RESUMEN
The poly(ADP-ribose)polymerases Tankyrase 1/2 (TNKS/TNKS2) catalyze the covalent linkage of ADP-ribose polymer chains onto target proteins, regulating their ubiquitylation, stability, and function. Dysregulation of substrate recognition by Tankyrases underlies the human disease cherubism. Tankyrases recruit specific motifs (often called RxxPDG "hexapeptides") in their substrates via an N-terminal region of ankyrin repeats. These ankyrin repeats form five domains termed ankyrin repeat clusters (ARCs), each predicted to bind substrate. Here we report crystal structures of a representative ARC of TNKS2 bound to targeting peptides from six substrates. Using a solution-based peptide library screen, we derive a rule-based consensus for Tankyrase substrates common to four functionally conserved ARCs. This 8-residue consensus allows us to rationalize all known Tankyrase substrates and explains the basis for cherubism-causing mutations in the Tankyrase substrate 3BP2. Structural and sequence information allows us to also predict and validate other Tankyrase targets, including Disc1, Striatin, Fat4, RAD54, BCR, and MERIT40.
Asunto(s)
Querubismo/metabolismo , Tanquirasas/química , Tanquirasas/metabolismo , Secuencia de Aminoácidos , Animales , Repetición de Anquirina , Cristalografía por Rayos X , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
In the ubiquitin-proteasome system (UPS), E2 enzymes mediate the conjugation of ubiquitin to substrates and thereby control protein stability and interactions. The E2 enzyme hCdc34 catalyzes the ubiquitination of hundreds of proteins in conjunction with the cullin-RING (CRL) superfamily of E3 enzymes. We identified a small molecule termed CC0651 that selectively inhibits hCdc34. Structure determination revealed that CC0651 inserts into a cryptic binding pocket on hCdc34 distant from the catalytic site, causing subtle but wholesale displacement of E2 secondary structural elements. CC0651 analogs inhibited proliferation of human cancer cell lines and caused accumulation of the SCF(Skp2) substrate p27(Kip1). CC0651 does not affect hCdc34 interactions with E1 or E3 enzymes or the formation of the ubiquitin thioester but instead interferes with the discharge of ubiquitin to acceptor lysine residues. E2 enzymes are thus susceptible to noncatalytic site inhibition and may represent a viable class of drug target in the UPS.
Asunto(s)
Aminoácidos/farmacología , Compuestos de Bifenilo/farmacología , Complejos de Ubiquitina-Proteína Ligasa/antagonistas & inhibidores , Sitio Alostérico , Secuencia de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Análisis Mutacional de ADN , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Enzimas Ubiquitina-Conjugadoras , Complejos de Ubiquitina-Proteína Ligasa/química , Complejos de Ubiquitina-Proteína Ligasa/genéticaRESUMEN
Protein-protein interactions (PPIs) are some of the most challenging target classes in drug discovery. Highly sensitive detection techniques are required for the identification of chemical modulators of PPIs. Here, we introduce PPI confocal nanoscanning (PPI-CONA), a miniaturized, microbead based high-resolution fluorescence imaging assay. We demonstrate the capabilities of PPI-CONA by detecting low affinity ternary complex formation between the human CDC34A ubiquitin-conjugating (E2) enzyme, ubiquitin, and CC0651, a small molecule enhancer of the CDC34A-ubiquitin interaction. We further exemplify PPI-CONA with an E2 enzyme binding study on CC0651 and a CDC34A binding specificity study of a series of CC0651 analogues. Our results indicate that CC0651 is highly selective toward CDC34A. We further demonstrate how PPI-CONA can be applied to screening very low affinity interactions. PPI-CONA holds potential for high-throughput screening for modulators of PPI targets and characterization of their affinity, specificity, and selectivity.
