RESUMEN
Autosomal recessive congenital ichthyosis (ARCI) is a diverse group of cornification diseases associated with severe clinical complications and decreased quality of life. Germline mutations in the TGM1 gene, which encodes the enzyme TGM1, are the predominant cause of ARCI. These TGM1 mutations trigger the abnormal epidermal differentiation and impaired cutaneous barrier function observed in patients with ARCI. Unfortunately, current ARCI therapies focus solely on symptomatic relief. Thus, there is a significant unmet need for therapeutic strategies aimed at correcting the TGM1 deficiency underlying ARCI. In this study, we investigated the ability of KB105, a gene therapy vector encoding full-length human TGM1, to deliver functional human TGM1 to keratinocytes. In vitro, KB105 efficiently infected TGM1-deficient human keratinocytes, produced TGM1 protein, and rescued transglutaminase enzyme function. In vivo studies demonstrated that both single and repeated topical KB105 administration induced TGM1 protein expression in the target epidermal layer without triggering fibrosis, necrosis, or acute inflammation. Toxicity and biodistribution assessments on repeat dosing indicated that KB105 was well-tolerated and restricted to the dose site. Overall, our results demonstrate that rescuing TGM1 deficiency in patients with ARCI through topical KB105 application represents a promising strategy for safely and noninvasively treating this debilitating disease.
Asunto(s)
Vectores Genéticos/administración & dosificación , Herpesvirus Humano 1/genética , Ictiosis Lamelar/terapia , Transglutaminasas/genética , Animales , Biopsia , Células Cultivadas , Pruebas de Enzimas , Femenino , Terapia Genética/métodos , Vectores Genéticos/genética , Mutación de Línea Germinal , Humanos , Ictiosis Lamelar/genética , Ictiosis Lamelar/patología , Queratinocitos , Masculino , Ratones , Modelos Animales , Cultivo Primario de Células , Calidad de Vida , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Piel/enzimología , Piel/patología , Transglutaminasas/metabolismoRESUMEN
CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection for bacteria and archaea against intruding genomic materials. The programmable nature of CRISPR-targeting mechanisms has enabled their adaptation as powerful genome engineering tools. Cas9, a type II CRISPR effector protein, has been widely used for gene-editing applications owing to the fact that a single-guide RNA can direct Cas9 to cleave desired genomic targets. An understanding of the role of different domains of the protein and guide RNA-induced conformational changes of Cas9 in selecting target DNA has been and continues to enable development of Cas9 variants with reduced off-targeting effects. It has been previously established that an arginine-rich bridge helix (BH) present in Cas9 is critical for its activity. In the present study, we show that two proline substitutions within a loop region of the BH of Streptococcus pyogenes Cas9 impair the DNA cleavage activity by accumulating nicked products and reducing target DNA linearization. This in turn imparts a higher selectivity in DNA targeting. We discuss the probable mechanisms by which the BH-loop contributes to target DNA recognition.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Edición Génica/métodos , Prolina/metabolismo , ARN Guía de Kinetoplastida/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteína 9 Asociada a CRISPR/química , Proteína 9 Asociada a CRISPR/genética , ADN/química , ADN/genética , ADN/metabolismo , División del ADN , Modelos Moleculares , Mutación Missense , Conformación de Ácido Nucleico , Prolina/química , Prolina/genética , Estructura Secundaria de Proteína , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/genéticaRESUMEN
CRISPR-Cas systems provide bacteria and archaea with sequence-specific protection against invading mobile genetic elements. In the presence of divalent metal ions, Cas9 and Cas12a (formerly Cpf1) proteins target and cleave DNA that is complementary to a cognate guide RNA. The recognition of a protospacer adjacent motif (PAM) sequence in the target DNA by Cas9 and Cas12a is essential for cleavage. This RNA-guided DNA targeting is widely used for gene-editing methods. Here, we show that Francisella tularensis novicida (Fno) Cas12a, FnoCas9, and Streptococcus pyogenes Cas9 (SpyCas9) cleave DNA without a guide RNA in the presence of Mn2+ ions. Substrate requirements for the RNA-independent activity vary. FnoCas9 preferentially nicks double-stranded plasmid, SpyCas9 degrades single-stranded plasmid, and FnoCas12a cleaves both substrates. These observations suggest that the identities and levels of intracellular metals, along with the Cas9/Cas12a ortholog employed, could have significant impacts in genome editing applications.
Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , División del ADN , ARN/metabolismo , Secuencia de Bases , Dominio Catalítico , ADN/química , ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Cinética , Manganeso/metabolismo , Conformación de Ácido Nucleico , Proteolisis , Especificidad por Sustrato , Factores de Tiempo , Tripsina/metabolismoRESUMEN
In a type II clustered regularly interspaced short palindromic repeats (CRISPR) system, RNAs that are encoded at the CRISPR locus complex with the CRISPR-associated (Cas) protein Cas9 to form an RNA-guided nuclease that cleaves double-stranded DNAs at specific sites. In recent years, the CRISPR-Cas9 system has been successfully adapted for genome engineering in a wide range of organisms. Studies have indicated that a series of conformational changes in Cas9, coordinated by the RNA and the target DNA, direct the protein into its active conformation, yet details on these conformational changes, as well as their roles in the mechanism of function of Cas9, remain to be elucidated. Here, nucleic acid-dependent conformational changes in Streptococcus pyogenes Cas9 (SpyCas9) were investigated using the method of site-directed spin labeling (SDSL). Single nitroxide spin labels were attached, one at a time, at one of the two native cysteine residues (Cys80 and Cys574) of SpyCas9, and the spin-labeled proteins were shown to maintain their function. X-band continuous-wave electron paramagnetic resonance spectra of the nitroxide attached at Cys80 revealed conformational changes of SpyCas9 that are consistent with a large-scale domain re-arrangement upon binding to its RNA partner. The results demonstrate the use of SDSL to monitor conformational changes in CRISPR-Cas9, which will provide key information for understanding the mechanism of CRISPR function.
Asunto(s)
Proteínas Bacterianas/química , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN/química , Endonucleasas/química , Óxidos de Nitrógeno/química , ARN Guía de Kinetoplastida/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR , Clonación Molecular , ADN/genética , ADN/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Endonucleasas/genética , Endonucleasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Marcadores de Spin , Coloración y Etiquetado/métodos , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismoRESUMEN
Changes in the phosphorylation status of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) correlate with the process of eukaryotic transcription. The yeast protein regulator of transcription 1 (Rtr1) and the human homolog RNAPII-associated protein 2 (RPAP2) may function as CTD phosphatases; however, crystal structures of Kluyveromyces lactis Rtr1 lack a consensus active site. We identified a phosphoryl transfer domain in Saccharomyces cerevisiae Rtr1 by obtaining and characterizing a 2.6 Å resolution crystal structure. We identified a putative substrate-binding pocket in a deep groove between the zinc finger domain and a pair of helices that contained a trapped sulfate ion. Because sulfate mimics the chemistry of a phosphate group, this structural data suggested that this groove represents the phosphoryl transfer active site. Mutagenesis of the residues lining this groove disrupted catalytic activity of the enzyme assayed in vitro with a fluorescent chemical substrate, and expression of the mutated Rtr1 failed to rescue growth of yeast lacking Rtr1. Characterization of the phosphatase activity of RPAP2 and a mutant of the conserved putative catalytic site in the same chemical assay indicated a conserved reaction mechanism. Our data indicated that the structure of the phosphoryl transfer domain and reaction mechanism for the phosphoryl transfer activity of Rtr1 is distinct from those of other phosphatase families.
