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1.
PLoS Negl Trop Dis ; 14(9): e0008555, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32976538

RESUMO

Junin virus (JUNV) is a New World arenavirus that is the causative agent of Argentine hemorrhagic fever (AHF). Candid#1 (Can) is a live-attenuated vaccine strain of JUNV that since its introduction has resulted in a marked decrease in AHF incidence within the endemic regions of the Pampas in Argentina. Originally, the viral determinants and mechanisms of Can attenuation were not well understood. Recent work has identified the glycoprotein as the major attenuating factor for Can. The establishment of attenuating strategies based on any of the other viral proteins, however, has not been pursued. Here, we document the role of Can Z resulting in incompatibilities with wild type JUNV that results in decreased growth in vitro. In addition, this incompatibility results in attenuation of the virus in the guinea pig model. Further, we identify a single mutation (V64G) in the Z protein that is able to confer this demonstrated attenuation. By establishing and characterizing a novel attenuation strategy for New World mammarenaviruses, we hope to aid future vaccine development for related emerging pathogens including Machupo virus (MACV), Guanarito virus (GTOV), and Sabia virus (SABV).


Assuntos
Glicoproteínas/genética , Febre Hemorrágica Americana/virologia , Vírus Junin/genética , Mutação , Animais , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Feminino , Glicoproteínas/metabolismo , Cobaias , Vírus Junin/crescimento & desenvolvimento , Células Vero , Proteínas Virais/genética , Proteínas Virais/metabolismo
2.
Gene ; 761: 145024, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32755659

RESUMO

Understanding how various pathologies of breast cancer respond to their environment may be imperative in the creation of novel therapeutic targets. Central to the organisation and behaviour of cells within the tumour microenvironment is the extracellular matrix (ECM), a meshwork of fibrous proteins and glycoproteins that directly influences cell behaviour and the bioavailability of signalling molecules. Our appreciation on how the composition of the ECM can influence cancer behaviour has evolved significantly and although we are highly cognisant of the dramatic impact the ECM can have on cancer cell behaviour, we continue to neglect this during diagnosis and treatment. In the following study, we aimed to identify how three breast cancer cell lines respond functionally and genetically to common components of the ECM. Using real time and end point assays we have identified similar patterns of behaviour among the three breast cancer cell lines in response to commonly found ECM components of the breast. Using a selected gene panel, we have been able to identify cell line specific changes in gene differentiation when breast cancer cells are in contact with these elements. Although the response of our cells to these elements differ at the genetic level, their functional responses are consistent. This work adds to the growing arguments that highlight a need for histologically assessing ECM composition of breast tumours. In particular monitoring of fibrous protein deposition at the site of malignancy could provide critical information during clinical assessment influencing disease prognosis and treatment decisions for breast cancer patients.


Assuntos
Neoplasias da Mama/genética , Colágeno/genética , Fibronectinas/genética , Mama/patologia , Linhagem Celular Tumoral , Colágeno/metabolismo , Colágeno Tipo I/genética , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/genética , Feminino , Fibronectinas/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Genótipo , Glicoproteínas/genética , Humanos , Fenótipo , Prognóstico , Transdução de Sinais , Microambiente Tumoral
3.
PLoS One ; 15(6): e0232111, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32579557

RESUMO

Glaucoma is the second leading cause of irreversible blindness worldwide. Primary open angle glaucoma (POAG), the most common form of glaucoma, is often associated with elevation of intraocular pressure (IOP) due to the dysfunction of trabecular meshwork (TM) tissues. Currently, an ex vivo human anterior segment perfusion cultured system is widely used to study the effects of glaucoma factors and disease modifying drugs on physiological parameters like aqueous humor (AH) dynamics and IOP homeostasis. This system requires the use of freshly enucleated intact human eyes, which are sparsely available at very high cost. In this study, we explored the feasibility of using human donor corneoscleral segments for modeling morphological and biochemical changes associated with POAG. Among the number of corneas donated each year, many are deemed ineligible for transplantation due to stringent acceptance criteria. These ineligible corneoscleral segments were obtained from the Lions Eye Bank, Tampa, Florida. Each human donor anterior corneoscleral segment was dissected into four equal quadrants and cultured for 7 days by treating with the glaucoma factors dexamethasone (Dex) or recombinant transforming growth factor (TGF) ß2 or transduced with lentiviral expression vectors containing wild type (WT) and mutant myocilin. Hematoxylin and Eosin (H&E) staining analysis revealed that the TM structural integrity is maintained after 7 days in culture. Increased TUNEL positive TM cells were observed in corneoscleral quadrants treated with glaucoma factors compared to their respective controls. However, these TUNEL positive cells were mainly confined to the scleral region adjacent to the TM. Treatment of corneoscleral quadrants with Dex or TGFß2 resulted in glaucomatous changes at the TM, which included increased extracellular matrix (ECM) proteins and induction of endoplasmic reticulum (ER) stress. Western blot analysis of the conditioned medium showed an increase in ECM (fibronectin and collagen IV) levels in Dex- or TGFß2-treated samples compared to control. Lentiviral transduction of quadrants resulted in expression of WT and mutant myocilin in TM tissues. Western blot analysis of conditioned medium revealed decreased secretion of mutant myocilin compared to WT myocilin. Moreover, increased ECM deposition and ER stress induction was observed in the TM of mutant myocilin transduced quadrants. Our findings suggest that the ex-vivo cultured human corneoscleral segment model is cost-effective and can be used as a pre-screening tool to study the effects of glaucoma factors and anti-glaucoma therapeutics on the TM.


