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1.
Molecules ; 26(17)2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34500777

RESUMO

Human neutrophil elastase (HNE) is a uniquely destructive serine protease with the ability to unleash a wave of proteolytic activity by destroying the inhibitors of other proteases. Although this phenomenon forms an important part of the innate immune response to invading pathogens, it is responsible for the collateral host tissue damage observed in chronic conditions such as chronic obstructive pulmonary disease (COPD), and in more acute disorders such as the lung injuries associated with COVID-19 infection. Previously, a combinatorially selected activity-based probe revealed an unexpected substrate preference for oxidised methionine, which suggests a link to oxidative pathogen clearance by neutrophils. Here we use oxidised model substrates and inhibitors to confirm this observation and to show that neutrophil elastase is specifically selective for the di-oxygenated methionine sulfone rather than the mono-oxygenated methionine sulfoxide. We also posit a critical role for ordered solvent in the mechanism of HNE discrimination between the two oxidised forms methionine residue. Preference for the sulfone form of oxidised methionine is especially significant. While both host and pathogens have the ability to reduce methionine sulfoxide back to methionine, a biological pathway to reduce methionine sulfone is not known. Taken together, these data suggest that the oxidative activity of neutrophils may create rapidly cleaved elastase "super substrates" that directly damage tissue, while initiating a cycle of neutrophil oxidation that increases elastase tissue damage and further neutrophil recruitment.


Assuntos
Imunidade Inata , Elastase de Leucócito/metabolismo , Metionina/análogos & derivados , Neutrófilos/imunologia , Biocatálise , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico/genética , Ensaios Enzimáticos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/genética , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Metionina/metabolismo , Simulação de Dinâmica Molecular , Infiltração de Neutrófilos , Neutrófilos/enzimologia , Oxirredução/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , SARS-CoV-2/imunologia , Especificidade por Substrato/imunologia
2.
Adv Exp Med Biol ; 1288: 215-240, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34453739

RESUMO

Ubiquitination is one of the most diverse forms of protein post-translational modification that changes the function of the landscape of substrate proteins in response to stimuli, without the need for "de novo" protein synthesis. Ubiquitination is involved in almost all aspects of eukaryotic cell biology, from the best-studied role in promoting the removal of faulty or unnecessary proteins by the way of the ubiquitin proteasome system and autophagy-lysosome pathway to the recruitment of proteins in specific non-proteolytic signaling pathways, as emerged by the more recent discoveries about the protein signature with peculiar types of ubiquitin chains. Spermatogenesis, on its own, is a complex cellular developmental process in which mitosis, meiosis, and cell differentiation coexist so to result in the continuous formation of haploid spermatozoa. Successful spermatogenesis is thus at the same time a mixed result of the precise expression and correct intracellular destination of structural proteins and enzymes, from one hand, and the fine removal by targeted degradation of unfolded or damaged proteins as well as of obsolete, outlived proteins, from the other hand. In this minireview, I will focus on the importance of the ubiquitin system all over the spermatogenic process, discussing both proteolytic and non-proteolytic functions of protein ubiquitination. Alterations in the ubiquitin system have been in fact implicated in pathologies leading to male infertility. Notwithstanding several aspects of the multifaceted world of the ubiquitin system have been clarified, the physiological meaning of the so-called ubiquitin code remains still partially elusive. The studies reviewed in this chapter provide information that could aid the investigators to pursue new promising discoveries in the understanding of human and animal reproductive potential.


Assuntos
Espermatogênese , Ubiquitina , Animais , Humanos , Masculino , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitina/metabolismo , Ubiquitinação
3.
Mol Cell ; 81(16): 3246-3261.e11, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34352208

