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1.
J Proteome Res ; 23(3): 985-998, 2024 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-38306169

RESUMEN

This study aims to elucidate the cellular mechanisms behind the secretion of complement factor B (CFB), known for its dual roles as an early biomarker for pancreatic ductal adenocarcinoma (PDAC) and as the initial substrate for the alternative complement pathway (ACP). Using parallel reaction monitoring analysis, we confirmed a consistent ∼2-fold increase in CFB expression in PDAC patients compared with that in both healthy donors (HD) and chronic pancreatitis (CP) patients. Elevated ACP activity was observed in CP and other benign conditions compared with that in HD and PDAC patients, suggesting a functional link between ACP and PDAC. Protein-protein interaction analyses involving key complement proteins and their regulatory factors were conducted using blood samples from PDAC patients and cultured cell lines. Our findings revealed a complex control system governing the ACP and its regulatory factors, including Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation, adrenomedullin (AM), and complement factor H (CFH). Particularly, AM emerged as a crucial player in CFB secretion, activating CFH and promoting its predominant binding to C3b over CFB. Mechanistically, our data suggest that the KRAS mutation stimulates AM expression, enhancing CFH activity in the fluid phase through binding. This heightened AM-CFH interaction conferred greater affinity for C3b over CFB, potentially suppressing the ACP cascade. This sequence of events likely culminated in the preferential release of ductal CFB into plasma during the early stages of PDAC. (Data set ID PXD047043.).


Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Factor B del Complemento/genética , Factor B del Complemento/metabolismo , Vía Alternativa del Complemento , Proteínas Proto-Oncogénicas p21(ras) , Detección Precoz del Cáncer , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/genética
2.
Pest Manag Sci ; 80(4): 1991-2000, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38092527

RESUMEN

BACKGROUND: Mosquitoes are vectors of various diseases, posing significant health threats worldwide. Chemical pesticides, particularly pyrethroids like deltamethrin, are commonly used for mosquito control, but the emergence of resistant mosquito populations has become a concern. In the deltamethrin-resistant (DR) strain of Culex pipiens pallens, the highly expressed cytochrome P450 9 J34 (CYP9J34) gene is believed to play a role in resistance, yet the underlying mechanism remains unclear. RESULTS: Quantitative polymerase chain reaction with reverse transcription (qRT-PCR) analysis revealed that the expression of CYP9J34 was 14.6-fold higher in DR strains than in deltamethrin-susceptible (DS) strains. The recombinant production of CYP9J34 protein of Cx. pipiens pallens showed that the protein could directly metabolize deltamethrin, yielding the major metabolite 4'-OH deltamethrin. Through dual luciferase reporter assays and RNA interference, the transcription factor homeobox protein B-H2-like (B-H2) was identified to modulate the expression of the CYP9J34 gene, contributing to mosquito resistance to deltamethrin. CONCLUSIONS: Our findings demonstrate that the CYP9J34 protein could directly degrade deltamethrin, and the transcription factor B-H2 could regulate CYP9J34 expression, influencing the resistance of mosquitoes to deltamethrin. © 2023 Society of Chemical Industry.


Asunto(s)
Culex , Insecticidas , Piretrinas , Animales , Insecticidas/farmacología , Insecticidas/metabolismo , Factor B del Complemento/metabolismo , Resistencia a los Insecticidas/genética , Piretrinas/farmacología , Piretrinas/metabolismo , Nitrilos/farmacología , Nitrilos/metabolismo , Culex/genética , Culex/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo
3.
J Biomol Struct Dyn ; 42(2): 948-959, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-37139795

RESUMEN

The majority of the world population (around 25%) has latent Mycobacterium tuberculosis (Mtb) infection, among which only 5-10% of individuals develop active tuberculosis (TB), and 90-95% continue to have latent tuberculosis infection. This makes it the biggest global health concern. It has been reported that the resuscitation-promoting factor B (RpfB) is an exciting potential target for tuberculosis drug discovery due to its significant role in the reactivation of latent TB infection to an active infection. Several attempts have been made to investigate potential inhibitors against RpfB utilizing in-silico approaches. The present study also utilized a computational approach to investigate microbially derived natural compounds against the Mtb RpfB protein which is a very cost-effective This evaluation used structure-based virtual screening (SBVS), drug-likeness profiling, molecular docking, molecular dynamics simulation, and free-binding energy calculations. Six potential natural compounds, viz. Cyclizidine I, Boremexin C, Xenocoumacin 2, PM-94128, Cutinostatin B, and (+)1-O-demethylvariecolorquinone A were selected, which displayed a potential binding affinity between -52.39 and -60.87 Kcal/mol MMGBSA score and docking energy between -7.307 Kcal/mol to -6.972 Kcal/mol. All the complexes showed acceptable stability (<2.7 Å RMSD) during 100 ns MD simulation time except the RpfB protein-xenocoumacin 2 complex. This result exhibited that the selected compounds have high efficiency in inhibiting the Mtb RpfB and can be taken into account for additional in vitro and in vivo experimental validation.Communicated by Ramaswamy H. Sarma.


Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Humanos , Mycobacterium tuberculosis/metabolismo , Factor B del Complemento/metabolismo , Simulación del Acoplamiento Molecular , Proteínas Bacterianas/química , Simulación de Dinámica Molecular
4.
Mol Immunol ; 161: 25-32, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37481826

RESUMEN

Uncontrolled activation of the alternative pathway (AP) of complement, due to genetic and/or acquired defects, plays a primary pathogenetic role in C3 glomerulopathy (C3G), a rare and heterogeneous disease characterised by predominant C3 fragment deposition within the glomerulus, as well as glomerular damage. There are currently no approved disease-specific treatments for C3G, but new drugs that directly counteract AP dysregulation, targeting components of the pathway, have opened promising new perspectives for managing the disease. Complement factor B (FB), which is primarily synthesised by hepatocytes, is a key component of the AP, as it drives the central amplification loop of the complement system. In this study we used a GalNAc (N-Acetylgalactosamine)-conjugated siRNA to selectively target and suppress liver FB expression in two mouse models characterised by the complete (Cfh-/- mice) or partial (Cfh+/-) loss of function of complement factor H (FH). Homozygous deletion of FH induced a severe C3G phenotype, with strong dysregulation of the AP of complement, glomerular C3 deposition and almost complete C3 consumption. Mice with a heterozygous deletion of FH had intermediate C3 levels and exhibited slower disease progression, resembling human C3G more closely. Here we showed that FB siRNA treatment did not improve serum C3 levels, nor limit glomerular C3 deposition in Cfh-/- mice, while it did normalise circulating C3 levels, reduce glomerular C3 deposits, and limit mesangial electron-dense deposits in Cfh+/- mice. The present data provide important insights into the potential benefits and limitations of FB-targeted inhibition strategies and suggest RNA interference-mediated FB silencing in the liver as a possible therapeutic approach for treating C3G patients with FH haploinsufficiency.


Asunto(s)
Glomerulonefritis Membranoproliferativa , Enfermedades Renales , Humanos , Animales , Ratones , Factor B del Complemento/genética , Factor B del Complemento/metabolismo , Complemento C3 , Homocigoto , Eliminación de Secuencia , Factor H de Complemento/genética , Hígado/metabolismo , Vía Alternativa del Complemento/genética , Glomerulonefritis Membranoproliferativa/genética , Glomerulonefritis Membranoproliferativa/terapia , Glomerulonefritis Membranoproliferativa/metabolismo
5.
Neurosci Lett ; 810: 137318, 2023 07 27.
Artículo en Inglés | MEDLINE | ID: mdl-37271220

RESUMEN

BACKGROUND: The complement system plays a crucial role in cognitive impairment. The aim of this study is to investigate the correlation between the complement proteins levels in serum astrocyte-derived exosomes (ADEs) and mild cognitive impairment (MCI) in type 1 diabetes mellitus (T1DM) patients. METHODS: In this cross-sectional study, the patients with immune-mediated T1DM were enrolled. Healthy subjects matched for age and sex with T1DM patients were selected as controls. The cognitive function was evaluated by a Beijing version of the Montreal Cognitive Assessment (MoCA) questionnaire. The complement proteins including C5b-9, C3b and Factor B in serum ADEs were measured by ELISA kits. RESULTS: This study recruited 55 subjects immune-mediated T1DM patients without dementia, including 31 T1DM patients with MCI, 24 T1DM patients without MCI. 33 healthy subjects were enrolled as controls. The results showed higher complement proteins including C5b-9, C3b and Factor B levels in ADEs from T1DM patients with MCI than those in the controls (P < 0.001, P < 0.001, P = 0.006) and T1DM patients without MCI (P = 0.02, P = 0.02, P = 0.03). The C5b-9 levels in ADEs were independently associated with MCI in T1DM patients(OR: 1.20, 95% CI: 1.00-1.44, P = 0.04). The C5b-9 levels in ADEs were significantly correlated with global cognitive scores (ß = -0.360, P<0.001) and visuo-executive (ß = -0.132, P<0.001), language(ß = -0.036, P = 0.026) and delayed recall score (ß = -0.090,P = 0.007). There was no correlation between the C5b-9 levels in ADEs and the fasting glucose, HbA1c, fasting c-peptide and GAD65 antibody in T1DM patients. Furthermore, the C5b-9, C3b and Factor B levels in ADEs exhibited a fair combined diagnostic value for MCI, with an area under the curve of 0.76 (95% CI: 0.63-0.88, P = 0.001). CONCLUSION: The elevated C5b-9 levels in ADEswere significantly associated with theMCI in T1DM patients. The C5b-9 in ADEs may be used as a marker of MCI in T1DM patients.


