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1.
Pestic Biochem Physiol ; 202: 105936, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38879328

RESUMO

The fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), is one of the most destructive agricultural pests. The entomopathogenic fungus Beauveria bassiana (Hypocreales: Clavicipitaceae) is a biopesticide widely used for biocontrol of various pests. Secreted fungal proteases are critical for insect cuticle destruction and successful infection. We have previously shown that the serine protease BbAorsin in B. bassiana has entomopathogenic and antiphytopathogenic activities. However, the contribution of BbAorsin to fungal growth, conidiation, germination, virulence and antiphytopathogenic activities remains unclear. In this study, the deletion (ΔBbAorsin), complementation (Comp), and overexpression (BbAorsinOE) strains of B. bassiana were generated for comparative studies. The results showed that ΔBbAorsin exhibited slower growth, reduced conidiation, lower germination rate, and longer germination time compared to WT and Comp. In contrast, BbAorsinOE showed higher growth rate, increased conidiation, higher germination rate and shorter germination time. Injection of BbAorsinOE showed the highest virulence against S. frugiperda larvae, while injection of ΔBbAorsin showed the lowest virulence. Feeding BbAorsinOE resulted in lower pupation and adult eclosion rates and malformed adults. 16S rRNA sequencing revealed no changes in the gut microbiota after feeding either WT or BbAorsinOE. However, BbAorsinOE caused a disrupted midgut, leakage of gut microbiota into the hemolymph, and upregulation of apoptosis and immunity-related genes. BbAorsin can disrupt the cell wall of the phytopathogen Fusarium graminearum and alleviate symptoms in wheat seedlings and cherry tomatoes infected with F. graminearum. These results highlight the importance of BbAorsin for B. bassiana and its potential as a multifunctional biopesticide.


Assuntos
Beauveria , Beauveria/patogenicidade , Beauveria/genética , Beauveria/fisiologia , Animais , Virulência , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Spodoptera/microbiologia , Esporos Fúngicos , Larva/microbiologia , Serina Proteases/metabolismo , Serina Proteases/genética , Controle Biológico de Vetores , Fusarium/patogenicidade , Fusarium/genética
2.
Am J Pathol ; 194(7): 1162-1170, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38880601

RESUMO

The placenta plays a crucial role in pregnancy success. ΔNp63α (p63), a transcription factor from the TP53 family, is highly expressed in villous cytotrophoblasts (CTBs), the epithelial stem cells of the human placenta, and is involved in CTB maintenance and differentiation. We examined the mechanisms of action of p63 by identifying its downstream targets. Gene expression changes were evaluated following overexpression and knockdown of p63 in the JEG3 choriocarcinoma cell line, using microarray-based RNA profiling. High-temperature requirement A4 (HTRA4), a placenta-specific serine protease involved in trophoblast differentiation and altered in preeclampsia, was identified as a gene reciprocally regulated by p63, and its expression was characterized in primary human placental tissues by RNA-sequencing and in situ hybridization. Potential p63 DNA-binding motifs were identified in the HTRA4 promoter, and p63 occupancy at some of these sites was confirmed using chromatin immunoprecipitation, followed by quantitative PCR in both JEG3 and trophoblast stem cells. These data begin to identify members of the transcriptional network downstream of p63, thus laying the groundwork for probing mechanisms by which this important transcription factor regulates trophoblast stemness and differentiation.


Assuntos
Fatores de Transcrição , Trofoblastos , Humanos , Trofoblastos/metabolismo , Feminino , Gravidez , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Serina Endopeptidases/metabolismo , Serina Endopeptidases/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , Placenta/metabolismo , Serina Proteases/metabolismo , Serina Proteases/genética , Regiões Promotoras Genéticas/genética , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Transcrição Gênica
3.
Cancer Lett ; 596: 217004, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38838765

RESUMO

Long non-coding RNA (lncRNA) is closely related to a variety of human cancers, which may provide huge potential biomarkers for cancer diagnosis and treatment. However, the aberrant expression of most lncRNAs in colorectal cancer (CRC) remains elusive. This study aims to explore the clinical significance and potential mechanism of lncRNA ABHD11 antisense RNA 1 (ABHD11-AS1) in the colorectal cancer. Here, we demonstrated that lncRNA ABHD11-AS1 is high-expressed in colorectal cancer (CRC) patients, and strongly related with poor prognosis. Functionally, ABHD11-AS1 suppresses ferroptosis and promotes proliferation and migration in CRC both in vitro and in vivo. Mechanically, lncRNA ABHD11-AS1 interacted with insulin-like growing factor 2 mRNA-binding protein 2 (IGF2BP2) to enhance FOXM1 stability, forming an ABHD11-AS1/FOXM1 positive feedback loop. E3 ligase tripartite motif containing 21 (TRIM21) promotes the degradation of IGF2BP2 via the K48-ubiquitin-lysosome pathway and ABHD11-AS1 promotes the interaction between IGF2BP2 and TRIM21 as scaffold platform. Furthermore, N6 -adenosine-methyltransferase-like 3 (METTL3) upregulated the stabilization of ABHD11-AS1 through the m6A reader IGF2BP2. Our study highlights ABHD11-AS1 as a significant regulator in CRC and it may become a potential target in future CRC treatment.


