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1.
Nat Commun ; 11(1): 4393, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32879321

RESUMO

Rcr3 is a secreted protease of tomato that is targeted by fungal effector Avr2, a secreted protease inhibitor of the fungal pathogen Cladosporium fulvum. The Avr2-Rcr3 complex is recognized by receptor-like protein Cf-2, triggering hypersensitive cell death (HR) and disease resistance. Avr2 also targets Rcr3 paralog Pip1, which is not required for Avr2 recognition but contributes to basal resistance. Thus, Rcr3 acts as a guarded decoy in this interaction, trapping the fungus into a recognition event. Here we show that Rcr3 evolved > 50 million years ago (Mya), whereas Cf-2 evolved <6Mya by co-opting the pre-existing Rcr3 in the Solanum genus. Ancient Rcr3 homologs present in tomato, potato, eggplants, pepper, petunia and tobacco can be inhibited by Avr2 with the exception of tobacco Rcr3. Four variant residues in Rcr3 promote Avr2 inhibition, but the Rcr3 that co-evolved with Cf-2 lacks three of these residues, indicating that the Rcr3 co-receptor is suboptimal for Avr2 binding. Pepper Rcr3 triggers HR with Cf-2 and Avr2 when engineered for enhanced inhibition by Avr2. Nicotiana benthamiana (Nb) is a natural null mutant carrying Rcr3 and Pip1 alleles with deleterious frame-shift mutations. Resurrected NbRcr3 and NbPip1 alleles were active proteases and further NbRcr3 engineering facilitated Avr2 inhibition, uncoupled from HR signalling. The evolution of a receptor co-opting a conserved pathogen target contrasts with other indirect pathogen recognition mechanisms.


Assuntos
Cladosporium , Resistência à Doença/genética , Peptídeo Hidrolases/genética , Imunidade Vegetal/genética , Solanum , Tabaco , Cladosporium/genética , Cladosporium/metabolismo , Cladosporium/patogenicidade , Evolução Molecular , Proteínas Fúngicas/metabolismo , Genes de Plantas , Interações Hospedeiro-Parasita , Peptídeo Hidrolases/metabolismo , Filogenia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Inibidores de Proteases/metabolismo , Solanum/genética , Solanum/metabolismo , Solanum/microbiologia , Tabaco/genética , Tabaco/metabolismo , Tabaco/microbiologia
2.
Proc Natl Acad Sci U S A ; 117(29): 17409-17417, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32616567

RESUMO

Proteolytic cascades regulate immunity and development in animals, but these cascades in plants have not yet been reported. Here we report that the extracellular immune protease Rcr3 of tomato is activated by P69B and other subtilases (SBTs), revealing a proteolytic cascade regulating extracellular immunity in solanaceous plants. Rcr3 is a secreted papain-like Cys protease (PLCP) of tomato that acts both in basal resistance against late blight disease (Phytophthora infestans) and in gene-for-gene resistance against the fungal pathogen Cladosporium fulvum (syn. Passalora fulva) Despite the prevalent model that Rcr3-like proteases can activate themselves at low pH, we found that catalytically inactive proRcr3 mutant precursors are still processed into mature mRcr3 isoforms. ProRcr3 is processed by secreted P69B and other Asp-selective SBTs in solanaceous plants, providing robust immunity through SBT redundancy. The apoplastic effector EPI1 of P. infestans can block Rcr3 activation by inhibiting SBTs, suggesting that this effector promotes virulence indirectly by preventing the activation of Rcr3(-like) immune proteases. Rcr3 activation in Nicotiana benthamiana requires a SBT from a different subfamily, indicating that extracellular proteolytic cascades have evolved convergently in solanaceous plants or are very ancient in the plant kingdom. The frequent incidence of Asp residues in the cleavage region of Rcr3-like proteases in solanaceous plants indicates that activation of immune proteases by SBTs is a general mechanism, illuminating a proteolytic cascade that provides robust apoplastic immunity.


