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1.
Structure ; 28(2): 145-156.e5, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31831213

RESUMO

The class I phosphoinositide 3-kinases (PI3Ks) are key signaling enzymes composed of a heterodimer of a p110 catalytic subunit and a p85 regulatory subunit, with PI3K mutations being causative of multiple human diseases including cancer, primary immunodeficiencies, and developmental disorders. Mutations in the p85α regulatory subunit encoded by PIK3R1 can both activate PI3K through oncogenic truncations in the iSH2 domain, or inhibit PI3K through developmental disorder mutations in the cSH2 domain. Using a combined biochemical and hydrogen deuterium exchange mass spectrometry approach we have defined the molecular basis for how these mutations alter the activity of p110α/p110δ catalytic subunits. We find that the oncogenic Q572∗ truncation of PIK3R1 disrupts all p85-inhibitory inputs, with p110α being hyper-activated compared with p110δ. In addition, we find that the R649W mutation in the cSH2 of PIK3R1 decreases sensitivity to activation by receptor tyrosine kinases. This work reveals unique insight into isoform-specific regulation of p110s by p85α.


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Mutação , Domínio Catalítico , Classe I de Fosfatidilinositol 3-Quinases/química , Classe Ia de Fosfatidilinositol 3-Quinase/química , Ativação Enzimática , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Modelos Moleculares , Conformação Proteica , Domínios Proteicos
2.
J Mol Biol ; 430(18 Pt B): 3129-3142, 2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-30031006

RESUMO

Phosphatidylinositol 4-kinase IIIα (PI4KIIIα) is the lipid kinase primarily responsible for generating the lipid phosphatidylinositol 4-phosphate (PI4P) at the plasma membrane, which acts as the substrate for generation of the signaling lipids PIP2 and PIP3. PI4KIIIα forms a large heterotrimeric complex with two regulatory partners, TTC7 and FAM126. We describe using an integrated electron microscopy and hydrogen-deuterium exchange mass spectrometry (HDX-MS) approach to probe the architecture and dynamics of the complex of PI4KIIIα/TTC7/FAM126. HDX-MS reveals that the majority of the PI4KIIIα sequence was protected from exchange in short deuterium pulse experiments, suggesting presence of secondary structure, even in putative unstructured regions. Negative stain electron microscopy reveals the shape and architecture of the full-length complex, revealing an overall dimer of PI4KIIIα/TTC7/FAM126 trimers. HDX-MS reveals conformational changes in the TTC7/FAM126 complex upon binding PI4KIIIα, including both at the direct TTC7-PI4KIIIα interface and at the putative membrane binding surface. Finally, HDX-MS experiments of PI4KIIIα bound to the highly potent and selective inhibitor GSK-A1 compared to that bound to the non-specific inhibitor PIK93 revealed substantial conformational changes throughout an extended region of the kinase domain. Many of these changes were distant from the putative inhibitor binding site, showing a large degree of allosteric conformational changes that occur upon inhibitor binding. Overall, our results reveal novel insight into the regulation of PI4KIIIα by its regulatory proteins TTC7/FAM126, as well as additional dynamic information on how selective inhibition of PI4KIIIα is achieved.


Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas/metabolismo , 1-Fosfatidilinositol 4-Quinase/química , Regulação Alostérica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas/química , Proteínas Recombinantes
3.
Front Immunol ; 9: 575, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29616047

