RESUMEN
Drugs that can treat one disease may either be detrimental or beneficial toward another due to possible cross-interactions. Therefore, care in choosing a suitable drug for patients with multiple diseases is crucial in successful patient management. This study explores several currently available ophthalmic drugs used to treat common ocular diseases to understand how they can affect the amyloidogenesis of a transforming growth factor ß-induced protein (TGFBIp) peptide fragment found in abundance in the corneal protein aggregation deposits of lattice corneal dystrophy (LCD) patients. Results from this study provided supporting evidence that some drugs intended to treat other diseases can enhance or inhibit fibrillar aggregation of TGFBIp peptide, which may have potential implication of affecting the disease progression of LCD by either worsening or ameliorating it. Comparisons of the different properties of ophthalmic compounds explored in this study may also provide some guidance for future design of drugs geared toward the treatment of LCD.
Asunto(s)
Distrofias Hereditarias de la Córnea , Proteínas de la Matriz Extracelular , Factor de Crecimiento Transformador beta , Humanos , Proteínas de la Matriz Extracelular/metabolismo , Distrofias Hereditarias de la Córnea/metabolismo , Distrofias Hereditarias de la Córnea/tratamiento farmacológico , Factor de Crecimiento Transformador beta/metabolismo , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/metabolismo , Soluciones Oftálmicas , Amiloide/metabolismoRESUMEN
Transforming growth factor ß-induced protein (TGFBIp), as an extracellular matrix protein, is expressed TGF-ß in some types of cells. Experimental sepsis is mediated by expressed and released TGFBIp in primary human umbilical vein endothelial cells (HUVECs). Cornuside (CNS) is a bisiridoid glucoside compound found in the fruit of Cornus officinalis SIEB. et ZUCC. Based on the known functions of CNS, such as the immunomodulatory and anti-inflammatory activities, we tested whether TGFBIp-mediated septic responses were suppressed by CNS in human endothelial cells and mice and investigated the underlying anti-septic mechanisms of CNS. Data showed that the secretion of TGFBIp by lipopolysaccharide (LPS) and severe septic responses by TGFBIp were effectively inhibited by CNS. And, TGFBIp-mediated sepsis lethality and pulmonary injury were reduced by CNS. Therefore, the suppression of TGFBIp-mediated septic responses by CNS suggested that CNS may be used as a potential therapeutic agent for several vascular inflammatory diseases, with the inhibition of the TGFBIp signaling pathway as the mechanism of action.
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Glucósidos , Factor de Crecimiento Transformador beta , Animales , Glucósidos/farmacología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , PiranosRESUMEN
Protein aggregation in the outermost layers of the cornea, which can lead to cloudy vision and in severe cases blindness, is linked to mutations in the extracellular matrix protein transforming growth factor-ß-induced protein (TGFBIp). Among the most frequent pathogenic mutations are R124H and R555W, both associated with granular corneal dystrophy (GCD) characterized by the early-onset formation of amorphous aggregates. The molecular mechanisms of protein aggregation in GCD are largely unknown. In this study, we determined the crystal structures of R124H, R555W, and the lattice corneal dystrophy-associated A546T. Although there were no changes in the monomeric TGFBIp structure of any mutant that would explain their propensity to aggregate, R124H and R555W demonstrated a new dimer interface in the crystal packing, which is not present in wildtype TGFBIp or A546T. This interface, as seen in both the R124H and R555W structures, involves residue 124 of the first TGFBIp molecule and 555 in the second. The interface is not permitted by the Arg124 and Arg555 residues of wildtype TGFBIp and may play a central role in the aggregation exhibited by R124H and R555W in vivo. Using cross-linking mass spectrometry and in-line size exclusion chromatography-small-angle X-ray scattering, we characterized a dimer formed by wildtype and mutant TGFBIps in solution. Dimerization in solution also involves interactions between the N- and C-terminal domains of two TGFBIp molecules but was not identical to the crystal packing dimerization. TGFBIp-targeted interventions that disrupt the R124H/R555W crystal packing dimer interface might offer new therapeutic opportunities to treat patients with GCD.
