RESUMEN
As part of the efforts to understand nuclear IκB function in NF-κB-dependent gene expression, we report an X-ray crystal structure of the IκBζ ankyrin repeat domain in complex with the dimerization domain of the NF-κB p50 homodimer. IκBζ possesses an N-terminal α helix that conveys domain folding stability. Affinity and specificity of the complex depend on a small portion of p50 at the nuclear localization signal. The model suggests that only one p50 subunit supports binding with IκBζ, and biochemical experiments confirm that IκBζ associates with DNA-bound NF-κB p50:RelA heterodimers. Comparisons of IκBζ:p50 and p50:κB DNA complex crystallographic models indicate that structural rearrangement is necessary for ternary complex formation of IκBζ and p50 with DNA.
Asunto(s)
Modelos Moleculares , Subunidad p50 de NF-kappa B , Unión Proteica , Multimerización de Proteína , Humanos , Secuencia de Aminoácidos , Núcleo Celular/metabolismo , Cristalografía por Rayos X , ADN/metabolismo , ADN/química , Proteínas I-kappa B/metabolismo , Proteínas I-kappa B/química , Proteínas I-kappa B/genética , Subunidad p50 de NF-kappa B/metabolismo , Subunidad p50 de NF-kappa B/química , Subunidad p50 de NF-kappa B/genética , Factor de Transcripción ReIA/metabolismo , Factor de Transcripción ReIA/química , Factor de Transcripción ReIA/genéticaRESUMEN
Canonical NF-κB signaling through the inhibitor of κB kinase (IKK) complex requires induction of IKK2/IKKß subunit catalytic activity via specific phosphorylation within its activation loop. This process is known to be dependent upon the accessory ubiquitin (Ub)-binding subunit NF-κB essential modulator (NEMO)/IKKγ as well as poly-Ub chains. However, the mechanism through which poly-Ub binding serves to promote IKK catalytic activity is unclear. Here, we show that binding of NEMO/IKKγ to linear poly-Ub promotes a second interaction between NEMO/IKKγ and IKK2/IKKß, distinct from the well-characterized interaction of the NEMO/IKKγ N terminus to the "NEMO-binding domain" at the C terminus of IKK2/IKKß. We mapped the location of this second interaction to a stretch of roughly six amino acids immediately N-terminal to the zinc finger domain in human NEMO/IKKγ. We also showed that amino acid residues within this region of NEMO/IKKγ are necessary for binding to IKK2/IKKß through this secondary interaction in vitro and for full activation of IKK2/IKKß in cultured cells. Furthermore, we identified a docking site for this segment of NEMO/IKKγ on IKK2/IKKß within its scaffold-dimerization domain proximal to the kinase domain-Ub-like domain. Finally, we showed that a peptide derived from this region of NEMO/IKKγ is capable of interfering specifically with canonical NF-κB signaling in transfected cells. These in vitro biochemical and cell culture-based experiments suggest that, as a consequence of its association with linear poly-Ub, NEMO/IKKγ plays a direct role in priming IKK2/IKKß for phosphorylation and that this process can be inhibited to specifically disrupt canonical NF-κB signaling.
Asunto(s)
Quinasa I-kappa B , FN-kappa B , Poliubiquitina , Humanos , Quinasa I-kappa B/metabolismo , FN-kappa B/metabolismo , Poliubiquitina/metabolismo , Unión ProteicaRESUMEN
The IKK-NFκB complex is a key signaling node that facilitates activation of gene expression in response to extracellular signals. The kinase IKKß and the transcription factor RELA have been targeted by covalent modifiers that bind to surface exposed cysteine residues. A common feature in well characterized covalent modifiers of RELA and IKKß is the Michael acceptor containing α-methylene-γ-butyrolactone functionality. Through synthesis and evaluation of a focused set of α-methylene-γ-butyrolactone containing spirocyclic dimers (SpiDs) we identified SpiD3 as an anticancer agent with low nanomolar potency. Using cell-free and cell-based studies we show that SpiD3 is a covalent modifier that generates stable RELA containing high molecular weight complexes. SpiD3 inhibits TNFα-induced IκBα phosphorylation resulting in the blockade of RELA nuclear translocation. SpiD3 induces apoptosis, inhibits colony formation and migration of cancer cells. The NCI-60 cell line screen revealed that SpiD3 potently inhibits growth of leukemia cell lines, making it a suitable pre-therapeutic lead for hematological malignancies.
