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1.
Biochem Biophys Res Commun ; 529(2): 251-256, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32703419

RESUMO

The nucleocapsid protein is significant in the formation of viral RNA of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), accounting for the largest proportion of viral structural proteins. Here, we report for the first time that the 11S proteasomal activator PA28γ regulates the intracellular abundance of the SARS-CoV-2 N protein (nCoV N). Furthermore, we have identified proteasome activator PA28γ as a nCoV N binding protein by co-immunoprecipitation assay. As a result of their interaction, nCoV N could be degraded by PA28γ-20S in vitro degradation assay. This was also demonstrated by blocking de novo protein synthesis with cycloheximide. The stability of nCoV N in PA28γ-knockout cells was greater than in PA28γ-wildtype cells. Notably, immunofluorescence staining revealed that knockout of the PA28γ gene in cells led to the transport of nCoV N from the nucleus to the cytoplasm. Overexpression of PA28γ enhanced proteolysis of nCoV N compared to that in PA28γ-N151Y cells containing a dominant-negative PA28γ mutation, which reduced this process. These results suggest that PA28γ binding is important in regulating 20S proteasome activity, which in turn regulates levels of the critical nCoV N nucleocapsid protein of SARS-CoV-2, furthering our understanding of the pathogenesis of COVID-19.


Assuntos
Autoantígenos/metabolismo , Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Pneumonia Viral/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Infecções por Coronavirus/virologia , Células HEK293 , Humanos , Técnicas In Vitro , Pandemias , Pneumonia Viral/virologia , Ligação Proteica , Estabilidade Proteica , Transporte Proteico
3.
JCI Insight ; 5(14)2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32559180

RESUMO

BACKGROUNDReprogramming of host metabolism supports viral pathogenesis by fueling viral proliferation, by providing, for example, free amino acids and fatty acids as building blocks.METHODSTo investigate metabolic effects of SARS-CoV-2 infection, we evaluated serum metabolites of patients with COVID-19 (n = 33; diagnosed by nucleic acid testing), as compared with COVID-19-negative controls (n = 16).RESULTSTargeted and untargeted metabolomics analyses identified altered tryptophan metabolism into the kynurenine pathway, which regulates inflammation and immunity. Indeed, these changes in tryptophan metabolism correlated with interleukin-6 (IL-6) levels. Widespread dysregulation of nitrogen metabolism was also seen in infected patients, with altered levels of most amino acids, along with increased markers of oxidant stress (e.g., methionine sulfoxide, cystine), proteolysis, and renal dysfunction (e.g., creatine, creatinine, polyamines). Increased circulating levels of glucose and free fatty acids were also observed, consistent with altered carbon homeostasis. Interestingly, metabolite levels in these pathways correlated with clinical laboratory markers of inflammation (i.e., IL-6 and C-reactive protein) and renal function (i.e., blood urea nitrogen).CONCLUSIONIn conclusion, this initial observational study identified amino acid and fatty acid metabolism as correlates of COVID-19, providing mechanistic insights, potential markers of clinical severity, and potential therapeutic targets.FUNDINGBoettcher Foundation Webb-Waring Biomedical Research Award; National Institute of General and Medical Sciences, NIH; and National Heart, Lung, and Blood Institute, NIH.


Assuntos
Infecções por Coronavirus/metabolismo , Ácidos Graxos/metabolismo , Interleucina-6/metabolismo , Cinurenina/metabolismo , Estresse Oxidativo , Pneumonia Viral/metabolismo , Insuficiência Renal/metabolismo , Adulto , Idoso , Aminoácidos/metabolismo , Betacoronavirus , Glicemia/metabolismo , Estudos de Casos e Controles , Creatina/metabolismo , Creatinina/metabolismo , Cistina , Ácidos Graxos não Esterificados/metabolismo , Feminino , Humanos , Masculino , Metaboloma , Metabolômica , Metionina/análogos & derivados , Pessoa de Meia-Idade , Pandemias , Poliaminas/metabolismo , Proteólise , Triptofano/metabolismo
4.
Elife ; 92020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32452762

