Asunto(s)

Biosimilares Farmacéuticos/análisis , Control de Medicamentos y Narcóticos , Anticuerpos Neutralizantes/análisis , Biosimilares Farmacéuticos/efectos adversos , Control de Medicamentos y Narcóticos/métodos , Control de Medicamentos y Narcóticos/organización & administración , Humanos , Inmunidad , Inmunoensayo/métodos , Estados Unidos , United States Food and Drug Administration

RESUMEN

The endoribonuclease RNase-L is the terminal component of an interferon-regulated RNA decay pathway known as the 2'-5'-oligoadenylate (2-5A) system, whose established functions include antimicrobial and tumor suppressive activities. RNase-L activity requires binding of the small molecule 2-5A, leading to RNase-L dimerization and cleavage of single-stranded RNA. RNase-L expression is controlled post-transcriptionally by its 3'-untranslated region (3' UTR), which exerts a strong negative effect on RNase-L levels. MicroRNAs (miRNAs) are a class of small noncoding RNAs that repress expression of target genes by binding to regions of complementarity often in the 3' UTR. The miR-29 family acts as a tumor suppressor in several cancers, including acute and chronic myelogenous leukemia (CML), and has many oncogenic targets. We report that the miR-29 family represses RNase-L protein expression across several cell types. Using a luciferase reporter, we showed that miR-29 acts via 4 target sites within the RNASEL 3' UTR. Mutation of all sites is required for abrogation of miR-29 repression. In light of the reported tumor suppressive role of miR-29 in K562 CML cells and miR-29 repression of RNase-L in these cells, we generated K562 cells with stable RNase-L knockdown and demonstrated that loss of RNase-L inhibits proliferation in vitro as well as tumor growth in a xenograft model. Our findings identify a previously unknown miRNA regulator of RNase-L expression and support a novel oncogenic role for RNase-L in CML and potentially other hematopoietic malignancies.

Asunto(s)

Endorribonucleasas/genética , Regulación Leucémica de la Expresión Génica , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , MicroARNs/genética , Regiones no Traducidas 3'/genética , Animales , Sitios de Unión/genética , Western Blotting , Línea Celular Tumoral , Proliferación Celular , Endorribonucleasas/metabolismo , Femenino , Células HEK293 , Células HeLa , Humanos , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Ratones Desnudos , MicroARNs/metabolismo , Mutación , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Carga Tumoral/genética , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

MicroRNAs (miRs) play major roles in normal hematopoietic differentiation and hematopoietic malignancies. In this work, we report that miR-27a, and its coordinately expressed cluster (miR-23a∼miR-27a∼miR-24-2), was down-regulated in acute leukemia cell lines and primary samples compared to hematopoietic stem-progenitor cells (HSPCs). Decreased miR-23a cluster expression in some acute leukemia cell lines was mediated by c-MYC. Replacement of miR-27a in acute leukemia cell lines inhibited cell growth due, at least in part, to increased cellular apoptosis. We identified a member of the anti-apoptotic 14-3-3 family of proteins, which support cell survival by interacting with and negatively regulating pro-apoptotic proteins such as Bax and Bad, as a target of miR-27a. Specifically, miR-27a regulated 14-3-3θ at both the mRNA and protein levels. These data indicate that miR-27a contributes a tumor suppressor-like activity in acute leukemia cells via regulation of apoptosis, and that miR-27a and 14-3-3θ may be potential therapeutic targets.

Asunto(s)

Proteínas 14-3-3/genética , Leucemia/genética , MicroARNs/genética , Proteínas Supresoras de Tumor/genética , Proteínas 14-3-3/metabolismo , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Humanos , Leucemia/metabolismo , MicroARNs/metabolismo , Proteínas Supresoras de Tumor/metabolismo

