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1.
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
2.
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
3.
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
4.
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
5.
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
6.
Int J Mol Sci ; 22(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34281200

RESUMO

The best-characterized members of the M23 family are glycyl-glycine hydrolases, such as lysostaphin (Lss) from Staphylococcus simulans or LytM from Staphylococcus aureus. Recently, enzymes with broad specificities were reported, such as EnpACD from Enterococcus faecalis, that cleaves D,L peptide bond between the stem peptide and a cross-bridge. Previously, the activity of EnpACD was demonstrated only on isolated peptidoglycan fragments. Herein we report conditions in which EnpACD lyses bacterial cells live with very high efficiency demonstrating great bacteriolytic potential, though limited to a low ionic strength environment. We have solved the structure of the EnpACD H109A inactive variant and analyzed it in the context of related peptidoglycan hydrolases structures to reveal the bases for the specificity determination. All M23 structures share a very conserved ß-sheet core which constitutes the rigid bottom of the substrate-binding groove and active site, while variable loops create the walls of the deep and narrow binding cleft. A detailed analysis of the binding groove architecture, specificity of M23 enzymes and D,L peptidases demonstrates that the substrate groove, which is particularly deep and narrow, is accessible preferably for peptides composed of amino acids with short side chains or subsequent L and D-isomers. As a result, the bottom of the groove is involved in interactions with the main chain of the substrate while the side chains are protruding in one plane towards the groove opening. We concluded that the selectivity of the substrates is based on their conformations allowed only for polyglycine chains and alternating chirality of the amino acids.


Assuntos
Endopeptidases/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Peptídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Peptidoglicano/metabolismo , Prófagos/genética , Prófagos/metabolismo , Ligação Proteica , Staphylococcus/metabolismo , Staphylococcus aureus/metabolismo , Especificidade por Substrato
7.
Mol Cell ; 81(15): 3187-3204.e7, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34157307

RESUMO

OTULIN coordinates with LUBAC to edit linear polyubiquitin chains in embryonic development, autoimmunity, and inflammatory diseases. However, the mechanism by which angiogenesis, especially that of endothelial cells (ECs), is regulated by linear ubiquitination remains unclear. Here, we reveal that constitutive or EC-specific deletion of Otulin resulted in arteriovenous malformations and embryonic lethality. LUBAC conjugates linear ubiquitin chains onto Activin receptor-like kinase 1 (ALK1), which is responsible for angiogenesis defects, inhibiting ALK1 enzyme activity and Smad1/5 activation. Conversely, OTULIN deubiquitinates ALK1 to promote Smad1/5 activation. Consistently, embryonic survival of Otulin-deficient mice was prolonged by BMP9 pretreatment or EC-specific ALK1Q200D (constitutively active) knockin. Moreover, mutant ALK1 from type 2 hereditary hemorrhagic telangiectasia (HHT2) patients exhibited excessive linear ubiquitination and increased HOIP binding. As such, a HOIP inhibitor restricted the excessive angiogenesis of ECs derived from ALK1G309S-expressing HHT2 patients. These results show that OTULIN and LUBAC govern ALK1 activity to balance EC angiogenesis.


Assuntos
Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Endopeptidases/genética , Complexos Multiproteicos/metabolismo , Neovascularização Patológica/genética , Poliubiquitina/metabolismo , Adulto , Animais , Endopeptidases/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Fator 2 de Diferenciação de Crescimento/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos Mutantes , Mutação , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Fisiológica/genética , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad5/genética , Proteína Smad5/metabolismo , Telangiectasia Hemorrágica Hereditária , Ubiquitina-Proteína Ligases/metabolismo
8.
Trends Immunol ; 42(7): 590-603, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34074601

RESUMO

Tight control of inflammatory signaling pathways is an absolute requirement to avoid chronic inflammation and disease. One of the proteins responsible for such control is OTU deubiquitinase with linear linkage specificity (OTULIN), the only mammalian deubiquitinating enzyme (DUB) exclusively hydrolyzing linear ubiquitin chains from proteins modified by the linear ubiquitin chain assembly complex (LUBAC) described thus far. Recent findings show that loss-of-function mutations in OTULIN underlie a severe early-onset human autoinflammatory disease and severe pathology in experimental mouse models. Here, we review the molecular and cellular mechanisms by which OTULIN controls inflammation and discuss the involvement of OTULIN in inflammatory disease development. We also highlight several newly identified roles for OTULIN, including a ubiquitin-independent function.