Assuntos
Dexametasona/farmacologia , Limbo da Córnea/metabolismo , Malha Trabecular/efeitos dos fármacos , Fator de Crescimento Transformador beta2/farmacologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Glaucoma de Ângulo Aberto/patologia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Limbo da Córnea/citologia , Limbo da Córnea/efeitos dos fármacos , Malha Trabecular/metabolismo , Malha Trabecular/patologia , Regulação para Cima/efeitos dos fármacos
4.
PLoS One ; 15(6): e0234873, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32559232

RESUMO

OBJECTIVE: Comparison of human mRNA microarray results from tumor-associated and normal cervical fibroblasts revealed significant TFPI2 downregulation in tumor-associated fibroblasts isolated from cervical cancer, indicating that TFPI2 downregulation may play an important role in the pathogenesis of the disease. In the present work, we investigated the mechanism of TFPI2 downregulation in tumor-associated fibroblasts and tumor cells. METHODS: In vitro models of monocultures and co-cultures were established with tumor cells and fibroblasts to explore the changes of TFPI-2 expression and epigenetic modifications of the TFPI2 gene. RESULTS: The TFPI2 gene was hypermethylated only in tumor cells. Reduction of TFPI-2 protein levels in tumor-associated fibroblasts, although the gene was not methylated, suggested alternative regulatory mechanisms of gene expression, such as inhibition by microRNAs. The expression pattern of miR-23a, a gene thought to inhibit TFPI2 translation, showed changes strongly correlated to detected TFPI-2 protein alterations. Transfections with miR-23a mimics resulted in a decrease of TFPI-2 protein expression whereas miR-23a inhibitors increased the TFPI-2 amount. Due to downregulation of miR-23a expression by HPV in cancer cells, TFPI2 was silenced by promoter methylation. In contrary, miR-23a was active in HPV-free fibroblasts and inactivated TFPI2. CONCLUSION: These results indicate dual epigenetic inhibition of TFPI2 on the transcription level by promoter methylation in cancer cells and on the translation level by miR-23a in tumor-associated fibroblasts. As a consequence, inactivation of the TFPI2 gene plays a strategic role in the progression of cervical cancer.


Assuntos
Inativação Gênica , Glicoproteínas/genética , Neoplasias do Colo do Útero/genética , Adulto , Fibroblastos Associados a Câncer/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Células Tumorais Cultivadas
5.
Nat Biotechnol ; 38(7): 865-869, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32483365

RESUMO

We describe base editors that combine both cytosine and adenine base-editing functions. A codon-optimized fusion of the cytosine deaminase PmCDA1, the adenosine deaminase TadA and a Cas9 nickase (Target-ACEmax) showed a high median simultaneous C-to-T and A-to-G editing activity at 47 genomic targets. On-target as well as DNA and RNA off-target activities of Target-ACEmax were similar to those of existing single-function base editors.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , DNA/genética , Edição de Genes , Adenina/metabolismo , Adenosina Desaminase/genética , Citosina/metabolismo , Desoxirribonuclease I/genética , Genoma Humano/genética , Glicoproteínas/genética , Guanina/metabolismo , Células HEK293 , Humanos , Mutação/genética , Proteínas Nucleares/genética , RNA/genética
6.
Life Sci ; 256: 117922, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32522569