Asunto(s)
Disfunción Cognitiva , Diabetes Mellitus Tipo 1 , Exosomas , Humanos , Diabetes Mellitus Tipo 1/complicaciones , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Factor B del Complemento/metabolismo , Astrocitos/metabolismo , Exosomas/metabolismo , Estudios Transversales , Disfunción Cognitiva/diagnóstico
6.
Sci Rep ; 13(1): 5764, 2023 04 08.
Artículo en Inglés | MEDLINE | ID: mdl-37031266

RESUMEN

Defective regulation of the alternative complement pathway (AP) causes excessive activation and promotes the inflammation and renal injury observed in atypical hemolytic-uremic syndrome (aHUS). The usefulness of heat-inactivated Factor B (HFB) in reducing AP activation was evaluated in: fluid-phase reactions, using purified complement proteins and Factor H (FH)-depleted serum; and in surface-activated reactions using human endothelial cells (ECs). C3a and Ba levels, measured by quantitative Western blots, determined the extent of fluid-phase activation. In reactions using C3, FB, and Factor D proteins, HFB addition (2.5-fold FB levels), reduced C3a levels by 60% and Ba levels by 45%. In reactions using FH-depleted serum (supplemented with FH at 12.5% normal levels), Ba levels were reduced by 40% with HFB added at 3.5-fold FB levels. The effectiveness of HFB in limiting AP convertase formation on activated surfaces was evaluated using stimulated ECs. Fluorescent microscopy was used to quantify endogenously released C3, FB, and C5 attached to EC-secreted ultra-large VWF strings. HFB addition reduced attachment of C3b by 2.7-fold, FB by 1.5-fold and C5 by fourfold. Our data indicate that HFB may be of therapeutic value in preventing AP-mediated generation of C3a and C5a, and the associated inflammation caused by an overactive AP.


Asunto(s)
Factor B del Complemento , Factor de von Willebrand , Humanos , Factor B del Complemento/metabolismo , Factor de von Willebrand/metabolismo , Células Endoteliales/metabolismo , Calor , Factor H de Complemento/metabolismo , Inflamación/metabolismo , Activación de Complemento , Complemento C3b/metabolismo
7.
J Endocrinol ; 258(1)2023 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-37074365

RESUMEN

Obesity is caused by imbalanced energy intake and expenditure. Excessive energy intake and storage in adipose tissues are associated with many diseases. Several studies have demonstrated that vascular growth endothelial factor B (VEGFB) deficiency induces obese phenotypes. However, the roles of VEGFB isoforms VEGFB167 and VEGFB186 in adipose tissue development and function are still not clear. In this study, genetic mouse models of adipose-specific VEGFB167 and VEGFB186 overexpression (aP2-Vegfb167 tg/+and aP2-Vegfb186tg/+) were generated and their biologic roles were investigated. On regular chow, adipose-specific VEGFB186 is negatively associated with white adipose tissues (WATs) and positively regulates brown adipose tissues (BATs). VEGFB186 upregulates energy metabolism and metabolism-associated genes. In contrast, VEGFB167 has a nominal role in adipose development and function. On high-fat diet, VEGFB186 expression can reverse the phenotypes of VEGFB deletion. VEGFB186 overexpression upregulates BAT-associated genes and downregulates WAT-associated genes. VEGFB186 and VEGFB167 have very distinct roles in the regulation of adipose development and energy metabolism. As a key regulator of adipose tissue development and energy metabolism, VEGFB186 may be a target for obesity prevention and treatment.


Asunto(s)
Tejido Adiposo , Factor B del Complemento , Ratones , Animales , Factor B del Complemento/metabolismo , Tejido Adiposo/metabolismo , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , Obesidad/metabolismo , Metabolismo Energético/genética , Dieta Alta en Grasa/efectos adversos
8.
J Bacteriol ; 205(5): e0046622, 2023 05 25.
Artículo en Inglés | MEDLINE | ID: mdl-37098979

RESUMEN

The anaerobic pathogen Clostridioides difficile, which is a primary cause of antibiotic-associated diarrhea, faces a variety of stresses in the environment and in the mammalian gut. To cope with these stresses, alternative sigma factor B (σB) is employed to modulate gene transcription, and σB is regulated by an anti-sigma factor, RsbW. To understand the role of RsbW in C. difficile physiology, a rsbW mutant (ΔrsbW), in which σB is assumed to be "always on," was generated. ΔrsbW did not show fitness defects in the absence of stress but tolerated acidic environments and detoxified reactive oxygen and nitrogen species better compared to the parental strain. ΔrsbW was defective in spore and biofilm formation, but it displayed increased adhesion to human gut epithelia and was less virulent in a Galleria mellonella infection model. A transcriptomic analysis to understand the unique phenotype of ΔrsbW showed changes in expression of genes associated with stress responses, virulence, sporulation, phage, and several σB-controlled regulators, including the pleiotropic regulator sinRR'. While these profiles were distinct to ΔrsbW, changes in some σB-controlled stress-associated genes were similar to those reported in the absence of σB. Further analysis of ΔrsbW showed unexpected lower intracellular levels of σB, suggesting an additional post-translational control mechanism for σB in the absence of stress. Our study provides insight into the regulatory role of RsbW and the complexity of regulatory networks mediating stress responses in C. difficile. IMPORTANCE Pathogens like Clostridioides difficile face a range of stresses in the environment and within the host. Alternative transcriptional factors like sigma factor B (σB) enable the bacterium to respond quickly to different stresses. Anti-sigma factors like RsbW control sigma factors and therefore the activation of genes via these pathways. Some of these transcriptional control systems provide C. difficile with the ability to tolerate and detoxify harmful compounds. Here, we investigate the role of RsbW in C. difficile physiology. We demonstrate distinctive phenotypes for a rsbW mutant in growth, persistence, and virulence and suggest alternate σB control mechanisms in C. difficile. Understanding C. difficile responses to external stress is key to designing better strategies to combat this highly resilient bacterial pathogen.


