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1.
Nat Commun ; 11(1): 3965, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770022

RESUMO

Dysregulated Wnt/ß-catenin activation plays a critical role in cancer progression, metastasis, and drug resistance. Genotoxic agents such as radiation and chemotherapeutics have been shown to activate the Wnt/ß-catenin signaling although the underlying mechanism remains incompletely understood. Here, we show that genotoxic agent-activated Wnt/ß-catenin signaling is independent of the FZD/LRP heterodimeric receptors and Wnt ligands. OTULIN, a linear linkage-specific deubiquitinase, is essential for the DNA damage-induced ß-catenin activation. OTULIN inhibits linear ubiquitination of ß-catenin, which attenuates its Lys48-linked ubiquitination and proteasomal degradation upon DNA damage. The association with ß-catenin is enhanced by OTULIN Tyr56 phosphorylation, which depends on genotoxic stress-activated ABL1/c-Abl. Inhibiting OTULIN or Wnt/ß-catenin sensitizes triple-negative breast cancer xenograft tumors to chemotherapeutics and reduces metastasis. Increased OTULIN levels are associated with aggressive molecular subtypes and poor survival in breast cancer patients. Thus, OTULIN-mediated Wnt/ß-catenin activation upon genotoxic treatments promotes drug resistance and metastasis in breast cancers.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos , Endopeptidases/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Via de Sinalização Wnt , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Células HEK293 , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Biológicos , Metástase Neoplásica , Fosforilação , Fosfotirosina/metabolismo , Ubiquitinação , beta Catenina/metabolismo
2.
Proc Natl Acad Sci U S A ; 117(29): 16938-16948, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32616570

RESUMO

Despite nearly four decades of effort, broad inhibition of oncogenic RAS using small-molecule approaches has proven to be a major challenge. Here we describe the development of a pan-RAS biologic inhibitor composed of the RAS-RAP1-specific endopeptidase fused to the protein delivery machinery of diphtheria toxin. We show that this engineered chimeric toxin irreversibly cleaves and inactivates intracellular RAS at low picomolar concentrations terminating downstream signaling in receptor-bearing cells. Furthermore, we demonstrate in vivo target engagement and reduction of tumor burden in three mouse xenograft models driven by either wild-type or mutant RAS Intracellular delivery of a potent anti-RAS biologic through a receptor-mediated mechanism represents a promising approach to developing RAS therapeutics against a broad array of cancers.


Assuntos
Toxina Diftérica/metabolismo , Endopeptidases/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Proteólise , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Animais , Antineoplásicos/uso terapêutico , Células Cultivadas , Toxina Diftérica/química , Toxina Diftérica/genética , Endopeptidases/química , Endopeptidases/genética , Feminino , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Nus , Mutação , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/uso terapêutico , Proteínas ras/genética
3.
PLoS Pathog ; 16(7): e1008702, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32667958

RESUMO

The type I interferon response is an important innate antiviral pathway. Recognition of viral RNA by RIG-I-like receptors (RLRs) activates a signaling cascade that leads to type I interferon (IFN-α/ß) gene transcription. Multiple proteins in this signaling pathway (e.g. RIG-I, MDA5, MAVS, TBK1, IRF3) are regulated by (de)ubiquitination events. Most viruses have evolved mechanisms to counter this antiviral response. The leader protease (Lpro) of foot-and-mouth-disease virus (FMDV) has been recognized to reduce IFN-α/ß gene transcription; however, the exact mechanism is unknown. The proteolytic activity of Lpro is vital for releasing itself from the viral polyprotein and for cleaving and degrading specific host cell proteins, such as eIF4G and NF-κB. In addition, Lpro has been demonstrated to have deubiquitination/deISGylation activity. Lpro's deubiquitination/deISGylation activity and the cleavage/degradation of signaling proteins have both been postulated to be important for reduced IFN-α/ß gene transcription. Here, we demonstrate that TBK1, the kinase that phosphorylates and activates the transcription factor IRF3, is cleaved by Lpro in FMDV-infected cells as well as in cells infected with a recombinant EMCV expressing Lpro. In vitro cleavage experiments revealed that Lpro cleaves TBK1 at residues 692-694. We also observed cleavage of MAVS in HeLa cells infected with EMCV-Lpro, but only observed decreasing levels of MAVS in FMDV-infected porcine LFPK αVß6 cells. We set out to dissect Lpro's ability to cleave RLR signaling proteins from its deubiquitination/deISGylation activity to determine their relative contributions to the reduction of IFN-α/ß gene transcription. The introduction of specific mutations, of which several were based on the recently published structure of Lpro in complex with ISG15, allowed us to identify specific amino acid substitutions that separate the different proteolytic activities of Lpro. Characterization of the effects of these mutations revealed that Lpro's ability to cleave RLR signaling proteins but not its deubiquitination/deISGylation activity correlates with the reduced IFN-ß gene transcription.


