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1.
Alzheimers Res Ther ; 13(1): 183, 2021 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-34732252

RESUMO

BACKGROUND: The interaction between the brain and periphery might play a crucial role in the development of Alzheimer's disease (AD). METHODS: Using blood transcriptomic profile data from two independent AD cohorts, we performed expression quantitative trait locus (cis-eQTL) analysis of 29 significant genetic loci from a recent large-scale genome-wide association study to investigate the effects of the AD genetic variants on gene expression levels and identify their potential target genes. We then performed differential gene expression analysis of identified AD target genes and linear regression analysis to evaluate the association of differentially expressed genes with neuroimaging biomarkers. RESULTS: A cis-eQTL analysis identified and replicated significant associations in seven genes (APH1B, BIN1, FCER1G, GATS, MS4A6A, RABEP1, TRIM4). APH1B expression levels in the blood increased in AD and were associated with entorhinal cortical thickness and global cortical amyloid-ß deposition. CONCLUSION: An integrative analysis of genetics, blood-based transcriptomic profiles, and imaging biomarkers suggests that APH1B expression levels in the blood might play a role in the pathogenesis of AD.


Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Endopeptidases , Proteínas de Membrana , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/metabolismo , Atrofia/patologia , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Endopeptidases/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Proteínas de Membrana/genética , Transcriptoma
2.
Nat Commun ; 12(1): 5912, 2021 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-34625557

RESUMO

Linear ubiquitination regulates inflammatory and cell death signalling. Deficiency of the linear ubiquitin chain-specific deubiquitinase, OTULIN, causes OTULIN-related autoinflammatory syndrome (ORAS), a systemic inflammatory pathology affecting multiple organs including the skin. Here we show that mice with epidermis-specific OTULIN deficiency (OTULINE-KO) develop inflammatory skin lesions that are driven by TNFR1 signalling in keratinocytes and require RIPK1 kinase activity. OTULINE-KO mice lacking RIPK3 or MLKL have only very mild skin inflammation, implicating necroptosis as an important etiological mediator. Moreover, combined loss of RIPK3 and FADD fully prevents skin lesion development, showing that apoptosis also contributes to skin inflammation in a redundant function with necroptosis. Finally, MyD88 deficiency suppresses skin lesion development in OTULINE-KO mice, suggesting that toll-like receptor and/or IL-1 signalling are involved in mediating skin inflammation. Thus, OTULIN maintains homeostasis and prevents inflammation in the skin by inhibiting TNFR1-mediated, RIPK1 kinase activity-dependent keratinocyte death and primarily necroptosis.


Assuntos
Dermatite/prevenção & controle , Endopeptidases/metabolismo , Endopeptidases/farmacologia , Queratinócitos/metabolismo , Necroptose/efeitos dos fármacos , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Apoptose , Morte Celular , Dermatite/patologia , Endopeptidases/genética , Epiderme/metabolismo , Feminino , Homeostase , Inflamação , Queratinócitos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Receptores Tipo I de Fatores de Necrose Tumoral , Transdução de Sinais , Pele/patologia , Transcriptoma
3.
Nat Commun ; 12(1): 5913, 2021 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-34625556

RESUMO

OTULIN is a deubiquitinase that specifically cleaves linear ubiquitin chains. Here we demonstrate that the ablation of Otulin selectively in keratinocytes causes inflammatory skin lesions that develop into verrucous carcinomas. Genetic deletion of Tnfr1, knockin expression of kinase-inactive Ripk1 or keratinocyte-specific deletion of Fadd and Mlkl completely rescues mice with OTULIN deficiency from dermatitis and tumorigenesis, thereby identifying keratinocyte cell death as the driving force for inflammation. Single-cell RNA-sequencing comparing non-lesional and lesional skin reveals changes in epidermal stem cell identity in OTULIN-deficient keratinocytes prior to substantial immune cell infiltration. Keratinocytes lacking OTULIN display a type-1 interferon and IL-1ß response signature, and genetic or pharmacologic inhibition of these cytokines partially inhibits skin inflammation. Finally, expression of a hypomorphic mutant Otulin allele, previously shown to cause OTULIN-related autoinflammatory syndrome in humans, induces a similar inflammatory phenotype, thus supporting the importance of OTULIN for restraining skin inflammation and maintaining immune homeostasis.