RESUMO

Molecular mimicry is an evolutionary strategy adopted by viruses to exploit the host cellular machinery. We report that SARS-CoV-2 has evolved a unique S1/S2 cleavage site, absent in any previous coronavirus sequenced, resulting in the striking mimicry of an identical FURIN-cleavable peptide on the human epithelial sodium channel α-subunit (ENaC-α). Genetic alteration of ENaC-α causes aldosterone dysregulation in patients, highlighting that the FURIN site is critical for activation of ENaC. Single cell RNA-seq from 66 studies shows significant overlap between expression of ENaC-α and the viral receptor ACE2 in cell types linked to the cardiovascular-renal-pulmonary pathophysiology of COVID-19. Triangulating this cellular characterization with cleavage signatures of 178 proteases highlights proteolytic degeneracy wired into the SARS-CoV-2 lifecycle. Evolution of SARS-CoV-2 into a global pandemic may be driven in part by its targeted mimicry of ENaC-α, a protein critical for the homeostasis of airway surface liquid, whose misregulation is associated with respiratory conditions.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/virologia , Canais Epiteliais de Sódio/metabolismo , Mimetismo Molecular , Peptídeo Hidrolases/metabolismo , Pneumonia Viral/virologia , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/metabolismo , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Canais Epiteliais de Sódio/genética , Interações Hospedeiro-Patógeno , Humanos , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Proteólise , Especificidade por Substrato , Proteínas do Envelope Viral/genética , Proteínas Virais/genética
5.
Chemosphere ; 255: 126954, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32387908

RESUMO

Silica nanoparticles (SiNPs) are one of the most widely used types of nanoparticles across many industrial sectors, and are known to be present in the air year-round. In this study, we aimed to evaluate the potential adverse effects of SiNP exposure on pulmonary epithelial tight junctions, which serve as a critical barrier between the respiratory system and the circulatory system. In vivo studies confirmed that SiNPs decreased the protein expression levels of zonula occludens 1 (ZO-1), zonula occludens 2 (ZO-2), and occludin in the lungs of C57BL/6 mice. In vitro studies showed that SiNPs not only decreased the mRNA and protein expression of ZO-1 and ZO-2, but also decreased the protein expression of occludin in human bronchial epithelial (BEAS-2B) cells. In addition, SiNP exposure increased reactive oxygen species (ROS) production and activated extracellular regulated protein kinases (ERKs) and c-Jun N-terminal kinase (JNK). The inhibition of ROS and ERKs effectively protected the SiNP-induced downregulation of ZO-1 mRNA and protein expression, but had no effect on ZO-2 or occludin expression. SiNP-induced matrix metalloproteinase 9 (MMP9) protein expression appeared to be involved in occludin proteolytic degradation, in addition to SiNP-induced direct occludin protein degradation. The present study suggests that SiNPs disturb pulmonary epithelial tight junction structure and function via the ROS/ERK pathway and protein degradation.


Assuntos
Nanopartículas/toxicidade , Espécies Reativas de Oxigênio/química , Dióxido de Silício/toxicidade , Animais , Brônquios , Regulação para Baixo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Pulmão/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Ocludina , Fosfoproteínas/metabolismo , Proteólise , Espécies Reativas de Oxigênio/metabolismo , Junções Íntimas , Proteína da Zônula de Oclusão-1
6.
Bioresour Technol ; 311: 123496, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32438093

RESUMO

The mechanism that tannins alter microbial community to inhibit proteolysis and enhance silage quality is unclear. Neolamarckia cadamba leaf (NCL; rich in tannins) were ensiled alone or with addition of polyethylene glycol (PEG, tannins inactivator), and then fermentation quality, proteolysis activity and bacterial community were investigated during ensiling (Day 3, 7, 14 and 30). As a result, PEG addition increased lactic acid (1.09% vs 2.03%, on dry matter basis) and nonprotein-N (13.65% vs 17.59%, on crude protein basis) contents but decreased ammonia-N content (9.21% vs 2.29%, on crude protein basis) in NCL silage. Meanwhile, the dominant microbiome shifted from Cyanobacteria (60.92%-81.50%) to Firmicutes (48.96%-88.67%), where the unclassified genus (80.95%-85.71%) was substituted by Leuconstoc (42.03%-55.55%) and subsequently Lactobacillus (65.98%-82.43%). This study suggests that the intrinsic tannins inhibit lactic acid fermentation and protein degradation in NCL silage.


