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1.
J Vis Exp ; (166)2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33369607

RESUMO

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.


Assuntos
Elementos de DNA Transponíveis/genética , Estresse Oxidativo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Transfecção , Antioxidantes/farmacologia , Biomarcadores/metabolismo , Contagem de Células , Morte Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/química , Células Epiteliais/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/isolamento & purificação , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/isolamento & purificação , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/isolamento & purificação , Fatores de Crescimento Neural/metabolismo , Neuroproteção/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serpinas/genética , Serpinas/isolamento & purificação , Serpinas/metabolismo , Doadores de Tecidos , Proteína Desacopladora 2/genética , Proteína Desacopladora 2/metabolismo
2.
Nat Commun ; 11(1): 4571, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917871

RESUMO

Early therapeutic interventions are essential to prevent Alzheimer Disease (AD). The association of several inflammation-related genetic markers with AD and the early activation of pro-inflammatory pathways in AD suggest inflammation as a plausible therapeutic target. Inflammatory Caspase-1 has a significant impact on AD-like pathophysiology and Caspase-1 inhibitor, VX-765, reverses cognitive deficits in AD mouse models. Here, a one-month pre-symptomatic treatment of Swedish/Indiana mutant amyloid precursor protein (APPSw/Ind) J20 and wild-type mice with VX-765 delays both APPSw/Ind- and age-induced episodic and spatial memory deficits. VX-765 delays inflammation without considerably affecting soluble and aggregated amyloid beta peptide (Aß) levels. Episodic memory scores correlate negatively with microglial activation. These results suggest that Caspase-1-mediated inflammation occurs early in the disease and raise hope that VX-765, a previously Food and Drug Administration-approved drug for human CNS clinical trials, may be a useful drug to prevent the onset of cognitive deficits and brain inflammation in AD.


Assuntos
Envelhecimento/metabolismo , Doença de Alzheimer/metabolismo , Disfunção Cognitiva/metabolismo , Serpinas/metabolismo , Proteínas Virais/metabolismo , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Animais , Comportamento Animal , Disfunção Cognitiva/tratamento farmacológico , Citocinas/metabolismo , Dipeptídeos/sangue , Dipeptídeos/farmacologia , Modelos Animais de Doenças , Encefalite/metabolismo , Encefalite/patologia , Feminino , Humanos , Inflamação/metabolismo , Masculino , Transtornos da Memória/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Serpinas/sangue , Serpinas/farmacologia , Memória Espacial/fisiologia , Proteínas Virais/sangue , Proteínas Virais/farmacologia , para-Aminobenzoatos/sangue , para-Aminobenzoatos/farmacologia
3.
PLoS Negl Trop Dis ; 14(8): e0008510, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760059

RESUMO

Serine protease inhibitors (serpins) regulate proteolytic events within diverse biological processes, including digestion, coagulation, inflammation and immune responses. The presence of serpins in Fasciola hepatica excretory-secretory products indicates that the parasite exploits these to regulate proteases encountered during its development within vertebrate hosts. Interrogation of the F. hepatica genome identified a multi-gene serpin family of seven members that has expanded by gene duplication and divergence to create an array of inhibitors with distinct specificities. We investigated the molecular properties and functions of two representatives, FhSrp1 and FhSrp2, highly expressed in the invasive newly excysted juvenile (NEJ). Consistent with marked differences in the reactive centre loop (RCL) that executes inhibitor-protease complexing, the two recombinant F. hepatica serpins displayed distinct inhibitory profiles against an array of mammalian serine proteases. In particular, rFhSrp1 efficiently inhibited kallikrein (Ki = 40 nM) whilst rFhSrp2 was a highly potent inhibitor of chymotrypsin (Ki = 0.07 nM). FhSrp1 and FhSrp2 are both expressed on the NEJ surface, predominantly around the oral and ventral suckers, suggesting that these inhibitors protect the parasites from the harmful proteolytic effects of host proteases, such as chymotrypsin, during invasion. Furthermore, the unusual inhibition of kallikrein suggests that rFhSrp1 modulates host responses such as inflammation and vascular permeability by interfering with the kallikrein-kinin system. A vaccine combination of rFhSrp1 and rFhSrp2 formulated in the adjuvant Montanide ISA 206VG elicited modest but non-significant protection against a challenge infection in a rat model, but did induce some protection against liver pathogenesis when compared to a control group and a group vaccinated with two well-studied vaccine candidates, F. hepatica cathepsin L2 and L3. This work highlights the importance of F. hepatica serpins to regulate host responses that enables parasite survival during infection and, coupled with the vaccine data, encourages future vaccine trials in ruminants.


