Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 24
Filtrar
1.
Elife ; 102021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34524082

RESUMO

The SUV39 class of methyltransferase enzymes deposits histone H3 lysine 9 di- and trimethylation (H3K9me2/3), the hallmark of constitutive heterochromatin. How these enzymes are regulated to mark specific genomic regions as heterochromatic is poorly understood. Clr4 is the sole H3K9me2/3 methyltransferase in the fission yeast Schizosaccharomyces pombe, and recent evidence suggests that ubiquitination of lysine 14 on histone H3 (H3K14ub) plays a key role in H3K9 methylation. However, the molecular mechanism of this regulation and its role in heterochromatin formation remain to be determined. Our structure-function approach shows that the H3K14ub substrate binds specifically and tightly to the catalytic domain of Clr4, and thereby stimulates the enzyme by over 250-fold. Mutations that disrupt this mechanism lead to a loss of H3K9me2/3 and abolish heterochromatin silencing similar to clr4 deletion. Comparison with mammalian SET domain proteins suggests that the Clr4 SET domain harbors a conserved sensor for H3K14ub, which mediates licensing of heterochromatin formation.


Assuntos
Proteínas de Ciclo Celular , Heterocromatina , Código das Histonas/genética , Histona-Lisina N-Metiltransferase , Histonas , Proteínas de Schizosaccharomyces pombe , Domínio Catalítico/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Metilação de DNA/genética , Heterocromatina/química , Heterocromatina/genética , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Ubiquitinação/genética
2.
Sci Adv ; 7(35)2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34452907

RESUMO

The class IB phosphoinositide 3-kinase (PI3K), PI3Kγ, is a master regulator of immune cell function and a promising drug target for both cancer and inflammatory diseases. Critical to PI3Kγ function is the association of the p110γ catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G protein-coupled receptors. Here, we report the cryo-electron microscopy structure of a heterodimeric PI3Kγ complex, p110γ-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its Gßγ-binding domain, recruiting the heterodimer to the membrane and allowing for engagement of a secondary Gßγ-binding site in p110γ. Mutations at the p110γ-p101 and p110γ-adaptor binding domain interfaces enhanced Gßγ activation. A nanobody that specifically binds to the p101-Gßγ interface blocks activation, providing a novel tool to study and target p110γ-p101-specific signaling events in vivo.

3.
Nat Commun ; 12(1): 3788, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145271

RESUMO

Active host cell invasion by the obligate intracellular apicomplexan parasites relies on the formation of a moving junction, which connects parasite and host cell plasma membranes during entry. Invading Toxoplasma gondii tachyzoites secrete their rhoptry content and insert a complex of RON proteins on the cytoplasmic side of the host cell membrane providing an anchor to which the parasite tethers. Here we show that a rhoptry-resident kinase RON13 is a key virulence factor that plays a crucial role in host cell entry. Cryo-EM, kinase assays, phosphoproteomics and cellular analyses reveal that RON13 is a secretory pathway kinase of atypical structure that phosphorylates rhoptry proteins including the components of the RON complex. Ultimately, RON13 kinase activity controls host cell invasion by anchoring the moving junction at the parasite-host cell interface.


Assuntos
Membrana Celular/parasitologia , Proteínas de Protozoários/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Toxoplasma/metabolismo , Toxoplasmose/patologia , Transporte Biológico/fisiologia , Células Cultivadas , Interações Hospedeiro-Parasita , Humanos , Via Secretória/fisiologia , Fatores de Virulência
4.
Nat Commun ; 12(1): 1538, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33750786

RESUMO

Plasmodium vivax preferentially invades reticulocytes and recognition of these cells is mediated by P. vivax Reticulocyte Binding Protein 2b (PvRBP2b) binding to human Transferrin receptor 1 (TfR1) and Transferrin (Tf). Longitudinal cohort studies in Papua New Guinea, Thailand and Brazil show that PvRBP2b antibodies are correlated with protection against P. vivax infection and disease. Here, we isolate and characterize anti-PvRBP2b human monoclonal antibodies from two individuals in Cambodia with natural P. vivax infection. These antibodies bind with high affinities and map to different regions of PvRBP2b. Several human antibodies block PvRBP2b binding to reticulocytes and inhibit complex formation with human TfR1-Tf. We describe different structural mechanisms for functional inhibition, including either steric hindrance with TfR1-Tf or the reticulocyte membrane. These results show that naturally acquired human antibodies against PvRBP2b can inhibit its function which is important for P. vivax invasion.