RESUMO

To test the hypothesis of STC-1 participation in maintenance of glucose homeostasis in fed and fasting (48 h) rats, we investigated that this hormone may be implicated in the regulation of renal gluconeogenesis pathway from lactate and lactate oxidation in renal cortex and medulla. Our results demonstrate the hSTC-1 role on lactate metabolism in the renal cortex and medulla from fed and fasting rats. hSTC-1 increased the gluconeogenesis activity in fed state in renal cortex, and this increase was induced by raise in Pck1 gene expression. In fasting animals hSTC-1 increase the renal medulla gluconeogenesis activity, but Pck1 gene expression was not alter. The stimulatory effect of hSTC-1 on 14C-lactate oxidation occurred only in the renal cortex from fed rats. These findings show the hSTC-1 contribution to lactate homeostasis and supplies glucose to other tissues. This response may represent a strategy of action of STC-1 in response to fasting stress as postulated by different authors. On the other hand, hSTC-1 acts downstream of adenylcyclase pathway, decreasing the gluconeogenesis activity induced by cAMP intracellular increase or stimulating the phosphodiesterase activity in the renal cortex. However, no hSTC-1 effect on 14C-lactate oxidation was found after increase in the intracellular cAMP. The findings also revealed that the renal cortex and medulla respond differently to hSTC-1, possibly due to the higher level of STC-1 gene expression in inner renal medulla than in renal cortex.


Assuntos
Gluconeogênese/efeitos dos fármacos , Glicoproteínas/metabolismo , Hormônios/metabolismo , Rim/metabolismo , Lactatos/metabolismo , Proteínas Recombinantes/metabolismo , Adenilil Ciclases/metabolismo , Animais , Dióxido de Carbono/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Glicoproteínas/genética , Hormônios/genética , Humanos , Córtex Renal/metabolismo , Medula Renal/metabolismo , Masculino , Oxirredução , Diester Fosfórico Hidrolases/metabolismo , Ratos , Ratos Wistar , Proteínas Recombinantes/genética , Transdução de Sinais
7.
Mol Genet Genomics ; 295(5): 1253-1262, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32556999

RESUMO

Monogenic diabetes is a rare type of diabetes resulting from mutations in a single gene. To date, most cases remain genetically unexplained, posing a challenge for accurate diabetes treatment, which leads to on a molecular diagnosis. Therefore, a trio exome scan was performed in a lean, nonsyndromic Caucasian girl with diabetes onset at 2½ years who was negative for autoantibodies. The lean father had diabetes from age 11 years. A novel heterozygous mutation in EDEM2, c.1271G > A; p.Arg424His, was found in the proband and father. Downregulation of Edem2 in rat RIN-m ß-cells resulted in a decrease in insulin genes Ins1 to 67.9% (p = 0.006) and Ins2 to 16.8% (p < 0.001) and reduced insulin secretion by 60.4% (p = 0.0003). Real-time PCR revealed a major disruption of endocrine pancreas-specific genes, including Glut2 and Pxd1, with mRNA suppression to 54% (p < 0.001) and 85.7% (p = 0.01), respectively. No other expression changes related to stress or apoptotic genes were observed. Extended clinical follow-up involving ten family members showed that two healthy individuals carried the same mutation with no sign of diabetes in the clinical screen except for a slight increase in IA-2 antibody in one of them, suggesting incomplete penetrance. In conclusion, we describe EDEM2 as a likely/potential novel diabetes gene, in which inhibition in vitro reduces the expression of ß-cell genes involved in the glucose-stimulated insulin secretion (GSIS) pathway, leading to an overall suppression of insulin secretion but not apoptosis.


Assuntos
Diabetes Mellitus/genética , Regulação para Baixo , Transportador de Glucose Tipo 2/genética , Glicoproteínas/genética , Proteínas de Homeodomínio/genética , Mutação Puntual , Transativadores/genética , alfa-Manosidase/genética , Idade de Início , Idoso , Animais , Linhagem Celular , Diabetes Mellitus/metabolismo , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Inativação Gênica , Humanos , Insulina/genética , Insulina/metabolismo , Masculino , Pessoa de Meia-Idade , Linhagem , Ratos , Sequenciamento Completo do Exoma , Adulto Jovem
8.
PLoS One ; 15(5): e0233492, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469948

RESUMO

Glycosylation can affect various protein properties such as stability, biological activity, and immunogenicity. To produce human therapeutic proteins, a host that can produce glycoproteins with correct glycan structures is required. Microbial expression systems offer economical, rapid and serum-free production and are more amenable to genetic manipulation. In this study, we developed a protocol for CRISPR/Cas9 multiple gene knockouts and knockins in Kluyveromyces marxianus, a probiotic yeast with a rapid growth rate. As hyper-mannosylation is a common problem in yeast, we first knocked out the α-1,3-mannosyltransferase (ALG3) and α-1,6-mannosyltransferase (OCH1) genes to reduce mannosylation. We also knocked out the subunit of the telomeric Ku domain (KU70) to increase the homologous recombination efficiency of K. marxianus. In addition, we knocked in the MdsI (α-1,2-mannosidase) gene to reduce mannosylation and the GnTI (ß-1,2-N-acetylglucosaminyltransferase I) and GnTII genes to produce human N-glycan structures. We finally obtained two strains that can produce low amounts of the core N-glycan Man3GlcNAc2 and the human complex N-glycan Man3GlcNAc4, where Man is mannose and GlcNAc is N-acetylglucosamine. This study lays a cornerstone of glycosylation engineering in K. marxianus toward producing human glycoproteins.


