RESUMEN
Using N-Myc61-89 as a starting template we showcase the systematic use of truncation and maleimide constraining to develop peptidomimetic inhibitors of the N-Myc/Aurora-A protein-protein interaction (PPI); a potential anticancer drug discovery target. The most promising of these - N-Myc73-94-N85C/G89C-mal - is shown to favour a more Aurora-A compliant binding ensemble in comparison to the linear wild-type sequence as observed through fluorescence anisotropy competition assays, circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments. Further inâ silico investigation of this peptide in its Aurora-A bound state, by molecular dynamics (MD) simulations, imply (i)â the bound conformation is more stable as a consequence of the constraint, which likely suppresses dissociation and (ii)â the constraint may make further stabilizing interactions with the Aurora-A surface. Taken together this work unveils the first orthosteric N-Myc/Aurora-A inhibitor and provides useful insights on the biophysical properties and thus design of constrained peptides, an attractive therapeutic modality.
Asunto(s)
Peptidomiméticos , Peptidomiméticos/farmacología , Proteína Proto-Oncogénica N-Myc , Ciclización , Péptidos/química , Unión ProteicaRESUMEN
Protein kinases are key components in cellular signaling pathways as they carry out the phosphorylation of proteins, primarily on Ser, Thr, and Tyr residues. The catalytic activity of protein kinases is regulated, and they can be thought of as molecular switches that are controlled through protein-protein interactions and post-translational modifications. Protein kinases exhibit diverse structural mechanisms of regulation and have been fascinating subjects for structural biologists from the first crystal structure of a protein kinase over 30 years ago, to recent insights into kinase assemblies enabled by the breakthroughs in cryo-EM. Protein kinases are high-priority targets for drug discovery in oncology and other disease settings, and kinase inhibitors have transformed the outcomes of specific groups of patients. Most kinase inhibitors are ATP competitive, deriving potency by occupying the deep hydrophobic pocket at the heart of the kinase domain. Selectivity of inhibitors depends on exploiting differences between the amino acids that line the ATP site and exploring the surrounding pockets that are present in inactive states of the kinase. More recently, allosteric pockets outside the ATP site are being targeted to achieve high selectivity and to overcome resistance to current therapeutics. Here, we review the key regulatory features of the protein kinase family, describe the different types of kinase inhibitors, and highlight examples where the understanding of kinase regulatory mechanisms has gone hand in hand with the development of inhibitors.
Asunto(s)
Inhibidores de Proteínas Quinasas , Proteínas Quinasas , Adenosina Trifosfato/química , Descubrimiento de Drogas , Humanos , Fosforilación , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/químicaRESUMEN
A multiprotein complex containing TACC3, clathrin and other proteins has been implicated in mitotic spindle stability. To disrupt this complex in an anti-cancer context, we need to understand its composition and how it interacts with microtubules. Induced relocalization of proteins in cells is a powerful way to analyze protein-protein interactions and, additionally, monitor where and when these interactions occur. We used CRISPR/Cas9 gene editing to add tandem FKBP-GFP tags to each complex member. The relocalization of endogenous tagged protein from the mitotic spindle to mitochondria and assessment of the effect on other proteins allowed us to establish that TACC3 and clathrin are core complex members and that chTOG (also known as CKAP5) and GTSE1 are ancillary to the complex, binding respectively to TACC3 and clathrin, but not each other. We also show that PIK3C2A, a clathrin-binding protein that was proposed to stabilize the TACC3-chTOG-clathrin-GTSE1 complex during mitosis, is not a member of the complex. This work establishes that targeting the TACC3-clathrin interface or their microtubule-binding sites are the two strategies most likely to disrupt spindle stability mediated by this multiprotein complex.