Assuntos
Endopeptidases/metabolismo , Queratinócitos/metabolismo , Pele/metabolismo , Animais , Morte Celular/genética , Citocinas/metabolismo , Endopeptidases/genética , Proteína de Domínio de Morte Associada a Fas , Técnicas de Introdução de Genes , Homeostase , Inflamação/patologia , Interferon Tipo I , Interleucina-1beta , Camundongos , Necroptose , Fragmentos de Peptídeos , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Pele/patologia , Células-Tronco/metabolismo , Análise de Sistemas , Ubiquitina/metabolismo
4.
Int J Mol Sci ; 22(19)2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34638628

RESUMO

Disturbance in a differentiation program of skeletal stem cells leads to indecorous skeletogenesis. Growing evidence suggests that a fine-tuning of ubiquitin-mediated protein degradation is crucial for skeletal stem cells to maintain their stemness and osteogenic potential. Here, we demonstrate that the deubiquitinating enzyme (DUB) ubiquitin-specific protease 8 (USP8) stabilizes the Wnt receptor frizzled 5 (FZD5) by preventing its lysosomal degradation. This pathway is essential for Wnt/ß-catenin signaling and the differentiation of osteoprogenitors to mature osteoblasts. Accordingly, deletion of USP8 in osteoprogenitors (Usp8Osx) resulted in a near-complete blockade in skeletal mineralization, similar to that seen in mice with defective Wnt/ß-catenin signaling. Likewise, transplanting USP8-deficient osteoprogenitors under the renal capsule in wild-type secondary hosts did not to induce bone formation. Collectively, this study unveils an essential role for the DUB USP8 in Wnt/ß-catenin signaling in osteoprogenitors and osteogenesis during skeletal development.


Assuntos
Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Osteogênese/fisiologia , Ubiquitina Tiolesterase/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Diferenciação Celular/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Osteoblastos/fisiologia , Células-Tronco/metabolismo , Células-Tronco/fisiologia , beta Catenina/metabolismo
5.
PLoS One ; 16(10): e0256817, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34699529

RESUMO

The glycoside hydrolase 19 (GH19) is a bifunctional family of chitinases and endolysins, which have been studied for the control of plant fungal pests, the recycle of chitin biomass, and the treatment of multi-drug resistant bacteria. The GH19 domain-containing sequences (22,461) were divided into a chitinase and an endolysin subfamily by analyzing sequence networks, guided by taxonomy and the substrate specificity of characterized enzymes. The chitinase subfamily was split into seventeen groups, thus extending the previous classification. The endolysin subfamily is more diverse and consists of thirty-four groups. Despite their sequence diversity, twenty-six residues are conserved in chitinases and endolysins, which can be distinguished by two specific sequence patterns at six and four positions, respectively. Their location outside the catalytic cleft suggests a possible mechanism for substrate specificity that goes beyond the direct interaction with the substrate. The evolution of the GH19 catalytic domain was investigated by large-scale phylogeny. The inferred evolutionary history and putative horizontal gene transfer events differ from previous works. While no clear patterns were detected in endolysins, chitinases varied in sequence length by up to four loop insertions, causing at least eight distinct presence/absence loop combinations. The annotated GH19 sequences and structures are accessible via the GH19 Engineering Database (GH19ED, https://gh19ed.biocatnet.de). The GH19ED has been developed to support the prediction of substrate specificity and the search for novel GH19 enzymes from neglected taxonomic groups or in regions of the sequence space where few sequences have been described yet.