Assuntos
Proteína DEAD-box 58/metabolismo , Endopeptidases/metabolismo , Vírus da Febre Aftosa/metabolismo , Interferon Tipo I/biossíntese , Animais , Linhagem Celular , Endopeptidases/genética , Febre Aftosa/imunologia , Febre Aftosa/metabolismo , Vírus da Febre Aftosa/imunologia , Humanos , Proteólise
4.
Int J Food Microbiol ; 329: 108686, 2020 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-32516659

RESUMO

Clostridium tyrobutyricum has been identified as a major species associated with the late blowing defect (LBD) of semi-hard and hard cheeses, due to undesirable butyric acid fermentation. To find new strategies to control this spoilage bacterium, we investigated the delivery of a bacteriophage endolysin by a cheese starter culture. The nisin producer Lactococcus lactis subsp. lactis INIA 415 was engineered to produce the CTP1L endolysin, encoded by the virulent bacteriophage ΦCTP1 of C. tyrobutyricum and with a demonstrated lytic activity in vitro, to the cheese matrix. The presence of the nisRK two-component regulatory system in the host strain allowed constitutive expression of the endolysin under the control of the nisA promoter (PnisA), while the use of a signal peptide (SLPmod) led to successful secretion of the active endolysin to the surrounding media. Engineered lysins with a second cell wall binding domain were also tested and shown to have improved lytic activity. Transformation of L. lactis subsp. lactis INIA 415 with endolysin delivery plasmids had a detrimental effect on its ability to produce nisin in milk, but did not affect its acidifying capacity. Transformed L. lactis subsp. lactis INIA 415 were evaluated as starters in cheeses contaminated with spores of C. tyrobutyricum. Evolution of microbiological parameters, pH and dry matter of cheeses were studied, and Clostridium metabolism and LBD in cheeses were monitored by sensory and instrumental analyses during ripening. Cheese made with the parental strain L. lactis subsp. lactis INIA 415 delayed LBD by one month, attributable to the activity of the nisin, but it was not sufficient to arrest the growth of C. tyrobutyricum during ripening completely. The use of the endolysin-producing strains in cheese manufacture as single cultures also delayed the appearance of LBD by one month, attributable to the activity of the endolysin produced in situ during ripening, because nisin activity in these cheeses was very low at day 1 and undetectable from 15 days onwards. Endolysin was more effective than nisin in inhibiting Clostridium growth, since cheeses made with the CTP1L or the chimeric derivative producers only as starters showed lower LBD symptoms, higher lactic acid levels and lower concentrations of propionic and butyric acids (associated with off-flavours) than cheese made with the parental strain. Investigation of different promoters to maximise endolysin production may help to implement CTP1L as a tool to control C. tyrobutyricum by L. lactis cheese starter and reduce LBD even further.