Asunto(s)
Dominio Catalítico , Fosfoproteínas Fosfatasas/química , Dominios Proteicos , Estructura Secundaria de Proteína , Proteínas de Saccharomyces cerevisiae/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Sitios de Unión/genética , Biocatálisis/efectos de los fármacos , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Humanos , Himecromona/análogos & derivados , Himecromona/metabolismo , Cinética , Toxinas Marinas , Metales/química , Metales/metabolismo , Modelos Moleculares , Mutación , Oxazoles/farmacología , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Sulfatos/química , Sulfatos/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Post-translational modifications of the heptad repeat sequences in the C-terminal domain (CTD) of RNA polymerase II (Pol II) are well recognized for their roles in coordinating transcription with other nuclear processes that impinge upon transcription by the Pol II machinery; and this is primarily achieved through CTD interactions with the various nuclear factors. The identification of novel modifications on new regulatory sites of the CTD suggests that, instead of an independent action for all modifications on CTD, a combinatorial effect is in operation. In this review we focus on two well-characterized modifications of the CTD, namely serine phosphorylation and prolyl isomerization, and discuss the complex interplay between the enzymes modifying their respective regulatory sites. We summarize the current understanding of how the prolyl isomerization state of the CTD dictates the specificity of writers (CTD kinases), erasers (CTD phosphatases) and readers (CTD binding proteins) and how that correlates to transcription status. Subtle changes in prolyl isomerization states cannot be detected at the primary sequence level, we describe the methods that have been utilized to investigate this mode of regulation. Finally, a general model of how prolyl isomerization regulates the phosphorylation state of CTD, and therefore transcription-coupled processes, is proposed.
Asunto(s)
Procesamiento Proteico-Postraduccional/genética , ARN Polimerasa II/genética , Secuencias Repetitivas de Aminoácido/genética , Transcripción Genética , Fosforilación , Fosfotransferasas/química , Fosfotransferasas/genética , Unión Proteica , Estructura Terciaria de Proteína , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/genética , Serina/química , Serina/genéticaRESUMEN
Protein phosphatases, as the counterpart to protein kinases, are essential for homeostatic balance of cell signaling. Small chemical compounds that modulate the specific activity of phosphatases can be powerful tools to elucidate the biological functions of these enzymes. More importantly, many phosphatases are central players in the development of pathological pathways where inactivation can reverse or delay the onset of human diseases. Therefore, potent inhibitors for such phosphatases can be of great therapeutic benefit. In contrast to the seemingly identical enzymatic mechanism and structural characterization of eukaryotic protein kinases, protein phosphatases evolved from diverse ancestors, resulting in different domain architectures, reaction mechanisms and active site properties. In this review, we discuss for each family of serine/threonine protein phosphatases their involvement in biological processes and corresponding strategies for small chemical intervention. Recent advances in modern drug discovery technologies have markedly facilitated the identification of selective inhibitors for some members of the phosphatase family. Furthermore, the rapid growth in knowledge about structure-activity relationships related to possible new drug targets has aided the discovery of natural product inhibitors for the phosphatase family. This review summarizes the current state of investigation of the small molecules that regulate the function of serine/threonine phosphatases, the challenges presented and also strategies to overcome these obstacles.
Asunto(s)
Diseño de Fármacos , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/metabolismo , Animales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Relación Estructura-ActividadRESUMEN
