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1.
Int J Biol Macromol ; 266(Pt 2): 131065, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38521329

RESUMO

Protein C inhibitor (PCI) maintains hemostasis by inhibiting both procoagulant and anticoagulant serine proteases, and plays important roles in coagulation, fibrinolysis, reproduction, and anti-angiogenesis. The reactive site loop of PCI traps and irreversibly inhibits the proteases like APC (activating protein C), thrombin (FIIa) and factor Xa (FXa). Previous studies on antithrombin (ATIII) had identified Tyr253 and Glu255 as functional exosites that interact and aid in the inhibition of factor IXa and FXa. Presence of exosite in PCI is not known, however a sequence comparison with the PCI from different vertebrate species and ATIII identified Glu239 to be absolutely conserved. PCI residues analogous to ATIII exosite residues were mutated to R238A and E239A. Purified variant PCI in the presence of heparin (10 µg/ml) showed a 2-4 fold decrease in the rate of inhibition of the proteases. However, the stoichiometry of inhibition of FIIa, APC, and FXa by native PCI, R238A and E239A variants were found to be close to 1.0, which also indicated the formation of stable complexes based on SDS-PAGE and western blot analysis with thrombin and APC. Our findings revealed the possible presence of an exosite in PCI that influences the protease inhibition rates.


Assuntos
Heparina , Inibidor da Proteína C , Serina Proteases , Inibidor da Proteína C/química , Inibidor da Proteína C/metabolismo , Heparina/química , Heparina/farmacologia , Humanos , Serina Proteases/metabolismo , Serina Proteases/química , Trombina/metabolismo , Proteína C/metabolismo , Proteína C/química , Fator Xa/metabolismo , Fator Xa/química , Sequência de Aminoácidos , Ativação Enzimática/efeitos dos fármacos
2.
Biomolecules ; 13(12)2023 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-38136662

RESUMO

Pre-eclampsia (PE) is a severe pregnancy disorder that poses a significant health risk to both mother and fetus, with no preventive or therapeutic measures. Our previous research suggested an association between elevated SERPINA5 levels and PE features. This study investigated whether SERPINA5 could be a potential therapeutic target for PE. We established PE-like features in pregnant rats using L-NAME (75 mg/kg/d) treatment. Adenoviruses carrying overexpressed or suppressed SERPINA5 genes were intravenously injected into these PE rats on the fifth and seventh days of pregnancy. We evaluated the rats' systolic blood pressure, urine protein concentration, and placental and fetal metrics and histology. Placental gene expression following SERPINA5 overexpression was evaluated using mRNA sequencing. The L-NAME-induced PE rat model observed a significant increase in placental and peripheral SERPINA5 levels. The overexpression of SERPINA5 exacerbated L-NAME-induced hypertension and proteinuria in pregnant rats. A histology examination revealed a smaller placental junctional zone in L-NAME + overexpressing rats. Placental gene expression analysis in the L-NAME + overexpressing group indicated increased coagulation activation. L-NAME-induced hypertension and proteinuria were mitigated when SERPINA5 expression was suppressed. Additionally, placental development was improved in the SERPINA5-suppressed group. Our findings suggested that SERPINA5 may worsen L-NAME-induced PE-like features by promoting the activation of the coagulation cascade. Therefore, reducing SERPINA5 expression could potentially serve as a therapeutic strategy for PE.


Assuntos
Hipertensão , Pré-Eclâmpsia , Humanos , Ratos , Gravidez , Feminino , Animais , Pré-Eclâmpsia/induzido quimicamente , Pré-Eclâmpsia/genética , Placenta/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Proteinúria/metabolismo , Hipertensão/metabolismo , Inibidor da Proteína C/metabolismo , Inibidor da Proteína C/uso terapêutico
3.
Medicine (Baltimore) ; 102(24): e34017, 2023 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-37327267

