Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 17 de 17
Filtrar
Mais filtros








Base de dados
Intervalo de ano de publicação
1.
Nat Struct Mol Biol ; 31(7): 1028-1038, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38388830

RESUMO

The RAS-MAPK pathway regulates cell proliferation, differentiation and survival, and its dysregulation is associated with cancer development. The pathway minimally comprises the small GTPase RAS and the kinases RAF, MEK and ERK. Activation of RAF by RAS is notoriously intricate and remains only partially understood. There are three RAF isoforms in mammals (ARAF, BRAF and CRAF) and two related pseudokinases (KSR1 and KSR2). RAS-mediated activation of RAF depends on an allosteric mechanism driven by the dimerization of its kinase domain. Recent work on human RAFs showed that MEK binding to KSR1 promotes KSR1-BRAF heterodimerization, which leads to the phosphorylation of free MEK molecules by BRAF. Similar findings were made with the single Drosophila RAF homolog. Here we show that the fly scaffold proteins CNK and HYP stabilize the KSR-MEK interaction, which in turn enhances RAF-KSR heterodimerization and RAF activation. The cryogenic electron microscopy structure of the minimal KSR-MEK-CNK-HYP complex reveals a ring-like arrangement of the CNK-HYP complex allowing CNK to simultaneously engage KSR and MEK, thus stabilizing the binary interaction. Together, these results illuminate how CNK contributes to RAF activation by stimulating the allosteric function of KSR and highlight the diversity of mechanisms impacting RAF dimerization as well as the regulatory potential of the KSR-MEK interaction.


Assuntos
Proteínas de Drosophila , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Humanos , Microscopia Crioeletrônica , Quinases raf/metabolismo , Quinases raf/química , Ligação Proteica , Multimerização Proteica , Modelos Moleculares , Drosophila melanogaster/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases , Proteínas ras
2.
J Biol Chem ; 300(1): 105505, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38029963

RESUMO

Mutations in receptor guanylyl cyclase C (GC-C) cause severe gastrointestinal disease, including meconium ileus, early onset acute diarrhea, and pediatric inflammatory bowel disease that continues into adulthood. Agonists of GC-C are US Food and Drug Administration-approved drugs for the treatment of constipation and irritable bowel syndrome. Therapeutic strategies targeting GC-C are tested in preclinical mouse models, assuming that murine GC-C mimics human GC-C in its biochemical properties and downstream signaling events. Here, we reveal important differences in ligand-binding affinity and GC activity between mouse GC-C and human GC-C. We generated a series of chimeric constructs of various domains of human and mouse GC-C to show that the extracellular domain of mouse GC-C contributed to log-orders lower affinity of mouse GC-C for ligands than human GC-C. Further, the Vmax of the murine GC domain was lower than that of human GC-C, and allosteric regulation of the receptor by ATP binding to the intracellular kinase-homology domain also differed. These altered properties are reflected in the high concentrations of ligands required to elicit signaling responses in the mouse gut in preclinical models and the specificity of a GC inhibitor towards human GC-C. Therefore, our studies identify considerations in using the murine model to test molecules for therapeutic purposes that work as either agonists or antagonists of GC-C, and vaccines for the bacterial heat-stable enterotoxin that causes watery diarrhea in humans.


Assuntos
Receptores Acoplados a Guanilato Ciclase , Animais , Criança , Humanos , Camundongos , Diarreia , Enterotoxinas , Guanilato Ciclase/metabolismo , Ligantes , Receptores de Enterotoxina/genética , Receptores Acoplados a Guanilato Ciclase/antagonistas & inibidores , Receptores Acoplados a Guanilato Ciclase/genética , Receptores Acoplados a Guanilato Ciclase/metabolismo , Gastroenteropatias/tratamento farmacológico , Gastroenteropatias/metabolismo , Gastroenteropatias/patologia
3.
J Am Chem Soc ; 145(13): 7123-7135, 2023 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-36961978

