguished characteristic motions of the ligands in solution explaining the pronounced thermodynamic changes. The unexpected drastic change in protein ligand interaction highlights the importance of crystallographic control even for minor modifications such as the introduction of a methyl group. For an in-depth understanding of ligand binding behavior, MD simulations have shown to be a powerful tool.


Assuntos
Preparações Farmacêuticas/química , Proteínas Quinases/química , Calorimetria , Cristalografia por Raios X , Ligantes , Metilação , Simulação de Dinâmica Molecular , Ligação Proteica , Proteínas Quinases/metabolismo
20.
Nucleic Acids Res ; 47(20): e128, 2019 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-31504818

RESUMO

Chemical similarity searching is a basic research tool that can be used to find small molecules which are similar in shape to known active molecules. Despite its popularity, the retrieval of local molecular features that are critical to functional activity related to target binding often fails. To overcome this limitation, we developed a novel machine learning-based chemical binding similarity score by using various evolutionary relationships of binding targets. The chemical similarity was defined by the probability of chemical compounds binding to identical targets. Comprehensive and heterogeneous multiple target-binding chemical data were integrated into a paired data format and processed using multiple classification similarity-learning models with various levels of target evolutionary information. Encoding evolutionary information to chemical compounds through their binding targets substantially expanded available chemical-target interaction data and significantly improved model performance. The output probability of our integrated model, referred to as ensemble evolutionary chemical binding similarity (ensECBS), was effective for finding hidden chemical relationships. The developed method can serve as a novel chemical similarity tool that uses evolutionarily conserved target binding information.


Assuntos
Evolução Molecular , Aprendizado de Máquina , Inibidores de Proteínas Quinases/química , Proteínas Quinases/metabolismo , Análise de Sequência de Proteína/métodos , Bibliotecas de Moléculas Pequenas/química , Animais , Genes , Humanos , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Proteínas Quinases/genética , Relação Quantitativa Estrutura-Atividade , Bibliotecas de Moléculas Pequenas/farmacologia
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