RESUMO

We previously demonstrated that increased expression of the SERPINA5 gene is associated with hippocampal vulnerability in Alzheimer's disease (AD) brains. SERPINA5 was further demonstrated to be a novel tau-binding partner that colocalizes within neurofibrillary tangles. Our goal was to determine whether genetic variants in the SERPINA5 gene contributed to clinicopathologic phenotypes in AD. To screen for SERPINA5 variants, we sequenced 103 autopsy-confirmed young-onset AD cases with a positive family history of cognitive decline. To further assess the frequency of a rare missense variant, SERPINA5 p.E228Q, we screened an additional 1114 neuropathologically diagnosed AD cases. To provide neuropathologic context in AD, we immunohistochemically evaluated SERPINA5 and tau in a SERPINA5 p.E228Q variant carrier and a matched noncarrier. In the initial SERPINA5 screen, we observed 1 individual with a rare missense variant (rs140138746) that resulted in an amino acid change (p.E228Q). In our AD validation cohort, we identified an additional 5 carriers of this variant, resulting in an allelic frequency of 0.0021. There was no significant difference between SERPINA5 p.E228Q carriers and noncarriers in terms of demographic or clinicopathologic characteristics. Although not significant, on average SERPINA5 p.E228Q carriers were 5 years younger at age of disease onset than noncarriers (median: 66 [60-73] vs 71 [63-77] years, P = .351). In addition, SERPINA5 p.E228Q carriers exhibited a longer disease duration than noncarriers that approached significance (median: 12 [10-15]) vs 9 [6-12] years, P = .079). More severe neuronal loss was observed in the locus coeruleus, hippocampus, and amygdala of the SERPINA5 p.E228Q carrier compared to noncarrier, although no significant difference in SERPINA5-immunopositive lesions was observed. Throughout the AD brain in either carrier or noncarrier, areas with early pretangle pathology or burnt-out ghost tangle accumulation did not reveal SERPINA5-immunopositive neurons. Mature tangles and newly formed ghost tangles appeared to correspond well with SERPINA5-immunopositive tangle-bearing neurons. SERPINA5 gene expression was previously associated with disease phenotype; however, our findings suggest that SERPINA5 genetic variants may not be a contributing factor to clinicopathologic differences in AD. SERPINA5-immunopositive neurons appear to undergo a pathologic process that corresponded with specific levels of tangle maturity.


Assuntos
Doença de Alzheimer , Humanos , Doença de Alzheimer/metabolismo , Estudos Transversais , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologia , Encéfalo/patologia , Hipocampo/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo , Inibidor da Proteína C/metabolismo
4.
Transl Res ; 251: 14-26, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-35717024

RESUMO

Preeclampsia (PE) is the leading cause of maternal and fetal morbidity or mortality but lacks reliable methods for early diagnosis. In a previous study, serum SERPINA5 levels were higher in women with PE before the clinical manifestation of the disease. This study aimed to evaluate the efficacy of SERPINA5 in predicting PE and investigate its role in trophoblast cell biology. A multicenter, 2-stage observational case-control study was performed to develop and validate an early predictive PE model based on SERPINA5, maternal characteristics, and inflammatory factors. To further understand the relationship between SERPINA5 and PE, SERPINA5 was overexpressed or knocked down in extravillous trophoblast cells (EVT) and a pregnant rat model. After development and initial validation, a model that combined SERPINA5 and inflammatory factors had a high predictive ability for PE before 20 weeks gestation with an AUC of 0.90 (95% CI 0.83-0.96). It also demonstrated that SERPINA5 inhibited primary EVT cell invasion by disrupting the urokinase-type plasminogen activator/urokinase-type plasminogen activator receptor (uPA/uPAR) pathway, in turn, is involved in the development of PE. In vivo experiments also proved that overexpression of SERPINA5 induced a PE-like syndrome (hypertension and proteinuria) in pregnant rats. Therefore, serum SERPINA5 is a promising early biomarker of PE, suggesting that it may be involved in placental development through its action on the uPA/uPAR system prior to the clinical manifestation of PE.


Assuntos
Pré-Eclâmpsia , Inibidor da Proteína C , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Ativador de Plasminogênio Tipo Uroquinase , Animais , Feminino , Humanos , Gravidez , Ratos , Estudos de Casos e Controles , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Inibidor da Proteína C/metabolismo
5.
J Cell Mol Med ; 26(18): 4837-4846, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36000536

RESUMO

SERPINA5 belongs to the serine protease inhibitor superfamily and has been reported to be lowly expressed in a variety of malignancies. However, few report of SERPINA5 in gastric cancer has been found. The purpose of this study was to determine the role of SERPINA5 in GC and to investigate potential tumorigenic mechanisms. We performed qPCR to determine the level of SERPINA5 expression in GC. We used public databases to evaluate whether SERPINA5 could be utilized to predict overall survival and disease-free survival in GC patients. We also knocked down the expression of SERPINA5 and evaluated its effect on cell proliferation and migration. Furthermore, we explored the signal pathways and regulatory mechanisms related to SERPINA5 functions. According to our findings, SERPINA5 was shown to exhibit high expression in GC. Notably, SERPINA5 was prognostic in GC with high expression being unfavourable. SERPINA5 was further observed to promote GC tumorigenesis by modulating GC cell proliferation ability. Mechanically, SERPINA5 could inhibit CBL to regulate the PI3K/AKT/mTOR signalling pathway, thereby promoting GC carcinogenesis progression. These results highlight the important role of SERPINA5 in GC cell proliferation and suggest that SERPINA5 could be a novel target for GC treatment and a predictor for GC prognosis.