Assuntos
Quitinases/genética , Endopeptidases/genética , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Quitinases/química , Quitinases/metabolismo , Bases de Dados de Proteínas , Endopeptidases/química , Endopeptidases/metabolismo , Evolução Molecular , Fungos/química , Fungos/genética , Fungos/metabolismo , Humanos , Modelos Moleculares , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Conformação Proteica , Especificidade por Substrato
6.
Medicine (Baltimore) ; 100(35): e27162, 2021 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-34477172

RESUMO

ABSTRACT: Cancer-associated fibroblasts (CAFs) have been attracting attention in recent years, but their nature has not been fully elucidated. Although CAFs have been recognized as an important therapeutic target, therapeutic agents have not been developed to date. CAFs are characterized by their high migration rate and involvement in epithelial-to-mesenchymal transition with some displaying a dendritic morphology that is reminiscent of fascin expression.The present study was designed to immunohistochemically investigate fascin expression in lung adenocarcinoma including CAFs and compare the results with existing CAF markers.We immunohistochemically investigated fascin expression in not only cancer tissue but also CAFs from 26 autopsy cases of lung adenocarcinoma. Immunohistochemistry of α-smooth muscle actin and fibroblast activation protein was also performed.Fascin-positive staining in CAFs was observed in all cases, with a strong correlation observed with existing CAF markers α-smooth muscle actin and fibroblast activation protein (P < .001). In addition, the proportion of tumor cells showing fascin-positive staining was found to correlate with its expression in CAFs (P < .05).We propose that CAFs express fascin, and that fascin may mediate crosstalk between cancer tissue and CAFs. Fascin might be a novel therapeutic target for treatments that target the cancer stroma.


Assuntos
Adenocarcinoma/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Proteínas de Transporte/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas dos Microfilamentos/metabolismo , Actinas/metabolismo , Adenocarcinoma/patologia , Idoso , Idoso de 80 Anos ou mais , Endopeptidases/metabolismo , Feminino , Humanos , Pulmão/patologia , Neoplasias Pulmonares/patologia , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade
7.
Nanoscale ; 13(32): 13658-13664, 2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34477641

RESUMO

Multiplexing methods which are capable of measurement of multiple analytes in a single assay are of great importance in many fields. The conventional strategy for simultaneous detection of multiple species is to construct a sensor array. Herein, we report an innovative multiplex multi-analyte detection platform in a non-array format for protease measurement. By monitoring protease degradation of a single peptide substrate containing two cleavage sites for a disintegrin and metalloproteinase 10 (ADAM10) and a disintegrin and metalloproteinase 10 (ADAM17) in a single nanopore, simultaneous detection and quantification of these two model proteases in mixture samples could satisfactorily be accomplished. Our developed multiplexing sensing platform has the potential to be coupled with the traditional sensor array to further improve the multiplexing capability of the sensor, which may find useful applications in clinical diagnosis and prognosis.


Assuntos
Técnicas Biossensoriais , Nanoporos , Endopeptidases , Peptídeo Hidrolases , Peptídeos
8.
Int J Mol Sci ; 22(17)2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34502443

RESUMO

Clostridium botulinum is a Gram-positive, anaerobic, spore-forming bacterium capable of producing botulinum toxin and responsible for botulism of humans and animals. Phage-encoded enzymes called endolysins, which can lyse bacteria when exposed externally, have potential as agents to combat bacteria of the genus Clostridium. Bioinformatics analysis revealed in the genomes of several Clostridium species genes encoding putative N-acetylmuramoyl-l-alanine amidases with anti-clostridial potential. One such enzyme, designated as LysB (224-aa), from the prophage of C. botulinum E3 strain Alaska E43 was chosen for further analysis. The recombinant 27,726 Da protein was expressed and purified from E. coli Tuner(DE3) with a yield of 37.5 mg per 1 L of cell culture. Size-exclusion chromatography and analytical ultracentrifugation experiments showed that the protein is dimeric in solution. Bioinformatics analysis and results of site-directed mutagenesis studies imply that five residues, namely H25, Y54, H126, S132, and C134, form the catalytic center of the enzyme. Twelve other residues, namely M13, H43, N47, G48, W49, A50, L73, A75, H76, Q78, N81, and Y182, were predicted to be involved in anchoring the protein to the lipoteichoic acid, a significant component of the Gram-positive bacterial cell wall. The LysB enzyme demonstrated lytic activity against bacteria belonging to the genera Clostridium, Bacillus, Staphylococcus, and Deinococcus, but did not lyse Gram-negative bacteria. Optimal lytic activity of LysB occurred between pH 4.0 and 7.5 in the absence of NaCl. This work presents the first characterization of an endolysin derived from a C. botulinum Group II prophage, which can potentially be used to control this important pathogen.