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Córnea/ultraestructura , Distrofias Hereditarias de la Córnea/genética , Proteínas de la Matriz Extracelular/genética , Agregado de Proteínas/genética , Factor de Crecimiento Transformador beta/genética , Amiloide/genética , Amiloide/ultraestructura , Córnea/metabolismo , Distrofias Hereditarias de la Córnea/patología , Cristalografía por Rayos X , Proteínas de la Matriz Extracelular/ultraestructura , Humanos , Mutación Missense/genética , Multimerización de Proteína/genéticaRESUMEN
Aloin is the major anthraquinone glycoside obtained from the Aloe species. Transforming growth factor ß-induced protein (TGFBIp) is an extracellular matrix protein and released by primary human umbilical vein endothelial cells (HUVECs) and functions as a mediator of experimental sepsis. We hypothesized that aloin could reduce TGFBIp-mediated severe inflammatory responses in HUVECs and mice. Aloin effectively inhibited lipopolysaccharide (LPS)-induced release of TGFBIp and suppressed TGFBIp-mediated septic responses. Aloin suppressed TGFBIp-induced sepsis lethality and pulmonary injury. Therefore, aloin is a potential therapeutic agent for various severe vascular inflammatory diseases, with inhibition of the TGFBIp signaling pathway as the mechanism of action. [Formula: see text].
Asunto(s)
Proteínas de la Matriz Extracelular , Factor de Crecimiento Transformador beta , Animales , Emodina/análogos & derivados , Células Endoteliales de la Vena Umbilical Humana , Ratones , Ratones Endogámicos C57BL , Estructura MolecularRESUMEN
Corneal dystrophies are a group of genetically inherited disorders with mutations in the TGFBI gene affecting the Bowman's membrane and the corneal stroma. The mutant TGFBIp is highly aggregation-prone and is deposited in the cornea. Depending on the type of mutation the protein deposits may vary (amyloid, amorphous powdery aggregate or a mixed form of both), making the cornea opaque and thereby decreases visual acuity. The aggregation of the mutant protein is found to be specific with a unique aggregation mechanism distinct to the cornea. The proteolytic processing of the mutant protein is reported to be different compared to the WT protein. The proteolytic processing of mutant protein gives rise to highly amyloidogenic peptide fragments. The current treatment option, available for patients, is tissue replacement surgery that is associated with high recurrence rates. The clinical need for a simple treatment option for corneal dystrophy patients has become highly essential either to prevent the protein aggregation or to dissolve the preformed aggregates. Here, we report the screening of 2500 compounds from the Maybridge RO3 fragment library using weak affinity chromatography (WAC). The primary hits from WAC were validated by 15N-HSQC NMR assays and specific regions of binding were identified. The recombinant mutant proteins (4th FAS-1 domain of R555W and H572R) were subjected to limited proteolysis by trypsin together with the lead compounds identified by NMR assays. The lead compounds (MO07617, RJF00203 and, BTB05094) were effective to delay/prevent the generation of amyloidogenic peptides in the R555W mutant and compounds (RJF00203 and BTB05094) were effective to delay/prevent the generation of amyloidogenic peptides in the H572R mutant. Thus the lead compounds reported here upon further validation and/or modification might be proposed as a potential treatment option to prevent/delay aggregation by inhibiting the formation of amyloidogenic peptides in TGFBI-corneal dystrophy.