Assuntos
Silagem , Taninos , Bactérias , Fermentação , Proteólise
7.
Chem Biol Interact ; 325: 109088, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32360554

RESUMO

Osteoarthritis (OA) is one of the most common degenerative joint diseases in aging people. The activation of chondrocytes and their dysregulation are closely related to the pathogenesis of OA. GPR55 is an unique orphan G-receptor which binds to cannabinoids. In this study, we explored the role of GPR55 in advanced glycation end productions (AGEs)- induced chondrocytes activation in cultured cells. We showed that AGEs dose dependently induced GPR55 expression in ATDC5 chondrocytes. The blockage of GPR55 by its newly discovered antagonist-CID16020046 mitigated AGEs- induced increase in cellular ROS and decrease in antioxidant NRF2. Moreover, CID16020046 showed a dose-response suppressive effect on AGEs- induced expression of the major inflammatory mediators, including COX-2 and iNOS, and the production of NO and PGE2. CID16020046 also dose responsively inhibited AGEs- induced key effectors of cartilage degradation such as MMP-3 and MMP-13. In consequence, CID16020046 showed robust inhibition on AGEs- induced type II collagen degradation. Mechanistically, our data demonstrated that CID16020046 mediated GPR55 blockage ameliorated AGEs- induced NF-κB activation as revealed by its inhibition on IκBα, nuclear p65 translocation and NF-κB promoter activity. Collectively, our study demonstrates that GPR55 signaling mediates AGEs- induced chondrocyte activation, and the targeted blockage of GPR55 pathway could be therapeutic choice in the treatment of osteoarthritis.


Assuntos
Compostos Azabicíclicos/farmacologia , Benzoatos/farmacologia , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Produtos Finais de Glicação Avançada/farmacologia , Receptores de Canabinoides/metabolismo , Linhagem Celular , Condrócitos/metabolismo , Colágeno Tipo II/metabolismo , Ciclo-Oxigenase 2/genética , Dinoprostona/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 3 da Matriz/genética , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Proteólise/efeitos dos fármacos
8.
PLoS Genet ; 16(5): e1008797, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32392219

RESUMO

Sun-loving plants perceive the proximity of potential light-competing neighboring plants as a reduction in the red:far-red ratio (R:FR), which elicits a suite of responses called the "shade avoidance syndrome" (SAS). Changes in R:FR are primarily perceived by phytochrome B (phyB), whereas UV-B perceived by UV RESISTANCE LOCUS 8 (UVR8) elicits opposing responses to provide a counterbalance to SAS, including reduced shade-induced hypocotyl and petiole elongation. Here we show at the genome-wide level that UVR8 broadly suppresses shade-induced gene expression. A subset of this gene regulation is dependent on the UVR8-stabilized atypical bHLH transcription regulator LONG HYPOCOTYL IN FAR-RED 1 (HFR1), which functions in part redundantly with PHYTOCHROME INTERACTING FACTOR 3-LIKE 1 (PIL1). In parallel, UVR8 signaling decreases protein levels of the key positive regulators of SAS, namely the bHLH transcription factors PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and PIF5, in a COP1-dependent but HFR1-independent manner. We propose that UV-B antagonizes SAS via two mechanisms: degradation of PIF4 and PIF5, and HFR1- and PIL1-mediated inhibition of PIF4 and PIF5 function. This work highlights the importance of typical and atypical bHLH transcription regulators for the integration of light signals from different photoreceptors and provides further mechanistic insight into the crosstalk of UVR8 signaling and SAS.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/química , Raios Ultravioleta/efeitos adversos , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Estabilidade Proteica , Proteólise , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
9.
Artigo em Inglês | MEDLINE | ID: mdl-32361468