Assuntos
Clostridium botulinum tipo E/enzimologia , Endopeptidases/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Clostridium/efeitos dos fármacos , Clostridium/ultraestrutura , Endopeptidases/química , Endopeptidases/isolamento & purificação , Endopeptidases/farmacologia , Lipopolissacarídeos/metabolismo , Testes de Sensibilidade Microbiana , N-Acetil-Muramil-L-Alanina Amidase/química , N-Acetil-Muramil-L-Alanina Amidase/isolamento & purificação , N-Acetil-Muramil-L-Alanina Amidase/farmacologia , Prófagos/enzimologia , Ácidos Teicoicos/metabolismo
10.
J Biol Chem ; 297(4): 101184, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34509474

RESUMO

The deubiquitinating enzyme USP37 is known to contribute to timely onset of S phase and progression of mitosis. However, it is not clear if USP37 is required beyond S-phase entry despite expression and activity of USP37 peaking within S phase. We have utilized flow cytometry and microscopy to analyze populations of replicating cells labeled with thymidine analogs and monitored mitotic entry in synchronized cells to determine that USP37-depleted cells exhibited altered S-phase kinetics. Further analysis revealed that cells depleted of USP37 harbored increased levels of the replication stress and DNA damage markers γH2AX and 53BP1 in response to perturbed replication. Depletion of USP37 also reduced cellular proliferation and led to increased sensitivity to agents that induce replication stress. Underlying the increased sensitivity, we found that the checkpoint kinase 1 is destabilized in the absence of USP37, attenuating its function. We further demonstrated that USP37 deubiquitinates checkpoint kinase 1, promoting its stability. Together, our results establish that USP37 is required beyond S-phase entry to promote the efficiency and fidelity of replication. These data further define the role of USP37 in the regulation of cell proliferation and contribute to an evolving understanding of USP37 as a multifaceted regulator of genome stability.


Assuntos
Quinase 1 do Ponto de Checagem/metabolismo , Endopeptidases/metabolismo , Fase S , Quinase 1 do Ponto de Checagem/genética , Dano ao DNA , Replicação do DNA , Endopeptidases/genética , Estabilidade Enzimática , Instabilidade Genômica , Células HCT116 , Células HeLa , Histonas , Humanos , Células MCF-7 , Ubiquitinação
11.
Int J Mol Sci ; 22(18)2021 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-34576269

RESUMO

BMI-1, a polycomb ring finger oncogene, is highly expressed in multiple cancer cells and is involved in cancer cell proliferation, invasion, and apoptosis. BMI-1 represents a cancer stemness marker that is associated with the regulation of stem cell self-renewal. In this study, pharmacological inhibition (PTC596) or knockdown (siRNA) of BMI-1 reduced cancer stem-like cells and enhanced cancer cell death. Mechanistically, the inhibition of BMI-1 induced the downregulation of Mcl-1 protein, but not Mcl-1 mRNA. PTC596 downregulated Mcl-1 protein expression at the post-translational level through the proteasome-ubiquitin system. PTC596 and BMI-1 siRNA induced downregulation of DUB3 deubiquitinase, which was strongly linked to Mcl-1 destabilization. Furthermore, overexpression of Mcl-1 or DUB3 inhibited apoptosis by PTC596. Taken together, our findings reveal that the inhibition of BMI-1 induces Mcl-1 destabilization through downregulation of DUB3, resulting in the induction of cancer cell death.