Assuntos
Neoplasias Gástricas , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidor da Proteína C/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Neoplasias Gástricas/patologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
6.
Cell Oncol (Dordr) ; 45(5): 861-872, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35951287

RESUMO

BACKGROUND: Metastasis is still the major cause of endometrial cancer (EC)-related death. Because of their biological function and regenerative properties, exosomes have been applied to therapeutic regimens. SERPINA5 expression is downregulated in several tumors and linked to tumor cell migration and invasion. However, the expression and biological functions of SERPINA5 in EC remain unclear. METHODS: The levels of SERPINA5 in plasma exosomes were determined with ELISAs. SERPINA5 expression in EC and its relationship with survival outcomes were analyzed using the TCGA database and clinical EC tissue samples. The effect of SERPINA5 overexpression or exosomal SERPINA5 on EC metastasis was examined by cell migration and invasion assays in vitro. Mechanistically, overexpression of SERPINA5 or high exosomal SERPINA5 levels mediated the regulation of the integrin ß1/FAK signaling pathway in EC cell lines. The therapeutic effect of exosomal SERPINA5 was determined with xenograft models. RESULTS: This study revealed that the level of exosomal SERPINA5 was increased in the circulating plasma of EC patients. In addition, the expression of SERPINA5 was decreased in EC patients with distant metastasis, and low expression of SERPINA5 indicated worse survival. In addition, SERPINA5 was elevated in normal tissues adjacent to EC tumors. Moreover, overexpression of SERPINA5 inhibited metastatic potential of EC cell lines in vitro. Furthermore, SERPINA5 loaded on secreted exosomes reduced the metastatic ability of EC cells. Notably, overexpression of SERPINA5 or high exosomal SERPINA5 levels suppressed EC metastatic potential by suppressing integrin ß1/FAK signaling pathway activation. Finally, exosomal SERPINA5 impeded tumor growth and metastasis in xenograft models. CONCLUSIONS: Our findings revealed that a low level of SERPINA5 expression indicated poor survival outcomes in EC and that exogenous SERPINA5 loading of exosomes may be a novel therapeutic strategy for metastatic EC.


Assuntos
Neoplasias do Endométrio , Exossomos , MicroRNAs , Feminino , Humanos , Exossomos/metabolismo , Integrina beta1 , Linhagem Celular Tumoral , Movimento Celular , Transdução de Sinais , Neoplasias do Endométrio/metabolismo , MicroRNAs/metabolismo , Proliferação de Células , Inibidor da Proteína C/metabolismo
7.
Reprod Sci ; 29(8): 2350-2362, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35194761

RESUMO

Obtaining high-quality sperm is key to improving the success rate of assisted reproductive technology (ART). Although cytokines secreted by cumulus-oocyte complexes (COCs) bind to sperm surface receptors to improve sperm quality, the effects of adding mouse COCs to human tubal fluid (HTF) medium on sperm capacitation have not yet been explored. Eight-week-old ICR mouse COCs were added to HTF medium and crushed to obtain the post-modified HTF medium. Compared with using HTF medium, the fertilisation rate and number of sperm combined with the zona pellucida significantly increased after in vitro capacitation using the post-modified HTF medium (P < 0.01). Proteomic and Western blotting analyses showed that the level of SERPINA5 in sperm increased significantly following in vitro capacitation with the post-modified HTF medium (P < 0.05). Immunohistochemical staining analysis demonstrated that SERPINA5 protein was expressed in mouse cumulus cells. A SERPINA5 antibody was added in the post-modified HTF medium to block the effects of SERPINA5 after in vitro capacitation, which significantly decreased the fertilisation rate and the number of sperm combined with the zona pellucida (P < 0.05). Recombinant mouse SERPINA5 protein (1 ~ 2 µg/ml) was added to HTF medium and the fertilisation rate and the number of sperm combined with the zona pellucida significantly increased (P < 0.01). Moreover, recombinant human SERPINA5 protein (5 µg/ml) was added before human semen freezing. Compared with adding no SERPINA5 protein, the percentage of normal sperm morphology and the intact acrosome significantly increased (P < 0.05). Our study provides a reference method for optimising sperm quality in the process of in vitro capacitation.