Assuntos
Lycopersicon esculentum/metabolismo , Peptídeo Hidrolases/metabolismo , Doenças das Plantas/imunologia , Imunidade Vegetal , Proteólise , Cladosporium , Lycopersicon esculentum/genética , Peptídeo Hidrolases/genética , Phytophthora infestans , Doenças das Plantas/parasitologia , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/metabolismo , Isoformas de Proteínas , Virulência
3.
Am J Hum Genet ; 107(1): 158-163, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32516568

RESUMO

The discovery of genetic causes of inherited skin disorders has been pivotal to the understanding of epidermal differentiation, function, and renewal. Here we show via exome sequencing that mutations in ASPRV1 (aspartic peptidase retroviral-like 1) cause a dominant Mendelian disorder featuring palmoplantar keratoderma and lamellar ichthyosis, a phenotype that has otherwise been exclusively recessive. ASPRV1 encodes a mammalian-specific and stratified epithelia-specific protease important in processing of filaggrin, a critical component of the uppermost epidermal layer. Three different heterozygous ASPRV1 missense mutations in four unrelated ichthyosis kindreds segregate with disease and disrupt protein residues within close proximity to each other and autocatalytic cleavage sites. Expression of mutant ASPRV1 proteins demonstrates that all three mutations alter ASPRV1 auto-cleavage and filaggrin processing, a function vital to epidermal barrier integrity.


Assuntos
Hereditariedade/genética , Ictiose Lamelar/genética , Mutação de Sentido Incorreto/genética , Peptídeo Hidrolases/genética , Dermatopatias/genética , Heterozigoto , Humanos , Proteínas de Filamentos Intermediários/genética , Fenótipo , Sequenciamento Completo do Exoma/métodos
4.
Arch Microbiol ; 202(7): 1669-1675, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32285165

RESUMO

Archaea swim using archaella that are domain-specific rotary type IV pilus-like appendages. The structural components of the archaellum filament are archaellins, initially made as preproteins with type IV pilin-like signal peptides which are removed by signal peptidases that are homologues of prepilin peptidases that remove signal peptides from type IV pilins. N-terminal sequences of archaellins, including the signal peptide cleavage site, are conserved and various positions have been previously shown to be critical for signal peptide removal. Archaellins have an absolute conservation of glycine at the + 3 position from the signal peptide cleavage site. To investigate its role in signal peptide cleavage, I used archaellin variants in which the + 3 glycine was mutated to all other possibilities in in vitro cleavage reactions. Cleavage was observed with ten different amino acids at the + 3 position, indicating that the observed glycine conservation is not required for this essential processing step.


Assuntos
Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Mathanococcus/enzimologia , Mathanococcus/genética , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Proteínas Arqueais/química , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/enzimologia , Mathanococcus/metabolismo , Sinais Direcionadores de Proteínas
5.
Biochem Biophys Res Commun ; 526(1): 135-140, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: covidwho-9823

RESUMO

The new coronavirus (SARS-CoV-2) outbreak from December 2019 in Wuhan, Hubei, China, has been declared a global public health emergency. Angiotensin I converting enzyme 2 (ACE2), is the host receptor by SARS-CoV-2 to infect human cells. Although ACE2 is reported to be expressed in lung, liver, stomach, ileum, kidney and colon, its expressing levels are rather low, especially in the lung. SARS-CoV-2 may use co-receptors/auxiliary proteins as ACE2 partner to facilitate the virus entry. To identify the potential candidates, we explored the single cell gene expression atlas including 119 cell types of 13 human tissues and analyzed the single cell co-expression spectrum of 51 reported RNA virus receptors and 400 other membrane proteins. Consistent with other recent reports, we confirmed that ACE2 was mainly expressed in lung AT2, liver cholangiocyte, colon colonocytes, esophagus keratinocytes, ileum ECs, rectum ECs, stomach epithelial cells, and kidney proximal tubules. Intriguingly, we found that the candidate co-receptors, manifesting the most similar expression patterns with ACE2 across 13 human tissues, are all peptidases, including ANPEP, DPP4 and ENPEP. Among them, ANPEP and DPP4 are the known receptors for human CoVs, suggesting ENPEP as another potential receptor for human CoVs. We also conducted "CellPhoneDB" analysis to understand the cell crosstalk between CoV-targets and their surrounding cells across different tissues. We found that macrophages frequently communicate with the CoVs targets through chemokine and phagocytosis signaling, highlighting the importance of tissue macrophages in immune defense and immune pathogenesis.