RESUMEN

Endogenous mediators within the inflammatory milieu play a critical role in directing the scope, duration, and resolution of inflammation. High-molecular-weight extracellular matrix hyaluronan (HA) helps to maintain homeostasis. During inflammation, hyaluronan is broken down into fragments that induce chemokines and cytokines, thereby augmenting the inflammatory response. Tissue-derived adenosine, released during inflammation, inhibits inflammation via the anti-inflammatory A2 adenosine receptor (A2aR). We demonstrate that adenosine modulates HA-induced gene expression via the A2aR. A2aR stimulation inhibits HA fragment-induced pro-fibrotic genes TNF-alpha, keratinocyte chemoattractant (KC), macrophage inflammatory protein (MIP)-2, and MIP-1alpha while simultaneously synergizing with hyaluronan fragments to up-regulate the TH1 cytokine IL-12. Interestingly, A2aR stimulation mediates these affects via the novel cAMP-activated guanine nucleotide exchange factor EPAC. In addition, A2aR-null mice are more susceptible to bleomycin-induced lung injury, consistent with a role for endogenous adenosine in inhibiting the inflammation that may lead to fibrosis. Indeed, the bleomycin treated A2aR-null mice demonstrate increased lung inflammation, HA accumulation, and histologic damage. Overall, our data elucidate the opposing roles of tissue-derived HA fragments and adenosine in regulating noninfectious lung inflammation and support the pursuit of A2aR agonists as a means of pharmacologically inhibiting inflammation that may lead to fibrosis.

Asunto(s)

Adenosina/metabolismo , Ácido Hialurónico/metabolismo , Inflamación/metabolismo , Pulmón/metabolismo , Receptor de Adenosina A2A/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Bleomicina/farmacología , Línea Celular , Regulación de la Expresión Génica , Humanos , Ácido Hialurónico/genética , Interleucina-12/inmunología , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Receptor de Adenosina A2A/genética , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

BACKGROUND: The balance between reactive oxygen species (ROS) and endogenous anti-oxidants is important in maintaining healthy tissues. Excessive ROS states occur in diseases such as ARDS and Idiopathic Pulmonary Fibrosis. Redox imbalance breaks down the extracellular matrix component hyaluronan (HA) into fragments that activate innate immune responses and perpetuate tissue injury. HA fragments, via a TLR and NF-kappaB pathway, induce inflammatory gene expression in macrophages and epithelial cells. NAC and DMSO are potent anti-oxidants which may help balance excess ROS states. METHODS: We evaluated the effect of H2O2, NAC and DMSO on HA fragment induced inflammatory gene expression in alveolar macrophages and epithelial cells. RESULTS: NAC and DMSO inhibit HA fragment-induced expression of TNF-alpha and KC protein in alveolar and peritoneal macrophages. NAC and DMSO also show a dose dependent inhibition of IP-10 protein expression, but not IL-8 protein, in alveolar epithelial cells. In addition, H2O2 synergizes with HA fragments to induce inflammatory genes, which are inhibited by NAC. Mechanistically, NAC and DMSO inhibit HA induced gene expression by inhibiting NF-kappaB activation, but NAC had no influence on HA-fragment-AP-1 mediated gene expression. CONCLUSION: ROS play a central role in a pathophysiologic "vicious cycle" of inflammation: tissue injury generates ROS, which fragment the extracellular matrix HA, which in turn synergize with ROS to activate the innate immune system and further promote ROS, HA fragment generation, inflammation, tissue injury and ultimately fibrosis. The anti-oxidants NAC and DMSO, by inhibiting the HA induced inflammatory gene expression, may help re-balance excessive ROS induced inflammation.

RESUMEN

Upon tissue injury, high m.w. hyaluronan (HA), a ubiquitously distributed extracellular matrix component, is broken down into lower m.w. (LMW) fragments, which in turn activate an innate immune response. In doing so, LMW HA acts as an endogenous danger signal alerting the immune system of a breach in tissue integrity. In this report, we demonstrate that LMW HA activates the innate immune response via TLR-2 in a MyD88-, IL-1R-associated kinase-, TNFR-associated factor-6-, protein kinase Czeta-, and NF-kappaB-dependent pathway. Furthermore, we show that intact high m.w. HA can inhibit TLR-2 signaling. Finally, we demonstrate that LMW HA can act as an adjuvant promoting Ag-specific T cell responses in vivo in wild-type but not TLR-2(null) mice.