RESUMO

The Wnt/ß-catenin pathway is a highly conserved, frequently mutated developmental and cancer pathway. Its output is defined mainly by ß-catenin's phosphorylation- and ubiquitylation-dependent proteasomal degradation, initiated by the multi-protein ß-catenin destruction complex. The precise mechanisms underlying destruction complex function have remained unknown, largely because of the lack of suitable in vitro systems. Here we describe the in vitro reconstitution of an active human ß-catenin destruction complex from purified components, recapitulating complex assembly, ß-catenin modification, and degradation. We reveal that AXIN1 polymerization and APC promote ß-catenin capture, phosphorylation, and ubiquitylation. APC facilitates ß-catenin's flux through the complex by limiting ubiquitylation processivity and directly interacts with the SCFß-TrCP E3 ligase complex in a ß-TrCP-dependent manner. Oncogenic APC truncation variants, although part of the complex, are functionally impaired. Nonetheless, even the most severely truncated APC variant promotes ß-catenin recruitment. These findings exemplify the power of biochemical reconstitution to interrogate the molecular mechanisms of Wnt/ß-catenin signaling.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Proteína Axina/genética , beta Catenina/genética , Proteína da Polipose Adenomatosa do Colo/ultraestrutura , Proteína Axina/química , Proteína Axina/ultraestrutura , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Fosforilação/genética , Multimerização Proteica/genética , Proteólise , Ubiquitinação/genética , Via de Sinalização Wnt
4.
Molecules ; 26(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34443492

RESUMO

Muscle wasting and cachexia are prominent comorbidities in cancer. Treatment with polyphenolic compounds may partly revert muscle wasting. We hypothesized that treatment with curcumin or resveratrol in cancer cachectic mice may improve muscle phenotype and total body weight through attenuation of several proteolytic and signaling mechanisms in limb muscles. In gastrocnemius and soleus muscles of cancer cachectic mice (LP07 adenocarcinoma cells, N = 10/group): (1) LC-induced cachexia, (2) LC-cachexia+curcumin, and (3) LC-cachexia + resveratrol, muscle structure and damage (including blood troponin I), sirtuin-1, proteolytic markers, and signaling pathways (NF-κB and FoxO3) were explored (immunohistochemistry and immunoblotting). Compared to nontreated cachectic mice, in LC-cachexia + curcumin and LC-cachexia + resveratrol groups, body and muscle weights (gastrocnemius), limb muscle strength, muscle damage, and myofiber cross-sectional area improved, and in both muscles, sirtuin-1 increased, while proteolysis (troponin I), proteolytic markers, and signaling pathways were attenuated. Curcumin and resveratrol elicited beneficial effects on fast- and slow-twitch limb muscle phenotypes in cachectic mice through sirtuin-1 activation, attenuation of atrophy signaling pathways, and proteolysis in cancer cachectic mice. These findings have future therapeutic implications as these natural compounds, separately or in combination, may be used in clinical settings of muscle mass loss and dysfunction including cancer cachexia.


Assuntos
Caquexia/etiologia , Caquexia/fisiopatologia , Curcumina/farmacologia , Músculos/patologia , Músculos/fisiopatologia , Neoplasias/complicações , Proteólise , Resveratrol/farmacologia , Animais , Biomarcadores/metabolismo , Linhagem Celular , Feminino , Camundongos Endogâmicos BALB C , Proteínas Musculares/metabolismo , Músculos/efeitos dos fármacos , Atrofia Muscular/metabolismo , Fenótipo , Proteólise/efeitos dos fármacos , Transdução de Sinais , Sirtuína 1/metabolismo
5.
FASEB J ; 35(9): e21870, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34436790

RESUMO

COVID-19 is often characterized by dysregulated inflammatory and immune responses. It has been shown that the Traditional Chinese Medicine formulation Qing-Fei-Pai-Du decoction (QFPDD) is effective in the treatment of the disease, especially for patients in the early stage. Our network pharmacology analyses indicated that many inflammation and immune-related molecules were the targets of the active components of QFPDD, which propelled us to examine the effects of the decoction on inflammation. We found in the present study that QFPDD effectively alleviated dextran sulfate sodium-induced intestinal inflammation in mice. It inhibited the production of pro-inflammatory cytokines IL-6 and TNFα, and promoted the expression of anti-inflammatory cytokine IL-10 by macrophagic cells. Further investigations found that QFPDD and one of its active components wogonoside markedly reduced LPS-stimulated phosphorylation of transcription factor ATF2, an important regulator of multiple cytokines expression. Our data revealed that both QFPDD and wogonoside decreased the half-life of ATF2 and promoted its proteasomal degradation. Of note, QFPDD and wogonoside down-regulated deubiquitinating enzyme USP14 along with inducing ATF2 degradation. Inhibition of USP14 with the small molecular inhibitor IU1 also led to the decrease of ATF2 in the cells, indicating that QFPDD and wogonoside may act through regulating USP14 to promote ATF2 degradation. To further assess the importance of ubiquitination in regulating ATF2, we generated mice that were intestinal-specific KLHL5 deficiency, a CUL3-interacting protein participating in substrate recognition of E3s. In these mice, QFPDD mitigated inflammatory reaction in the spleen, but not intestinal inflammation, suggesting CUL3-KLHL5 may function as an E3 for ATF2 degradation.


Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Regulação para Baixo/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Flavanonas/farmacologia , Glucosídeos/farmacologia , Inflamação/tratamento farmacológico , Proteólise/efeitos dos fármacos , Ubiquitina Tiolesterase/deficiência , Animais , Linhagem Celular , Colite/induzido quimicamente , Colite/tratamento farmacológico , Proteínas Culina/metabolismo , Citocinas/metabolismo , Sulfato de Dextrana/farmacologia , Sulfato de Dextrana/uso terapêutico , Medicamentos de Ervas Chinesas/uso terapêutico , Flavanonas/uso terapêutico , Glucosídeos/uso terapêutico , Inflamação/induzido quimicamente , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Pirróis/farmacologia , Pirrolidinas/farmacologia , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitinação
6.
Nat Commun ; 12(1): 4794, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373456

RESUMO

The cellular NLRP3 protein level is crucial for assembly and activation of the NLRP3 inflammasome. Various posttranslational modifications (PTMs), including phosphorylation and ubiquitination, control NLRP3 protein degradation and inflammasome activation; however, the function of small ubiquitin-like modifier (SUMO) modification (called SUMOylation) in controlling NLRP3 stability and subsequent inflammasome activation is unclear. Here, we show that the E3 SUMO ligase tripartite motif-containing protein 28 (TRIM28) is an enhancer of NLRP3 inflammasome activation by facilitating NLRP3 expression. TRIM28 binds NLRP3, promotes SUMO1, SUMO2 and SUMO3 modification of NLRP3, and thereby inhibits NLRP3 ubiquitination and proteasomal degradation. Concordantly, Trim28 deficiency attenuates NLRP3 inflammasome activation both in vitro and in vivo. These data identify a mechanism by which SUMOylation controls the cellular NLRP3 level and inflammasome activation, and reveal correlations and interactions of NLRP3 SUMOylation and ubiquitination during inflammasome activation.


Assuntos
Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Sumoilação/fisiologia , Proteína 28 com Motivo Tripartido/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Proteólise , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação/genética , Proteína 28 com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ubiquitinas/metabolismo
7.
FASEB J ; 35(9): e21813, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34390512

RESUMO

Cell adhesion is tightly controlled in multicellular organisms, for example, through proteolytic ectodomain shedding of the adhesion-mediating cell surface transmembrane proteins. In the brain, shedding of cell adhesion proteins is required for nervous system development and function, but the shedding of only a few adhesion proteins has been studied in detail in the mammalian brain. One such adhesion protein is the transmembrane protein endoglycan (PODXL2), which belongs to the CD34-family of highly glycosylated sialomucins. Here, we demonstrate that endoglycan is broadly expressed in the developing mouse brains and is proteolytically shed in vitro in mouse neurons and in vivo in mouse brains. Endoglycan shedding in primary neurons was mediated by the transmembrane protease a disintegrin and metalloprotease 10 (ADAM10), but not by its homolog ADAM17. Functionally, endoglycan deficiency reduced the branching of neurites extending from primary neurons in vitro, whereas deletion of ADAM10 had the opposite effect and increased neurite branching. Taken together, our study discovers a function for endoglycan in neurite branching, establishes endoglycan as an ADAM10 substrate and suggests that ADAM10 cleavage of endoglycan may contribute to neurite branching.