Asunto(s)
Clatrina , Proteínas Asociadas a Microtúbulos , Huso Acromático , Clatrina/genética , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos , MitosisRESUMEN
Cysteine-rich receptor-like kinases (CRKs) are a large family of plasma membrane-bound receptors ubiquitous in higher plants. However, despite their prominence, their biological roles have remained largely elusive so far. In this study we report the characterization of an Arabidopsis mutant named crk10-A397T in which alanine 397 has been replaced by a threonine in the αC helix of the kinase domain of CRK10, known to be a crucial regulatory module in mammalian kinases. The crk10-A397T mutant is a dwarf that displays collapsed xylem vessels in the root and hypocotyl, whereas the vasculature of the inflorescence develops normally. In situ phosphorylation assays with His-tagged wild type and crk10-A397T versions of the CRK10 kinase domain revealed that both alleles are active kinases capable of autophosphorylation, with the newly introduced threonine acting as an additional phosphorylation site in crk10-A397T. Transcriptomic analysis of wild type and crk10-A397T mutant hypocotyls revealed that biotic and abiotic stress-responsive genes are constitutively up-regulated in the mutant, and a root-infection assay with the vascular pathogen Fusarium oxysporum demonstrated that the mutant has enhanced resistance to this pathogen compared with wild type plants. Taken together our results suggest that crk10-A397T is a gain-of-function allele of CRK10, the first such mutant to have been identified for a CRK in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Mutación Puntual , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Variants of the oncogenic EML4-ALK fusion protein contain a similar region of ALK encompassing the kinase domain, but different portions of EML4. Here, we show that EML4-ALK V1 and V3 proteins form cytoplasmic foci that contain components of the MAPK, PLCγ and PI3K signalling pathways. The ALK inhibitors ceritinib and lorlatinib dissolve these foci and EML4-ALK V3 but not V1 protein re-localises to microtubules, an effect recapitulated in a catalytically inactive EML4-ALK mutant. Mutations that promote a constitutively active ALK stabilise the cytoplasmic foci even in the presence of these inhibitors. In contrast, the inhibitor alectinib increases foci formation of both wild-type and catalytically inactive EML4-ALK V3 proteins, but not a Lys-Glu salt bridge mutant. We propose that EML4-ALK foci formation occurs as a result of transient association of stable EML4-ALK trimers mediated through an active conformation of the ALK kinase domain. Our results demonstrate the formation of EML4-ALK cytoplasmic foci that orchestrate oncogenic signalling and reveal that their assembly depends upon the conformational state of the catalytic domain and can be differentially modulated by structurally divergent ALK inhibitors.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Quinasa de Linfoma Anaplásico/genética , Humanos , Neoplasias Pulmonares/genética , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Conformación Proteica , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
How cellular functions are regulated through protein phosphorylation events that promote or inhibit protein-protein interactions (PPIs) is key to understanding regulatory molecular mechanisms. Whilst phosphorylation can orthosterically or allosterically influence protein recognition, phospho-driven changes in the conformation of recognition motifs are less well explored. We recently discovered that clathrin heavy chain recognizes phosphorylated TACC3 through a helical motif that, in the unphosphorylated protein, is disordered. However, it was unclear whether and how phosphorylation could stabilize a helix in a broader context. In the current manuscript, we address this challenge using poly-Ala-based model peptides and a suite of circular dichroism and nuclear magnetic resonance spectroscopies. We show that phosphorylation of a Ser residue stabilizes the α-helix in the context of an Arg(i-3)pSeri Lys(i+4) triad through charge-reinforced side chain interactions with positive co-operativity, whilst phosphorylation of Thr induces an opposing response. This is significant as it may represent a general method for control of PPIs by phosphorylation; basic kinase-substrate motifs are common with 55 human protein kinases recognizing an Arg at a position -3 from the phosphorylated Ser, whilst the Arg(i-3)Seri Lys(i+4) is a motif found in over 2000 human proteins.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Asociadas a Microtúbulos , Dicroismo Circular , Humanos , Fosforilación , Fosfoserina , Conformación Proteica en Hélice alfaRESUMEN
Aurora-A regulates the recruitment of TACC3 to the mitotic spindle through a phospho-dependent interaction with clathrin heavy chain (CHC). Here, we describe the structural basis of these interactions, mediated by three motifs in a disordered region of TACC3. A hydrophobic docking motif binds to a previously uncharacterized pocket on Aurora-A that is blocked in most kinases. Abrogation of the docking motif causes a delay in late mitosis, consistent with the cellular distribution of Aurora-A complexes. Phosphorylation of Ser558 engages a conformational switch in a second motif from a disordered state, needed to bind the kinase active site, into a helical conformation. The helix extends into a third, adjacent motif that is recognized by a helical-repeat region of CHC, not a recognized phospho-reader domain. This potentially widespread mechanism of phospho-recognition provides greater flexibility to tune the molecular details of the interaction than canonical recognition motifs that are dominated by phosphate binding.
