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1.
Methods Mol Biol ; 2597: 89-104, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36374416

RESUMO

The mechanisms underlying nervous system injury, such as spinal cord injury (SCI), traumatic brain injury (TBI), and peripheral nerve injury are complex and not well understood. Following acute tissue damage and cell death, inflammatory processes cause ongoing damage. Many factors regulate this inflammation, including factors that modulate chemokine expression. Serine proteases, including those of the thrombotic and thrombolytic pathways (e.g., thrombin, tPA, uPA) are upregulated during nervous system damage and can modulate the release and bioavailability of many chemokines. Virus-derived immunomodulators, such as Serp-1, a serine protease inhibitor (serpin), have protective effects by reducing inflammation and tissue damage. However, the precise mechanisms of Serp-1 neuroprotection are still being studied. Compartmentalized in vitro neuron culture systems, such as the Campenot trichamber, are useful for such mechanistic studies. This chapter provides a protocol for assembling and culturing rodent embryonic superior cervical ganglion (SCG) and dorsal root ganglion (DRG) neurons in Campenot trichambers, as well as instructive examples of the types of experiments enabled by these methods.


Assuntos
Serpinas , Humanos , Serpinas/farmacologia , Serpinas/metabolismo , Inflamação/metabolismo , Inibidores de Serino Proteinase , Fibrinolíticos , Serina Endopeptidases/metabolismo , Gânglios Espinais/metabolismo
2.
Cell Death Dis ; 13(11): 945, 2022 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-36351890

RESUMO

Pancreatic cancer (PC) is one of the most malignant types of cancer, and is characterized by early metastasis, limited response to chemotherapeutics, and poor prognosis. Therefore, there is an urgent need to explore new therapeutic strategies for PC treatment. Human rhomboid-like 2 (RHBDL2) is differentially expressed in cervical and breast cancer. However, the correlation between RHBDL2 and PC remains unclear. We found that RHBDL2 is highly expressed in human PC cells and tissues and is significantly associated with distant metastasis and poor survival of patients with PC. Gain- and loss-of-function assays indicated that RHBDL2 could accelerate PC cell proliferation and mobility in vitro and in vivo. The RNA-Seq results suggest that RHBDL2 may be involved in the activation of Notch signaling pathway. IMR-1 could restore the proliferation and metastatic capacity of PC cells mediated by RHBDL2. RHBDL2 interacted with and cleaved Notch1, resulting in the release of N1ICD. RHBDL2 decreased the ubiquitination level of N1ICD and collaborated with Ovarian tumor domain-containing 7B (OTUD7B) to stabilize N1ICD via the ubiquitin-proteasome pathway. RHBDL2 facilitated PC cell proliferation and mobility by stabilizing the N1ICD via the OTUD7B and activating the Notch signaling pathway. Thus, targeting this novel pathway may be a potential therapeutic strategy for PC.


Assuntos
Neoplasias Pancreáticas , Transdução de Sinais , Humanos , Proliferação de Células , Neoplasias Pancreáticas/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Endopeptidases/metabolismo , Serina Endopeptidases/metabolismo
3.
Sci Rep ; 12(1): 19268, 2022 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-36357523

RESUMO

Transgenic animals with increased or abrogated target gene expression are powerful tools for drug discovery research. Here, we developed a CRISPR-based Rosa26-LSL-dCas9-VPR mouse model for targeted induction of endogenous gene expression using different Adeno-associated virus (AAV) capsid variants for tissue-specific gRNAs delivery. To show applicability of the model, we targeted low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9), either individually or together. We induced up to ninefold higher expression of hepatocellular proteins. In consequence of LDLR upregulation, plasma LDL levels almost abolished, whereas upregulation of PCSK9 led to increased plasma LDL and cholesterol levels. Strikingly, simultaneous upregulation of both LDLR and PCSK9 resulted in almost unaltered LDL levels. Additionally, we used our model to achieve expression of all α1-Antitrypsin (AAT) gene paralogues simultaneously. These results show the potential of our model as a versatile tool for optimized targeted gene expression, alone or in combination.