RESUMO

The design of PROteolysis-TArgeting Chimeras (PROTACs) requires bringing an E3 ligase into proximity with a target protein to modulate the concentration of the latter through its ubiquitination and degradation. Here, we present a method for generating high-accuracy structural models of E3 ligase-PROTAC-target protein ternary complexes. The method is dependent on two computational innovations: adding a "silent" convolution term to an efficient protein-protein docking program to eliminate protein poses that do not have acceptable linker conformations and clustering models of multiple PROTACs that use the same E3 ligase and target the same protein. Results show that the largest consensus clusters always have high predictive accuracy and that the ensemble of models can be used to predict the dissociation rate and cooperativity of the ternary complex that relate to the degrading activity of the PROTAC. The method is demonstrated by applications to known PROTAC structures and a blind test involving PROTACs against BRAF mutant V600E. The results confirm that PROTACs function by stabilizing a favorable interaction between the E3 ligase and the target protein but do not necessarily exploit the most energetically favorable geometry for interaction between the proteins.


Assuntos
Proteínas , Ubiquitina-Proteína Ligases , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Proteínas/metabolismo , Ubiquitinação
4.
J Med Chem ; 65(19): 12725-12746, 2022 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-36117290

RESUMO

Targeted protein degradation (TPD) strategies exploit bivalent small molecules to bridge substrate proteins to an E3 ubiquitin ligase to induce substrate degradation. Few E3s have been explored as degradation effectors due to a dearth of E3-binding small molecules. We show that genetically induced recruitment to the GID4 subunit of the CTLH E3 complex induces protein degradation. An NMR-based fragment screen followed by structure-guided analog elaboration identified two binders of GID4, 16 and 67, with Kd values of 110 and 17 µM in vitro. A parallel DNA-encoded library (DEL) screen identified five binders of GID4, the best of which, 88, had a Kd of 5.6 µM in vitro and an EC50 of 558 nM in cells with strong selectivity for GID4. X-ray co-structure determination revealed the basis for GID4-small molecule interactions. These results position GID4-CTLH as an E3 for TPD and provide candidate scaffolds for high-affinity moieties that bind GID4.


Assuntos
DNA , Ubiquitina-Proteína Ligases , DNA/metabolismo , Humanos , Proteólise , Ubiquitina-Proteína Ligases/metabolismo
5.
Front Mol Biosci ; 8: 671011, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34150847

RESUMO

Small-angle X-ray scattering (SAXS) experiments are important in structural biology because they are solution methods, and do not require crystallization of protein complexes. Structure determination from SAXS data, however, poses some difficulties. Computation of a SAXS profile from a protein model is expensive in CPU time. Hence, rather than directly refining against the data, most computational methods generate a large number of conformers and then filter the structures based on how well they satisfy the SAXS data. To address this issue in an efficient manner, we propose here a Bayesian model for SAXS data and use it to directly drive a Monte Carlo simulation. We show that the automatic weighting of SAXS data is the key to finding optimal structures efficiently. Another key problem with obtaining structures from SAXS data is that proteins are often flexible and the data represents an average over a structural ensemble. To address this issue, we first characterize the stability of the best model with extensive molecular dynamics simulations. We analyse the resulting trajectories further to characterize a dynamic structural ensemble satisfying the SAXS data. The combination of methods is applied to a tandem of domains from the protein PTPN4, which are connected by an unstructured linker. We show that the SAXS data contain information that supports and extends other experimental findings. We also show that the conformation obtained by the Bayesian analysis is stable, but that a minor conformation is present. We propose a mechanism in which the linker may maintain PTPN4 in an inhibited enzymatic state.

6.
Nat Commun ; 11(1): 6233, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33277478

RESUMO

The KEOPS complex, which is conserved across archaea and eukaryotes, is composed of four core subunits; Pcc1, Kae1, Bud32 and Cgi121. KEOPS is crucial for the fitness of all organisms examined. In humans, pathogenic mutations in KEOPS genes lead to Galloway-Mowat syndrome, an autosomal-recessive disease causing childhood lethality. Kae1 catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine, but the precise roles of all other KEOPS subunits remain an enigma. Here we show using structure-guided studies that Cgi121 recruits tRNA to KEOPS by binding to its 3' CCA tail. A composite model of KEOPS bound to tRNA reveals that all KEOPS subunits form an extended tRNA-binding surface that we have validated in vitro and in vivo to mediate the interaction with the tRNA substrate and its modification. These findings provide a framework for understanding the inner workings of KEOPS and delineate why all KEOPS subunits are essential.


