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1.
Nature ; 583(7818): 819-824, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32699411

RESUMO

The thalamic reticular nucleus (TRN), the major source of thalamic inhibition, regulates thalamocortical interactions that are critical for sensory processing, attention and cognition1-5. TRN dysfunction has been linked to sensory abnormality, attention deficit and sleep disturbance across multiple neurodevelopmental disorders6-9. However, little is known about the organizational principles that underlie its divergent functions. Here we performed an integrative study linking single-cell molecular and electrophysiological features of the mouse TRN to connectivity and systems-level function. We found that cellular heterogeneity in the TRN is characterized by a transcriptomic gradient of two negatively correlated gene-expression profiles, each containing hundreds of genes. Neurons in the extremes of this transcriptomic gradient express mutually exclusive markers, exhibit core or shell-like anatomical structure and have distinct electrophysiological properties. The two TRN subpopulations make differential connections with the functionally distinct first-order and higher-order thalamic nuclei to form molecularly defined TRN-thalamus subnetworks. Selective perturbation of the two subnetworks in vivo revealed their differential role in regulating sleep. In sum, our study provides a comprehensive atlas of TRN neurons at single-cell resolution and links molecularly defined subnetworks to the functional organization of thalamocortical circuits.


Assuntos
Redes Reguladoras de Genes , Núcleos Talâmicos/citologia , Núcleos Talâmicos/metabolismo , Animais , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Hibridização in Situ Fluorescente , Metaloendopeptidases/metabolismo , Camundongos , Vias Neurais , Neurônios/metabolismo , Osteopontina/metabolismo , Técnicas de Patch-Clamp , RNA-Seq , Análise de Célula Única , Sono/genética , Sono/fisiologia , Núcleos Talâmicos/fisiologia , Transcriptoma
2.
PLoS One ; 15(5): e0233059, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32433687

RESUMO

Complex extracellular structures exist throughout phylogeny, but the dynamics of their formation and dissolution are often opaque. One example is the pharyngeal grinder of the nematode Caenorhabditis elegans, an extracellular structure that ruptures bacteria during feeding. During each larval transition stage, called lethargus, the grinder is replaced with one of a larger size. Here, we characterize at the ultrastructural level the deconstruction of the larval grinder and the construction of the adult grinder during the fourth larval stage (L4)-to-adult transition. Early in L4 lethargus, pharyngeal muscle cells trans-differentiate from contractile to secretory cells, as evidenced by the appearance of clear and dense core vesicles and disruptions in sarcomere organization. This is followed, within minutes, by the dissolution of the L4 grinder and the formation and maturation of the adult grinder. Components of the nascent adult grinder are deposited basally, and are separated from the dissolving larval grinder by a visible apical layer. The complete grinder is a lamellated extracellular matrix comprised of five layers. Following grinder formation, pharyngeal muscle cells regain ultrastructural contractile properties, and muscle contractions resume. Our findings add to our understanding of how complex extracellular structures assemble and dissemble.


Assuntos
Caenorhabditis elegans/fisiologia , Muda , Erupção Dentária , Animais , Caenorhabditis elegans/ultraestrutura , Proteínas de Caenorhabditis elegans/metabolismo , Larva , Metaloendopeptidases/metabolismo , Microscopia Eletrônica de Transmissão , Músculos Faríngeos/ultraestrutura , Sono , Imagem com Lapso de Tempo
3.
Cardiovasc Drugs Ther ; 34(3): 345-356, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32236861

RESUMO

PURPOSE: Mitochondrial dysfunction plays a vital role in the pathophysiologic process of heart failure (HF). As a quality control system, mitochondrial fusion and fission are under control of mitochondrial fusion and fission-related proteins. The objective of this study was to investigate the effects of common variants in mitochondrial fusion and fission-related genes on the prognosis of HF. METHODS: We performed whole exome sequencing (WES) with 1000 HF patients; the statistically significant variant was further genotyped in the replicated population with 2324 HF patients. A series of function analysis including western blot, cell proliferation assay, and in vitro OMA1 activity assay were conducted to illuminate the underlying mechanism. RESULTS: We identified a missense variant rs17117699 associated with the prognosis of HF in group without ß-blocker use rather than with ß-blocker use in two-stage population: adjusted P = 0.79, HR = 0.88 (0.36-2.13) in group with ß-blocker use and adjusted P = 0.016, HR = 1.43 (1.07-1.91) in group without ß-blocker in first-stage population; adjusted P = 0.42, HR = 0.85 (0.56-1.28) in group with ß-blocker use and adjusted P = 0.015, HR = 1.39 (1.06-1.82) in group without ß-blocker in replicated stage. Functional analysis indicated that rs17117699-G allele increased the activity of OMA1 assessed by the ratio of S-OPA1 to L-OPA1 and suppressed cells proliferation under ISO treatment when compared with rs17117699-T allele. Furthermore, OMA1 functioned downstream of ß-adrenergic receptor signaling and ISO-induced OPA1 cleavage is dependent on OMA1. CONCLUSIONS: Our findings demonstrate that rs17117699T>G in OMA1 increases the risk of HF mortality via enhancing its OPA1 cleavage activity. It is a promising potential treatment target for HF. CLINICAL TRIAL REGISTRATION: NCT03461107. https://www.clinicaltrials.gov/ct2/show/NCT03461107?term=03461107&cond=Heart+Failure&cntry=CN&rank=1.