Assuntos
Fasciola hepatica/metabolismo , Inibidores de Serino Proteinase/metabolismo , Serpinas/metabolismo , Sequência de Aminoácidos , Animais , Fasciola hepatica/genética , Regulação da Expressão Gênica , Interações Hospedeiro-Parasita
4.
Proc Natl Acad Sci U S A ; 117(31): 18574-18581, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32661155

RESUMO

Many vertebrates have distinctive blue-green bones and other tissues due to unusually high biliverdin concentrations-a phenomenon called chlorosis. Despite its prevalence, the biochemical basis, biology, and evolution of chlorosis are poorly understood. In this study, we show that the occurrence of high biliverdin in anurans (frogs and toads) has evolved multiple times during their evolutionary history, and relies on the same mechanism-the presence of a class of serpin family proteins that bind biliverdin. Using a diverse combination of techniques, we purified these serpins from several species of nonmodel treefrogs and developed a pipeline that allowed us to assemble their complete amino acid and nucleotide sequences. The described proteins, hereafter named biliverdin-binding serpins (BBS), have absorption spectra that mimic those of phytochromes and bacteriophytochromes. Our models showed that physiological concentration of BBSs fine-tune the color of the animals, providing the physiological basis for crypsis in green foliage even under near-infrared light. Additionally, we found that these BBSs are most similar to human glycoprotein alpha-1-antitrypsin, but with a remarkable functional diversification. Our results present molecular and functional evidence of recurrent evolution of chlorosis, describe a biliverdin-binding protein in vertebrates, and introduce a function for a member of the serpin superfamily, the largest and most ubiquitous group of protease inhibitors.


Assuntos
Anuros/fisiologia , Biliverdina/metabolismo , Serpinas/metabolismo , Pigmentação da Pele/fisiologia , Animais , Anuros/classificação , Anuros/genética , Biliverdina/química , Mimetismo Biológico/fisiologia , Serpinas/química , Serpinas/genética , Pigmentação da Pele/genética
5.
Arch Insect Biochem Physiol ; 105(1): e21727, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32677094

RESUMO

Here, we assessed the effect of a systematic change in reactive center loop (RCL) length, N-terminal to the reactive center, on the inhibitory activity of the recombinant Apserpin-6. The domain prediction results indicated that the RCL is located between the amino acid numbered 359-379 at the C-terminal of Apserpin-6. The N-terminal variable region for amino acid positions P7-P1 of the RCL of Apserpin-6 was truncated or extended by residue deletion or insertion using site-directed mutagenesis. The recombinant Apserpin-6 with one or two residues insertion in RCL had no effect on prophenoloxidase (proPO) activity, whereas deletion of one or two residues in RCL lowered the efficiency of inhibition of Apserpin-6. The results of this study will facilitate the understanding of inhibition mechanism of RCL on proPO activity.


Assuntos
Proteínas de Insetos/genética , Mariposas/genética , Inibidores de Serino Proteinase/genética , Serpinas/genética , Animais , Proteínas de Insetos/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Mariposas/crescimento & desenvolvimento , Mariposas/metabolismo , Inibidores de Serino Proteinase/metabolismo , Serpinas/metabolismo
6.
Mol Carcinog ; 59(8): 955-966, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32391971

RESUMO

Maspin repression is frequently observed in prostate cancer; however, the molecular mechanism(s) causing the loss is not completely understood. Here, we demonstrate that inhibition of class I histone deacetylases (HDACs) mediates re-expression of maspin which plays an essential role in suppressing proliferation and migration capability in prostate cancer cells. Human prostate cancer LNCaP and DU145 cells treated with HDAC inhibitors, sodium butyrate, and trichostatin A, resulted in maspin re-expression. Interestingly, an exploration into the molecular mechanisms demonstrates that maspin repression in prostate tumor and human prostate cancer cell lines occurs via epigenetic silencing through an increase in HDAC activity/expression, independent of promoter DNA hypermethylation. Furthermore, transcriptional activation of maspin was accompanied with the suppression of HDAC1 and HDAC8 with significant p53 enrichment at the maspin promoter associated with an increase in histone H3/H4 acetylation. Our results provide evidence of maspin induction as a critical epigenetic event altered by class I HDACs in the restoration of balance to delay proliferation and migration ability of prostate cancer cells.