Assuntos
Apoptose , Benzimidazóis/farmacologia , Regulação para Baixo , Endopeptidases/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Complexo Repressor Polycomb 1/antagonistas & inibidores , Pirazinas/farmacologia , Células A549 , Índice de Massa Corporal , Caspase 3/metabolismo , Morte Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fragmentação do DNA , Ativação Enzimática , Células HeLa , Humanos , Células-Tronco Neoplásicas/metabolismo , RNA Interferente Pequeno/metabolismo , Ubiquitina/química
12.
Sci Rep ; 11(1): 17925, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504197

RESUMO

Ras-specific proteases to degrade RAS within cancer cells are under active development as an innovative strategy to treat tumorigenesis. The naturally occurring biological toxin effector called RAS/RAP1-specific endopeptidase (RRSP) is known to cleave all RAS within a cell, including HRAS, KRAS, NRAS and mutant KRAS G13D. Yet, our understanding of the mechanisms by which RRSP drives growth inhibition are unknown. Here, we demonstrate, using isogenic mouse fibroblasts expressing a single isoform of RAS or mutant KRAS, that RRSP equally inactivates all isoforms of RAS as well as the major oncogenic KRAS mutants. To investigate how RAS processing might lead to varying outcomes in cell fate within cancer cells, we tested RRSP against four colorectal cancer cell lines with a range of cell fates. While cell lines highly susceptible to RRSP (HCT116 and SW1463) undergo apoptosis, RRSP treatment of GP5d and SW620 cells induces G1 cell cycle arrest. In some cell lines, growth effects were dictated by rescued expression of the tumor suppressor protein p27 (Kip1). The ability of RRSP to irreversibly inhibit cancer cell growth highlights the antitumor potential of RRSP, and further warrants investigation as a potential anti-tumor therapeutic.


Assuntos
Neoplasias Colorretais/metabolismo , Endopeptidases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Pontos de Checagem do Ciclo Celular , Sobrevivência Celular , Senescência Celular , Fibroblastos , Células HCT116 , Humanos , Camundongos
13.
Theranostics ; 11(16): 7755-7766, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335962

RESUMO

Background: Myocardial infarction (MI) evokes an organized remodeling process characterized by the activation and transdifferentiation of quiescent cardiac fibroblasts to generate a stable collagen rich scar. Early fibroblast activation may be amenable to targeted therapy, but is challenging to identify in vivo. We aimed to non-invasively image active fibrosis by targeting the fibroblast activation protein (FAP) expressed by activated (myo)fibroblasts, using a novel positron emission tomography (PET) radioligand [68Ga]MHLL1 after acute MI. Methods: One-step chemical synthesis and manual as well as module-based radiolabeling yielded [68Ga]MHLL1. Binding characteristics were evaluated in murine and human FAP-transfected cells, and stability tested in human serum. Biodistribution in healthy animals was interrogated by dynamic PET imaging, and metabolites were measured in blood and urine. The temporal pattern of FAP expression was determined by serial PET imaging at 7 d and 21 d after coronary artery ligation in mice as percent injected dose per gram (%ID/g). PET measurements were validated by ex vivo autoradiography and immunostaining for FAP and inflammatory macrophages. Results: [68Ga]MHLL1 displayed specific uptake in murine and human FAP-positive cells (p = 0.0208). In healthy mice the tracer exhibited favorable imaging characteristics, with low blood pool retention and dominantly renal clearance. At 7 d after coronary artery ligation, [68Ga]MHLL1 uptake was elevated in the infarct relative to the non-infarcted remote myocardium (1.3 ± 0.3 vs. 1.0 ± 0.2 %ID/g, p < 0.001) which persisted to 21 d after MI (1.3 ± 0.4 vs. 1.1 ± 0.4 %ID/g, p = 0.013). Excess unlabeled compound blocked tracer accumulation in both infarct and non-infarct remote myocardium regions (p < 0.001). Autoradiography and histology confirmed the regional uptake of [68Ga]MHLL1 in the infarct and especially border zone regions, as identified by Masson trichrome collagen staining. Immunostaining further delineated persistent FAP expression at 7 d and 21 d post-MI in the border zone, consistent with tracer distribution in vivo. Conclusion: The simplified synthesis of [68Ga]MHLL1 bears promise for non-invasive characterization of fibroblast activation protein early in remodeling after MI.