Assuntos
Betacoronavirus/fisiologia , Receptores Virais/genética , Análise de Sequência de RNA , Análise de Célula Única , Coronavirus , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Humanos , Macrófagos/metabolismo , Especificidade de Órgãos , Pandemias , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/isolamento & purificação , Peptidil Dipeptidase A/genética , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Receptores Virais/isolamento & purificação
6.
Biochem Biophys Res Commun ; 526(1): 135-140, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32199615

RESUMO

The new coronavirus (SARS-CoV-2) outbreak from December 2019 in Wuhan, Hubei, China, has been declared a global public health emergency. Angiotensin I converting enzyme 2 (ACE2), is the host receptor by SARS-CoV-2 to infect human cells. Although ACE2 is reported to be expressed in lung, liver, stomach, ileum, kidney and colon, its expressing levels are rather low, especially in the lung. SARS-CoV-2 may use co-receptors/auxiliary proteins as ACE2 partner to facilitate the virus entry. To identify the potential candidates, we explored the single cell gene expression atlas including 119 cell types of 13 human tissues and analyzed the single cell co-expression spectrum of 51 reported RNA virus receptors and 400 other membrane proteins. Consistent with other recent reports, we confirmed that ACE2 was mainly expressed in lung AT2, liver cholangiocyte, colon colonocytes, esophagus keratinocytes, ileum ECs, rectum ECs, stomach epithelial cells, and kidney proximal tubules. Intriguingly, we found that the candidate co-receptors, manifesting the most similar expression patterns with ACE2 across 13 human tissues, are all peptidases, including ANPEP, DPP4 and ENPEP. Among them, ANPEP and DPP4 are the known receptors for human CoVs, suggesting ENPEP as another potential receptor for human CoVs. We also conducted "CellPhoneDB" analysis to understand the cell crosstalk between CoV-targets and their surrounding cells across different tissues. We found that macrophages frequently communicate with the CoVs targets through chemokine and phagocytosis signaling, highlighting the importance of tissue macrophages in immune defense and immune pathogenesis.


Assuntos
Betacoronavirus/fisiologia , Receptores Virais/genética , Análise de Sequência de RNA , Análise de Célula Única , Coronavirus , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Humanos , Macrófagos/metabolismo , Especificidade de Órgãos , Pandemias , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/isolamento & purificação , Peptidil Dipeptidase A/genética , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Receptores Virais/isolamento & purificação
7.
Microb Cell Fact ; 19(1): 52, 2020 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111210

RESUMO

BACKGROUND: Bacillus subtilis is an important industrial workhorse applied in the production of many different commercially relevant proteins, especially enzymes. Virtually all of these proteins are secreted via the general secretion (Sec) pathway. Studies from different laboratories have demonstrated essential or non-essential contributions of various Sec machinery components to protein secretion in B. subtilis. However, a systematic comparison of the impact of each individual Sec machinery component under conditions of high-level protein secretion was so far missing. RESULTS: In the present study, we have compared the contributions of non-essential Sec pathway components and cell envelope-associated proteases on the secretion efficiency of three proteins expressed at high level. This concerned the α-amylases AmyE from B. subtilis and AmyL from Bacillus licheniformis, and the serine protease BPN' from Bacillus amyloliquefaciens. We compared the secretion capacity of mutant strains in shake flask cultures, and the respective secretion kinetics by pulse-chase labeling experiments. The results show that secDF, secG or rasP mutations severely affect AmyE, AmyL and BPN' secretion, but the actual effect size depends on the investigated protein. Additionally, the chaperone DnaK is important for BPN' secretion, while AmyE or AmyL secretion are not affected by a dnaK deletion. Further, we assessed the induction of secretion stress responses in mutant strains by examining AmyE- and AmyL-dependent induction of the quality control proteases HtrA and HtrB. Interestingly, the deletion of certain sip genes revealed a strong differential impact of particular signal peptidases on the magnitude of the secretion stress response. CONCLUSIONS: The results of the present study highlight the importance of SecDF, SecG and RasP for protein secretion and reveal unexpected differences in the induction of the secretion stress response in different mutant strains.