Assuntos
Bacteriófagos , Queijo/microbiologia , Clostridium tyrobutyricum/efeitos dos fármacos , Endopeptidases/genética , Endopeptidases/farmacologia , Microbiologia de Alimentos/métodos , Lactococcus lactis/genética , Bacteriófagos/enzimologia , Bacteriófagos/genética , Lactococcus lactis/enzimologia , Nisina/farmacologia , Organismos Geneticamente Modificados
5.
Proc Natl Acad Sci U S A ; 117(27): 15947-15954, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32576686

RESUMO

The cytosolic DNA sensor cGMP-AMP synthase (cGAS) synthesizes the noncanonical cyclic dinucleotide 2'3'-cGAMP to activate the adaptor protein stimulator of IFN genes (STING), thus awakening host immunity in response to DNA pathogen infection. However, dengue virus (DENV), an RNA virus without a DNA stage in its life cycle, also manipulates cGAS-STING-mediated innate immunity by proteolytic degradation of STING. Here, we found that the sensitivity of STING to DENV protease varied with different human STING haplotypes. Exogenous DNA further enhanced DENV protease's ability to interact and cleave protease-sensitive STING. DNA-enhanced STING cleavage was reduced in cGAS-knockdown cells and triggered by the cGAS product 2'3'-cGAMP. The source of DNA may not be endogenous mitochondrial DNA but rather exogenous reactivated viral DNA. Cells producing 2'3'-cGAMP by overexpressing cGAS or with DNA virus reactivation enhanced STING cleavage in neighboring cells harboring DENV protease. DENV infection reduced host innate immunity in cells with the protease-sensitive STING haplotype, whose homozygote genotype frequency was found significantly reduced in Taiwanese people with dengue fever. Therefore, the human STING genetic background and DNA pathogen coinfection may be the missing links contributing to DENV pathogenesis.


Assuntos
Dengue/enzimologia , Endopeptidases/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Nucleotídeos Cíclicos/metabolismo , Células A549 , DNA Viral/genética , Dengue/imunologia , Endopeptidases/genética , Haplótipos , Humanos , Evasão da Resposta Imune , Imunidade Inata , Nucleotídeos Cíclicos/genética
6.
Bioresour Technol ; 312: 123397, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32526667

RESUMO

This study reported a novel pretreatment approach with combination of alkaline protease (AP) and pH 10 for enhancing short-chain fatty acids (SCFAs) production from waste activated sludge (WAS). Through the AP-based pretreatment, WAS flocs were disintegrated with cell lysis, leading to release of biodegradable organic matters. At the external AP dosage of 5%, SCOD of 5363.7 mg/L (SCOD/TCOD = 32.5%) was achievable after 2-h pretreatment. More than 66% of SCOD was composed of proteins and carbohydrates. Considerable SCFAs of 607 mg COD/g VSS was produced over a short-term anaerobic fermentation of 3 days, which was 5.4 times higher than that in the control. Acetic and propionic acids accounted for 74.1% of the SCFAs. The AP-based approach increased endogenous protease and α-glucosidase activities, facilitating biodegradation of dissolved organic matters and SCFAs production. Such approach is promising for WAS disposal and carbon recovery, the produced SCFAs might supply 60% of carbon gap in wastewater.


Assuntos
Ácidos Graxos Voláteis , Esgotos , Anaerobiose , Proteínas de Bactérias , Endopeptidases , Fermentação , Concentração de Íons de Hidrogênio
7.
Science ; 368(6495): 1132-1135, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32499443

RESUMO

The lumicrine system is a postulated signaling system in which testis-derived (upstream) secreted factors enter the male reproductive tract to regulate epididymal (downstream) pathways required for sperm maturation. Until now, no lumicrine factors have been identified. We demonstrate that a testicular germ-cell-secreted epidermal growth factor-like protein, neural epidermal growth factor-like-like 2 (NELL2), specifically binds to an orphan receptor tyrosine kinase, c-ros oncogene 1 (ROS1), and mediates the differentiation of the initial segment (IS) of the caput epididymis. Male mice in which Nell2 had been knocked out were infertile. The IS-specific secreted proteases, ovochymase 2 (OVCH2) and A disintegrin and metallopeptidase 28 (ADAM28), were expressed upon IS maturation, and OVCH2 was required for processing of the sperm surface protein ADAM3, which is required for sperm fertilizing ability. This work identifies a lumicrine system essential for testis-epididymis-spermatozoa (NELL2-ROS1-OVCH2-ADAM3) signaling and male fertility.