Assuntos
Biomarcadores Tumorais/metabolismo , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Histona Desacetilases/metabolismo , Neoplasias da Próstata/patologia , Serpinas/genética , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Epigênese Genética , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/química , Histona Desacetilases/genética , Histonas , Humanos , Ácidos Hidroxâmicos/farmacologia , Masculino , Prognóstico , Regiões Promotoras Genéticas , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Serpinas/metabolismo , Células Tumorais Cultivadas
7.
Proc Natl Acad Sci U S A ; 117(21): 11450-11458, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32385162

RESUMO

Dynamic remodeling of the extracellular matrix affects many cellular processes, either directly or indirectly, through the regulation of soluble ligands; however, the mechanistic details of this process remain largely unknown. Here we propose that type I collagen remodeling regulates the receptor-binding activity of pigment epithelium-derived factor (PEDF), a widely expressed secreted glycoprotein that has multiple important biological functions in tissue and organ homeostasis. We determined the crystal structure of PEDF in complex with a disulfide cross-linked heterotrimeric collagen peptide, in which the α(I) chain segments-each containing the respective PEDF-binding region (residues 930 to 938)-are assembled with an α2α1α1 staggered configuration. The complex structure revealed that PEDF specifically interacts with a unique amphiphilic sequence, KGHRGFSGL, of the type I collagen α1 chain, with its proposed receptor-binding sites buried extensively. Molecular docking demonstrated that the PEDF-binding surface of type I collagen contains the cross-link-susceptible Lys930 residue of the α1 chain and provides a good foothold for stable docking with the α1(I) N-telopeptide of an adjacent triple helix in the fibril. Therefore, the binding surface is completely inaccessible if intermolecular crosslinking between two crosslink-susceptible lysyl residues, Lys9 in the N-telopeptide and Lys930, is present. These structural analyses demonstrate that PEDF molecules, once sequestered around newly synthesized pericellular collagen fibrils, are gradually liberated as collagen crosslinking increases, making them accessible for interaction with their target cell surface receptors in a spatiotemporally regulated manner.


Assuntos
Colágeno Tipo I/metabolismo , Proteínas do Olho/química , Proteínas do Olho/metabolismo , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/metabolismo , Serpinas/química , Serpinas/metabolismo , Sítios de Ligação , Dicroísmo Circular , Colágeno Tipo I/química , Cristalografia por Raios X , Dissulfetos/química , Lisina/química , Simulação de Acoplamento Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Transdução de Sinais , Análise Espaço-Temporal
8.
Vet Parasitol ; 279: 109064, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32143012

RESUMO

Tick serpins are involved in enzyme activity, food digestion, blood-feeding, immune response and anticoagulation. Little is known about the potential roles of serpins in tick reproduction. RHS8, a serpin from the tick Rhipicephalus haemaphysaloides, has an open reading frame 1212 bp long and encodes a protein that has 404 amino acids and a predicted molecular weight of 45 kDa. RHS8 exhibits 89.58 % amino acid identity with RmS15 in Rhipicephalus microplus. RHS8 was expressed primarily in larvae and nymphs. RHS8 mRNA expression in the ovaries, fat bodies and salivary glands were up-regulated from feeding to ovipositing ticks. RNAi results showed that RHS8 dsRNA-injected ticks had a lower body weight, longer feeding time, fewer eggs laid and lower egg hatchability. Tick reproduction, such as egg laying and hatching, was disrupted by RNAi. Compared with the control group, ovaries of the RHS8 interference group were light brown color, indicating a reduction in yolk granule accumulation. Western blot results showed that the expression of RHVg3 and RHVg4 proteins in ovaries was reduced in the RHS8 dsRNA-injected group. These results indicate that RHS8 is related to tick reproduction and its interference affects vitellogenesis.


Assuntos
Proteínas de Artrópodes/genética , Rhipicephalus/fisiologia , Serpinas/genética , Vitelogênese/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Feminino , Larva/crescimento & desenvolvimento , Larva/fisiologia , Ninfa/crescimento & desenvolvimento , Ninfa/fisiologia , Filogenia , Interferência de RNA , Rhipicephalus/genética , Rhipicephalus/crescimento & desenvolvimento , Alinhamento de Sequência , Serpinas/química , Serpinas/metabolismo
9.
Oxid Med Cell Longev ; 2020: 8941057, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32215180