Assuntos
Bacillus subtilis/enzimologia , Membrana Celular/enzimologia , Peptídeo Hidrolases/biossíntese , Via Secretória , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Peptídeo Hidrolases/genética , Transporte Proteico , Canais de Translocação SEC/genética , Canais de Translocação SEC/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , alfa-Amilases/genética
8.
J Fish Dis ; 43(5): 571-581, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32196698

RESUMO

Nocardia seriolae, a Gram-positive bacterium, is the main pathogen of fish nocardiosis. Protein NlpC/P60 is a cell-wall peptidase and a potential virulence factor of N. seriolae. Subcellular localization research revealed that both NlpC/P60-GFP and NlpC/P60Δsig-GFP fusion proteins were evenly distributed in the whole cell of fathead minnow (FHM) cells. Furthermore, typical apoptotic features, such as nuclear pyrosis and apoptotic bodies, were observed in the transfected FHM cells and grouper spleen cells by the overexpression of protein NlpC/P60. Then, quantitative assays of mitochondrial membrane potential (ΔΨm) value, caspase-3 activity and apoptosis-related gene (Bax, BNIP3, TNF1 and TNF6) mRNA expression were conducted. The results showed that ΔΨm was decreased, caspase-3 was significantly activated, and the mRNA expression of pro-apoptotic genes (Bax and BNIP3) and tumour necrosis factors (TNF1 and TNF6) was up-regulated in NlpC/P60-overexpressed cells. Taken together, the results indicated that the protein NlpC/P60 of N. seriolae might involve in apoptosis regulation. This study may lay the foundation for further study on the function of N. seriolae NlpC/P60 and promote the understanding of the virulence factors and pathogenic mechanism of N. seriolae.


Assuntos
Apoptose , Proteínas de Bactérias/genética , Cyprinidae , Nocardia/genética , Peptídeo Hidrolases/genética , Fatores de Virulência/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Doenças dos Peixes/microbiologia , Nocardia/enzimologia , Nocardiose/microbiologia , Nocardiose/veterinária , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Filogenia , Alinhamento de Sequência , Fatores de Virulência/química , Fatores de Virulência/metabolismo
9.
J Agric Food Chem ; 68(9): 2757-2764, 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32026695

RESUMO

Aspergillus oryzae 3.042 was mutagenized using atmospheric and room-temperature plasma (ARTP) technology to enhance its salt-tolerant proteases activity. Compared to the starting strain, mutant H8 subjected to 180 s of ARTP treatment exhibited excellent genetic stability (15 generations), growth rate, and significantly increased activities of neutral proteases, alkaline proteases, and aspartyl aminopeptidase during fermentation. Mutant H8 significantly enhanced the contents of 1-5 kDa peptides, aspartic acid, serine, threonine, and cysteine in soy sauce by 16.61, 7.69, 17.30, 8.61, and 45.00%, respectively, but it had no effects on the contents of the other 14 free amino acids (FAAs) due to its slightly enhanced acidic proteases activity. Analyses of transcriptional expressions of salt-tolerant alkaline protease gene (AP, gi: 217809) and aspartyl aminopeptidase gene (AAP, gi: 6165646) indicated that their expression levels were increased by approximately 30 and 27%, respectively. But no mutation was found in the sequences of AP and AAP expression cassettes, suggesting that the increased activities of proteases in mutant H8 should be partially attributed to the increased expression of proteases. ARTP technology showed great potential in enhancing the activities of salt-tolerant proteases from A. oryzae.


