RESUMO
Idiopathic pulmonary fibrosis (IPF) is a chronic inflammatory and fibrotic pathological condition with undefined effective therapies and a poor prognosis, partly due to the lack of specific and effective therapies. Galectin 3 (Gal-3), a pro-fibrotic ß-galactoside binding lectin, was upregulated in the early stages of the pathology, suggesting that it may be considered a marker of active fibrosis. In the present in vitro study, we use Hylach®, a lactose-modified hyaluronic acid able to bind Gal-3, to prevent the activation of lung myofibroblast and the consequent excessive ECM protein cell expression. Primary human pulmonary fibroblasts obtained from normal and IPF subjects activated with TGF-ß were used, and changes in cell viability, fibrotic components, and pro-inflammatory mediator expression at both gene and protein levels were analyzed. Hylach compounds with a lactosylation degree of about 10% and 30% (Hylach1 and Hylach 2), administrated to TGF-ß-stimulated lung fibroblast cultures, significantly downregulated α-smooth muscle actin (α-SMA) gene expression and decreased collagen type I, collagen type III, elastin, fibronectin gene and protein expression to near baseline values. This anti-fibrotic activity is accompanied by a strong anti-inflammatory effect and by a downregulation of the gene expression of Smad2 for both Hylachs in comparison to the native HA. In conclusion, the Gal-3 binding molecules Hylachs attenuated inflammation and TGF-ß-induced over-expression of α-SMA and ECM protein expression by primary human lung fibroblasts, providing a new direction for the treatment of pulmonary fibrotic diseases.
RESUMO
BACKGROUND: Activated factor IX (FIXa) is an inefficient enzyme that needs activated factor VIII (FVIII) for full activity. Recently, we identified a network of FVIII-driven changes in FIXa employing hydrogen-deuterium eXchange mass spectrometry (HDX-MS). Some changes also occurred in active-site inhibited FIXa, but others were not cofactor-driven, in particular those within the 220-loop (in chymotrypsin numbering). OBJECTIVE: The aim of this work is to better understand the zymogen-to-enzyme transition in FIX, with specific focus on substrate-driven changes at the catalytic site. METHODS: Footprinting mass spectrometry by HDX and Tandem-Mass Tags (TMT) labelling were used to explore changes occurring upon the conversion from FIX into FIXa. Mutagenesis and kinetic studies served to assess the role of the 220-loop. RESULTS: HDX-MS displayed remarkably few differences between FIX and FIXa. In comparison with FIX, FIXa did exhibit decreased deuterium uptake at the N-terminus region. This was more prominent when the FIXa active site was occupied by an irreversible inhibitor. TMT-labelling showed that the N-terminus is largely protected from labelling, and that inhibitor binding increases protection to a minor extent. Occupation of the active site also reduced deuterium uptake within the 220-loop backbone. Mutagenesis within the 220-loop revealed that a putative H-bond network contributes to FIXa activity. TMT labeling of the N-terminus suggested that these 220-loop variants are more zymogen-like than wild-type FIXa. CONCLUSION: In the absence of cofactor and substrate, FIXa is predominantly zymogen-like. Stabilization in its enzyme-like form involves, apart from FVIII-binding, also interplay between the 220-loop, N-terminus, and the substrate binding site.
Assuntos
Fator IX , Fator IXa , Fator IX/genética , Fator IX/metabolismo , Fator IXa/metabolismo , Fator VIIIa , Humanos , Cinética , Espectrometria de MassasRESUMO
Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was employed to gain insight into the changes in factor VIII (FVIII) that occur upon its activation and assembly with activated factor IX (FIXa) on phospholipid membranes. HDX-MS analysis of thrombin-activated FVIII (FVIIIa) revealed a marked increase in deuterium incorporation of amino acid residues along the A1-A2 and A2-A3 interface. Rapid dissociation of the A2 domain from FVIIIa can explain this observation. In the presence of FIXa, enhanced deuterium incorporation at the interface of FVIIIa was similar to that of FVIII. This is compatible with the previous finding that FIXa contributes to A2 domain retention in FVIIIa. A2 domain region Leu631-Tyr637, which is not part of the interface between the A domains, also showed a marked increase in deuterium incorporation in FVIIIa compared with FVIII. Deuterium uptake of this region was decreased in the presence of FIXa beyond that observed in FVIII. This implies that FIXa alters the conformation or directly interacts with this region in FVIIIa. Replacement of Val634 in FVIII by alanine using site-directed mutagenesis almost completely impaired the ability of the activated cofactor to enhance the activity of FIXa. Surface plasmon resonance analysis revealed that the rates of A2 domain dissociation from FVIIIa and FVIIIa-Val634Ala were indistinguishable. HDX-MS analysis showed, however, that FIXa was unable to retain the A2 domain in FVIIIa-Val634Ala. The combined results of this study suggest that the local structure of Leu631-Tyr637 is altered by FIXa and that this region contributes to the cofactor function of FVIII.