Assuntos
Endopeptidases , NF-kappa B , Animais , Morte Celular , Endopeptidases/metabolismo , Camundongos , NF-kappa B/metabolismo , Transdução de Sinais , Ubiquitina
9.
Methods Mol Biol ; 2268: 223-232, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085272

RESUMO

Split-TEV assay enables the identification of protein-protein interaction in mammalian cells. This method is based on the split of tobacco etch virus (TEV) protease in two fragments, where each fragment is fused to the candidate proteins predicted to interact. If there is indeed an interaction between both proteins, TEV protease reconstitutes its proteolytic activity and this activity is used to induce the expression of some reporter genes. However, some studies have detected unspecific interaction between membrane proteins due to its higher tendency to aggregate. Here we describe a variation of the Split-TEV method developed with the aim to increase the specificity in the study of G protein-coupled receptor (GPCR) interacting proteins. This approach for monitoring interactions between GPCRs is an easy and robust assay and offers good perspectives in drug discovery.


Assuntos
Bioensaio/métodos , Endopeptidases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Potyvirus/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Cultivadas , Genes Reporter , Humanos , Imagem Molecular/métodos , Potyvirus/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Análise de Célula Única/métodos
10.
Aging (Albany NY) ; 13(11): 14999-15012, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34081623

RESUMO

The ubiquitin-specific protease 8 (USP8) is a prototypic multidomain deubiquitinating enzyme with pleiotropic functions. We investigated the role of USP8 in hepatocellular carcinoma (HCC) by analyzing expression patterns of USP8 in HCC patients, and evaluating its functions and underlying signaling. Among 20 HCC patients investigated, we found that USP8 protein upregulation was a common phenomenon (17 out of 20) in HCC compared to normal liver tissue. Furthermore, the upregulation of USP8 was not associated with any clinicopathology. USP8 inhibition via genetic and pharmacological approaches resulted in growth inhibition and apoptosis induction in both sensitive and doxorubicin-resistant HCC cells. Of note, USP8 inhibition significantly enhanced doxorubicin or sorafenib's efficacy in HCC cells and mouse models. We further found that USP8 inhibition decreased levels of multiple receptor tyrosine kinases (RTKs) by ~90%, such as epidermal growth factor receptor (EGFR) and c-Met. Consistently, the downstream signaling regulated by RTKs was disrupted in HCC cells after USP8 inhibition, as shown by the decreased p-Akt, p-STAT3 and p-Raf. Our findings demonstrate that USP8 is a novel therapeutic target in HCC. Inhibiting USP8 has potential to overcome current drug resistance, particularly on HCC patients with high USP8 expression.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Complexos Endossomais de Distribuição Requeridos para Transporte/antagonistas & inibidores , Neoplasias Hepáticas/tratamento farmacológico , Receptores Proteína Tirosina Quinases/metabolismo , Ubiquitina Tiolesterase/antagonistas & inibidores , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Regulação para Baixo/genética , Endopeptidases/genética , Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos SCID , Transdução de Sinais , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Regulação para Cima/genética
11.
Food Chem ; 362: 130098, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34090041

RESUMO

The specificity of pepsin, the major protease of gastric digestion, has been previously investigated, but only regarding the primary sequence of the protein substrates. The present study aimed to consider in addition physicochemical and structural characteristics, at the molecular and sub-molecular scales. For six different proteins submitted to in vitro gastric digestion, the peptide bonds cleaved were determined from the peptides released and identified by LC-MS/MS. An original statistical approach, based on propensity scores calculated for each amino acid residue on both sides of the peptide bonds, concluded that preferential cleavage occurred after Leu and Phe, and before Ile. Moreover, reliable statistical models developed for predicting peptide bond cleavage, highlighted the predominant role of the amino acid residues at the N-terminal side of the peptide bonds, up to the seventh position (P7 and P7'). The significant influence of hydrophobicity, charge and structural constraints around the peptide bonds was also evidenced.