Assuntos
Anticorpos Bloqueadores , Anticorpos Monoclonais/imunologia , Proteínas de Membrana/metabolismo , Plasmodium vivax/metabolismo , Proteínas de Protozoários/metabolismo , Reticulócitos/metabolismo , Anticorpos Antiprotozoários/imunologia , Antígenos CD , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Camboja , Cristalografia por Raios X , Humanos , Estudos Longitudinais , Malária Vivax/imunologia , Malária Vivax/parasitologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Plasmodium vivax/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Receptores da Transferrina
5.
Sci Signal ; 13(656)2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33144519

RESUMO

The PI3Kγ isoform is activated by Gi-coupled GPCRs in myeloid cells, but the extent to which the two endogenous complexes of PI3Kγ, p101/p110γ and p84/p110γ, receive direct regulation through Gßγ or indirect regulation through RAS and the sufficiency of those inputs is controversial or unclear. We generated mice with point mutations that prevent Gßγ binding to p110γ (RK552DD) or to p101 (VVKR777AAAA) and investigated the effects of these mutations in primary neutrophils and in mouse models of neutrophilic inflammation. Loss of Gßγ binding to p110γ substantially reduced the activation of both p101/p110γ and p84/p110γ in neutrophils by various GPCR agonists. Loss of Gßγ binding to p101 caused more variable effects, depending on both the agonist and cellular response, with the biggest reductions seen in PIP3 production by primary neutrophils in response to LTB4 and MIP-2 and in the migration of neutrophils during thioglycolate-induced peritonitis or MIP2-induced ear pouch inflammation. We also observed that p101VVKR777AAAA neutrophils showed enhanced p84-dependent ROS responses to fMLP and C5a, suggesting that competition may exist between p101/p110γ and p84/p110γ for Gßγ subunits downstream of GPCR activation. GPCRs did not activate p110γ in neutrophils from mice lacking both the p101 and p84 regulatory subunits, indicating that RAS binding to p110γ is insufficient to support GPCR activation in this cell type. These findings define a direct role for Gßγ subunits in activating both of the endogenous PI3Kγ complexes and indicate that the regulatory PI3Kγ subunit biases activation toward different GPCRs.

6.
PLoS One ; 15(9): e0238089, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32903266

RESUMO

A novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) is the source of a current pandemic (COVID-19) with devastating consequences in public health and economic stability. Using a peptide array to map the antibody response of plasma from healing patients (12) and heathy patients (6), we identified three immunodominant linear epitopes, two of which correspond to key proteolytic sites on the spike protein (S1/S2 and S2') known to be critical for cellular entry. We show biochemical evidence that plasma positive for the epitope adjacent to the S1/S2 cleavage site inhibits furin-mediated proteolysis of spike.


Assuntos
Infecções por Coronavirus/patologia , Epitopos/química , Pneumonia Viral/patologia , Sequência de Aminoácidos , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Betacoronavirus/isolamento & purificação , COVID-19 , Infecções por Coronavirus/virologia , Mapeamento de Epitopos , Epitopos/sangue , Epitopos/imunologia , Furina/metabolismo , Humanos , Pandemias , Ácidos Nucleicos Peptídicos/química , Peptídeos/química , Pneumonia Viral/virologia , Análise Serial de Proteínas , Estrutura Terciária de Proteína , Proteólise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , SARS-CoV-2 , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
7.
RNA Biol ; 17(5): 637-650, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32050838