Assuntos
Kluyveromyces/genética , Kluyveromyces/metabolismo , Engenharia Metabólica/métodos , Polissacarídeos/biossíntese , Polissacarídeos/química , Biotecnologia , Sistemas CRISPR-Cas , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Genes Fúngicos , Glicoproteínas/biossíntese , Glicoproteínas/química , Glicoproteínas/genética , Glicosilação , Humanos , Manosidases/genética , Manosidases/metabolismo , Manosiltransferases/antagonistas & inibidores , Manosiltransferases/genética , Manosiltransferases/metabolismo , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
9.
DNA Cell Biol ; 39(6): 938-948, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32283037

RESUMO

Diabetic nephropathy (DN) is characterized by excessive accumulation of extracellular matrix leading to early thickening of glomerular and tubular basement membrane. C1q/tumor necrosis factor (TNF)-related protein-9 (CTRP9) was recently identified as an adiponectin paralog of superior prominence. CTRP9 is an anti-inflammatory, antioxidant, vasodilation and atheroprotective adipose cytokine that share a similar metabolic regulatory function as adiponectin. Additionally, CTRP9 inhibits apoptosis of endothelial cells, decreases blood glucose level, and increases insulin sensitivity. However, the renoprotective effects of CTRP9 and the underlying molecular mechanisms in DN have not been explored. This study examined the effects of CTRP9 on DN in diabetic db/db mice through adenovirus-mediated overexpression. From the results, CTRP9 ameliorated renal dysfunction and injury at the structural and functional level in diabetic db/db mice. Additionally, CTRP9 inhibited glomerular and tubular glycogen accumulation, fibrosis, relieved hyperglycemia-mediated oxidative stress, and apoptosis. This is the first study to report on therapeutic effects of CTRP9 on DN, presenting a potentially effective clinical treatment method for DN patients.


Assuntos
Adiponectina/metabolismo , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Glicoproteínas/metabolismo , Rim/patologia , Adiponectina/genética , Animais , Apoptose , Fibrose , Regulação da Expressão Gênica , Glicoproteínas/genética , Sistema de Sinalização das MAP Quinases , Camundongos , Estresse Oxidativo , Receptores de Adiponectina/metabolismo , Fator de Crescimento Transformador beta/metabolismo
10.
J Vet Sci ; 21(2): e22, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32233130

RESUMO

Rabid raccoon dogs (Nyctereutes procyonoides koreensis) have been responsible for animal rabies in South Korea since the 1990s. A recombinant rabies vaccine strain, designated as ERAGS, was constructed for use as a bait vaccine. Therefore, new means of differentiating ERAGS from other rabies virus (RABV) strains will be required in biological manufacturing and diagnostic service centers. In this study, we designed two specific primer sets for differentiation between ERAGS and other RABVs based on mutation in the RABV glycoprotein gene. Polymerase chain reaction analysis of the glycoprotein gene revealed two DNA bands of 383 bp and 583 bp in the ERAGS strain but a single DNA band of 383 bp in the field strains. The detection limits of multiplex reverse transcription polymerase chain reaction (RT-PCR) were 80 and 8 FAID50/reaction for the ERAGS and Evelyn-Rokitnicki-Abelseth strains, respectively. No cross-reactions were detected in the non-RABV reference viruses, including canine distemper virus, parvovirus, canine adenovirus type 1 and 2, and parainfluenza virus. The results of multiplex RT-PCR were 100% consistent with those of the fluorescent antibody test. Therefore, one-step multiplex RT-PCR is likely useful for differentiation between RABVs with and those without mutation at position 333 of the RABV glycoprotein gene.


Assuntos
Glicoproteínas/genética , Reação em Cadeia da Polimerase Multiplex/veterinária , Vírus da Raiva/isolamento & purificação , Raiva/veterinária , Cães Guaxinins , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Animais , Cães , Glicoproteínas/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Mutação , Vírus da Raiva/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
11.
Arch Virol ; 165(5): 1207-1210, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32140837

RESUMO

Bovine ephemeral fever virus (BEFV) is an economically important arbovirus affecting cattle and water buffalo. Currently, isolates can be separated into three phylogenetic groups, differentiated by the place of isolation, namely, East Asia, Australia, and the Middle East. BEFV surface glycoprotein (G) genes from 14 South African field strains collected between 1968 and 1999 were sequenced and compared to 154 published sequences. The BEFV isolates from South Africa were found to be phylogenetically distinct from those from other parts of the world.