Assuntos
Proteínas Arqueais/química , Methanocaldococcus/metabolismo , Complexos Multiproteicos/química , RNA de Transferência/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Cristalografia por Raios X , Methanocaldococcus/genética , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA de Transferência de Lisina/química , RNA de Transferência de Lisina/genética , RNA de Transferência de Lisina/metabolismo
7.
Nat Chem Biol ; 16(11): 1170-1178, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32778845

RESUMO

The RAF family kinases function in the RAS-ERK pathway to transmit signals from activated RAS to the downstream kinases MEK and ERK. This pathway regulates cell proliferation, differentiation and survival, enabling mutations in RAS and RAF to act as potent drivers of human cancers. Drugs targeting the prevalent oncogenic mutant BRAF(V600E) have shown great efficacy in the clinic, but long-term effectiveness is limited by resistance mechanisms that often exploit the dimerization-dependent process by which RAF kinases are activated. Here, we investigated a proteolysis-targeting chimera (PROTAC) approach to BRAF inhibition. The most effective PROTAC, termed P4B, displayed superior specificity and inhibitory properties relative to non-PROTAC controls in BRAF(V600E) cell lines. In addition, P4B displayed utility in cell lines harboring alternative BRAF mutations that impart resistance to conventional BRAF inhibitors. This work provides a proof of concept for a substitute to conventional chemical inhibition to therapeutically constrain oncogenic BRAF.


Assuntos
Antineoplásicos , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas B-raf , Talidomida , Ubiquitina , Animais , Feminino , Humanos , Camundongos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Moleculares , Estrutura Molecular , Terapia de Alvo Molecular , Mutação , Fosforilação/efeitos dos fármacos , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteólise , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Transdução de Sinais , Relação Estrutura-Atividade , Talidomida/análogos & derivados , Talidomida/química , Ubiquitina/química
8.
J Biol Chem ; 295(14): 4526-4540, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32086379

RESUMO

The small GTPases H, K, and NRAS are molecular switches indispensable for proper regulation of cellular proliferation and growth. Several mutations in the genes encoding members of this protein family are associated with cancer and result in aberrant activation of signaling processes caused by a deregulated recruitment of downstream effector proteins. In this study, we engineered variants of the Ras-binding domain (RBD) of the C-Raf proto-oncogene, Ser/Thr kinase (CRAF). These variants bound with high affinity with the effector-binding site of Ras in an active conformation. Structural characterization disclosed how the newly identified RBD mutations cooperate and thereby enhance affinity with the effector-binding site in Ras compared with WT RBD. The engineered RBD variants closely mimicked the interaction mode of naturally occurring Ras effectors and acted as dominant-negative affinity reagents that block Ras signal transduction. Experiments with cancer cells showed that expression of these RBD variants inhibits Ras signaling, reducing cell growth and inducing apoptosis. Using these optimized RBD variants, we stratified patient-derived colorectal cancer organoids with known Ras mutational status according to their response to Ras inhibition. These results revealed that the presence of Ras mutations was insufficient to predict sensitivity to Ras inhibition, suggesting that not all of these tumors required Ras signaling for proliferation. In summary, by engineering the Ras/Raf interface of the CRAF-RBD, we identified potent and selective inhibitors of Ras in its active conformation that outcompete binding of Ras-signaling effectors.


Assuntos
Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas ras/metabolismo , Apoptose , Sítios de Ligação , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Simulação de Dinâmica Molecular , Mutagênese , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Transdução de Sinais , Proteínas ras/antagonistas & inibidores , Proteínas ras/genética
9.
ACS Med Chem Lett ; 10(7): 1074-1080, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31312411

RESUMO

One effective means to achieve inhibitor specificity for RAF kinases, an important family of cancer drug targets, has been to target the monomeric inactive state conformation of the kinase domain, which, unlike most other kinases, can accommodate sulfonamide-containing drugs such as vemurafenib and dabrafenib because of the presence of a unique pocket specific to inactive RAF kinases. We previously reported an alternate strategy whereby rigidification of a nonselective pyrazolo[3,4-d]pyrimidine-based inhibitor through ring closure afforded moderate but appreciable increases in selectivity for RAF kinases. Here, we show that a further application of the rigidification strategy to a different pyrazolopyrimidine-based scaffold dramatically improved selectivity for RAF kinases. Crystal structure analysis confirmed our inhibitor design hypothesis revealing that 2l engages an active-like state conformation of BRAF normally associated with poorly discriminating inhibitors. When screened against a panel of distinct cancer cell lines, the optimized inhibitor 2l primarily inhibited the proliferation of the expected BRAFV600E-harboring cell lines consistent with its kinome selectivity profile. These results suggest that rigidification could be a general and powerful strategy for enhancing inhibitor selectivity against protein kinases, which may open up therapeutic opportunities not afforded by other approaches.