RESUMO

The present study aimed to evaluate the effect of the immobilization method of trypsin on biochar on the hydrolysis of casein from different sources, when compared to the process using trypsin in native form, to obtain bioactive peptides. The modification of the surface of biochar with glutaraldehyde was effective, as shown by the results of FTIR assay and the texture profile of the materials. Both activated and functionalized biochar showed high immobilization efficiency (greater than 87%) and high binding capacity (greater than 91 mg/g). During hydrolysis, the biocatalyst obtained by enzyme immobilization on the functionalized biochar presented a higher hydrolysis capacity for the different caseins when compared to the enzyme immobilized by adsorption, with values of 3.05 and 2.73 U/mg for goat casein, 2.36 and 1.85 U/mg for bovine casein, and 2.60 and 2.37 U/mg for buffalo, casein, respectively, with 60 min of reaction. The results of inhibitory activity in this study ranged from 93.5% and 25.5% for trypsin in its free form and immobilized on functionalized activated carbon, respectively, under the same reaction conditions. The immobilization methods were efficient, presenting high immobilization capacity. The proteolytic activity of trypsin immobilized via covalent binding was higher when compared the immobilization by adsorption. Thus, the functionalized biochar has proven to be potential support for enzyme immobilization, and the biocatalyst can be reused for more than 4 cycles. Despite lower ACE inhibition values of hydrolyzed obtained with the immobilized enzymes compared to free enzymes, biocatalysts present advantage due to the possibility of reuse.


Assuntos
Caseínas/química , Carvão Vegetal/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Tripsina/química , Tripsina/metabolismo , Adsorção , Animais , Biocatálise , Bovinos , Estabilidade Enzimática , Glutaral/química , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Ácidos Fosfóricos/química , Proteólise , Propriedades de Superfície , Temperatura
10.
PLoS One ; 15(5): e0232253, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32365084

RESUMO

Proteases have been implicated in the tumorigenesis and aggressiveness of a variety of cancer types. In fact, proteases have proven to be very clinically useful as tumor biomarkers in the blood of patients. Proteases are typically involved in complex systems of substrates, activators, and inhibitors, thus making our ability to establish their exact function in cancer more difficult. Trypsin, perhaps the most famous of proteases, has been shown to play a role in cancer progression, but its functional role in ovarian cancer has not been much studied. PAR2, a transmembrane receptor that is known to be activated by trypsin, has been reported to be associated with ovarian cancer. Here, we found that stimulation of ovarian cancer cell lines with trypsin or PAR2 activating peptide markedly increased MAPK signaling and cell proliferation. Additionally, HE4, a WAP-family glycoprotein and ovarian cancer biomarker, was found to inhibit trypsin degradation, thereby retaining its activity. Patient data seemed to support this phenomenon, as the serum of ovarian cancer patients with high HE4 expression, revealed significantly elevated trypsin levels. These data support the hypothesis that trypsin plays a tumorigenic role in ovarian cancer, which can be mediated by its receptor PAR2, and potentiated by HE4.


Assuntos
Carcinoma Epitelial do Ovário/genética , Neoplasias Ovarianas/genética , Receptor PAR-2/metabolismo , Tripsina/genética , Proteína 2 do Domínio Central WAP de Quatro Dissulfetos/metabolismo , Carcinoma Epitelial do Ovário/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases , Neoplasias Ovarianas/metabolismo , Proteólise , Receptor PAR-2/genética , Tripsina/química , Tripsina/metabolismo , Regulação para Cima
11.
Nat Biomed Eng ; 4(5): 560-571, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32393891

RESUMO

The oral administration of peptide drugs is hampered by their metabolic instability and limited intestinal uptake. Here, we describe a method for the generation of small target-specific peptides (less than 1,600 Da in size) that resist gastrointestinal proteases. By using phage display to screen large libraries of genetically encoded double-bridged peptides on protease-resistant fd bacteriophages, we generated a peptide inhibitor of the coagulation Factor XIa with nanomolar affinity that resisted gastrointestinal proteases in all regions of the gastrointestinal tract of mice after oral administration, enabling more than 30% of the peptide to remain intact, and small quantities of it to reach the blood circulation. We also developed a gastrointestinal-protease-resistant peptide antagonist for the interleukin-23 receptor, which has a role in the pathogenesis of Crohn's disease and ulcerative colitis. The de novo generation of targeted peptides that resist proteolytic degradation in the gastrointestinal tract should help the development of effective peptides for oral delivery.