RESUMO

Choroidal neovascularization (CNV) is a sight-threatening disease and is characterized by the formation of pathological neovascularization in the choroid which extends into the subretinal space. Exudative age-related macular degeneration (AMD) is the formation of CNV in the macular area which leads to irreversible blindness. Continuous leakage and hemorrhage of the CNV lesion may eventually result in scarring or later fibrosis, which could result in photoreceptor cell atrophy. The current strategy for treating CNV is the use of antivascular endothelial growth factor (VEGF) agents. Many studies have demonstrated the efficacy of intravitreal anti-VEGF therapy. Other studies have also reported the side effects of single anti-VEGF treatment. And long-term inhibition of a single system may result in collateral damage to other visual elements. Pigment epithelium-derived factor (PEDF) is a 50 kDa protein that was first isolated from the conditioned medium of human RPE cells. PEDF has both antiangiogenesis and neuroprotective functions for photoreceptor cells. It may be a potential ocular antiangiogenic agent. This review outlines the distribution of PEDF in the eye, the mechanism of antiangiogenesis, the protective effect on the retina, and the relationship between PEDF and VEGF.


Assuntos
Inibidores da Angiogênese/uso terapêutico , Neovascularização de Coroide/tratamento farmacológico , Proteínas do Olho/uso terapêutico , Fatores de Crescimento Neural/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Serpinas/uso terapêutico , Animais , Permeabilidade Capilar/efeitos dos fármacos , Neovascularização de Coroide/genética , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Humanos , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Retina/efeitos dos fármacos , Retina/metabolismo , Retina/patologia , Serpinas/genética , Serpinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo
10.
J Endocrinol ; 245(2): 291-300, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32171180

RESUMO

Polycystic ovary syndrome (PCOS), one of the most common female endocrine disorder, is a prevalent cause of infertility. Hyperandrogenism is a key feature in PCOS and is correlated with increased expression of VEGF and cytokines in the ovaries. We have previously shown that pigment epithelium-derived factor (PEDF), an endogenous protein, presents potent anti-angiogenic and anti-inflammatory activities in the ovary and negates the effects of cytokines and VEGF. Additionally, PEDF plays a role in both pathophysiology and treatment of ovarian-hyperstimulation syndrome (OHSS), frequently seen in PCOS patients. We established hyperandrogenic-PCOS models, both in vivo, using mice exposed prenatally to dihydrotestosterone (DHT) and, in vitro, using human primary granulosa cells (hpGCs) and human granulosa cell line (KGN). In PCOS-induced mice, the mRNA levels of I l-6, V egf and Amh were higher than those of control; yet, treatment with rPEDF decreased these levels. Moreover, treating OHSS-induced PCOS-mice with rPEDF alleviated all OHSS symptoms. Stimulation of hpGCs with DHT resulted in downregulation of PEDF mRNA expression, concomitantly with a significant increase in IL-6 and IL-8 mRNAs expression. However, co-stimulation of DHT with rPEDF attenuated the increase in cytokines expression. The anti-inflammatory effect of PEDF was found to be mediated via PPARγ pathway. Our findings suggest that rPEDF treatment may normalize the ovarian angiogenic-inflammatory imbalance, induced by PCOS-associated hyperandrogenism. Moreover, the therapeutic potency of PEDF in preventing OHSS symptomes offers a rationale for using PEDF as novel physiological treatment for PCOS sequels.


Assuntos
Anti-Inflamatórios/metabolismo , Proteínas do Olho/metabolismo , Hiperandrogenismo/metabolismo , Fatores de Crescimento Neural/metabolismo , Síndrome do Ovário Policístico/metabolismo , Serpinas/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Di-Hidrotestosterona , Modelos Animais de Doenças , Feminino , Células da Granulosa/metabolismo , Humanos , Hiperandrogenismo/induzido quimicamente , Hiperandrogenismo/complicações , Camundongos , Ovário/metabolismo , Síndrome do Ovário Policístico/induzido quimicamente
11.
Cancer Res ; 80(7): 1475-1485, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32019869

RESUMO

Metastasis causes most cancer-related deaths, and one poorly understood aspect of metastatic cancer is the adaptability of cells from a primary tumor to create new niches and survive in multiple, different secondary sites. We used quantitative mass spectrometry to analyze the extracellular matrix (ECM), a critical component of metastatic niches, in metastases to the brain, lungs, liver, and bone marrow, all derived from parental MDA-MB-231 triple-negative breast cancer cells. Tumor and stromal cells cooperated in forming niches; stromal cells produced predominantly core, structural ECM proteins and tumor cells produced a diverse array of ECM-associated proteins, including secreted factors and modulators of the matrix. In addition, tumor and stromal cells together created distinct niches in each tissue. Downregulation of SERPINB1, a protein elevated in brain metastases, led to a reduction in brain metastasis, suggesting that some niche-specific ECM proteins may be involved in metastatic tropism. SIGNIFICANCE: Tumor and stromal cells together create distinct ECM niches in breast cancer metastases to various tissues, providing new insight into how tumor cells adapt to survive in different tissue environments.