Assuntos
Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Moléculas de Adesão Celular/metabolismo , Desintegrinas/metabolismo , Proteínas de Membrana/metabolismo , Neuritos/metabolismo , Neurônios/metabolismo , Sialoglicoproteínas/metabolismo , Proteína ADAM17/metabolismo , Animais , Encéfalo/metabolismo , Adesão Celular/fisiologia , Linhagem Celular , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurogênese/fisiologia , Proteólise
8.
Curr Protoc ; 1(8): e219, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34370399

RESUMO

Targeted protein depletion using a conditional degron is a powerful method to probe the role of proteins in living cells because of the speed with which depletion can be induced and its reversibility. The auxin-inducible degron (AID) is one of the most common degron-based technologies used in cell biology. We recently established an improved system, called AID2, which involves expressing a mutant E3 ligase subunit, OsTIR1(F74G), and fusing a protein of interest to the mini-AID (mAID) tag, and that employs a new and more potent ligand, 5-phenyl-indole-3-acetic acid (5-Ph-IAA). The AID2 system overcomes some of the drawbacks associated with the original AID system, i.e., leaky degradation without auxin and the requirement of high auxin doses. With AID2 it is, therefore, now possible to control a degron-fused protein more precisely, enabling target proteins to be degraded with a half-life of 10 to 45 min via the addition of a low dose of 5-Ph-IAA. Importantly, in AID2, it is not necessary to control the expression of OsTIR1(F74G) for suppressing leaky degradation and a parental cell line constitutively expressing OsTIR1(F74G) can be used for the generation of multiple mAID-tagged proteins. Here, we describe a protocol for the tagging of endogenous proteins with mAID in diploid HCT116 cells. Our protocol can be applied to other mammalian cell lines and will enhance the utility of AID2 for studying protein functions in living cells. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Generation of a parental HCT116 cell line expressing OsTIR1(F74G) Basic Protocol 2: Construction of CRISPR and donor plasmids for tagging endogenous genes Basic Protocol 3: Generation of cell lines expressing a protein of interest fused with mAID.


Assuntos
Ácidos Indolacéticos , Proteínas , Animais , Células HCT116 , Humanos , Ácidos Indolacéticos/farmacologia , Proteólise , Ubiquitina-Proteína Ligases/genética
9.
Nat Commun ; 12(1): 5010, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408146

RESUMO

Poly(ADP)-ribosylation (PARylation) regulates chromatin structure and recruits DNA repair proteins. Using single-molecule fluorescence microscopy to track topoisomerase I (TOP1) in live cells, we found that sustained PARylation blocked the repair of TOP1 DNA-protein crosslinks (TOP1-DPCs) in a similar fashion as inhibition of the ubiquitin-proteasome system (UPS). PARylation of TOP1-DPC was readily revealed by inhibiting poly(ADP-ribose) glycohydrolase (PARG), indicating the otherwise transient and reversible PARylation of the DPCs. As the UPS is a key repair mechanism for TOP1-DPCs, we investigated the impact of TOP1-DPC PARylation on the proteasome and found that the proteasome is unable to associate with and digest PARylated TOP1-DPCs. In addition, PARylation recruits the deubiquitylating enzyme USP7 to reverse the ubiquitylation of PARylated TOP1-DPCs. Our work identifies PARG as repair factor for TOP1-DPCs by enabling the proteasomal digestion of TOP1-DPCs. It also suggests the potential regulatory role of PARylation for the repair of a broad range of DPCs.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , DNA/química , DNA/metabolismo , Dano ao DNA , Reparo do DNA , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/genética , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Células HEK293 , Humanos , Poli ADP Ribosilação , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/genética , Proteólise , Ubiquitinação
10.
Nutrients ; 13(8)2021 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-34444950

RESUMO

The purpose of this research was to investigate the prophylactic effects of glutamine on muscle protein synthesis and degradation in rats with ethanol-induced liver injury. For the first 2 weeks, Wistar rats were divided into two groups and fed a control (n = 16) or glutamine-containing diet (n = 24). For the following 6 weeks, rats fed the control diet were further divided into two groups (n = 8 per group) according to whether their diet contained no ethanol (CC) or did contain ethanol (CE). Rats fed the glutamine-containing diet were also further divided into three groups (n = 8 per group), including a GG group (glutamine-containing diet without ethanol), GE group (control diet with ethanol), and GEG group (glutamine-containing diet with ethanol). After 6 weeks, results showed that hepatic fatty change, inflammation, altered liver function, and hyperammonemia had occurred in the CE group, but these were attenuated in the GE and GEG groups. Elevated intestinal permeability and a higher plasma endotoxin level were observed in the CE group, but both were lower in the GE and GEG groups. The level of a protein synthesis marker (p70S6K) was reduced in the CE group but was higher in both the GE and GEG groups. In conclusion, glutamine supplementation might elevate muscle protein synthesis by improving intestinal health and ameliorating liver damage in rats with chronic ethanol intake.