Assuntos
Pepsina A/metabolismo , Proteínas/metabolismo , Sequência de Aminoácidos , Aminoácidos , Cromatografia Líquida , Endopeptidases/metabolismo , Modelos Estatísticos , Peptídeos/metabolismo , Proteínas/química , Proteólise , Especificidade por Substrato , Espectrometria de Massas em Tandem
12.
PLoS Pathog ; 17(6): e1009680, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34161398

RESUMO

Positive-strand (+)RNA viruses take advantage of the host cells by subverting a long list of host protein factors and transport vesicles and cellular organelles to build membranous viral replication organelles (VROs) that support robust RNA replication. How RNA viruses accomplish major recruitment tasks of a large number of cellular proteins are intensively studied. In case of tomato bushy stunt virus (TBSV), a single viral replication protein, named p33, carries out most of the recruitment duties. Yet, it is currently unknown how the viral p33 replication protein, which is membrane associated, is capable of the rapid and efficient recruitment of numerous cytosolic host proteins to facilitate the formation of large VROs. In this paper, we show that, TBSV p33 molecules do not recruit each cytosolic host factor one-by-one into VROs, but p33 targets a cytosolic protein interaction hub, namely Rpn11, which interacts with numerous other cytosolic proteins. The highly conserved Rpn11, called POH1 in humans, is the metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates. However, TBSV takes advantage of a noncanonical function of Rpn11 by exploiting Rpn11's interaction with highly abundant cytosolic proteins and the actin network. We provide supporting evidence that the co-opted Rpn11 in coordination with the subverted actin network is used for delivering cytosolic proteins, such as glycolytic and fermentation enzymes, which are readily subverted into VROs to produce ATP locally in support of VRO formation, viral replicase complex assembly and viral RNA replication. Using several approaches, including knockdown of Rpn11 level, sequestering Rpn11 from the cytosol into the nucleus in plants or temperature-sensitive mutation in Rpn11 in yeast, we show the inhibition of recruitment of glycolytic and fermentation enzymes into VROs. The Rpn11-assisted recruitment of the cytosolic enzymes by p33, however, also requires the combined and coordinated role of the subverted actin network. Accordingly, stabilization of the actin filaments by expression of the Legionella VipA effector in yeast and plant, or via a mutation of ACT1 in yeast resulted in more efficient and rapid recruitment of Rpn11 and the selected glycolytic and fermentation enzymes into VROs. On the contrary, destruction of the actin filaments via expression of the Legionella RavK effector led to poor recruitment of Rpn11 and glycolytic and fermentation enzymes. Finally, we confirmed the key roles of Rpn11 and the actin filaments in situ ATP production within TBSV VROs via using a FRET-based ATP-biosensor. The novel emerging theme is that TBSV targets Rpn11 cytosolic protein interaction hub driven by the p33 replication protein and aided by the subverted actin filaments to deliver several co-opted cytosolic pro-viral factors for robust replication within VROs.


Assuntos
Citoesqueleto de Actina/metabolismo , Endopeptidases/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Tombusvirus/fisiologia , Replicação Viral/fisiologia , Citosol/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo
13.
J Cell Biol ; 220(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34081091

RESUMO

The step-by-step process of chromosome segregation defines the stages of the cell cycle. In eukaryotes, signals controlling these steps converge upon the kinetochore, a multiprotein assembly that connects spindle microtubules to chromosomal centromeres. Kinetochores control and adapt to major chromosomal transactions, including replication of centromeric DNA, biorientation of sister centromeres on the metaphase spindle, and transit of sister chromatids into daughter cells during anaphase. Although the mechanisms that ensure tight microtubule coupling at anaphase are at least partly understood, kinetochore adaptations that support other cell cycle transitions are not. We report here a mechanism that enables regulated control of kinetochore sumoylation. A conserved surface of the Ctf3/CENP-I kinetochore protein provides a binding site for Ulp2, the nuclear enzyme that removes SUMO chains from modified substrates. Ctf3 mutations that disable Ulp2 recruitment cause elevated inner kinetochore sumoylation and defective chromosome segregation. The location of the site within the assembled kinetochore suggests coordination between sumoylation and other cell cycle-regulated processes.


Assuntos
Segregação de Cromossomos , Cromossomos Fúngicos , Endopeptidases/metabolismo , Cinetocoros/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Microscopia Crioeletrônica , Endopeptidases/química , Endopeptidases/genética , Microscopia de Fluorescência , Modelos Moleculares , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Sumoilação
14.
Int J Mol Sci ; 22(11)2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073633

RESUMO

Clostridioides difficile is a spore-forming enteric pathogen causing life-threatening diarrhoea and colitis. Microbial disruption caused by antibiotics has been linked with susceptibility to, and transmission and relapse of, C. difficile infection. Therefore, there is an urgent need for novel therapeutics that are effective in preventing C. difficile growth, spore germination, and outgrowth. In recent years bacteriophage-derived endolysins and their derivatives show promise as a novel class of antibacterial agents. In this study, we recombinantly expressed and characterized a cell wall hydrolase (CWH) lysin from C. difficile phage, phiMMP01. The full-length CWH displayed lytic activity against selected C. difficile strains. However, removing the N-terminal cell wall binding domain, creating CWH351-656, resulted in increased and/or an expanded lytic spectrum of activity. C. difficile specificity was retained versus commensal clostridia and other bacterial species. As expected, the putative cell wall binding domain, CWH1-350, was completely inactive. We also observe the effect of CWH351-656 on preventing C. difficile spore outgrowth. Our results suggest that CWH351-656 has therapeutic potential as an antimicrobial agent against C. difficile infection.