Asunto(s)
Antineoplásicos , Isatina , 4-Butirolactona/análogos & derivados , Antineoplásicos/farmacología , Línea Celular Tumoral , Quinasa I-kappa B/metabolismo , Isatina/farmacología , FN-kappa B/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismoRESUMEN
The NF-κB family of dimeric transcription factors regulates transcription by selectively binding to DNA response elements present within promoters or enhancers of target genes. The DNA response elements, collectively known as κB sites or κB DNA, share the consensus 5'-GGGRNNNYCC-3' (where R, Y and N are purine, pyrimidine and any nucleotide base, respectively). In addition, several DNA sequences that deviate significantly from the consensus have been shown to accommodate binding by NF-κB dimers. X-ray crystal structures of NF-κB in complex with diverse κB DNA have helped elucidate the chemical principles that underlie target selection in vitro. However, NF-κB dimers encounter additional impediments to selective DNA binding in vivo. Work carried out during the past decades has identified some of the barriers to sequence selective DNA target binding within the context of chromatin and suggests possible mechanisms by which NF-κB might overcome these obstacles. In this review, we first highlight structural features of NF-κB:DNA complexes and how distinctive features of NF-κB proteins and DNA sequences contribute to specific complex formation. We then discuss how native NF-κB dimers identify DNA binding targets in the nucleus with support from additional factors and how post-translational modifications enable NF-κB to selectively bind κB sites in vivo.
Asunto(s)
ADN/genética , Genoma Humano/genética , FN-kappa B/genética , Elementos de Respuesta/genética , Cromatina/genética , Cristalografía por Rayos X , ADN/química , Humanos , Modelos Moleculares , FN-kappa B/química , Regiones Promotoras Genéticas/genética , Factores de Transcripción/genéticaRESUMEN
The ability to precisely control protein complex formation has high utility in the expanding field of biomaterials. Driving protein-protein binding through metal-ligand bridging interactions is a promising method of achieving this goal. Furthermore, the capacity to precisely regulate both complex formation and dissociation enables additional control not available with constitutive protein complexes. Here we describe the design of three metal-controlled protein dimers that are completely monomeric in the absence of metal yet form high-affinity symmetric homodimers in the presence of zinc sulfate. The scaffold used for the designed dimers is the ß1 domain of streptococcal protein G. In addition to forming high-affinity dimers in the presence of metal, the complexes also dissociate upon addition of EDTA. Biophysical characterization revealed that the proteins maintain relatively high thermal stability, bind with high affinity, and are completely monodisperse in the monomeric and dimeric states. High-resolution crystal structures revealed that the dimers adopt the target structure and that the designed metal-binding histidine residues successfully bind zinc and function to drive dimer formation.
Asunto(s)
Proteínas Bacterianas/química , Metales/química , Dominios Proteicos , Multimerización de Proteína , Proteínas Bacterianas/metabolismo , Unión Competitiva , Dicroismo Circular , Cristalografía por Rayos X , Diseño de Fármacos , Metales/metabolismo , Modelos Moleculares , Unión Proteica , Sulfato de Zinc/química , Sulfato de Zinc/metabolismoRESUMEN
The NF-κB (Nuclear Factor kappa B) transcription factor plays crucial roles in the regulation of numerous biological processes including development of the immune system, inflammation, and innate and adaptive immune responses. Control over the immune cell functions of NF-κB results from signaling through one of two different routes: the canonical and noncanonical NF-κB signaling pathways. Present at the end of both pathways are the proteins NF-κB, IκB, and the IκB kinase (IKK). These proteins work together to deliver the myriad outcomes that influence context-dependent transcriptional control in immune cells. In the present chapter, we review the structural information available on NF-κB, IκB, and IKK, the critical terminal components of the NF-κB signaling, in relation to their physiological function.