Asunto(s)
Clostridioides difficile , Factor sigma , Animales , Humanos , Factor sigma/genética , Factor sigma/metabolismo , Clostridioides difficile/metabolismo , Clostridioides/metabolismo , Factor B del Complemento/genética , Factor B del Complemento/metabolismo , Factores de Transcripción/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Mamíferos/metabolismo
9.
J Neuroinflammation ; 20(1): 66, 2023 Mar 09.
Artículo en Inglés | MEDLINE | ID: mdl-36895046

RESUMEN

BACKGROUND: Helicobacter pylori (Hp) infects the stomach of 50% of the world's population. Importantly, chronic infection by this bacterium correlates with the appearance of several extra-gastric pathologies, including neurodegenerative diseases. In such conditions, brain astrocytes become reactive and neurotoxic. However, it is still unclear whether this highly prevalent bacterium or the nanosized outer membrane vesicles (OMVs) they produce, can reach the brain, thus affecting neurons/astrocytes. Here, we evaluated the effects of Hp OMVs on astrocytes and neurons in vivo and in vitro. METHODS: Purified OMVs were characterized by mass spectrometry (MS/MS). Labeled OMVs were administered orally or injected into the mouse tail vein to study OMV-brain distribution. By immunofluorescence of tissue samples, we evaluated: GFAP (astrocytes), ßIII tubulin (neurons), and urease (OMVs). The in vitro effect of OMVs in astrocytes was assessed by monitoring NF-κB activation, expression of reactivity markers, cytokines in astrocyte-conditioned medium (ACM), and neuronal cell viability. RESULTS: Urease and GroEL were prominent proteins in OMVs. Urease (OMVs) was present in the mouse brain and its detection coincided with astrocyte reactivity and neuronal damage. In vitro, OMVs induced astrocyte reactivity by increasing the intermediate filament proteins GFAP and vimentin, the plasma membrane αVß3 integrin, and the hemichannel connexin 43. OMVs also produced neurotoxic factors and promoted the release of IFNγ in a manner dependent on the activation of the transcription factor NF-κB. Surface antigens on reactive astrocytes, as well as secreted factors in response to OMVs, were shown to inhibit neurite outgrowth and damage neurons. CONCLUSIONS: OMVs administered orally or injected into the mouse bloodstream reach the brain, altering astrocyte function and promoting neuronal damage in vivo. The effects of OMVs on astrocytes were confirmed in vitro and shown to be NF-κB-dependent. These findings suggest that Hp could trigger systemic effects by releasing nanosized vesicles that cross epithelial barriers and access the CNS, thus altering brain cells.


Asunto(s)
Helicobacter pylori , Ratones , Animales , Helicobacter pylori/metabolismo , Astrocitos , Ureasa/metabolismo , Ureasa/farmacología , FN-kappa B/metabolismo , Factor B del Complemento/metabolismo , Factor B del Complemento/farmacología , Modelos Animales de Enfermedad , Espectrometría de Masas en Tándem , Neuronas
10.
Blood ; 141(25): 3109-3121, 2023 06 22.
Artículo en Inglés | MEDLINE | ID: mdl-36947859

RESUMEN

Inhibitors of complement and coagulation are present in the saliva of a variety of blood-feeding arthropods that transmit parasitic and viral pathogens. Here, we describe the structure and mechanism of action of the sand fly salivary protein lufaxin, which inhibits the formation of the central alternative C3 convertase (C3bBb) and inhibits coagulation factor Xa (fXa). Surface plasmon resonance experiments show that lufaxin stabilizes the binding of serine protease factor B (FB) to C3b but does not detectably bind either C3b or FB alone. The crystal structure of the inhibitor reveals a novel all ß-sheet fold containing 2 domains. A structure of the lufaxin-C3bB complex obtained via cryo-electron microscopy (EM) shows that lufaxin binds via its N-terminal domain at an interface containing elements of both C3b and FB. By occupying this spot, the inhibitor locks FB into a closed conformation in which proteolytic activation of FB by FD cannot occur. C3bB-bound lufaxin binds fXa at a separate site in its C-terminal domain. In the cryo-EM structure of a C3bB-lufaxin-fXa complex, the inhibitor binds to both targets simultaneously, and lufaxin inhibits fXa through substrate-like binding of a C-terminal peptide at the active site as well as other interactions in this region. Lufaxin inhibits complement activation in ex vivo models of atypical hemolytic uremic syndrome (aHUS) and paroxysmal nocturnal hemoglobinuria (PNH) as well as thrombin generation in plasma, providing a rationale for the development of a bispecific inhibitor to treat complement-related diseases in which thrombosis is a prominent manifestation.