RESUMEN
Granular corneal dystrophy type 2 (GCD2) is the most common form of transforming growth factor ß-induced (TGFBI) gene-linked corneal dystrophy and is pathologically characterized by the corneal deposition of mutant-TGFBIp. The defective autophagic degradation of pathogenic mutant-TGFBIp has been shown in GCD2; however, its exact mechanisms are unknown. To address this, we investigated lysosomal functions using corneal fibroblasts. Levels of cathepsins K and L (CTSK and CTSL) were significantly decreased in GCD2 cells, but of cathepsins B and D (CTSB and CTSD) did not change. The maturation of the pro-enzymes to their active forms (CTSB, CTSK and CTSL) was inhibited in GCD2 cells. CTSL enzymes directly degraded both LC3 (autophagosomes marker) and mutant-TGFBIp. Exogenous CTSL expression dramatically reduced mutant-TGFBIp in GCD2 cells, but not TGFBIp in WT cells. An increased lysosomal pH and clustered lysosomal perinuclear position were found in GCD2 cells. Transcription factor EB (TFEB) levels were significantly reduced in GCD2 cells, compared to WT. Notably, exogenous TFEB expression improved mutant-TGFBIp clearance and lysosomal abnormalities in GCD2 cells. Taken together, lysosomal dysfunction in the corneal fibroblasts underlies the pathogenesis of GCD2, and TFEB has a therapeutic potential in the treatment of GCD2.
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Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Córnea/patología , Distrofias Hereditarias de la Córnea/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Lisosomas/metabolismo , Apoptosis , Catepsinas/metabolismo , Núcleo Celular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Proteínas Mutantes/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Transforming growth factor-ß-induced protein (TGFBIp), an extracellular matrix protein, is the second most abundant protein in the corneal stroma. In this review, we summarize the current knowledge concerning the expression, molecular structure, binding partners, and functions of human TGFBIp. To date, 74 mutations in the transforming growth factor-ß-induced gene (TGFBI) are associated with amyloid and amorphous protein deposition in TGFBI-linked corneal dystrophies. We discuss the current understanding of the biochemical mechanisms of TGFBI-linked corneal dystrophies and propose that mutations leading to granular corneal dystrophy (GCD) decrease the solubility of TGFBIp and affect the interactions between TGFBIp and components of the corneal stroma, whereas mutations associated with lattice corneal dystrophy (LCD) lead to a destabilization of the protein that disrupts proteolytic turnover, especially by the serine protease HtrA1. Future research should focus on TGFBIp function in the cornea, confirmation of the biochemical mechanisms in vivo, and the development of disease models. Future therapies for TGFBI-linked corneal dystrophies might include topical agents that regulate protein aggregation or gene therapy that targets the mutant allele by CRISPR/Cas9 technology.
Asunto(s)
Distrofias Hereditarias de la Córnea/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Agregación Patológica de Proteínas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteína 9 Asociada a CRISPR , Distrofias Hereditarias de la Córnea/genética , Distrofias Hereditarias de la Córnea/terapia , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Terapia Genética , Humanos , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/genéticaRESUMEN
PURPOSE: Corneal dystrophies (CD) are classified as rare eye diseases that results in visual impairment and requires corneal transplant in advanced stages. Ocular surface inflammatory status in different types of CD remains underexplored. Hence, we studied the levels of tear soluble factors in the tears of patients with various types of corneal dystrophies. METHODS: 17 healthy subjects and 30 CD subjects (including epithelial, stromal and endothelial CD) were included in the study. Schirmer's strips were used to collect the tear fluid in all subjects. 27 soluble factors including cytokines, chemokines, soluble cell adhesion molecules and growth factors were measured in the eluted tears by multiplex ELISA or single analyte sandwich ELISA. RESULTS: Percentages of subjects with detectable levels of tear soluble factors were significantly higher in CD compared to controls. Significant higher level of IL-2 was observed in both epithelial and stromal CD. IL-4, TGFß1 and IgE were significantly higher in stromal CD. VCAM, IL-13 and Fractalkine were significantly elevated in epithelial and macular CD. IL-1α, IL-8, IL-12, ANG, Eotaxin, MCP1, RANTES, ICAM1, L-selectin and P-selectin were significantly higher in epithelial CD. TGFBIp was significantly elevated in lattice CD and endothelial CD. CONCLUSION: Distinct set of the tear soluble factors were dysregulated in various types of CD. Increase in tear inflammatory factors was observed in majority of the CD subjects depending on their sub-types. This suggests a plausible role of aberrant inflammation in CD pathobiology. Hence, modulating inflammation could be a potential strategy in improving the prognosis of CD.