Assuntos
Comunicação Celular/fisiologia , Endopeptidases/metabolismo , Epididimo/metabolismo , Fertilidade , Infertilidade Masculina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Espermatozoides/metabolismo , Testículo/metabolismo , Proteínas ADAM/metabolismo , Animais , Comunicação Celular/genética , Endopeptidases/genética , Infertilidade Masculina/genética , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo
8.
Mol Cell ; 79(1): 155-166.e9, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32454028

RESUMO

To understand gene function, the encoding DNA or mRNA transcript can be manipulated and the consequences observed. However, these approaches do not have a direct effect on the protein product of the gene, which is either permanently abrogated or depleted at a rate defined by the half-life of the protein. We therefore developed a single-component system that could induce the rapid degradation of the specific endogenous protein itself. A construct combining the RING domain of ubiquitin E3 ligase RNF4 with a protein-specific camelid nanobody mediates target destruction by the ubiquitin proteasome system, a process we describe as antibody RING-mediated destruction (ARMeD). The technique is highly specific because we observed no off-target protein destruction. Furthermore, bacterially produced nanobody-RING fusion proteins electroporated into cells induce degradation of target within minutes. With increasing availability of protein-specific nanobodies, this method will allow rapid and specific degradation of a wide range of endogenous proteins.


Assuntos
Endopeptidases/metabolismo , Proteína NEDD8/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Anticorpos de Domínio Único/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Endopeptidases/imunologia , Células HeLa , Humanos , Proteína NEDD8/imunologia , Proteínas Nucleares/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Proteólise , Anticorpos de Domínio Único/imunologia , Fatores de Transcrição/imunologia , Ubiquitinação
9.
Nucleic Acids Res ; 48(11): 6353-6366, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32396195

RESUMO

Most eukaryotic mRNAs harbor a characteristic 5' m7GpppN cap that promotes pre-mRNA splicing, mRNA nucleocytoplasmic transport and translation while also protecting mRNAs from exonucleolytic attacks. mRNA caps are eliminated by Dcp2 during mRNA decay, allowing 5'-3' exonucleases to degrade mRNA bodies. However, the Dcp2 decapping enzyme is poorly active on its own and requires binding to stable or transient protein partners to sever the cap of target mRNAs. Here, we analyse the role of one of these partners, the yeast Pby1 factor, which is known to co-localize into P-bodies together with decapping factors. We report that Pby1 uses its C-terminal domain to directly bind to the decapping enzyme. We solved the structure of this Pby1 domain alone and bound to the Dcp1-Dcp2-Edc3 decapping complex. Structure-based mutant analyses reveal that Pby1 binding to the decapping enzyme is required for its recruitment into P-bodies. Moreover, Pby1 binding to the decapping enzyme stimulates growth in conditions in which decapping activation is compromised. Our results point towards a direct connection of Pby1 with decapping and P-body formation, both stemming from its interaction with the Dcp1-Dcp2 holoenzyme.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Endorribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Trifosfato de Adenosina/metabolismo , Domínio Catalítico , Proteínas de Ligação a DNA/química , Endopeptidases/química , Endopeptidases/metabolismo , Endorribonucleases/química , Holoenzimas/química , Holoenzimas/metabolismo , Ligases/metabolismo , Modelos Moleculares , Organelas/enzimologia , Organelas/metabolismo , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química
10.
Proc Natl Acad Sci U S A ; 117(21): 11692-11702, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32393643

RESUMO

Most bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over; the latter process is mediated by PG cleavage enzymes, for example, the endopeptidases (EPs). EPs themselves are essential for growth but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., ß-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated in vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogen Vibrio cholerae Our data suggest that ShyA assumes two drastically different conformations: a more open form that allows for substrate binding and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity in vitro and in vivo, and these results were recapitulated in EPs from the divergent pathogens Neisseria gonorrheae and Escherichia coli Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain 1 from the M23 active site, likely through conformational rearrangement in vivo.