RESUMEN
FBXW7, which encodes a substrate-specific receptor of an SCF E3 ligase complex, is a frequently mutated human tumor suppressor gene known to regulate the post-translational stability of various proteins involved in cellular proliferation. Here, using genome-wide CRISPR screens, we report a novel synthetic lethal genetic interaction between FBXW7 and CCNL1 and describe CCNL1 as a new substrate of the SCF-FBXW7 E3 ligase. Further analysis showed that the CCNL1-CDK11 complex is critical at the G2-M phase of the cell cycle since defective CCNL1 accumulation, resulting from FBXW7 mutation, leads to shorter mitotic time. Cells harboring FBXW7 loss-of-function mutations are hypersensitive to treatment with a CDK11 inhibitor, highlighting a genetic vulnerability that could be leveraged for cancer treatment.
Asunto(s)
Ciclinas , Proteína 7 que Contiene Repeticiones F-Box-WD , Ubiquitina-Proteína Ligasas , Humanos , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Mutación , Ubiquitina-Proteína Ligasas/genética , Ciclinas/metabolismo , UbiquitinaciónRESUMEN
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.
Asunto(s)
Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas B-raf/química , Proteínas Proto-Oncogénicas B-raf/metabolismo , Regulación Alostérica , Cristalografía por Rayos X , Activación Enzimática , Humanos , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , Modelos Moleculares , Fosforilación , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de SeñalRESUMEN
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.
Asunto(s)
Proteínas , Ubiquitina-Proteína Ligasas , Proteolisis , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas/metabolismo , UbiquitinaciónRESUMEN
BRCC36 is a Zn(2+)-dependent deubiquitinating enzyme (DUB) that hydrolyzes lysine-63-linked ubiquitin chains as part of distinct macromolecular complexes that participate in either interferon signaling or DNA-damage recognition. The MPN(+) domain protein BRCC36 associates with pseudo DUB MPN(-) proteins KIAA0157 or Abraxas, which are essential for BRCC36 enzymatic activity. To understand the basis for BRCC36 regulation, we have solved the structure of an active BRCC36-KIAA0157 heterodimer and an inactive BRCC36 homodimer. Structural and functional characterizations show how BRCC36 is switched to an active conformation by contacts with KIAA0157. Higher-order association of BRCC36 and KIAA0157 into a dimer of heterodimers (super dimers) was required for DUB activity and interaction with targeting proteins SHMT2 and RAP80. These data provide an explanation of how an inactive pseudo DUB allosterically activates a cognate DUB partner and implicates super dimerization as a new regulatory mechanism underlying BRCC36 DUB activity, subcellular localization, and biological function.
Asunto(s)
Hormigas/enzimología , Proteínas de Insectos/química , Proteínas Asociadas a Matriz Nuclear/química , Proteasas Ubiquitina-Específicas/química , Animales , Dominio Catalítico , Cristalografía por Rayos X , Enzimas Desubicuitinizantes , Células HEK293 , Células HeLa , Humanos , Proteínas de Insectos/fisiología , Cinética , Proteínas de la Membrana/química , Modelos Moleculares , Proteínas Asociadas a Matriz Nuclear/fisiología , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteasas Ubiquitina-Específicas/fisiologíaRESUMEN
KEOPS (Kinase, Endopeptidase and Other Proteins of Small size) is a five-subunit protein complex that is highly conserved in eukaryotes and archaea and is essential for the fitness of cells and for animal development. In humans, mutations in KEOPS genes underlie Galloway-Mowat syndrome, which manifests in severe microcephaly and renal dysfunction that lead to childhood death. The Kae1 subunit of KEOPS catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine (t6A), while the auxiliary subunits Cgi121, the kinase/ATPase Bud32, Pcc1 and Gon7 play a supporting role. Kae1 orthologs are also present in bacteria and mitochondria but function in distinct complexes with proteins that are not related in structure or function to the auxiliary subunits of KEOPS. Over the past 15 years since its discovery, extensive study in the KEOPS field has provided many answers towards understanding the roles that KEOPS plays in cells and in human disease and how KEOPS carries out these functions. In this review, we provide an overview into recent advances in the study of KEOPS and illuminate exciting future directions.