Asunto(s)
Distrofias Hereditarias de la Córnea , Citocinas , Ensayo de Inmunoadsorción Enzimática , Ojo , Humanos , LágrimasRESUMEN
Transforming growth factor ß-induced protein (TGFBIp) is an extracellular matrix protein; its expression by several cell types is greatly increased by TGF-ß. TGFBIp is released by primary human umbilical vein endothelial cells (HUVECs) and functions as a mediator of experimental sepsis. 2,2'-Bipyridine-containing natural products are generally accepted to have antimicrobial, cytotoxic and anti-inflammatory properties. We hypothesized that a 2,2'-bipyridine containing natural product, collismycin C, could reduce TGFBIp-mediated severe inflammatory responses in human endothelial cells and mice. Here we investigated the effects and underlying mechanisms of collismycin C against TGFBIp-mediated septic responses. Collismycin C effectively inhibited lipopolysaccharide-induced release of TGFBIp and suppressed TGFBIp-mediated septic responses. In addition, collismycin C suppressed TGFBIp-induced sepsis lethality and pulmonary injury. This suppression of TGFBIp-mediated and CLP-induced septic responses indicates that collismycin C is a potential therapeutic agent for various severe vascular inflammatory diseases, with inhibition of the TGFBIp signaling pathway as the mechanism of action.
Asunto(s)
2,2'-Dipiridil/uso terapéutico , Antiinflamatorios/uso terapéutico , Proteínas de la Matriz Extracelular/uso terapéutico , Inflamación/tratamiento farmacológico , Sepsis/tratamiento farmacológico , Factor de Crecimiento Transformador beta/uso terapéutico , 2,2'-Dipiridil/farmacología , Animales , Proteínas de la Matriz Extracelular/farmacología , Humanos , Ratones , Ratones Endogámicos C57BL , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
The serine protease high-temperature requirement protein A1 (HtrA1) is associated with protein-misfolding disorders such as Alzheimer's disease and transforming growth factor ß-induced protein (TGFBIp)-linked corneal dystrophy. In this study, using several biochemical and biophysical approaches, including recombinant protein expression, LC-MS/MS and 2DE analyses, and thioflavin T (ThT) fluorescence assays for amyloid fibril detection, and FTIR assays, we investigated the role of HtrA1 both in normal TGFBIp turnover and in corneal amyloid formation. We show that HtrA1 can cleave WT TGFBIp but prefers amyloidogenic variants. Corneal TGFBIp is extensively processed in healthy people, resulting in C-terminal degradation products spanning the FAS1-4 domain of TGFBIp. We show here that HtrA1 cleaves the WT FAS1-4 domain only inefficiently, whereas the amyloidogenic FAS1-4 mutations transform this domain into a considerably better HTRA1 substrate. Moreover, HtrA1 cleavage of the mutant FAS1-4 domains generated peptides capable of forming in vitro amyloid aggregates. Significantly, these peptides have been previously identified in amyloid deposits in vivo, supporting the idea that HtrA1 is a causative agent for TGFBIp-associated amyloidosis in corneal dystrophy. In summary, our results indicate that TGFBIp is an HtrA1 substrate and that some mutations in the gene encoding TGFBIp cause aberrant HtrA1-mediated processing that results in amyloidogenesis in corneal dystrophies.