Assuntos
Vírus da Febre Efêmera Bovina/classificação , Vírus da Febre Efêmera Bovina/isolamento & purificação , Febre Efêmera/virologia , Variação Genética , Glicoproteínas/genética , Filogenia , Proteínas Virais/genética , Animais , Bovinos , Vírus da Febre Efêmera Bovina/genética , África do Sul
12.
Arch Virol ; 165(5): 1109-1120, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32189084

RESUMO

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease with a mortality rate of up to 50% in humans. To avoid safety concerns associated with the use of live virus in virus neutralization assays and to detect human serum neutralizing antibodies, we prepared lentiviral particles containing the CCHF glycoprotein (lenti-CCHFV-GP). Incorporation of the GP into the lentiviral particle was confirmed by electron microscopy and Western blotting. Lenti-CCHFV-GP was found to be able to infect a wide range of cell lines, including BHK-21, HeLa, HepG2, and AsPC-1 cells. In addition, lenti-CCHFV-GP was successfully used as an alternative to CCHFV for the detection of neutralizing antibodies. Sera collected from CCHF survivors neutralized lenti-CCHFV-GP particles in a dose-dependent manner. Our results suggest that the lenti-CCHFV-GP pseudovirus can be used as a safe tool for neutralization assays in low-containment laboratories.


Assuntos
Técnicas de Visualização da Superfície Celular , Glicoproteínas/imunologia , Vírus da Febre Hemorrágica da Crimeia-Congo/imunologia , Lentivirus/crescimento & desenvolvimento , Testes de Neutralização/métodos , Proteínas Virais/imunologia , Internalização do Vírus , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linhagem Celular , Vetores Genéticos , Glicoproteínas/genética , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Especificidade de Hospedeiro , Humanos , Lentivirus/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Virais/genética
13.
Zhonghua Gan Zang Bing Za Zhi ; 28(2): 135-140, 2020 Feb 20.
Artigo em Chinês | MEDLINE | ID: mdl-32164064

RESUMO

Objective: To investigate the mechanism of occurrence and development of zinc-alpha-2-glycoprotein (AZGP1) in the activated hepatic stellate cells (HSCs) and liver fibrosis. Methods: The activated human hepatic stellate cell line LX2 was induced by the stimulation of transforming growth factor - ß1 to construct carbon tetrachloride liver fibrosis mice model. The situation expression of AZGP1 in liver cells and tissues were observed. Plasmid transfection method was used to detect the activation, proliferation, apoptotic functions and changes in related factors of LX2 cells, respectively, after the overexpression and inhibition of AZGP1expression. Univariate analysis of variance was used for multiple group comparison. Results: The results of immunofluorescence staining showed that AZGP1 protein was decreased and α-smooth muscle actin was increased in the activated LX2 cells, and the two were negatively correlated. AZGP1 gene and protein were significantly under-expressed in activated LX2 cells and liver tissues of mice with carbon tetrachloride liver fibrosis. Collagen I, matrix metalloproteinase-2, and α-smooth muscle actin genes and proteins were significantly down-regulated in LX2 cells after over-expression of AZGP1. Cell fluorescence showed that AZGP1-overexpressing cells were activated and α-smooth muscle actin protein was reduced. In addition, the proliferative activity and G1/S-specific cyclin D1 protein of LX2 cells were significantly reduced after overexpression of AZGP1, while cell cycle experiments showed that the proportion of cells overexpressing AZGP1 was significantly increased in the G0/G1 phase, and the proportion of S phase was significantly reduced. AZGP1 had no significant effect on the apoptosis of LX2 cells. Conclusion: AZGP1 can reverse liver fibrosis by inhibiting the activation and proliferation of hepatic stellate cells, and thereby overexpression of AZGP1 is expected to become a new target for liver fibrosis treatment.


Assuntos
Tetracloreto de Carbono/toxicidade , Proliferação de Células/efeitos dos fármacos , Glicoproteínas/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Actinas , Animais , Glicoproteínas/genética , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática , Metaloproteinase 2 da Matriz , Camundongos , Zinco
14.
Nat Commun ; 11(1): 1368, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170195

RESUMO

MCFD2 and ERGIC-53, which are the products of causative genes of combined factor V and factor VIII deficiency, form a cargo receptor complex responsible for intracellular transport of these coagulation factors in the early secretory pathway. In this study, using an NMR technique, we successfully identified an MCFD2-binding segment from factor VIII composed of a 10 amino acid sequence that enhances its secretion. This prompted us to examine possible effects of attaching this sequence to recombinant glycoproteins on their secretion. We found that the secretion level of recombinant erythropoietin was significantly increased simply by tagging it with the passport sequence. Our findings not only provide molecular basis for the intracellular trafficking of coagulation factors and their genetic deficiency but also offer a potentially useful tool for increasing the production yields of recombinant glycoproteins of biopharmaceutical interest.


Assuntos
Proteínas de Transporte/metabolismo , Glicoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Polissacarídeos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Retículo Endoplasmático/fisiologia , Eritropoetina/metabolismo , Fator V , Fator VIII/metabolismo , Glicoproteínas/genética , Complexo de Golgi/fisiologia , Humanos , Lectinas de Ligação a Manose/metabolismo , Transporte Proteico , Via Secretória
15.
Proc Natl Acad Sci U S A ; 117(14): 7764-7775, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32205440

RESUMO

The cell surface proteome, the surfaceome, is the interface for engaging the extracellular space in normal and cancer cells. Here we apply quantitative proteomics of N-linked glycoproteins to reveal how a collection of some 700 surface proteins is dramatically remodeled in an isogenic breast epithelial cell line stably expressing any of six of the most prominent proliferative oncogenes, including the receptor tyrosine kinases, EGFR and HER2, and downstream signaling partners such as KRAS, BRAF, MEK, and AKT. We find that each oncogene has somewhat different surfaceomes, but the functions of these proteins are harmonized by common biological themes including up-regulation of nutrient transporters, down-regulation of adhesion molecules and tumor suppressing phosphatases, and alteration in immune modulators. Addition of a potent MEK inhibitor that blocks MAPK signaling brings each oncogene-induced surfaceome back to a common state reflecting the strong dependence of the oncogene on the MAPK pathway to propagate signaling. Cell surface protein capture is mediated by covalent tagging of surface glycans, yet current methods do not afford sequencing of intact glycopeptides. Thus, we complement the surfaceome data with whole cell glycoproteomics enabled by a recently developed technique called activated ion electron transfer dissociation (AI-ETD). We found massive oncogene-induced changes to the glycoproteome and differential increases in complex hybrid glycans, especially for KRAS and HER2 oncogenes. Overall, these studies provide a broad systems-level view of how specific driver oncogenes remodel the surfaceome and the glycoproteome in a cell autologous fashion, and suggest possible surface targets, and combinations thereof, for drug and biomarker discovery.


Assuntos
Neoplasias da Mama/genética , Glicoproteínas de Membrana/genética , Proteoma/genética , Proteômica , Biomarcadores Tumorais/genética , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Receptores ErbB/genética , Feminino , Glicoproteínas/genética , Humanos , MAP Quinase Quinase Quinases/genética , Proteína Oncogênica v-akt/genética , Oncogenes/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptor ErbB-2/genética , Transdução de Sinais/genética
16.
Hum Cell ; 33(3): 512-520, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32130677

RESUMO

Pre-eclampsia (PE) is a disorder of pregnancy characterized by proteinuria and high blood pressure, affecting 2-8% of pregnancies worldwide. Previous studies have shown that PE is closely associated with trophoblast cell dysfunction. Here, we investigated the role of tissue factor pathway inhibitor-2 (TFPI-2) in regulating the biological processes of trophoblast cells. The TFPI-2 levels in plasma samples and placental tissues were tested by ELISA, immunohistochemistry, qRT-PCR, and western blot. HTR8/Svneo cell line was used to simulate the primary trophoblast cells and H/R culture was applied to mimic the oxidative stress state of PE. MTT assay, Annexin V/propidium iodide (PI) apoptosis assay, and transwell assay were used to determine the cell proliferation, apoptosis, and invasion. The expression levels of matrix metalloproteinases (MMPs) were evaluated by western blot. The expression of TFPI-2 was remarkably up-regulated in both the serum and placenta of PE patients. Hypoxia/reoxygenation increased the expression of TFPI-2 in HTR-8/SVneo cell line. TFPI-2 promoted that cell proliferation and inhibited the cell apoptosis of HTR8/SVneo cells in H/R condition. In addition, downregulation of TFPI-2 increased the cell invasion and the expression of MMP2 and MMP9. This study reveals that TFPI-2 plays a crucial role in monitoring the biological function of trophoblast cells, which might provide theoretical basis and therapeutic targets for the treatment of PE.