Assuntos
Inibidor da Proteína C , Sêmen , Animais , Feminino , Fertilização , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Oócitos , Inibidor da Proteína C/metabolismo , Proteômica , Capacitação Espermática , Interações Espermatozoide-Óvulo , Espermatozoides/metabolismo , Zona Pelúcida , Glicoproteínas da Zona Pelúcida
8.
Proteomics Clin Appl ; 16(3): e2100117, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34964303

RESUMO

PURPOSE: Nowadays, there is no clinically applicable biomarker for osteoarthritis (OA). Therefore, the aim of the study is to discover a potential biomarker for OA. EXPERIMENTAL DESIGN: We performed a proteomics of eight cartilage samples (four damaged cartilage and four macroscopically intact cartilage) from four OA patients without any comorbidities to search for valuable OA biomarkers. Four rats underwent bilateral ovariectomy to induce the OA (OVX-OA) model, while another four underwent a sham procedure wherein the ovaries were exteriorized but not removed (SHAM). Selected candidate proteins were further verified in the patients and the OVX-OA animal model. RESULTS: A comprehensive cartilage proteome profile of patients with OA was constructed. Additionally, the complement and coagulation cascades were found to be significantly altered, and serpinA5 was chosen as a protein of interest based on biological information analysis. The reduction of serpinA5 in locally damaged cartilage and serum of patients with OA compared to the control group was determined. Furthermore, we found that serpinA5 was decreased in OVX-OA rats compared to that in SHAM rats. CONCLUSIONS AND CLINICAL RELEVANCE: Our results suggest that there is dyscoagulation in the OA process and that serpinA5 can serve as a potentially valuable OA biomarker.


Assuntos
Cartilagem Articular , Osteoartrite , Animais , Biomarcadores/metabolismo , Cartilagem Articular/metabolismo , Feminino , Humanos , Osteoartrite/metabolismo , Ovariectomia , Inibidor da Proteína C/metabolismo , Proteômica/métodos , Ratos
9.
Proc Natl Acad Sci U S A ; 118(45)2021 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-34740972

RESUMO

Serine proteases are essential for many physiological processes and require tight regulation by serine protease inhibitors (SERPINs). A disturbed SERPIN-protease balance may result in disease. The reactive center loop (RCL) contains an enzymatic cleavage site between the P1 through P1' residues that controls SERPIN specificity. This RCL can be modified to improve SERPIN function; however, a lack of insight into sequence-function relationships limits SERPIN development. This is complicated by more than 25 billion mutants needed to screen the entire P4 to P4' region. Here, we developed a platform to predict the effects of RCL mutagenesis by using α1-antitrypsin as a model SERPIN. We generated variants for each of the residues in P4 to P4' region, mutating them into each of the 20 naturally occurring amino acids. Subsequently, we profiled the reactivity of the resulting 160 variants against seven proteases involved in coagulation. These profiles formed the basis of an in silico prediction platform for SERPIN inhibitory behavior with combined P4 to P4' RCL mutations, which were validated experimentally. This prediction platform accurately predicted SERPIN behavior against five out of the seven screened proteases, one of which was activated protein C (APC). Using these findings, a next-generation APC-inhibiting α1-antitrypsin variant was designed (KMPR/RIRA; / indicates the cleavage site). This variant attenuates blood loss in an in vivo hemophilia A model at a lower dosage than the previously developed variant AIKR/KIPP because of improved potency and specificity. We propose that this SERPIN-based RCL mutagenesis approach improves our understanding of SERPIN behavior and will facilitate the design of therapeutic SERPINs.


Assuntos
Desenho de Fármacos , Modelos Moleculares , Inibidor da Proteína C/genética , Engenharia de Proteínas , alfa 1-Antitripsina/genética , Animais , Testes de Coagulação Sanguínea , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Hemofilia A/tratamento farmacológico , Humanos , Camundongos , Inibidor da Proteína C/metabolismo , Inibidor da Proteína C/uso terapêutico , Especificidade por Substrato , alfa 1-Antitripsina/metabolismo , alfa 1-Antitripsina/uso terapêutico
10.
Nat Commun ; 12(1): 2311, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33875655

RESUMO

Selective vulnerability of different brain regions is seen in many neurodegenerative disorders. The hippocampus and cortex are selectively vulnerable in Alzheimer's disease (AD), however the degree of involvement of the different brain regions differs among patients. We classified corticolimbic patterns of neurofibrillary tangles in postmortem tissue to capture extreme and representative phenotypes. We combined bulk RNA sequencing with digital pathology to examine hippocampal vulnerability in AD. We identified hippocampal gene expression changes associated with hippocampal vulnerability and used machine learning to identify genes that were associated with AD neuropathology, including SERPINA5, RYBP, SLC38A2, FEM1B, and PYDC1. Further histologic and biochemical analyses suggested SERPINA5 expression is associated with tau expression in the brain. Our study highlights the importance of embracing heterogeneity of the human brain in disease to identify disease-relevant gene expression.