Assuntos
Insuficiência Cardíaca/genética , Metaloendopeptidases/genética , Mitocôndrias Cardíacas/genética , Dinâmica Mitocondrial/genética , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Antagonistas Adrenérgicos beta/uso terapêutico , Adulto , Idoso , Proliferação de Células , Feminino , GTP Fosfo-Hidrolases/metabolismo , Estudos de Associação Genética , Células HEK293 , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/mortalidade , Humanos , Masculino , Metaloendopeptidases/metabolismo , Pessoa de Meia-Idade , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Prognóstico , Sequenciamento Completo do Exoma
4.
Am J Physiol Renal Physiol ; 318(5): F1147-F1159, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32174142

RESUMO

Meprin metalloproteases have been implicated in the progression of kidney injury. Previous work from our group has shown that meprins proteolytically process the catalytic subunit of protein kinase A (PKA-C), resulting in decreased PKA-C kinase activity. The goal of the present study was to determine the PKA-C isoforms impacted by meprin-ß and whether meprin-ß expression affects downstream mediators of the PKA signaling pathway in ischemia-reperfusion (IR)-induced kidney injury. IR was induced in 12-wk-old male wild-type (WT) and meprin-ß knockout (ßKO) mice. Madin-Darby canine kidney cells transfected with meprin-ß cDNA were also subjected to 2 h of hypoxia. Western blot analysis was used to evaluate levels of total PKA-C, PKA-Cα, PKA-Cß, phosphorylated (p-)PKA-C, and p-ERK1/2. Meprin-ß expression enhanced kidney injury as indicated by levels of neutrophil gelatinase-associated lipocalin and cystatin C. IR-associated decreases were observed in levels of p-PKA-C in kidney tissue from WT mice but not ßKO mice, suggesting that meprin-ß expression/activity is responsible for the in vivo reduction in kinase activity. Significant increases in levels of PKA-Cß were observed in kidney lysates for WT mice but not ßKO mice at 6 h post-IR. Proximal tubule PKA-Cß increases in WT but not ßKO kidneys were demonstrated by fluorescent microscopy. Furthermore, IR-induced injury was associated with significant increases in p-ERK levels for both genotypes. The present data demonstrate that meprin-ß enhances IR-induced kidney injury in part by modulating mediators of the PKA-Cß signaling pathway.


Assuntos
Lesão Renal Aguda/enzimologia , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Rim/enzimologia , Metaloendopeptidases/metabolismo , Traumatismo por Reperfusão/enzimologia , Lesão Renal Aguda/genética , Lesão Renal Aguda/patologia , Animais , Hipóxia Celular , Modelos Animais de Doenças , Cães , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Rim/patologia , Células Madin Darby de Rim Canino , Masculino , Metaloendopeptidases/deficiência , Metaloendopeptidases/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais
5.
Nature ; 579(7799): 427-432, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32132707

RESUMO

In mammalian cells, mitochondrial dysfunction triggers the integrated stress response, in which the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) results in the induction of the transcription factor ATF41-3. However, how mitochondrial stress is relayed to ATF4 is unknown. Here we show that HRI is the eIF2α kinase that is necessary and sufficient for this relay. In a genome-wide CRISPR interference screen, we identified factors upstream of HRI: OMA1, a mitochondrial stress-activated protease; and DELE1, a little-characterized protein that we found was associated with the inner mitochondrial membrane. Mitochondrial stress stimulates OMA1-dependent cleavage of DELE1 and leads to the accumulation of DELE1 in the cytosol, where it interacts with HRI and activates the eIF2α kinase activity of HRI. In addition, DELE1 is required for ATF4 translation downstream of eIF2α phosphorylation. Blockade of the OMA1-DELE1-HRI pathway triggers an alternative response in which specific molecular chaperones are induced. The OMA1-DELE1-HRI pathway therefore represents a potential therapeutic target that could enable fine-tuning of the integrated stress response for beneficial outcomes in diseases that involve mitochondrial dysfunction.