Asunto(s)
Amiloide/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Serina Peptidasa A1 que Requiere Temperaturas Altas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Anciano de 80 o más Años , Cromatografía Líquida de Alta Presión , Córnea/metabolismo , Enfermedades de la Córnea/metabolismo , Enfermedades de la Córnea/patología , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Serina Peptidasa A1 que Requiere Temperaturas Altas/genética , Humanos , Mutagénesis Sitio-Dirigida , Péptidos/análisis , Péptidos/metabolismo , Dominios Proteicos , Pliegue de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Espectrometría de Masas en Tándem , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/genéticaRESUMEN
SCOPE: The purpose of this study is to identify and visualize the spatial distribution of proteins present in amyloid corneal deposits of TGFBI-CD patients using Mass Spectrometry Imaging (MSI) and compare it with healthy control cornea. Corneal Dystrophies (CD) constitute a group of genetically inherited protein aggregation disorders that affects different layers of the cornea. With accumulated protein deposition, the cornea becomes opaque with decreased visual acuity. CD affecting the stroma and Bowman's membrane, is associated with mutations in transforming growth factor ß-induced (TGFBI) gene. METHODS: MALDI-Mass Spectrometry Imaging (MSI) is performed on 2 patient corneas and is compared with 1 healthy control cornea using a 7T-MALDI-FTICR. Molecular images obtained are overlaid with congo-red stained sections to visualize the proteins associated with the corneal amyloid aggregates. RESULTS: MALDI-MSI provides a relative abundance and two dimensional spatial protein signature of key proteins (TGFBIp, Apolipoprotein A-I, Apolipoprotein A-IV, Apolipoprotein E, Kaliocin-1, Pyruvate Kinase and Ras related protein Rab-10) in the patient deposits compared to the control. This is the first report of the anatomical localization of key proteins on corneal tissue section from CD patients. This may provide insight in understanding the mechanism of amyloid fibril formation in TGFBI-corneal dystrophy.
Asunto(s)
Córnea/metabolismo , Imagen Molecular , Mutación , Proteómica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factor de Crecimiento Transformador beta/genética , Córnea/diagnóstico por imagen , Distrofias Hereditarias de la Córnea/diagnóstico por imagen , Distrofias Hereditarias de la Córnea/genética , Distrofias Hereditarias de la Córnea/metabolismo , Humanos , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
We describe the histological changes in the collagen fibers of a 50-year-old male who presented keratoconus with secondary corneal amyloidosis. Corneal tissue from the patient was obtained following a penetrating keratoplasty and was subjected to histochemical analysis using Masson's trichrome staining, Congo red staining, anti-lactoferrin antibody, and anti-transforming growth factor-beta-induced protein (TGFBIp) antibody. A Congo red-positive region was detected in the anterior half of the stroma in the center and inferior cornea. Although hemotoxylin and eosin staining revealed irregularity in the Congo red-positive region, other parts of the stroma did not show any abnormalities. Positive staining both by anti-TGFBIp and anti-lactoferrin antibodies was observed in the Congo red-positive region. Interestingly, all the layers of the corneal stroma, including the peripheral region, were positively stained by anti-TFGBIp antibody, even in the Congo red-negative area. Masson's trichrome staining also showed irregular staining throughout the corneal stroma, even outside of the Congo red-positive region. Additionally, Bowman's layer, which consists of collagen type IV, was damaged. TGFBIp was strongly expressed and Masson's trichrome staining was reduced throughout the entire keratoconic stroma. The constant qualitative changes in keratoconic collagen fibers, along with the observed abnormality in the Bowman's membrane, might point to the pathogenesis of secondary corneal amyloidosis in keratoconus.
RESUMEN
Numerous mutations in the corneal protein TGFBIp lead to opaque extracellular deposits and corneal dystrophies (CDs). Here we elucidate the molecular origins underlying TGFBIp's mutation-induced increase in aggregation propensity through comprehensive biophysical and bioinformatic analyses of mutations associated with every major subtype of TGFBIp-linked CDs including lattice corneal dystrophy (LCD) and three subtypes of granular corneal dystrophy (GCD 1-3). LCD mutations at buried positions in the C-terminal Fas1-4 domain lead to decreased stability. GCD variants show biophysical profiles distinct from those of LCD mutations. GCD 1 and 3 mutations reduce solubility rather than stability. Half of the 50 positions within Fas1-4 most sensitive to mutation are associated with at least one known disease-causing mutation, including 10 of the top 11 positions. Thus, TGFBIp aggregation is driven by mutations that despite their physico-chemical diversity target either the stability or solubility of Fas1-4 in predictable ways, suggesting straightforward general therapeutic strategies.