Assuntos
Endopeptidases/metabolismo , Radioisótopos de Gálio/farmacologia , Proteínas de Membrana/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Animais , Autorradiografia/métodos , Linhagem Celular Tumoral , Endopeptidases/fisiologia , Fibroblastos/metabolismo , Fibrose/diagnóstico por imagem , Radioisótopos de Gálio/metabolismo , Humanos , Masculino , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Imagem Molecular/métodos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Distribuição Tecidual/fisiologia , Tomografia Computadorizada por Raios X/métodos
14.
Int J Mol Sci ; 22(15)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34361120

RESUMO

A major limiting factor for systemically delivered gene therapies is the lack of novel tissue specific AAV (Adeno-associated virus) derived vectors. Bispecific antibodies can be used to redirect AAVs to specific target receptors. Here, we demonstrate that the insertion of a short linear epitope "2E3" derived from human proprotein-convertase subtilisin/kexin type 9 (PCSK9) into different surface loops of the VP capsid proteins can be used for AAV de-targeting from its natural receptor(s), combined with a bispecific antibody-mediated retargeting. We chose to target a set of distinct disease relevant membrane proteins-fibroblast activation protein (FAP), which is upregulated on activated fibroblasts within the tumor stroma and in fibrotic tissues, as well as programmed death-ligand 1 (PD-L1), which is strongly upregulated in many cancers. Upon incubation with a bispecific antibody recognizing the 2E3 epitope and FAP or PD-L1, the bispecific antibody/rAAV complex was able to selectively transduce receptor positive cells. In summary, we developed a novel, rationally designed vector retargeting platform that can target AAVs to a new set of cellular receptors in a modular fashion. This versatile platform may serve as a valuable tool to investigate the role of disease relevant cell types and basis for novel gene therapy approaches.


Assuntos
Anticorpos Biespecíficos/imunologia , Proteínas do Capsídeo/imunologia , Capsídeo/imunologia , Dependovirus/genética , Endopeptidases/imunologia , Epitopos/imunologia , Vetores Genéticos/administração & dosagem , Proteínas de Membrana/imunologia , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/imunologia , Pró-Proteína Convertase 9/metabolismo , Transdução Genética
15.
Viruses ; 13(7)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34372584

RESUMO

Double-stranded DNA bacteriophages end their lytic cycle by disrupting the host cell envelope, which allows the release of the virion progeny. Each phage must synthesize lysis proteins that target each cell barrier to phage release. In addition to holins, which permeabilize the cytoplasmic membrane, and endolysins, which disrupt the peptidoglycan (PG), mycobacteriophages synthesize a specific lysis protein, LysB, capable of detaching the outer membrane from the complex cell wall of mycobacteria. The family of LysB proteins is highly diverse, with many members presenting an extended N-terminus. The N-terminal region of mycobacteriophage Ms6 LysB shows structural similarity to the PG-binding domain (PGBD) of the φKZ endolysin. A fusion of this region with enhanced green fluorescent protein (Ms6LysBPGBD-EGFP) was shown to bind to Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BGC and Mycobacterium tuberculosis H37Ra cells pretreated with SDS or Ms6 LysB. In pulldown assays, we demonstrate that Ms6 LysB and Ms6LysBPGBD-EGFP bind to purified peptidoglycan of M. smegmatis, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, demonstrating affinity to PG of the A1γ chemotype. An infection assay with an Ms6 mutant producing a truncated version of LysB lacking the first 90 amino acids resulted in an abrupt lysis. These results clearly demonstrate that the N-terminus of Ms6 LysB binds to the PG.