Assuntos
Glutamina/administração & dosagem , Hepatopatias Alcoólicas/prevenção & controle , Proteínas Musculares/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Animais , Suplementos Nutricionais , Modelos Animais de Doenças , Etanol , Inflamação , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Hepatopatias Alcoólicas/etiologia , Ratos , Ratos Wistar
11.
Molecules ; 26(15)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34361849

RESUMO

Proteases play a central role in various biochemical pathways catalyzing and regulating key biological events. Proteases catalyze an irreversible post-translational modification called proteolysis by hydrolyzing peptide bonds in proteins. Given the destructive potential of proteolysis, protease activity is tightly regulated. Dysregulation of protease activity has been reported in numerous disease conditions, including cancers, neurodegenerative diseases, inflammatory conditions, cardiovascular diseases, and viral infections. The proteolytic profile of a cell, tissue, or organ is governed by protease activation, activity, and substrate specificity. Thus, identifying protease substrates and proteolytic events under physiological conditions can provide crucial information about how the change in protease regulation can alter the cellular proteolytic landscape. In recent years, mass spectrometry-based techniques called N-terminomics have become instrumental in identifying protease substrates from complex biological mixtures. N-terminomics employs the labeling and enrichment of native and neo-N-termini peptides, generated upon proteolysis followed by mass spectrometry analysis allowing protease substrate profiling directly from biological samples. In this review, we provide a brief overview of N-terminomics techniques, focusing on their strengths, weaknesses, limitations, and providing specific examples where they were successfully employed to identify protease substrates in vivo and under physiological conditions. In addition, we explore the current trends in the protease field and the potential for future developments.


Assuntos
Peptídeo Hidrolases/química , Peptídeos/química , Proteólise , Proteômica , Humanos , Espectrometria de Massas , Especificidade por Substrato
12.
Molecules ; 26(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34443430

RESUMO

Parkinson's disease (PD) is a currently incurable neurodegenerative disorder characterized by the loss of dopaminergic (DAergic) neurons in the substantia nigra pars compacta and α-synuclein aggregation. Accumulated evidence indicates that the saponins, especially from ginseng, have neuroprotective effects against neurodegenerative disorders. Interestingly, saponin can also be found in marine organisms such as the sea cucumber, but little is known about its effect in neurodegenerative disease, including PD. In this study, we investigated the anti-Parkinson effects of frondoside A (FA) from Cucumaria frondosa and ginsenoside Rg3 (Rg3) from Panax notoginseng in C. elegans PD model. Both saponins were tested for toxicity and optimal concentration by food clearance assay and used to treat 6-OHDA-induced BZ555 and transgenic α-synuclein NL5901 strains in C. elegans. Treatment with FA and Rg3 significantly attenuated DAergic neurodegeneration induced by 6-OHDA in BZ555 strain, improved basal slowing rate, and prolonged lifespan in the 6-OHDA-induced wild-type strain with downregulation of the apoptosis mediators, egl-1 and ced-3, and upregulation of sod-3 and cat-2. Interestingly, only FA reduced α-synuclein aggregation, rescued lifespan in NL5901, and upregulated the protein degradation regulators, including ubh-4, hsf-1, hsp-16.1 and hsp-16.2. This study indicates that both FA and Rg3 possess beneficial effects in rescuing DAergic neurodegeneration in the 6-OHDA-induced C. elegans model through suppressing apoptosis mediators and stimulating antioxidant enzymes. In addition, FA could attenuate α-synuclein aggregation through the protein degradation process.


Assuntos
Caenorhabditis elegans/fisiologia , Ginsenosídeos/farmacologia , Glicosídeos/farmacologia , Doença de Parkinson/patologia , Triterpenos/farmacologia , Animais , Animais Geneticamente Modificados , Apoptose/efeitos dos fármacos , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Ginsenosídeos/química , Ginsenosídeos/toxicidade , Glicosídeos/química , Glicosídeos/toxicidade , Longevidade/efeitos dos fármacos , Degeneração Neural/complicações , Degeneração Neural/patologia , Oxidopamina , Doença de Parkinson/complicações , Proteólise/efeitos dos fármacos , Triterpenos/química , Triterpenos/toxicidade , alfa-Sinucleína/metabolismo
13.
Molecules ; 26(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34443447