Assuntos
Peptídeos/administração & dosagem , Peptídeos/uso terapêutico , Proteólise , Administração Oral , Sequência de Aminoácidos , Animais , Técnicas de Visualização da Superfície Celular , Cristalografia por Raios X , Feminino , Trato Gastrointestinal/metabolismo , Humanos , Isomerismo , Camundongos Endogâmicos BALB C , Modelos Moleculares , Peptídeo Hidrolases/metabolismo , Biblioteca de Peptídeos , Peptídeos/química , Estabilidade Proteica , Estrutura Secundária de Proteína , Receptores de Interleucina/antagonistas & inibidores , Receptores de Interleucina/metabolismo
12.
Anticancer Res ; 40(5): 2449-2456, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32366388

RESUMO

The maintenance of genome integrity is essential for cellular survival and propagation. It relies upon the accurate and timely replication of the genetic material, as well as the rapid sensing and repairing of damage to DNA. Uncontrolled DNA replication and unresolved DNA lesions contribute to genomic instability and can lead to cancer. Chromatin licensing and DNA replication factor 1 (Cdt1) is essential for loading the minichromosome maintenance 2-7 helicase complex onto chromatin exclusively during the G1 phase of the cell cycle, thus limiting DNA replication to once per cell cycle. Upon DNA damage, Cdt1 rapidly accumulates to sites of damage and is subsequently poly-ubiquitinated by the cullin 4-RING E3 ubiquitin ligase complex, in conjunction with the substrate recognition factor Cdt2 (CRL4Cdt2), and targeted for degradation. We here discuss the cellular functions of Cdt1 and how it may interlink cell cycle regulation and DNA damage response pathways, contributing to genome stability.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Dano ao DNA , Animais , Cromatina/genética , Cromatina/metabolismo , Replicação do DNA , Instabilidade Genômica , Humanos , Proteínas Nucleares/metabolismo , Proteólise
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.
Science ; 368(6494)2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32299851

RESUMO

Cytotoxic lymphocyte-mediated immunity relies on granzymes. Granzymes are thought to kill target cells by inducing apoptosis, although the underlying mechanisms are not fully understood. Here, we report that natural killer cells and cytotoxic T lymphocytes kill gasdermin B (GSDMB)-positive cells through pyroptosis, a form of proinflammatory cell death executed by the gasdermin family of pore-forming proteins. Killing results from the cleavage of GSDMB by lymphocyte-derived granzyme A (GZMA), which unleashes its pore-forming activity. Interferon-γ (IFN-γ) up-regulates GSDMB expression and promotes pyroptosis. GSDMB is highly expressed in certain tissues, particularly digestive tract epithelia, including derived tumors. Introducing GZMA-cleavable GSDMB into mouse cancer cells promotes tumor clearance in mice. This study establishes gasdermin-mediated pyroptosis as a cytotoxic lymphocyte-killing mechanism, which may enhance antitumor immunity.


Assuntos
Granzimas/metabolismo , Células Matadoras Naturais/imunologia , Proteínas de Neoplasias/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Piroptose/imunologia , Linfócitos T Citotóxicos/enzimologia , Animais , Granzimas/química , Células HEK293 , Humanos , Interferon gama , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Domínios Proteicos , Proteólise
15.
Adv Exp Med Biol ; 1233: 101-115, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274754