Assuntos
Aspergillus oryzae/enzimologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Cloreto de Sódio/metabolismo , Aspergillus oryzae/química , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Estabilidade Enzimática , Fermentação , Proteínas Fúngicas/química , Mutagênese , Peptídeo Hidrolases/química , Alimentos de Soja/análise , Alimentos de Soja/microbiologia
10.
J Food Sci ; 85(3): 535-544, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32027028

RESUMO

In this study, we characterized protease activities of 23 Ficus carica cultivars. Extracts of fruit, branch, and leaf of Masui Dauphine, one of the most representative F. carica cultivars in Japan, exhibited gelatin-hydrolyzing activity, both in the absence and presence of a cysteine protease-specific inhibitor, E-64, suggesting that not only ficin (classified as cysteine protease) but also collagenase (classified as serine protease) were involved in the digestion of gelatin. In the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-l-Lys-l-Pro-l-Leu-Gly-l-Leu-[N3 -(2,4-dinitrophenyl)-l-2,3-diaminopropionyl]-l-Ala-l-Arg-NH2 , all branch extracts of 23 F. carica cultivars exhibited the activity both in the absence and presence of cysteine protease-specific inhibitor E-64, indicating that they contain ficin and collagenase. During digestion of acid-solubilized type I collagen by the branch extract of Masui Dauphine at 40-55 °C, collagen was completely digested in the absence of E-64, while it was partially digested in the presence of the inhibitor, indicating that the manner of digestion differed between ficin and collagenase contained in the extract. These results suggest that F. carica is attractive for industrial use to digest collagen. PRACTICAL APPLICATION: The industrial use of F. carica might be enhanced by efficiently utilizing these proteases and/or selecting the appropriate F. carica cultivar. Collagen is one of the targets to which our results might be applied. It is widely accepted today that collagen and its digestion products could be useful as functional food. F. carica is a potential candidate for use in not only complete but also partial digestion of collagen.


Assuntos
Ficus/enzimologia , Peptídeo Hidrolases/química , Proteínas de Plantas/química , Biocatálise , Colágeno/química , Ficus/química , Ficus/classificação , Ficus/genética , Frutas/química , Frutas/enzimologia , Frutas/genética , Japão , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Folhas de Planta/química , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteólise
11.
Proc Natl Acad Sci U S A ; 117(8): 4358-4367, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32029587

RESUMO

When nutrients in their environment are exhausted, bacterial cells become arrested for growth. During these periods, a primary challenge is maintaining cellular integrity with a reduced capacity for renewal or repair. Here, we show that the heat-shock protease FtsH is generally required for growth arrest survival of Pseudomonas aeruginosa, and that this requirement is independent of a role in regulating lipopolysaccharide synthesis, as has been suggested for Escherichia coli We find that ftsH interacts with diverse genes during growth and overlaps functionally with the other heat-shock protease-encoding genes hslVU, lon, and clpXP to promote survival during growth arrest. Systematic deletion of the heat-shock protease-encoding genes reveals that the proteases function hierarchically during growth arrest, with FtsH and ClpXP having primary, nonredundant roles, and HslVU and Lon deploying a secondary response to aging stress. This hierarchy is partially conserved during growth at high temperature and alkaline pH, suggesting that heat, pH, and growth arrest effectively impose a similar type of proteostatic stress at the cellular level. In support of this inference, heat and growth arrest act synergistically to kill cells, and protein aggregation appears to occur more rapidly in protease mutants during growth arrest and correlates with the onset of cell death. Our findings suggest that protein aggregation is a major driver of aging and cell death during growth arrest, and that coordinated activity of the heat-shock response is required to ensure ongoing protein quality control in the absence of growth.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Temperatura Alta , Concentração de Íons de Hidrogênio , Viabilidade Microbiana , Peptídeo Hidrolases/genética , Pseudomonas aeruginosa/genética
12.
Enzyme Microb Technol ; 134: 109468, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32044021

RESUMO

Transglutaminases (TGases) are a class of transferases widely used in the food and biotechnology industries. In this work, we describe the production of recombinant Bacillus amyloliquefaciens TGase in Escherichia coli, obtaining the protein in its soluble and active form. In order to reduce TGase activity inside host cells and consequently its toxicity, we constructed a bicistronic plasmid containing the B. amyloliquefaciens TGase gene fused to the inhibitory Streptomyces caniferus prodomain. To make the enzyme active and avoid the need of prodomain removal in vitro, we also cloned the 3C protease gene into the same plasmid. After a fast single-step purification protocol, we obtained a partially purified recombinant TGase with 37 mU/mg protein activity, that crosslinked bovine serum albumin (BSA). This is the first report on the expression of B. amyloliquefaciens TGase in E. coli in its mature and active form.