The C-terminal domain of RNA polymerase II (CTD) modulates the process of transcription through sequential phosphorylation/dephosphorylation of its heptide repeats, through which it recruits various transcription regulators. Ssu72 is the first characterized cis-specific CTD phosphatase that dephosphorylates Ser5 with a requirement for the adjacent Pro6 in a cis conformation. The recent discovery of Thr4 phosphorylation in the CTD calls into question whether such a modification can interfere with Ssu72 binding via the elimination of a conserved intramolecular hydrogen bond in the CTD that is potentially essential for recognition. To test if Thr4 phosphorylation will abolish Ser5 dephosphorylation by Ssu72, we determined the kinetic and structural properties of Drosophila Ssu72-symplekin in complex with the CTD peptide with consecutive phosphorylated Thr4 and Ser5. Our mass spectrometric and kinetic data established that Ssu72 does not dephosphorylate Thr4, but the existence of phosphoryl-Thr4 next to Ser5 reduces the activity of Ssu72 toward the CTD peptide by 4-fold. To our surprise, even though the intramolecular hydrogen bond is eliminated due to the phosphorylation of Thr4, the CTD adopts an almost identical conformation to be recognized by Ssu72 with Ser5 phosphorylated alone or both Thr4/Ser5 phosphorylated. Our results indicate that Thr4 phosphorylation will not abolish the essential Ssu72 activity, which is needed for cell survival. Instead, the phosphatase activity of Ssu72 is fine-tuned by Thr4 phosphorylation and eventually may lead to changes in transcription. Overall, we report the first case of structural and kinetic effects of phosphorylated Thr4 on CTD modifying enzymes. Our results support a model in which a combinatorial cascade of CTD modification can modulate transcription.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Proteínas Tirosina Fosfatasas/metabolismo , ARN Polimerasa II/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Proteínas de Drosophila/química , Drosophila melanogaster/química , Drosophila melanogaster/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Fosforilación , Conformación Proteica , Proteínas Tirosina Fosfatasas/química , ARN Polimerasa II/química , Factores de Escisión y Poliadenilación de ARNm/químicaRESUMEN
The human asparaginase-like protein 1 (hASRGL1) catalyzes the hydrolysis of l-asparagine and isoaspartyl-dipeptides. As an N-terminal nucleophile (Ntn) hydrolase superfamily member, the active form of hASRGL1 is generated by an intramolecular cleavage step with Thr168 as the catalytic residue. However, in vitro, autoprocessing is incomplete (~50%), fettering the biophysical characterization of hASRGL1. We circumvented this obstacle by constructing a circularly permuted hASRGL1 that uncoupled the autoprocessing reaction, allowing us to kinetically and structurally characterize this enzyme and the precursor-like hASRGL1-Thr168Ala variant. Crystallographic and biochemical evidence suggest an activation mechanism where a torsional restraint on the Thr168 side chain helps drive the intramolecular processing reaction. Cleavage and formation of the active site releases the torsional restriction on Thr168, which is facilitated by a small conserved Gly-rich loop near the active site that allows the conformational changes necessary for activation.
Asunto(s)
Amidohidrolasas/química , Amidohidrolasas/metabolismo , Asparaginasa/química , Asparaginasa/metabolismo , Autoantígenos/química , Autoantígenos/metabolismo , Amidohidrolasas/genética , Asparaginasa/genética , Asparagina/metabolismo , Autoantígenos/genética , Dominio Catalítico , Cristalografía por Rayos X , Activación Enzimática , Humanos , Hidrólisis , Modelos Moleculares , Mutación Puntual , Conformación ProteicaRESUMEN
The cytoskeletal protein talin activates integrin receptors by binding of its FERM domain to the cytoplasmic tail of ß-integrin. Talin also couples integrins to the actin cytoskeleton, largely by binding to and activating the cytoskeletal protein vinculin, which binds to F-actin through the agency of its five-helix bundle tail (Vt) domain. Talin activates vinculin by means of buried amphipathic α-helices coined vinculin binding sites (VBSs) that reside within numerous four- and five-helix bundle domains that comprise the central talin rod, which are released from their buried locales by means of mechanical tension on the integrin:talin complex. In turn, these VBSs bind to the N-terminal seven-helix bundle (Vh1) domain of vinculin, creating an entirely new helix bundle that severs its head-tail interactions. Interestingly, talin harbors a second integrin binding site coined IBS2 that consists of two five-helix bundle domains that also contain a VBS (VBS50). Here we report the crystal structure of VBS50 in complex with vinculin at 2.3 Å resolution and show that intramolecular interactions of VBS50 within IBS2 are much more extensive versus its interactions with vinculin. Indeed, the IBS2-vinculin interaction only occurs at physiological temperature and the affinity of VBS50 for vinculin is about 30 times less than other VBSs. The data support a model where integrin binding destabilizes IBS2 to allow it to bind to vinculin.