Asunto(s)
Aurora Quinasa A/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Huso Acromático/metabolismo , Línea Celular , Humanos , Proteínas Asociadas a Microtúbulos/genética , Conformación Proteica en Hélice alfaRESUMEN
EML4-ALK is an oncogenic fusion present in â¼5% of non-small cell lung cancers. However, alternative breakpoints in the EML4 gene lead to distinct variants of EML4-ALK with different patient outcomes. Here, we show that, in cell models, EML4-ALK variant 3 (V3), which is linked to accelerated metastatic spread, causes microtubule stabilization, formation of extended cytoplasmic protrusions and increased cell migration. EML4-ALK V3 also recruits the NEK9 and NEK7 kinases to microtubules via the N-terminal EML4 microtubule-binding region. Overexpression of wild-type EML4, as well as constitutive activation of NEK9, also perturbs cell morphology and accelerates migration in a microtubule-dependent manner that requires the downstream kinase NEK7 but does not require ALK activity. Strikingly, elevated NEK9 expression is associated with reduced progression-free survival in EML4-ALK patients. Hence, we propose that EML4-ALK V3 promotes microtubule stabilization through NEK9 and NEK7, leading to increased cell migration. This represents a novel actionable pathway that could drive metastatic disease progression in EML4-ALK lung cancer.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/genética , Humanos , Neoplasias Pulmonares/genética , Microtúbulos , Quinasas Relacionadas con NIMA/genética , Proteínas de Fusión Oncogénica/genética , Proteínas Tirosina Quinasas ReceptorasRESUMEN
Nek7 is a serine/threonine-protein kinase required for proper spindle formation and cytokinesis. Elevated Nek7 levels have been observed in several cancers, and inhibition of Nek7 might provide a route to the development of cancer therapeutics. To date, no selective and potent Nek7 inhibitors have been identified. Nek7 crystal structures exhibit an improperly formed regulatory-spine (R-spine), characteristic of an inactive kinase. We reasoned that the preference of Nek7 to crystallise in this inactive conformation might hinder attempts to capture Nek7 in complex with Type I inhibitors. Here, we have introduced aromatic residues into the R-spine of Nek7 with the aim to stabilise the active conformation of the kinase through R-spine stacking. The strong R-spine mutant Nek7SRS retained catalytic activity and was crystallised in complex with compound 51, an ATP-competitive inhibitor of Nek2 and Nek7. Subsequently, we obtained the same crystal form for wild-type Nek7WT in apo form and bound to compound 51. The R-spines of the three well-ordered Nek7WT molecules exhibit variable conformations while the R-spines of the Nek7SRS molecules all have the same, partially stacked configuration. Compound 51 bound to Nek2 and Nek7 in similar modes, but differences in the precise orientation of a substituent highlights features that could be exploited in designing inhibitors that are selective for particular Nek family members. Although the SRS mutations are not required to obtain a Nek7-inhibitor structure, we conclude that it is a useful strategy for restraining the conformation of a kinase in order to promote crystallogenesis.
