RESUMO
The ancient arm of innate immunity known as the complement system is a blood proteolytic cascade involving dozens of membrane-bound and solution-phase components. Although many of these components serve as regulatory molecules to facilitate controlled activation of the cascade, C1 esterase inhibitor (C1-INH) is the sole canonical complement regulator belonging to a superfamily of covalent inhibitors known as serine protease inhibitors (SERPINs). In addition to its namesake role in complement regulation, C1-INH also regulates proteases of the coagulation, fibrinolysis, and contact pathways. Despite this, the structural basis for C1-INH recognition of its target proteases has remained elusive. In this study, we present the crystal structure of the Michaelis-Menten (M-M) complex of the catalytic domain of complement component C1s and the SERPIN domain of C1-INH at a limiting resolution of 3.94 Å. Analysis of the structure revealed that nearly half of the protein/protein interface is formed by residues outside of the C1-INH reactive center loop. The contribution of these residues to the affinity of the M-M complex was validated by site-directed mutagenesis using surface plasmon resonance. Parallel analysis confirmed that C1-INH-interfacing residues on C1s surface loops distal from the active site also drive affinity of the M-M complex. Detailed structural comparisons revealed differences in substrate recognition by C1s compared with C1-INH recognition and highlight the importance of exosite interactions across broader SERPIN/protease systems. Collectively, this study improves our understanding of how C1-INH regulates the classical pathway of complement, and it sheds new light on how SERPINs recognize their cognate protease targets.
Assuntos
Proteína Inibidora do Complemento C1 , Complemento C1s , Proteína Inibidora do Complemento C1/metabolismo , Complemento C1s/metabolismo , Complemento C1s/química , Humanos , Cristalografia por Raios X , Domínio Catalítico , Ligação Proteica , Modelos Moleculares , Conformação ProteicaRESUMO
Parkinson's disease (PD) pathology is characterized by alpha-synuclein (α-syn) aggregates, degeneration of dopamine neurons in the substantia nigra pars compacta (SNpc), and neuroinflammation. The presence of reactive glia correlates with deposition of pathological α-syn in early-stage PD. Thus, understanding the neuroinflammatory response of microglia and astrocytes to synucleinopathy may identify therapeutic targets. Here we characterized the neuroinflammatory gene expression profile of reactive microglia and astrocytes in the SNpc during early synucleinopathy in the rat α-syn pre-formed fibril (PFF) model. Rats received intrastriatal injection of α-syn PFFs and expression of immune genes was quantified with droplet digital PCR (ddPCR), after which fluorescent in situ hybridization (FISH) was used to localize gene expression to microglia or astrocytes in the SNpc. Genes previously associated with reactive microglia (Cd74, C1qa, Stat1, Axl, Casp1, Il18, Lyz2) and reactive astrocytes (C3, Gbp2, Serping1) were significantly upregulated in the SN of PFF injected rats. Localization of gene expression to SNpc microglia near α-syn aggregates identified a unique α-syn aggregate microglial gene expression profile characterized by upregulation of Cd74, Cxcl10, Rt-1a2, Grn, Csf1r, Tyrobp, C3, C1qa, Serping1 and Fcer1g. Importantly, significant microglial upregulation of Cd74 and C3 were only observed following injection of α-syn PFFs, not α-syn monomer, confirming specificity to α-syn aggregation. Serping1 expression also localized to astrocytes in the SNpc. Interestingly, C3 expression in the SNpc localized to microglia at 2- and 4-months post-PFF, but to astrocytes at 6-months post-PFF. We also observed expression of Rt1-a2 and Cxcl10 in SNpc dopamine neurons. Cumulatively our results identify a dynamic, yet reproducible gene expression profile of reactive microglia and astrocytes associated with early synucleinopathy in the rat SNpc.