Assuntos
Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Camundongos , Animais , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertases/metabolismo , Serina Endopeptidases/metabolismo , Receptores de LDL/metabolismo , Modelos Animais de Doenças , Descoberta de Drogas
4.
Sci Rep ; 12(1): 19185, 2022 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-36357546

RESUMO

The cleavage reactions of catalytic antibodies are mediated by a serine protease mechanism involving a catalytic triad composed of His, Ser, and Asp residues, which reside in the variable region. Recently, we discovered a catalytic antibody, H34 wild type (H34wt), that is capable of enzymatically cleaving an immune-check point PD-1 peptide and recombinant PD-1; however, H34wt does not contain His residues in the variable region. To clarify the reason behind the catalytic features of H34wt and the amino acid residues involved in the catalytic reaction, we performed site-directed mutagenesis focusing on the amino acid residues involved in the cleavage reaction, followed by catalytic activity tests, immunological reactivity evaluation, and molecular modeling. The results revealed that the cleavage reaction by H34wt proceeds through the action of a new catalytic site composed of Arg, Thr, and Gln. This new scheme differs from that of the serine protease mechanism of catalytic antibodies.


Assuntos
Anticorpos Catalíticos , Domínio Catalítico , Anticorpos Catalíticos/química , Anticorpos Catalíticos/metabolismo , Sequência de Aminoácidos , Receptor de Morte Celular Programada 1 , Cadeias Leves de Imunoglobulina/metabolismo , Serina Endopeptidases/metabolismo , Aminoácidos
5.
J Pharmacol Sci ; 150(4): 267-274, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36344049

RESUMO

The pathogenesis of hypertensive disorder of pregnancy (HDP), which affects about 10% of pregnant women, is still incompletely understood. Our previous study showed that endoplasmic reticulum (ER) stress influences high-temperature requirement A serine peptidase 1 (HTRA1) expression and trophoblast invasion. However, the involvement of ER stress in the regulation of HTRA subtype expression and pathophysiology of HDP has not been characterized in extravillous trophoblasts (EVTs). To investigate this, HTR8/SVneo EVTs cell line was treated with the ER stress inducers Thapsigargin (Thap) or Tunicamycin (Tuni). Treatment with either Thap or Tuni inhibited trophoblast invasion, reduced HTRA1 and HTRA3 expression, but did not alter HTRA2 or HTRA4 expression. Knockdown of HTRA1 or HTRA3 also inhibited trophoblast invasion. Furthermore, treatment with either ER stress inducer or HTRA1 silencing increased the ratio of soluble fms-like tyrosine kinase-1/placental growth factor (sFLT1/PlGF), which is a marker of HDP. Immunohistochemical analysis revealed that HTRA1 is localized to EVTs and the endometrial decidua in the placenta of patients with HDP. These results suggest that factors that cause ER stress could result in the inhibition of EVTs invasion via HTRA1.


Assuntos
Trofoblastos , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Humanos , Feminino , Gravidez , Trofoblastos/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Estresse do Retículo Endoplasmático/genética , Temperatura , Fator de Crescimento Placentário , Placenta/química , Placenta/metabolismo , Movimento Celular/genética , Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/análise , Serina Endopeptidases/metabolismo , Serina Proteases/genética , Serina Proteases/metabolismo
6.
Biomolecules ; 12(11)2022 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-36358970

RESUMO

Bone is a highly vascularized tissue and relies on the angiogenesis and response of cells in the immediate environmental niche at the defect site for regeneration. Hence, the ability to control angiogenesis and cellular responses during osteogenesis has important implications in tissue-engineered strategies. Self-assembling ionic-complementary peptides have received much interest as they mimic the natural extracellular matrix. Three-dimensional (3D)-printed biphasic calcium phosphate (BCP) scaffolds coated with self-assembling DAR 16-II peptide provide a support template with the ability to recruit and enhance the adhesion of cells. In vitro studies demonstrated prompt the adhesion of both human umbilical vein endothelial cells (HUVEC) and human mesenchymal stem cells (hMSC), favoring endothelial cell activation toward an angiogenic phenotype. The SEM-EDS and protein micro bicinchoninic acid (BCA) assays demonstrated the efficacy of the coating. Whole proteomic analysis of DAR 16-II-treated HUVECs demonstrated the upregulation of proteins involved in cell adhesion (HABP2), migration (AMOTL1), cytoskeletal re-arrangement (SHC1, TMOD2), immuno-modulation (AMBP, MIF), and morphogenesis (COL4A1). In vivo studies using DAR-16-II-coated scaffolds provided an architectural template, promoting cell colonization, osteogenesis, and angiogenesis. In conclusion, DAR 16-II acts as a proactive angiogenic factor when adsorbed onto BCP scaffolds and provides a simple and effective functionalization step to facilitate the translation of tailored 3D-printed BCP scaffolds for clinical applications.