Asunto(s)
Inhibidores Enzimáticos/metabolismo , Quinasas Relacionadas con NIMA/química , Quinasas Relacionadas con NIMA/metabolismo , Catálisis , Inhibidores Enzimáticos/química , Humanos , Cinética , Mutación , Quinasas Relacionadas con NIMA/genética , Unión Proteica , Conformación Proteica , Ingeniería de ProteínasRESUMEN
Zika virus is a mosquito-borne Flavivirus originally isolated from humans in 1952. Following its re-emergence in Brazil in 2015, an increase in the number of babies born with microcephaly to infected mothers was observed. Microcephaly is a neurodevelopmental disorder, characterised phenotypically by a smaller than average head size, and is usually developed in utero. The 2015 outbreak in the Americas led to the World Health Organisation declaring Zika a Public Health Emergency of International Concern. Since then, much research into the effects of Zika has been carried out. Studies have investigated the structure of the virus, its effects on and evasion of the immune response, cellular entry including target receptors, its transmission from infected mother to foetus and its cellular targets. This review discusses current knowledge and novel research into these areas, in hope of developing a further understanding of how exposure of pregnant women to the Zika virus can lead to impaired brain development of their foetus. Although no longer considered an epidemic in the Americas, the mechanism by which Zika acts is still not comprehensively and wholly understood, and this understanding will be crucial in developing effective vaccines and treatments.
Asunto(s)
Infección por el Virus Zika/fisiopatología , Infección por el Virus Zika/transmisión , Virus Zika/metabolismo , Encéfalo/embriología , Encéfalo/virología , Brasil/epidemiología , Brotes de Enfermedades , Femenino , Humanos , Lactante , Microcefalia/epidemiología , Microcefalia/virología , Embarazo , Salud Pública , Virus Zika/patogenicidad , Infección por el Virus Zika/epidemiologíaRESUMEN
Historically, chemists have explored chemical space in a highly uneven and unsystematic manner. As an example, the shape diversity of existing fragment sets does not generally reflect that of all theoretically possible fragments. To assess experimentally the added value of increased three dimensionality, a shape-diverse fragment set was designed and collated. The set was assembled by both using commercially available fragments and harnessing unified synthetic approaches to sp3 -rich molecular scaffolds. The resulting set of 80 fragments was highly three-dimensional, and its shape diversity was significantly enriched by twenty synthesised fragments. The fragment set was screened by high-throughput protein crystallography against Aurora-A kinase, revealing four hits that targeted the binding site of allosteric regulators. In the longer term, it is envisaged that the fragment set could be screened against a range of functionally diverse proteins, allowing the added value of more shape-diverse screening collections to be more fully assessed.
Asunto(s)
Aurora Quinasa A/metabolismo , Diseño de Fármacos , Inhibidores de Proteínas Quinasas/síntesis química , Regulación Alostérica , Aurora Quinasa A/antagonistas & inhibidores , Sitios de Unión , Cristalografía por Rayos X , Bases de Datos de Compuestos Químicos , Simulación del Acoplamiento Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Estructura Terciaria de ProteínaRESUMEN
Sulfation is a common modification of extracelluar glycans and tyrosine residues on proteins, which is important in many signalling pathways and interactions. Existing methods for studying sulfotransferases, the enzymes that catalyse sulfation, are cumbersome and low-throughput. Recent studies published in the Biochemical Journal have repurposed established biochemical assays from the kinase field and applied them to the characterisation of sulfotransferases. Biochemical screening of a library of kinase inhibitors revealed that compounds that target RAF kinases may also be repurposed to inhibit sulfotransferases. Together with the available structures of sulfotransferases, these studies open the door to the development of chemical tools to probe the biological functions of these important enzymes.