RESUMO

RNA helicases are fundamental players in RNA metabolism: they remodel RNA secondary structures and arrange ribonucleoprotein complexes. While DExH-box RNA helicases function in ribosome biogenesis and splicing in eukaryotes, information is scarce about bacterial homologs. HrpB is the only bacterial DExH-box protein whose structure is solved. Besides the catalytic core, HrpB possesses three accessory domains, conserved in all DExH-box helicases, plus a unique C-terminal extension (CTE). The function of these auxiliary domains remains unknown. Here, we characterize genetically and biochemically Pseudomonas aeruginosa HrpB homolog. We reveal that the auxiliary domains shape HrpB RNA preferences, affecting RNA species recognition and catalytic activity. We show that, among several types of RNAs, the single-stranded poly(A) and the highly structured MS2 RNA strongly stimulate HrpB ATPase activity. In addition, deleting the CTE affects only stimulation by structured RNAs like MS2 and rRNAs, while deletion of accessory domains results in gain of poly(U)-dependent activity. Finally, using hydrogen-deuterium exchange, we dissect the molecular details of HrpB interaction with poly(A) and MS2 RNAs. The catalytic core interacts with both RNAs, triggering a conformational change that reorients HrpB. Regions within the accessory domains and CTE are, instead, specifically responsive to MS2. Altogether, we demonstrate that in bacteria, like in eukaryotes, DExH-box helicase auxiliary domains are indispensable for RNA handling.


Assuntos
Proteínas de Bactérias/química , RNA Helicases DEAD-box/química , RNA/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Modelos Moleculares , Mutação , Fenótipo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Pseudomonas aeruginosa , RNA/metabolismo , Deleção de Sequência , Relação Estrutura-Atividade
8.
Mol Cell ; 71(2): 343-351.e4, 2018 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-30029007

RESUMO

Class II phosphoinositide 3-kinases (PI3K-C2) are large multidomain enzymes that control cellular functions ranging from membrane dynamics to cell signaling via synthesis of 3'-phosphorylated phosphoinositides. Activity of the alpha isoform (PI3K-C2α) is associated with endocytosis, angiogenesis, and glucose metabolism. How PI3K-C2α activity is controlled at sites of endocytosis remains largely enigmatic. Here we show that the lipid-binding PX-C2 module unique to class II PI3Ks autoinhibits kinase activity in solution but is essential for full enzymatic activity at PtdIns(4,5)P2-rich membranes. Using HDX-MS, we show that the PX-C2 module folds back onto the kinase domain, inhibiting its basal activity. Destabilization of this intramolecular contact increases PI3K-C2α activity in vitro and in cells, leading to accumulation of its lipid product, increased recruitment of the endocytic effector SNX9, and facilitated endocytosis. Our studies uncover a regulatory mechanism in which coincident binding of phosphoinositide substrate and cofactor selectively activate PI3K-C2α at sites of endocytosis.


Assuntos
Classe II de Fosfatidilinositol 3-Quinases/metabolismo , Classe II de Fosfatidilinositol 3-Quinases/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Animais , Domínios C2/fisiologia , Células COS , Chlorocebus aethiops , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/fisiologia , Clatrina/fisiologia , Endocitose/fisiologia , Células HEK293 , Homeostase , Humanos , Lipídeos/fisiologia , Espectrometria de Massas , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Ligação Proteica , Domínios Proteicos , Transdução de Sinais
9.
Dev Cell ; 43(4): 522-529.e4, 2017 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-29161595

RESUMO

Clathrin-mediated endocytosis occurs by bending and remodeling of the membrane underneath the coat. Bin-amphiphysin-rvs (BAR) domain proteins are crucial for endocytic membrane remodeling, but how their activity is spatiotemporally controlled is largely unknown. We demonstrate that the membrane remodeling activity of sorting nexin 9 (SNX9), a late-acting endocytic PX-BAR domain protein required for constriction of U-shaped endocytic intermediates, is controlled by an allosteric structural switch involving coincident detection of the clathrin adaptor AP2 and phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2) at endocytic sites. Structural, biochemical, and cell biological data show that SNX9 is autoinhibited in solution. Binding to PI(3,4)P2 via its PX-BAR domain, and concomitant association with AP2 via sequences in the linker region, releases SNX9 autoinhibitory contacts to enable membrane constriction. Our results reveal a mechanism for restricting the latent membrane remodeling activity of BAR domain proteins to allow spatiotemporal coupling of membrane constriction to the progression of the endocytic pathway.