10.
Structure ; 27(6): 1000-1012.e6, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31056421

RESUMO

Pseudoenzymes have been identified across a diverse range of enzyme classes and fulfill important cellular functions. Examples of pseudoenzymes exist within ubiquitin conjugating and deubiquitinase (DUB) protein families. Here we characterize FAM105A/OTULINL, the only putative pseudodeubiquitinase of the ovarian tumor protease (OTU domain) family in humans. The crystal structure of FAM105A revealed that the OTU domain possesses structural deficiencies in both active site and substrate-binding infrastructure predicted to impair normal DUB function. We confirmed the absence of catalytic function against all ubiquitin linkages and an inability of FAM105A to bind ubiquitin compared with catalytically active FAM105B/OTULIN. FAM105A co-localized with KDEL markers and Lamin B1 at the endoplasmic reticulum (ER) and nuclear envelope, respectively. Accordingly, the FAM105A interactome exhibited significant enrichment in proteins localized to the ER/outer nuclear, Golgi and vesicular membranes. In light of undetectable deubiquitinase activity, we posit that FAM105A/OTULINL functions through its ability to mediate protein-protein interactions.


Assuntos
Enzimas Desubiquitinantes/química , Endopeptidases/química , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Sequência de Aminoácidos , Animais , Domínio Catalítico , Linhagem Celular Tumoral , Cristalografia por Raios X , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Homologia de Sequência de Aminoácidos , Ubiquitina/química , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
11.
Nature ; 554(7693): 549-553, 2018 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-29433126

RESUMO

RAF family kinases have prominent roles in cancer. Their activation is dependent on dimerization of their kinase domains, which has emerged as a hindrance for drug development. In mammals, RAF family kinases include three catalytically competent enzymes (ARAF, BRAF and CRAF) and two pseudokinases (KSR1 and KSR2) that have been described as scaffolds owing to their apparent ability to bridge RAF isoforms and their substrate, mitogen-activated protein kinase kinase (MEK). Kinase suppressor of Ras (KSR) pseudokinases were also shown to dimerize with kinase-competent RAFs to stimulate catalysis allosterically. Although GTP-bound RAS can modulate the dimerization of RAF isoforms by engaging their RAS-binding domains, KSR1 and KSR2 lack an RAS-binding domain and therefore the regulatory principles underlying their dimerization with other RAF family members remain unknown. Here we show that the selective heterodimerization of BRAF with KSR1 is specified by direct contacts between the amino-terminal regulatory regions of each protein, comprising in part a novel domain called BRS in BRAF and the coiled-coil-sterile α motif (CC-SAM) domain in KSR1. We also discovered that MEK binding to the kinase domain of KSR1 asymmetrically drives BRAF-KSR1 heterodimerization, resulting in the concomitant stimulation of BRAF catalytic activity towards free MEK molecules. These findings demonstrate that KSR-MEK complexes allosterically activate BRAF through the action of N-terminal regulatory region and kinase domain contacts and challenge the accepted role of KSR as a scaffold for MEK recruitment to RAF.


Assuntos
Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/metabolismo , Regulação Alostérica , Cristalografia por Raios X , Ativação Enzimática , Humanos , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Modelos Moleculares , Fosforilação , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais
12.
Eur J Med Chem ; 146: 519-528, 2018 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-29407977

RESUMO

Established strategies for discovering selective kinase inhibitors are target-centric as they often target certain structural or reactive features in the target kinase. In the absence of such prominent features, there is a lack of general methods for discovering selective inhibitors. Here we describe a new strategy that exploits conformational flexibility of kinases for achieving selective kinase inhibition. Through ring closure, we designed and synthesized a panel of isoquinoline-containing compounds as rigidified analogs of an amidophenyl-containing parent compound. These analogs potently inhibit kinases including Abl and BRAF but have diminished inhibition against some other kinases compared to the parent compound. Sequence analysis reveals that many of the kinases that are potently inhibited by the isoquonoline-containing compounds contain a long insertion within their catalytic domains. A crystal structure of one rigid compound bound to BRAF confirmed its binding mode. Our findings highlight the potential of a novel strategy of rigidification for improving the selectivity of kinase inhibitors.