Assuntos
Células-Tronco Mesenquimais , Tecidos Suporte , Humanos , Proteômica , Porosidade , Engenharia Tecidual/métodos , Osteogênese , Células Endoteliais da Veia Umbilical Humana , Neovascularização Patológica/metabolismo , Impressão Tridimensional , Proteínas de Membrana/metabolismo , Serina Endopeptidases/metabolismo
7.
BMC Endocr Disord ; 22(1): 280, 2022 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-36380313

RESUMO

Transmembrane protease serine 4 (TMPRSS4) is a cancer-associated protease associated with prognosis in various types of cancer. Mechanistically, TMPRSS4 mainly regulates malignant phenotypes, such as tumor invasion and metastasis, by either the epithelial to mesenchymal transition (EMT) program or promoting the proliferation of cancer cells. To date, TMPRSS4 and immune infiltration in thyroid carcinoma (TC) are largely unknown. Thus, this paper evaluated the expression of TMPRSS4 in tumor tissue through the Tumor Immune Estimation Resource (TIMER) database, and Oncomine, and its correlation with clinical parameters by UALCAN databases. Furthermore, we analyzed its prognostic value from Kaplan-Meier Plotter database, and the relationship between TMPRSS4 and the abundance of tumor-infiltrating lymphocytes (TILs) in TC in TISIDB, screening potential immune targets to explore novel mechanisms for the clinical management of TC. Finally, we assessed the correlation between TMPRSS4 and some immune markers to uncover a potential immune-related biomarker in TC patients by TIMER2.0. The results revealed that TMPRSS4 was highly expressed in TC and was also associated with lymphatic metastasis, advanced stage, histological subtype, and favorable clinical outcome. The stratified analysis based on immune cell content showed that decreased TMPRSS4 had worse prognosis in CD8+ T cell-enriched TC patients. TMPRSS4 was positively correlated with tumor immune infiltration and the expression of gene markers of immune cells. Notably, its expression was lower in the lymphocyte-depleted subtype than in other immunosubtypes in TC. Moreover, TMPRSS4 was closely related to chemokines as well as their receptors and the immunosuppressive checkpoints CTLA-4, PD-1, and HLA-G. In conclusion, TMPRSS4 may act as a novel biomarker predicting prognosis and immune infiltration in TC.


Assuntos
Transição Epitelial-Mesenquimal , Neoplasias da Glândula Tireoide , Humanos , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Peptídeo Hidrolases , Serina , Proteínas de Membrana/genética , Prognóstico , Biomarcadores , Biomarcadores Tumorais/genética
8.
Biol Pharm Bull ; 45(11): 1596-1601, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36328494

RESUMO

Vibrio vulnificus is a Gram-negative estuarine bacterium that causes infection in immuno-compromised patients, eels, and shrimp. V. vulnificus NCIMB2137, a metalloprotease-negative strain isolated from a diseased eel, produces a 45-kDa chymotrypsin-like alkaline serine protease known as VvsA. The gene encoding vvsA also includes another gene, vvsB with an unknown function; however, it is assumed to be an essential molecular chaperone for the maturation of VvsA. In the present study, we used an in vitro cell-free translation system to examine the maturation pathway of VvsA. We individually expressed the vvsA and vvsB genes and detected their mRNAs. However, the sample produced from vvsA did not exhibit protease activity. A sodium dodecyl sulfate (SDS) analysis detected the VvsB protein, but not the VvsA protein. A Western blotting analysis using a histidine (His)-tag at the amino terminus of proteins also showed no protein production by vvsA. These results suggested the translation, but not the transcription of vvsA. Factors derived from Escherichia coli were used in the in vitro cell-free translation system employed in the present study. The operon of the serine protease gene containing vvsA and vvsB was expressed in E. coli. Although serine proteases were produced, they were cleaved at different sites and no active mature forms were detected. These results indicate that the operon encoding vvsA and vvsB is a gene constructed to be specifically expressed in V. vulnificus.


Assuntos
Vibrio vulnificus , Humanos , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo , Serina Proteases/genética , Serina Proteases/metabolismo , Escherichia coli/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo
9.
Structure ; 30(11): 1467-1469, 2022 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-36332609

RESUMO

In this issue of Structure, Maso et al. (2022) discover nanobodies that inhibit the SOS response of Escherichia coli by targeting the LexA repressor-protease. High-resolution structures of the novel LexA-nanobody complexes reveal they function by stabilizing LexA in its inactive conformation and preventing co-proteolysis by RecA∗.


Assuntos
Camelídeos Americanos , Resposta SOS em Genética , Animais , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Camelídeos Americanos/metabolismo , Lã/metabolismo , Proteínas de Bactérias/genética , Serina Endopeptidases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Bactérias/metabolismo
10.
Viruses ; 14(10)2022 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-36298651

RESUMO

SARS-CoV-2 cell-cell fusion and syncytiation is an emerging pathomechanism in COVID-19, but the precise factors contributing to the process remain ill-defined. In this study, we show that metalloproteases promote SARS-CoV-2 spike protein-induced syncytiation in the absence of established serine proteases using in vitro cell-cell fusion assays. We also show that metalloproteases promote S2'-activation of the SARS-CoV-2 spike protein, and that metalloprotease inhibition significantly reduces the syncytiation of SARS-CoV-2 variants of concern. In the presence of serine proteases, however, metalloprotease inhibition does not reduce spike protein-induced syncytiation and a combination of metalloprotease and serine protease inhibition is necessitated. Moreover, we show that the spike protein induces metalloprotease-dependent ectodomain shedding of the ACE2 receptor and that ACE2 shedding contributes to spike protein-induced syncytiation. These observations suggest a benefit to the incorporation of pharmacological inhibitors of metalloproteases into treatment strategies for patients with COVID-19.


Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Humanos , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Fusão Celular , Serina Endopeptidases/metabolismo , Metaloproteases , Serina Proteases
11.
Circ Res ; 131(11): 873-889, 2022 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-36263780

RESUMO

BACKGROUND: Activated macrophages contribute to the pathogenesis of vascular disease. Vein graft failure is a major clinical problem with limited therapeutic options. PCSK9 (proprotein convertase subtilisin/kexin 9) increases low-density lipoprotein (LDL)-cholesterol levels via LDL receptor (LDLR) degradation. The role of PCSK9 in macrophage activation and vein graft failure is largely unknown, especially through LDLR-independent mechanisms. This study aimed to explore a novel mechanism of macrophage activation and vein graft disease induced by circulating PCSK9 in an LDLR-independent fashion. METHODS: We used Ldlr-/- mice to examine the LDLR-independent roles of circulating PCSK9 in experimental vein grafts. Adeno-associated virus (AAV) vector encoding a gain-of-function mutant of PCSK9 (rAAV8/D377Y-mPCSK9) induced hepatic PCSK9 overproduction. To explore novel inflammatory targets of PCSK9, we used systems biology in Ldlr-/- mouse macrophages. RESULTS: In Ldlr-/- mice, AAV-PCSK9 increased circulating PCSK9, but did not change serum cholesterol and triglyceride levels. AAV-PCSK9 promoted vein graft lesion development when compared with control AAV. In vivo molecular imaging revealed that AAV-PCSK9 increased macrophage accumulation and matrix metalloproteinase activity associated with decreased fibrillar collagen, a molecular determinant of atherosclerotic plaque stability. AAV-PCSK9 induced mRNA expression of the pro-inflammatory mediators IL-1ß (interleukin-1 beta), TNFα (tumor necrosis factor alpha), and MCP-1 (monocyte chemoattractant protein-1) in peritoneal macrophages underpinned by an in vitro analysis of Ldlr-/- mouse macrophages stimulated with endotoxin-free recombinant PCSK9. A combination of unbiased global transcriptomics and new network-based hyperedge entanglement prediction analysis identified the NF-κB (nuclear factor-kappa B) signaling molecules, lectin-like oxidized LOX-1 (LDL receptor-1), and SDC4 (syndecan-4) as potential PCSK9 targets mediating pro-inflammatory responses in macrophages. CONCLUSIONS: Circulating PCSK9 induces macrophage activation and vein graft lesion development via LDLR-independent mechanisms. PCSK9 may be a potential target for pharmacologic treatment for this unmet medical need.


Assuntos
Ativação de Macrófagos , Pró-Proteína Convertase 9 , Animais , Camundongos , Colesterol , Lipoproteínas LDL/metabolismo , NF-kappa B , Pró-Proteína Convertase 9/genética , Receptores de LDL/genética , Receptores de LDL/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Subtilisinas
12.
Biomed Res Int ; 2022: 3865844, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36246974

RESUMO

Matriptases are cell surface proteolytic enzymes belonging to the type II transmembrane serine protease family that mediate inflammatory skin disorders and cancer progression. Matriptases may affect the development of periodontitis via protease-activated receptor-2 activity. However, the cellular mechanism by which matriptases are involved in periodontitis is unknown. In this study, we examined the antiperiodontitis effects of matriptase on Porphyromonas gingivalis-derived lipopolysaccharide (PG-LPS)-stimulated human gingival fibroblasts (HGFs). Matriptase small interfering RNA-transfected HGFs were treated with PG-LPS. The mRNA and protein levels of proinflammatory cytokines and matrix metalloproteinase 1 (MMP-1) were evaluated using the quantitative real-time polymerase chain reaction (qRT-PCR) and an enzyme-linked immunosorbent assay (ELISA), respectively. Western blot analyses were performed to measure the levels of Toll-like receptor 4 (TLR4)/interleukin-1 (IL-1) receptor-associated kinase (IRAK)/transforming growth factor ß-activated kinase 1 (TAK1), p65, and p50 in PG-LPS-stimulated HGFs. Matriptase downregulation inhibited LPS-induced proinflammatory cytokine expression, including the expression of IL-6, IL-8, tumor necrosis factor-α (TNF-α), and IL-Iß. Moreover, matriptase downregulation inhibited PG-LPS-stimulated MMP-1 expression. Additionally, we confirmed that the mechanism underlying the effects of matriptase downregulation involves the suppression of PG-LPS-induced IRAK1/TAK1 and NF-κB. These results suggest that downregulation of matriptase PG-LPS-induced MMP-1 and proinflammatory cytokine expression via TLR4-mediated IRAK1/TAK1 and NF-κB signaling pathways in HGFs.


Assuntos
Fibroblastos , Metaloproteinase 1 da Matriz , Periodontite , Serina Endopeptidases , Citocinas/metabolismo , Regulação para Baixo , Fibroblastos/metabolismo , Humanos , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipopolissacarídeos/toxicidade , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , NF-kappa B/metabolismo , Periodontite/genética , Periodontite/metabolismo , Porphyromonas gingivalis , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores Ativados por Proteinase/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
13.
Appl Microbiol Biotechnol ; 106(21): 7063-7072, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36195703

RESUMO

Protein expression with a fusion partner followed by the removal of the fusion partner via in vitro processing with a specific endoprotease is a favored method for the efficient production of intact recombinant proteins. Due to the high cost of commercial endoproteases, this process is restricted to laboratories. Kex2p is a membrane-bound serine protease that cleaves after dibasic residues of substrates in the late Golgi network. Although Kex2p is a very efficient endoprotease with exceptional specificity, it has not yet been used for the in vitro processing of fusion proteins due to its autolysis and high production cost. In this study, we developed an alternative endoprotease, autolysis-proof Kex2p, via site-directed mutagenesis of truncated KEX2 from Candida albicans (CaKEX2). Secretory production of manipulated CaKex2p was improved by employing target protein-specific translational fusion partner in Saccharomyces cerevisiae. The mass production of autolysis-proof Kex2p could facilitate the use of Kex2p for the large-scale production of recombinant proteins. KEY POINTS: • A soluble and active CaKex2p variant was produced by autocatalytic cleavage of the pro-peptide after truncation of C-terminus • Autolysis-proof CaKex2p was developed by site-directed mutagenesis • Secretion of autolysis-proof CaKex2p was improved by employing optimal translational fusion partner in Saccharomyces cerevisiae.


Assuntos
Proteínas Fúngicas , Pró-Proteína Convertases , Saccharomyces cerevisiae , Candida albicans/enzimologia , Candida albicans/genética , Peptídeo Hidrolases/metabolismo , Peptídeos/metabolismo , Pró-Proteína Convertases/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Serina Endopeptidases/metabolismo , Subtilisinas/metabolismo , Proteínas Fúngicas/biossíntese
14.
Structure ; 30(11): 1479-1493.e9, 2022 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-36240773

RESUMO

Antimicrobial resistance threatens the eradication of infectious diseases and impairs the efficacy of available therapeutics. The bacterial SOS pathway is a conserved response triggered by genotoxic stresses and represents one of the principal mechanisms that lead to resistance. The RecA recombinase acts as a DNA-damage sensor inducing the autoproteolysis of the transcriptional repressor LexA, thereby derepressing SOS genes that mediate DNA repair, survival to chemotherapy, and hypermutation. The inhibition of such pathway represents a promising strategy for delaying the evolution of antimicrobial resistance. We report the identification, via llama immunization and phage display, of nanobodies that bind LexA with sub-micromolar affinity and block autoproteolysis, repressing SOS response in Escherichia coli. Biophysical characterization of nanobody-LexA complexes revealed that they act by trapping LexA in an inactive conformation and interfering with RecA engagement. Our studies pave the way to the development of new-generation antibiotic adjuvants for the treatment of bacterial infections.


Assuntos
Resposta SOS em Genética , Anticorpos de Domínio Único , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Antibacterianos/farmacologia
15.
Int J Biol Macromol ; 222(Pt A): 1326-1342, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36242508

RESUMO

We recently described the production of a detergent-biocompatible crude protease from Streptomyces mutabilis strain TN-X30. Here, we describe the purification, characterization, and immobilization of the serine alkaline protease (named SPSM), as well as the cloning, sequencing, and over-expression of its corresponding gene (spSM). Pure enzyme was obtained after ammonium sulphate precipitation followed by heat-treatment and Sephacryl® S-200 column purification. The sequence of the first 26 NH2-terminal residues of SPSM showed a high sequence identity to subtilisin-like serine proteases produced by actinobacteria. The spSM gene was heterologously expressed in Escherichia coli BL21(DE3)pLysS and E. coli BL21-AI™ strains using pTrc99A (rSPSM) and Gateway™ pDEST™ 17 [(His)6-tagged SPSM] vectors, respectively. Results obtained indicated that the (His)6-tagged SPSM showed the highest stability. The SPSM was immobilized using encapsulation and adsorption-encapsulation approaches and three different carriers. Features of SPSM in soluble and immobilized forms were analyzed by Fourier transform infrared (FTIR) spectroscopy in attenuated total reflection (ATR) mode, X-ray diffraction (XRD), zeta potential measurements, and field emission scanning electron microscopy (FE-SEM). The white clay and kaolin used in this study are eco-friendly binders to alginate-SPSM and show great potential for application of the immobilized SPSM in various industries. Molecular modeling and docking of N-succinyl-l-Phe-l-Ala-l-Ala-l-Phe-p-nitroanilide in the active site of SPSM revealed the involvement of 21 amino acids in substrate binding.


Assuntos
Detergentes , Streptomyces , Simulação de Acoplamento Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Estabilidade Enzimática , Serina/genética , Proteínas de Bactérias/química , Serina Endopeptidases/metabolismo , Subtilisinas/metabolismo , Clonagem Molecular , Concentração de Íons de Hidrogênio
16.
ACS Chem Biol ; 17(9): 2631-2642, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36070465

RESUMO

Factor VII Activating protease (FSAP) has a protective effect in diverse disease conditions as inferred from studies in FSAP-/- mice and humans deficient in FSAP activity due to single-nucleotide polymorphism. The zymogen form of FSAP in plasma is activated by extracellular histones that are released during tissue injury or inflammation or by positively charged surfaces. However, it is not clear whether this activation mechanism is specific and amenable to manipulation. Using a phage display approach, we have identified a Cys-constrained 11 amino acid peptide, NNKC9/41, that activates pro-FSAP in plasma. The synthetic linear peptide has a propensity to cyclize through the terminal Cys groups, of which the antiparallel cyclic dimer, but not the monocyclic peptide, is the active component. Other commonly found zymogens in the plasma, related to the hemostasis system, were not activated. Binding studies with FSAP domain deletion mutants indicate that the N-terminus of FSAP is the key interaction site of this peptide. In a monoclonal antibody screen, we identified MA-FSAP-38C7 that prevented the activation of pro-FSAP by the peptide. This antibody bound to the LESLDP sequence (amino acids 30-35) in an intrinsically disordered stretch in the N-terminus of FSAP. The plasma clotting time was shortened by NNKC9/41, and this was reversed by MA-FSAP-38C7, demonstrating the utility of this peptide. Peptide NNKC9/41 will be useful as a tool to delineate the molecular mechanism of activation of pro-FSAP, elucidate its biological role, and provide a starting point for the pharmacological manipulation of FSAP activity.


Assuntos
Bacteriófagos , Fator VII , Animais , Humanos , Camundongos , Aminoácidos , Anticorpos Monoclonais/metabolismo , Bacteriófagos/metabolismo , Precursores Enzimáticos/metabolismo , Fator VII/metabolismo , Histonas , Peptídeo Hidrolases/metabolismo , Peptídeos/metabolismo , Serina Endopeptidases/metabolismo
17.
Proc Natl Acad Sci U S A ; 119(38): e2209514119, 2022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-36048924

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry starts with membrane attachment and ends with spike (S) protein-catalyzed membrane fusion depending on two cleavage steps, namely, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time three-dimensional single-virion tracking, we show that fusion and genome penetration require virion exposure to an acidic milieu of pH 6.2 to 6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2-overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2-expressing cells in the acidic milieu of the nasal cavity.


Assuntos
COVID-19 , Cavidade Nasal , SARS-CoV-2 , Serina Endopeptidases , Internalização do Vírus , COVID-19/virologia , Furina/genética , Furina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cavidade Nasal/química , Cavidade Nasal/virologia , SARS-CoV-2/fisiologia , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo
18.
Int Immunopharmacol ; 112: 109237, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36152535

RESUMO

Fibroblast activation protein (FAPα) is a tumor stromal antigen expressed by cancer-associated fibroblasts (CAFs) in more than 90 % of malignant epithelial carcinomas. FAPα-based immunotherapy has been reported and showed that FAPα-specific immune response can remold immune microenvironment and contribute to tumor regression. Many FAPα-based vaccines have been investigated in preclinical trials, which can elicit strong and durable cytolytic T lymphocytes (CTL) with good safety. However, epitope-based FAPα vaccines are rarely reported. To break tolerance against self-antigens, analogue epitopes with modified peptides at the anchor residues are typically used to improve epitope immunogenicity. To investigate the feasibility of a FAPα epitope-based vaccine for cancer immunotherapy in vivo, we conducted a preclinical study to identify a homologous CTL epitope of human and mouse FAPα and obtained its analogue epitope in BALB/c mice, and explored the anti-tumor activity of their minigene vaccines in 4 T1 tumor-bearing mice. By using in silico epitope prediction tools and immunogenicity assays, immunodominant epitope FAP.291 (YYFSWLTWV) and its analogue epitope FAP.291I9 (YYFSWLTWI) were identified. The FAP.291-based epitope minigene vaccine successfully stimulated CTLs targeting CAFs and exhibited anti-tumor activity in a 4 T1 murine breast cancer model. Furthermore, although the analogue epitope FAP.291I9 enhanced FAP.291-specific immune responses, improvement of anti-tumor immunity effects was not observed. Check of immunosuppressive factors revealed that the high levels of IL-10, IL-13, myeloid-derived suppressor cells and iNOS induced by FAP.291I9 increased, which considered the main cause of the failure of the analogue epitope-based vaccine. Thus, we demonstrated for the first time that the FAP.291 minigene vaccine could induce mouse CTLs and also function as a tumor regression antigen, providing the basis for future studies of FAPα epitope-based vaccines. This study may also be valuable for further improvement of the immunogenicity of analogue epitope vaccines.


Assuntos
Neoplasias da Mama , Vacinas Anticâncer , Camundongos , Humanos , Animais , Feminino , Gelatinases/metabolismo , Interleucina-10 , Serina Endopeptidases/metabolismo , Interleucina-13 , Epitopos , Epitopos Imunodominantes , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Camundongos Endogâmicos BALB C , Antígenos de Neoplasias , Imunidade , Autoantígenos , Microambiente Tumoral
19.
J Lipid Res ; 63(10): 100272, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36067830

RESUMO

Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) regulates the cell-surface localization of LDL receptors in hepatocytes and is associated with LDL and lipoprotein(a) [Lp(a)] uptake, reducing blood concentrations. However, the connection between PCSK9 and HDL is unclear. Here, we investigated the association of plasma PCSK9 with HDL subpopulations and examined the effects of PCSK9 on the atheroprotective function of HDL. We examined the association of PCSK9 with HDL in apoB-depleted plasma by ELISA, native PAGE, and immunoblotting. Our analyses showed that upon apoB-depletion, total circulating PCSK9 levels were 32% of those observed in normolipidemic plasma, and only 6% of PCSK9 in the apoB-depleted plasma, including both the mature and furin-cleaved forms, was associated with HDL. We also show human recombinant PCSK9 abolished the capacity of reconstituted HDL to reduce the formation of ROS in endothelial cells, while a PCSK9-blocking antibody enhanced the capacity of human HDL (in apoB-depleted plasma) to reduce ROS formation in endothelial cells and promote endothelial cell migration. Overall, our findings suggest that PCSK9 is only minimally associated with HDL particles, but PCSK9 in apoB-depleted plasma can affect the atheroprotective properties of HDL related to preservation of endothelial function. This study contributes to the elucidation of the pathophysiological role of plasma PCSK9 and highlights further the anti-atherosclerotic effect of PCSK9 inhibition.


Assuntos
Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Humanos , Apolipoproteínas B , Células Endoteliais/metabolismo , Furina , Lipoproteína(a) , Pró-Proteína Convertases/metabolismo , Espécies Reativas de Oxigênio , Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Subtilisinas
20.
Psychiatry Res ; 317: 114838, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36103758

RESUMO

Major depressive disorder (MDD) is a devastating mental illness and the leading cause of disability worldwide. Previous studies have suggested that synaptic plasticity in the hippocampus plays an important role in depression pathogenesis. Reelin is expressed mainly in the frontal lobe and hippocampus, and is closely associated with neurodevelopment and synaptic plasticity. However, few studies have investigated its role in MDD combining clinical trials and animal experiments. We show that in a clinical trial, plasma reelin levels decreased in patients with first-episode drug-naïve MDD and increased after treatment; further, plasma reelin levels allowed to distinguish drug-naïve patients with first-episode MDD from healthy individuals. In rats, chronic mild and unpredictable stress led to a decrease in both reelin mRNA and protein levels in the hippocampus, which could be reversed by vortioxetine. Subsequent experiments confirmed that the reelin-ApoER2-NR2A /NR2B pathway regulates hippocampal synaptic plasticity and may be involved in depression or antidepressant responses. Our work contributes to a deeper understanding of MDD pathogenesis and provides new evidence that reelin should be considered a potential therapeutic target for MDD.


Assuntos
Moléculas de Adesão Celular Neuronais , Transtorno Depressivo Maior , Animais , Ratos , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Depressão , Transtorno Depressivo Maior/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteína Reelina , Roedores/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Ensaios Clínicos como Assunto
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