Assuntos
Citosol/metabolismo , Metaloendopeptidases/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Estresse Fisiológico , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/biossíntese , Fator 4 Ativador da Transcrição/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Citosol/enzimologia , Ativação Enzimática , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Masculino , Proteínas Mitocondriais/química , Chaperonas Moleculares/metabolismo , Fosforilação , Ligação Proteica
6.
Nature ; 579(7799): 433-437, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32132706

RESUMO

Mitochondrial fidelity is tightly linked to overall cellular homeostasis and is compromised in ageing and various pathologies1-3. Mitochondrial malfunction needs to be relayed to the cytosol, where an integrated stress response is triggered by the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) in mammalian cells4,5. eIF2α phosphorylation is mediated by the four eIF2α kinases GCN2, HRI, PERK and PKR, which are activated by diverse types of cellular stress6. However, the machinery that communicates mitochondrial perturbation to the cytosol to trigger the integrated stress response remains unknown1,2,7. Here we combine genome engineering and haploid genetics to unbiasedly identify genes that affect the induction of C/EBP homologous protein (CHOP), a key factor in the integrated stress response. We show that the mitochondrial protease OMA1 and the poorly characterized protein DELE1, together with HRI, constitute the missing pathway that is triggered by mitochondrial stress. Mechanistically, stress-induced activation of OMA1 causes DELE1 to be cleaved into a short form that accumulates in the cytosol, where it binds to and activates HRI via its C-terminal portion. Obstruction of this pathway can be beneficial or adverse depending on the type of mitochondrial perturbation. In addition to the core pathway components, our comparative genetic screening strategy identifies a suite of additional regulators. Together, these findings could be used to inform future strategies to modulate the cellular response to mitochondrial dysfunction in the context of human disease.


Assuntos
Citosol/metabolismo , Citosol/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Ativação Enzimática , Fator de Iniciação 2 em Eucariotos/metabolismo , Genoma Humano/genética , Humanos , Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Fosforilação , Ligação Proteica , Estresse Fisiológico , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/metabolismo
7.
Mol Med Rep ; 21(3): 1285-1295, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32016477

RESUMO

Of the different types of lung cancer, lung squamous cell cancer (LUSC) has the second highest rates of morbidity and mortality, which have been increasing in recent years. Epigenetic abnormalities may serve as potential biomarkers and diagnostic and/or therapeutic targets, which may help to monitor and improve the prognosis of patients with cancer. In the present study, data were obtained from The Cancer Genome Atlas database and survival and joint survival analyses were conducted using the R MethylMix package. Peptidase, mitochondrial processing a subunit pseudogene 1 (PMPCAP1), sosondowah ankyrin repeat domain family member C (SOWAHC) and zinc finger protein (ZNF) 454 were identified as independent prognosis­related hub methylation­driven genes (MDGs). Of these three genes, PMPCAP1 and SOWAHC, characterized by hypomethylation and high expression levels, were associated with poor prognosis in patients with LUSC, whilst ZNF454 was associated with an improved prognosis. In addition, pathway enrichment analysis suggested that PMPCAP1, SOWAHC and ZNF454 were primarily involved in gene expression or transcription pathways. Furthermore, 5, 1 and 10 key methylation sites of PMPCAP1, SOWAHC and ZNF454, respectively, were confirmed to be significantly relevant to gene expression, establishing a basis for further investigation into the mechanisms and more precise targets of these 3 genes. In conclusion, the MDGs PMPCAP1, SOWAHC and ZNF454 may be potential prognostic biomarkers of LUSC for guiding diagnosis and therapy options, as well as providing a theoretical basis for further investigation.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma de Células Escamosas/diagnóstico , Proteínas de Ligação a DNA/metabolismo , Neoplasias Pulmonares/diagnóstico , Metaloendopeptidases/metabolismo , Proteínas/metabolismo , Algoritmos , Biomarcadores/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Metilação de DNA , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Metaloendopeptidases/genética , Prognóstico , Proteínas/genética , Análise de Sobrevida , Dedos de Zinco
8.
Artigo em Inglês | MEDLINE | ID: mdl-32083973