Assuntos
Proteínas de Bactérias , Endopeptidases , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Modelos Moleculares , Mutação/genética , Neisseria gonorrhoeae/enzimologia , Neisseria gonorrhoeae/genética , Conformação Proteica , Vibrio cholerae/enzimologia , Vibrio cholerae/genética
11.
Am J Hematol ; 95(7): 863-869, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32350907

RESUMO

Ecarin is derived from venom of Echis carinatus, and will activate prothrombin into meizothrombin which will then cleave fibrinogen to result in clot formation. Ecarin based testing has been described for decades, but these assays were typically restricted to reference or speciality coagulation laboratories. This test was initially described for the assessment of direct thrombin inhibitors (eg, bivalirudin lepirudin, or argatroban) and was not affected by heparins or heparinoids. Ecarin based assays were rarely used for anticoagulation monitoring until the emergence of the direct oral thrombin inhibitor dabigatran etexilate in 2010. As this test was mentioned in the prescribing information for dabigatran etexilate, there was increased interest for use by clinical laboratories as the preferred method for assessing the anticoagulant effect of this drug. The purpose of this document is to review the current status of ecarin based assays for assessing dabigatran. This is with the understanding that these methods can also be exploited for determining the anticoagulation effect of parenteral direct thrombin inhibitors, such as argatroban and bivalirudin.


Assuntos
Antitrombinas/farmacocinética , Dabigatrana/farmacocinética , Monitoramento de Medicamentos , Endopeptidases/química , Antitrombinas/uso terapêutico , Testes de Coagulação Sanguínea , Dabigatrana/uso terapêutico , Humanos
12.
Proc Natl Acad Sci U S A ; 117(23): 13023-13032, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32461361

RESUMO

Clear cell renal cell carcinoma (ccRCC) is characterized by loss of tumor suppressor Von Hippel Lindau (VHL) function, which leads to accumulation of hypoxia inducible factor α (including HIF1α and HIF2α). HIF2α was previously reported to be one of the major oncogenic drivers in ccRCC, however, its therapeutic targets remain challenging. Here we performed a deubiquitinase (DUB) complementary DNA (cDNA) library binding screen and discovered that ubiquitin-specific peptidase 37 (USP37) is a DUB that binds HIF2α and promotes HIF2α deubiquitination. As a result, USP37 promotes HIF2α protein stability in an enzymatically dependent manner, and depletion of USP37 leads to HIF2α down-regulation in ccRCC. Functionally, USP37 depletion causes decreased cell proliferation measured by MTS, two-dimensional (2D) colony formation as well as three-dimensional (3D) anchorage- independent growth. USP37 is also essential for maintaining kidney tumorigenesis in an orthotopic xenograft model and its depletion leads to both decreased primary kidney tumorigenesis and spontaneous lung metastasis. Our results suggest that USP37 is a potential therapeutic target in ccRCC.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma de Células Renais/patologia , Endopeptidases/metabolismo , Neoplasias Renais/patologia , Animais , Carcinogênese , Carcinoma de Células Renais/genética , Linhagem Celular Tumoral , Sequenciamento de Cromatina por Imunoprecipitação , Regulação para Baixo , Endopeptidases/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/genética , Camundongos , Estabilidade Proteica , RNA Interferente Pequeno/metabolismo , RNA-Seq , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto
13.
PLoS One ; 15(5): e0232755, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32401766

RESUMO

The quality control of intracellular proteins is achieved by degrading misfolded proteins which cannot be refolded by molecular chaperones. In eukaryotes, such degradation is handled primarily by the ubiquitin-proteasome system. However, it remained unclear whether and how protein quality control deploys various deubiquitinases. To address this question, we screened deletions or mutation of the 20 deubiquitinase genes in Saccharomyces cerevisiae and discovered that almost half of the mutations slowed the removal of misfolded proteins whereas none of the remaining mutations accelerated this process significantly. Further characterization revealed that Ubp6 maintains the level of free ubiquitin to promote the elimination of misfolded cytosolic proteins, while Ubp3 supports the degradation of misfolded cytosolic and ER luminal proteins by different mechanisms.