Assuntos
Isoquinolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Relação Dose-Resposta a Droga , Humanos , Isoquinolinas/síntese química , Isoquinolinas/química , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade
13.
Sci Rep ; 7(1): 7875, 2017 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-28801650

RESUMO

Human protein tyrosine phosphatase non-receptor type 4 (PTPN4) has been shown to prevent cell death. The active form of human PTPN4 consists of two globular domains, a PDZ (PSD-95/Dlg/ZO-1) domain and a phosphatase domain, tethered by a flexible linker. Targeting its PDZ domain abrogates this protection and triggers apoptosis. We previously demonstrated that the PDZ domain inhibits the phosphatase activity of PTPN4 and that the mere binding of a PDZ ligand is sufficient to release the catalytic inhibition. We demonstrate here that the linker connecting the PDZ domain and the phosphatase domain is involved in the regulation of the phosphatase activity in both PDZ-related inhibition and PDZ ligand-related activation events. We combined bioinformatics and kinetic studies to decipher the role of the linker in the PTPN4 activity. By comparing orthologous sequences, we identified a conserved patch of hydrophobic residues in the linker. We showed that mutations in this patch affect the regulation of the PTPN4 bidomain indicating that the PDZ-PDZ ligand regulation of PTPN4 is a linker-mediated mechanism. However, the mutations do not alter the binding of the PDZ ligand. This study strengthens the notion that inter-domain linker can be of functional importance in enzyme regulation of large multi-domain proteins.


Assuntos
Mutação , Domínios PDZ/genética , Monoéster Fosfórico Hidrolases/genética , Proteína Tirosina Fosfatase não Receptora Tipo 4/genética , Regulação Alostérica/genética , Sequência de Aminoácidos , Sítios de Ligação/genética , Biocatálise , Humanos , Cinética , Ligantes , Monoéster Fosfórico Hidrolases/metabolismo , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 4/metabolismo , Proteólise , Homologia de Sequência de Aminoácidos
14.
Nucleic Acids Res ; 45(2): 805-817, 2017 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-27903914

RESUMO

The KEOPS/EKC complex is a tRNA modification complex involved in the biosynthesis of N6-threonylcarbamoyladenosine (t6A), a universally conserved tRNA modification found on ANN-codon recognizing tRNAs. In archaea and eukaryotes, KEOPS is composed of OSGEP/Kae1, PRPK/Bud32, TPRKB/Cgi121 and LAGE3/Pcc1. In fungi, KEOPS contains an additional subunit, Gon7, whose orthologs outside of fungi, if existent, remain unidentified. In addition to displaying defective t6A biosynthesis, Saccharomyces cerevisiae strains harboring KEOPS mutations are compromised for telomere homeostasis, growth and transcriptional co-activation. To identify a Gon7 ortholog in multicellular eukaryotes as well as to uncover KEOPS-interacting proteins that may link t6A biosynthesis to the diverse set of KEOPS mutant phenotypes, we conducted a proteomic analysis of human KEOPS. This work identified 152 protein interactors, one of which, C14ORF142, interacted strongly with all four KEOPS subunits, suggesting that it may be a core component of human KEOPS. Further characterization of C14ORF142 revealed that it shared a number of biophysical and biochemical features with fungal Gon7, suggesting that C14ORF142 is the human ortholog of Gon7. In addition, our proteomic analysis identified specific interactors for different KEOPS subcomplexes, hinting that individual KEOPS subunits may have additional functions outside of t6A biosynthesis.