Asunto(s)
Coagulación Sanguínea , Factor B del Complemento , Microscopía por Crioelectrón , Factor B del Complemento/química , Factor B del Complemento/metabolismo , Activación de Complemento , Serina Endopeptidasas , Complemento C3b/química
11.
J Biol Chem ; 299(3): 102930, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36682494

RESUMEN

Hyperactivation of the complement system, a major component of innate immunity, has been recognized as one of the core clinical features in severe covid-19 patients. However, how the virus escapes the targeted elimination by the network of activated complement pathways still remains an enigma. Here, we identified SARS-CoV-2-encoded ORF8 protein as one of the major binding partners of human complement C3/C3b components and their metabolites. Our results demonstrated that preincubation of ORF8 with C3/C3b in the fluid phase has two immediate functional consequences in the alternative pathway; this preincubation inhibits factor I-mediated proteolysis and blocks factor B zymogen activation into active Bb. ORF8 binding results in the occlusion of both factor H and factor B from C3b, rendering the complexes resistant to factor I-mediated proteolysis and inhibition of pro-C3-convertase (C3bB) formation, respectively. We also confirmed the complement inhibitory activity of ORF8 in our hemolysis-based assay, where ORF8 prevented human serum-induced lysis of rabbit erythrocytes with an IC50 value of about 2.3 µM. This inhibitory characteristic of ORF8 was also supported by in-silico protein-protein docking analysis, as it appeared to establish primary interactions with the ß-chain of C3b, orienting itself near the C3b CUB (C1r/C1s, Uegf, Bmp1) domain like a peptidomimetic compound, sterically hindering the binding of essential cofactors required for complement amplification. Thus, ORF8 has characteristics to act as an inhibitor of critical regulatory steps in the alternative pathway, converging to hasten the decay of C3-convertase and thereby, attenuating the complement amplification loop.


Asunto(s)
COVID-19 , Factor B del Complemento , Animales , Humanos , Conejos , Activación de Complemento , Convertasas de Complemento C3-C5/metabolismo , Complemento C3b/metabolismo , Factor B del Complemento/metabolismo , Factor H de Complemento/metabolismo , Vía Alternativa del Complemento/fisiología , SARS-CoV-2/metabolismo , Unión Proteica , Simulación por Computador
12.
Int J Mol Sci ; 23(20)2022 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-36293087

RESUMEN

Complement pathway proteins are reported to be increased in polycystic ovary syndrome (PCOS) and may be affected by obesity and insulin resistance. To investigate this, a proteomic analysis of the complement system was undertaken, including inhibitory proteins. In this cohort study, plasma was collected from 234 women (137 with PCOS and 97 controls). SOMALogic proteomic analysis was undertaken for the following complement system proteins: C1q, C1r, C2, C3, C3a, iC3b, C3b, C3d, C3adesArg, C4, C4a, C4b, C5, C5a, C5b-6 complex, C8, properdin, factor B, factor D, factor H, factor I, mannose-binding protein C (MBL), complement decay-accelerating factor (DAF) and complement factor H-related protein 5 (CFHR5). The alternative pathway of the complement system was primarily overexpressed in PCOS, with increased C3 (p < 0.05), properdin and factor B (p < 0.01). In addition, inhibition of this pathway was also seen in PCOS, with an increase in CFHR5, factor H and factor I (p < 0.01). Downstream complement factors iC3b and C3d, associated with an enhanced B cell response, and C5a, associated with an inflammatory cytokine release, were increased (p < 0.01). Hyperandrogenemia correlated positively with properdin and iC3b, whilst insulin resistance (HOMA-IR) correlated with iC3b and factor H (p < 0.05) in PCOS. BMI correlated positively with C3d, factor B, factor D, factor I, CFHR5 and C5a (p < 0.05). This comprehensive evaluation of the complement system in PCOS revealed the upregulation of components of the complement system, which appears to be offset by the concurrent upregulation of its inhibitors, with these changes accounted for in part by BMI, hyperandrogenemia and insulin resistance.


Asunto(s)
Resistencia a la Insulina , Lectina de Unión a Manosa , Síndrome del Ovario Poliquístico , Femenino , Humanos , Properdina/metabolismo , Factor H de Complemento , Factor B del Complemento/metabolismo , Antígenos CD55 , Factor D del Complemento , Estudios de Cohortes , Proteómica , Complemento C1q , Complemento C3b , Fibrinógeno , Citocinas
13.
Protein Sci ; 31(10): e4432, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-36173177

RESUMEN

Structure determination of macromolecular complexes is challenging if subunits can dissociate during crystallization or preparation of electron microscopy grids. We present an approach where a labile complex is stabilized by linking subunits though introduction of a peptide tag in one subunit that is recognized by a nanobody tethered to a second subunit. This allowed crystal structure determination at 3.9 Å resolution of the highly non-globular 320 kDa proconvertase formed by complement components C3b, factor B, and properdin. Whereas the binding mode of properdin to C3b is preserved, an internal rearrangement occurs in the zymogen factor B von Willebrand domain type A domain compared to the proconvertase not bound to properdin. The structure emphasizes the role of two noncanonical loops in thrombospondin repeats 5 and 6 of properdin in augmenting the activity of the C3 convertase. We suggest that linking of subunits through peptide specific tethered nanobodies represents a simple alternative to approaches like affinity maturation and chemical cross-linking for the stabilization of large macromolecular complexes. Besides applications for structural biology, nanobody bridging may become a new tool for biochemical analysis of unstable macromolecular complexes and in vitro selection of highly specific binders for such complexes.


Asunto(s)
Properdina , Anticuerpos de Dominio Único , Convertasas de Complemento C3-C5/química , Convertasas de Complemento C3-C5/metabolismo , Factor B del Complemento/química , Factor B del Complemento/metabolismo , Precursores Enzimáticos , Sustancias Macromoleculares , Properdina/química , Properdina/metabolismo , Trombospondinas
14.
Front Immunol ; 13: 942482, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35958553

RESUMEN

Multidrug-resistant Acinetobacter baumannii is known to be one of the leading pathogens that cause severe nosocomial infections. To overcome eradication by the innate immune system during infection, A. baumannii developed a number of immune evasion strategies. Previously, we identified CipA as a plasminogen-binding and complement-inhibitory protein. Here we show that CipA inhibits all three complement activation pathways and interacts with key complement components C3, C3b, C4b, C5, Factor B, Factor D, and in particular Factor I. CipA also targets function of the C5 convertase as cleavage of C5 was impaired. Systematic screening of CipA variants identified two separate binding sites for C3b and a Factor I-interacting domain located at the C-terminus. Structure predictions using AlphaFold2 and binding analyses employing CipA variants lacking Factor I-binding capability confirmed that the orientation of the C-terminal domain is essential for the interaction with Factor I. Hence, our analyses point to a novel Factor I-dependent mechanisms of complement inactivation mediated by CipA of A. baumannii. Recruitment of Factor I by CipA initiates the assembly of a quadripartite complex following binding of either Factor H or C4b-binding protein to degrade C3b and C4b, respectively. Loss of Factor I binding in a CipA-deficient strain, or a strain producing a CipA variant lacking Factor I-binding capability, correlated with a higher susceptibility to human serum, indicating that recruitment of Factor I enables A. baumannii to resist complement-mediated killing.


Asunto(s)
Acinetobacter baumannii , Activación de Complemento , Convertasas de Complemento C3-C5/metabolismo , Factor B del Complemento/metabolismo , Fibrinógeno/metabolismo , Humanos
15.
Life Sci ; 300: 120569, 2022 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-35472453

RESUMEN

Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disease characterized by involuntary bizarre movements, psychiatric symptoms, dementia, and early death. Several studies suggested neuroprotective activities of inosine; however its role in HD is yet to be elucidated. The current study aimed to demonstrate the neuroprotective effect of inosine in 3-nitropropionic acid (3-NP)-induced neurotoxicity in rats while investigating possible underlying mechanisms. Rats were randomly divided into five groups; group 1 received i.p. injections of 1% DMSO, whereas groups 2, 3, 4, and 5 received 3-NP (10 mg/kg, i.p.) for 14 days, concomitantly with inosine (200 mg/kg., i.p.) in groups 3, 4, and 5, SCH58261, a selective adenosine 2A receptor (A2AR) antagonist, (0.05 mg/kg, i.p.) in group 4, and PD98059, an extracellular signal-regulated kinase (ERK) inhibitor, (0.3 mg/kg, i.p.) in group 5. Treatment with inosine mitigated 3-NP-induced motor abnormalities and body weight loss. Moreover, inosine boosted the striatal brain-derived neurotrophic factor (BDNF) level, p-tropomyosin receptor kinase B (TrKB), p-ERK, and p-cAMP response element-binding protein (CREB) expression, which subsequently suppressed oxidative stress biomarkers (malondialdehyde and nitric oxide) and pro-inflammatory cytokines (tumor necrosis factor alpha and interleukin-1ß) and replenished the glutathione content. Similarly, histopathological analyses revealed decreased striatal injury score, the expression of the glial fibrillary acidic protein, and neuronal loss after inosine treatment. These effects were attenuated by the pre-administration of SCH58261 or PD98059. In conclusion, inosine attenuated 3-NP-induced HD-like symptoms in rats, at least in part, via the activation of the A2AR/BDNF/TrKB/ERK/CREB signaling pathway.


Asunto(s)
Enfermedad de Huntington , Enfermedades Neurodegenerativas , Fármacos Neuroprotectores , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor B del Complemento/metabolismo , Factor B del Complemento/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Enfermedad de Huntington/inducido químicamente , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/metabolismo , Inosina/farmacología , Fármacos Neuroprotectores/uso terapéutico , Nitrocompuestos , Propionatos/farmacología , Ratas , Transducción de Señal
16.
J Immunol ; 208(7): 1772-1781, 2022 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-35277417

RESUMEN

Alternative pathway complement dysregulation with abnormal glomerular C3 deposits and glomerular damage is a key mechanism of pathology in C3 glomerulopathy (C3G). No disease-specific treatments are currently available for C3G. Therapeutics inhibiting complement are emerging as a potential strategy for the treatment of C3G. In this study, we investigated the effects of N-acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) targeting the C3 component of complement that inhibits liver C3 expression in the C3G model of mice with heterozygous deficiency of factor H (Cfh +/- mice). We showed a duration of action for GalNAc-conjugated C3 siRNA in reducing the liver C3 gene expression in Cfh +/- mice that were dosed s.c. once a month for up to 7 mo. C3 siRNA limited fluid-phase alternative pathway activation, reducing circulating C3 fragmentation and activation of factor B. Treatment with GalNAc-conjugated C3 siRNA reduced glomerular C3d deposits in Cfh +/- mice to levels similar to those of wild-type mice. Ultrastructural analysis further revealed the efficacy of the C3 siRNA in slowing the formation of mesangial and subendothelial electron-dense deposits. The present data indicate that RNA interference-mediated C3 silencing in the liver may be a relevant therapeutic strategy for treating patients with C3G associated with the haploinsufficiency of complement factor H.


Asunto(s)
Glomerulonefritis Membranoproliferativa , Enfermedades Renales , Animales , Complemento C3/genética , Complemento C3/metabolismo , Factor B del Complemento/metabolismo , Factor H de Complemento/genética , Vía Alternativa del Complemento/genética , Glomerulonefritis Membranoproliferativa/patología , Humanos , Ratones , ARN Interferente Pequeño/genética
17.
Exp Eye Res ; 218: 108982, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35183540

RESUMEN

Age-related macular degeneration (AMD) has been associated with both complement activation and increased levels of circulating cytokines. Here, we sougth to investigate if cytokine-preexposure of retinal pigment epithelial (RPE) leads to increased complement activation and deposition of membrane attack complex (MAC). Primary human RPE and the ARPE19 cell line cultured in serum-free conditions were preexposed to 100 ng/ml interferon-gamma (IFNγ) and 20 ng/ml tumor necrosis factor-alpha (TNFα) for 48 h followed by exposure to diluted serum from healthy donors or complement factor B deficient (CFBd) serum for 70 min. Deposition of membrane attack complexes (MAC) was examined by use of a MAC-ELISA kit and by immunofluorescence. Eculizumab (anti-C5) was examined for its ability to prevent deposition of MAC on RPE cells exposed to serum. Lactatdehydrogenase (LDH) and thiazolyl blue tetrazolium bromide (MTT) assays were used to assess cellular metabolism and survival. MAC was deposited only on RPE preexposed to both IFNγ and TNFα. Lack of complement factor B or inhibition of C5 abrogated the MAC-deposition on RPE cells, while reconstitution of CFBd serum with CFB resulted in MAC-deposition. MAC-deposition resulted in RPE-release of LDH, but unaltered mitochondrial activity estimated by MTT. We conclude that preexposure of primary RPE and ARPE19 with inflammatory cytokines promoted alternative pathway activation of complement and deposition of MAC. This implies that circulating inflammatory mediators may increase susceptibility to local complement activation and MAC-deposition, which may represent an early event in the pathogenesis leading to AMD development.


Asunto(s)
Degeneración Macular , Factor de Necrosis Tumoral alfa , Activación de Complemento/fisiología , Factor B del Complemento/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Humanos , Interferón gamma/metabolismo , Interferón gamma/farmacología , Degeneración Macular/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Pigmentos Retinianos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología
18.
Bioengineered ; 12(2): 12854-12866, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34898340

RESUMEN

Stromal and immune cells are major components of tumor microenvironment (TME) and affect the growth and development of thyroid carcinoma (THCA). However, data on the exact mechanisms that define the relationship between the TME and THCA remain scant. We calculated stromal and immune cells scores and the proportion of tumor-infiltrating immune cells (TICs) by CIBERSORT and ESTIMATE based on the THCA gene expression data from the Cancer Genome Atlas (TCGA). In addition, we evaluated differentially expressed genes (DEGs) from high- and low-score groups and performed functional enrichment analysis. Furthermore, our data show a significant correlation between plasma complement factor B (CFB) and PTC development and prognosis. Gene Set Enrichment Analysis (GSEA) demonstrated that the CFB was mainly enriched in immune response pathways. The expression of CFB was positively correlated with T cells CD8, Macrophages M1, Plasma cells, T cells CD4 memory activated, T cells follicular helper and T cells regulatory (Tregs), whereas negatively correlated with Eosinophils, Macrophages M0, Macrophages M2, Mast cells resting, T cells CD4 memory resting in the TME. Finally, the expression level of CFB was verified by other cohorts from Gene Expression Omnibus (GEO) database and quantitative Real-Time PCR (qRT-PCR) analyses, which was consistent with the results of bioinformatic analysis. Taken together, our data demonstrated that the CFB could be a prognostic marker for THCA and its expression influences the infiltration of immune cells.


Asunto(s)
Factor B del Complemento/metabolismo , Neoplasias de la Tiroides/sangre , Neoplasias de la Tiroides/diagnóstico , Bases de Datos Factuales , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Ontología de Genes , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Persona de Mediana Edad , Pronóstico , Modelos de Riesgos Proporcionales , Mapas de Interacción de Proteínas/genética , Células del Estroma/metabolismo , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/inmunología
19.
J Proteome Res ; 20(12): 5315-5328, 2021 12 03.
Artículo en Inglés | MEDLINE | ID: mdl-34766501

RESUMEN

Although plasma complement factor B (CFB, NX_P00751), both alone and in combination with CA19-9 (i.e., the ComB-CAN), previously exhibited a reliable diagnostic ability for pancreatic cancer (PC), its detectability of the early stages and the cancer detection mechanism remained elusive. We first evaluated the diagnostic accuracy of ComB-CAN using plasma samples from healthy donors (HDs), patients with chronic pancreatitis (CP), and patients with different PC stages (I/II vs III/IV). An analysis of the area under the curve (AUC) by PanelComposer using logistic regression revealed that ComB-CAN has a superior diagnostic ability for early-stage PC (97.1.% [95% confidence interval (CI): (97.1-97.2)]) compared with CFB (94.3% [95% CI: 94.2-94.4]) or CA19-9 alone (34.3% [95% CI: 34.1-34.4]). In the comparisons of all stages of patients with PC vs CP and HDs, the AUC values of ComB-CAN, CFB, and CA19-9 were 0.983 (95% CI: 0.983-0.983), 0.950 (95% CI: 0.950-0.951), and 0.873 (95% CI: 0.873-0.874), respectively. We then investigated the molecular mechanism underlying the detection of early-stage PC by using stable cell lines of CFB knockdown and CFB overexpression. A global transcriptomic analysis coupled to cell invasion assays of both CFB-modulated cell lines suggested that CFB plays a tumor-promoting role in PC, which likely initiates the PI3K-AKT cancer signaling pathway. Thus our study establishes ComB-CAN as a reliable early diagnostic marker for PC that can be clinically applied for early PC screening in the general public.


Asunto(s)
Factor B del Complemento , Neoplasias Pancreáticas , Biomarcadores de Tumor/genética , Antígeno CA-19-9 , Factor B del Complemento/metabolismo , Humanos , Fosfatidilinositol 3-Quinasas
20.
JCI Insight ; 6(19)2021 10 08.
Artículo en Inglés | MEDLINE | ID: mdl-34622800

RESUMEN

The role and mechanisms for upregulating complement factor B (CFB) expression in podocyte dysfunction in diabetic kidney disease (DKD) are not fully understood. Here, analyzing Gene Expression Omnibus GSE30528 data, we identified genes enriched in mTORC1 signaling, CFB, and complement alternative pathways in podocytes from patients with DKD. In mouse models, podocyte mTOR complex 1 (mTORC1) signaling activation was induced, while blockade of mTORC1 signaling reduced CFB upregulation, alternative complement pathway activation, and podocyte injury in the glomeruli. Knocking down CFB remarkably alleviated alternative complement pathway activation and DKD in diabetic mice. In cultured podocytes, high glucose treatment activated mTORC1 signaling, stimulated STAT1 phosphorylation, and upregulated CFB expression, while blockade of mTORC1 or STAT1 signaling abolished high glucose-upregulated CFB expression. Additionally, high glucose levels downregulated protein phosphatase 2Acα (PP2Acα) expression, while PP2Acα deficiency enhanced high glucose-induced mTORC1/STAT1 activation, CFB induction, and podocyte injury. Taken together, these findings uncover a mechanism by which CFB mediates podocyte injury in DKD.


Asunto(s)
Factor B del Complemento/genética , Nefropatías Diabéticas/genética , Hiperglucemia/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Podocitos/metabolismo , Factor de Transcripción STAT1/metabolismo , Animales , Células Cultivadas , Factor B del Complemento/metabolismo , Vía Alternativa del Complemento , Bases de Datos Genéticas , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Técnicas de Silenciamiento del Gen , Glucosa/farmacología , Humanos , Hiperglucemia/metabolismo , Hiperglucemia/patología , Riñón/metabolismo , Riñón/patología , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Ratones , Podocitos/ultraestructura , Proteína Fosfatasa 2C/genética , Proteína Fosfatasa 2C/metabolismo , Factor de Transcripción STAT1/antagonistas & inhibidores
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