Assuntos
Doença de Parkinson , Sinucleinopatias , Animais , Ratos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Proteína Inibidora do Complemento C1/genética , Proteína Inibidora do Complemento C1/metabolismo , Neurônios Dopaminérgicos/metabolismo , Hibridização in Situ Fluorescente , Neuroglia/metabolismo , Doenças Neuroinflamatórias , Doença de Parkinson/metabolismo , Substância Negra/metabolismo , Sinucleinopatias/patologia , TranscriptomaRESUMO
The role of contact system activation has been clearly established in the pathogenesis of hereditary angioedema due to C1 inhibitor deficiency (HAE-C1INH). C1 inhibitor (C1INH)-protease complexes, levels of functional C1INH, plasma kallikrein activation, and cleavage of high-molecular-weight kininogen have each been associated with disease activity. More recently, HAE with normal levels of C1INH (HAE-nl-C1INH) has been recognized. Six genetic mutations have been identified which are linked to HAE-nl-C1INH phenotypes. The majority of individuals with HAE-nl-C1INH fall into the unknown category. There is substantial evidence that bradykinin generation underlies the recurrent attacks of swelling in some of these cohorts.
Assuntos
Biomarcadores , Bradicinina , Proteína Inibidora do Complemento C1 , Humanos , Bradicinina/metabolismo , Proteína Inibidora do Complemento C1/metabolismo , Angioedema/diagnóstico , Angioedema/metabolismo , Angioedema/etiologia , Angioedemas Hereditários/diagnóstico , Angioedemas Hereditários/metabolismo , Angioedemas Hereditários/etiologia , MutaçãoRESUMO
Introduction: Hereditary angioedema (HAE) is a rare, life-threatening autosomal dominant genetic disorder caused by a deficient and/or dysfunctional C1 esterase inhibitor (C1-INH) (type 1 and type 2) leading to recurrent episodes of edema. This study aims to explore HAE patients' metabolomic profiles and identify novel potential diagnostic biomarkers for HAE. The study also examined distinguishing HAE from idiopathic angioedema (AE). Methods: Blood plasma samples from 10 HAE (types 1/2) patients, 15 patients with idiopathic AE, and 20 healthy controls were collected in Latvia and analyzed using LC-MS based targeted metabolomics workflow. T-test and fold change calculation were used to identify metabolites with significant differences between diseases and control groups. ROC analysis was performed to evaluate metabolite based classification model. Results: A total of 33 metabolites were detected and quantified. The results showed that isovalerylcarnitine, cystine, and hydroxyproline were the most significantly altered metabolites between the disease and control groups. Aspartic acid was identified as a significant metabolite that could differentiate between HAE and idiopathic AE. The mathematical combination of metabolites (hydroxyproline * cystine)/(creatinine * isovalerylcarnitine) was identified as the diagnosis signature for HAE. Furthermore, glycine/asparagine ratio could differentiate between HAE and idiopathic AE. Conclusion: Our study identified isovalerylcarnitine, cystine, and hydroxyproline as potential biomarkers for HAE diagnosis. Identifying new biomarkers may offer enhanced prospects for accurate, timely, and economical diagnosis of HAE, as well as tailored treatment selection for optimal patient care.