Assuntos
Complexos Multiproteicos , Fases de Leitura Aberta , Subunidades Proteicas , Proteômica , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Complexos Multiproteicos/química , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/química
15.
J Biol Chem ; 291(32): 16699-708, 2016 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-27246854

RESUMO

The human protein tyrosine phosphatase non-receptor type 4 (PTPN4) prevents cell death induction in neuroblastoma and glioblastoma cell lines in a PDZ·PDZ binding motifs-dependent manner, but the cellular partners of PTPN4 involved in cell protection are unknown. Here, we described the mitogen-activated protein kinase p38γ as a cellular partner of PTPN4. The main contribution to the p38γ·PTPN4 complex formation is the tight interaction between the C terminus of p38γ and the PDZ domain of PTPN4. We solved the crystal structure of the PDZ domain of PTPN4 bound to the p38γ C terminus. We identified the molecular basis of recognition of the C-terminal sequence of p38γ that displays the highest affinity among all endogenous partners of PTPN4. We showed that the p38γ C terminus is also an efficient inducer of cell death after its intracellular delivery. In addition to recruiting the kinase, the binding of the C-terminal sequence of p38γ to PTPN4 abolishes the catalytic autoinhibition of PTPN4 and thus activates the phosphatase, which can efficiently dephosphorylate the activation loop of p38γ. We presume that the p38γ·PTPN4 interaction promotes cellular signaling, preventing cell death induction.


Assuntos
Proteína Quinase 12 Ativada por Mitógeno/metabolismo , Complexos Multienzimáticos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 4/metabolismo , Transdução de Sinais/fisiologia , Morte Celular , Linhagem Celular Tumoral , Humanos , Proteína Quinase 12 Ativada por Mitógeno/genética , Complexos Multienzimáticos/genética , Proteína Tirosina Fosfatase não Receptora Tipo 4/genética
16.
Prog Biophys Mol Biol ; 119(1): 53-9, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25748547

RESUMO

PDZ (PSD-95/Dlg/ZO-1) domains play a major role in neuronal homeostasis in which they act as scaffold domains regulating cellular trafficking, self-association and catalytic activity of essential proteins such as kinases and phosphatases. Because of their central role in cell signaling, cellular PDZ-containing proteins are preferential targets of viruses to hijack cellular function to their advantage. Here, we describe how the viral G protein of the rabies virus specifically targets the PDZ domain of neuronal enzymes during viral infection. By disrupting the complexes formed by cellular enzymes and their ligands, the virus triggers drastic effect on cell signaling and commitment of the cell to either survival (virulent strains) or death (vaccinal strains). We provide structural and biological evidences that the viral proteins act as competitors endowed with specificity and affinity in an essential cellular process by mimicking PDZ binding motif of cellular partners. Disruption of critical endogenous protein-protein interactions by viral protein drastically alters intracellular protein trafficking and catalytic activity of cellular proteins that control cell homeostasis. This work opens up many perspectives to mimic viral sequences and developing innovative therapies to manipulate cellular homeostasis.


Assuntos
Neurônios/metabolismo , Domínios PDZ , Vírus da Raiva/fisiologia , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/enzimologia , Neurônios/virologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 4/metabolismo , Vírus da Raiva/metabolismo
17.
FEBS J ; 281(21): 4852-65, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25158884

RESUMO

The human protein tyrosine phosphatase non-receptor type 4 (PTPN4) prevents cells death. Targeting its PDZ domain abrogates this protection and triggers apoptosis. We demonstrate here that the PDZ domain inhibits the phosphatase activity of PTPN4. The mere binding of a PDZ ligand is sufficient to release the catalytic inhibition. We combined analytical ultracentrifugation, small angle X-ray scattering and NMR to understand how the PDZ domain controls PTPN4 activity. We show that the physiologically active PTPN4 two-domain, encompassing the PDZ and the phosphatase domains, adopts a predominant compact conformation in solution. The PDZ ligand binding restores the catalytic competence of PTPN4 disrupting the transient interdomain communication. This study strengthens the emerging notion that PDZ domains can act as regulators of enzyme activity and therefore are active players in the dynamic regulation of signaling pathways.


Assuntos
Proteína Tirosina Fosfatase não Receptora Tipo 4/metabolismo , Catálise , Humanos , Cinética , Ligantes , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Domínios PDZ , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 4/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 4/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Espalhamento a Baixo Ângulo , Transdução de Sinais , Soluções , Relação Estrutura-Atividade , Difração de Raios X
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA