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1.
Plant Physiol Biochem ; 186: 310-321, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-35932655

RESUMO

The serine carboxypeptidase-like protein (SCPL) family plays a key part in plant growth, development and stress responses. However, the serine carboxypeptidase-like (SCPL) proteins in Brassica napus L. (B. napus) have not been reported yet. Here, we identified a total of 117 putative SCPL genes in B. napus, which were unevenly distributed on all 19 chromosomes and were divided into three groups (carboxypeptidase Ⅰ to Ⅲ) according to their phylogenetic relationships. Synteny and duplication analysis revealed that the SCPL gene family of B. napus was amplified during allopolyploidization, in which the whole genome triplication and dispersed duplication played critical roles. After the separation of Brassica and Arabidopsis lineages, orthologous gene analysis showed that many SCPL genes were lost during the evolutionary process in B. rapa, B. oleracea and B. napus. Subsequently, the analyses of the gene structure, conserved motifs, cis-element and expression patterns showed that the members in the same group were highly conserved. Furthermore, candidate gene based association study suggested the role of BnSCPL52 in controlling seed number per silique, seed weight and silique length and a CAPS marker was developed to distinguish different haplotypes. Our results provide an overview of rapeseed SCPL genes that enable us for further functional research and benefit the marker-assisted breeding in Brassica napus.


Assuntos
Arabidopsis , Brassica napus , Arabidopsis/genética , Arabidopsis/metabolismo , Brassica napus/genética , Brassica napus/metabolismo , Carboxipeptidases/genética , Carboxipeptidases/metabolismo , Regulação da Expressão Gênica de Plantas , Genoma de Planta/genética , Família Multigênica , Filogenia , Melhoramento Vegetal , Proteínas de Plantas/metabolismo
2.
PLoS One ; 17(7): e0270920, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35867642

RESUMO

Angiotensin-converting enzyme 2 (ACE2) is the carboxypeptidase to degrade angiotensin II (Ang II) to angiotensin 1-7 (Ang 1-7) and improves the pathologies of cardiovascular disease and acute respiratory distress syndrome (ARDS)/acute lung injury. B38-CAP is a bacteria-derived ACE2-like carboxypeptidase as potent as human ACE2 and ameliorates hypertension, heart failure and SARS-CoV-2-induced lung injury in mice. Recombinant B38-CAP is prepared with E. coli protein expression system more efficiently than recombinant soluble human ACE2. Here we show therapeutic effects of B38-CAP on abdominal sepsis- or acid aspiration-induced acute lung injury. ACE2 expression was downregulated in the lungs of mice with cecal ligation puncture (CLP)-induced sepsis or acid-induced lung injury thereby leading to upregulation of Ang II levels. Intraperitoneal injection of B38-CAP significantly decreased Ang II levels while upregulated angiotensin 1-7 levels. B38-CAP improved survival rate of the mice under sepsis. B38-CAP suppressed the pathologies of lung inflammation, improved lung dysfunction and downregulated elevated cytokine mRNA levels in the mice with acute lung injury. Thus, systemic treatment with an ACE2-like enzyme might be a potential therapeutic strategy for the patients with severe sepsis or ARDS.


Assuntos
Lesão Pulmonar Aguda , COVID-19 , Síndrome do Desconforto Respiratório , Sepse , Lesão Pulmonar Aguda/patologia , Angiotensina II/metabolismo , Enzima de Conversão de Angiotensina 2 , Animais , Carboxipeptidases/metabolismo , Escherichia coli/metabolismo , Humanos , Pulmão/patologia , Camundongos , Peptidil Dipeptidase A/metabolismo , Sistema Renina-Angiotensina , Síndrome do Desconforto Respiratório/tratamento farmacológico , SARS-CoV-2 , Sepse/complicações , Sepse/tratamento farmacológico , Sepse/metabolismo
3.
Cancer Lett ; 544: 215802, 2022 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-35732215

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with poor prognosis. Its fibrotic tumor microenvironment (TME) plays a crucial role in promoting tumor invasion and metastasis, which eventually leads to a dismal 5-year survival rate in PDAC patients. Aortic carboxypeptidase-like protein (ACLP) promotes tissue fibrosis in benign diseases. However, its role in cancer-associated fibrosis remains unelucidated. Here, we show that ACLP was mainly expressed in cancer-associated fibroblasts (CAFs) but not in cancer cells and highly expressed in PDAC tissues. High ACLP expression was correlated with poor overall survival. Moreover, ACLP expression in PDAC patients with liver metastases was higher than that in PDAC patients without liver metastases. By detecting activation marker expression and CAF contractility and motility, we found that ACLP promoted CAF activation in PDAC, leading to TME fibrosis. Furthermore, ACLP-activated CAFs could promote cancer cell invasion in vitro and tumor metastasis in vivo. Mechanistically, ACLP promotes the expressions of MMP1 and MMP3 in CAFs, thus promoting PDAC invasion and metastasis. Intriguingly, we identified an ACLP-PPARγ-ACLP feedback loop in PDAC CAFs. Abatement of this feedback loop might be a promising approach in CAF-targeting PDAC treatment.


Assuntos
Fibroblastos Associados a Câncer , Carboxipeptidases/metabolismo , Carcinoma Ductal Pancreático , Neoplasias Hepáticas , Neoplasias Pancreáticas , Proteínas Repressoras/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Retroalimentação , Fibrose , Humanos , Neoplasias Hepáticas/patologia , PPAR gama/genética , PPAR gama/metabolismo , Neoplasias Pancreáticas/patologia , Microambiente Tumoral
4.
J Ind Microbiol Biotechnol ; 49(4)2022 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-35648451

RESUMO

D, D-carboxypeptidase DacA plays an important role in the synthesis and stabilization of Escherichia coli cell wall peptidoglycan. The production level of extracellular recombinant proteins in E. coli can be enhanced by high D, D-carboxypeptidase activity. Construction of expression systems under optimal promoters is one of the main strategies to realize high protein production in E. coli. In this study, the promoter PdacA-3 from DacA on the genome of E. coli BL21 (DE3) was verified to be efficient for recombinant green fluorescent protein using the plasmid mutant pET28a-PdacA with PdacA-3. Meanwhile, the promoter PdacA-3 was engineered to increase the production level of proteins via inserting one or two Shine-Dalgarno (SD) sequences between the promoter PdacA-3 and the target genes. The expression level of dacA on the genome was increased by the improved transcription of the engineered promoters (especially after inserting one additional SD sequence). The engineered promoters increased cell membrane permeabilities to significantly enhance the secretion production of extracellular recombinant proteins in E. coli. Among them, the extracellular recombinant amylase activities in E. coli BL21::1SD-pET28a-amyK and E. coli BL21::2SD-pET28a-amyK were increased by 2.0- and 1.6-fold that of the control (E. coli BL21-pET28a-amyK), respectively. Promoter engineering also affected the morphology and growth of the E. coli mutants. It was indicated that the engineered promoters enhanced the expression of dacA on the genome to disturb the synthesis and structural stability of cell wall peptidoglycans.


Assuntos
Escherichia coli , Peptidoglicano , Carboxipeptidases , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Peptidoglicano/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
5.
J Econ Entomol ; 115(4): 1285-1293, 2022 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-35640220

RESUMO

The M14 family metal carboxypeptidase genes play an important role in digestion and pathogenic infections in the gut of insects. However, the roles of these genes in Antheraea pernyi (Guérin-Méneville, 1855) remain to be analyzed. In the present study, we cloned a highly expressed M14 metal carboxypeptidase gene (ApMCP1) found in the gut and discovered that it contained a 1,194 bp open reading frame encoding a 397-amino acid protein with a predicted molecular weight of 45 kDa. Furthermore, 14 members of the M14 family metal carboxypeptidases (ApMCP1-ApMCP14) were identified in the A. pernyi genome, with typical Zn_pept domains and two Zn-anchoring motifs, and were further classified into M14A, M14B, and M14D subfamilies. Expression analysis indicated that ApMCP1 and ApMCP9 were mainly expressed in the gut. Additionally, we observed that ApMCP1 and ApMCP9 displayed opposite expression patterns after starvation, highlighting their functional divergence during digestion. Following natural infection with baculovirus NPV, their expression was significantly upregulated in the gut of A. pernyi. Our results suggest that the M14 family metal carboxypeptidase genes are conservatively digestive enzymes and evolutionarily involved in exogenous pathogenic infections.


Assuntos
Proteínas de Insetos , Mariposas , Animais , Carboxipeptidases/genética , Carboxipeptidases/metabolismo , Proteínas de Insetos/metabolismo , Insetos , Mariposas/genética , Mariposas/metabolismo
6.
Clin. transl. oncol. (Print) ; 24(5): 757-769, mayo 2022.
Artigo em Inglês | IBECS | ID: ibc-203779

RESUMO

Colorectal cancer (CRC) is one of the leading causes of mortality among cancers. Many aspects of this cancer are under investigation to find established markers of diagnosis, prognosis, and also potential drug targets. In this review article, we are going to discuss the possible solution to all these aims by investigating the literature about cancer-associated fibroblasts (CAFs) involved in CRC. Moreover, we are going to review their interaction with the tumor microenvironment (TME) and vitamin D and their role in tumorigenesis and metastasis. Moreover, we are going to expand more on some markers produced by them or related to them including FAP, a-SMA, CXCL12, TGF- β, POSTN, and β1-Integrin. Some signaling pathways related to CAFs are as follows: FAK, AKT, activin A, and YAP/TAZ. Some genes related to the CAFs which are found to be possible therapeutic targets include COL3A1, JAM3, AEBP1 and, CAF-derived TGFB3, WNT2, and WNT54.


Assuntos
Humanos , Fibroblastos Associados a Câncer/metabolismo , Carboxipeptidases/metabolismo , Carcinogênese/patologia , Neoplasias Colorretais/patologia , Microambiente Tumoral , Fibroblastos/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Crescimento Endotelial/metabolismo
7.
Protein Expr Purif ; 197: 106112, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35598696

RESUMO

CCP6 is a member of cytosolic carboxypeptidases (CCPs) family, an eraser of a reversible protein posttranslational modification - polyglutamylation, and represents a potential therapeutic target. Currently, production of CCPs mainly depends on eukaryotic expression system, which is time-consuming and costly. Here, we reported that mouse origin full-length CCP6 can be successfully expressed in the soluble fraction of bacteria ArcticExpress (DE3) strain. However, the recombinant mCCP6 was initially co-purified with Cpn60 in a stoichiometric ratio of roughly 1:7 and exhibited no enzyme activity. When coupled with a step to promote the release of the substrate protein from the chaperonins by treatment with ATP/Mg2+/K+, the recombinant CCP6 with deglutamylation activity was obtained, though still partially associated with Cpn60. This is the first report, to our knowledge, that the successful expression and purification of active recombinant mammalian CCPs using a bacterial system was achieved.


Assuntos
Carboxipeptidases , Escherichia coli , Animais , Carboxipeptidases/genética , Carboxipeptidases/isolamento & purificação , Carboxipeptidases/metabolismo , Chaperonina 60/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Mamíferos , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
8.
Plant J ; 111(1): 117-133, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35437852

RESUMO

Serine carboxypeptidase-like acyltransferases (SCPL-ATs) play a vital role in the diversification of plant metabolites. Galloylated flavan-3-ols highly accumulate in tea (Camellia sinensis), grape (Vitis vinifera), and persimmon (Diospyros kaki). To date, the biosynthetic mechanism of these compounds remains unknown. Herein, we report that two SCPL-AT paralogs are involved in galloylation of flavan-3-ols: CsSCPL4, which contains the conserved catalytic triad S-D-H, and CsSCPL5, which has the alternative triad T-D-Y. Integrated data from transgenic plants, recombinant enzymes, and gene mutations showed that CsSCPL4 is a catalytic acyltransferase, while CsSCPL5 is a non-catalytic companion paralog (NCCP). Co-expression of CsSCPL4 and CsSCPL5 is likely responsible for the galloylation. Furthermore, pull-down and co-immunoprecipitation assays showed that CsSCPL4 and CsSCPL5 interact, increasing protein stability and promoting post-translational processing. Moreover, phylogenetic analyses revealed that their homologs co-exist in galloylated flavan-3-ol- or hydrolyzable tannin-rich plant species. Enzymatic assays further revealed the necessity of co-expression of those homologs for acyltransferase activity. Evolution analysis revealed that the mutations of the CsSCPL5 catalytic residues may have taken place about 10 million years ago. These findings show that the co-expression of SCPL-ATs and their NCCPs contributes to the acylation of flavan-3-ols in the plant kingdom.


Assuntos
Diospyros , Vitis , Acilação , Aciltransferases/metabolismo , Carboxipeptidases/genética , Carboxipeptidases/metabolismo , Flavonoides , Filogenia , Plantas/metabolismo , Polifenóis , Vitis/metabolismo
9.
Science ; 376(6595): eabn6020, 2022 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-35482892

RESUMO

The detyrosination-tyrosination cycle involves the removal and religation of the C-terminal tyrosine of α-tubulin and is implicated in cognitive, cardiac, and mitotic defects. The vasohibin-small vasohibin-binding protein (SVBP) complex underlies much, but not all, detyrosination. We used haploid genetic screens to identify an unannotated protein, microtubule associated tyrosine carboxypeptidase (MATCAP), as a remaining detyrosinating enzyme. X-ray crystallography and cryo-electron microscopy structures established MATCAP's cleaving mechanism, substrate specificity, and microtubule recognition. Paradoxically, whereas abrogation of tyrosine religation is lethal in mice, codeletion of MATCAP and SVBP is not. Although viable, defective detyrosination caused microcephaly, associated with proliferative defects during neurogenesis, and abnormal behavior. Thus, MATCAP is a missing component of the detyrosination-tyrosination cycle, revealing the importance of this modification in brain formation.


Assuntos
Carboxipeptidases , Proteínas Associadas aos Microtúbulos , Microtúbulos , Processamento de Proteína Pós-Traducional , Tubulina (Proteína) , Tirosina , Animais , Carboxipeptidases/genética , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/química , Tubulina (Proteína)/química , Tirosina/química
10.
Biochem Biophys Res Commun ; 606: 17-22, 2022 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-35338854

RESUMO

The worldwide pandemic of Coronavirus disease 2019 (COVID-19) is triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and further worsened by the emergence of a variety of SARS-CoV-2 variants. Angiotensin-converting enzyme 2 (ACE2), a carboxypeptidase of M32 family, serves as the receptor of SARS-CoV-2 and key regulator of host renin-angiotensin system (RAS), both of which are mainly mediated via the carboxypeptidase domain of ACE2 (sACE2) or its activity. sACE2 is thus promising in the treatment of COVID-19 but unfortunately weakened by its unstrigent substrate preference and complex interplay with host RAS. B38-CAP, an isoenzyme of ACE2, partically compensates these defects but still encounters the problem related to carboxypeptidase activity and specificity. In this study, we firstly determined the crystal structure of B38-CAP at a resolution of 2.44 Å which exists in dimeric form with the non-crystallographic two-fold axis being in coincidence with the crystallographic two-fold axis. Further structural analysis revealed the structural conservatism feature among M32 family, particularly the catalytic core and moreover lead us to hypothesize that conformational flexibility might play an pivotal role in the catalysis of B38-CAP and ACE2. The work provided here presents key features of the M32 family carboxypeptidase and provides structural basis for further development of B38-CAP-based anti-SARS-CoV-2 drugs.


Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Enzima de Conversão de Angiotensina 2/química , Carboxipeptidases , Humanos , Isoenzimas , Sistema Renina-Angiotensina , SARS-CoV-2
11.
Clin Chim Acta ; 531: 4-11, 2022 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-35283094

RESUMO

BACKGROUND: COVID-19 patients experience several features of dysregulated immune system observed in sepsis. We previously showed a dysregulation of several proline-selective peptidases such as dipeptidyl peptidase 4 (DPP4), fibroblast activation protein alpha (FAP), prolyl oligopeptidase (PREP) and prolylcarboxypeptidase (PRCP) in sepsis. In this study, we investigated whether these peptidases are similarly dysregulated in hospitalized COVID-19 patients. METHODS: Fifty-six hospitalized COVID-19 patients and 32 healthy controls were included. Enzymatic activities of DPP4, FAP, PREP and PRCP were measured in samples collected shortly after hospital admission and in longitudinal follow-up samples. RESULTS: Compared to healthy controls, both DPP4 and FAP activities were significantly lower in COVID-19 patients at hospital admission and FAP activity further decreased significantly in the first week of hospitalization. While PRCP activity remained unchanged, PREP activity was significantly increased in COVID-19 patients at hospitalization and further increased during hospital stay and stayed elevated until the day of discharge. CONCLUSION: The changes in activities of proline-selective peptidases in plasma are very similar in COVID-19 and septic shock patients. The pronounced decrease in FAP activity deserves further investigation, both from a pathophysiological viewpoint and as its utility as a part of a biomarker panel.