Assuntos
Bacillus amyloliquefaciens/genética , Clonagem Molecular , Vetores Genéticos , Plasmídeos/genética , Transglutaminases/genética , Bacillus amyloliquefaciens/enzimologia , Escherichia coli/genética , Indústria Alimentícia , Expressão Gênica , Peptídeo Hidrolases/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Transglutaminases/biossíntese
13.
Artigo em Inglês | MEDLINE | ID: mdl-31982542

RESUMO

Cyclophosphamide (CPA) is an alkylating agent used for cancer chemotherapy, organ transplantation, and autoimmune disease treatment. Here, mRNA sequencing and high-resolution respirometry were performed to evaluate the alterations of Drosophila melanogaster gene expression fed with CPA under acute (0.1 mg/mL, for 24 h) and chronic (0.05 mg/mL, for 35 days) treatments. Differential expression analysis was performed using Cufflinks-Cuffdiff, DESeq2, and edgeR software. CPA affected genes are involved in several biological functions, including stress response and immune-related pathways, oxi-reduction and apoptotic processes, and cuticle and vitelline membrane formation. In particular, this is the first report of CPA-induced mitochondrial dysfunction caused by the downregulation of genes involved with mitochondria constituents. CPA treatment also changed the transcription pattern of transposable elements (TEs) from the gypsy and copia superfamilies. The results presented here provided evidence of CPA mitochondrial toxicity mechanisms and that CPA can modify TEs transcription in Drosophila flies.


Assuntos
Ciclofosfamida/toxicidade , Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Expressão Gênica , Mitocôndrias , Animais , Apoptose , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Oxirredução , Peptídeo Hidrolases/genética , Retroelementos/genética
14.
PLoS One ; 15(1): e0227917, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31978183

RESUMO

Experimental studies highlight the important role of the extracellular matrix (ECM) in the regulation of neuronal excitability and synaptic connectivity in the nervous system. In its turn, the neural ECM is formed in an activity-dependent manner. Its maturation closes the so-called critical period of neural development, stabilizing the efficient configurations of neural networks in the brain. ECM is locally remodeled by proteases secreted and activated in an activity-dependent manner into the extracellular space and this process is important for physiological synaptic plasticity. We ask if ECM remodeling may be exaggerated under pathological conditions and enable activity-dependent switches between different regimes of ECM expression. We consider an analytical model based on known mechanisms of interaction between neuronal activity and expression of ECM, ECM receptors and ECM degrading proteases. We demonstrate that either inhibitory or excitatory influence of ECM on neuronal activity may lead to the bistability of ECM expression, so two stable stationary states are observed. Noteworthy, only in the case when ECM has predominant inhibitory influence on neurons, the bistability is dependent on the activity of proteases. Excitatory ECM-neuron feedback influences may also result in spontaneous oscillations of ECM expression, which may coexist with a stable stationary state. Thus, ECM-neuronal interactions support switches between distinct dynamic regimes of ECM expression, possibly representing transitions into disease states associated with remodeling of brain ECM.