Asunto(s)
Adenosina/análogos & derivados , Proteína 1 de Intercambio de Anión de Eritrocito/genética , Hernia Hiatal/genética , Microcefalia/genética , Nefrosis/genética , ARN de Transferencia/genética , Proteínas de Saccharomyces cerevisiae/genética , Adenosina/metabolismo , Animales , Proteína 1 de Intercambio de Anión de Eritrocito/química , Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Archaea/genética , Archaea/metabolismo , Secuencia Conservada , Regulación de la Expresión Génica , Hernia Hiatal/metabolismo , Hernia Hiatal/patología , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Microcefalia/metabolismo , Microcefalia/patología , Modelos Moleculares , Nefrosis/metabolismo , Nefrosis/patología , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conformación Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The oligoadenylate synthetase (OAS)-RNase L system is an IFN-inducible antiviral pathway activated by viral infection. Viral double-stranded (ds) RNA activates OAS isoforms that synthesize the second messenger 2-5A, which binds and activates the pseudokinase-endoribonuclease RNase L. In cells, OAS activation is tamped down by ADAR1, an adenosine deaminase that destabilizes dsRNA. Mutation of ADAR1 is one cause of Aicardi-Goutières syndrome (AGS), an interferonopathy in children. ADAR1 deficiency in human cells can lead to RNase L activation and subsequent cell death. To evaluate RNase L as a possible therapeutic target for AGS, we sought to identify small-molecule inhibitors of RNase L. A 500-compound library of protein kinase inhibitors was screened for modulators of RNase L activity in vitro. We identified ellagic acid (EA) as a hit with 10-fold higher selectivity against RNase L compared with its nearest paralog, IRE1. SAR analysis identified valoneic acid dilactone (VAL) as a superior inhibitor of RNase L, with 100-fold selectivity over IRE1. Mechanism-of-action analysis indicated that EA and VAL do not bind to the pseudokinase domain of RNase L despite acting as ATP competitive inhibitors of the protein kinase CK2. VAL is nontoxic and functional in cells, although with a 1,000-fold decrease in potency, as measured by RNA cleavage activity in response to treatment with dsRNA activator or by rescue of cell lethality resulting from self dsRNA induced by ADAR1 deficiency. These studies lay the foundation for understanding novel modes of regulating RNase L function using small-molecule inhibitors and avenues of therapeutic potential.
Asunto(s)
Adenosina Desaminasa/deficiencia , Enfermedades Autoinmunes del Sistema Nervioso/enzimología , Endorribonucleasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Malformaciones del Sistema Nervioso/enzimología , Fenol/farmacología , 2',5'-Oligoadenilato Sintetasa/genética , 2',5'-Oligoadenilato Sintetasa/metabolismo , Nucleótidos de Adenina/metabolismo , Adenosina Desaminasa/genética , Enfermedades Autoinmunes del Sistema Nervioso/genética , Enfermedades Autoinmunes del Sistema Nervioso/fisiopatología , Muerte Celular/efectos de los fármacos , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Inhibidores Enzimáticos/química , Humanos , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/fisiopatología , Oligorribonucleótidos/metabolismo , Fenol/química , Proteínas de Unión al ARN/genéticaRESUMEN
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.