RESUMO

Matrix metalloproteinases (MMPs) are proteolytic enzymes that break down extracellular matrix (ECM) components and have shown to be highly active in the myocardial infarction (MI) landscape. In addition to breaking down ECM products, MMPs modulate cytokine signaling and mediate leukocyte cell physiology. MMP-2, -7, -8, -9, -12, -14, and -28 are well studied as effectors of cardiac remodeling after MI. Whereas 13 MMPs have been evaluated in the MI setting, 13 MMPs have not been investigated during cardiac remodeling. Here, we measure the remaining MMPs across the MI time continuum to provide the full catalog of MMP expression in the left ventricle after MI in mice. We found that MMP-10, -11, -16, -24, -25, and -27 increase after MI, whereas MMP-15, -17, -19, -21, -23b, and -26 did not change with MI. For the MMPs increased with MI, the macrophage was the predominant cell source. This work provides targets for investigation to understand the full complement of specific MMP roles in cardiac remodeling.NEW & NOTEWORTHY To date, a number of matrix metalloproteinases (MMPs) have not been evaluated in the left ventricle after myocardial infarction (MI). This article supplies the missing knowledge to provide a complete MI MMP compendium.


Assuntos
Ventrículos do Coração/metabolismo , Metaloendopeptidases/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Animais , Modelos Animais de Doenças , Camundongos , Remodelação Ventricular/fisiologia
9.
Toxicon ; 178: 1-3, 2020 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-32094098

RESUMO

Binding of two P-III snake venom metalloproteinase (SVMPs), one procoagulant and one hemorrhagic, to microvessels was compared in an ex vivo model. The procoagulant SVMP did not bind to the microvasculature, in contrast to the clear localization on microvessels of the hemorrhagic SVMP. Deglycosylation of the procoagulant enzyme did not enable this toxin to bind to microvessels, suggesting that glycosylation is not interfering with binding. These observations suggest that procoagulant SVMPs lack exosites for interaction with microvessels components.


Assuntos
Músculos Abdominais/fisiologia , Metaloendopeptidases/metabolismo , Venenos de Serpentes/metabolismo , Animais , Camundongos , Venenos de Serpentes/enzimologia
10.
J Vet Diagn Invest ; 32(2): 259-267, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31924132

RESUMO

Two putative zinc metalloproteases encoded by Clostridium perfringens have been implicated in the pathogenesis of necrotic enteritis, an economically significant poultry disease that is caused by this anaerobic bacterium. These proteases have ~64% amino acid identity and are encoded by the zmpA and zmpB genes. We screened 83 C. perfringens isolates by PCR for the presence of these genes. The first gene, zmpB, is chromosomally located and was present in all screened strains of C. perfringens, regardless of their origin and virulence. The second gene, zmpA, is plasmid-borne and was only found in isolates derived from chickens with necrotic enteritis. We describe the generation of insertionally inactivated mutants of both zmpA and zmpB in a virulent C. perfringens isolate. For each mutant, a significant (p < 0.001) reduction in virulence was observed in a chicken necrotic enteritis disease model. Examples of each mutant strain were characterized by whole genome sequencing, which showed that there were a few off-site mutations with the potential to affect the virulence of these strains. To confirm the importance of these genes, independently derived zmpA and zmpB mutants were constructed in different virulent C. perfringens isolates and shown to have reduced virulence in the experimental disease induction model. A zmpA-zmpB double mutant also was generated and shown to have significantly reduced virulence, to the same extent as the respective single mutants. Our results provide evidence that both putative zinc metalloproteases play an important role in disease pathogenesis.


Assuntos
Proteínas de Bactérias/genética , Infecções por Clostridium/veterinária , Clostridium perfringens/fisiologia , Clostridium perfringens/patogenicidade , Enterocolite Necrosante/veterinária , Metaloendopeptidases/genética , Doenças das Aves Domésticas/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Infecções por Clostridium/microbiologia , Clostridium perfringens/enzimologia , Enterocolite Necrosante/microbiologia , Metaloendopeptidases/metabolismo , Virulência
11.
Cancer Sci ; 111(1): 59-71, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31729097