Assuntos
Membrana Celular/metabolismo , Endocitose/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sítio Alostérico , Animais , Humanos , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/metabolismo , Ligação Proteica/fisiologia , Nexinas de Classificação/metabolismo , Proteínas de Transporte Vesicular/química
10.
Proc Natl Acad Sci U S A ; 114(8): 1982-1987, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28167755

RESUMO

Activated PI3K Delta Syndrome (APDS) is a primary immunodeficiency disease caused by activating mutations in either the leukocyte-restricted p110δ catalytic (PIK3CD) subunit or the ubiquitously expressed p85α regulatory (PIK3R1) subunit of class IA phosphoinositide 3-kinases (PI3Ks). There are two classes of APDS: APDS1 that arises from p110δ mutations that are analogous to oncogenic mutations found in the broadly expressed p110α subunit and APDS2 that occurs from a splice mutation resulting in p85α with a central deletion (Δ434-475). As p85 regulatory subunits associate with and inhibit all class IA catalytic subunits, APDS2 mutations are expected to similarly activate p110α, ß, and δ, yet APDS2 largely phenocopies APDS1 without dramatic effects outside the immune system. We have examined the molecular mechanism of activation of both classes of APDS mutations using a combination of biochemical assays and hydrogen-deuterium exchange mass spectrometry. Intriguingly, we find that an APDS2 mutation in p85α leads to substantial basal activation of p110δ (>300-fold) and disrupts inhibitory interactions from the nSH2, iSH2, and cSH2 domains of p85, whereas p110α is only minimally basally activated (∼2-fold) when associated with mutated p85α. APDS1 mutations in p110δ (N334K, E525K, E1021K) mimic the activation mechanisms previously discovered for oncogenic mutations in p110α. All APDS mutations were potently inhibited by the Food and Drug Administration-approved p110δ inhibitor idelalisib. Our results define the molecular basis of how PIK3CD and PIK3R1 mutations result in APDS and reveal a potential path to treatment for all APDS patients.


Assuntos
Domínio Catalítico/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Inibidores Enzimáticos/farmacologia , Síndromes de Imunodeficiência/genética , Fosfatidilinositol 3-Quinases/genética , Purinas/farmacologia , Quinazolinonas/farmacologia , Membrana Celular/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Classe Ia de Fosfatidilinositol 3-Quinase , Ensaios Enzimáticos , Inibidores Enzimáticos/uso terapêutico , Mutação com Ganho de Função , Humanos , Síndromes de Imunodeficiência/tratamento farmacológico , Espectrometria de Massas/métodos , Modelos Moleculares , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Doenças da Imunodeficiência Primária , Conformação Proteica , Purinas/uso terapêutico , Quinazolinonas/uso terapêutico , Ensaios Clínicos Controlados Aleatórios como Assunto , Deleção de Sequência
11.
Biochem Soc Trans ; 43(5): 773-86, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26517882

RESUMO

Many cellular signalling events are controlled by the selective recruitment of protein complexes to membranes. Determining the molecular basis for how lipid signalling complexes are recruited, assembled and regulated on specific membrane compartments has remained challenging due to the difficulty of working in conditions mimicking native biological membrane environments. Enzyme recruitment to membranes is controlled by a variety of regulatory mechanisms, including binding to specific lipid species, protein-protein interactions, membrane curvature, as well as post-translational modifications. A powerful tool to study the regulation of membrane signalling enzymes and complexes is hydrogen deuterium exchange-MS (HDX-MS), a technique that allows for the interrogation of protein dynamics upon membrane binding and recruitment. This review will highlight the theory and development of HDX-MS and its application to examine the molecular basis of lipid signalling enzymes, specifically the regulation and activation of phosphoinositide 3-kinases (PI3Ks).


Assuntos
Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Modelos Moleculares , Transdução de Sinais , Animais , Aniversários e Eventos Especiais , Distinções e Prêmios , Bioquímica , Membrana Celular/química , Membrana Celular/enzimologia , Classe I de Fosfatidilinositol 3-Quinases/química , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Medição da Troca de Deutério , Humanos , Espectrometria de Massas , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutação , Conformação Proteica , Dobramento de Proteína , Transporte Proteico , Sociedades Científicas , Reino Unido
12.
Biochem J ; 469(1): 59-69, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-26173259