Asunto(s)
Antineoplásicos , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas B-raf , Talidomida , Ubiquitina , Animales , Femenino , Humanos , Ratones , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Resistencia a Antineoplásicos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Modelos Moleculares , Estructura Molecular , Terapia Molecular Dirigida , Mutación , Fosforilación/efectos de los fármacos , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Proteolisis , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Transducción de Señal , Relación Estructura-Actividad , Talidomida/análogos & derivados , Talidomida/química , Ubiquitina/químicaAsunto(s)
Biotecnología/historia , Biología Molecular/historia , Agricultura/historia , Animales , Bovinos , Clonación Molecular , ADN Complementario/genética , Hormona del Crecimiento/historia , Hormona del Crecimiento/aislamiento & purificación , Historia del Siglo XX , Humanos , Israel , Poli A/química , Poli A/aislamiento & purificación , ARN Mensajero/química , ARN Mensajero/aislamiento & purificaciónRESUMEN
DNA double-strand breaks (DSBs) elicit a histone modification cascade that controls DNA repair. This pathway involves the sequential ubiquitination of histones H1 and H2A by the E3 ubiquitin ligases RNF8 and RNF168, respectively. RNF168 ubiquitinates H2A on lysine 13 and lysine 15 (refs 7, 8) (yielding H2AK13ub and H2AK15ub, respectively), an event that triggers the recruitment of 53BP1 (also known as TP53BP1) to chromatin flanking DSBs. 53BP1 binds specifically to H2AK15ub-containing nucleosomes through a peptide segment termed the ubiquitination-dependent recruitment motif (UDR), which requires the simultaneous engagement of histone H4 lysine 20 dimethylation (H4K20me2) by its tandem Tudor domain. How 53BP1 interacts with these two histone marks in the nucleosomal context, how it recognizes ubiquitin, and how it discriminates between H2AK13ub and H2AK15ub is unknown. Here we present the electron cryomicroscopy (cryo-EM) structure of a dimerized human 53BP1 fragment bound to a H4K20me2-containing and H2AK15ub-containing nucleosome core particle (NCP-ubme) at 4.5 Å resolution. The structure reveals that H4K20me2 and H2AK15ub recognition involves intimate contacts with multiple nucleosomal elements including the acidic patch. Ubiquitin recognition by 53BP1 is unusual and involves the sandwiching of the UDR segment between ubiquitin and the NCP surface. The selectivity for H2AK15ub is imparted by two arginine fingers in the H2A amino-terminal tail, which straddle the nucleosomal DNA and serve to position ubiquitin over the NCP-bound UDR segment. The structure of the complex between NCP-ubme and 53BP1 reveals the basis of 53BP1 recruitment to DSB sites and illuminates how combinations of histone marks and nucleosomal elements cooperate to produce highly specific chromatin responses, such as those elicited following chromosome breaks.
Asunto(s)
Microscopía por Crioelectrón , Histonas/química , Histonas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Nucleosomas/metabolismo , Nucleosomas/ultraestructura , Roturas del ADN de Doble Cadena , Reparación del ADN , Humanos , Metilación , Modelos Moleculares , Nucleosomas/química , Nucleosomas/genética , Docilidad , Multimerización de Proteína , Estructura Terciaria de Proteína , Especificidad por Sustrato , Proteína 1 de Unión al Supresor Tumoral P53 , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
RNase L is an ankyrin repeat domain-containing dual endoribonuclease-pseudokinase that is activated by unusual 2,'5'-oligoadenylate (2-5A) second messengers and which impedes viral infections in higher vertebrates. Despite its importance in interferon-regulated antiviral innate immunity, relatively little is known about its precise mechanism of action. Here we present a functional characterization of 2.5 Å and 3.25 Å X-ray crystal and small-angle X-ray scattering structures of RNase L bound to a natural 2-5A activator with and without ADP or the nonhydrolysable ATP mimetic AMP-PNP. These studies reveal how recognition of 2-5A through interactions with the ankyrin repeat domain and the pseudokinase domain, together with nucleotide binding, imposes a rigid intertwined dimer configuration that is essential for RNase catalytic and antiviral functions. The involvement of the pseudokinase domain of RNase L in 2-5A sensing, nucleotide binding, dimerization, and ribonuclease functions highlights the evolutionary adaptability of the eukaryotic protein kinase fold.