RESUMO

The signaling lipid phosphatidylinositol 3,4,5, trisphosphate (PIP3) is an essential mediator of many vital cellular processes, including growth, survival, and metabolism. PIP3 is generated through the action of the class I phosphoinositide 3-kinases (PI3K), and their activity is tightly controlled through interactions with regulatory proteins and activating stimuli. The class IA PI3Ks are composed of three distinct p110 catalytic subunits (p110α, p110ß, and p110δ), and they play different roles in specific tissues due to disparities in both expression and engagement downstream of cell-surface receptors. Disruption of PI3K regulation is a frequent driver of numerous human diseases. Activating mutations in the PIK3CA gene encoding the p110α catalytic subunit of class IA PI3K are frequently mutated in several cancer types, and mutations in the PIK3CD gene encoding the p110δ catalytic subunit have been identified in primary immunodeficiency patients. All class IA p110 subunits interact with p85 regulatory subunits, and mutations/deletions in different p85 regulatory subunits have been identified in both cancer and primary immunodeficiencies. In this review, we will summarize our current understanding for the molecular basis of how class IA PI3K catalytic activity is regulated by p85 regulatory subunits, and how activating mutations in the PI3K catalytic subunits PIK3CA and PIK3CD (p110α, p110δ) and regulatory subunits PIK3R1 (p85α) mediate PI3K activation and human disease.


Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/genética , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Suscetibilidade a Doenças , Síndromes de Imunodeficiência/etiologia , Síndromes de Imunodeficiência/metabolismo , Mutação , Neoplasias/etiologia , Neoplasias/metabolismo , Oncogenes , Animais , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Deficiências do Desenvolvimento/etiologia , Deficiências do Desenvolvimento/metabolismo , Regulação da Expressão Gênica , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais
4.
Science ; 358(6366): 1056-1059, 2017 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-29074584

RESUMO

Newly transcribed eukaryotic precursor messenger RNAs (pre-mRNAs) are processed at their 3' ends by the ~1-megadalton multiprotein cleavage and polyadenylation factor (CPF). CPF cleaves pre-mRNAs, adds a polyadenylate tail, and triggers transcription termination, but it is unclear how its various enzymes are coordinated and assembled. Here, we show that the nuclease, polymerase, and phosphatase activities of yeast CPF are organized into three modules. Using electron cryomicroscopy, we determined a 3.5-angstrom-resolution structure of the ~200-kilodalton polymerase module. This revealed four ß propellers, in an assembly markedly similar to those of other protein complexes that bind nucleic acid. Combined with in vitro reconstitution experiments, our data show that the polymerase module brings together factors required for specific and efficient polyadenylation, to help coordinate mRNA 3'-end processing.


Assuntos
Processamento de Terminações 3' de RNA , RNA Polimerase II/química , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Fatores de Poliadenilação e Clivagem de mRNA/química , Microscopia Crioeletrônica , Polinucleotídeo Adenililtransferase/metabolismo , Conformação Proteica , RNA Polimerase II/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Fatores de Poliadenilação e Clivagem de mRNA/ultraestrutura
6.
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
7.
Nature ; 538(7625): 344-349, 2016 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-27602946

RESUMO

Antimalarial drugs have thus far been chiefly derived from two sources-natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.


Assuntos
Antimaláricos/síntese química , Antimaláricos/farmacologia , Azetidinas/uso terapêutico , Descoberta de Drogas , Estágios do Ciclo de Vida/efeitos dos fármacos , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimento , Animais , Antimaláricos/administração & dosagem , Antimaláricos/uso terapêutico , Compostos Azabicíclicos/administração & dosagem , Compostos Azabicíclicos/síntese química , Compostos Azabicíclicos/farmacologia , Compostos Azabicíclicos/uso terapêutico , Azetidinas/administração & dosagem , Azetidinas/efeitos adversos , Azetidinas/farmacologia , Citosol/enzimologia , Modelos Animais de Doenças , Feminino , Fígado/efeitos dos fármacos , Fígado/parasitologia , Macaca mulatta/parasitologia , Malária Falciparum/prevenção & controle , Malária Falciparum/transmissão , Masculino , Camundongos , Fenilalanina-tRNA Ligase/antagonistas & inibidores , Compostos de Fenilureia/administração & dosagem , Compostos de Fenilureia/síntese química , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/uso terapêutico , Plasmodium falciparum/citologia , Plasmodium falciparum/enzimologia , Segurança
8.
J Med Chem ; 59(5): 1830-9, 2016 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-26885694