Asunto(s)
Quinasa I-kappa B , Proteínas I-kappa B , Sistema Inmunológico , FN-kappa B , Transducción de Señal , Animales , Humanos , Quinasa I-kappa B/inmunología , Proteínas I-kappa B/inmunología , Sistema Inmunológico/enzimología , FN-kappa B/inmunología , Fosforilación , Transducción de Señal/inmunologíaRESUMEN
Transcription activator proteins typically contain two functional domains: a DNA binding domain (DBD) that binds to DNA with sequence specificity and an activation domain (AD) whose established function is to recruit RNA polymerase. In this report, we show that purified recombinant nuclear factor κB (NF-κB) RelA dimers bind specific κB DNA sites with an affinity significantly lower than that of the same dimers from nuclear extracts of activated cells, suggesting that additional nuclear cofactors might facilitate DNA binding by the RelA dimers. Additionally, recombinant RelA binds DNA with relatively low affinity at a physiological salt concentration in vitro. The addition of p53 or RPS3 (ribosomal protein S3) increases RelA:DNA binding affinity 2- to >50-fold depending on the protein and ionic conditions. These cofactor proteins do not form stable ternary complexes, suggesting that they stabilize the RelA:DNA complex through dynamic interactions. Surprisingly, the RelA-DBD alone fails to bind DNA under the same solution conditions even in the presence of cofactors, suggesting an important role of the RelA-AD in DNA binding. Reduced RelA:DNA binding at a physiological ionic strength suggests that multiple cofactors might be acting simultaneously to mitigate the electrolyte effect and stabilize the RelA:DNA complex in vivo. Overall, our observations suggest that the RelA-AD and multiple cofactor proteins function cooperatively to prime the RelA-DBD and stabilize the RelA:DNA complex in cells. Our study provides a mechanism for nuclear cofactor proteins in NF-κB-dependent gene regulation.
Asunto(s)
Coenzimas/química , Proteínas de Unión al ADN/química , FN-kappa B/química , Factor de Transcripción ReIA/química , Línea Celular , Núcleo Celular/química , Núcleo Celular/genética , Coenzimas/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Humanos , FN-kappa B/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Dominios Proteicos , Proteínas Ribosómicas/química , Factor de Transcripción ReIA/genética , Proteína p53 Supresora de Tumor/químicaRESUMEN
Cysteinyl leukotrienes (CysLTs) are a small family of biological signaling lipids produced by active leukocytes that contribute to diverse inflammatory disease states as a consequence of their engagement with dedicated G protein-coupled receptors. Immunization of mice with a CysLT-modified hapten carrier protein yielded novel monoclonal antibodies that display variable binding affinity to CysLTs. Solution binding assays indicated differing specificities among the antibodies tested, with antibody 10G4 displaying a preference for leukotriene C4 (LTC4). X-ray crystallography of a humanized 10G4 Fab fragment in complex with LTC4 revealed that binding induces a hook-like conformation within the hydrocarbon tail of the lipid arachidonic acid moiety. Specific hydrogen bonding to the LTC4 carboxylate groups further stabilized the complex, while a water molecule mediated a hydrogen bond network that connected the N-terminal arm of l-glutathione to both the arachidonyl carboxylate of LTC4 and the antibody heavy chain. Prophylactic administration of two anti-CysLT antibodies in mice followed by challenge with LTC4 demonstrated their in vivo efficacy against acute inflammation in a vascular permeability model. 10G4 ameliorated the effects of acute dextran sulfate sodium-induced colitis, suggesting that anti-CysLT antibodies could provide a therapeutic benefit in the treatment of inflammatory diseases.