Asunto(s)
Nucleótidos de Adenina/química , Endorribonucleasas/química , Oligorribonucleótidos/química , Adenosina Difosfato/química , Adenilil Imidodifosfato/química , Animales , Repetición de Anquirina , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Virus de la Encefalomiocarditis , Endorribonucleasas/genética , Endorribonucleasas/fisiología , Células HeLa , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Picornaviridae , Estructura Terciaria de Proteína , Dispersión de Radiación , Relación Estructura-Actividad , Sus scrofaRESUMEN
Drugs that reverse epigenetic silencing, such as the DNA methyltransferase inhibitor (DNMTi) 5-azacytidine (AZA), have profound effects on transcription and tumor cell survival. AZA is an approved drug for myelodysplastic syndromes and acute myeloid leukemia, and is under investigation for different solid malignant tumors. AZA treatment generates self, double-stranded RNA (dsRNA), transcribed from hypomethylated repetitive elements. Self dsRNA accumulation in DNMTi-treated cells leads to type I IFN production and IFN-stimulated gene expression. Here we report that cell death in response to AZA treatment occurs through the 2',5'-oligoadenylate synthetase (OAS)-RNase L pathway. OASs are IFN-induced enzymes that synthesize the RNase L activator 2-5A in response to dsRNA. Cells deficient in RNase L or OAS1 to 3 are highly resistant to AZA, as are wild-type cells treated with a small-molecule inhibitor of RNase L. A small-molecule inhibitor of c-Jun NH2-terminal kinases (JNKs) also antagonizes RNase L-dependent cell death in response to AZA, consistent with a role for JNK in RNase L-induced apoptosis. In contrast, the rates of AZA-induced and RNase L-dependent cell death were increased by transfection of 2-5A, by deficiencies in ADAR1 (which edits and destabilizes dsRNA), PDE12 or AKAP7 (which degrade 2-5A), or by ionizing radiation (which induces IFN-dependent signaling). Finally, OAS1 expression correlates with AZA sensitivity in the NCI-60 set of tumor cell lines, suggesting that the level of OAS1 can be a biomarker for predicting AZA sensitivity of tumor cells. These studies may eventually lead to pharmacologic strategies for regulating the antitumor activity and toxicity of AZA and related drugs.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/metabolismo , Azacitidina/farmacología , Desmetilación del ADN , Endorribonucleasas/metabolismo , Inmunidad Innata , Células A549 , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Muerte Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Isoenzimas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Radiación Ionizante , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
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.
Asunto(s)
Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas ras/metabolismo , Apoptosis , Sitios de Unión , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Simulación de Dinámica Molecular , Mutagénesis , Fosfatidilinositol 3-Quinasas/metabolismo , Unión Proteica , Dominios Proteicos , Estructura Terciaria de Proteína , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-raf/química , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Transducción de Señal , Proteínas ras/antagonistas & inhibidores , Proteínas ras/genéticaRESUMEN
Ubiquitylation entails the concerted action of E1, E2, and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Here we elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizes ubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme. Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibited E2 complex. Lys48 of donor ubiquitin lies near the OTUB1 catalytic site and the C terminus of free ubiquitin, a configuration that mimics the products of Lys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chain recognition to suppress ubiquitin conjugation and the DNA damage response.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Proteínas Ubiquitinadas/metabolismo , Sustitución de Aminoácidos , Línea Celular , Cristalografía por Rayos X , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Enzimas Desubicuitinizantes , Humanos , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Organismos Modificados Genéticamente , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitinación , Levaduras/genética , Levaduras/crecimiento & desarrolloRESUMEN
IpaH enzymes are secreted bacterial effectors that function within host cells as E3 ubiquitin (Ub) ligases. Catalytic activity is imparted by a conserved novel E3 ligase (NEL) domain that is unique to Gram-negative pathogens and whose activity is repressed by a flanking substrate-binding leucine-rich repeat (LRR) domain when substrate is absent. How the NEL domain catalyzes the conjugation of Ub onto substrates, recognizes host E2s, and maintains its autoinhibited state remain poorly understood. Here we used mutagenesis and enzyme kinetic analyses to address these gaps in knowledge. Mutagenesis of conserved residues on two remote surfaces of the NEL domain identified functional clusters proximal to and distal to the active site cysteine. By analyzing the kinetics of Ub charging and discharging, we identified proximal active site residues that function as either the catalytic acid or catalytic base for aminolysis. Further analysis revealed that distal site residues mediate the direct binding of E2. In studying the full-length protein, we also have uncovered that IpaH family autoinhibition is achieved by a short-circuiting mechanism wherein the LRR domain selectively blocks productive aminolysis, but not the nonproductive discharge of Ub from the E3 to solvent. This mode of autoinhibition, which is not shared by the HECT domain ligase Smurf2, leads to the unanticipated depletion of E2â¼Ub and thus a concomitant dominant-negative effect on other E3s in vitro, raising the possibility that short circuiting also may serve to restrict the function of host E3s in cells.