Assuntos
Neoplasias Colorretais , Ferroptose , Proteína Forkhead Box M1 , Regulação Neoplásica da Expressão Gênica , RNA Longo não Codificante , Proteínas de Ligação a RNA , Ribonucleoproteínas , Humanos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Ferroptose/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proliferação de Células , Animais , Camundongos , Retroalimentação Fisiológica , Progressão da Doença , Linhagem Celular Tumoral , Masculino , Movimento Celular/genética , Feminino , Camundongos Nus , Prognóstico , Adenosina/análogos & derivados , Serina Proteases
4.
World J Microbiol Biotechnol ; 40(7): 224, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38822201

RESUMO

Nematophagous fungi have been widely evaluated in the biological control of parasitic helminths in animals, both through their direct use and the use of their derived products. Fungal bioproducts can include extracellular enzymes, silver nanoparticles (AgNPs), as well as secondary metabolites. The aim of this study was to conduct a systematic review covering the evaluation of products derived from nematophagous fungi in the biological control of parasitic helminths in animals. In total, 33 studies met the inclusion criteria and were included in this review. The majority of the studies were conducted in Brazil (72.7%, 24/33), and bioproducts derived from the fungus Duddingtonia flagrans were the most commonly evaluated (36.3%, 12/33). The studies involved the production of extracellular enzymes (48.4%, 16/33), followed by crude enzymatic extract (27.2%, 9/33), secondary metabolites (15.1%, 5/33) and biosynthesis of AgNPs (9.1%, 3/33). The most researched extracellular enzymes were serine proteases (37.5%, 6/16), with efficacies ranging from 23.9 to 85%; proteases (31.2%, 5/16), with efficacies from 41.4 to 95.4%; proteases + chitinases (18.7%, 3/16), with efficacies from 20.5 to 43.4%; and chitinases (12.5%, 2/16), with efficacies ranging from 12 to 100%. In conclusion, extracellular enzymes are the most investigated derivatives of nematophagous fungi, with proteases being promising strategies in the biological control of animal helminths. Further studies under in vivo and field conditions are needed to explore the applicability of these bioproducts as tools for biological control.


Assuntos
Helmintos , Animais , Agentes de Controle Biológico/metabolismo , Brasil , Duddingtonia/metabolismo , Fungos/metabolismo , Nanopartículas Metálicas/química , Controle Biológico de Vetores/métodos , Serina Proteases/metabolismo , Prata/metabolismo
5.
Int J Biol Macromol ; 270(Pt 1): 132286, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38735612

RESUMO

Microbial proteases have proven their efficiency in various industrial applications; however, their application in accelerating the wound healing process has been inconsistent in previous studies. In this study, heterologous expression was used to obtain an over-yielding of the serine alkaline protease. The serine protease-encoding gene aprE was isolated from Bacillus safensis lab 418 and expressed in E. coli BL21 (DE3) using the pET28a (+) expression vector. The gene sequence was assigned the accession number OP610065 in the NCBI GenBank. The open reading frame of the recombinant protease (aprEsaf) was 383 amino acids, with a molecular weight of 35 kDa. The yield of aprEsaf increased to 300 U/mL compared with the native serine protease (SAFWD), with a maximum yield of 77.43 U/mL after optimization conditions. aprEsaf was immobilized on modified amine-functionalized films (MAFs). By comparing the biochemical characteristics of immobilized and free recombinant enzymes, the former exhibited distinctive biochemical characteristics: improved thermostability, alkaline stability over a wider pH range, and efficient reusability. The immobilized serine protease was effectively utilized to expedite wound healing. In conclusion, our study demonstrates the suitability of the immobilized recombinant serine protease for wound healing, suggesting that it is a viable alternative therapeutic agent for wound management.