Assuntos
Doença de Alzheimer/genética , Córtex Cerebral/metabolismo , Perfilação da Expressão Gênica/métodos , Hipocampo/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/diagnóstico , Autopsia , Córtex Cerebral/patologia , Feminino , Hipocampo/patologia , Humanos , Aprendizado de Máquina , Masculino , Emaranhados Neurofibrilares/genética , Emaranhados Neurofibrilares/metabolismo , Inibidor da Proteína C/genética , Inibidor da Proteína C/metabolismo , RNA-Seq/métodos , Proteínas tau/genética , Proteínas tau/metabolismo
11.
Exp Cell Res ; 391(1): 111987, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32240661

RESUMO

BACKGROUND: The protein plasminogen activator inhibitor-1 (PAI-1), an inhibitor specific for urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA), has been shown to have a key role in cancer metastases. Currently, it is unknown as to whether the exocellular inhibition of PAI-1 can inhibit the migration of cancer cells. METHODS: By fusing the mutated serine protease domain (SPD) of uPA and human serum albumin (HSA), PAItrap3, a protein that traps PAI-1, was synthesized and experiments were conducted to determine if exocellular PAItrap3 attenuates PAI-1-induced cancer cell migration in vitro. RESULTS: PAItrap3 (0.8 µM) significantly inhibited the motility of MCF-7, MDA-MB-231, HeLa and 4T1 cancer cells, by 90%, 50%, 30% and 20%, respectively, without significantly altering their proliferation. The PAI-1-induced rearrangement of F-actin was significantly inhibited by PAItrap3, which produced a decrease in the number of cell protrusions by at least 20%. CONCLUSIONS: In vitro, PAItrap3 inhibited PAI-1-induced cancer cell migration, mainly through inhibiting the rearrangement of F-actin. Overall, these results, provided they can be extrapolated to humans, suggest that the PAItrap3 protein could be used as an exocellular inhibitor to attenuate cancer metastases.


Assuntos
Actinas/genética , Movimento Celular/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Inibidor da Proteína C/farmacologia , Actinas/antagonistas & inibidores , Actinas/metabolismo , Sítios de Ligação , Linhagem Celular , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Células HeLa , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Histidina/genética , Histidina/metabolismo , Humanos , Células MCF-7 , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Pichia/genética , Pichia/metabolismo , Inibidor 1 de Ativador de Plasminogênio/química , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ligação Proteica , Inibidor da Proteína C/química , Inibidor da Proteína C/genética , Inibidor da Proteína C/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
12.
Proc Natl Acad Sci U S A ; 117(9): 5039-5048, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32071217

RESUMO

Thrombin, a procoagulant protease, cleaves and activates protease-activated receptor-1 (PAR1) to promote inflammatory responses and endothelial dysfunction. In contrast, activated protein C (APC), an anticoagulant protease, activates PAR1 through a distinct cleavage site and promotes anti-inflammatory responses, prosurvival, and endothelial barrier stabilization. The distinct tethered ligands formed through cleavage of PAR1 by thrombin versus APC result in unique active receptor conformations that bias PAR1 signaling. Despite progress in understanding PAR1 biased signaling, the proteins and pathways utilized by thrombin versus APC signaling to induce opposing cellular functions are largely unknown. Here, we report the global phosphoproteome induced by thrombin and APC signaling in endothelial cells with the quantification of 11,266 unique phosphopeptides using multiplexed quantitative mass spectrometry. Our results reveal unique dynamic phosphoproteome profiles of thrombin and APC signaling, an enrichment of associated biological functions, including key modulators of endothelial barrier function, regulators of gene transcription, and specific kinases predicted to mediate PAR1 biased signaling. Using small interfering RNA to deplete a subset of phosphorylated proteins not previously linked to thrombin or APC signaling, a function for afadin and adducin-1 actin binding proteins in thrombin-induced endothelial barrier disruption is unveiled. Afadin depletion resulted in enhanced thrombin-promoted barrier permeability, whereas adducin-1 depletion completely ablated thrombin-induced barrier disruption without compromising p38 signaling. However, loss of adducin-1 blocked APC-induced Akt signaling. These studies define distinct thrombin and APC dynamic signaling profiles and a rich array of proteins and biological pathways that engender PAR1 biased signaling in endothelial cells.


Assuntos
Proteômica , Receptor PAR-1/metabolismo , Transdução de Sinais , Trombina/metabolismo , Proteínas de Ligação a Calmodulina , Proteínas de Transporte , Células Endoteliais/metabolismo , Humanos , Proteínas dos Microfilamentos , Fosforilação , Inibidor da Proteína C/metabolismo
13.
Genes (Basel) ; 10(7)2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31336797

RESUMO

Up to 30% of men with normal semen parameters suffer from infertility and the reason for this is unknown. Altered expression of sperm proteins may be a major cause of infertility in these men. Proteomic profiling was performed on pooled semen samples from eight normozoospermic fertile men and nine normozoospermic infertile men using LC-MS/MS. Furthermore, key differentially expressed proteins (DEPs) related to the fertilization process were selected for validation using Western blotting. A total of 1139 and 1095 proteins were identified in normozoospermic fertile and infertile men, respectively. Of these, 162 proteins were identified as DEPs. The canonical pathway related to free radical scavenging was enriched with upregulated DEPs in normozoospermic infertile men. The proteins associated with reproductive system development and function, and the ubiquitination pathway were underexpressed in normozoospermic infertile men. Western blot analysis revealed the overexpression of annexin A2 (ANXA2) (2.03 fold change; P = 0.0243), and underexpression of sperm surface protein Sp17 (SPA17) (0.37 fold change; P = 0.0205) and serine protease inhibitor (SERPINA5) (0.32 fold change; P = 0.0073) in men with unexplained male infertility (UMI). The global proteomic profile of normozoospermic infertile men is different from that of normozoospermic fertile men. Our data suggests that SPA17, ANXA2, and SERPINA5 may potentially serve as non-invasive protein biomarkers associated with the fertilization process of the spermatozoa in UMI.


Assuntos
Anexina A2/metabolismo , Antígenos de Superfície/metabolismo , Biomarcadores/metabolismo , Proteínas de Transporte/metabolismo , Infertilidade Masculina/metabolismo , Inibidor da Proteína C/metabolismo , Western Blotting , Proteínas de Ligação a Calmodulina , Cromatografia Líquida , Humanos , Infertilidade Masculina/etiologia , Masculino , Proteínas de Membrana , Proteoma , Análise do Sêmen , Espectrometria de Massas em Tandem
14.
Adv Exp Med Biol ; 966: 93-101, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28639251

RESUMO

It is generally accepted that the phospholipid bilayer of the cell membrane is impermeable for proteins and peptides and that these molecules require special mechanisms for their transport from the extra- to the intracellular space. Recently there is increasing evidence that certain proteins/peptides can also directly cross the phospholipid membrane. SERPINA5 (protein C inhibitor) is a secreted protease inhibitor with broad protease reactivity and wide tissue distribution. It binds glycosaminoglycans and certain phospoholipids, which can modulate its inhibitory activity. SERPINA5 has been shown to be internalized by platelets, granulocytes, HL-60 promyelocytic leukemia cells, and by Jurkat lymphoma cells. Once inside the cell it can translocate to the nucleus. There are several indications that SERPINA5 can directly cross the phospholipid bilayer of the cell membrane. In this review we will describe what is known so far about the conditions, as well as the cellular and molecular requirements for SERPINA5 translocation through the cell membrane and for its penetration of pure phospholipid vesicles.


Assuntos
Permeabilidade da Membrana Celular , Membrana Celular/metabolismo , Inibidor da Proteína C/metabolismo , Animais , Humanos , Inibidor da Proteína C/química , Conformação Proteica , Transporte Proteico , Relação Estrutura-Atividade
15.
Semin Cell Dev Biol ; 62: 187-193, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27989561

RESUMO

SERPINA5 (proteinC inhibitor, plasminogen activator inhibitor-3) is a secreted, extracellular clade A serpin. Its main characteristics are broad protease reactivity and wide tissue distribution (in man). SERPINA5 has originally been described as an inhibitor of activated protein C and independently as an inhibitor of the plasminogen activator urokinase. SERPINA5 binds glycosaminoglycans, phospholipids, and retinoic acid. Glycosaminoglycans and certain phospholipids can modulate its inhibitory activity and specificity. Studies suggest that SERPINA5 may play a role in hemostasis, in male reproduction, in host defense, and as a tumor suppressor. However, its biological role has not yet been defined. So far SERPINA5 deficiency has not been described in man. Mouse models are of limited value, since in mice serpinA5 is almost exclusively expressed in the reproductive tract. Consistently the only obvious phenotype of serpinA5-knockout mice is infertility of homozygous males. SERPINA5 can be internalized by cells and translocated to the nucleus. The internalization is dependent on the phospholipid phosphatidylethanolamine and on the intact N-terminus of SERPINA5, which functions as a cell penetrating peptide. Further functional analysis of intracellular SERPINA5 will contribute to our understanding of the biological role of this molecule.


Assuntos
Inibidor da Proteína C/metabolismo , Animais , Núcleo Celular/metabolismo , Endocitose , Humanos , Ligantes , Modelos Animais , Transporte Proteico
16.
PLoS One ; 10(11): e0143137, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26580551