Assuntos
Bacteriófagos , Clostridioides difficile , Endopeptidases/metabolismo , Esporos Bacterianos , Proteínas Virais/metabolismo , Bacteriófagos/enzimologia , Bacteriófagos/genética , Clostridioides difficile/enzimologia , Clostridioides difficile/genética , Clostridioides difficile/virologia , Endopeptidases/genética , Endopeptidases/farmacologia , Enterocolite Pseudomembranosa/tratamento farmacológico , Humanos , Esporos Bacterianos/enzimologia , Esporos Bacterianos/genética , Esporos Bacterianos/virologia , Proteínas Virais/genética , Proteínas Virais/farmacologia
15.
Arch Microbiol ; 203(6): 3551-3555, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33942156

RESUMO

The antibacterial activity of the monoterpene estragole was evaluated against two strains of bacteria with an efflux pump mechanism, which are Staphylococcus aureus 1199B and S. aureus K2068, which have a NorA and MepA pump, respectively. For that, the methodology proposed by CLSI with modifications was followed, and to evaluate the reversal of the efflux pump, subinhibitory concentrations (MIC/8) of estragole and standard pump inhibitors, CCCP and Chlorpromazine were used and it was verified whether they managed to modulate the action of Norfloxacin, Ciprofloxacin and Ethidium Bromide, an indicator of an efflux pump. It was observed that estragole positively modulated norfloxacin and ethidium bromide against the strain of S. aureus 1199B and that it also managed to reduce the MIC of ethidium bromide against the strain of S. aureus K2068. In the non-clinical acute toxicity tests with estragole, the animals treated with the dose of 625 mg/kg/v.o. showed no clinical signs of toxicity, according to the parameters evaluated. These results are promising, since it places estragole as a possible inhibitor of the efflux pump, thus managing to inhibit this mechanism of action in the strains tested.


Assuntos
Derivados de Alilbenzenos , Anisóis , Staphylococcus aureus , Derivados de Alilbenzenos/farmacologia , Animais , Anisóis/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Endopeptidases/metabolismo , Testes de Sensibilidade Microbiana , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo
16.
J Virol ; 95(15): e0036121, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980594

RESUMO

Foot-and-mouth disease virus (FMDV) is the pathogen of foot-and-mouth disease (FMD), which is a highly contagious disease in cloven-hoofed animals. To survive in the host, FMDV has evolved multiple strategies to antagonize host innate immune responses. In this study, we showed that the leader protease (Lpro) of FMDV, a papain-like proteinase, promoted viral replication by evading the antiviral interferon response through counteracting the 2',5'-oligoadenylate synthetase (OAS)/RNase L system. Specifically, we observed that the titers of Lpro deletion virus were significantly lower than those of wild-type FMDV (FMDV-WT) in cultured cells. Our mechanistic studies demonstrated that Lpro interfered with the OAS/RNase L pathway by interacting with the N-terminal domain of swine RNase L (sRNase L). Remarkably, Lpro of FMDV exhibited species-specific binding to RNase L in that the interaction was observed only in swine cells, not human, monkey, or canine cells. Lastly, we presented evidence that by interacting with sRNase L, FMDV Lpro inhibited cellular apoptosis. Taken together, these results demonstrate a novel mechanism that Lpro utilizes to escape the OAS/RNase L-mediated antiviral defense pathway. IMPORTANCE FMDV is a picornavirus that causes a significant disease in agricultural animals. FMDV has developed diverse strategies to escape the host interferon response. Here, we show that Lpro of FMDV antagonizes the OAS/RNase L pathway, an important interferon effector pathway, by interacting with the N-terminal domain of sRNase L. Interestingly, such a virus-host interaction is species-specific because the interaction is detected only in swine cells, not in human, monkey, or canine cells. Furthermore, Lpro inhibits apoptosis through interacting with sRNase L. This study demonstrates a novel mechanism by which FMDV has evolved to inhibit host innate immune responses.


Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Endopeptidases/metabolismo , Endorribonucleases/metabolismo , Vírus da Febre Aftosa/imunologia , Evasão da Resposta Imune/imunologia , Imunidade Inata/imunologia , Animais , Apoptose/imunologia , Linhagem Celular , Cricetinae , Cães , Endopeptidases/genética , Endopeptidases/imunologia , Endorribonucleases/genética , Febre Aftosa/imunologia , Febre Aftosa/virologia , Células HEK293 , Haplorrinos , Humanos , Evasão da Resposta Imune/genética , Células Madin Darby de Rim Canino , Domínios Proteicos , Suínos
17.
Food Chem ; 360: 130026, 2021 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-34023711

RESUMO

The proteolytic activity of some soybean endogenous proteases have been clarified in the previous studies, but the information concerning the roles of these proteases and some other unknown ones during soybean processing are scarce. Herein, 16 endopeptidases, 13 exopeptidases, 24 inhibitors (two serpin-ZX and one subtilisin inhibitor firstly identified), and one glutamate decarboxylase were identified in the soybean water extract by the liquid chromatography tandem mass spectrometry analysis. Amongst the identified endopeptidases, just the aspartic endopeptidases (optimal at pH 2.5-3 and 35-45 °C) showed the detectable proteolytic activity by the tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis and protease inhibitor assay analyses, whereas serine, cysteine, and metallo- endopeptidases (except P34 probable thiol protease) did not. Free amino acid analysis showed that the exopeptidases and glutamate decarboxylase were optimal at pH 6 and 45 °C, and by 6 h incubation, the free amino acids and γ-aminobutyric acid almost doubled.


Assuntos
Endopeptidases/metabolismo , Exopeptidases/metabolismo , Glutamato Descarboxilase/metabolismo , Soja/enzimologia , Água/química , Alérgenos/metabolismo , Inibidores de Proteases/farmacologia , Proteólise
18.
BMC Cancer ; 21(1): 492, 2021 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-33941102

RESUMO

BACKGROUND: Mycosis fungoides (MF) is a primary cutaneous T-cell lymphoma (CTCL) that transforms from mature, skin-homing T cells and progresses during the early stages in the skin. The role of the skin microenvironment in MF development is unclear, but recent findings in a variety of cancers have highlighted the role of stromal fibroblasts in promoting or inhibiting tumorigenesis. Stromal fibroblasts are an important part of the cutaneous tumor microenvironment (TME) in MF. Here we describe studies into the interaction of TME-fibroblasts and malignant T cells to gain insight into their role in CTCL. METHODS: Skin from normal (n = 3) and MF patients (n = 3) were analyzed for FAPα by immunohistochemistry. MyLa is a CTCL cell line that retains expression of biomarkers TWIST1 and TOX that are frequently detected in CTCL patients. MyLa cells were cultured in the presence or absence of normal or MF skin derived fibroblasts for 5 days, trypsinized to detached MyL a cells, and gene expression analyzed by RT-PCR for MF biomarkers (TWIST1 and TOX), Th1 markers (IFNG, TBX21), Th2 markers (GATA3, IL16), and proliferation marker (MKI67). Purified fibroblasts were assayed for VIM and ACTA2 gene expression. Cellular senescence assay was performed to assess senescence. RESULTS: MF skin fibroblast showed increased expression of FAP-α with increasing stage compared to normal. Normal fibroblasts co-cultured with MyLa cells suppressed expression of TWIST1 (p < 0.0006), and TOX (p < 0.03), GATA3 (p < 0.02) and IL16 (p < 0.03), and increased expression of IFNG (p < 0.03) and TBX21 (p < 0.03) in MyLa cells. In contrast, MyLa cells cultured with MF fibroblasts retained high expression of TWIST1, TOX and GATA3. MF fibroblasts co-culture with MyLa cells increased expression of IL16 (p < 0.01) and IL4 (p < 0.02), and suppressed IFNG and TBX21 in MyLa cells. Furthermore, expression of MKI67 in MyLa cells was suppressed by normal fibroblasts compared to MF fibroblasts. CONCLUSION: Skin fibroblasts represent important components of the TME in MF. In co-culture model, normal and MF fibroblasts have differential influence on T-cell phenotype in modulating expression of Th1 cytokine and CTCL biomarker genes to reveal distinct roles with implications in MF progression.