Assuntos
Matriz Extracelular/genética , Neurogênese/genética , Peptídeo Hidrolases/genética , Receptores de Superfície Celular/genética , Potenciais de Ação/genética , Animais , Encéfalo/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Modelos Teóricos , Rede Nervosa/metabolismo , Plasticidade Neuronal/genética , Neurônios/metabolismo , Sinapses/genética
16.
Biochim Biophys Acta Biomembr ; 1862(4): 183193, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31945321

RESUMO

Intramembrane proteases (IMPs) are proteolytic enzymes embedded in the lipid bilayer, where they cleave transmembrane substrates. The importance of IMPs relies on their role in a wide variety of cellular processes and diseases. In order to study the activity and function of IMPs, their purified form is often desired. The production of pure and active IMPs has proven to be a challenging task. This process unavoidably requires the use of solubilizing agents that will, to some extent, alter the native environment of these proteases. In this review we present the current solubilization and reconstitution techniques that have been applied to IMPs. In addition, we describe how these techniques had an influence on the activity and structural studies of IMPs, focusing on rhomboid proteases and γ-secretase.


Assuntos
Secretases da Proteína Precursora do Amiloide/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Peptídeo Hidrolases/isolamento & purificação , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/genética , Membrana Celular/química , Membrana Celular/enzimologia , Membrana Celular/genética , Microambiente Celular/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética
17.
Biochem J ; 477(2): 525-540, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31942933

RESUMO

Subtilisin-like serine peptidases (subtilases) play important roles in the life cycle of many organisms, including the protozoan parasites that are the causative agent of malaria, Plasmodium spp. As with other peptidases, subtilase proteolytic activity has to be tightly regulated in order to prevent potentially deleterious uncontrolled protein degradation. Maturation of most subtilases requires the presence of an N-terminal propeptide that facilitates folding of the catalytic domain. Following its proteolytic cleavage, the propeptide acts as a transient, tightly bound inhibitor until its eventual complete removal to generate active protease. Here we report the identification of a stand-alone malaria parasite propeptide-like protein, called SUB1-ProM, encoded by a conserved gene that lies in a highly syntenic locus adjacent to three of the four subtilisin-like genes in the Plasmodium genome. Template-based modelling and ab initio structure prediction showed that the SUB1-ProM core structure is most similar to the X-ray crystal structure of the propeptide of SUB1, an essential parasite subtilase that is discharged into the parasitophorous vacuole (PV) to trigger parasite release (egress) from infected host cells. Recombinant Plasmodium falciparum SUB1-ProM was found to be a fast-binding, potent inhibitor of P. falciparum SUB1, but not of the only other essential blood-stage parasite subtilase, SUB2, or of other proteases examined. Mass-spectrometry and immunofluorescence showed that SUB1-ProM is expressed in the PV of blood stage P. falciparum, where it may act as an endogenous inhibitor to regulate SUB1 activity in the parasite.


Assuntos
Malária Falciparum/genética , Plasmodium falciparum/genética , Serina Proteases/química , Subtilisina/genética , Sequência de Aminoácidos/genética , Animais , Eritrócitos/parasitologia , Genoma/genética , Humanos , Estágios do Ciclo de Vida/genética , Malária Falciparum/enzimologia , Malária Falciparum/parasitologia , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Plasmodium falciparum/patogenicidade , Proteólise , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Serina Proteases/genética , Subtilisina/química , Vacúolos/parasitologia
18.
Proc Natl Acad Sci U S A ; 117(5): 2519-2525, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31964807

RESUMO

The highly conserved COP9 signalosome (CSN), composed of 8 subunits (Cops1 to Cops8), has been implicated in pluripotency maintenance of human embryonic stem cells (ESCs). Yet, the mechanism for the CSN to regulate pluripotency remains elusive. We previously showed that Cops2, independent of the CSN, is essential for the pluripotency maintenance of mouse ESCs. In this study, we set out to investigate how Cops5 and Cops8 regulate ESC differentiation and tried to establish Cops5 and Cops8 knockout (KO) ESC lines by CRISPR/Cas9. To our surprise, no Cops5 KO ESC clones were identified out of 127 clones, while three Cops8 KO ESC lines were established out of 70 clones. We then constructed an inducible Cops5 KO ESC line. Cops5 KO leads to decreased expression of the pluripotency marker Nanog, proliferation defect, G2/M cell-cycle arrest, and apoptosis of ESCs. Further analysis revealed dual roles of Cops5 in maintaining genomic stability of ESCs. On one hand, Cops5 suppresses the autophagic degradation of Mtch2 to direct cellular metabolism toward glycolysis and minimize reactive oxygen species (ROS) production, thereby reducing endogenous DNA damage. On the other hand, Cops5 is required for high DNA damage repair (DDR) activities in ESCs. Without Cops5, elevated ROS and reduced DDR activities lead to DNA damage accumulation in ESCs. Subsequently, p53 is activated to trigger G2/M arrest and apoptosis. Altogether, our studies reveal an essential role of Cops5 in maintaining genome integrity and self-renewal of ESCs by regulating cellular metabolism and DDR pathways.