RESUMO

Protein C inhibitor is a secreted, non-specific serine protease inhibitor with broad protease reactivity. It binds glycosaminoglycans and anionic phospholipids, which can modulate its activity. Anionic phospholipids, such as phosphatidylserine are normally localized to the inner leaflet of the plasma membrane, but are exposed on activated and apoptotic cells and on plasma membrane-derived microparticles. In this report we show by flow cytometry that microparticles derived from cultured cells and activated platelets incorporated protein C inhibitor during membrane blebbing. Moreover, protein C inhibitor is present in/on microparticles circulating in normal human plasma as judged from Western blots, ELISAs, flow cytometry, and mass spectrometry. These plasma microparticles are mainly derived from megakaryocytes. They seem to be saturated with protein C inhibitor, since they do not bind added fluorescence-labeled protein C inhibitor. Heparin partially removed microparticle-bound protein C inhibitor, supporting our assumption that protein C inhibitor is bound via phospholipids. To assess the biological role of microparticle-bound protein C inhibitor we performed protease inhibition assays and co-precipitated putative binding partners on microparticles with anti-protein C inhibitor IgG. As judged from amidolytic assays microparticle-bound protein C inhibitor did not inhibit activated protein C or thrombin, nor did microparticles modulate the activity of exogenous protein C inhibitor. Among the proteins co-precipitating with protein C inhibitor, complement factors, especially complement factor 3, were most striking. Taken together, our data do not support a major role of microparticle-associated protein C inhibitor in coagulation, but rather suggest an interaction with proteins of the complement system present on these phospholipid vesicles.


Assuntos
Plaquetas/química , Membrana Celular/química , Micropartículas Derivadas de Células/química , Megacariócitos/química , Inibidor da Proteína C/química , Proteína C/antagonistas & inibidores , Adulto , Plaquetas/citologia , Membrana Celular/metabolismo , Micropartículas Derivadas de Células/metabolismo , Feminino , Heparina/química , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Células Jurkat , Masculino , Megacariócitos/citologia , Pessoa de Meia-Idade , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Fator Plaquetário 3/química , Fator Plaquetário 3/metabolismo , Ligação Proteica , Proteína C/metabolismo , Inibidor da Proteína C/metabolismo , Trombina/química , Trombina/metabolismo
17.
J Mol Neurosci ; 57(1): 48-62, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25982926

RESUMO

Neuroinflammation is thought to contribute to cell death in neurodegenerative disorders, but the factors involved in the inflammatory process are not completely understood. Proteinase-activated receptor-2 (PAR2) expression in brain is increased in Alzheimer's disease and multiple sclerosis, but the status of PAR2 in Parkinson's disease is unknown. This study examined expression of PAR2 and endogenous proteinase activators (trypsin-2, mast cell tryptase) and proteinase inhibitors (serpin-A5, serpin-A13) in areas vulnerable and resistant to neurodegeneration in Parkinson's disease at different Braak α-synuclein stages of the disease in post-mortem brain. In normal aged brain, expression of PAR-2, trypsin-2, and serpin-A5 and serpin-A13 was found in neurons and microglia, and alterations in the amount of immunoreactivity for these proteins were found in some brain regions. Namely, there was a decrease in neurons positive for serpin-A5 in the dorsal motor nucleus, and serpin-A13 expression was reduced in the locus coeruleus and primary motor cortex, while expression of PAR2, trypsin-2 and both serpins was reduced in neurons within the substantia nigra. There was an increased number of microglia that expressed serpin-A5 in the dorsal motor nucleus of vagus and elevated numbers of microglia that expressed serpin-A13 in the substantia nigra of late Parkinson's disease cases. The number of microglia that expressed trypsin-2 increased in primary motor cortex of incidental Lewy body disease cases. Analysis of Parkinson's disease cases alone indicated that serpin-A5 and serpin-A13, and trypsin-2 expression in midbrain and cerebral cortex was different in cases with a high incidence of L-DOPA-induced dyskinesia and psychosis compared to those with low levels of these treatment-induced side effects. This study showed that there was altered expression in brain of PAR2 and some proteins that can control its function in Parkinson's disease. Given the role of PAR2 in neuroinflammation, drugs that mitigate these changes may be neuroprotective when administered to patients with Parkinson's disease.


Assuntos
Doença de Parkinson/metabolismo , Inibidor da Proteína C/metabolismo , Receptor PAR-2/metabolismo , Serpinas/metabolismo , Tripsina/metabolismo , Tripsinogênio/metabolismo , Idoso , Idoso de 80 Anos ou mais , Encéfalo/citologia , Encéfalo/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Microglia/metabolismo , Pessoa de Meia-Idade , Neurônios/metabolismo , Especificidade de Órgãos , Inibidor da Proteína C/genética , Receptor PAR-2/genética , Serpinas/genética , Tripsina/genética , Tripsinogênio/genética
18.
J Biol Chem ; 290(5): 3081-91, 2015 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-25488662