RESUMO

Low vitamin D status is associated with progression in patients with renal cell carcinoma (RCC). The present study found that vimentin, a mesenchymal marker, was accordingly upregulated, and E-cadherin, an epithelial marker, was downregulated in RCC patients with low vitamin D status. Thus, we investigated the effects of calcitriol or vitamin D3, an active form of vitamin D, on epithelial-mesenchymal transition (EMT) in RCC cells. RCC cells were treated by two models. In model 1, three RCC cell lines, ACHN, 786-O and CAKI-2, were incubated with either LPS (2.0 µg/mL) or transforming growth factor (TGF)-ß1 (10 ng/mL) in the presence or absence of calcitriol (200 nmol/L). In model 2, two RCC cell lines, ACHN and CAKI-2, were incubated with calcitriol (200 nmol/L) only. Calcitriol inhibited migration and invasion not only in TGF-ß1-stimulated but also in TGF-ß1-unstimulated RCC cells. Moreover, calcitriol suppressed E-cadherin downregulation and vimentin upregulation not only in TGF-ß1-stimulated but also in TGF-ß1-unstimulated ACHN and CAKI-2 cells. Calcitriol attenuated LPS-induced upregulation of MMP-2, MMP-7, MMP-9, MMP-26 and urokinase-type plasminogen activator (u-PA) in ACHN cells. In addition, calcitriol blocked TGF-ß1-induced nuclear translocation of ZEB1, Snail and Twist1 in ACHN and CAKI-2 cells. Mechanistically, calcitriol suppressed EMT through different signaling pathways: (i) calcitriol suppressed Smad2/3 phosphorylation by reinforcing physical interaction between vitamin D receptor (VDR) and Smad3 in TGF-ß1-stimulated RCC cells; (ii) calcitriol inhibited signal transducer and activator of transcription (STAT)3 activation in LPS-stimulated RCC cells; (iii) calcitriol inhibited ß-catenin/TCF-4 activation by promoting integration of VDR with ß-catenin in TGF-ß1-unstimulated RCC cells. Taken together, calcitriol inhibits migration and invasion of RCC cells partially by suppressing Smad2/3-, STAT3- and ß-catenin-mediated EMT.


Assuntos
Calcitriol/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Renais/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Adulto , Idoso , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Renais/metabolismo , Masculino , Metaloendopeptidases/metabolismo , Pessoa de Meia-Idade , Fator de Transcrição STAT3/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , beta Catenina/metabolismo
12.
Microbiol Immunol ; 64(2): 87-98, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31769530

RESUMO

Burkholderia cepacia complex (Bcc) are opportunistic pathogens implicated with nosocomial infections, and high rates of morbidity and mortality, especially in individuals with cystic fibrosis (CF). B. cepacia are naturally resistant to different classes of antibiotics, and can subvert the host innate immune responses by producing quorum sensing (QS) controlled virulence factors and biofilms. It still remains a conundrum as to how exactly the bacterium survives the intracellular environment within the host cells of CF patients and immunocompromised individuals although the bacterium can invade human lung epithelial cells, neutrophils, and murine macrophages. The mechanisms associated with intracellular survival in the airway epithelial cells and the role of QS and virulence factors in B. cepacia infections in cystic fibrosis remain largely unclear. The current review focuses on understanding the role of QS-controlled virulence factors and biofilms, and provides additional impetus to understanding the potentials of QS-inhibitory strategies against B. cepacia.


Assuntos
Biofilmes , Infecções por Burkholderia , Burkholderia cepacia/patogenicidade , Fibrose Cística/microbiologia , Percepção de Quorum/imunologia , Animais , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Infecções por Burkholderia/etiologia , Infecções por Burkholderia/imunologia , Burkholderia cepacia/crescimento & desenvolvimento , Complexo Burkholderia cepacia/patogenicidade , Doenças Transmissíveis Emergentes , Infecção Hospitalar/imunologia , Fibrose Cística/complicações , Fibrose Cística/imunologia , Síndrome da Liberação de Citocina , Farmacorresistência Bacteriana Múltipla , Humanos , Evasão da Resposta Imune , Hospedeiro Imunocomprometido , Inflamação , Lipase/metabolismo , Lipopolissacarídeos/metabolismo , Pulmão/microbiologia , Macrófagos/microbiologia , Metaloendopeptidases/metabolismo , Camundongos , Neutrófilos/imunologia , Sideróforos/metabolismo , Fatores de Virulência/metabolismo
13.
Methods Mol Biol ; 2043: 113-123, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31463907