Assuntos
Bacteriólise/fisiologia , Micobacteriófagos/metabolismo , Proteínas Virais/genética , Membrana Celular/metabolismo , Parede Celular/metabolismo , Endopeptidases , Hidrólise , Mycobacterium/metabolismo , Mycobacterium/virologia , Peptidoglicano/metabolismo , Ligação Proteica
16.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360587

RESUMO

In the present study, we analyzed the activity of several aminopeptidases (angiotensinases) involved in the metabolism of various angiotensin peptides, in pituitary and adrenal glands of untreated Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) or treated with the antihypertensive drugs captopril and propranolol or with the L-Arginine hypertensive analogue L-NG-Nitroarginine Methyl Ester (L-NAME). Intra- and inter-gland correlations between angiotensinase activities were also calculated. Membrane-bound alanyl-, cystinyl-, and glutamyl-aminopeptidase activities were determined fluorometrically using aminoacyl-ß-naphthylamide as substrates. Depending on the type of angiotensinase analyzed, the results reflect a complex picture showing substantial differences between glands, strains, and treatments. Alanyl-aminopeptidase responsible for the metabolism of Ang III to Ang IV appears to be the most active angiotensinase in both pituitary and adrenals of WKY and particularly in SHR. Independently of treatment, most positive correlations are observed in the pituitary gland of WKY whereas such positive correlations are predominant in adrenals of SHR. Negative inter-gland correlations were observed in control SHR and L-NAME treated WKY. Positive inter-gland correlations were observed in captopril-treated SHR and propranolol-treated WKY. These results may reflect additional mechanisms for increasing or decreasing systolic blood pressure in WKY or SHR.


Assuntos
Glândulas Suprarrenais/metabolismo , Anti-Hipertensivos/farmacologia , Endopeptidases/metabolismo , Hipertensão/metabolismo , Hipotensão/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Hipófise/metabolismo , Glândulas Suprarrenais/efeitos dos fármacos , Animais , Captopril/farmacologia , Endopeptidases/genética , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica , Hipertensão/tratamento farmacológico , Hipertensão/patologia , Hipotensão/tratamento farmacológico , Hipotensão/patologia , Masculino , Hipófise/efeitos dos fármacos , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY
17.
J Microbiol ; 59(9): 840-847, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34383247

RESUMO

Endolysin, a peptidoglycan hydrolase derived from bacteriophage, has been suggested as an alternative antimicrobial agent. Many endolysins on staphylococcal phages have been identified and applied extensively against Staphylococcus spp. Among them, LysK-like endolysin, a well-studied staphylococcal endolysin, accounts for most of the identified endolysins. However, relatively little interest has been paid to LysKunlike endolysin and a few of them has been characterized. An endolysin LysSAP33 encoded on bacteriophage SAP33 shared low homology with LysK-like endolysin in sequence by 41% and domain composition (CHAP-unknown CBD). A green fluorescence assay using a fusion protein for LysSAP33_CBD indicated that the CBD domain (157-251 aa) was bound to the peptidoglycan of S. aureus. The deletion of LysSAP33_CBD at the C-terminal region resulted in a significant decrease in lytic activity and efficacy. Compared to LysK-like endolysin, LysSAP33 retained its lytic activity in a broader range of temperature, pH, and NaCl concentrations. In addition, it showed a higher activity against biofilms than LysK-like endolysin. This study could be a helpful tool to develop our understanding of staphylococcal endolysins not belonging to LysK-like endolysins and a potential biocontrol agent against biofilms.


Assuntos
Endopeptidases/metabolismo , Fagos de Staphylococcus/enzimologia , Staphylococcus aureus/virologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Parede Celular/metabolismo , Parede Celular/virologia , Endopeptidases/química , Endopeptidases/genética , Peptidoglicano/metabolismo , Alinhamento de Sequência , Fagos de Staphylococcus/química , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/fisiologia , Staphylococcus aureus/metabolismo , Proteínas Virais/química , Proteínas Virais/genética
18.
Food Res Int ; 147: 110461, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34399461

RESUMO

Salmonella enterica and Shiga toxin-producing (or verotoxin-producing) Escherichia coli are major foodborne pathogens, posing substantial food safety risks. Due to the negative effects of chemical treatment against foodborne pathogens, the application of enzyme-based techniques is currently receiving great attention. Here, we evaluated the inhibitory properties of Flavourzyme, a commercial peptidase, against these two foodborne pathogens. We noticed 4.0 and 5.5 log inhibition of biofilm formation by S. Typhimurium and E. coli, respectively, while treated with sub-minimum inhibitory concentrations of Flavourzyme for 24 h. For both bacteria, the enzyme exhibited quorum-quenching activity, preventing autoinducer-2 production completely by E. coli. In addition, Flavourzyme significantly suppressed the relative expression levels of biofilm-forming, quorum sensing, and virulence regulatory genes as measured by qRT-PCR. Based on our results, we suggest the use of Flavourzyme as a preventive agent against foodborne pathogens that possibly acts by inhibiting bacterial self-defense mechanisms following disruption of cellular proteins. This finding may shed light on how enzymes can be applied as a novel weapon to control foodborne illnesses to ensure food safety and public health.