RESUMO

Protein C inhibitor (PCI) is a serpin with broad protease reactivity. It binds glycosaminoglycans and certain phospholipids that can modulate its inhibitory activity. PCI can penetrate through cellular membranes via binding to phosphatidylethanolamine. The exact mechanism of PCI internalization and the intracellular role of the serpin are not well understood. Here we showed that testisin, a glycosylphosphatidylinositol-anchored serine protease, cleaved human PCI and mouse PCI (mPCI) at their reactive sites as well as at sites close to their N terminus. This cleavage was observed not only with testisin in solution but also with cell membrane-anchored testisin on U937 cells. The cleavage close to the N terminus released peptides rich in basic amino acids. Synthetic peptides corresponding to the released peptides of human PCI (His(1)-Arg(11)) and mPCI (Arg(1)-Ala(18)) functioned as cell-penetrating peptides. Because intact mPCI but not testisin-cleaved mPCI was internalized by Jurkat T cells, a truncated mPCI mimicking testisin-cleaved mPCI was created. The truncated mPCI lacking 18 amino acids at the N terminus was not taken up by Jurkat T cells. Therefore our model suggests that testisin or other proteases could regulate the internalization of PCI by removing its N terminus. This may represent one of the mechanisms regulating the intracellular functions of PCI.


Assuntos
Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Inibidor da Proteína C/química , Inibidor da Proteína C/metabolismo , Animais , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/fisiologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Camundongos , Serina Endopeptidases/metabolismo , Células U937
19.
J Biomol Struct Dyn ; 33(1): 85-92, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-24251463

RESUMO

Activated Protein C (APC) is a multifunctional serine protease, primarily known for its anticoagulant function in the coagulation system. Several studies have already elucidated its role in counteracting apoptosis and inflammation in cells, while significant effort is still ongoing for defining its involvement in sepsis. Earlier literature has shown that the antiseptic function of APC is mediated by its binding to leukocyte integrins, which is due to the presence of the integrin binding motif Arg-Gly-Asp at the N-terminus of the APC catalytic chain. Many natural mutants have been identified in patients with Protein C deficiency diagnosis including a variant of specificity pocket (Gly216Asp). In this work, we present a molecular model of the complex of APC with αVß3 integrin obtained by protein-protein docking approach. A computational analysis of this variant is hereby presented, based on molecular dynamics and docking simulations, aiming at investigating the effects of the Gly216Asp mutation on the protein conformation and inferring its functional implications. Our study shows that such mutation is likely to impair the protease activity while preserving the overall protein fold. Moreover, superposition of the integrin binding motifs in wild-type and mutant forms suggests that the interaction with integrin can still occur and thus the mutant is likely to retain its antiseptic function related to the neutrophyl integrin binding. Therapeutic applications could result in this APC mutant which retains antiseptic function without anticoagulant side effects.


Assuntos
Integrina alfaVbeta3/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeo Hidrolases/química , Proteína C/química , Anti-Infecciosos Locais/química , Anti-Infecciosos Locais/metabolismo , Sítios de Ligação/genética , Humanos , Integrina alfaVbeta3/metabolismo , Mutação de Sentido Incorreto , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Ligação Proteica , Proteína C/genética , Proteína C/metabolismo , Inibidor da Proteína C/química , Inibidor da Proteína C/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína
20.
PLoS One ; 9(7): e101794, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25000564

RESUMO

Protein C Inhibitor (PCI) is a secreted serine protease inhibitor, belonging to the family of serpins. In addition to activated protein C PCI inactivates several other proteases of the coagulation and fibrinolytic systems, suggesting a regulatory role in hemostasis. Glycosaminoglycans and certain negatively charged phospholipids, like phosphatidylserine, bind to PCI and modulate its activity. Phosphatidylerine (PS) is exposed on the surface of apoptotic cells and known as a phagocytosis marker. We hypothesized that PCI might bind to PS exposed on apoptotic cells and thereby influence their removal by phagocytosis. Using Jurkat T-lymphocytes and U937 myeloid cells, we show here that PCI binds to apoptotic cells to a similar extent at the same sites as Annexin V, but in a different manner as compared to live cells (defined spots on ∼10-30% of cells). PCI dose dependently decreased phagocytosis of apoptotic Jurkat cells by U937 macrophages. Moreover, the phagocytosis of PS exposing, activated platelets by human blood derived monocytes declined in the presence of PCI. In U937 cells the expression of PCI as well as the surface binding of PCI increased with time of phorbol ester treatment/macrophage differentiation. The results of this study suggest a role of PCI not only for the function and/or maturation of macrophages, but also as a negative regulator of apoptotic cell and activated platelets removal.


Assuntos
Apoptose , Plaquetas/fisiologia , Fagocitose , Fosfatidilserinas/metabolismo , Ativação Plaquetária , Inibidor da Proteína C/metabolismo , Apoptose/efeitos dos fármacos , Camptotecina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Microesferas , Fagocitose/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Poliestirenos/química , Poliestirenos/metabolismo , Ligação Proteica , Transporte Proteico/efeitos dos fármacos
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