RESUMO

Class IB phosphoinositide 3-kinases γ (PI3Kγ) are second-messenger-generating enzymes downstream of signalling cascades triggered by G-protein-coupled receptors (GPCRs). PI3Kγ variants have one catalytic p110γ subunit that can form two different heterodimers by binding to one of a pair of non-catalytic subunits, p87 or p101. Growing experimental data argue for a different regulation of p87-p110γ and p101-p110γ allowing integration into distinct signalling pathways. Pharmacological tools enabling distinct modulation of the two variants are missing. The ability of an anti-p110γ monoclonal antibody [mAb(A)p110γ] to block PI3Kγ enzymatic activity attracted us to characterize this tool in detail using purified proteins. In order to get insight into the antibody-p110γ interface, hydrogen-deuterium exchange coupled to MS (HDX-MS) measurements were performed demonstrating binding of the monoclonal antibody to the C2 domain in p110γ, which was accompanied by conformational changes in the helical domain harbouring the Gßγ-binding site. We then studied the modulation of phospholipid vesicles association of PI3Kγ by the antibody. p87-p110γ showed a significantly reduced Gßγ-mediated phospholipid recruitment as compared with p101-p110γ. Concomitantly, in the presence of mAb(A)p110γ, Gßγ did not bind to p87-p110γ. These data correlated with the ability of the antibody to block Gßγ-stimulated lipid kinase activity of p87-p110γ 30-fold more potently than p101-p110γ. Our data argue for differential regulatory functions of the non-catalytic subunits and a specific Gßγ-dependent regulation of p101 in PI3Kγ activation. In this scenario, we consider the antibody as a valuable tool to dissect the distinct roles of the two PI3Kγ variants downstream of GPCRs.


Assuntos
Anticorpos Monoclonais Murinos/química , Classe Ib de Fosfatidilinositol 3-Quinase , Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Animais , Classe Ib de Fosfatidilinositol 3-Quinase/química , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Medição da Troca de Deutério , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Células Sf9 , Spodoptera
13.
Proc Natl Acad Sci U S A ; 110(47): 18862-7, 2013 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-24190998

RESUMO

Phosphoinositide 3-kinase gamma (PI3Kγ) has profound roles downstream of G-protein-coupled receptors in inflammation, cardiac function, and tumor progression. To gain insight into how the enzyme's activity is shaped by association with its p101 adaptor subunit, lipid membranes, and Gßγ heterodimers, we mapped these regulatory interactions using hydrogen-deuterium exchange mass spectrometry. We identify residues in both the p110γ and p101 subunits that contribute critical interactions with Gßγ heterodimers, leading to PI3Kγ activation. Mutating Gßγ-interaction sites of either p110γ or p101 ablates G-protein-coupled receptor-mediated signaling to p110γ/p101 in cells and severely affects chemotaxis and cell transformation induced by PI3Kγ overexpression. Hydrogen-deuterium exchange mass spectrometry shows that association with the p101 regulatory subunit causes substantial protection of the RBD-C2 linker as well as the helical domain of p110γ. Lipid interaction massively exposes that same helical site, which is then stabilized by Gßγ. Membrane-elicited conformational change of the helical domain could help prepare the enzyme for Gßγ binding. Our studies and others identify the helical domain of the class I PI3Ks as a hub for diverse regulatory interactions that include the p101, p87 (also known as p84), and p85 adaptor subunits; Rab5 and Gßγ heterodimers; and the ß-adrenergic receptor kinase.


Assuntos
Classe Ib de Fosfatidilinositol 3-Quinase/química , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Modelos Moleculares , Fosfatidilinositol 3-Quinases/metabolismo , Conformação Proteica , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Animais , Quimiotaxia , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Medição da Troca de Deutério , Ativação Enzimática , Células HEK293 , Humanos , Espectrometria de Massas , Camundongos , Microscopia Confocal , Células NIH 3T3 , Receptores Acoplados a Proteínas G/agonistas , Transdução de Sinais/genética , Proteínas ras/metabolismo
14.
Science ; 342(6160): 866-71, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-24136356

RESUMO

Genetic mutations cause primary immunodeficiencies (PIDs) that predispose to infections. Here, we describe activated PI3K-δ syndrome (APDS), a PID associated with a dominant gain-of-function mutation in which lysine replaced glutamic acid at residue 1021 (E1021K) in the p110δ protein, the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ), encoded by the PIK3CD gene. We found E1021K in 17 patients from seven unrelated families, but not among 3346 healthy subjects. APDS was characterized by recurrent respiratory infections, progressive airway damage, lymphopenia, increased circulating transitional B cells, increased immunoglobulin M, and reduced immunoglobulin G2 levels in serum and impaired vaccine responses. The E1021K mutation enhanced membrane association and kinase activity of p110δ. Patient-derived lymphocytes had increased levels of phosphatidylinositol 3,4,5-trisphosphate and phosphorylated AKT protein and were prone to activation-induced cell death. Selective p110δ inhibitors IC87114 and GS-1101 reduced the activity of the mutant enzyme in vitro, which suggested a therapeutic approach for patients with APDS.