Assuntos
Salmonella typhimurium , Escherichia coli Shiga Toxigênica , Biofilmes , Endopeptidases , Percepção de Quorum , Salmonella typhimurium/genética , Escherichia coli Shiga Toxigênica/genética , Virulência/genética
19.
Microb Pathog ; 160: 105137, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34390765

RESUMO

Chlamydia trachomatis urogenital tract infection causes pelvic inflammatory disease and infertility, increases the risk of co-infection with HPV and HIV. Chlamydial vaccination is considered the most promising approach to prevent and control its infection. Among various chlamydial vaccine candidates, chlamydial protease-like activity factor (CPAF) have been reported to provide robust protective immunity against genital chlamydial infection in mice with reduced vaginal shedding and oviduct pathology. However, CPAF is a serine protease which has enzymatical activity to degrade a large number of substrates. In order to increase the safety of CPAF vaccine, in this study, we used a mutant CPAF that is deficient in enzymatical activity to determine whether proteolytic activity of CPAF affect its vaccine efficacy. The wild type or mutant CPAF immunization causes a significant lower chlamydial shedding from the vaginal and resolve the infection as early as day 20, compared to day 28 in adjuvant control mice. More important, reduced upper reproductive tract pathology were also observed in these two groups. The mutant or wild type CPAF immunization induced not only robust splenic IFN-γ and serum IgG2a but also sIgA secretion in the vaginal fluids. Furthermore, neutralization of chlamydia with immune sera did not provide protection against oviduct pathology. However, adoptive transfer of CD4+ splenocytes isolated from the mutant or wild type CPAF immunized mice resulted in a significant and comparable reduced oviduct pathology. Our results indicate mutant CPAF vaccination is as same efficacy as wild type, and the protection relies on CD4+ T cells, which will further promote the development of CPAF as clinical chlamydial vaccine.


Assuntos
Infecções por Chlamydia , Chlamydia muridarum , Infecções do Sistema Genital , Administração Intranasal , Animais , Vacinas Bacterianas , Infecções por Chlamydia/prevenção & controle , Endopeptidases/genética , Feminino , Camundongos , Vacinação
20.
Microb Pathog ; 159: 105135, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34390766

RESUMO

Vibrio alginolyticus is a common opportunistic pathogen that can cause vibriosis of marine aquatic animals. The application of phages or particularly associated protein products for the treatment of vibriosis has shown prominent advantages compared with the treatment with traditional antibiotics. In this study, the function of a holin-endolysin system from V. alginolyticus phage HH109 was characterized by examining the effect of their overexpression on Escherichia coli and V. alginolyticus. Our data revealed that the endolysin of the phage HH109 has stronger bactericidal activity than the holin, as evidenced by observing more cell death and severe structural damage of cells in the endolysin-expressing E. coli. Furthermore, the two proteins displayed the synergistic effect when the holA and lysin were co-expressed in E. coli, although no interaction between them was detected using the bacterial two-hybrid assay. Transmission electron microscopy observation revealed disruptions of cell envelopes accompanied by leakage of intracellular contents. Similarly, the bactericidal activity of the holin and endolysin against V. alginolyticus was also examined whatever the host is sensitive or resistant to phage HH109. Together, our study contributes to a better understanding of the mechanism of phage HH109 destroying the bacterial cell wall to lyse their host and may offer alternative applications potentially for vibriosis treatment.


Assuntos
Bacteriófagos , Animais , Bacteriófagos/genética , Endopeptidases/genética , Escherichia coli , Vibrio alginolyticus
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