Assuntos
COVID-19 , Choque Séptico , Carboxipeptidases , Dipeptidil Peptidase 4 , Endopeptidases , Gelatinases , Humanos , Proteínas de Membrana , Peptídeo Hidrolases , Prolina , Serina Endopeptidases
12.
Cells ; 11(3)2022 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-35159379

RESUMO

Carboxypeptidase A3 (CPA3) is a specific mast cell (MC) protease with variable expression. This protease is one of the preformed components of the secretome. During maturation of granules, CPA3 becomes an active enzyme with a characteristic localization determining the features of the cytological and ultrastructural phenotype of MC. CPA3 takes part in the regulation of a specific tissue microenvironment, affecting the implementation of innate immunity, the mechanisms of angiogenesis, the processes of remodeling of the extracellular matrix, etc. Characterization of CPA3 expression in MC can be used to refine the MC classification, help in a prognosis, and increase the effectiveness of targeted therapy.


Assuntos
Mastócitos , Peptídeo Hidrolases , Carboxipeptidases/metabolismo , Contagem de Células , Endopeptidases/metabolismo , Mastócitos/metabolismo , Peptídeo Hidrolases/metabolismo , Fenótipo
13.
Int J Mol Sci ; 23(4)2022 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-35216380

RESUMO

Left ventricular hypertrophy (LVH) is a major risk factor for adverse cardiovascular events. Recently, a novel candidate gene encoding the carboxypeptidase X member 2 (CPXM2) was found to be associated with hypertension-induced LVH. CPXM2 belongs to the M14 family of metallocarboxypeptidases, yet it lacks detectable enzyme activity, and its function remains unknown. Here, we investigated the impact of micro (mi)RNA-29b, miRNA-195, and miRNA-497 on the posttranscriptional expression control of CPXM2. Candidate miRNAs for CPXM2 expression control were identified in silico. CPXM2 expression in rat cardiomyocytes (H9C2) was characterized via real-time PCR, Western blotting, and immunofluorescence. Direct miRNA/target mRNA interaction was analysed by dual luciferase assay. CPXM2 was expressed in H9C2 and co-localised with z-disc associated protein PDZ and LIM domain 3 (Pdlim3). Transfection of H9C2 with miRNA-29b, miRNA-195, and miRNA-497 led to decreased levels of CPXM2 mRNA and protein, respectively. Results of dual luciferase assays revealed that miRNA-29b and miRNA-497, but not miRNA-195, directly regulated CPXM2 expression on a posttranscriptional level via binding to the 3'UTR of CPXM2 mRNA. We identified two miRNAs capable of the direct posttranscriptional expression control of CPXM2 expression in rat cardiomyocytes. This novel data may help to shed more light on the-so far-widely unexplored expression control of CPXM2 and its potential role in LVH.


Assuntos
Carboxipeptidases/genética , Hipertrofia Ventricular Esquerda/genética , MicroRNAs/genética , Regiões 3' não Traduzidas/genética , Animais , Células Cultivadas , Regulação da Expressão Gênica/genética , Hipertensão/genética , Miócitos Cardíacos/patologia , RNA Mensageiro/genética , Ratos
14.
PLoS Pathog ; 18(1): e1010235, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-35007297