Assuntos
Bacillus , Proteínas de Bactérias , Clonagem Molecular , Endopeptidases , Estabilidade Enzimática , Enzimas Imobilizadas , Proteínas Recombinantes , Cicatrização , Clonagem Molecular/métodos , Cicatrização/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Bacillus/enzimologia , Bacillus/genética , Endopeptidases/genética , Endopeptidases/química , Endopeptidases/metabolismo , Endopeptidases/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/isolamento & purificação , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Serina Proteases/genética , Serina Proteases/química , Serina Proteases/isolamento & purificação , Serina Proteases/metabolismo , Concentração de Íons de Hidrogênio , Expressão Gênica , Escherichia coli/genética , Temperatura , Sequência de Aminoácidos
6.
Microb Pathog ; 192: 106683, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38735447

RESUMO

Bacteria possess the ability to develop diverse and ingenious strategies to outwit the host immune system, and proteases are one of the many weapons employed by bacteria. This study sought to identify S. agalactiae additional serine protease and determine its role in virulence. The S. agalactiae THN0901 genome features one S8 family serine peptidase B (SfpB), acting as a secreted and externally exposed entity. A S8 family serine peptidase mutant strain (ΔsfpB) and complement strain (CΔsfpB) were generated through homologous recombination. Compared to the wild-type strain THN0901, the absorption of EtBr dyes was significantly reduced (P < 0.01) in ΔsfpB, implying an altered cell membrane permeability. In addition, the ΔsfpB strain had a significantly lower survival rate in macrophages (P < 0.01) and a 61.85 % lower adhesion ability to the EPC cells (P < 0.01) compared to THN0901. In the in vivo colonization experiment using tilapia as a model, 210 fish were selected and injected with different bacterial strains at a concentration of 3 × 106 CFU/tail. At 6, 12, 24, 48, 72 and 96 h post-injection, three fish were randomly selected from each group and their brain, liver, spleen, and kidney tissues were isolated. Subsequently, it was demonstrated that the ΔsfpB strain exhibited a markedly diminished capacity for colonization in tilapia. Additionally, the cumulative mortality of ΔsfpB in fish after intraperitoneal injection was reduced by 19.92-23.85 %. In conclusion, the findings in this study have demonstrated that the SfpB plays a significant role in S. agalactiae cell membrane stability and immune evasion. The immune evasion is fundamental for the development and transmission of invasive diseases, the serine protease SfpB may be a promising candidate for the development of antimicrobial agents to reduce the transmission of S. agalactiae.


Assuntos
Membrana Celular , Doenças dos Peixes , Evasão da Resposta Imune , Infecções Estreptocócicas , Streptococcus agalactiae , Streptococcus agalactiae/genética , Streptococcus agalactiae/patogenicidade , Streptococcus agalactiae/enzimologia , Streptococcus agalactiae/imunologia , Animais , Virulência , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/imunologia , Membrana Celular/metabolismo , Doenças dos Peixes/microbiologia , Doenças dos Peixes/imunologia , Aderência Bacteriana , Macrófagos/microbiologia , Macrófagos/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Serina Proteases/genética , Serina Proteases/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Camundongos
7.
Microb Pathog ; 192: 106706, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38763316

RESUMO

Mannheimiahaemolytica is an opportunistic agent of the respiratory tract of bovines, a member of the Pasteurellaceae family, and the causal agent of fibrinous pleuropneumonia. This bacterium possesses different virulence factors, allowing it to colonize and infect its host. The present work describes the isolation and characterization of a serine protease secreted by M. haemolytica serotype 1. This protease was isolated from M. haemolytica cultured media by precipitation with 50 % methanol and ion exchange chromatography on DEAE-cellulose. It is a 70-kDa protease able to degrade sheep and bovine fibrinogen or porcine gelatin but not bovine IgG, hemoglobin, or casein. Mass spectrometric analysis indicates its identity with protease IV of M. haemolytica. The proteolytic activity was active between pH 5 and 9, with an optimal pH of 8. It was stable at 50 °C for 10 min but inactivated at 60 °C. The sera of bovines with chronic or acute pneumonia recognized this protease. Still, it showed no cross-reactivity with rabbit hyperimmune serum against the secreted metalloprotease from Actinobacilluspleuropneumoniae, another member of the Pasteurellaceae family. M. haemolytica secreted proteases could contribute to the pathogenesis of this bacterium through fibrinogen degradation, a characteristic of this fibrinous pleuropneumonia.


Assuntos
Fibrinogênio , Mannheimia haemolytica , Serina Proteases , Animais , Mannheimia haemolytica/enzimologia , Ovinos , Bovinos , Fibrinogênio/metabolismo , Concentração de Íons de Hidrogênio , Serina Proteases/metabolismo , Serina Proteases/isolamento & purificação , Temperatura , Proteólise , Peso Molecular , Gelatina/metabolismo , Estabilidade Enzimática , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/isolamento & purificação , Espectrometria de Massas , Cromatografia por Troca Iônica , Suínos , Fatores de Virulência/metabolismo , Fatores de Virulência/isolamento & purificação
8.
Placenta ; 152: 53-64, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38805949