Asunto(s)
Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Especificidad de Anticuerpos , Colitis/inmunología , Colitis/terapia , Cisteína/inmunología , Leucotrienos/inmunología , Enfermedad Aguda , Animales , Anticuerpos Monoclonales Humanizados/química , Vasos Sanguíneos/metabolismo , Colitis/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunización , Ratones , Modelos Moleculares , Permeabilidad , Conformación ProteicaRESUMEN
Cytochrome P450BM3 is a heme-containing enzyme from Bacillus megaterium that exhibits high monooxygenase activity and has a self-sufficient electron transfer system in the full-length enzyme. Its potential synthetic applications drive protein engineering efforts to produce variants capable of oxidizing nonnative substrates such as pharmaceuticals and aromatic pollutants. However, promiscuous P450BM3 mutants often exhibit lower stability, thereby hindering their industrial application. This study demonstrated that the heme domain R47L/F87V/L188Q/E267V/F81I pentuple mutant (PM) is destabilized because of the disruption of hydrophobic contacts and salt bridge interactions. This was directly observed from crystal structures of PM in the presence and absence of ligands (palmitic acid and metyrapone). The instability of the tertiary structure and heme environment of substrate-free PM was confirmed by pulse proteolysis and circular dichroism, respectively. Binding of the inhibitor, metyrapone, significantly stabilized PM, but the presence of the native substrate, palmitic acid, had no effect. On the basis of high-temperature molecular dynamics simulations, the lid domain, ß-sheet 1, and Cys ligand loop (a ß-bulge segment connected to the heme) are the most labile regions and, thus, potential sites for stabilizing mutations. Possible approaches to stabilization include improvement of hydrophobic packing interactions in the lid domain and introduction of new salt bridges into ß-sheet 1 and the heme region. An understanding of the molecular factors behind the loss of stability of P450BM3 variants therefore expedites site-directed mutagenesis studies aimed at developing thermostability.
Asunto(s)
Bacillus megaterium/enzimología , Proteínas Bacterianas/química , Sistema Enzimático del Citocromo P-450/química , Metirapona/metabolismo , Proteínas Mutantes/química , Mutación/genética , NADPH-Ferrihemoproteína Reductasa/química , Ácido Palmítico/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Dicroismo Circular , Cristalización , Cristalografía por Rayos X , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Transporte de Electrón , Inhibidores Enzimáticos/metabolismo , Hidroxilación , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , NADPH-Ferrihemoproteína Reductasa/genética , NADPH-Ferrihemoproteína Reductasa/metabolismo , Oxidación-Reducción , Conformación ProteicaRESUMEN
Activation of the IκB kinase (IKK) is central to NF-κB signaling. However, the precise activation mechanism by which catalytic IKK subunits gain the ability to induce NF-κB transcriptional activity is not well understood. Here we report a 4 Å x-ray crystal structure of human IKK2 (hIKK2) in its catalytically active conformation. The hIKK2 domain architecture closely resembles that of Xenopus IKK2 (xIKK2). However, whereas inactivated xIKK2 displays a closed dimeric structure, hIKK2 dimers adopt open conformations that permit higher order oligomerization within the crystal. Reversible oligomerization of hIKK2 dimers is observed in solution. Mutagenesis confirms that two of the surfaces that mediate oligomerization within the crystal are also critical for the process of hIKK2 activation in cells. We propose that IKK2 dimers transiently associate with one another through these interaction surfaces to promote trans auto-phosphorylation as part of their mechanism of activation. This structure-based model supports recently published structural data that implicate strand exchange as part of a mechanism for IKK2 activation via trans auto-phosphorylation. Moreover, oligomerization through the interfaces identified in this study and subsequent trans auto-phosphorylation account for the rapid amplification of IKK2 phosphorylation observed even in the absence of any upstream kinase.