Asunto(s)
Sulfotransferasas/metabolismo , Animales , Carbohidratos/análisis , Humanos , Resonancia Magnética Nuclear Biomolecular/métodos , Sulfotransferasas/análisis , Tirosina/análisis , Tirosina/metabolismoRESUMEN
Myc family proteins promote cancer by inducing widespread changes in gene expression. Their rapid turnover by the ubiquitin-proteasome pathway is regulated through phosphorylation of Myc Box I and ubiquitination by the E3 ubiquitin ligase SCFFbxW7 However, N-Myc protein (the product of the MYCN oncogene) is stabilized in neuroblastoma by the protein kinase Aurora-A in a manner that is sensitive to certain Aurora-A-selective inhibitors. Here we identify a direct interaction between the catalytic domain of Aurora-A and a site flanking Myc Box I that also binds SCFFbxW7 We determined the crystal structure of the complex between Aurora-A and this region of N-Myc to 1.72-Å resolution. The structure indicates that the conformation of Aurora-A induced by compounds such as alisertib and CD532 is not compatible with the binding of N-Myc, explaining the activity of these compounds in neuroblastoma cells and providing a rational basis for the design of cancer therapeutics optimized for destabilization of the complex. We also propose a model for the stabilization mechanism in which binding to Aurora-A alters how N-Myc interacts with SCFFbxW7 to disfavor the generation of Lys48-linked polyubiquitin chains.
Asunto(s)
Aurora Quinasa A/química , Proteína Proto-Oncogénica N-Myc/química , Neoplasias/tratamiento farmacológico , Proteínas Ligasas SKP Cullina F-box/química , Aurora Quinasa A/genética , Azepinas/farmacología , Sitios de Unión , Dominio Catalítico/efectos de los fármacos , Cristalografía por Rayos X , Humanos , Proteína Proto-Oncogénica N-Myc/genética , Neoplasias/genética , Neoplasias/patología , Compuestos de Fenilurea/farmacología , Fosforilación/efectos de los fármacos , Poliubiquitina/química , Poliubiquitina/genética , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Proteínas Ligasas SKP Cullina F-box/genéticaRESUMEN
Screening a 3-aminopyridin-2-one based fragment library against a 26-kinase panel representative of the human kinome identified 3-amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-2(1H)-one (2) and 3-amino-5-(pyridin-4-yl)pyridin-2(1H)-one (3) as ligand efficient inhibitors of the mitotic kinase Monopolar Spindle 1 (MPS1) and the Aurora kinase family. These kinases are well recognised as attractive targets for therapeutic intervention for treating cancer. Elucidation of the binding mode of these fragments and their analogues has been carried out by X-ray crystallography. Structural studies have identified key interactions with a conserved lysine residue and have highlighted potential regions of MPS1 which could be targeted to improve activity and selectivity.
Asunto(s)
Aminopiridinas/química , Sistemas de Liberación de Medicamentos , Fragmentos de Péptidos/síntesis química , Inhibidores de Proteínas Quinasas , Aminopiridinas/síntesis química , Aminopiridinas/farmacología , Cristalografía por Rayos X , Concentración 50 Inhibidora , Estructura Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Biblioteca de Péptidos , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
The essential mammalian gene TACC3 is frequently mutated and amplified in cancers and its fusion products exhibit oncogenic activity in glioblastomas. TACC3 functions in mitotic spindle assembly and chromosome segregation. In particular, phosphorylation on S558 by the mitotic kinase, Aurora-A, promotes spindle recruitment of TACC3 and triggers the formation of a complex with ch-TOG-clathrin that crosslinks and stabilises kinetochore microtubules. Here we map the Aurora-A-binding interface in TACC3 and show that TACC3 potently activates Aurora-A through a domain centered on F525. Vertebrate cells carrying homozygous F525A mutation in the endogenous TACC3 loci exhibit defects in TACC3 function, namely perturbed localization, reduced phosphorylation and weakened interaction with clathrin. The most striking feature of the F525A cells however is a marked shortening of mitosis, at least in part due to rapid spindle assembly. F525A cells do not exhibit chromosome missegregation, indicating that they undergo fast yet apparently faithful mitosis. By contrast, mutating the phosphorylation site S558 to alanine in TACC3 causes aneuploidy without a significant change in mitotic duration. Our work has therefore defined a regulatory role for the Aurora-A-TACC3 interaction beyond the act of phosphorylation at S558. We propose that the regulatory relationship between Aurora-A and TACC3 enables the transition from the microtubule-polymerase activity of TACC3-ch-TOG to the microtubule-crosslinking activity of TACC3-ch-TOG-clathrin complexes as mitosis progresses. Aurora-A-dependent control of TACC3 could determine the balance between these activities, thereby influencing not only spindle length and stability but also the speed of spindle formation with vital consequences for chromosome alignment and segregation.