Assuntos
Neoplasias da Medula Óssea/secundário , Neoplasias Encefálicas/secundário , Matriz Extracelular/patologia , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/secundário , Neoplasias de Mama Triplo Negativas/patologia , Medula Óssea/patologia , Encéfalo/patologia , Sobrevivência Celular , Progressão da Doença , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Fígado/patologia , Pulmão/patologia , Células-Tronco Neoplásicas/patologia , Proteômica , Serpinas/genética , Serpinas/metabolismo , Células Estromais/patologia , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Cancer Res ; 80(7): 1461-1474, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32029550

RESUMO

The prognosis for pancreatic ductal adenocarcinoma (PDAC) remains poor despite decades of effort. The abundant extracellular matrix (ECM) in PDAC comprises a major fraction of the tumor mass and plays various roles in promoting resistance to therapies. However, nonselective depletion of ECM has led to poor patient outcomes. Consistent with that observation, we previously showed that individual matrisome proteins derived from stromal cells correlate with either long or short patient survival. In marked contrast, those derived from cancer cells correlate strongly with poor survival. Here, we studied three cancer cell-derived matrisome proteins that are significantly overrepresented during PDAC progression, AGRN (agrin), SERPINB5 (serine protease inhibitor B5), and CSTB (cystatin B). Using both overexpression and knockdown experiments, we demonstrate that all three are promoters of PDAC metastasis. Furthermore, these proteins operate at different metastatic steps. AGRN promoted epithelial-to-mesenchymal transition in primary tumors, whereas SERPINB5 and CSTB enhanced late steps in the metastatic cascade by elevating invadopodia formation and in vivo extravasation. All three genes were associated with a poor prognosis in human patients and high levels of SERPINB5, secreted by cancer cells and deposited in the ECM, correlated with poor patient prognosis. This study provides strong evidence that cancer cell-derived matrisome proteins can be causal in promoting tumorigenesis and metastasis and lead to poor patient survival. Therefore, compared with the bulk matrix, mostly made by stromal cells, precise interventions targeting cancer cell-derived matrisome proteins, such as AGRN, SERPINB5, and CSTB, may represent preferred potential therapeutic targets. SIGNIFICANCE: This study provides insights into the biological roles of cancer cell-derived matrisome proteins in PDAC and supports the notion that these proteins are protumorigenic and better therapeutic targets.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinogênese/patologia , Carcinoma Ductal Pancreático/patologia , Proteínas da Matriz Extracelular/metabolismo , Neoplasias Pancreáticas/patologia , Agrina/genética , Agrina/metabolismo , Animais , Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/cirurgia , Linhagem Celular Tumoral , Movimento Celular , Cistatina B/genética , Cistatina B/metabolismo , Progressão da Doença , Transição Epitelial-Mesenquimal , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Proteínas da Matriz Extracelular/genética , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Invasividade Neoplásica/patologia , Metástase Neoplásica/patologia , Pâncreas/patologia , Pâncreas/cirurgia , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/cirurgia , Prognóstico , Serpinas/genética , Serpinas/metabolismo , Células Estromais/metabolismo , Células Estromais/patologia , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Exp Cell Res ; 389(2): 111891, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32035134

RESUMO

Pigment epithelium-derived factor (PEDF) is an endogenous human glycoprotein first identified as a neurotrophic factor in retinal pigmented epithelium cells. PEDF has since been shown to play a central role in mediating cellular protection against oxidative stress, by promoting cell survival, reducing inflammation, and inhibiting pathological angiogenesis in a range of cell types and tissues. PEDF is a well-established neurotrophic factor which supports neurogenesis and provides neuroprotection in response to cellular stress, with numerous studies demonstrating the ability of PEDF to promote neuronal survival and growth following injury. PEDF is an essential component of the stem cell microenvironment and bone extracellular matrix, where it regulates the differentiation of osteoblast precursor cells to promote normal bone development. Accumulating evidence indicates that PEDF maintains stem cell populations and promotes neuronal growth and bone formation by directing cell fate and regulating cell cycle progression. The ability of PEDF to promote neurogenesis, osteogenesis, and stemness indicates therapeutic potential in diseases characterised by tissue degeneration. In this review, we provide a current summary of the role of PEDF in regulating cellular survival and differentiation in bone, the central nervous system, and other stem cell niches, and highlight the emerging potential of PEDF as a regenerative therapeutic agent.