RESUMO

The Epstein-Barr virus (EBV) BGLF2 protein is a tegument protein with multiple effects on the cellular environment, including induction of SUMOylation of cellular proteins. Using affinity-purification coupled to mass-spectrometry, we identified the miRNA-Induced Silencing Complex (RISC), essential for miRNA function, as a top interactor of BGLF2. We confirmed BGLF2 interaction with the Ago2 and TNRC6 components of RISC in multiple cell lines and their co-localization in cytoplasmic bodies that also contain the stress granule marker G3BP1. In addition, BGLF2 expression led to the loss of processing bodies in multiple cell types, suggesting disruption of RISC function in mRNA regulation. Consistent with this observation, BGLF2 disrupted Ago2 association with multiple miRNAs. Using let-7 miRNAs as a model, we tested the hypothesis that BGLF2 interfered with the function of RISC in miRNA-mediated mRNA silencing. Using multiple reporter constructs with 3'UTRs containing let-7a regulated sites, we showed that BGLF2 inhibited let-7a miRNA activity dependent on these 3'UTRs, including those from SUMO transcripts which are known to be regulated by let-7 miRNAs. In keeping with these results, we showed that BGLF2 increased the cellular level of unconjugated SUMO proteins without affecting the level of SUMO transcripts. Such an increase in free SUMO is known to drive SUMOylation and would account for the effect of BGLF2 in inducing SUMOylation. We further showed that BGLF2 expression inhibited the loading of let-7 miRNAs into Ago2 proteins, and conversely, that lytic infection with EBV lacking BGLF2 resulted in increased interaction of let-7a and SUMO transcripts with Ago2, relative to WT EBV infection. Therefore, we have identified a novel role for BGLF2 as a miRNA regulator and shown that one outcome of this activity is the dysregulation of SUMO transcripts that leads to increased levels of free SUMO proteins and SUMOylation.


Assuntos
Carboxipeptidases/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/metabolismo , Interações Hospedeiro-Parasita/fisiologia , MicroRNAs/metabolismo , Proteínas Virais de Fusão/metabolismo , Linhagem Celular , Infecções por Vírus Epstein-Barr/metabolismo , Humanos , Sumoilação
15.
Thromb Res ; 210: 78-86, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35030422

RESUMO

INTRODUCTION: Thrombolysis using recombinant tissue-type plasminogen activator (rt-PA) is the pharmacological treatment of choice in acute thrombotic events. However, a narrow therapeutic window and bleeding complications limit its use. We describe the role of carboxypeptidase inhibitor from potato tuber (PTCI), an inhibitor of activated thrombin-activatable fibrinolysis inhibitor (TAFIa), on Glu-plasminogen accumulation and microthrombus dynamics in vivo and demonstrate its influence on rt-PA-mediated thrombolysis. MATERIALS AND METHODS: In conjunction with real-time intravital two-photon excitation fluorescence microscopy, we produced and imaged laser-induced microthrombi in the mesenteric venules of Green Fluorescent Protein (GFP)-expressing mice. We examined microthrombus dynamics and thrombolysis patterns in vivo by measuring the changes in the fluorescence intensity of labeled Glu-plasminogen following administration of epsilon aminocaproic acid (EACA), PTCI, and rt-PA. RESULTS: PTCI enhanced Glu-plasminogen accumulation at the core of the thrombus by inhibiting TAFIa, while EACA inhibited this process. Exogenous rt-PA effectively triggered Glu-plasminogen activation within the thrombus and promoted thrombolysis. Administration of PTCI and rt-PA together showed no significant benefit on thrombolysis compared to rt-PA administration alone. However, early-phase systemic administration of PTCI before thrombolytic therapy by rt-PA expedited clot lysis as evidenced by significantly faster time to reach peak Glu-plasminogen fluorescence intensity and shorter time to achieve near-complete clot lysis (P = 0.014 and P = 0.003, respectively). CONCLUSIONS: PTCI potentiates rt-PA-mediated thrombolysis when administered early in acute thrombotic events. Further studies are warranted to explore the potential of TAFI inhibitors as adjunct agents in thrombolysis or thromboprophylaxis.