RESUMO

Biotinylation is a versatile technique that has been used to label proteins for a variety of applications. Under alkaline conditions, the N-hydroxylsuccinimide (NHS) ester present on the biotinylation reagent reacts with primary amines such as the side chain of lysine residues or the N-termini of proteins to yield stable amide bonds. However, the effect of biotinylation on enzyme structure and function has not been generally appreciated. In this chapter, I describe specific issues involving biotinylation of proteoglycanases (e.g., ADAMTS-1, -4, and -5). Taking ADAMTS-5 as an example, I show how high incorporation of biotin molecules causes a decrease in aggrecanase activity, most likely by disrupting exosites present in the cysteine-rich and spacer domains. Such an effect is not evident when enzymatic activity is measured with synthetic peptides, since exosites are not strictly required for peptidolytic activity. Therefore, extreme care must be taken when labeling proteoglycanases and the appropriate enzyme/biotin ratio must be determined experimentally for each enzyme.


Assuntos
Biotina/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Biotinilação , Lisina/metabolismo , Peptídeos/metabolismo , Domínios Proteicos , Coloração e Rotulagem
14.
PLoS One ; 14(12): e0225666, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31805094

RESUMO

The hatching enzymes or choriolysins are key proteases in fish life cycle controlling the release of larvae to surrounding environment that have been suggested as target for novel biotechnological uses. Due to the large amounts of eggs released by the flatfish Solea senegalensis, during the spawning season, the hatching liquid properties and choriolysin-encoding genes were investigated in this species. A genomic analysis identified four putative genes referred to as SseHCEa, SseHCEb, SseLCE and SseHE. The phylogenetic analysis classified these paralogs into two clades, the clade I containing SseHCE paralogs and the clade II containing two well-supported subclades named as HE and LCE. The two SseHCE paralogs were intron-less and both genes were tandemly arrayed very close in the genome. The synteny and gene rearrangement identified in the flatfish lineage indicated that the duplication of these two paralogs occurred recently and they are under divergent evolution. The genes SseHE and SseLCE were structured in 8 exons and 7 introns and the synteny was conserved in teleosts. Expression studies confirmed that the four genes were expressed in the hatching gland cells and they migrate co-ordinately from the head to around the yolk sac close to the hatch with specific temporal and intensity expression profiles. Although the mRNA levels of the four genes peaked in the hours previous to larval hatching, the SseHCE and SseLCE paralogs kept a longer expression than SseHE after hatching. These expression patterns were consistent even when larvae were incubated at different temperatures that modified hatching times. The analysis of hatching-liquid using SDS-PAGE and zymography analyses of hatching liquid identified a major band of expected choriolysin size. The optimal pH for protease activity was 8.5 and inhibition assays using EDTA demonstrated that most of the activity in the hatching liquid was due to metalloproteases with Ca2+ ions acting as the most effective metal to restore the activity. All these data provide new clues about the choriolysin evolution and function in flatfish with impact in the aquaculture and the blue cosmetic industry.


Assuntos
Evolução Molecular , Linguados/metabolismo , Metaloendopeptidases , Animais , Linguados/genética , Regulação da Expressão Gênica no Desenvolvimento , Larva/genética , Metaloendopeptidases/classificação , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Filogenia , RNA Mensageiro/genética
15.
Cells ; 9(1)2019 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-31877874

RESUMO

The extracellular matrix (ECM) is a complex and specialized three-dimensional macromolecular network, present in nearly all tissues, that also interacts with cell surface receptors on joint resident cells. Changes in the composition and physical properties of the ECM lead to the development of many diseases, including osteoarthritis (OA). OA is a chronic degenerative rheumatic disease characterized by a progressive loss of synovial joint function as a consequence of the degradation of articular cartilage, also associated with alterations in the synovial membrane and subchondral bone. During OA, ECM-degrading enzymes, including urokinase-type plasminogen activator (uPA), matrix metalloproteinases (MMPs), and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs), cleave ECM components, such as fibronectin (Fn), generating fibronectin fragments (Fn-fs) with catabolic properties. In turn, Fn-fs promote activation of these proteinases, establishing a degradative and inflammatory feedback loop. Thus, the aim of this review is to update the contribution of ECM-degrading proteinases to the physiopathology of OA as well as their modulation by Fn-fs.