RESUMO

INTRODUCTION: The placenta differs greatly among species, and deep extra-villous trophoblast (EVT) invasion is a unique feature of placentation of higher primates including humans. We reported serine protease HtrA4 being found predominantly in human placentas with aberrant expression linked to preeclampsia. However, it remains unclear where HtrA4 is produced in the placenta, how it is expressed in other species, and whether it is essential for human placentation. METHODS: We first compared HtrA4 protein sequences of over 100 species, then scrutinized the key characteristics of HtrA4 in the human, rhesus macaque and mouse, and determined cellular localization in the placenta. We next investigated functional significance of HtrA4 in EVT differentiation using human trophoblast stem cells (TSCs). RESULTS: Across broader species HtrA4 is well conserved only in higher primates. In humans, only the placenta expressed HtrA4, localising to trophoblasts of villous as well as extra-villous lineages. Rhesus macaques produced HtrA4 but again only in placentas, whereas mice showed no abundant HtrA4 expression anywhere including the placenta, yet it was an active protease if produced. The functional importance of HtrA4 in human EVT was demonstrated using TSCs, which expressed low levels of HtrA4 but significantly up-regulated it during EVT differentiation, and knockdown of HtrA4 severely inhibited the differentiation process. DISCUSSION: HtrA4 is expressed in placentas of humans and macaques but not mice; it is critical for human EVT differentiation. Together with previous reports showing HtrA4 is also indispensable for syncytialization, this study further revealed HtrA4 as a functionally important protease for human placentation.


Assuntos
Diferenciação Celular , Macaca mulatta , Serina Endopeptidases , Trofoblastos , Animais , Trofoblastos/metabolismo , Humanos , Feminino , Gravidez , Diferenciação Celular/fisiologia , Camundongos , Serina Endopeptidases/metabolismo , Serina Endopeptidases/genética , Placenta/metabolismo , Placentação/fisiologia , Serina Proteases
9.
Arq Neuropsiquiatr ; 82(5): 1-8, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38763144

RESUMO

BACKGROUND: Neuronal ceroid lipofuscinoses (NCL) are a group of autosomal recessive, inherited, lysosomal, and neurodegenerative diseases that causes progressive dementia, seizures, movement disorders, language delay/regression, progressive visual failure, and early death. Neuronal ceroid lipofuscinosis type 2 (CLN2), caused by biallelic pathogenic variants of the TPP1 gene, is the only NCL with an approved targeted therapy. The laboratory diagnosis of CLN2 is established through highly specific tests, leading to diagnostic delays and eventually hampering the provision of specific treatment for patients with CLN2. Epilepsy is a common and clinically-identifiable feature among NCLs, and seizure onset is the main driver for families to seek medical care. OBJECTIVE: To evaluate the results of the Latin America Epilepsy and Genetics Program, an epilepsy gene panel, as a comprehensive tool for the investigation of CLN2 among other genetic causes of epilepsy. METHODS: A total of 1,284 patients with epilepsy without a specific cause who had at least 1 symptom associated with CLN2 were screened for variants in 160 genes associated with epilepsy or metabolic disorders presenting with epilepsy through an epilepsy gene panel. RESULTS: Variants of the TPP1 gene were identified in 25 individuals (1.9%), 21 of them with 2 variants. The 2 most frequently reported variants were p.Arg208* and p.Asp276Val, and 2 novel variants were detected in the present study: p.Leu308Pro and c.89 + 3G > C Intron 2. CONCLUSION: The results suggest that these genetic panels can be very useful tools to confirm or exclude CLN2 diagnosis and, if confirmed, provide disease-specific treatment for the patients.


ANTECEDENTES: As lipofuscinoses ceroides neuronais (neuronal ceroid lipofuscinoses, NCLs, em inglês) são um grupo de doenças autossômicas recessivas, hereditárias, lisossomais e neurodegenerativas que causam demência progressiva, crises epiléticas, distúrbios de movimento, atraso/regressão da linguagem, deficiência visual progressiva e morte precoce. A lipofuscinose ceroide neuronal tipo 2 (neuronal ceroid lipofuscinosis type 2, CLN2, em inglês), causada por variantes patogênicas bialélicas do gene TPP1, é a única com terapia-alvo aprovada. O diagnóstico laboratorial é realizado por testes específicos, o que leva a atrasos diagnósticos e, consequentemente, prejudica a disponibilização de tratamento. A epilepsia é uma característica comum e clinicamente identificável entre as NCLs, e o início das convulsões é o principal motivo para as famílias buscarem atendimento médico. OBJETIVO: Avaliar os resultados do Programa de Epilepsia e Genética da América Latina, um painel genético, como uma ferramenta abrangente para a investigação de CLN2 entre outras causas genéticas de epilepsia. MéTODOS: Um total de 1.284 pacientes com epilepsia sem uma causa específica e que tinham pelo menos 1 sintoma associado à CLN2 foram rastreados em busca de variantes em 160 genes associados à epilepsia ou a distúrbios metabólicos que apresentam epilepsia, por meio de um painel genético. RESULTADOS: Variantes do gene TPP1 foram identificadas em 25 indivíduos (1,9%), sendo que ; 21 apresentavam duas variantes. As duas variantes mais frequentes foram p.Arg208* e p.Asp276Val, e duas variantes novas foram detectadas neste: p.Leu308Pro e c.89 + 3G > C Intron 2. CONCLUSãO: Os resultados sugerem que os painéis genéticos de epilepsia podem ser uma ferramenta útil para confirmar ou excluir o diagnóstico de CLN2 e, se confirmado, fornecer tratamento específico para os pacientes.