Asunto(s)

Ácido Hialurónico/fisiología , Mediadores de Inflamación/fisiología , Transducción de Señal/inmunología , Receptor Toll-Like 2/metabolismo , Proteínas Adaptadoras Transductoras de Señales/fisiología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/metabolismo , Adyuvantes Inmunológicos/fisiología , Animales , Línea Celular , Femenino , Regulación de la Expresión Génica/inmunología , Humanos , Ácido Hialurónico/administración & dosificación , Ácido Hialurónico/metabolismo , Mediadores de Inflamación/administración & dosificación , Mediadores de Inflamación/metabolismo , Isoenzimas/metabolismo , Isoenzimas/fisiología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Peso Molecular , Factor 88 de Diferenciación Mieloide , Proteína Quinasa C/metabolismo , Proteína Quinasa C/fisiología , Transducción de Señal/genética , Receptor Toll-Like 2/antagonistas & inhibidores , Receptor Toll-Like 2/fisiología

RESUMEN

Determination of protein oligomerization state can be technically challenging. We have combined the methods of expressed protein ligation (EPL) and fluorescence resonance energy transfer (FRET) for the analysis of protein homo-oligomerization states. We have attached fluorescein (donor) and rhodamine (acceptor) chromophores via dipeptide linkages to the C-termini of three recombinant proteins and examined the potential for FRET between mixtures of these semisynthetic proteins. The known protein dimer (glutathione S-transferase) showed evidence of FRET and the known protein monomer (SH2 domain phosphatase-1) did not display FRET. Using this method, the previously uncharacterized circadian rhythm enzyme, serotonin N-acetyltransferase, displayed significant FRET, indicating its likely propensity for dimerization or more complex oligomerization. These results establish the potential of the union of EPL and FRET in the analysis of protein-protein interactions and provide insight into the unusual enzymatic behavior of a key circadian rhythm enzyme.

Asunto(s)

Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Arilamina N-Acetiltransferasa/química , Arilamina N-Acetiltransferasa/genética , Arilamina N-Acetiltransferasa/metabolismo , Escherichia coli/genética , Fluoresceína/química , Expresión Génica , Glutatión Transferasa/química , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Unión Proteica , Proteína Fosfatasa 1 , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Rodaminas/química , Proteínas Tirosina Fosfatasas con Dominio SH2 , Espectrometría de Fluorescencia

RESUMEN

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase (AANAT)) is a critical enzyme in the light-mediated regulation of melatonin production and circadian rhythm. It is a member of the GNAT (GCN-5-related N-acetyltransferase) superfamily of enzymes, which catalyze a diverse array of biologically important acetyl transfer reactions from antibiotic resistance to chromatin remodeling. In this study, we probed the functional properties of two histidines (His-120 and His-122) and a tyrosine (Tyr-168) postulated to be important in the mechanism of AANAT based on prior x-ray structural and biochemical studies. Using a combination of steady-state kinetic measurements of microviscosity effects and pH dependence on the H122Q, H120Q, and H120Q/H122Q AANAT mutants, we show that His-122 (with an apparent pK(a) of 7.3) contributes approximately 6-fold to the acetyltransferase chemical step as either a remote catalytic base or hydrogen bond donor. Furthermore, His-120 and His-122 appear to contribute redundantly to this function. By analysis of the Y168F AANAT mutant, it was demonstrated that Tyr-168 contributes approximately 150-fold to the acetyltransferase chemical step and is responsible for the basic limb of the pH-rate profile with an apparent (subnormal) pK(a) of 8.5. Paradoxically, Y168F AANAT showed 10-fold enhanced apparent affinity for acetyl-CoA despite the loss of a hydrogen bond between the Tyr phenol and the CoA sulfur atom. The X-ray crystal structure of Y168F AANAT bound to a bisubstrate analog inhibitor showed no significant structural perturbation of the enzyme compared with the wild-type complex, but revealed the loss of dual inhibitor conformations present in the wild-type complex. Taken together with kinetic measurements, these crystallographic studies allow us to propose the relevant structural conformations related to the distinct alkyltransferase and acetyltransferase reactions catalyzed by AANAT. These findings have significant implications for understanding GNAT catalysis and the design of potent and selective inhibitors.

Asunto(s)

Arilamina N-Acetiltransferasa/metabolismo , Arilamina N-Acetiltransferasa/química , Arilamina N-Acetiltransferasa/genética , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli , Cinética , Modelos Químicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación Proteica , Relación Estructura-Actividad