Asunto(s)
Actinas/química , Integrinas/química , Complejos Multiproteicos/química , Talina/química , Vinculina/química , Sitios de Unión , Calorimetría , Adhesiones Focales/química , Humanos , Modelos Moleculares , Unión Proteica , Mapeo de Interacción de Proteínas , Estabilidad Proteica , Estructura Terciaria de Proteína , Receptores de Superficie Celular/química , Difracción de Rayos XRESUMEN
The merlin-1 tumor suppressor is encoded by the Neurofibromatosis-2 (Nf2) gene and loss-of-function Nf2 mutations lead to nervous system tumors in man and to several tumor types in mice. Merlin is an ERM (ezrin, radixin, moesin) family cytoskeletal protein that interacts with other ERM proteins and with components of cell-cell adherens junctions (AJs). Merlin stabilizes the links of AJs to the actin cytoskeleton. Thus, its loss destabilizes AJs, promoting cell migration and invasion, which in Nf2(+/-) mice leads to highly metastatic tumors. Paradoxically, the "closed" conformation of merlin-1, where its N-terminal four-point-one, ezrin, radixin, moesin (FERM) domain binds to its C-terminal tail domain, directs its tumor suppressor functions. Here we report the crystal structure of the human merlin-1 head domain when crystallized in the presence of its tail domain. Remarkably, unlike other ERM head-tail interactions, this structure suggests that binding of the tail provokes dimerization and dynamic movement and unfurling of the F2 motif of the FERM domain. We conclude the "closed" tumor suppressor conformer of merlin-1 is in fact an "open" dimer whose functions are disabled by Nf2 mutations that disrupt this architecture.
Asunto(s)
Neurofibromina 2/química , Secuencias de Aminoácidos , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Multimerización de Proteína , Estructura Terciaria de ProteínaRESUMEN
The cytoskeletal proteins talin and vinculin are localized at cell-matrix junctions and are key regulators of cell signaling, adhesion, and migration. Talin couples integrins via its FERM domain to F-actin and is an important regulator of integrin activation and clustering. The 220 kDa talin rod domain comprises several four- and five-helix bundles that harbor amphipathic α-helical vinculin binding sites (VBSs). In its inactive state, the hydrophobic VBS residues involved in binding to vinculin are buried within these helix bundles, and the mechanical force emanating from bound integrin receptors is thought necessary for their release and binding to vinculin. The crystal structure of a four-helix bundle of talin that harbors one of these VBSs, coined VBS33, was recently determined. Here we report the crystal structure of VBS33 in complex with vinculin at 2 Å resolution. Notably, comparison of the apo and vinculin bound structures shows that intermolecular interactions of the VBS33 α-helix with vinculin are more extensive than the intramolecular interactions of the VBS33 within the talin four-helix bundle.
Asunto(s)
Talina/química , Vinculina/química , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Talina/genética , Talina/metabolismo , Vinculina/genética , Vinculina/metabolismoRESUMEN
IL-3, a cytokine secreted by activated T lymphocytes, stimulates the proliferation, differentiation and survival of pluripotent hematopoietic stem cells. In this study, we investigated the mechanism of inhibitory action of IL-3 on osteoclast differentiation. We show here that IL-3 significantly inhibits receptor activator of NF-kappaB (RANK) ligand (RANKL)-induced activation of c-Jun N-terminal kinase (JNK). IL-3 down-regulates expression of c-Fos and nuclear factor of activated T cells (NFATc1) transcription factors. In addition, IL-3 down-regulates RANK expression posttranscriptionally in both purified osteoclast precursors and whole bone marrow cells. Furthermore, the inhibitory effect of IL-3 on RANK expression was irreversible. Interestingly, IL-3 inhibits in vivo RANK expression in mice. Thus, we provide the first evidence that IL-3 irreversibly inhibits RANK expression that results in inhibition of important signaling molecules induced by RANKL.
Asunto(s)
Diferenciación Celular , Interleucina-3/metabolismo , Osteoclastos/citología , Ligando RANK/metabolismo , Receptor Activador del Factor Nuclear kappa-B/biosíntesis , Animales , Células de la Médula Ósea/citología , Células Cultivadas , Interleucina-3/farmacología , MAP Quinasa Quinasa 4/antagonistas & inhibidores , MAP Quinasa Quinasa 4/biosíntesis , Ratones , Factores de Transcripción NFATC/metabolismo , Osteoclastos/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Receptor Activador del Factor Nuclear kappa-B/antagonistas & inhibidoresRESUMEN
Cells require distinct adhesion complexes to form contacts with their neighbors or the extracellular matrix, and vinculin links these complexes to the actin cytoskeleton. Metavinculin, an isoform of vinculin that harbors a unique 68-residue insert in its tail domain, has distinct actin bundling and oligomerization properties and plays essential roles in muscle development and homeostasis. Moreover, patients with sporadic or familial mutations in the metavinculin-specific insert invariably develop fatal cardiomyopathies. Here we report the high resolution crystal structure of the metavinculin tail domain, as well as the crystal structures of full-length human native metavinculin (1,134 residues) and of the full-length cardiomyopathy-associated DeltaLeu954 metavinculin deletion mutant. These structures reveal that an alpha-helix (H1') and extended coil of the metavinculin insert replace alpha-helix H1 and its preceding extended coil found in the N-terminal region of the vinculin tail domain to form a new five-helix bundle tail domain. Further, biochemical analyses demonstrate that this helix replacement directs the distinct actin bundling and oligomerization properties of metavinculin. Finally, the cardiomyopathy associated DeltaLeu954 and Arg975Trp metavinculin mutants reside on the replaced extended coil and the H1' alpha-helix, respectively. Thus, a helix replacement mechanism directs metavinculin's unique functions.