RESUMO

Okara is a soybean transformation agri-food by-product, the massive production of which currently poses severe disposal issues. However, its composition is rich in seed storage proteins, which, once extracted, can represent an interesting source of bioactive peptides. Antimicrobial and antifungal proteins and peptides have been described in plant seeds; thus, okara is a valuable source of compounds, exploitable for integrated pest management. The aim of this work is to describe a rapid and economic procedure to isolate proteins from okara, and to produce an enzymatic proteolyzed product, active against fungal plant pathogens. The procedure allowed the isolation and recovery of about 30% of okara total proteins. Several proteolytic enzymes were screened to identify the proper procedure to produce antifungal compounds. Antifungal activity of the protein digested for 24 h with pancreatin against Fusarium and R. solani mycelial growth and Pseudomonas spp was assessed. A dose-response inhibitory activity was established against fungi belonging to the Fusarium genus. The exploitation of okara to produce antifungal bioactive peptides has the potential to turn this by-product into a paradigmatic example of circular economy, since a field-derived food waste is transformed into a source of valuable compounds to be used in field crops protection.


Assuntos
Antifúngicos/farmacologia , Enzimas/metabolismo , Proteínas de Plantas/metabolismo , Plantas/microbiologia , Polissacarídeos/metabolismo , Liofilização , Fusarium/efeitos dos fármacos , Fusarium/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Peso Molecular , Proteólise/efeitos dos fármacos , Alimentos de Soja , Espectrofotometria Ultravioleta , Tripsina/metabolismo , Inibidores da Tripsina/farmacologia
14.
Nutrients ; 13(8)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34444848

RESUMO

Flavonoids are a major group of dietary plant polyphenols and have a positive health impact, but their modification and degradation in the human gut is still widely unknown. Due to the rise of metagenome data of the human gut microbiome and the assembly of hundreds of thousands of bacterial metagenome-assembled genomes (MAGs), large-scale screening for potential flavonoid-modifying enzymes of human gut bacteria is now feasible. With sequences of characterized flavonoid-transforming enzymes as queries, the Unified Human Gastrointestinal Protein catalog was analyzed and genes encoding putative flavonoid-modifying enzymes were quantified. The results revealed that flavonoid-modifying enzymes are often encoded in gut bacteria hitherto not considered to modify flavonoids. The enzymes for the physiologically important daidzein-to-equol conversion, well studied in Slackiaisoflavoniconvertens, were encoded only to a minor extent in Slackia MAGs, but were more abundant in Adlercreutzia equolifaciens and an uncharacterized Eggerthellaceae species. In addition, enzymes with a sequence identity of about 35% were encoded in highly abundant MAGs of uncultivated Collinsella species, which suggests a hitherto uncharacterized daidzein-to-equol potential in these bacteria. Of all potential flavonoid modification steps, O-deglycosylation (including derhamnosylation) was by far the most abundant in this analysis. In contrast, enzymes putatively involved in C-deglycosylation were detected less often in human gut bacteria and mainly found in Agathobacter faecis (formerly Roseburia faecis). Homologs to phloretin hydrolase, flavanonol/flavanone-cleaving reductase and flavone reductase were of intermediate abundance (several hundred MAGs) and mainly prevalent in Flavonifractor plautii. This first comprehensive insight into the black box of flavonoid modification in the human gut highlights many hitherto overlooked and uncultured bacterial genera and species as potential key organisms in flavonoid modification. This could lead to a significant contribution to future biochemical-microbiological investigations on gut bacterial flavonoid transformation. In addition, our results are important for individual nutritional recommendations and for biotechnological applications that rely on novel enzymes catalyzing potentially useful flavonoid modification reactions.


Assuntos
Proteínas de Bactérias/metabolismo , Flavonoides/metabolismo , Microbioma Gastrointestinal/fisiologia , Fenômenos Fisiológicos da Nutrição/fisiologia , Simulação por Computador , Equol/metabolismo , Genoma Bacteriano , Humanos , Isoflavonas/metabolismo , Metagenoma , Peptídeo Hidrolases/metabolismo , Proteólise
15.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445480