Assuntos
Coagulação Sanguínea/genética , Medição da Troca de Deutério/métodos , Deutério/química , Fator IXa/química , Fator VIIIa/química , Hemofilia A/genética , Fator IXa/genética , Humanos , Leucina , Espectrometria de Massas , Conformação Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Ressonância de Plasmônio de Superfície , TirosinaRESUMO
The assembly of the enzyme-activated factor IX (FIXa) with its cofactor, activated factor VIII (FVIIIa) is a crucial event in the coagulation cascade. The absence or dysfunction of either enzyme or cofactor severely compromises hemostasis and causes hemophilia. FIXa is a notoriously inefficient enzyme that needs FVIIIa to drive its hemostatic potential, by a mechanism that has remained largely elusive to date. In this study, we employed hydrogen-deuterium exchange-mass spectrometry (HDX-MS) to investigate how FIXa responds to assembly with FVIIIa in the presence of phospholipids. This revealed a complex pattern of changes that partially overlaps with those changes that occur upon occupation of the substrate-binding site by an active site-directed inhibitor. Among the changes driven by both cofactor and substrate, HDX-MS highlighted several surface loops that have been implicated in allosteric networks in related coagulation enzymes. Inspection of FVIIIa-specific changes indicated that 3 helices are involved in FIXa-FVIIIa assembly. These are part of a basic interface that is also known as exosite II. Mutagenesis of basic residues herein, followed by functional studies, identified this interface as an extended FVIIIa-interactive patch. HDX-MS was also applied to recombinant FIXa variants that are associated with severe hemophilia B. This revealed that single amino acid substitutions can silence the extended network of FVIIIa-driven allosteric changes. We conclude that HDX-MS has the potential to visualize the functional impact of disease-associated mutations on enzyme-cofactor complexes in the hemostatic system.
Assuntos
Medição da Troca de Deutério , Fator IXa/química , Fator VIII/química , Espectrometria de Massas , Mutação , Regulação Alostérica/genética , Fator IXa/genética , Fator IXa/metabolismo , Fator VIII/genética , Fator VIII/metabolismo , Hemofilia B/genética , Hemofilia B/metabolismo , Humanos , Conformação Proteica em alfa-Hélice , Domínios ProteicosRESUMO
BACKGROUND: The identity of the amino acid regions of factor VIII (FVIII) that contribute to factor IXa (FIXa) and von Willebrand factor (VWF) binding has not been fully resolved. Previously, we observed that replacing the FVIII C1 domain for the one of factor V (FV) markedly reduces VWF binding and cofactor function. Compared to the FV C1 domain, this implies that the FVIII C1 domain comprises unique surface-exposed elements involved in VWF and FIXa interaction. OBJECTIVE: The aim of this study is to identify residues in the FVIII C1 domain that contribute to VWF and FIXa binding. METHODS: Structures and primary sequences of FVIII and FV were compared to identify surface-exposed residues unique to the FVIII C1 domain. The identified residues were replaced with alanine residues to identify their role in FIXa and VWF interaction. This role was assessed employing surface plasmon resonance analysis studies and enzyme kinetic assays. RESULTS: Five surface-exposed hydrophobic residues unique to the FVIII C1 domain, ie, F2035, F2068, F2127, V2130, I2139 were identified. Functional analysis indicated that residues F2068, V2130, and especially F2127 contribute to VWF and/or FIXa interaction. Substitution into alanine of the also surface-exposed V2125, which is spatially next to F2127, affected only VWF binding. CONCLUSION: The surface-exposed hydrophobic residues in C1 domain contribute to cofactor function and VWF binding. These findings provide novel information on the fundamental role of the C1 domain in FVIII life cycle.
Assuntos
Hemostáticos , Fator de von Willebrand , Fator IXa , Fator VIII , Humanos , Domínios ProteicosRESUMO
Infection with high risk Human papillomavirus (HPV) is the main known cause of cervical cancer. HPV induces different grades of lesions: among them, Atypical squamous cells of undetermined significance are abnormal lesions that could evolve in pre-cancer lesions or spontaneously regress. The mannose binding lectin (MBL) is an innate immunity serum protein also found in cervico-vaginal mucosa, whose expression is known to be affected by polymorphisms in exon 1 and promoter of the MBL2 gene. In the present study the possible association between MBL2 functional polymorphisms and susceptibility to develop atypical squamous cells of undetermined significance was investigated in a group of women from North-East of Italy, stratified for HPV infection status. The MBL2 D and O alleles and the deficient producer combined genotypes, responsible for low MBL production, were more represented among atypical squamous cells of undetermined significance positive women than healthy controls and the results were confirmed when only HPV negative samples were considered. These results suggest a possible involvement of MBL2 functional polymorphisms in atypical squamous cells of undetermined significance susceptibility.
Assuntos
Células Escamosas Atípicas do Colo do Útero , Predisposição Genética para Doença , Lectina de Ligação a Manose/genética , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Feminino , Frequência do Gene , Humanos , Itália , Adulto JovemRESUMO
The role of the human beta-defensin 1 (hBD-1) in the susceptibility to the onset of the Atypical Squamous Cells of Undetermined Significance (ASCUS) lesion, in the presence or not of HPV infection, is still unknown. In the current study, the three functional single nucleotide polymorphisms (SNPs) -52G > A, -44C > G, and -20G > A at the 5' un-translated region (UTR) of DEFB1 gene, encoding hBD-1, were analyzed in ASCUS lesion gynecological patients and healthy women from the north-east of Italy (Trieste). Cervical samples from 249 European-Caucasian women were collected, screened for HPV and cytologically evaluated; DEFB1 genotyping has been performed by direct sequencing. No significant differences were found for -52G > A, -44C > G, and -20G > A SNPs allele and genotype frequencies between women with and without ASCUS lesions. DEFB1 minor haplotypes were significantly more frequent in ASCUS lesion positive than negative women, associating with an increased risk of this type of lesion. When women were stratified according to HPV infection status, significant differences in the distribution of -52G > A SNP genotype frequencies were found: the presence of the A allele in the homozygous genotype A/A associated with a lower risk of developing ASCUS lesions in HPV negative women. DEFB1 minor haplotypes were also associated with an increased risk of developing ASCUS lesions, being significantly more frequent in HPV negative women with lesions, than without lesions. Although these results highlight the possible involvement of DEFB1, further studies are needed to support the role of DEFB1 in the modulation of the susceptibility to ASCUS lesions.