RESUMO

The most common form of pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC), has a dismal 5-year survival rate of less than 5%. Radical surgical resection, in combination with adjuvant chemotherapy, provides the best option for long-term patient survival. However, only approximately 20% of patients are resectable at the time of diagnosis, due to locally advanced or metastatic disease. There is an urgent need for the identification of new, specific, and more sensitive biomarkers for diagnosis, prognosis, and prediction to improve the treatment options for pancreatic cancer patients. Dysregulation of proteostasis is linked to many pathophysiological conditions, including various types of cancer. In this review, we report on findings relating to the main cellular protein degradation systems, the ubiquitin-proteasome system (UPS) and autophagy, in pancreatic cancer. The expression of several components of the proteolytic network, including E3 ubiquitin-ligases and deubiquitinating enzymes, are dysregulated in PDAC, which accounts for approximately 90% of all pancreatic malignancies. In the future, a deeper understanding of the emerging role of proteostasis in pancreatic cancer has the potential to provide clinically relevant biomarkers and new strategies for combinatorial therapeutic options to better help treat the patients.


Assuntos
Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteólise , Proteostase , Biomarcadores Tumorais , Humanos , Ubiquitina/metabolismo
16.
Adv Exp Med Biol ; 1233: 329-348, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274765

RESUMO

During lifetime, the molecular mechanisms that are responsible for cellular defense against adverse conditions such as oxidative and heat stress tend to be less efficient, thus gradually leading to the natural phenomenon of aging. Aging is linked to increased oxidative stress and is characterized by the accumulation of damaged macromolecules. The accumulation of oxidized and misfolded proteins is also accusable for various neurodegenerative pathologies that are linked to aging. Among self-defense mechanisms of cells, proteostasis network is responsible for the proper biogenesis/folding/trafficking of proteins and their elimination through proteolysis. The ubiquitin-proteasome system (UPS) is the major proteolytic mechanism that has attracted the interest of many researchers as an antiaging target. Interestingly, many natural compounds have been identified as potent UPS activators. Given that diet is a manageable environmental factor that affects aging, consumption of natural dietary products that may potentially enhance the UPS function, would contribute to increased health span and delayed onset or progression of age-related disorders. Herein, we summarize natural compounds and extracts derived from edible products that have exhibited antiaging and anti-aggregation properties and the beneficial properties have been linked to the UPS modulation.


Assuntos
Envelhecimento/metabolismo , Dieta Saudável , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Humanos , Estresse Oxidativo , Proteólise , Proteostase
17.
Food Chem ; 321: 126689, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32259732

RESUMO

Peptides and free amino acids are naturally generated in dry-cured ham as a consequence of proteolysis phenomenon exerted by muscle peptidases. The generation of bioactive peptides in different types of dry-cured ham produced in Spain, Italy and China is reviewed in this manuscript. Major muscle proteins are extensively hydrolysed firstly by endogenous endo-peptidases followed by the successive action of exo-peptidases, mainly, tri- and di-peptidylpeptidases, aminopeptidases and carboxypeptidases. Such proteolysis is very intense and consists of the generation of large amounts of free amino acids and a good number of peptides with different sequences and lengths, some of them exerting relevant bioactivities like angiotensin converting enzyme inhibitory activity, antioxidant activity, di-peptidylpeptidase IV inhibitory activity among other and in vivo antihypertensive, hypoglycemic or anti-inflammatory activity. This manuscript reviews the recent findings showing that dry-cured ham constitutes a good source of natural bioactive peptides that have potential benefit for human health.


Assuntos
Carne de Porco , Aminoácidos/metabolismo , Animais , Hidrólise , Produtos da Carne/análise , Peptídeo Hidrolases/metabolismo , Peptídeos/química , Proteólise , Suínos
18.
PLoS Pathog ; 16(4): e1008426, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32282833