Assuntos
Aminopeptidases , Epilepsia , Lipofuscinoses Ceroides Neuronais , Serina Proteases , Tripeptidil-Peptidase 1 , Humanos , Lipofuscinoses Ceroides Neuronais/genética , Feminino , Masculino , Epilepsia/genética , Aminopeptidases/genética , Serina Proteases/genética , Criança , Adolescente , Adulto , Adulto Jovem , Pré-Escolar , Proteínas de Ligação a Telômeros/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Mutação , Testes Genéticos/métodos , Pessoa de Meia-Idade , Lactente
10.
Parasit Vectors ; 17(1): 242, 2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38812022

RESUMO

BACKGROUND: Proteases produced by Acanthamoeba spp. play an important role in their virulence and may be the key to understanding Acanthamoeba pathogenesis; thus, increasing attention has been directed towards these proteins. The present study aimed to investigate the lytic factors produced by Acanthamoeba castellanii during the first hours of in vitro co-culture with human corneal epithelial cells (HCECs). METHODS: We used one old and one recent Acanthamoeba isolate, both from patients with severe keratitis, and subsets of these strains with enhanced pathogenic potential induced by sequential passaging over HCEC monolayers. The proteolytic profiles of all strains and substrains were examined using 1D in-gel zymography. RESULTS: We observed the activity of additional proteases (ranging from 33 to 50 kDa) during the early interaction phase between amoebae and HCECs, which were only expressed for a short time. Based on their susceptibilities to protease inhibitors, these proteases were characterized as serine proteases. Protease activities showed a sharp decline after 4 h of co-incubation. Interestingly, the expression of Acanthamoeba mannose-binding protein did not differ between amoebae in monoculture and those in co-culture. Moreover, we observed the activation of matrix metalloproteinases in HCECs after contact with Acanthamoeba. CONCLUSIONS: This study revealed the involvement of two novel serine proteases in Acanthamoeba pathogenesis and suggests a pivotal role of serine proteases during Acanthamoeba-host cell interaction, contributing to cell adhesion and lysis.


Assuntos
Acanthamoeba castellanii , Técnicas de Cocultura , Células Epiteliais , Epitélio Corneano , Peptídeo Hidrolases , Humanos , Acanthamoeba castellanii/enzimologia , Acanthamoeba castellanii/genética , Células Epiteliais/parasitologia , Epitélio Corneano/parasitologia , Epitélio Corneano/enzimologia , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/genética , Ceratite por Acanthamoeba/parasitologia , Serina Proteases/metabolismo , Serina Proteases/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Virulência
11.
Int J Mol Sci ; 25(10)2024 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-38791530

RESUMO

Neutrophil-derived proteases are critical to the pathology of many inflammatory lung diseases, both chronic and acute. These abundant enzymes play roles in key neutrophil functions, such as neutrophil extracellular trap formation and reactive oxygen species release. They may also be released, inducing tissue damage and loss of tissue function. Historically, the neutrophil serine proteases (NSPs) have been the main subject of neutrophil protease research. Despite highly promising cell-based and animal model work, clinical trials involving the inhibition of NSPs have shown mixed results in lung disease patients. As such, the cutting edge of neutrophil-derived protease research has shifted to proteases that have had little-to-no research in neutrophils to date. These include the cysteine and serine cathepsins, the metzincins and the calpains, among others. This review aims to outline the previous work carried out on NSPs, including the shortcomings of some of the inhibitor-orientated clinical trials. Our growing understanding of other proteases involved in neutrophil function and neutrophilic lung inflammation will then be discussed. Additionally, the potential of targeting these more obscure neutrophil proteases will be highlighted, as they may represent new targets for inhibitor-based treatments of neutrophil-mediated lung inflammation.