Assuntos
Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Osteoartrite/metabolismo , Proteínas ADAMTS/metabolismo , Proteínas ADAMTS/fisiologia , Animais , Cartilagem Articular/metabolismo , Endopeptidases/metabolismo , Matriz Extracelular/fisiologia , Fibronectinas/fisiologia , Humanos , Metaloproteinases da Matriz/metabolismo , Metaloendopeptidases/metabolismo , Metaloendopeptidases/fisiologia , Osteoartrite/fisiopatologia , Peptídeo Hidrolases/metabolismo
16.
Integr Biol (Camb) ; 11(10): 384-393, 2019 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-31851360

RESUMO

In order to perform critical immune functions at sites of inflammation, circulatory T lymphocytes must be able to arrest, adhere, migrate and transmigrate on the endothelial surface. This progression of steps is coordinated by cellular adhesion molecules (CAMs), chemokines, and selectins presented on the endothelium. Two important interactions are between Lymphocyte Function-associated Antigen-1 (LFA-1) and Intracellular Adhesion Molecule-1 (ICAM-1) and also between Very Late Antigen-4 (VLA-4) and Vascular Cell Adhesion Molecule-1 (VCAM-1). Recent studies have shown that T lymphocytes and other cell types can migrate upstream (against the direction) of flow through the binding of LFA-1 to ICAM-1. Since upstream migration of T cells depends on a specific adhesive pathway, we hypothesized that mechanotransduction is critical to migration, and that signals might allow T-cells to remember their direction of migration after the flow is terminated. Cells on ICAM-1 surfaces migrate against the shear flow, but the upstream migration reverts to random migration after the flow is stopped. Cells on VCAM-1 migrate with the direction of flow. However, on surfaces that combine ICAM-1 and VCAM-1, cells crawl upstream at a shear rate of 800 s-1 and continue migrating in the upstream direction for at least 30 minutes after the flow is terminated-we call this 'migrational memory'. Post-flow upstream migration on VCAM-1/ICAM-1 surfaces is reversed upon the inhibition of PI3K, but conserved with cdc42 and Arp2/3 inhibitors. Using an antibody against VLA-4, we can block migrational memory on VCAM-1/ICAM-1 surfaces. Using a soluble ligand for VLA-4 (sVCAM-1), we can promote migrational memory on ICAM-1 surfaces. These results indicate that, while upstream migration under flow requires LFA-1 binding to immobilized ICAM-1, signaling from VLA-4 and PI3K activity is required for the migrational memory of CD4+ T cells. These results indicate that crosstalk between integrins potentiates the signal of upstream migration.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Linfócitos T CD4-Positivos/citologia , Movimento Celular , Integrinas/metabolismo , Metaloendopeptidases/metabolismo , Anticorpos/química , Linfócitos T CD8-Positivos/citologia , Adesão Celular , Quimiocinas , Endotélio Vascular/metabolismo , Humanos , Inflamação , Integrina alfa4beta1/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Ligantes , Antígeno-1 Associado à Função Linfocitária/metabolismo , Mecanotransdução Celular , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Molécula 1 de Adesão de Célula Vascular/metabolismo
17.
Nature ; 575(7782): 361-365, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31695197

RESUMO

Reprogramming of mitochondria provides cells with the metabolic flexibility required to adapt to various developmental transitions such as stem cell activation or immune cell reprogramming, and to respond to environmental challenges such as those encountered under hypoxic conditions or during tumorigenesis1-3. Here we show that the i-AAA protease YME1L rewires the proteome of pre-existing mitochondria in response to hypoxia or nutrient starvation. Inhibition of mTORC1 induces a lipid signalling cascade via the phosphatidic acid phosphatase LIPIN1, which decreases phosphatidylethanolamine levels in mitochondrial membranes and promotes proteolysis. YME1L degrades mitochondrial protein translocases, lipid transfer proteins and metabolic enzymes to acutely limit mitochondrial biogenesis and support cell growth. YME1L-mediated mitochondrial reshaping supports the growth of pancreatic ductal adenocarcinoma (PDAC) cells as spheroids or xenografts. Similar changes to the mitochondrial proteome occur in the tumour tissues of patients with PDAC, suggesting that YME1L is relevant to the pathophysiology of these tumours. Our results identify the mTORC1-LIPIN1-YME1L axis as a post-translational regulator of mitochondrial proteostasis at the interface between metabolism and mitochondrial dynamics.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Metabolismo dos Lipídeos , Metaloendopeptidases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Hipóxia Celular , Linhagem Celular , Proliferação de Células , Humanos , Lipídeos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metaloendopeptidases/genética , Proteínas Mitocondriais/genética , Proteólise
18.
Arch Biochem Biophys ; 677: 108164, 2019 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-31678046