RESUMO

Human cytomegalovirus (HCMV) is the most frequent viral cause of congenital defects and can trigger devastating disease in immune-suppressed patients. Cytotoxic lymphocytes (CD8+ T cells and NK cells) control HCMV infection by releasing interferon-γ and five granzymes (GrA, GrB, GrH, GrK, GrM), which are believed to kill infected host cells through cleavage of intracellular death substrates. However, it has recently been demonstrated that the in vivo killing capacity of cytotoxic T cells is limited and multiple T cell hits are required to kill a single virus-infected cell. This raises the question whether cytotoxic lymphocytes can use granzymes to control HCMV infection in a noncytotoxic manner. Here, we demonstrate that (primary) cytotoxic lymphocytes can block HCMV dissemination independent of host cell death, and interferon-α/ß/γ. Prior to killing, cytotoxic lymphocytes induce the degradation of viral immediate-early (IE) proteins IE1 and IE2 in HCMV-infected cells. Intriguingly, both IE1 and/or IE2 are directly proteolyzed by all human granzymes, with GrB and GrM being most efficient. GrB and GrM cleave IE1 after Asp398 and Leu414, respectively, likely resulting in IE1 aberrant cellular localization, IE1 instability, and functional impairment of IE1 to interfere with the JAK-STAT signaling pathway. Furthermore, GrB and GrM cleave IE2 after Asp184 and Leu173, respectively, resulting in IE2 aberrant cellular localization and functional abolishment of IE2 to transactivate the HCMV UL112 early promoter. Taken together, our data indicate that cytotoxic lymphocytes can also employ noncytotoxic ways to control HCMV infection, which may be explained by granzyme-mediated targeting of indispensable viral proteins during lytic infection.


Assuntos
Infecções por Citomegalovirus/enzimologia , Citomegalovirus/metabolismo , Granzimas/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Células Matadoras Naturais/enzimologia , Transativadores/metabolismo , Motivos de Aminoácidos , Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Granzimas/genética , Interações Hospedeiro-Patógeno , Humanos , Proteínas Imediatamente Precoces/genética , Proteólise , Linfócitos T Citotóxicos/enzimologia , Transativadores/genética
19.
Science ; 368(6489)2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32327568

RESUMO

Misfolded luminal endoplasmic reticulum (ER) proteins undergo ER-associated degradation (ERAD-L): They are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome. ERAD-L is mediated by the Hrd1 complex (composed of Hrd1, Hrd3, Der1, Usa1, and Yos9), but the mechanism of retrotranslocation remains mysterious. Here, we report a structure of the active Hrd1 complex, as determined by cryo-electron microscopy analysis of two subcomplexes. Hrd3 and Yos9 jointly create a luminal binding site that recognizes glycosylated substrates. Hrd1 and the rhomboid-like Der1 protein form two "half-channels" with cytosolic and luminal cavities, respectively, and lateral gates facing one another in a thinned membrane region. These structures, along with crosslinking and molecular dynamics simulation results, suggest how a polypeptide loop of an ERAD-L substrate moves through the ER membrane.


Assuntos
Proteínas de Transporte/química , Degradação Associada com o Retículo Endoplasmático , Glicoproteínas de Membrana/química , Proteínas de Membrana/química , Complexos Multiproteicos/química , Proteólise , Proteínas de Saccharomyces cerevisiae/química , Ubiquitina-Proteína Ligases/química , Proteínas de Transporte/metabolismo , Microscopia Crioeletrônica , Retículo Endoplasmático/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , Complexos Multiproteicos/metabolismo , Domínios Proteicos , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Nat Struct Mol Biol ; 27(4): 319-322, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251415

RESUMO

Thalidomide-dependent degradation of the embryonic transcription factor SALL4 by the CRL4CRBN E3 ubiquitin ligase is a plausible major driver of thalidomide teratogenicity. The structure of the second zinc finger of SALL4 in complex with pomalidomide, cereblon and DDB1 reveals the molecular details of recruitment. Sequence differences and a shifted binding position relative to Ikaros offer a path to the rational design of cereblon-binding drugs with reduced teratogenic risk.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Ligação a DNA/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Fatores de Transcrição/ultraestrutura , Proteínas Adaptadoras de Transdução de Sinal/genética , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Ligação Proteica , Conformação Proteica , Proteólise/efeitos dos fármacos , Especificidade por Substrato , Talidomida/análogos & derivados , Talidomida/química , Talidomida/farmacologia , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/ultraestrutura , Ubiquitinação/genética
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