RESUMO

Multivesicular body (MVB)-mediated endosomal sorting and macroautophagy are the main pathways mediating the transport of cellular components to the vacuole and are essential for maintaining cellular homeostasis. The interplay of these two pathways remains poorly understood in plants. In this study, we show that FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1), which was previously identified as a plant-specific component of the endosomal sorting complex required for transport (ESCRT), essential for MVB biogenesis and plant growth, can be transported to the vacuole for degradation in response to iron deficiency. The vacuolar transport of ubiquitinated FREE1 protein is mediated by the autophagy pathway. As a consequence, the autophagy deficient mutants, atg5-1 and atg7-2, accumulate more endogenous FREE1 protein and display hypersensitivity to iron deficiency. Furthermore, under iron-deficient growth condition autophagy related genes are upregulated to promote the autophagic degradation of FREE1, thereby possibly relieving the repressive effect of FREE1 on iron absorption. Collectively, our findings demonstrate a unique regulatory mode of protein turnover of the ESCRT machinery through the autophagy pathway to respond to iron deficiency in plants.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Proteínas Relacionadas à Autofagia/genética , Ferro/metabolismo , Proteínas de Transporte Vesicular/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Autofagia , Transporte Biológico , Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos/metabolismo , Mutação , Proteólise , Ubiquitinação
16.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445501

RESUMO

Lipid dysregulation in diabetes mellitus escalates endothelial dysfunction, the initial event in the development and progression of diabetic atherosclerosis. In addition, lipid-laden macrophage accumulation in the arterial wall plays a significant role in the pathology of diabetes-associated atherosclerosis. Therefore, inhibition of endothelial dysfunction and enhancement of macrophage cholesterol efflux is the important antiatherogenic mechanism. Rosmarinic acid (RA) possesses beneficial properties, including its anti-inflammatory, antioxidant, antidiabetic and cardioprotective effects. We previously reported that RA effectively inhibits diabetic endothelial dysfunction by inhibiting inflammasome activation in endothelial cells. However, its effect on cholesterol efflux remains unknown. Therefore, in this study, we aimed to assess the effect of RA on cholesterol efflux and its underlying mechanisms in macrophages. RA effectively reduced oxLDL-induced cholesterol contents under high glucose (HG) conditions in macrophages. RA enhanced ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1) expression, promoting macrophage cholesterol efflux. Mechanistically, RA differentially regulated ABCA1 expression through JAK2/STAT3, JNK and PKC-p38 and ABCG1 expression through JAK2/STAT3, JNK and PKC-ERK1/2/p38 in macrophages. Moreover, RA primarily stabilized ABCA1 rather than ABCG1 protein levels by impairing protein degradation. These findings suggest RA as a candidate therapeutic to prevent atherosclerotic cardiovascular disease complications related to diabetes by regulating cholesterol efflux in macrophages.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Colesterol/metabolismo , Cinamatos/farmacologia , Depsídeos/farmacologia , Glucose/efeitos adversos , Lipoproteínas LDL/efeitos adversos , Macrófagos/citologia , Transportador 1 de Cassete de Ligação de ATP/química , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Modelos Biológicos , Proteólise/efeitos dos fármacos , Transdução de Sinais , Células THP-1
17.
Int J Mol Sci ; 22(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34360560

RESUMO

Among autophagy-related molecules, p62/SQSTM1 is an adaptor for identifying and delivering intracellular cargo for degradation. Since ubiquitination is reversible, it has a switch role in autophagy. Ubiquitination is also involved in regulating autophagy in a timely manner. This study aimed to elucidate how p62-mediated autophagy is regulated in human endothelial cells and macrophages under atherosclerotic conditions, focusing on the lysosomal and proteasomal pathways. Co-cultured HUVECs and THP-1 cells were exposed to oxLDL (50 µg/mL) and autophagy was assessed. To downregulate p62, siRNA was administered, and the E3 ligases were inhibited by Heclin or MLN4924 treatment under the condition that cellular inflammatory processes were stimulated by oxLDL simultaneously initiated autophagy. Downregulating p62 induced an alternative degradation system, and the E3 ligases were found to be involved in the progression of atherosclerosis. Collectively, the present study demonstrated that the endothelial lipid accumulation under atherosclerotic conditions was caused by lysosomal dysfunction associated with autophagy.