Assuntos
Complexo do Signalossomo COP9/metabolismo , Reparo do DNA , Células-Tronco Embrionárias/enzimologia , Instabilidade Genômica , Peptídeo Hidrolases/metabolismo , Animais , Apoptose , Complexo do Signalossomo COP9/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular , Técnicas de Inativação de Genes , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Fosforilação Oxidativa , Peptídeo Hidrolases/genética , Espécies Reativas de Oxigênio/metabolismo
19.
Arch Virol ; 165(1): 21-31, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31624917

RESUMO

To obtain insight into the sequence diversity of strawberry latent ringspot virus (SLRSV), isolates from collections and diagnostic samples were sequenced by high-throughput sequencing. For five SLRSV isolates, the complete genome sequences were determined, and for 18 other isolates nearly complete genome sequences were determined. The sequence data were analysed in relation to sequences of SLRSV and related virus isolates available in the NCBI GenBank database. The genome sequences were annotated, and sequences of the protease-polymerase (Pro-Pol) region and coat proteins (CPs) (large and small CP together) were used for phylogenetic analysis. The amino acid sequences of the Pro-Pol region were very similar, whereas the nucleotide sequences of this region were more variable. The amino acid sequences of the CPs were less similar, which was corroborated by the results of a serological comparison performed using antisera raised against different isolates of SLRSV. Based on these results, we propose that SLRSV and related unassigned viruses be assigned to a new genus within the family Secoviridae, named "Stralarivirus". Based on the phylogenetic analysis, this genus should include at least three viruses, i.e., SLRSV-A, SLRSV-B and lychnis mottle virus. The newly generated sequence data provide a basis for designing molecular tests to screen for SLRSV.


Assuntos
Fragaria/virologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Secoviridae/classificação , Análise de Sequência de RNA/métodos , Proteínas do Capsídeo/genética , RNA Polimerases Dirigidas por DNA/genética , Variação Genética , Anotação de Sequência Molecular , Peptídeo Hidrolases/genética , Filogenia , Vírus de Plantas/classificação , Vírus de Plantas/genética , Vírus de Plantas/isolamento & purificação , RNA Viral/genética , Secoviridae/genética , Secoviridae/isolamento & purificação
20.
Phytopathology ; 110(1): 187-193, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31516080

RESUMO

Potyviral helper component protease (HC-Pro), as a major determinant of symptom expression in susceptible plants, is a likely target candidate in the production of attenuated strains for cross-protection. In this study, single or double mutations of Lys (K) to Glu (E) in the Lys-Ile-Thr-Cys motif and Arg (R) to Ile (I) in the Phe-Arg-Asn-Lys motif of the HC-Pro from the severe papaya leaf distortion mosaic virus strain DF (PLDMV-DF) reduced symptom expression and virus accumulation in infected papaya (Carica papaya) plants. The papaya plants infected with the attenuated double mutant of PLDMV-EI presented as symptomless. PLDMV-EI provided effective protection against PLDMV-DF infection in three papaya cultivars and had no effect on plant growth and development. Our result showed that PLDMV-EI is a promising mild strain for the practical use of cross-protection in the field.


Assuntos
Motivos de Aminoácidos , Carica , Peptídeo Hidrolases , Potyvirus , Motivos de Aminoácidos/genética , Carica/virologia , Mutação/genética , Peptídeo Hidrolases/genética , Potyvirus/enzimologia , Potyvirus/genética
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