Asunto(s)
Vinculina/química , Vinculina/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Cardiomiopatías/genética , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
The translational machinery of the cell relocalizes to focal adhesions following the activation of integrin receptors. This response allows for rapid, local production of components needed for adhesion complex assembly and signaling. Vinculin links focal adhesions to the actin cytoskeleton following its activation by integrin signaling, which severs intramolecular interactions of vinculin's head and tail (Vt) domains. Our vinculin:raver1 crystal structures and binding studies show that activated Vt selectively interacts with one of the three RNA recognition motifs of raver1, that the vinculin:raver1 complex binds to F-actin, and that raver1 binds selectively to RNA, including a sequence found in vinculin mRNA. Further, mutation of residues that mediate interaction of raver1 with vinculin abolish their colocalization in cells. These findings suggest a feed-forward model where vinculin activation at focal adhesions provides a scaffold for recruitment of raver1 and its mRNA cargo to facilitate the production of components of adhesion complexes.
Asunto(s)
Proteínas Portadoras/metabolismo , Adhesiones Focales/fisiología , Proteínas Nucleares/metabolismo , ARN Mensajero/metabolismo , ARN/metabolismo , Vinculina/metabolismo , Actinas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Portadoras/química , Proteínas Portadoras/aislamiento & purificación , Adhesión Celular/fisiología , Secuencia Conservada , Cristalización , Citoesqueleto/metabolismo , Vectores Genéticos , Células HeLa , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/aislamiento & purificación , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN Mensajero/química , Ribonucleoproteínas , Homología de Secuencia de Aminoácido , Electricidad Estática , Transfección , Vinculina/química , Vinculina/aislamiento & purificaciónRESUMEN
IL-3, a cytokine secreted by activated T cells is well known to regulate the proliferation, differentiation, and survival of pluripotent hematopoietic stem cells. IL-3 functions as a link between the immune and the hematopoietic system. In this study, we suggest an important new role of IL-3 in inhibition of TNF-alpha-induced bone resorption in vitro and prevention of inflammatory arthritis in mice. We show here that IL-3 potently and irreversibly inhibits TNF-alpha-induced bone resorption in hematopoietic precursors of monocyte/macrophage lineage. IL-3 showed an inhibitory effect on TNF-alpha-induced bone resorption even in the presence of proinflammatory cytokines such as IL-1alpha, TGF-beta(1), TGF-beta(3), IL-6, and PGE(2). We found that IL-3 prevented TNF-alpha-induced c-fos nuclear translocation and AP-1 DNA-binding activity. Interestingly, IL-3 pretreatment prevented the development of inflammatory arthritis in mice induced by a mixture of anti-type II collagen mAbs and LPS. Furthermore, IL-3 prevented cartilage and bone loss in the joints indirectly through inhibition of inflammation. Thus, we provide the first evidence that IL-3, a strong regulator of hematopoiesis, also plays an important role in inhibition of TNF-alpha-induced bone resorption and prevention of inflammatory arthritis in mice.
