RESUMEN
Infectious mammalian prions can be formed de novo from purified recombinant prion protein (PrP) substrate through a pathway that requires the sequential addition of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and RNA cofactor molecules. Recent studies show that the initial interaction between PrP and POPG causes widespread and persistent conformational changes to form an insoluble intermediate species, termed PrP(Int1). Here, we characterize the mechanism and functional consequences of the interaction between POPG and PrP. Negative-stain electron microscopy of PrP(Int1) revealed the presence of amorphous aggregates. Pull-down and photoaffinity label experiments indicate that POPG induces the formation of a PrP(C) polybasic-domain-binding neoepitope within PrP(Int1). The ongoing presence of POPG is not required to maintain PrP(Int1) structure, as indicated by the absence of stoichiometric levels of POPG in solid-state NMR measurements of PrP(Int1). Together, these results show that a transient interaction with POPG cofactor unmasks a PrP(C) binding site, leading to PrP(Int1) aggregation.
Asunto(s)
Fosfatidilgliceroles/química , Priones/química , Animales , Ratones , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/químicaRESUMEN
BACKGROUND: Invasive aspergillosis (IA) remains a leading cause of mortality in immunocompromised patients, in part due to the difficulty of diagnosing this infection. METHODS: Using thermal desorption-gas chromatography/mass spectrometry, we characterized the in vitro volatile metabolite profile of Aspergillus fumigatus, the most common cause of IA, and other pathogenic aspergilli. We prospectively collected breath samples from patients with suspected invasive fungal pneumonia from 2011 to 2013, and assessed whether we could discriminate patients with proven or probable IA from patients without aspergillosis, as determined by European Organization for Research and Treatment of Cancer/Mycoses Study Group consensus definitions, by direct detection of fungal volatile metabolites in these breath samples. RESULTS: The monoterpenes camphene, α- and ß-pinene, and limonene, and the sesquiterpene compounds α- and ß-trans-bergamotene were distinctive volatile metabolites of A. fumigatus in vitro, distinguishing it from other pathogenic aspergilli. Of 64 patients with suspected invasive fungal pneumonia based on host risk factors, clinical symptoms, and radiologic findings, 34 were diagnosed with IA, whereas 30 were ultimately diagnosed with other causes of pneumonia, including other invasive mycoses. Detection of α-trans-bergamotene, ß-trans-bergamotene, a ß-vatirenene-like sesquiterpene, or trans-geranylacetone identified IA patients with 94% sensitivity (95% confidence interval [CI], 81%-98%) and 93% specificity (95% CI, 79%-98%). CONCLUSIONS: In patients with suspected fungal pneumonia, an Aspergillus secondary metabolite signature in breath can identify individuals with IA. These results provide proof-of-concept that direct detection of exogenous fungal metabolites in breath can be used as a novel, noninvasive, pathogen-specific approach to identifying the precise microbial cause of pneumonia.
Asunto(s)
Aspergilosis/diagnóstico , Aspergilosis/metabolismo , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/patogenicidad , Adulto , Anciano , Monoterpenos Bicíclicos , Compuestos Bicíclicos con Puentes/análisis , Ciclohexenos/análisis , Femenino , Cromatografía de Gases y Espectrometría de Masas , Humanos , Limoneno , Masculino , Persona de Mediana Edad , Monoterpenos/análisis , Estudios Prospectivos , Sesquiterpenos/análisis , Terpenos/análisisRESUMEN
Fibrosis following tissue injury is distinguished from normal repair by the accumulation of pathogenic and apoptosis-resistant myofibroblasts (MFs), which arise primarily by differentiation from resident fibroblasts. Endogenous molecular brakes that promote MF dedifferentiation and clearance during spontaneous resolution of experimental lung fibrosis may provide insights that could inform and improve the treatment of progressive pulmonary fibrosis in patients. MAPK phosphatase 1 (MKP1) influences the cellular phenotype and fate through precise and timely regulation of MAPK activity within various cell types and tissues, yet its role in lung fibroblasts and pulmonary fibrosis has not been explored. Using gain- and loss-of-function studies, we found that MKP1 promoted lung MF dedifferentiation and restored the sensitivity of these cells to apoptosis - effects determined to be mainly dependent on MKP1's dephosphorylation of p38α MAPK (p38α). Fibroblast-specific deletion of MKP1 following peak bleomycin-induced lung fibrosis largely abrogated its subsequent spontaneous resolution. Such resolution was restored by treating these transgenic mice with the p38α inhibitor VX-702. We conclude that MKP1 is a critical antifibrotic brake whose inhibition of pathogenic p38α in lung fibroblasts is necessary for fibrosis resolution following lung injury.