Assuntos
Neutrófilos , Pneumonia , Humanos , Neutrófilos/metabolismo , Neutrófilos/enzimologia , Neutrófilos/imunologia , Animais , Pneumonia/metabolismo , Pneumonia/enzimologia , Pneumonia/patologia , Serina Proteases/metabolismo , Peptídeo Hidrolases/metabolismo
12.
Int J Mol Sci ; 25(10)2024 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-38791221

RESUMO

Snakebite accidents, neglected tropical diseases per the WHO, pose a significant public health threat due to their severity and frequency. Envenomation by Bothrops genus snakes leads to severe manifestations due to proteolytic enzymes. While the antibothropic serum produced by the Butantan Institute saves lives, its efficacy is limited as it fails to neutralize certain serine proteases. Hence, developing new-generation antivenoms, like monoclonal antibodies, is crucial. This study aimed to explore the inhibitory potential of synthetic peptides homologous to the CDR3 regions of a monoclonal antibody targeting a snake venom thrombin-like enzyme (SVTLE) from B. atrox venom. Five synthetic peptides were studied, all stable against hydrolysis by venoms and serine proteases. Impressively, four peptides demonstrated uncompetitive SVTLE inhibition, with Ki values ranging from 10-6 to 10-7 M. These findings underscore the potential of short peptides homologous to CDR3 regions in blocking snake venom toxins, suggesting their promise as the basis for new-generation antivenoms. Thus, this study offers potential advancements in combatting snakebites, addressing a critical public health challenge in tropical and subtropical regions.


Assuntos
Anticorpos Monoclonais , Bothrops , Peptídeos , Serina Proteases , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Serina Proteases/química , Serina Proteases/metabolismo , Antivenenos/química , Antivenenos/imunologia , Antivenenos/farmacologia , Regiões Determinantes de Complementaridade/química , Venenos de Crotalídeos/antagonistas & inibidores , Venenos de Crotalídeos/imunologia , Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/química , Sequência de Aminoácidos , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia
13.
Folia Neuropathol ; 62(1): 21-31, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38741434

RESUMO

Neuronal ceroid lipofuscinoses (NCLs) are a growing group of neurodegenerative storage diseases, in which specific features are sought to facilitate the creation of a universal diagnostic algorithm in the future. In our ultrastructural studies, the group of NCLs was represented by the CLN2 disease caused by a defect in the TPP1 gene encoding the enzyme tripeptidyl-peptidase 1. A 3.5-year-old girl was affected by this disease. Due to diagnostic difficulties, the spectrum of clinical, enzymatic, and genetic tests was extended to include analysis of the ultrastructure of cells from a rectal biopsy. The aim of our research was to search for pathognomonic features of CLN2 and to analyse the mitochondrial damage accompanying the disease. In the examined cells of the rectal mucosa, as expected, filamentous deposits of the curvilinear profile (CVP) type were found, which dominated quantitatively. Mixed deposits of the CVP/fingerprint profile (FPP) type were observed less frequently in the examined cells. A form of inclusions of unknown origin, not described so far in CLN2 disease, were wads of osmophilic material (WOMs). They occurred alone or co-formed mixed deposits. In addition, atypically damaged mitochondria were observed in muscularis mucosae. Their deformed cristae had contact with inclusions that looked like CVPs. Considering the confirmed role of the c subunit of the mitochondrial ATP synthase in the formation of filamentous lipopigment deposits in the group of NCLs, we suggest the possible significance of other mitochondrial proteins, such as mitochondrial contact site and cristae organizing system (MICOS), in the formation of these deposits. The presence of WOMs in the context of searching for ultrastructural pathognomonic features in CLN2 disease also requires further research.


Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases , Corpos de Inclusão , Mitocôndrias , Lipofuscinoses Ceroides Neuronais , Tripeptidil-Peptidase 1 , Lipofuscinoses Ceroides Neuronais/patologia , Lipofuscinoses Ceroides Neuronais/genética , Humanos , Feminino , Pré-Escolar , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Corpos de Inclusão/patologia , Corpos de Inclusão/ultraestrutura , Biópsia , Reto/patologia , Serina Proteases/genética , Aminopeptidases/genética
15.
Physiol Rep ; 12(9): e15977, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38697929

RESUMO

FAM111A gene mutations cause Kenney-Caffey syndrome (KCS) and Osteocraniostenosis (OCS), conditions characterized by short stature, low serum ionized calcium (Ca2+), low parathyroid hormone (PTH), and bony abnormalities. The molecular mechanism mediating this phenotype is unknown. The c-terminal domain of FAM111A harbors all the known disease-causing variations and encodes a domain with high homology to serine proteases. However, whether this serine protease domain contributes to the maintenance of Ca2+ homeostasis is not known. We hypothesized the disruption of the serine protease domain of FAM111A would disrupt Ca2+ homeostasis. To test this hypothesis, we generated with CRISPR/Cas9, mice with a frameshift insertion (c.1450insA) or large deletion (c.1253-1464del) mutation in the Fam111a serine protease domain. Serum-ionized Ca2+ and PTH levels were not significantly different between wild type, heterozygous, or homozygous Fam111a mutant mice. Additionally, there were no significant differences in fecal or urine Ca2+ excretion, intestinal Ca2+ absorption or overall Ca2+ balance. Only female homozygous (c.1450insA), but not heterozygous mice displayed differences in bone microarchitecture and mineral density compared to wild-type animals. We conclude that frameshift mutations that disrupt the c-terminal serine protease domain do not induce a KCS or OCS phenotype in mice nor alter Ca2+ homeostasis.