RESUMO

Type III phosphatidylinositol 4-kinase (PI4KIIIß) is an essential enzyme in mediating membrane trafficking and is implicated in a variety of pathogenic processes. It is a key host factor mediating replication of RNA viruses. The design of potent and specific inhibitors of this enzyme will be essential to define its cellular roles and may lead to novel antiviral therapeutics. We previously reported the PI4K inhibitor PIK93, and this compound has defined key functions of PI4KIIIß. However, this compound showed high cross reactivity with class I and III PI3Ks. Using structure-based drug design, we have designed novel potent and selective (>1000-fold over class I and class III PI3Ks) PI4KIIIß inhibitors. These compounds showed antiviral activity against hepatitis C virus. The co-crystal structure of PI4KIIIß bound to one of the most potent compounds reveals the molecular basis of specificity. This work will be vital in the design of novel PI4KIIIß inhibitors, which may play significant roles as antiviral therapeutics.


Assuntos
Antivirais/farmacologia , Desenho de Fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Antivirais/síntese química , Antivirais/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Hepacivirus/efeitos dos fármacos , Hepacivirus/fisiologia , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Inibidores de Proteínas Quinases/síntese química , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos
9.
Biochem Soc Trans ; 44(1): 260-6, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26862213

RESUMO

Many important cellular functions are regulated by the selective recruitment of proteins to intracellular membranes mediated by specific interactions with lipid phosphoinositides. The enzymes that generate lipid phosphoinositides therefore must be properly positioned and regulated at their correct cellular locations. Phosphatidylinositol 4 kinases (PI4Ks) are key lipid signalling enzymes, and they generate the lipid species phosphatidylinositol 4-phosphate (PI4P), which plays important roles in regulating physiological processes including membrane trafficking, cytokinesis and organelle identity. PI4P also acts as the substrate for the generation of the signalling phosphoinositides phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). PI4Ks also play critical roles in a number of pathological processes including mediating replication of a number of pathogenic RNA viruses, and in the development of the parasite responsible for malaria. Key to the regulation of PI4Ks is their regulation by a variety of both host and viral protein-binding partners. We review herein our current understanding of the structure, regulatory interactions and role in disease of the type III PI4Ks.


Assuntos
1-Fosfatidilinositol 4-Quinase/química , 1-Fosfatidilinositol 4-Quinase/metabolismo , Doença , Transdução de Sinais , Animais , Humanos , Modelos Moleculares , Ligação Proteica
10.
Mol Cell ; 54(5): 858-69, 2014 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-24905007

RESUMO

Fanconi anaemia (FA) is a cancer predisposition syndrome characterized by cellular sensitivity to DNA interstrand crosslinkers. The molecular defect in FA is an impaired DNA repair pathway. The critical event in activating this pathway is monoubiquitination of FANCD2. In vivo, a multisubunit FA core complex catalyzes this step, but its mechanism is unclear. Here, we report purification of a native avian FA core complex and biochemical reconstitution of FANCD2 monoubiquitination. This demonstrates that the catalytic FANCL E3 ligase subunit must be embedded within the complex for maximal activity and site specificity. We genetically and biochemically define a minimal subcomplex comprising just three proteins (FANCB, FANCL, and FAAP100) that functions as the monoubiquitination module. Residual FANCD2 monoubiquitination activity is retained in cells defective for other FA core complex subunits. This work describes the in vitro reconstitution and characterization of this multisubunit monoubiquitin E3 ligase, providing key insight into the conserved FA DNA repair pathway.


Assuntos
Proteínas Aviárias/metabolismo , Galinhas/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Ubiquitinação , Animais , Proteínas Aviárias/química , Proteínas Aviárias/genética , Linhagem Celular , Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/química , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/química , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Humanos , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo
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