Assuntos
Citosol/enzimologia , Endopeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Aneuploidia , Degradação Associada com o Retículo Endoplasmático , Testes Genéticos , Saccharomyces cerevisiae/genética , Ubiquitina/metabolismo
14.
Arch Microbiol ; 202(7): 1617-1626, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32253452

RESUMO

In this study, the genome of a new strain of lytic Staphylococcus aureus Herelleviridae, vBSM-A1, was characterized and annotated. The phage was isolated from sewage samples collected in Xinjiang Province, China. The genome of vBSM-A1 was found to comprise a linear double-stranded DNA of 140,654 bp length, with a G + C content of 30.33%. A total of 215 ORFs were detected in the phage DNA, 74 of which were functionally assigned. The 3D structure model of endolysin LysK (ORF 143) was created using Phyre2.


Assuntos
Genoma Viral/genética , Fagos de Staphylococcus/genética , Composição de Bases , China , DNA Viral/química , DNA Viral/genética , Endopeptidases/química , Endopeptidases/genética , Modelos Moleculares , Fases de Leitura Aberta , Estrutura Terciária de Proteína , Análise de Sequência de DNA , Fagos de Staphylococcus/isolamento & purificação
15.
Plant Biol (Stuttg) ; 22(4): 563-572, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32233097

RESUMO

A balance between the synthesis and degradation of active proteins governs diverse cellular processes in plants, spanning from cell-cycle progression and circadian rhythm to the outcome of several hormone signalling pathways. Ubiquitin-mediated post-translational modification determines the degradative fate of the target proteins, thereby altering the output of cellular processes. An equally important, and perhaps under-appreciated, aspect of this pathway is the antagonistic process of de-ubiquitination. De-ubiquitinases (DUBs), a group of processing enzymes, play an important role in maintaining cellular ubiquitin homeostasis by hydrolyzing ubiquitin poly-proteins and free poly-ubiquitin chains into mono-ubiquitin. Further, DUBs rescue the cellular proteins from 26S proteasome-mediated degradation to their active form by cleaving the poly-ubiquitin chain from the target protein. Any perturbation in DUB activity is likely to affect proteostasis and downstream cellular processes. This review illustrates recent findings on the biological significance and mechanisms of action of the DUBs in Arabidopsis thaliana, with an emphasis on ubiquitin-specific proteases (UBPs), the largest family among the DUBs. We focus on the putative roles of various protein-protein interaction interfaces in DUBs and their generalized function in ubiquitin recycling, along with their pre-eminent role in plant development.


Assuntos
Botânica , Endopeptidases , Plantas , Ubiquitina , Arabidopsis/enzimologia , Botânica/tendências , Endopeptidases/metabolismo , Plantas/enzimologia , Processamento de Proteína Pós-Traducional , Ubiquitina/metabolismo , Ubiquitinação
16.
PLoS Pathog ; 16(4): e1008465, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32271834

RESUMO

Roundworm parasite infections are a major cause of human and livestock disease worldwide and a threat to global food security. Disease control currently relies on anthelmintic drugs to which roundworms are becoming increasingly resistant. An alternative approach is control by vaccination and 'hidden antigens', components of the worm gut not encountered by the infected host, have been exploited to produce Barbervax, the first commercial vaccine for a gut dwelling nematode of any host. Here we present the structure of H-gal-GP, a hidden antigen from Haemonchus contortus, the Barber's Pole worm, and a major component of Barbervax. We demonstrate its novel architecture, subunit composition and topology, flexibility and heterogeneity using cryo-electron microscopy, mass spectrometry, and modelling. Importantly, we demonstrate that complexes with the same architecture are present in other Strongylid roundworm parasites including human hookworm. This suggests a common ancestry and the potential for development of a unified hidden antigen vaccine.