Assuntos
Carboxipeptidases/antagonistas & inibidores , Trombose , Tromboembolia Venosa , Animais , Anticoagulantes/uso terapêutico , Carboxipeptidase B2/antagonistas & inibidores , Fibrinólise , Humanos , Microscopia Intravital , Camundongos , Terapia Trombolítica , Trombose/tratamento farmacológico , Ativador de Plasminogênio Tecidual/farmacologia , Ativador de Plasminogênio Tecidual/uso terapêutico , Tromboembolia Venosa/tratamento farmacológico
16.
Clin Transl Oncol ; 24(5): 757-769, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-34839457

RESUMO

Colorectal cancer (CRC) is one of the leading causes of mortality among cancers. Many aspects of this cancer are under investigation to find established markers of diagnosis, prognosis, and also potential drug targets. In this review article, we are going to discuss the possible solution to all these aims by investigating the literature about cancer-associated fibroblasts (CAFs) involved in CRC. Moreover, we are going to review their interaction with the tumor microenvironment (TME) and vitamin D and their role in tumorigenesis and metastasis. Moreover, we are going to expand more on some markers produced by them or related to them including FAP, a-SMA, CXCL12, TGF- ß, POSTN, and ß1-Integrin. Some signaling pathways related to CAFs are as follows: FAK, AKT, activin A, and YAP/TAZ. Some genes related to the CAFs which are found to be possible therapeutic targets include COL3A1, JAM3, AEBP1 and, CAF-derived TGFB3, WNT2, and WNT54.


Assuntos
Fibroblastos Associados a Câncer , Neoplasias Colorretais , Fibroblastos Associados a Câncer/metabolismo , Carboxipeptidases/metabolismo , Carcinogênese/patologia , Neoplasias Colorretais/patologia , Fibroblastos/metabolismo , Humanos , Prognóstico , Proteínas Repressoras/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral
17.
Hum Mol Genet ; 31(7): 1082-1095, 2022 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-34850884

RESUMO

Osteonecrosis of the femoral head (ONFH) involves necrosis of bone and bone marrow of the femoral head caused by ischemia with unknown etiology. Previous genetic studies on ONFH failed to produce consistent results, presumably because ONFH has various causes with different genetic backgrounds and the underlying diseases confounded the associations. Steroid-associated ONFH (S-ONFH) accounts for one-half of all ONFH, and systemic lupus erythematosus (SLE) is a representative disease underlying S-ONFH. We performed a genome-wide association study (GWAS) to identify genetic risk factors for S-ONFH in patients with SLE. We conducted a two-staged GWAS on 636 SLE patients with S-ONFH and 95 588 non-SLE controls. Among the novel loci identified, we determined S-ONFH-specific loci by comparing allele frequencies between SLE patients without S-ONFH and non-SLE controls. We also used Korean datasets comprising 148 S-ONFH cases and 37 015 controls to assess overall significance. We evaluated the functional annotations of significant variants by in silico analyses. The Japanese GWAS identified 4 significant loci together with 12 known SLE susceptibility loci. The four significant variants showed comparable effect sizes on S-ONFH compared with SLE controls and non-SLE controls. Three of the four loci, MIR4293/MIR1265 [odds ratio (OR) = 1.99, P-value = 1.1 × 10-9)], TRIM49/NAALAD2 (OR = 1.65, P-value = 4.8 × 10-8) and MYO16 (OR = 3.91, P-value = 4.9 × 10-10), showed significant associations in the meta-analysis with Korean datasets. Bioinformatics analyses identified MIR4293, NAALAD2 and MYO16 as candidate causal genes. MIR4293 regulates a PPARG-related adipogenesis pathway relevant to S-ONFH. We identified three novel susceptibility loci for S-ONFH in SLE.


Assuntos
Necrose da Cabeça do Fêmur , Lúpus Eritematoso Sistêmico , Esteroides , Carboxipeptidases/genética , Proteínas de Transporte/genética , Cabeça do Fêmur , Necrose da Cabeça do Fêmur/induzido quimicamente , Necrose da Cabeça do Fêmur/complicações , Necrose da Cabeça do Fêmur/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Lúpus Eritematoso Sistêmico/genética , MicroRNAs/genética , Cadeias Pesadas de Miosina/genética , Polimorfismo de Nucleotídeo Único , Esteroides/efeitos adversos
18.
J Thromb Haemost ; 20(1): 238-244, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34626062