Assuntos
Diferenciação Celular , Proteínas do Olho/metabolismo , Fatores de Crescimento Neural/metabolismo , Neurônios/citologia , Medicina Regenerativa , Serpinas/metabolismo , Células-Tronco/citologia , Animais , Morte Celular , Humanos , Neurônios/metabolismo , Células-Tronco/metabolismo
14.
Invest Ophthalmol Vis Sci ; 61(2): 6, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32031578

RESUMO

Purpose: To determine whether priming of bone marrow mesenchymal stem cells (MSCs) by signals from injured retina, particularly tumor necrosis factor α (TNFα), increase their exosomes' neuroprotective efficacy on retinal ganglion cells (RGCs). Methods: MSCs were primed with retinal cell culture conditioned medium, with or without the TNFα blocker etanercept or TNFα prior to isolation of exosomes. MSC conditioned medium or exosomes were added to rat retinal cultures or human stem cell-derived retinal ganglion cell (hRGC) cultures, and RGC neuroprotective effects were quantified. Luminex assays were used to compare primed versus unprimed exosomes. Results: MSC conditioned medium and exosomes exerted a significant neuroprotective effect on injured rat and hRGC. This effect was significantly increased after MSCs were primed with retinal conditioned medium or TNFα. Blocking of TNFα signaling with etanercept prevented priming-induced RGC neuroprotective efficacy. Priming increased PEDF and VEGF-AA exosomal abundance. Conclusions: MSC exosomes promote RGC survival not just in rodent retinal cultures but also with hRGC. Their efficacy can be further enhanced through TNFα priming with the mechanism of action potentially mediated, at least in part, through increased levels of PEDF and VEGF-AA.


Assuntos
Células-Tronco Mesenquimais/fisiologia , Fármacos Neuroprotetores/farmacologia , Células Ganglionares da Retina/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Células Cultivadas , Etanercepte/farmacologia , Exossomos , Proteínas do Olho/metabolismo , Feminino , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Fatores de Crescimento Neural/metabolismo , Neuroproteção/efeitos dos fármacos , Ratos Sprague-Dawley , Serpinas/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo
15.
J Biomed Sci ; 27(1): 30, 2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32005234

RESUMO

BACKGROUND: The main strategy against nasopharyngeal carcinoma (NPC) is radiotherapy. However, radioresistance mediated recurrence is a leading clinical bottleneck in NPC. Revealing the mechanism of NPC radioresistance will help improve the therapeutic effect. METHODS: In this study, the role of TRIM21 (tripartite motif-containing 21) in NPC receiving ionizing radiation was firstly examined both in vivo and in vitro. Mass spectrometry analysis was performed to identify the downstream targets of TRIM21. NPC cells with TRIM21 or SERPINB5 (serpin family B member 5) overexpression or knockout were used to determine the epistatic relationship among SERPINB5, GMPS (guanine monophosphate synthase) and TRIM21. Flow cytometry, co-immunoprecipitation, western blot and immunofluorescence were employed to strengthen the results. Finally, immunohistochemistry using 4 radiosensitive and 8 radioresistent NPC patient samples was perform to examine the association between SERPINB5 or GMPS expression and patient radio-sensitivity. RESULTS: As an E3 ligase, TRIM21 was highly expressed in NPC. After ionizing radiation, TRIM21 repressed TP53 expression by mediating GMPS ubiquitination and degradation. Overexpression of TRIM21 protected NPC cells from radiation mediated cell apoptosis in vitro and in vivo. Further analysis revealed that TRIM21 mediated GMPS repression was dependent on SERPINB5, and SERPINB5 served as an adaptor which prevented GMPS from entering into the nucleus and introduced TRIM21 for GMPS ubiquitination. Moreover, the in vitro and in vivo results validated the finding that SERPINB5 promoted NPC cell radioresistance, and the radioresistant patients had higher SERPINB5 expression. CONCLUSIONS: Overall, our data showed that TRIM21-SERPINB5-mediated GMPS degradation facilitated TP53 repression, which promoted the radioresistance of NPC cells. This novel working model related to TP53 suppression provided new insight into NPC radioresistence clinically.