Asunto(s)
Quinasa I-kappa B/química , Quinasa I-kappa B/metabolismo , Cromatografía en Gel , Cristalografía por Rayos X , Activación Enzimática , Células HEK293 , Humanos , Modelos Moleculares , Fosforilación , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Soluciones , Relación Estructura-Actividad , TransfecciónRESUMEN
Catalytic subunits of the IκB kinase (IKK), IKK1/IKKα, and IKK2/IKKß function in vivo as dimers in association with the necessary scaffolding subunit NEMO/IKKγ. Recent X-ray crystal structures of IKK2 suggested that dimerization might be mediated by a smaller protein-protein interaction than previously thought. Here, we report that removal of a portion of the scaffold dimerization domain (SDD) of human IKK2 yields a kinase subunit that remains monomeric in solution. Expression in baculovirus-infected Sf9 insect cells and purification of this engineered monomeric human IKK2 enzyme allows for in vitro analysis of its substrate specificity and mechanism of activation. We find that the monomeric enzyme, which contains all of the amino-terminal kinase and ubiquitin-like domains as well as the more proximal portions of the SDD, functions in vitro to direct phosphorylation exclusively to residues S32 and S36 of its IκBα substrate. Thus, the NF-κB-inducing potential of IKK2 is preserved in the engineered monomer. Furthermore, we observe that our engineered IKK2 monomer readily autophosphorylates activation loop serines 177 and 181 in trans. However, when residues that were previously observed to interfere with IKK2 trans autophosphorylation in transfected cells are mutated within the context of the monomer, the resulting Sf9 cell expressed and purified proteins were significantly impaired in their trans autophosphorylation activity in vitro. This study further defines the determinants of substrate specificity and provides additional evidence in support of a model in which activation via trans autophosphorylation of activation loop serines in IKK2 requires transient assembly of higher-order oligomers.
Asunto(s)
Quinasa I-kappa B/química , Quinasa I-kappa B/genética , Ingeniería de Proteínas/métodos , Cristalografía por Rayos X , Activación Enzimática/genética , Humanos , Quinasa I-kappa B/metabolismo , Fosforilación/genética , Unión Proteica/genética , Multimerización de Proteína/genética , Especificidad por Sustrato/genéticaRESUMEN
Rapid and high-fidelity phosphorylation of two serines (S32 and S36) of IκBα by a prototype Ser/Thr kinase IKK2 is critical for fruitful canonical NF-κB activation. Here, we report that IKK2 is a dual specificity Ser/Thr kinase that autophosphorylates itself at tyrosine residues in addition to its activation loop serines. Mutation of one such tyrosine, Y169, located in proximity to the active site, to phenylalanine, renders IKK2 inactive for phosphorylation of S32 of IκBα. Surprisingly, auto-phosphorylated IKK2 relayed phosphate group(s) to IκBα without ATP when ADP is present. We also observed that mutation of K44, an ATP-binding lysine conserved in all protein kinases, to methionine renders IKK2 inactive towards specific phosphorylation of S32 or S36 of IκBα, but not non-specific substrates. These observations highlight an unusual evolution of IKK2, in which autophosphorylation of tyrosine(s) in the activation loop and the invariant ATP-binding K44 residue define its signal-responsive substrate specificity ensuring the fidelity of NF-κB activation.
RESUMEN
The molecular motor myosin is post-translationally modified in its globular head, its S2 hinge, and its thick filament domain during human skeletal muscle aging. To determine the importance of such modifications, we performed an integrative analysis of transgenic Drosophila melanogaster expressing myosin containing post-translational modification mimic mutations. We determined effects on muscle function, myofibril structure, and myosin biochemistry. Modifications in the homozygous state decreased jump muscle function by a third at 3 weeks of age and reduced indirect flight muscle function to negligible levels in young flies, with severe effects on flight muscle myofibril assembly and/or maintenance. Expression of mimic mutations in the heterozygous state or in a wild-type background yielded significant, but less severe, age-dependent effects upon flight muscle structure and function. Modification of the residue in the globular head disabled ATPase activity and in vitro actin filament motility, whereas the S2 hinge mutation reduced actin-activated ATPase activity by 30%. The rod modification diminished filament formation in vitro. The latter mutation also reduced proteostasis, as demonstrated by enhanced accumulation of polyubiquitinated proteins. Overall, we find that mutation of amino acids at sites that are chemically modified during human skeletal muscle aging can disrupt myosin ATPase, myosin filament formation, and/or proteostasis, providing a mechanistic basis for the observed muscle defects. We conclude that age-specific post-translational modifications present in human skeletal muscle are likely to act in a dominant fashion to affect muscle structure and function and may therefore be implicated in degeneration and dysfunction associated with sarcopenia.