Assuntos
Angioedemas Hereditários , Biomarcadores , Metabolômica , Humanos , Feminino , Masculino , Angioedemas Hereditários/diagnóstico , Angioedemas Hereditários/sangue , Adulto , Biomarcadores/sangue , Metabolômica/métodos , Pessoa de Meia-Idade , Metaboloma , Adulto Jovem , Estudos de Casos e Controles , Proteína Inibidora do Complemento C1/genética , Proteína Inibidora do Complemento C1/metabolismo , AdolescenteRESUMO
Background: Hereditary angioedema (HAE) is a rare disease characterized by localized and self-limited angioedema (AE) attacks. A local increase of bradykinin (BK) mediates AE attacks in HAE, however the role of inflammation in HAE has been poorly explored We aim to analyze the role of inflammatory mediators in HAE patients during AE attacks. Methods: Patients with a confirmed HAE diagnosis due to C1 inhibitor deficiency (HAE-C1INH) or patients F12 gene mutations (HAE-FXII) attending to our outpatient clinic between November-2019 and May-2022 were included. Demographic and clinical characteristics were analyzed. Blood samples were collected both during symptom-free periods (baseline) and during HAE attacks, and acute phase reactants (APR), such as serum amyloid A (SAA), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), D-Dimer and white blood cells were measured. Results: Seventy-eight patients were enrolled in the study, with a predominant representation of women (76%, n=59), and a mean age of 47.8 years (range 6-88). Among them, 67% (n=52) of patients had HAE-C1INH (46 classified as type 1 and 6 as type 2) while 33% (n=26) had HAE-FXII. During attack-free periods, the majority of patients exhibited normal levels of SAA, ESR, D-dimer, ACE and WCC. However, in a subset of patients (16% for SAA, 18% for ESR, and 14.5% for D-dimer), elevations were noted at baseline. Importantly, during HAE attacks, significant increases were observed in SAA in 88% of patients (p< 0.0001 vs. baseline), in ESR in 65% (p= 0.003 vs. baseline) and D-dimer in 71% (p=0.001 vs. baseline) of the patients. A comparison between baseline and acute attack levels in 17 patients revealed significant differences in SAA AA (p<0. 0001), ESR (p<0.0001) and D-dimer (p= 0.004). No significant differences were observed in CRP (p=0.7), ACE (p=0.67) and WCC (p=0.54). These findings remained consistent regardless of HAE type, disease activity or location of angioedema. Conclusion: The systemic increase in APR observed during HAE attacks suggests that inflammation extends beyond the localized edematous area. This finding underscores the potential involvement of inflammatory pathways in HAE and highlights the need for further investigation into their role in the pathophysiology of HAE.
Assuntos
Angioedemas Hereditários , Biomarcadores , Inflamação , Humanos , Feminino , Masculino , Adulto , Angioedemas Hereditários/sangue , Angioedemas Hereditários/diagnóstico , Pessoa de Meia-Idade , Biomarcadores/sangue , Idoso , Inflamação/sangue , Adolescente , Criança , Adulto Jovem , Idoso de 80 Anos ou mais , Proteína Inibidora do Complemento C1/genética , Proteína Inibidora do Complemento C1/metabolismo , Proteína Amiloide A Sérica/metabolismo , Fator XII/genética , Fator XII/metabolismo , Sedimentação Sanguínea , Mediadores da Inflamação/sangue , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Produtos de Degradação da Fibrina e do Fibrinogênio/análiseRESUMO
In this article, three cases of hereditary angioedema (HAE) type III (estrogen-dependent or with normal C1 inhibitor) are reported. The HAE was initially described in women of the same family in association with high-leveled estrogenic conditions such as the use of oral contraceptives and pregnancy. There is no change in the C1 inhibitor as happens in other types of hereditary angioedema, and mutations are observed in the encoding gene of the XII factor of coagulation in several patients. The current diagnosis is mainly clinical and treatment consists in the suspension of the triggering factors and control of acute symptoms. A brief review of physiopathology, clinical features, genetic alterations and treatment are also presented.
Neste artigo são relatados três casos de angioedema hereditário do tipo III (estrógeno-dependente ou com inibidor de C1 normal), que foi inicialmente descrito em mulheres da mesma família, em associação com condições de alto nível estrogênico, como uso de anticoncepcionais orais e gravidez. Não há alteração do inibidor de C1, como acontece nos outros tipos de angioedema hereditário, e são observadas mutações no gene codificador do fator XII da coagulação em várias pacientes. O diagnóstico atualmente é eminentemente clínico e o tratamento consiste na suspensão dos fatores desencadeantes e controle dos sintomas agudos. Também é apresentada breve revisão da fisiopatogenia, quadro clínico, alterações genéticas e tratamento.