Assuntos
Apoptose , Carcinoma Nasofaríngeo/metabolismo , Ribonucleoproteínas/genética , Serpinas/genética , Proteína Supressora de Tumor p53/genética , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Humanos , Ribonucleoproteínas/metabolismo , Serpinas/metabolismo , Proteína Supressora de Tumor p53/metabolismo
16.
Biochim Biophys Acta Proteins Proteom ; 1868(4): 140363, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31954927

RESUMO

Neuroserpin (NS) is predominantly expressed in brain and inhibits tissue-type plasminogen activator (tPA) with implications in brain development and memory. Nature of conformational change in pathological variants in strand 6B and helix B of NS that cause a relatively mild to severe epilepsy (and/or dementia) remains largely elusive. MD simulation with wild type (WT) NS, strand 6B and helix B variants indicated that substitution in this region affects the conformation of the strands 5B, 5A and reactive centre loop. Therefore, we designed variants of NS in strand 6B (I46D and F48S) and helix B (A54F, L55A and L55P) to investigate their role in tPA inhibition mechanism and propensity to aggregate. An interaction analysis showed disturbance of a hydrophobic patch centered at strands 5B, 6B and helix B in I46D and F48S but not in A54F, L55A, L55P and WT NS. Purified I46D, F48S and L55P variants showed decrease in fluorescence emission intensity but have similar α-helical content, however results of A54F and L55A were comparable to WT NS. Analysis of tPA inhibition showed marginal effect on A54F and L55A variant with tPA-NS complex formation. In contrast, I46D, F48S and L55P variants showed massive decrease in tPA inhibition, with no tPA-NS complex formation. Analysis of native PAGE under under polymerization condition showed prompt conversion of I46D, F48S and L55P to latent conformation but not A54F and L55A variants. Identification of these novel conformational changes will aid in the understanding of variable clinical phenotype of shutter region NS variants and other serpins.


Assuntos
Neuropeptídeos/química , Serpinas/química , Epilepsias Mioclônicas/genética , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Mutação , Neuropeptídeos/genética , Neuropeptídeos/isolamento & purificação , Neuropeptídeos/metabolismo , Fenótipo , Polimerização , Agregados Proteicos , Conformação Proteica , Conformação Proteica em alfa-Hélice , Serpinas/genética , Serpinas/isolamento & purificação , Serpinas/metabolismo , Ativador de Plasminogênio Tecidual/antagonistas & inibidores
17.
FASEB J ; 34(1): 619-630, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914706

RESUMO

Tannerella forsythia is a periodontopathogen that expresses miropin, a protease inhibitor in the serpin superfamily. In this study, we show that miropin is also a specific and efficient inhibitor of plasmin; thus, it represents the first proteinaceous plasmin inhibitor of prokaryotic origin described to date. Miropin inhibits plasmin through the formation of a stable covalent complex triggered by cleavage of the Lys368-Thr369 (P2-P1) reactive site bond with a stoichiometry of inhibition of 3.8 and an association rate constant (kass) of 3.3 × 105 M-1s-1. The inhibition of the fibrinolytic activity of plasmin was nearly as effective as that exerted by α2-antiplasmin. Miropin also acted in vivo by reducing blood loss in a mice tail bleeding assay. Importantly, intact T. forsythia cells or outer membrane vesicles, both of which carry surface-associated miropin, strongly inhibited plasmin. In intact bacterial cells, the antiplasmin activity of miropin protects envelope proteins from plasmin-mediated degradation. In summary, in the environment of periodontal pockets, which are bathed in gingival crevicular fluid consisting of 70% of blood plasma, an abundance of T. forsythia in the bacterial biofilm can cause local inhibition of fibrinolysis, which could have possible deleterious effects on the tooth-supporting structures of the periodontium.


Assuntos
Antifibrinolíticos/farmacologia , Fibrinólise/efeitos dos fármacos , Doenças Periodontais/tratamento farmacológico , Serpinas/efeitos dos fármacos , Animais , Bactérias/metabolismo , Domínio Catalítico , Feminino , Fibrinolisina/metabolismo , Fibrinolisina/farmacologia , Humanos , Camundongos Endogâmicos C57BL , Inibidores de Proteases/farmacologia , Serpinas/metabolismo
18.
Life Sci ; 243: 117285, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31926241