Assuntos
Endopeptidases/metabolismo , Endopeptidases/ultraestrutura , Haemonchus/imunologia , Proteínas de Helminto/metabolismo , Proteínas de Helminto/ultraestrutura , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/ultraestrutura , Animais , Anti-Helmínticos/farmacologia , Anticorpos Anti-Helmínticos , Antígenos de Helmintos/imunologia , Microscopia Crioeletrônica , Endopeptidases/imunologia , Haemonchus/patogenicidade , Proteínas de Helminto/imunologia , Glicoproteínas de Membrana/imunologia , Parasitos , Vacinação , Vacinas/imunologia
17.
Nat Commun ; 11(1): 2060, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345978

RESUMO

Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces are generated and sensed in biological tissues. However, testing these mechanical hypotheses on proteins in their natural environment remains inaccesible to conventional tools. To address this limitation, here we demonstrate a mouse model carrying a HaloTag-TEV insertion in the protein titin, the main determinant of myocyte stiffness. Using our system, we specifically sever titin by digestion with TEV protease, and find that the response of muscle fibers to length changes requires mechanical transduction through titin's intact polypeptide chain. In addition, HaloTag-based covalent tethering enables examination of titin dynamics under force using magnetic tweezers. At pulling forces < 10 pN, titin domains are recruited to the unfolded state, and produce 41.5 zJ mechanical work during refolding. Insertion of the HaloTag-TEV cassette in mechanical proteins opens opportunities to explore the molecular basis of cellular force generation, mechanosensing and mechanotransduction.


Assuntos
Conectina/metabolismo , Endopeptidases/genética , Especificidade de Órgãos , Animais , Fenômenos Biomecânicos , Conectina/química , Feminino , Proteínas Imobilizadas/metabolismo , Magnetismo , Camundongos , Músculos/metabolismo , Músculos/ultraestrutura , Pinças Ópticas , Fenótipo , Dobramento de Proteína , Análise Espectral
18.
Life Sci ; 253: 117659, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32283055

RESUMO

Abdominal aortic aneurysm (AAA) is a chronic vascular degenerative disease featured by progressive dilation and remodeling of the vascular wall, which may lead to aortic rupture and high mortality. The occurrence and development of AAA involve multiple mechanisms, including extracellular matrix degradation, chronic inflammation, oxidative stress, apoptosis of vascular smooth muscle cells and innate immunity. Extracellular matrix degradation is considered as the most important mechanism causing AAA. Matrix metalloproteinases (MMPs) are key factors in this process, contributing greatly to the occurrence and development of AAA. But whether the zinc-dependent endopeptidases (ADAM/ADAMTS) are involved in this process is very little known. This study is a review about the role of MMPs and ADAM/ADAMT as well as the existing MMP inhibitors in abdominal aortic aneurysm, with the purpose of providing reference for the clinical treatment of abdominal aortic aneurysm.


Assuntos
Proteínas ADAM/metabolismo , Aneurisma da Aorta Abdominal/tratamento farmacológico , Inibidores de Metaloproteinases de Matriz/farmacologia , Metaloproteinases da Matriz/metabolismo , Animais , Endopeptidases/metabolismo , Humanos , Miócitos de Músculo Liso/citologia , Proteólise , Transdução de Sinais
19.
Clin Implant Dent Relat Res ; 22(3): 424-450, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32319195

RESUMO

BACKGROUND AND OBJECTIVE: Although periimplantitis and periodontitis share similar features, particularly clinical features, they are two different diseases and should be analyzed separately. Thus far, few omics-level differences in periimplantitis and periodontitis have been reported. This study was aimed at exploring the differential effects of expression mRNAs, lncRNAs, and miRNAs in periodontitis and periimplantitis by high-throughput sequencing and competitive endogenous RNA (ceRNA) analysis. METHODS: Gingival tissues of healthy individuals (HI) and periimplantitis (PI) and periodontitis (P) patients were collected and used for genome-wide sequencing. The differentially expressed genes (DEGs) were screened and visualized by R software. The functions and pathways of DEGs were analyzed using Metascape, and the ceRNA network was constructed using the Cytoscape software. Finally, gene set enrichment analysis (GSEA) was used to predict the function of key nodes in ceRNA. RESULTS AND CONCLUSION: By constructing the regulated ceRNA network, six genes (FAM126B, SORL1, PRLR, CPEB2, RAP2C, and YOD1) and 16 miRNAs (hsa-miR-338-5p, hsa-miR-650, hsa-miR-9-5p, hsa-miR-1290, hsa-miR-544a, hsa-miR-3179, hsa-miR-1269a, hsa-miR-3679-5p, hsa-miR-149-5p, hsa-miR-615-3p, hsa-miR-33b-5p, hsa-miR-31-5p, hsa-miR-4639-5p, hsa-miR-204-5p, hsa-miR-5588-5p, and hsa-mir-196a-5p) were detected. Five long non-coding RNAs (lnc-CORO2B-1, lnc-MBL2-7, lnc-TRIM45-1, lnc-CHST10-2, and lnc-TNP1-6) were found to target these miRNAs in this ceRNA network. The ceRNA network based on transcriptome data revealed that FAM126B, SORL1, PRLR, CPEB2, RAP2C, and YOD1 were crucial proteins of differential effects in periodontitis and periimplantitis. The lncRNA-miRNA-mRNA interaction involved the regulation of the Hippo signaling pathway, Wnt signaling pathway, Toll-like receptor signaling pathway, NOD signaling pathway, oxidative stress, and innate immune process. These regulated pathways and biological processes may be factors contributing to the pathogenesis of periimplantitis being distinct from that of periodontitis.