Asunto(s)
Artritis Experimental/inmunología , Artritis Experimental/prevención & control , Resorción Ósea/inmunología , Resorción Ósea/prevención & control , Mediadores de Inflamación/fisiología , Interleucina-3/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Transporte Activo de Núcleo Celular/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Artritis Experimental/metabolismo , Resorción Ósea/patología , Cartílago Articular/inmunología , Cartílago Articular/metabolismo , Cartílago Articular/patología , Núcleo Celular/inmunología , Núcleo Celular/metabolismo , Células Cultivadas , Colágeno Tipo II/inmunología , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Mediadores de Inflamación/administración & dosificación , Interleucina-3/administración & dosificación , Lipopolisacáridos/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Técnicas de Cultivo de Órganos , Osteocondritis/inmunología , Osteocondritis/metabolismo , Osteocondritis/prevención & control , Unión Proteica/genética , Unión Proteica/inmunología , Proteínas Proto-Oncogénicas c-fos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-fos/metabolismo , Factor de Transcripción AP-1/antagonistas & inhibidores , Factor de Transcripción AP-1/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
Osteoclasts, the multinucleated cells that resorb bone, differentiate from hemopoietic precursors of monocyte/macrophage lineage, which also give rise to macrophages or dendritic cells. In this study we investigated the mechanism by which interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibit tumor necrosis factor (TNF)-alpha-induced osteoclast differentiation in mouse osteoclast precursors. We show here that both IL-3 and GM-CSF potently inhibits TNF-alpha-induced osteoclast differentiation by direct action on osteoclast precursors. The inhibitory effect of IL-3 and GM-CSF on osteoclast differentiation was completely neutralized by anti-IL-3 and anti-GM-CSF antibodies, respectively. In addition, the inhibitory effect of IL-3 and GM-CSF on TNF-alpha-induced osteoclast differentiation was irreversible. In osteoclast precursors, IL-3 and GM-CSF inhibited c-Fms expression post-transcriptionally. Interestingly, IL-3 and GM-CSF down-regulated both mRNA and surface expression of TNF receptor 1 (TNFR1) and TNFR2. Furthermore, cells in the presence of IL-3 and GM-CSF showed high expression of macrophage antigen CD11b, and low expression of dendritic cells antigen CD11c and prolong exposure of osteoclast precursors to GM-CSF increased the CD11c expression compare with IL-3. In summary, we provide the first evidence that IL-3 and GM-CSF block TNF-alpha-induced osteoclast differentiation by down-regulation of mRNA and surface expression of TNFR1 and TNFR2.
Asunto(s)
Regulación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Interleucina-3/farmacología , Osteoclastos/efectos de los fármacos , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Diferenciación Celular/efectos de los fármacos , Regulación hacia Abajo , Genes fms , Ratones , Ratones Endogámicos BALB C , Osteoclastos/citología , Células Madre/efectos de los fármacosRESUMEN
Osteoclasts, the multinucleated cells that resorb bone, differentiate from hemopoietic precursors of the monocyte/macrophage lineage in the presence of M-CSF and receptor activator of NF-kappaB ligand (RANKL). In this study we investigated the role of IL-3 in osteoclast differentiation. We show here that IL-3, a cytokine secreted by activated T lymphocytes, inhibits RANKL-induced osteoclast differentiation by a direct action on early osteoclast precursors. Anti-IL-3 Ab neutralized the inhibitory effect of IL-3 on osteoclast differentiation. In addition, IL-3 inhibits TNF-alpha-induced osteoclast differentiation in bone marrow-derived macrophages. However, IL-3 has no inhibitory effect on mature osteoclasts. In osteoclast precursors, IL-3 prevents RANKL-induced nuclear translocation of NF-kappaB by inhibiting the phosphorylation and degradation of IkappaB. RT-PCR analysis revealed that IL-3 down-regulated c-Fos transcription. Interestingly, the osteoclast precursors in the presence of IL-3 showed strong expression of macrophage markers such as Mac-1, MOMA-2, and F4/80. Furthermore, the inhibitory effect of IL-3 on osteoclast differentiation was irreversible, and the osteoclast precursors preincubated in IL-3 were resistant to RANKL action. Thus, our results reveal for the first time that IL-3 acts directly on early osteoclast precursors and irreversibly blocks RANKL-induced osteoclast differentiation by diverting the cells to macrophage lineage.