Assuntos
Fibroblastos Associados a Câncer/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Linfoma Cutâneo de Células T/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Cutâneas/metabolismo , Microambiente Tumoral , Proteína 1 Relacionada a Twist/metabolismo , Actinas/genética , Actinas/metabolismo , Idoso , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Senescência Celular , Técnicas de Cocultura , Endopeptidases/genética , Endopeptidases/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-16/genética , Interleucina-16/metabolismo , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Linfoma Cutâneo de Células T/genética , Linfoma Cutâneo de Células T/patologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Micose Fungoide/genética , Micose Fungoide/metabolismo , Micose Fungoide/patologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/citologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Linfócitos T/metabolismo , Proteína 1 Relacionada a Twist/genética , Vimentina/genética , Vimentina/metabolismo
19.
Cell Death Dis ; 12(6): 543, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34035230

RESUMO

Fibroblast activation protein alpha (FAP) is a marker of cancer-associated fibroblast, which is also expressed in cancer epithelial cells. However, the role of FAP in colorectal cancer (CRC) cells remains to be elucidated. Here we investigate the expression pattern of FAP in CRC tissues and cells to prove that FAP is upregulated in CRC cells. Loss- of and gain-of-function assays identified FAP promotes migration and invasion instead of an effect on cell proliferation. Microarray assays are adopted to identify the different expressed genes after FAP knockdown and gene set enrichment analysis (GSEA) is used to exploit the involved signaling pathway. Our works reveal FAP exerts a function dependent on NF-κB signaling pathway and FAP expression is associated with NF-κB signaling pathway in clinical samples. Our work shows FAP is secreted by CRC cells and soluble FAP could promote metastasis. To investigate the mechanism of FAP influencing the NF-κB signaling pathway, LC/MS is performed to identify the proteins interacting with FAP. We find that FAP binds to ENO1 and activates NF-κB signaling pathway dependent on ENO1. Blocking ENO1 could partially reverse the pro-metastatic effect mediated by FAP. We also provide evidences that both FAP and ENO1 are associated with CRC stages, and high levels of FAP and ENO1 predict a poor survival in CRC patients. In summary, our work could provide a novel mechanism of FAP in CRC cells and a potential strategy for treatment of metastatic CRC.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA/metabolismo , Endopeptidases/metabolismo , Proteínas de Membrana/metabolismo , Fosfopiruvato Hidratase/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Biomarcadores Tumorais/genética , Movimento Celular/genética , Proliferação de Células/genética , Células Cultivadas , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Proteínas de Ligação a DNA/genética , Endopeptidases/genética , Células HCT116 , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , NF-kappa B/metabolismo , Metástase Neoplásica , Fosfopiruvato Hidratase/genética , Ligação Proteica , Transdução de Sinais/genética , Proteínas Supressoras de Tumor/genética
20.
Exp Cell Res ; 405(1): 112646, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34029571

RESUMO

The deubiquitinating enzyme USP46 (ubiquitin-specific protease 46) is implicated in various cancers. However, its role and regulatory mechanism in HCC (hepatocellular carcinoma) are still unknown. In this study, we showed that USP46 is downregulated in HCC tissues and that low USP46 levels are associated with poor prognosis in HCC patients. In functional experiments, overexpression of USP46 impaired proliferation and metastasis of HCC cells, whereas knockdown of USP46 enhanced cell proliferation and invasiveness in vitro and in vivo. Furthermore, we found that USP46 suppresses HCC cell proliferation and metastasis by inhibiting YAP1. Ectopic expression of YAP1 rescued the inhibition of cell proliferation and metastasis caused by USP46 overexpression. Mechanistically, USP46 promotes the degradation of YAP1 by increasing expression of MST1, and the increase in MST1 protein antagonizes YAP1 to suppress HCC progression. Finally, we demonstrated that USP46 stabilizes the MST1 protein by directly binding to it and decreasing its ubiquitination. Taken together, our results demonstrated that USP46 may be a novel tumor suppressor in HCC. Moreover, USP46 acts as a deubiquitinating enzyme of MST1 to potentiate MST1 kinase activity to suppress tumor growth and metastasis, indicating that USP46 activation may represent a potential treatment strategy for HCC.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/patologia , Endopeptidases/metabolismo , Regulação Neoplásica da Expressão Gênica , Fator de Crescimento de Hepatócito/metabolismo , Neoplasias Hepáticas/patologia , Proteínas Proto-Oncogênicas/metabolismo , Ubiquitinação , Animais , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Movimento Celular , Proliferação de Células , Endopeptidases/genética , Feminino , Fator de Crescimento de Hepatócito/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Fosforilação , Prognóstico , Proteínas Proto-Oncogênicas/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
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