Assuntos
Predisposição Genética para Doença , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/patologia , Fosfatidilinositol 3-Quinases/genética , Infecções Respiratórias/genética , Infecções Respiratórias/patologia , Classe I de Fosfatidilinositol 3-Quinases , Humanos , Síndromes de Imunodeficiência/imunologia , Linfócitos/imunologia , Mutação , Linhagem , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Infecções Respiratórias/imunologia
15.
Sci Signal ; 5(253): ra89, 2012 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-23211529

RESUMO

Synergistic activation by heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) and receptor tyrosine kinases distinguishes p110ß from other class IA phosphoinositide 3-kinases (PI3Ks). Activation of p110ß is specifically implicated in various physiological and pathophysiological processes, such as the growth of tumors deficient in phosphatase and tensin homolog deleted from chromosome 10 (PTEN). To determine the specific contribution of GPCR signaling to p110ß-dependent functions, we identified the site in p110ß that binds to the Gßγ subunit of G proteins. Mutation of this site eliminated Gßγ-dependent activation of PI3Kß (a dimer of p110ß and the p85 regulatory subunit) in vitro and in cells, without affecting basal activity or phosphotyrosine peptide-mediated activation. Disrupting the p110ß-Gßγ interaction by mutation or with a cell-permeable peptide inhibitor blocked the transforming capacity of PI3Kß in fibroblasts and reduced the proliferation, chemotaxis, and invasiveness of PTEN-null tumor cells in culture. Our data suggest that specifically targeting GPCR signaling to PI3Kß could provide a therapeutic approach for tumors that depend on p110ß for growth and metastasis.


Assuntos
Transformação Celular Neoplásica/metabolismo , Fibroblastos/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Linhagem Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Classe I de Fosfatidilinositol 3-Quinases , Fibroblastos/patologia , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Neoplasias/terapia , Fosfatidilinositol 3-Quinases/genética , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/genética
16.
Proc Natl Acad Sci U S A ; 109(38): 15259-64, 2012 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-22949682

RESUMO

The p110α catalytic subunit (PIK3CA) is one of the most frequently mutated genes in cancer. We have examined the activation of the wild-type p110α/p85α and a spectrum of oncogenic mutants using hydrogen/deuterium exchange mass spectrometry (HDX-MS). We find that for the wild-type enzyme, the natural transition from an inactive cytosolic conformation to an activated form on membranes entails four distinct events. Analysis of oncogenic mutations shows that all up-regulate the enzyme by enhancing one or more of these dynamic events. We provide the first insight into the activation mechanism by mutations in the linker between the adapter-binding domain (ABD) and the Ras-binding domain (RBD) (G106V and G118D). These mutations, which are common in endometrial cancers, enhance two of the natural activation events: movement of the ABD and ABD-RBD linker relative to the rest of the catalytic subunit and breaking the C2-iSH2 interface on binding membranes. C2 domain mutants (N345K and C420R) also mimic these events, even in the absence of membranes. A third event is breaking the nSH2-helical domain contact caused by phosphotyrosine-containing peptides binding to the enzyme, which is mimicked by a helical domain mutation (E545K). Interaction of the C lobe of the kinase domain with membranes is the fourth activation event, and is potentiated by kinase domain mutations (e.g., H1047R). All mutations increased lipid binding and basal activity, even mutants distant from the membrane surface. Our results elucidate a unifying mechanism in which diverse PIK3CA mutations stimulate lipid kinase activity by facilitating allosteric motions required for catalysis on membranes.