RESUMO

Vaspin, an insulin-sensitizing adipokine, has been associated with type 2 diabetes (T2D). The present study aimed to investigate the distribution of genotypes and high-risk alleles of vaspin genetic variants (rs77060950 G/T and rs2236242 A/T), in Gujarat subpopulation (India). Genomic DNA isolated from PBMCs was used to genotype vaspin polymorphisms by PCR-RFLP and ARMS-PCR from 502 controls and 478 patients. RNA isolated from visceral adipose tissue (VAT) of 22 controls and 20 patients was used to assess vaspin transcript levels by qPCR while the vaspin titre of the subjects was assayed using ELISA. Phenotypic characteristics of Fasting Blood Glucose (FBG), BMI and plasma lipid profile were estimated and analyzed for the genotype-phenotype correlation. We identified a significant association of rs2236242 A/T with T2D as the TT genotype conferred a 3.087-fold increased risk. The TT genotype showed association with increased FBG, BMI and Triglycerides levels. Increased GA, GT and TA haplotype frequencies, decreased VAT transcript and vaspin protein levels in T2D patients was observed, which were further negatively correlated with FBG and BMI. In conclusion, rs2274907 A/T polymorphism is strongly associated with reduced vaspin transcript and protein levels, and related metabolic alterations that may play a role in the advancement of T2D.


Assuntos
Diabetes Mellitus Tipo 2/fisiopatologia , Íntrons , Polimorfismo de Nucleotídeo Único , Serpinas/genética , Diabetes Mellitus Tipo 2/genética , Genótipo , Humanos , RNA Mensageiro/genética , Serpinas/sangue , Serpinas/metabolismo
19.
Nutrients ; 12(1)2020 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-31968655

RESUMO

Non-alcoholic fatty liver disease (NAFLD), the leading cause of chronic liver diseases worldwide, ranges from simple steatosis to steatohepatitis, with the risk for progressive fibrosis or even cirrhosis. While simple steatosis is a relatively benign condition, the buildup of toxic lipid metabolites can induce chronic inflammation, ultimately triggering disease progression. Pigment epithelium-derived factor (PEDF) is a secreted, multifunctional glycoprotein with lipid metabolic activities. PEDF promotes lipolysis through binding to adipose triglyceride lipase (ATGL), a key enzyme for triglyceride breakdown. In the current study, we aimed to delineate how changes in PEDF expression affect hepatic lipid accumulation. Our data revealed that hepatic PEDF was downregulated in a mouse NAFLD model. We further showed that decreased PEDF levels in hepatocytes in vitro resulted in elevated fatty acid uptake and lipid droplet formation, with concomitant upregulation of fatty acid transport proteins CD36 and fatty acid binding protein 1 (FABP1). RNA sequencing analysis of PEDF knocked down hepatocytes revealed an alteration in gene expression profile toward lipid accumulation. Additionally, decreased PEDF promotes mobilization of fatty acids, an observation distinct from blocking ATGL activity. Taken together, our data suggest that hepatic PEDF downregulation causes molecular changes that favor triglyceride accumulation, which may further lead to NAFLD progression.


Assuntos
Proteínas do Olho/metabolismo , Ácidos Graxos/metabolismo , Hepatócitos/metabolismo , Gotículas Lipídicas/metabolismo , Fígado/metabolismo , Fatores de Crescimento Neural/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Serpinas/metabolismo , Animais , Antígenos CD36/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Progressão da Doença , Regulação para Baixo , Proteínas do Olho/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Hepatócitos/patologia , Humanos , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/genética , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Serpinas/genética
20.
Int J Pharm ; 576: 118999, 2020 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-31893541

RESUMO

Colorectal cancer (CRC) is a major cause of cancer-related mortality worldwide. Moreover, metastasis is one of the main causes of death in CRC patients. Nanotechnology-based gene therapy has shown significant therapeutic benefits in recent clinical trials for cancer treatment. Recent studies have shown that pigment epithelium-derived factor (PEDF) protein can inhibit tumor growth and metastasis by anti-angiogenesis and pro-apoptosis. In this study, we prepared a PEDF-DNA-loaded liposome for cancer-targeted gene therapy for metastatic CRC using an iRGD peptide. Our results showed that cancer-targeted PEDF-DNA liposomes (R-LP/PEDF) exhibited enhanced inhibitory effects on invasion, migration, and pro-apoptosis of CRC cells in vitro. In addition, it reduced metastasis tumor nodules in lung and prolonged the survival time in a mouse model of metastatic CRC.


Assuntos
Peptídeos Penetradores de Células/metabolismo , Neoplasias Colorretais/terapia , Proteínas do Olho/genética , Marcação de Genes , Neoplasias Pulmonares/prevenção & controle , Fatores de Crescimento Neural/genética , Oligopeptídeos/metabolismo , Serpinas/genética , Animais , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Peptídeos Penetradores de Células/química , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas do Olho/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fatores de Crescimento Neural/metabolismo , Oligopeptídeos/química , Receptores Imunológicos/metabolismo , Receptores de Peptídeos/metabolismo , Serpinas/metabolismo , Carga Tumoral
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