Assuntos
Proliferação de Células/genética , Glicoproteínas/fisiologia , Pré-Eclâmpsia/patologia , Trofoblastos/patologia , Células Cultivadas , Feminino , Expressão Gênica/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Terapia de Alvo Molecular , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/terapia , Gravidez , Regulação para Cima/genética
17.
Cancer Res ; 80(9): 1861-1874, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32132110

RESUMO

Skeletal muscle wasting is a devastating consequence of cancer that contributes to increased complications and poor survival, but is not well understood at the molecular level. Herein, we investigated the role of Myocilin (Myoc), a skeletal muscle hypertrophy-promoting protein that we showed is downregulated in multiple mouse models of cancer cachexia. Loss of Myoc alone was sufficient to induce phenotypes identified in mouse models of cancer cachexia, including muscle fiber atrophy, sarcolemmal fragility, and impaired muscle regeneration. By 18 months of age, mice deficient in Myoc showed significant skeletal muscle remodeling, characterized by increased fat and collagen deposition compared with wild-type mice, thus also supporting Myoc as a regulator of muscle quality. In cancer cachexia models, maintaining skeletal muscle expression of Myoc significantly attenuated muscle loss, while mice lacking Myoc showed enhanced muscle wasting. Furthermore, we identified the myocyte enhancer factor 2 C (MEF2C) transcription factor as a key upstream activator of Myoc whose gain of function significantly deterred cancer-induced muscle wasting and dysfunction in a preclinical model of pancreatic ductal adenocarcinoma (PDAC). Finally, compared with noncancer control patients, MYOC was significantly reduced in skeletal muscle of patients with PDAC defined as cachectic and correlated with MEF2c. These data therefore identify disruptions in MEF2c-dependent transcription of Myoc as a novel mechanism of cancer-associated muscle wasting that is similarly disrupted in muscle of patients with cachectic cancer. SIGNIFICANCE: This work identifies a novel transcriptional mechanism that mediates skeletal muscle wasting in murine models of cancer cachexia that is disrupted in skeletal muscle of patients with cancer exhibiting cachexia.


Assuntos
Caquexia/complicações , Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Músculo Esquelético/metabolismo , Doenças Musculares/metabolismo , Síndrome de Emaciação/etiologia , Animais , Composição Corporal , Caquexia/metabolismo , Carcinoma Ductal Pancreático/complicações , Carcinoma Ductal Pancreático/metabolismo , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Diafragma/fisiologia , Modelos Animais de Doenças , Regulação para Baixo , Proteínas do Olho/genética , Feminino , Glicoproteínas/deficiência , Glicoproteínas/genética , Xenoenxertos , Humanos , Fatores de Transcrição MEF2/metabolismo , Masculino , Camundongos , Músculo Esquelético/patologia , Atrofia Muscular , Doenças Musculares/etiologia , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/complicações , Neoplasias Pancreáticas/metabolismo , RNA Mensageiro/metabolismo , Regeneração , Corrida , Sarcolema , Síndrome de Emaciação/metabolismo , Síndrome de Emaciação/prevenção & controle
18.
Ann Otol Rhinol Laryngol ; 129(7): 653-656, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32028786

RESUMO

OBJECTIVES: Congenital midline cervical cleft (CMCC) is a rare congenital anterior neck anatomical anomaly. We present the case of two related patients (grandchild and maternal grandmother) who were both born with a congenital midline cervical cleft along with genetic analysis. METHODS: Clinical examination of both patients and surgical excision of the grandchild was performed. Genetic analysis with exome sequencing (ES) was conducted for both patients. RESULTS: Genetic analysis with exome sequencing (ES) revealed apparently novel single nucleotide variants in 66 genes present in both proband and grandmother. Five of these variants are predicted to cause frameshifting in the coding region of the respective genes and truncated proteins (OVGP1, TYW1B, ZAN, SSPO, FOLR3). Two of these genes (TYW1B and SSPO) have homozygous indel mutations in both patients. CONCLUSIONS: To our knowledge, this is the first case of two related patients with a congenital midline cervical cleft. The results of our genetic analysis reveal potential relevance to CMCC development.


Assuntos
Região Branquial/anormalidades , Proteínas de Transporte/genética , Moléculas de Adesão Celular Neuronais/genética , Anormalidades Craniofaciais/genética , Glicoproteínas/genética , Pescoço/anormalidades , Doenças Faríngeas/genética , Região Branquial/cirurgia , Anormalidades Craniofaciais/cirurgia , Feminino , Mutação da Fase de Leitura , Avós , Humanos , Mutação INDEL , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Oxigenases de Função Mista/genética , Pescoço/cirurgia , Doenças Faríngeas/cirurgia , Polimorfismo de Nucleotídeo Único , Sequenciamento Completo do Exoma
19.
Am J Physiol Renal Physiol ; 318(3): F809-F816, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32068457