Assuntos
Aterosclerose/patologia , Autofagia , Células Endoteliais/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteína Sequestossoma-1/metabolismo , Ubiquitinação , Aterosclerose/metabolismo , Células Endoteliais/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Proteína Sequestossoma-1/genética , Transdução de Sinais , Ubiquitina/metabolismo
18.
Science ; 373(6558): 998-1004, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34446601

RESUMO

In eukaryotic cells, half of all proteins function as subunits within multiprotein complexes. Imbalanced synthesis of subunits leads to unassembled intermediates that must be degraded to minimize cellular toxicity. Here, we found that excess PSMC5, a subunit of the proteasome base, was targeted for degradation by the HERC1 ubiquitin ligase in mammalian cells. HERC1 identified unassembled PSMC5 by its cognate assembly chaperone PAAF1. Because PAAF1 only dissociates after assembly, HERC1 could also engage later assembly intermediates such as the PSMC4-PSMC5-PAAF1 complex. A missense mutant of HERC1 that causes neurodegeneration in mice was impaired in the recognition and ubiquitination of the PSMC5-PAAF1 complex. Thus, proteasome assembly factors can serve as adaptors for ubiquitin ligases to facilitate elimination of unassembled intermediates and maintain protein homeostasis.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Calmodulina/metabolismo , Humanos , Células MCF-7 , Camundongos , Mutação , Mutação de Sentido Incorreto , Doenças Neurodegenerativas/genética , Mutação Puntual , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/metabolismo , Proteólise , Proteínas Proto-Oncogênicas/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
19.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360709

RESUMO

Toxoplasma gondii (T. gondii) is an important human and veterinary pathogen causing life-threatening disease in immunocompromised patients. The UBL-UBA shuttle protein family are important components of the ubiquitin-proteasome system. Here, we identified a novel UBL-UBA shuttle protein DSK2b that is charactered by an N-terminal ubiquitin-like domain (UBL) and a C-terminal ubiquitin-associated domain (UBA). DSK2b was localized in the cytoplasm and nucleus. The deletion of dsk2b did not affect the degradation of ubiquitinated proteins, parasite growth in vitro or virulence in mice. The double-gene knockout of dsk2b and its paralogs dsk2a (ΔΔdsk2adsk2b) results in a significant accumulation of ubiquitinated proteins and the asynchronous division of T. gondii. The growth of ΔΔdsk2adsk2b was significantly inhibited in vitro, while virulence in mice was not attenuated. In addition, autophagy occurred in the ΔΔdsk2adsk2b, which was speculated to degrade the accumulated ubiquitinated proteins in the parasites. Overall, DSK2b is a novel UBL-UBA shuttle protein contributing to the degradation of ubiquitinated proteins and is important for the synchronous cell division of T. gondii.


Assuntos
Divisão Celular , Proteólise , Proteínas de Protozoários/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Toxoplasma/metabolismo , Ubiquitinação , Animais , Deleção de Genes , Humanos , Camundongos , Proteínas de Protozoários/genética , Receptores Citoplasmáticos e Nucleares/genética , Toxoplasma/genética , Toxoplasma/patogenicidade
20.
Planta ; 254(3): 43, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34355288

RESUMO

MAIN CONCLUSION: A synthetic peptide from the C-terminal end of C4-phosphoenolpyruvate carboxylase is implicated in the proteolysis of the enzyme, and Glc-6P or phosphorylation of the enzyme modulate this effect. Phosphoenolpyruvate carboxylase (PEPC) is a cytosolic, homotetrameric enzyme that performs a variety of functions in plants. Among them, it is primarily responsible for CO2 fixation in the C4 photosynthesis pathway (C4-PEPC). Here we show that proteolysis of C4-PEPC by cathepsin proteases present in a semi-purified PEPC fraction was enhanced by the presence of a synthetic peptide containing the last 19 amino acids from the C-terminal end of the PEPC subunit (pC19). Threonine (Thr)944 and Thr948 in the peptide are important requirements for the pC19 effect. C4-PEPC proteolysis in the presence of pC19 was prevented by the PEPC allosteric effector glucose 6-phosphate (Glc-6P) and by phosphorylation of the enzyme. The role of these elements in the regulation of PEPC proteolysis is discussed in relation to the physiological context.


Assuntos
Fosfoenolpiruvato Carboxilase , Sorghum , Glucose-6-Fosfato , Peptídeos , Fosfoenolpiruvato Carboxilase/metabolismo , Fosforilação , Fotossíntese , Proteólise , Sorghum/metabolismo
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