Asunto(s)
Distrofias Hereditarias de la Córnea/genética , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Mutación , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/genética , Dicroismo Circular , Predisposición Genética a la Enfermedad , Humanos , Modelos Moleculares , Dominios Proteicos , Estabilidad Proteica , Estructura Secundaria de Proteína , SolubilidadRESUMEN
TGFBIp is a constituent of the extracellular matrix in many human tissues including the cornea, where it is one of the most abundant proteins expressed. TGFBIp interacts with Type I, II, IV, VI, and XII collagens as well as several members of the integrin family, suggesting it plays an important role in maintaining structural integrity and possibly corneal transparency as well. Significantly, more than 60 point mutations within the TGFBI gene have been reported to result in aberrant TGFBIp folding and aggregation in the cornea, resulting in severe visual impairment and blindness. Several studies have focused on targeting TGFBIp in the cornea as a therapeutic approach to treat TGFBI-linked corneal dystrophies, but the effect of this approach on corneal homeostasis and matrix integrity remained unknown. In the current study, we evaluated the histological and proteomic profiles of corneas from TGFBI-deficient mice as well as potential redundant functions of the paralogous protein POSTN. The absence of TGFBIp in mouse corneas did not grossly affect the collagen scaffold, and POSTN is unable to compensate for loss of TGFBIp. Proteomic comparison of wild-type and TGFBI-/- mice revealed 11 proteins were differentially regulated, including Type VI and XII collagens. However, as these alterations did not manifest at the macroscopic and behavioral levels, these data support partial or complete TGFBI knockdown as a potential therapy against TGFBI-linked corneal dystrophies. Lastly, in situ hybridization verified TGFBI mRNA in the epithelial cells but not in other cell types, supportive of a therapy directed specifically at this lineage.
Asunto(s)
Córnea/metabolismo , Distrofias Hereditarias de la Córnea/metabolismo , Matriz Extracelular/metabolismo , Proteómica/métodos , Factor de Crecimiento Transformador beta/deficiencia , Anciano , Anciano de 80 o más Años , Animales , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Córnea/ultraestructura , Distrofias Hereditarias de la Córnea/genética , Distrofias Hereditarias de la Córnea/terapia , Células Epiteliales/metabolismo , Femenino , Expresión Génica , Humanos , Masculino , Ratones , Ratones Noqueados , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Zingerone (ZGR), a phenolic alkanone isolated from ginger, has been reported to possess various pharmacological activities. Transforming growth factor ß-induced protein (TGFBIp) is an extracellular matrix protein whose expression in several cell types is greatly increased by TGF-ß. TGFBIp is released by human umbilical vein endothelial cells and functions as a mediator of experimental sepsis. We hypothesized that ZGR could reduce TGFBIp-mediated severe inflammatory responses in human endothelial cells and mice. Here, we investigated the anti-septic effects and underlying mechanisms of ZGR against TGFBIp-mediated septic responses. ZGR effectively inhibited lipopolysaccharide-induced release of TGFBIp and suppressed TGFBIp-mediated septic responses. In addition, ZGR suppressed TGFBIp-induced sepsis lethality and pulmonary injury. In conclusion, ZGR suppressed TGFBIp-mediated and CLP-induced septic responses. Therefore, ZGR could be a potential therapeutic agent for treatment of various severe vascular inflammatory diseases via inhibition of the TGFBIp signaling pathway.
Asunto(s)
Guayacol/análogos & derivados , Sepsis/tratamiento farmacológico , Sepsis/metabolismo , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/biosíntesis , Animales , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Guayacol/farmacología , Guayacol/uso terapéutico , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Resultado del TratamientoRESUMEN
Transforming growth factor-ß-induced protein (TGFBIp), an extracellular protein, is expressed on several cell types in response to TGF-ß stimulation. Human umbilical vein endothelial cell (HUVEC)-derived TGFBIp functions as a mediator of sepsis. Screening of bioactive compound libraries is an effective approach for repositioning FDA-approved drugs or discovering new treatments for human diseases (drug repositioning). Dabrafenib (DAB), a B-Raf inhibitor, was initially used for treating metastatic melanoma. The present study determined whether DAB modulated TGFBIp-mediated septic responses in HUVECs and in mice. Antiseptic functions of DAB were examined by measuring permeability, leukocyte adhesion and migration, and proinflammatory protein activation in TGFBIp-stimulated HUVECs and mice. In addition, beneficial effects of DAB on survival rate were examined using a mouse model of sepsis. We found that DAB inhibited TGFBIp-induced vascular barrier disruption, cell adhesion molecule (CAM) expression, and neutrophil adhesion/transendothelial migration toward human endothelial cells. DAB also suppressed TGFBIp-induced hyperpermeability and leukocyte migration in vivo. These results suggest that DAB exerts anti-inflammatory effects by inhibiting hyperpermeability, CAM expression, and leukocyte adhesion and migration, indicating its utility for treating vascular inflammatory diseases.