RESUMO

Pediatric sepsis is a leading cause of morbidity and mortality in children. One of the most common and devastating morbidities is sepsis-related acute kidney injury (AKI). AKI was traditionally thought to be related to low perfusion and acute tubular necrosis. However, little acute tubular necrosis can be found following septic AKI, and little is known about the mechanism of septic AKI. Olfactomedin-4 (OLFM4) is a secreted glycoprotein that marks a subset of neutrophils. Increased expression of OLFM4 in the blood is associated with worse outcomes in sepsis. Here, we investigated a pediatric model of murine sepsis using murine pups to investigate the mechanisms of OLFM4 in sepsis. When sepsis was induced in murine pups, survival was significantly increased in OLFM4-null pups. Immunohistochemistry at 24 h after the induction of sepsis demonstrated increased expression of OLFM4 in the kidney, which was localized to the loop of Henle. Renal cell apoptosis and plasma creatinine were significantly increased in wild-type versus OLFM4-null pups. Finally, bone marrow transplant suggested that increased OLFM4 in the kidney reflects local production rather than filtered from the plasma. These results demonstrate renal expression of OLFM4 for the first time and suggest that a kidney-specific mechanism may contribute to survival differences in OLFM4-null animals.


Assuntos
Lesão Renal Aguda/metabolismo , Glicoproteínas/metabolismo , Sepse/imunologia , Animais , Transplante de Medula Óssea , Regulação da Expressão Gênica/imunologia , Predisposição Genética para Doença , Glicoproteínas/genética , Masculino , Camundongos , Camundongos Knockout , Neutrófilos/metabolismo , Peritonite , Sepse/etiologia , Sepse/genética
20.
mBio ; 11(1)2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32098811

RESUMO

Lassa virus (LASV) is endemic in Western Africa and is estimated to infect hundreds of thousands of individuals annually. A considerable number of these infections result in Lassa fever (LF), which is associated with significant morbidity and a case-fatality rate as high as 69% among hospitalized confirmed patients. U.S. Food and Drug Administration-approved LF vaccines are not available. Current antiviral treatment is limited to off-label use of a nucleoside analogue, ribavirin, that is only partially effective and associated with significant side effects. We generated and characterized a recombinant LASV expressing a codon-deoptimized (CD) glycoprotein precursor gene (GPC), rLASV-GPC/CD. Comparison of growth kinetics and peak titers showed that rLASV-GPC/CD is slightly attenuated in cell culture compared to wild-type (WT) recombinant LASV (rLASV-WT). However, rLASV-GPC/CD is highly attenuated in strain 13 and Hartley guinea pigs, as reflected by the absence of detectable clinical signs in animals inoculated with rLASV-GPC/CD. Importantly, a single subcutaneous dose of rLASV-GPC/CD provides complete protection against an otherwise lethal exposure to LASV. Our results demonstrate the feasibility of implementing a CD approach for developing a safe and effective LASV live-attenuated vaccine candidate. Moreover, rLASV-GPC/CD might provide investigators with a tool to safely study LASV outside maximum (biosafety level 4) containment, which could accelerate the elucidation of basic aspects of the molecular and cell biology of LASV and the development of novel LASV medical countermeasures.IMPORTANCE Lassa virus (LASV) infects several hundred thousand people in Western Africa, resulting in many lethal Lassa fever (LF) cases. Licensed LF vaccines are not available, and anti-LF therapy is limited to off-label use of the nucleoside analog ribavirin with uncertain efficacy. We describe the generation of a novel live-attenuated LASV vaccine candidate. This vaccine candidate is based on mutating wild-type (WT) LASV in a key region of the viral genome, the glycoprotein precursor (GPC) gene. These mutations do not change the encoded GPC but interfere with its production in host cells. This mutated LASV (rLASV-GPC/CD) behaves like WT LASV (rLASV-WT) in cell culture, but in contrast to rLASV-WT, does not cause disease in inoculated guinea pigs. Guinea pigs immunized with rLASV-GPC/CD were protected against an otherwise lethal exposure to WT LASV. Our results support the testing of this candidate vaccine in nonhuman primate models ofLF.


Assuntos
Febre Lassa/prevenção & controle , Vírus Lassa/genética , Vírus Lassa/imunologia , Vacinas Atenuadas/imunologia , Vacinas Virais/imunologia , Células A549 , África Ocidental , Sequência de Aminoácidos , Animais , Arenaviridae , Arenavirus , Bunyaviridae , Chlorocebus aethiops , Códon , Modelos Animais de Doenças , Feminino , Genes Virais/genética , Genoma Viral , Glicoproteínas/genética , Cobaias , Humanos , Febre Lassa/imunologia , Febre Lassa/virologia , Masculino , Ribavirina , Vacinas Atenuadas/genética , Células Vero
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