Asunto(s)
Aurora Quinasa A/metabolismo , Proteínas Portadoras/genética , Proteínas Fetales/genética , Proteínas Asociadas a Microtúbulos/genética , Huso Acromático/metabolismo , Factores de Transcripción/genética , Proteínas de Xenopus/genética , Aneuploidia , Animales , Sitios de Unión/genética , Línea Celular Tumoral , Pollos , Segregación Cromosómica/genética , Clatrina/metabolismo , Células HeLa , Humanos , Cinetocoros , Ratones , Microtúbulos/metabolismo , Mitosis/genética , Fosforilación/genética , Unión Proteica/genética , Estructura Terciaria de Proteína , Huso Acromático/genética , Xenopus laevisRESUMEN
Interleukin-16 (IL-16) is reported to be a chemoattractant cytokine and modulator of T-cell activation, and has been proposed as a ligand for the co-receptor CD4. The secreted active form of IL-16 has been detected at sites of TH1-mediated inflammation, such as those seen in autoimmune diseases, ischemic reperfusion injury (IRI), and tissue transplant rejection. Neutralization of IL-16 recruitment to its receptor, using an anti-IL16 antibody, has been shown to significantly attenuate inflammation and disease pathology in IRI, as well as in some autoimmune diseases. The 14.1 antibody is a monoclonal anti-IL-16 antibody, which when incubated with CD4(+) cells is reported to cause a reduction in the TH1-type inflammatory response. Secreted IL-16 contains a characteristic PDZ domain. PDZ domains are typically characterized by a defined globular structure, along with a peptide-binding site located in a groove between the αB and ßB structural elements and a highly conserved carboxylate-binding loop. In contrast to other reported PDZ domains, the solution structure previously reported for IL-16 reveals a tryptophan residue obscuring the recognition groove. We have solved the structure of the 14.1Fab fragment in complex with IL-16, revealing that binding of the antibody requires a conformational change in the IL-16 PDZ domain. This involves the rotation of the αB-helix, accompanied movement of the peptide groove obscuring tryptophan residue, and consequent opening up of the binding site for interaction. Our study reveals a surprising mechanism of action for the antibody and identifies new opportunities for the development of IL-16-targeted therapeutics, including small molecules that mimic the interaction of the antibody.
Asunto(s)
Anticuerpos Monoclonales/química , Sitios de Unión de Anticuerpos , Fragmentos Fab de Inmunoglobulinas/química , Interleucina-16/química , Cristalografía por Rayos X , Humanos , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
The Myc proteins comprise a family of ubiquitous regulators of gene expression implicated in over half of all human cancers. They interact with a large number of other proteins, such as transcription factors, chromatin-modifying enzymes and kinases. Remarkably, few of these interactions have been characterized structurally. This is at least in part due to the intrinsically disordered nature of Myc proteins, which adopt a defined conformation only in the presence of binding partners. Owing to this behaviour, crystallographic studies on Myc proteins have been limited to short fragments in complex with other proteins. Most recently, we determined the crystal structure of Aurora-A kinase domain bound to a 28-amino acid fragment of the N-Myc transactivation domain. The structure reveals an α-helical segment within N-Myc capped by two tryptophan residues that recognize the surface of Aurora-A. The kinase domain acts as a molecular scaffold, independently of its catalytic activity, upon which this region of N-Myc becomes ordered. The binding site for N-Myc on Aurora-A is disrupted by certain ATP-competitive inhibitors, such as MLN8237 (alisertib) and CD532, and explains how these kinase inhibitors are able to disrupt the protein-protein interaction to affect Myc destabilization. Structural studies on this and other Myc complexes will lead to the design of protein-protein interaction inhibitors as chemical tools to dissect the complex pathways of Myc regulation and function, which may be developed into Myc inhibitors for the treatment of cancer.