Assuntos
Peri-Implantite , Periodontite , RNA Longo não Codificante , Endopeptidases , Humanos , Proteínas dos Microfilamentos , RNA Mensageiro , Proteínas de Ligação a RNA , Proteínas Repressoras , Tioléster Hidrolases , Transcriptoma , Proteínas ras
20.
Infect Dis Poverty ; 9(1): 33, 2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: covidwho-13772

RESUMO

BACKGROUND: Coronavirus can cross the species barrier and infect humans with a severe respiratory syndrome. SARS-CoV-2 with potential origin of bat is still circulating in China. In this study, a prediction model is proposed to evaluate the infection risk of non-human-origin coronavirus for early warning. METHODS: The spike protein sequences of 2666 coronaviruses were collected from 2019 Novel Coronavirus Resource (2019nCoVR) Database of China National Genomics Data Center on Jan 29, 2020. A total of 507 human-origin viruses were regarded as positive samples, whereas 2159 non-human-origin viruses were regarded as negative. To capture the key information of the spike protein, three feature encoding algorithms (amino acid composition, AAC; parallel correlation-based pseudo-amino-acid composition, PC-PseAAC and G-gap dipeptide composition, GGAP) were used to train 41 random forest models. The optimal feature with the best performance was identified by the multidimensional scaling method, which was used to explore the pattern of human coronavirus. RESULTS: The 10-fold cross-validation results showed that well performance was achieved with the use of the GGAP (g = 3) feature. The predictive model achieved the maximum ACC of 98.18% coupled with the Matthews correlation coefficient (MCC) of 0.9638. Seven clusters for human coronaviruses (229E, NL63, OC43, HKU1, MERS-CoV, SARS-CoV, and SARS-CoV-2) were found. The cluster for SARS-CoV-2 was very close to that for SARS-CoV, which suggests that both of viruses have the same human receptor (angiotensin converting enzyme II). The big gap in the distance curve suggests that the origin of SARS-CoV-2 is not clear and further surveillance in the field should be made continuously. The smooth distance curve for SARS-CoV suggests that its close relatives still exist in nature and public health is challenged as usual. CONCLUSIONS: The optimal feature (GGAP, g = 3) performed well in terms of predicting infection risk and could be used to explore the evolutionary dynamic in a simple, fast and large-scale manner. The study may be beneficial for the surveillance of the genome mutation of coronavirus in the field.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus , Coronavirus/imunologia , Reservatórios de Doenças/virologia , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral , Receptores Virais/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Algoritmos , Aminoácidos/genética , Animais , Betacoronavirus/genética , China , Chlorocebus aethiops , Coronavirus/genética , Coronavirus/isolamento & purificação , Infecções por Coronavirus/genética , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Endopeptidases/genética , Endopeptidases/metabolismo , Genoma/genética , Genoma Viral/genética , Humanos , Pandemias/prevenção & controle , Peptidil Dipeptidase A/genética , Filogenia , Pneumonia Viral/genética , Pneumonia Viral/transmissão , Pneumonia Viral/virologia , Receptores Virais/metabolismo , Medição de Risco
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