RESUMO

BACKGROUND: Kallikrein is generated when the contact system is activated, subsequently cleaving high molecular weight kininogen to bradykinin (BK). BK binds to bradykinin receptor 2, causing vascular leakage. BK is inactivated by proteolysis by the plasma carboxypeptidase B2 and N (CPB2 and CPN). CPN is constitutively active but CPB2 is generated from its zymogen, proCPB2. OBJECTIVES: Determine the role of CPB2 and CPN in the regulation of vascular leakage. METHODS: Mice deficient in CPB2, CPN, or both (Cpb2-/- , Cpn-/- , and Cpb2-/- /Cpn-/- ) were compared with wild-type mice (WT) in a model of vascular leakage caused by skin irritation. In some experiments, mice were pretreated with antibodies that prevent activation of proCPB2. RESULTS: Skin irritation increased vascular leakage most in Cpb2-/- /Cpn-/- , less in Cpb2-/- and Cpn-/- , and least in WT mice. There was no difference in vascular leakage without the challenge. Antibodies inhibiting activation of proCPB2 by plasmin, but not by the thrombin/thrombomodulin complex, increased vascular leakage to the level seen in Cpb2-/- mice. There was no change in levels of markers of coagulation and fibrinolysis. CONCLUSIONS: Bradykinin is inactivated by both CPB2 and CPN independently. Plasmin is the activator of proCPB2 in this model. Mice lacking both plasma carboxypeptidases have more vascular leak than those lacking either alone. Although BK levels were not determined, BK is the likely substrate for CPB2 and CPN in this model.


Assuntos
Carboxipeptidase B2 , Animais , Carboxipeptidases/genética , Fibrinolisina/metabolismo , Fibrinólise , Lisina Carboxipeptidase/genética , Camundongos
19.
Int J Biol Macromol ; 198: 77-86, 2022 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-34963626

RESUMO

This study aimed to partially characterize the three main serine carboxypeptidases (SCP3, SCP20, and SCP47) from Nepenthes mirabilis. Furthermore, one peptidase (SCP3) was chosen for further heterologous expression in Escherichia coli Shuffle®T7. SCP3 also was characterized in terms of its allergenic potential using bioinformatics tools. SCP3, SCP20, and SCP47 showed very similar 3D structures and mechanistic features to other plant serine peptidases belonging to clan SC and family S10. Although SCP3 was obtained in its soluble form, using 1% ethanol during induction with 0.5 mM IPTG at 16 °C for 18 h, it did not show proteolytic activity by zymography or in vitro analysis. SCP3 presented a few allergenic peptides and several cleavage sites for digestive enzymes. This work describes additional features of these enzymes, opening new perspectives for further studies for characterization and analysis of heterologous expression, as well as their potential biotechnological applications.


Assuntos
Carboxipeptidases
20.
Hypertens Res ; 45(2): 292-307, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34916661

RESUMO

Treatment of hypertension-mediated cardiac damage with left ventricular (LV) hypertrophy (LVH) and heart failure remains challenging. To identify novel targets, we performed comparative transcriptome analysis between genetic models derived from stroke-prone spontaneously hypertensive rats (SHRSP). Here, we identified carboxypeptidase X 2 (Cpxm2) as a genetic locus affecting LV mass. Analysis of isolated rat cardiomyocytes and cardiofibroblasts indicated Cpxm2 expression and intrinsic upregulation in genetic hypertension. Immunostaining indicated that CPXM2 associates with the t-tubule network of cardiomyocytes. The functional role of Cpxm2 was further investigated in Cpxm2-deficient (KO) and wild-type (WT) mice exposed to deoxycorticosterone acetate (DOCA). WT and KO animals developed severe and similar systolic hypertension in response to DOCA. WT mice developed severe LV damage, including increases in LV masses and diameters, impairment of LV systolic and diastolic function and reduced ejection fraction. These changes were significantly ameliorated or even normalized (i.e., ejection fraction) in KO-DOCA animals. LV transcriptome analysis showed a molecular cardiac hypertrophy/remodeling signature in WT but not KO mice with significant upregulation of 1234 transcripts, including Cpxm2, in response to DOCA. Analysis of endomyocardial biopsies from patients with cardiac hypertrophy indicated significant upregulation of CPXM2 expression. These data support further translational investigation of CPXM2.


Assuntos
Hipertensão , Animais , Carboxipeptidases , Cardiomegalia/genética , Humanos , Hipertrofia Ventricular Esquerda , Camundongos , Miócitos Cardíacos , Ratos
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