Asunto(s)
Envejecimiento , Drosophila melanogaster , Músculo Esquelético , Miofibrillas , Procesamiento Proteico-Postraduccional , Proteostasis , Animales , Miofibrillas/metabolismo , Proteostasis/fisiología , Drosophila melanogaster/metabolismo , Humanos , Envejecimiento/metabolismo , Músculo Esquelético/metabolismo , Miosinas del Músculo Esquelético/metabolismo , Miosinas del Músculo Esquelético/genética , Animales Modificados GenéticamenteRESUMEN
Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant unusually preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employ static and dynamic structural methods and observe that, compared to R132H, the R132Q active site adopts a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling reveals a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.
Asunto(s)
Dominio Catalítico , Isocitrato Deshidrogenasa , Mutación , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Humanos , Cinética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Resistencia a Antineoplásicos/genética , Inhibidores Enzimáticos/farmacologíaRESUMEN
Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant uniquely preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employed static and dynamic structural methods and found that, compared to R132H, the R132Q active site adopted a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling revealed a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.
RESUMEN
Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant uniquely preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employed static and dynamic structural methods and found that, compared to R132H, the R132Q active site adopted a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling revealed a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.
RESUMEN
Boron in the ocean is generally considered a nonbiological element due to its relatively high concentration (0.4 mM) and depth independent concentration profile. Here we report an unexpected role for boron in the iron transport system of the marine bacterium Marinobacter algicola. Proteome analysis under varying boron concentrations revealed that the periplasmic ferric binding protein (Mb-FbpA) was among the proteins whose expression was most affected, strongly implicating the involvement of boron in iron utilization. Here we show that boron facilitates Fe(3+) sequestration by Mb-FbpA at pH 8 (oceanic pH) by acting as a synergistic anion (B(OH)4(1-)). Fe(3+) sequestration does not occur at pH 6.5 where boric acid (B(OH)3; pK(a) = 8.55) is the predominant species. Borate anion is also shown to bind to apo-Mb-FbpA with mM affinity at pH 8, consistent with the biological relevance implied from boron's oceanic concentration (0.4 mM). Borate is among those synergistic anions tested which support the strongest Fe(3+) binding to Mb-FbpA, where the range of anion dependent affinity constants is log K'(eff) = 21-22. Since the pKa of boric acid (8.55) lies near the pH of ocean water, changes in oceanic pH, as a consequence of fluctuations in atmospheric CO2, may perturb iron uptake in many marine heterotrophic bacteria due to a decrease in oceanic borate anion concentration.