Asunto(s)
Proteínas Bacterianas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Bacterianas/genética , Catálisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonella/enzimología , Shigella/enzimología , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
RAS GTPases are important mediators of oncogenesis in humans. However, pharmacological inhibition of RAS has proved challenging. Here we describe a functionally critical region, located outside the effector lobe of RAS, that can be targeted for inhibition. We developed NS1, a synthetic binding protein (monobody) that bound with high affinity to both GTP- and GDP-bound states of H-RAS and K-RAS but not N-RAS. NS1 potently inhibited growth factor signaling and oncogenic H-RAS- and K-RAS-mediated signaling and transformation but did not block oncogenic N-RAS, BRAF or MEK1. NS1 bound the α4-ß6-α5 region of RAS, which disrupted RAS dimerization and nanoclustering and led to blocking of CRAF-BRAF heterodimerization and activation. These results establish the importance of the α4-ß6-α5 interface in RAS-mediated signaling and define a previously unrecognized site in RAS for inhibiting RAS function.
Asunto(s)
Sitio Alostérico/efectos de los fármacos , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Proteínas ras/antagonistas & inhibidores , Proteínas ras/química , Animales , Anticuerpos Monoclonales/química , Células COS , Células Cultivadas , Chlorocebus aethiops , Células HEK293 , Humanos , Ratones , Células 3T3 NIH , Proteínas ras/metabolismoRESUMEN
53BP1 (also called TP53BP1) is a chromatin-associated factor that promotes immunoglobulin class switching and DNA double-strand-break (DSB) repair by non-homologous end joining. To accomplish its function in DNA repair, 53BP1 accumulates at DSB sites downstream of the RNF168 ubiquitin ligase. How ubiquitin recruits 53BP1 to break sites remains unknown as its relocalization involves recognition of histone H4 Lys 20 (H4K20) methylation by its Tudor domain. Here we elucidate how vertebrate 53BP1 is recruited to the chromatin that flanks DSB sites. We show that 53BP1 recognizes mononucleosomes containing dimethylated H4K20 (H4K20me2) and H2A ubiquitinated on Lys 15 (H2AK15ub), the latter being a product of RNF168 action on chromatin. 53BP1 binds to nucleosomes minimally as a dimer using its previously characterized methyl-lysine-binding Tudor domain and a carboxy-terminal extension, termed the ubiquitination-dependent recruitment (UDR) motif, which interacts with the epitope formed by H2AK15ub and its surrounding residues on the H2A tail. 53BP1 is therefore a bivalent histone modification reader that recognizes a histone 'code' produced by DSB signalling.
Asunto(s)
Daño del ADN , Histonas/química , Histonas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisina/metabolismo , Ubiquitina/metabolismo , Ubiquitinación , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/deficiencia , Proteínas Cromosómicas no Histona/genética , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleosomas/química , Nucleosomas/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Transducción de Señal , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
A complex interaction of signalling events, including the Wnt pathway, regulates sprouting of blood vessels from pre-existing vasculature during angiogenesis. Here we show that two distinct mutations in the (uro)chordate-specific gumby (also called Fam105b) gene cause an embryonic angiogenic phenotype in gumby mice. Gumby interacts with disheveled 2 (DVL2), is expressed in canonical Wnt-responsive endothelial cells and encodes an ovarian tumour domain class of deubiquitinase that specifically cleaves linear ubiquitin linkages. A crystal structure of gumby in complex with linear diubiquitin reveals how the identified mutations adversely affect substrate binding and catalytic function in line with the severity of their angiogenic phenotypes. Gumby interacts with HOIP (also called RNF31), a key component of the linear ubiquitin assembly complex, and decreases linear ubiquitination and activation of NF-κB-dependent transcription. This work provides support for the biological importance of linear (de)ubiquitination in angiogenesis, craniofacial and neural development and in modulating Wnt signalling.