Asunto(s)
Fosfatasa 1 de Especificidad Dual , Pulmón , Proteína Quinasa 14 Activada por Mitógenos , Miofibroblastos , Fibrosis Pulmonar , Animales , Ratones , Fosfatasa 1 de Especificidad Dual/metabolismo , Fosfatasa 1 de Especificidad Dual/genética , Miofibroblastos/patología , Miofibroblastos/metabolismo , Miofibroblastos/enzimología , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/genética , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Fibrosis Pulmonar/patología , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/enzimología , Fibrosis Pulmonar/inducido químicamente , Pulmón/patología , Pulmón/metabolismo , Bleomicina/toxicidad , Humanos , Ratones Noqueados , Ratones Transgénicos , ApoptosisRESUMEN
PGE2 and PGI2 receptors are potential targets for the treatment of chronic lung disease.
RESUMEN
There is a paucity of information about potential molecular brakes on the activation of fibroblasts that drive tissue fibrosis. The transcription factor Krüppel-like factor 4 (KLF4) is best known as a determinant of cell stemness and a tumor suppressor. We found that its expression was diminished in fibroblasts from fibrotic lung. Gain- and loss-of-function studies showed that KLF4 inhibited fibroblast proliferation, collagen synthesis, and differentiation to myofibroblasts, while restoring their sensitivity to apoptosis. Conditional deletion of KLF4 from fibroblasts potentiated the peak degree of pulmonary fibrosis and abrogated the subsequent spontaneous resolution in a model of transient fibrosis. A small molecule inducer of KLF4 was able to restore its expression in fibrotic fibroblasts and elicit resolution in an experimental model characterized by more clinically relevant persistent pulmonary fibrosis. These data identify KLF4 as a pivotal brake on fibroblast activation whose induction represents a therapeutic approach in fibrosis of the lung and perhaps other organs.
Asunto(s)
Fibrosis Pulmonar , Fibroblastos/metabolismo , Fibrosis , Humanos , Factor 4 Similar a Kruppel/metabolismo , Miofibroblastos/metabolismo , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patologíaRESUMEN
Myofibroblasts are the major cellular source of collagen, and their accumulation - via differentiation from fibroblasts and resistance to apoptosis - is a hallmark of tissue fibrosis. Clearance of myofibroblasts by dedifferentiation and restoration of apoptosis sensitivity has the potential to reverse fibrosis. Prostaglandin E2 (PGE2) and mitogens such as FGF2 have each been shown to dedifferentiate myofibroblasts, but - to our knowledge - the resultant cellular phenotypes have neither been comprehensively characterized or compared. Here, we show that PGE2 elicited dedifferentiation of human lung myofibroblasts via cAMP/PKA, while FGF2 utilized MEK/ERK. The 2 mediators yielded transitional cells with distinct transcriptomes, with FGF2 promoting but PGE2 inhibiting proliferation and survival. The gene expression pattern in fibroblasts isolated from the lungs of mice undergoing resolution of experimental fibrosis resembled that of myofibroblasts treated with PGE2 in vitro. We conclude that myofibroblast dedifferentiation can proceed via distinct programs exemplified by treatment with PGE2 and FGF2, with dedifferentiation occurring in vivo most closely resembling the former.