Assuntos
Cálcio , Proteínas de Transporte , Homeostase , Animais , Cálcio/metabolismo , Camundongos , Hormônio Paratireóideo/metabolismo , Feminino , Masculino , Serina Proteases/metabolismo , Serina Proteases/genética , Camundongos Endogâmicos C57BL
16.
Int J Med Mushrooms ; 26(6): 53-68, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38801087

RESUMO

The purification of a fibrinolytic enzyme from the fruiting bodies of wild-growing medicinal mushroom, Pycnoporus coccineus was achieved through a two-step procedure, resulting in its homogeneity. This purification process yielded a significant 4.13-fold increase in specific activity and an 8.0% recovery rate. The molecular weight of P. coccineus fibrinolytic enzyme (PCFE) was estimated to be 23 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. PCFE demonstrated its optimal activity at a temperature of 40 °C and pH 8. Notably, the enzymatic activity was inhibited by the presence of zinc or copper metal ions, as well as serine protease inhibitors, such as phenylmethylsulfonyl fluoride and 4-amidinophenylmethanesulfonyl fluoride. PCFE exhibited remarkable specificity towards a synthetic chromogenic substrate for thrombin. The enzyme demonstrated the Michaelis-Menten constant (Km), maximal velocity (V ), and catalytic rate constant (Kcat) values of 3.01 mM, 0.33 mM min-1 µg-1, and 764.1 s-1, respectively. In vitro assays showed PCFE's ability to effectively degrade fibrin and blood clots. The enzyme induced alterations in the density and structural characteristics of fibrin clots. PCFE exhibited significant effects on various clotting parameters, including recalcification time, activated partial thromboplastin time, prothrombin time, serotonin secretion from thrombin-activated platelets, and thrombin-induced acute thromboembolism. These findings suggest that P. coccineus holds potential as an antithrombotic biomaterials and resources for cardiovascular research.


Assuntos
Fibrinolíticos , Pycnoporus , Serina Proteases , Fibrinolíticos/farmacologia , Fibrinolíticos/isolamento & purificação , Fibrinolíticos/química , Serina Proteases/isolamento & purificação , Serina Proteases/farmacologia , Serina Proteases/metabolismo , Serina Proteases/química , Animais , Pycnoporus/enzimologia , Peso Molecular , Carpóforos/química , Concentração de Íons de Hidrogênio , Temperatura , Humanos , Fibrina/metabolismo , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/química , Proteínas Fúngicas/farmacologia
17.
PLoS Pathog ; 20(5): e1012214, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38722857

RESUMO

Epithelial cells function as the primary line of defense against invading pathogens. However, bacterial pathogens possess the ability to compromise this barrier and facilitate the transmigration of bacteria. Nonetheless, the specific molecular mechanism employed by Mycobacterium tuberculosis (M.tb) in this process is not fully understood. Here, we investigated the role of Rv2569c in M.tb translocation by assessing its ability to cleave E-cadherin, a crucial component of cell-cell adhesion junctions that are disrupted during bacterial invasion. By utilizing recombinant Rv2569c expressed in Escherichia coli and subsequently purified through affinity chromatography, we demonstrated that Rv2569c exhibited cell wall-associated serine protease activity. Furthermore, Rv2569c was capable of degrading a range of protein substrates, including casein, fibrinogen, fibronectin, and E-cadherin. We also determined that the optimal conditions for the protease activity of Rv2569c occurred at a temperature of 37°C and a pH of 9.0, in the presence of MgCl2. To investigate the function of Rv2569c in M.tb, a deletion mutant of Rv2569c and its complemented strains were generated and used to infect A549 cells and mice. The results of the A549-cell infection experiments revealed that Rv2569c had the ability to cleave E-cadherin and facilitate the transmigration of M.tb through polarized A549 epithelial cell layers. Furthermore, in vivo infection assays demonstrated that Rv2569c could disrupt E-cadherin, enhance the colonization of M.tb, and induce pathological damage in the lungs of C57BL/6 mice. Collectively, these results strongly suggest that M.tb employs the serine protease Rv2569c to disrupt epithelial defenses and facilitate its systemic dissemination by crossing the epithelial barrier.