Asunto(s)
Aurora Quinasa A/antagonistas & inhibidores , Aurora Quinasa A/química , Azepinas/farmacología , Compuestos de Fenilurea/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/química , Pirimidinas/farmacología , Aurora Quinasa A/metabolismo , Azepinas/uso terapéutico , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Compuestos de Fenilurea/uso terapéutico , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/uso terapéutico , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-myc/metabolismo , Pirimidinas/uso terapéuticoRESUMEN
Serine phosphorylation is a key post-translational modification that regulates diverse biological processes. Powerful analytical methods have identified thousands of phosphorylation sites, but many of their functions remain to be deciphered. A key to understanding the function of protein phosphorylation is access to phosphorylated proteins, but this is often challenging or impossible. Here we evolve an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair that directs the efficient incorporation of phosphoserine (pSer (1)) into recombinant proteins in Escherichia coli. Moreover, combining the orthogonal pair with a metabolically engineered E. coli enables the site-specific incorporation of a nonhydrolyzable analog of pSer. Our approach enables quantitative decoding of the amber stop codon as pSer, and we purify, with yields of several milligrams per liter of culture, proteins bearing biologically relevant phosphorylations that were previously challenging or impossible to access--including phosphorylated ubiquitin and the kinase Nek7, which is synthetically activated by a genetically encoded phosphorylation in its activation loop.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Fosfoserina/metabolismo , Procesamiento Proteico-Postraduccional , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/metabolismo , Secuencia de Bases , Codón de Terminación/química , Codón de Terminación/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Código Genético , Modelos Moleculares , Datos de Secuencia Molecular , Quinasas Relacionadas con NIMA , Conformación de Ácido Nucleico , Fosforilación , Fosfoserina/química , Ingeniería de Proteínas , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitina/química , Ubiquitina/genética , Ubiquitina/metabolismoRESUMEN
Mitosis is controlled by multiple protein kinases, many of which are abnormally expressed in human cancers. Nek2, Nek6, Nek7, and Nek9 are NIMA-related kinases essential for proper mitotic progression. We determined the atomic structure of Nek7 and discovered an autoinhibited conformation that suggests a regulatory mechanism not previously described in kinases. Additionally, Nek2 adopts the same conformation when bound to a drug-like molecule. In both structures, a tyrosine side chain points into the active site, interacts with the activation loop, and blocks the alphaC helix. Tyrosine mutants of Nek7 and the related kinase Nek6 are constitutively active. The activity of Nek6 and Nek7, but not the tyrosine mutant, is increased by interaction with the Nek9 noncatalytic C-terminal domain, suggesting a mechanism in which the tyrosine is released from its autoinhibitory position. The autoinhibitory conformation is common to three Neks and provides a potential target for selective kinase inhibitors.
Asunto(s)
Ciclo Celular , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Tirosina/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Dominio Catalítico , Ciclo Celular/efectos de los fármacos , Línea Celular , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Quinasas Relacionadas con NIMA , Unión Proteica/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
A fusion between the EML4 (echinoderm microtubule-associated protein-like) and ALK (anaplastic lymphoma kinase) genes was identified in non-small cell lung cancer (NSCLC) in 2007 and there has been rapid progress in applying this knowledge to the benefit of patients. However, we have a poor understanding of EML4 and ALK biology and there are many challenges to devising the optimal strategy for treating EML4-ALK NSCLC patients. In this review, we describe the biology of EML4 and ALK, explain the main features of EML4-ALK fusion proteins and outline the therapies that target EML4-ALK. In particular, we highlight the recent advances in our understanding of the structures of EML proteins, describe the molecular mechanisms of resistance to ALK inhibitors and assess current thinking about combinations of ALK drugs with inhibitors that target other kinases or Hsp90.