Asunto(s)
Proteínas Bacterianas/metabolismo , Boratos/metabolismo , Proteínas de Unión a Hierro/metabolismo , Marinobacter/metabolismo , Aniones/metabolismo , Boro/metabolismo , Hierro/metabolismo , Modelos MolecularesRESUMEN
Biophysical and structural studies on muscle myosin rely upon milligram quantities of extremely pure material. However, many biologically interesting myosin isoforms are expressed at levels that are too low for direct purification from primary tissues. Efforts aimed at recombinant expression of functional striated muscle myosin isoforms in bacterial or insect cell culture have largely met with failure, although high level expression in muscle cell culture has recently been achieved at significant expense. We report a novel method for the use of strains of the fruit fly Drosophila melanogaster genetically engineered to produce histidine-tagged recombinant muscle myosin isoforms. This method takes advantage of the single muscle myosin heavy chain gene within the Drosophila genome, the high level of expression of accessible myosin in the thoracic indirect flight muscles, the ability to knock out endogenous expression of myosin in this tissue and the relatively low cost of fruit fly colony production and maintenance. We illustrate this method by expressing and purifying a recombinant histidine-tagged variant of embryonic body wall skeletal muscle myosin II from an engineered fly strain. The recombinant protein shows the expected ATPase activity and is of sufficient purity and homogeneity for crystallization. This system may prove useful for the expression and isolation of mutant myosins associated with skeletal muscle diseases and cardiomyopathies for their biochemical and structural characterization.
Asunto(s)
Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Vuelo Animal , Expresión Génica , Miosinas/genética , Miosinas/aislamiento & purificación , Transgenes/genética , Animales , Cromatografía de Afinidad/métodos , Técnicas de Inactivación de Genes , Miosinas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismoRESUMEN
Though originally characterized as an inactive or transcriptionally repressive factor, the NF-κB p50 homodimer has become appreciated as a physiologically relevant driver of specific target gene expression. By virtue of its low affinity for cytoplasmic IκB protein inhibitors, p50 accumulates in the nucleus of resting cells, where it is a binding target for the transcriptional co-activator IκBζ. In this study, we employed X-ray crystallography to analyze the structure of the p50 homodimer on κB DNA from the promoters of human interleukin-6 (IL-6) and neutrophil-gelatinase-associated lipocalin (NGAL) genes, both of which respond to IκBζ. The NF-κB p50 homodimer binds 11-bp on IL-6 κB DNA, while, on NGAL κB DNA, the spacing is 12-bp. This begs the question: what DNA binding mode is preferred by NF-κB p50 homodimer? To address this, we engineered a "Test" κB-like DNA containing the core sequence 5'-GGGGAATTCCCC-3' and determined its X-ray crystal structure in complex with p50. This revealed that, when presented with multiple options, NF-κB p50 homodimer prefers to bind 11-bp, which necessarily imposes asymmetry on the complex despite the symmetry inherent in both the protein and its target DNA, and that the p50 dimerization domain can contact DNA via distinct modes.
Asunto(s)
Interleucina-6 , Subunidad p50 de NF-kappa B , FN-kappa B , Humanos , Cristalografía por Rayos X , ADN , Lipocalina 2 , Rayos X , Subunidad p50 de NF-kappa B/química , Subunidad p50 de NF-kappa B/fisiologíaRESUMEN
Disease recurrence in high-grade serous ovarian cancer may be due to cancer stem-like cells (CSC) that are resistant to chemotherapy and capable of reestablishing heterogeneous tumors. The alternative NF-κB signaling pathway is implicated in this process; however, the mechanism is unknown. Here we show that TNF-like weak inducer of apoptosis (TWEAK) and its receptor, Fn14, are strong inducers of alternative NF-κB signaling and are enriched in ovarian tumors following chemotherapy treatment. We further show that TWEAK enhances spheroid formation ability, asymmetric division capacity, and expression of SOX2 and epithelial-to-mesenchymal transition genes VIM and ZEB1 in ovarian cancer cells, phenotypes that are enhanced when TWEAK is combined with carboplatin. Moreover, TWEAK in combination with chemotherapy induces expression of the CSC marker CD117 in CD117- cells. Blocking the TWEAK-Fn14-RelB signaling cascade with a small-molecule inhibitor of Fn14 prolongs survival following carboplatin chemotherapy in a mouse model of ovarian cancer. These data provide new insights into ovarian cancer CSC biology and highlight a signaling axis that should be explored for therapeutic development. IMPLICATIONS: This study identifies a unique mechanism for the induction of ovarian cancer stem cells that may serve as a novel therapeutic target for preventing relapse.