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Proteínas de la Matriz Extracelular/metabolismo , Imidazoles/farmacología , Imidazoles/uso terapéutico , Leucocitos/efectos de los fármacos , Oximas/farmacología , Oximas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Sepsis/tratamiento farmacológico , Factor de Crecimiento Transformador beta/metabolismo , Animales , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/farmacología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Leucocitos/citología , Leucocitos/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Permeabilidad/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Sepsis/mortalidad , Sepsis/patología , Tasa de Supervivencia , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Glycosaminoglycans (GAGs) are related to multiple biological functions and diseases. There is growing evidence that GAG concentration and sulfate content increase with age. The destabilizing mutation A546T in the corneal protein TGFBIp leads to lattice-type corneal dystrophy, but symptoms only appear in the fourth decade of life. We hypothesize that this delayed phenotype can be explained by increased GAG sulfation over time. Using in vitro assays with the C-terminal TGFIBIp domain Fas1-4, previously shown to recapitulate many properties of full-length TGFBIp, we find that only long GAGs with multiple sulfate groups on each repeating unit increase the amount of worm-like aggregates and induce long, straight fibrils in A546T. In contrast, GAGs did not induce aggregation of wildtype Fas1-4, suggesting that the finding might be specific for lattice corneal dystrophy mutants. Our results highlight a possible role of changing GAG sulfation in the accumulation of amyloid, which also may have implications for the development of neurodegenerative diseases.
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Amiloide/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Proteínas Mutantes/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Envejecimiento , Proteínas de la Matriz Extracelular/genética , Humanos , Proteínas Mutantes/genética , Mutación Puntual , Factor de Crecimiento Transformador beta/genéticaRESUMEN
TGFBI-associated corneal dystrophies are inherited disorders caused by TGFBI gene variants that promote deposition of mutant protein (TGFBIp) as insoluble aggregates in the cornea. Depending on the type and position of amino acid substitution, the aggregates may be amyloid fibrillar, amorphous globular or both, but the molecular mechanisms that drive these different patterns of aggregation are not fully understood. In the current study, we report the protein composition of amyloid corneal aggregates from lattice corneal dystrophy patients of Asian origin with H626R and R124C mutation and compared it with healthy corneal tissues via LC-MS/MS. We identified several amyloidogenic, nonfibrillar amyloid associated proteins and TGFBIp as the major components of the deposits. Our data indicates that apolipoprotein A-IV, apolipoprotein E, and serine protease HTRA1 were significantly enriched in patient deposits compared to healthy controls. HTRA1 was also found to be 7-fold enriched in the amyloid deposits of patients compared to the controls. Peptides sequences (G511DNRFSMLVAAIQSAGLTETLNR533 and Y571HIGDEILVSGGIGALVR588) derived from the fourth FAS-1 domain of TGFBIp were enriched in the corneal aggregates in a mutation-specific manner. Biophysical studies of these two enriched sequences revealed high propensity to form amyloid fibrils under physiological conditions. Our data suggests a possible proteolytic processing mechanism of mutant TGFBIp by HTRA1 and peptides generated by mutant protein may form the ß-amyloid core of corneal aggregates in dystrophic patients.