RESUMO

Excessive degradation of the cartilage articular extracellular matrix (ECM) in chondrocytes has been considered as an important pathological characteristics of OA. In the present study, we demonstrate that the G protein-coupled receptor GPR39 is expressed on SW1353 chondrocytes and is significantly downregulated in response to advanced glycation end products (AGEs). Our findings show that agonism of GPR39 exerts significant protective effects against AGE-induced degradation of articular extracellular matrix. Agonism of GPR39 rescued degradation of type II collagen by decreasing expression of the collagen-degrading enzymes matrix metalloproteinase (MMP)-3 and MMP-13. Additionally, agonism of GPR39 rescued AGE-induced suppression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. Agonism of GPR39 prevented degradation of aggrecan by downregulating AGE-induced expression of a disintegrin and metalloproteinase with type I thrombospondin motif (ADAMTS)-4 and ADAMTS-5. Finally, we demonstrate that the effects of GPR39 are mediated through the p38 mitogen activated protein kinase (MAPK)/nuclear factor-κB (NF-κB) cellular signaling pathway. Taken together, our findings show for the first time that targeted therapies involving GPR39 may provide a novel approach for the prevention and treatment of osteoarthritis.


Assuntos
Matriz Extracelular/efeitos dos fármacos , Produtos Finais de Glicação Avançada/farmacologia , Substâncias Protetoras/farmacologia , Pirimidinas/farmacologia , Receptores Acoplados a Proteínas-G/agonistas , Sulfonamidas/farmacologia , Agrecanas/química , Agrecanas/metabolismo , Linhagem Celular Tumoral , Colágeno Tipo II/química , Colágeno Tipo II/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloendopeptidases/metabolismo , Osteoartrite/tratamento farmacológico , Proteólise/efeitos dos fármacos , Receptores Acoplados a Proteínas-G/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Zinco/metabolismo
19.
Carbohydr Res ; 485: 107815, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31622943

RESUMO

Tripodal nonameric mannoside glycodendrimer 1 with carbohydrate tethered triazole linked with the TRIS-glycine-ß-alanine dipeptidic aromatic centered core was synthesized. Glycodendrimer 1 demonstrated potential in vitro anti-leishmanial activity. The bio-activity data was substantiated with molecular modelling and docking studies of 1 with the three-dimensional protein structure of Leishmanolysin.


Assuntos
Dipeptídeos/síntese química , Dipeptídeos/farmacologia , Glicina/química , Leishmania/efeitos dos fármacos , Manosídeos/química , Triazóis/química , beta-Alanina/química , Antiprotozoários/síntese química , Antiprotozoários/química , Antiprotozoários/metabolismo , Antiprotozoários/farmacologia , Técnicas de Química Sintética , Dendrímeros/química , Dipeptídeos/química , Dipeptídeos/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Conformação Proteica
20.
Cancer Metastasis Rev ; 38(3): 347-356, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31482488

RESUMO

A crucial step for tumor cell extravasation and metastasis is the migration through the extracellular matrix, which requires proteolytic activity. Hence, proteases, particularly matrix metalloproteases (MMPs), have been discussed as therapeutic targets and their inhibition should diminish tumor growth and metastasis. The metalloproteases meprin α and meprin ß are highly abundant on intestinal enterocytes and their expression was associated with different stages of colorectal cancer. Due to their ability to cleave extracellular matrix (ECM) components, they were suggested as pro-tumorigenic enzymes. Additionally, both meprins were shown to have pro-inflammatory activity by cleaving cytokines and their receptors, which correlates with chronic intestinal inflammation and associated conditions. On the other hand, meprin ß was identified as an essential enzyme for the detachment and renewal of the intestinal mucus, important to prevent bacterial overgrowth and infection. Considering this, it is hard to estimate whether high activity of meprins is generally detrimental or if these enzymes have also protective functions in certain cancer types. For instance, for colorectal cancer, patients with high meprin ß expression in tumor tissue exhibit a better survival prognosis, which is completely different to prostate cancer. This demonstrates that the very same enzyme may have contrary effects on tumor initiation and growth, depending on its tissue and subcellular localization. Hence, precise knowledge about proteolytic enzymes is required to design the most efficient therapeutic options for cancer treatment. In this review, we summarize the current findings on meprins' functions, expression, and cancer-associated variants with possible implications for tumor progression and metastasis.


Assuntos
Metaloendopeptidases/metabolismo , Neoplasias/enzimologia , Neoplasias/patologia , Animais , Humanos , Metaloendopeptidases/biossíntese , Metástase Neoplásica , Microambiente Tumoral
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