Assuntos
Proteínas de Bactérias , Caderinas , Células Epiteliais , Mycobacterium tuberculosis , Serina Proteases , Caderinas/metabolismo , Mycobacterium tuberculosis/patogenicidade , Mycobacterium tuberculosis/metabolismo , Animais , Humanos , Camundongos , Serina Proteases/metabolismo , Serina Proteases/genética , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Células A549 , Tuberculose/microbiologia , Tuberculose/metabolismo , Feminino
18.
Molecules ; 29(7)2024 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-38611800

RESUMO

4-Chloroisocoumarin compounds have broad inhibitory properties against serine proteases. Here, we show that selected 3-alkoxy-4-chloroisocoumarins preferentially inhibit the activity of the conserved serine protease High-temperature requirement A of Chlamydia trachomatis. The synthesis of a new series of isocoumarin-based scaffolds has been developed, and their anti-chlamydial properties were investigated. The structure of the alkoxy substituent was found to influence the potency of the compounds against High-temperature requirement A, and modifications to the C-7 position of the 3-alkoxy-4-chloroisocoumarin structure attenuate anti-chlamydial properties.


Assuntos
Álcoois , Chlamydia trachomatis , Inibidores de Proteases , Inibidores de Proteases/farmacologia , Terapia Enzimática , Isocumarinas , Serina Endopeptidases , Serina Proteases
19.
Eur J Med Chem ; 270: 116389, 2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38593588

RESUMO

Dipeptidyl peptidases (DPP) 8 and 9 are intracellular serine proteases that play key roles in various biological processes and recent findings highlight DPP8 and DPP9 as potential therapeutic targets for hematological and inflammasome-related diseases. Despite the substantial progress, the precise biological functions of these proteases remain elusive, and the lack of selective chemical tools hampers ongoing research. In this paper, we describe the synthesis and biochemical evaluation of the first active site-directed DPP8/9 probes which are derived from DPP8/9 inhibitors developed in-house. Specifically, we synthesized fluorescent inhibitors containing nitrobenzoxadiazole (NBD), dansyl (DNS) and cyanine-3 (Cy3) reporters to visualize intracellular DPP8/9. We demonstrate that the fluorescent inhibitors have high affinity and selectivity towards DPP8/9 over related S9 family members. The NBD-labeled DPP8/9 inhibitors were nominated as the best in class compounds to visualize DPP8/9 in human cells. Furthermore, a method has been developed for selective labeling and visualization of active DPP8/9 in vitro by fluorescence microscopy. A collection of potent and selective biotinylated DPP8/9-targeting probes was also prepared by replacing the fluorescent reporter with a biotin group. The present work provides the first DPP8/9-targeting fluorescent compounds as useful chemical tools for the study of DPP8 and DPP9's biological functions.


Assuntos
Dipeptidases , Dipeptidil Peptidase 4 , Humanos , Dipeptidil Peptidase 4/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases , Domínio Catalítico , Serina Endopeptidases , Serina Proteases , Dipeptidases/metabolismo
20.
Sci Rep ; 14(1): 8581, 2024 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-38615036

RESUMO

Parkinson's disease (PD) is the second most frequently diagnosed neurodegenerative disease, and it is characterized by the intracellular and extracellular accumulation of α-synuclein (α-syn) and Tau, which are major components of cytosolic protein inclusions called Lewy bodies, in the brain. Currently, there is a lack of effective methods that preventing PD progression. It has been suggested that the plasminogen activation system, which is a major extracellular proteolysis system, is involved in PD pathogenesis. We investigated the functional roles of plasminogen in vitro in an okadaic acid-induced Tau hyperphosphorylation NSC34 cell model, ex vivo using brains from normal controls and methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, and in vivo in a widely used MPTP-induced PD mouse model and an α-syn overexpression mouse model. The in vitro, ex vivo and in vivo results showed that the administered plasminogen crossed the blood‒brain barrier (BBB), entered cells, and migrated to the nucleus, increased plasmin activity intracellularly, bound to α-syn through lysine binding sites, significantly promoted α-syn, Tau and TDP-43 clearance intracellularly and even intranuclearly in the brain, decreased dopaminergic neurodegeneration and increased the tyrosine hydroxylase levels in the substantia nigra and striatum, and improved motor function in PD mouse models. These findings indicate that plasminogen plays a wide range of pivotal protective roles in PD and therefore may be a promising drug candidate for PD treatment.


Assuntos
Doenças Neurodegenerativas , Doença de Parkinson , Plasminogênio , Animais , Camundongos , alfa-Sinucleína , Modelos Animais de Doenças , Proteínas de Ligação a DNA/metabolismo , Dopamina , Doenças Neurodegenerativas/metabolismo , Doença de Parkinson/metabolismo , Plasminogênio/metabolismo , Serina Proteases , Proteínas tau/metabolismo , Neurônios Dopaminérgicos/patologia
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