Asunto(s)
Amiloide/análisis , Serina Peptidasa A1 que Requiere Temperaturas Altas/análisis , Mutación , Agregación Patológica de Proteínas/genética , Proteómica/métodos , Factor de Crecimiento Transformador beta1/genética , Adulto , Secuencia de Aminoácidos , Amiloide/química , Amiloide/metabolismo , Apolipoproteínas A/análisis , Apolipoproteínas E/análisis , Pueblo Asiatico , Estudios de Casos y Controles , Cromatografía Liquida , Distrofias Hereditarias de la Córnea/genética , Distrofias Hereditarias de la Córnea/metabolismo , Distrofias Hereditarias de la Córnea/patología , Femenino , Humanos , Masculino , Espectrometría de Masas en TándemRESUMEN
Corneal stromal dystrophies are a group of genetic disorders that may be caused by mutations in the transforming growth factor ß-induced (TGFBI) gene which results in the aggregation and deposition of mutant proteins in various layers of the cornea. The type of amino acid substitution dictates the age of onset, anatomical location of the deposits, morphological features of deposits (amyloid, amorphous powder or a mixture of both forms) and the severity of disease presentation. It has been suggested that abnormal turnover and aberrant proteolytic processing of the mutant proteins result in the accumulation of insoluble protein deposits. Using mass spectrometry, we identified increased abundance of a 32 amino acid-long peptide in the 4th fasciclin-like domain-1 (FAS-1) domain of transforming growth factor ß-induced protein (amino acid 611-642) in the amyloid deposits of the patients with lattice corneal dystrophies (LCD). In vitro studies demonstrated that the peptide readily formed amyloid fibrils under physiological conditions. Clinically relevant substitution (M619K, N622K, N622H, G623R and H626R) of the truncated peptide resulted in profound changes in the kinetics of amyloid formation, thermal stability of the amyloid fibrils and cytotoxicity of fibrillar aggregates, depending on the position and the type of the amino acid substitution. The results suggest that reduction in the overall net charge, nature and position of cationic residue substitution determines the amyloid aggregation propensity and thermal stability of amyloid fibrils.
Asunto(s)
Córnea/metabolismo , Distrofias Hereditarias de la Córnea/metabolismo , Proteínas del Ojo/metabolismo , Fragmentos de Péptidos/metabolismo , Mutación Puntual , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia Arriba , Sustitución de Aminoácidos , Amiloide/química , Amiloide/genética , Amiloide/metabolismo , Amiloide/ultraestructura , Proteínas Amiloidogénicas/química , Proteínas Amiloidogénicas/genética , Proteínas Amiloidogénicas/metabolismo , Células Cultivadas , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Córnea/citología , Córnea/patología , Córnea/ultraestructura , Distrofias Hereditarias de la Córnea/genética , Distrofias Hereditarias de la Córnea/patología , Proteínas del Ojo/química , Proteínas del Ojo/genética , Humanos , Cinética , Microscopía Electrónica de Transmisión , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta1/química , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
Transforming growth factor ß induced protein (TGFBIp) is an extracellular matrix protein expressed in several cell types in response to TGF-ß. TGFBIp is released by human umbilical vein endothelial cells (HUVECs) and functions as a mediator of experimental sepsis. Pelargonidin (PEL) is a well-known red pigment found in plants, and has been reported as having important biological activities that are potentially beneficial for human health. This study was undertaken to investigate whether PEL can modulate TGFBIp-mediated inflammatory responses in HUVECs and in mice. The anti-inflammatory activities of PEL were determined by measuring permeability, leukocyte adhesion and migration, and activation of proinflammatory proteins in TGFBIp-activated HUVECs and mice. In addition, the beneficial effects of PEL on survival rate in a mouse sepsis model were tested. We found that PEL inhibited TGFBIp-induced barrier disruption, expression of cell adhesion molecules and adhesion/transendothelial migration of neutrophils to human endothelial cells. PEL also suppressed TGFBIp-induced hyperpermeability and leukocyte migration in vivo. These results suggest that PEL possesses anti-inflammatory properties that result in inhibition of hyperpermeability, expression of cell adhesion molecules, and adhesion and migration of leukocytes, thereby endorsing its usefulness as a therapy for vascular inflammatory diseases.