Assuntos
Mutação , Fosfatidilinositol 3-Quinases/genética , Sítio Alostérico , Animais , Catálise , Domínio Catalítico , Classe I de Fosfatidilinositol 3-Quinases , Citosol/metabolismo , Neoplasias do Endométrio/metabolismo , Ativação Enzimática , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Cinética , Lipídeos/química , Modelos Moleculares , Conformação Molecular , Movimento (Física) , Fosfatidilinositol 3-Quinases/química , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais
18.
Sci Signal ; 4(195): re2, 2011 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-22009150

RESUMO

Phosphoinositide 3-kinases (PI3Ks) are implicated in a broad spectrum of cellular activities, such as growth, proliferation, differentiation, migration, and metabolism. Activation of class I PI3Ks by mutation or overexpression correlates with the development and maintenance of various human cancers. These PI3Ks are heterodimers, and the activity of the catalytic subunits is tightly controlled by the associated regulatory subunits. Although the same p85 regulatory subunits associate with all class IA PI3Ks, the functional outcome depends on the isotype of the catalytic subunit. New PI3K partners that affect the signaling by the PI3K heterodimers have been uncovered, including phosphate and tensin homolog (PTEN), cyclic adenosine monophosphate-dependent protein kinase (PKA), and nonstructural protein 1. Interactions with PI3K regulators modulate the intrinsic membrane affinity and either the rate of phosphoryl transfer or product release. Crystal structures for the class I and class III PI3Ks in complexes with associated regulators and inhibitors have contributed to developing isoform-specific inhibitors and have shed light on the numerous regulatory mechanisms controlling PI3K activation and inhibition.


Assuntos
Fosfatidilinositol 3-Quinases/química , Multimerização Proteica , Estrutura Terciária de Proteína , Ativação Enzimática/efeitos dos fármacos , Humanos , Indazóis/farmacologia , Modelos Moleculares , Neoplasias/enzimologia , Neoplasias/patologia , Neoplasias/prevenção & controle , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia
19.
Structure ; 19(8): 1127-37, 2011 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-21827948

RESUMO

Phosphoinositide 3-kinase δ is upregulated in lymphocytic leukemias. Because the p85-regulatory subunit binds to any class IA subunit, it was assumed there is a single universal p85-mediated regulatory mechanism; however, we find isozyme-specific inhibition by p85α. Using deuterium exchange mass spectrometry (DXMS), we mapped regulatory interactions of p110δ with p85α. Both nSH2 and cSH2 domains of p85α contribute to full inhibition of p110δ, the nSH2 by contacting the helical domain and the cSH2 via the C terminus of p110δ. The cSH2 inhibits p110ß and p110δ, but not p110α, implying that p110α is uniquely poised for oncogenic mutations. Binding RTK phosphopeptides disengages the SH2 domains, resulting in exposure of the catalytic subunit. We find that phosphopeptides greatly increase the affinity of the heterodimer for PIP2-containing membranes measured by FRET. DXMS identified regions decreasing exposure at membranes and also regions gaining exposure, indicating loosening of interactions within the heterodimer at membranes.


Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/química , Lipídeos de Membrana/química , Fosfatidilinositol 3-Quinases/química , Substituição de Aminoácidos , Animais , Classe I de Fosfatidilinositol 3-Quinases , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Medição da Troca de Deutério , Humanos , Lipossomos/química , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Receptores do Fator de Crescimento Derivado de Plaquetas/química , Propriedades de Superfície
20.
Mol Cell ; 41(5): 567-78, 2011 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-21362552

RESUMO

Phosphoinositide 3-kinases (PI3Ks) are essential for cell growth, migration, and survival. The structure of a p110ß/p85ß complex identifies an inhibitory function for the C-terminal SH2 domain (cSH2) of the p85 regulatory subunit. Mutagenesis of a cSH2 contact residue activates downstream signaling in cells. This inhibitory contact ties up the C-terminal region of the p110ß catalytic subunit, which is essential for lipid kinase activity. In vitro, p110ß basal activity is tightly restrained by contacts with three p85 domains: the cSH2, nSH2, and iSH2. RTK phosphopeptides relieve inhibition by nSH2 and cSH2 using completely different mechanisms. The binding site for the RTK's pYXXM motif is exposed on the cSH2, requiring an extended RTK motif to reach and disrupt the inhibitory contact with p110ß. This contrasts with the nSH2 where the pY-binding site itself forms the inhibitory contact. This establishes an unusual mechanism by which p85 SH2 domains contribute to RTK signaling specificities.


Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Regulação Enzimológica da Expressão Gênica , Motivos de Aminoácidos , Animais , Sítios de Ligação , Humanos , Ligação de Hidrogênio , Insetos , Camundongos , Mutagênese , Mutação , Fosforilação , Conformação Proteica , Estrutura Terciária de Proteína , Domínios de Homologia de src
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...