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1.
Gene ; 766: 145157, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-32949697

RESUMO

Glycolytic potential (GP) calculated based on glucose, glycogen, glucose-6-phosphate, and lactate contents is a critical factor for multiple meat quality characteristics. However, the genetic basis of glycolytic metabolism is still unclear. In this study, we constructed six RNA-Seq libraries using longissimus dorsi (LD) muscles from pigs divergent for GP phenotypic values and generated the whole genome-wide gene expression profiles. Furthermore, we identified 25,880 known and 220 novel genes from these skeletal muscle libraries, and 222 differentially expressed genes (DEGs) between the higher and lower GP groups. Notably, we found that the Lactate dehydrogenase B (LDHB) and Fructose-2, 6-biphosphatase 3 (PFKFB3) expression levels were higher in the higher GP group than the lower GP group, and positively correlated with GP and lactic acid (LA), and reversely correlated with pH value at 45 min postmortem (pH45min). Besides, LDHB and PFKFB3 expression were positively correlated with drip loss measured at 48 h postmortem (DL48h) and drip loss measured at 24 h postmortem (DL24h). Collectively, we identified a serial of DEGs as the potential key candidate genes affecting GP and found that LDHB and PFKFB3 are closely related to GP and GP-related traits. Our results lay a solid basis for in-depth studies of the regulatory mechanisms on GP and GP-related traits in pigs.


Assuntos
Glicólise/genética , Músculo Esquelético/metabolismo , Suínos/genética , Transcriptoma/genética , Animais , Perfilação da Expressão Gênica/métodos , Glucose/genética , Glicogênio/genética , Isoenzimas/genética , L-Lactato Desidrogenase/genética , Ácido Láctico/metabolismo , Carne , Fenótipo , Fosfofrutoquinase-2/genética , Suínos/metabolismo
2.
Mol Cell ; 80(2): 279-295.e8, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33065020

RESUMO

The PTEN tumor suppressor controls cell death and survival by regulating functions of various molecular targets. While the role of PTEN lipid-phosphatase activity on PtdIns(3,4,5)P3 and inhibition of PI3K pathway is well characterized, the biological relevance of PTEN protein-phosphatase activity remains undefined. Here, using knockin (KI) mice harboring cancer-associated and functionally relevant missense mutations, we show that although loss of PTEN lipid-phosphatase function cooperates with oncogenic PI3K to promote rapid mammary tumorigenesis, the additional loss of PTEN protein-phosphatase activity triggered an extensive cell death response evident in early and advanced mammary tumors. Omics and drug-targeting studies revealed that PI3Ks act to reduce glucocorticoid receptor (GR) levels, which are rescued by loss of PTEN protein-phosphatase activity to restrain cell survival. Thus, we find that the dual regulation of GR by PI3K and PTEN functions as a rheostat that can be exploited for the treatment of PTEN loss-driven cancers.


Assuntos
Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , PTEN Fosfo-Hidrolase/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Carcinogênese , Morte Celular , Linhagem Celular Tumoral , Proliferação de Células , Dexametasona/farmacologia , Feminino , Humanos , Isoenzimas/metabolismo , Camundongos , Modelos Biológicos , Mutação/genética , Organoides/patologia , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Estabilidade Proteica , Proteoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo
3.
PLoS Pathog ; 16(9): e1008842, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898178

RESUMO

Signaling through retinoic acid inducible gene I (RIG-I) like receptors (RLRs) is tightly regulated, with activation occurring upon sensing of viral nucleic acids, and suppression mediated by negative regulators. Under homeostatic conditions aberrant activation of melanoma differentiation-associated protein-5 (MDA5) is prevented through editing of endogenous dsRNA by RNA editing enzyme Adenosine Deaminase Acting on RNA (ADAR1). In addition, ADAR1 is postulated to play pro-viral and antiviral roles during viral infections that are dependent or independent of RNA editing activity. Here, we investigated the importance of ADAR1 isoforms in modulating influenza A virus (IAV) replication and revealed the opposing roles for ADAR1 isoforms, with the nuclear p110 isoform restricting versus the cytoplasmic p150 isoform promoting IAV replication. Importantly, we demonstrate that p150 is critical for preventing sustained RIG-I signaling, as p150 deficient cells showed increased IFN-ß expression and apoptosis during IAV infection, independent of RNA editing activity. Taken together, the p150 isoform of ADAR1 is important for preventing sustained RIG-I induced IFN-ß expression and apoptosis during viral infection.


Assuntos
Adenosina Desaminase/metabolismo , Apoptose , Proteína DEAD-box 58/metabolismo , Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Replicação Viral , Células A549 , Adenosina Desaminase/genética , Proteína DEAD-box 58/genética , Células HEK293 , Humanos , Influenza Humana/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Ligação a RNA/genética
4.
Mol Cell ; 80(1): 87-101.e5, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32931746

RESUMO

Studies in three mouse models of breast cancer identified profound discrepancies between cell-autonomous and systemic Akt1- or Akt2-inducible deletion on breast cancer tumorigenesis and metastasis. Although systemic Akt1 deletion inhibits metastasis, cell-autonomous Akt1 deletion does not. Single-cell mRNA sequencing revealed that systemic Akt1 deletion maintains the pro-metastatic cluster within primary tumors but ablates pro-metastatic neutrophils. Systemic Akt1 deletion inhibits metastasis by impairing survival and mobilization of tumor-associated neutrophils. Importantly, either systemic or neutrophil-specific Akt1 deletion is sufficient to inhibit metastasis of Akt-proficient tumors. Thus, Akt1-specific inhibition could be therapeutic for breast cancer metastasis regardless of primary tumor origin. Systemic Akt2 deletion does not inhibit and exacerbates mammary tumorigenesis and metastasis, but cell-autonomous Akt2 deletion prevents breast cancer tumorigenesis by ErbB2. Elevated circulating insulin level induced by Akt2 systemic deletion hyperactivates tumor Akt, exacerbating ErbB2-mediated tumorigenesis, curbed by pharmacological reduction of the elevated insulin.


Assuntos
Neoplasias Mamárias Animais/enzimologia , Neoplasias Mamárias Animais/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Carcinogênese/patologia , Feminino , Deleção de Genes , Humanos , Insulina/metabolismo , Isoenzimas/metabolismo , Metástase Neoplásica , Neutrófilos/metabolismo , Receptor ErbB-2/metabolismo
5.
Eur J Pharmacol ; 886: 173454, 2020 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-32763298

RESUMO

Antimalaria drugs such as chloroquine (CQ) and hydroxychloroquine (HCQ) have been administered to several inflammatory diseases including rheumatoid arthritis and systemic lupus erythematosus, and infectious diseases such as acquired immune deficiency syndrome and influenza. Recently, several patients infected with novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were given HCQ, and showed a discrepant response. HCQ inhibits SARS-CoV-2 cell entry, and inflammatory cascade by interfering with lysosomal and endosomal activities, and autophagy, impeding virus-membrane fusion, and inhibiting cytokine production resulted from inflammatory pathways activation. Despite ongoing administration of HCQ in a wide spectrum of disorders, there are some reports about several side effects, especially retinopathy in some patients treated with HCQ. Cytochrome P450 (CYP450) and its isoforms are the main metabolizers of HCQ and CQ. Pharmacokinetic properties of CYP enzymes are influenced by CYP polymorphism, non-coding RNAs, and epigenetic mechanisms such as DNA methylation, and histone acetylation. Accumulating evidence about side effects of HCQ in some patients raise the possibility that different response of patients to HCQ might be due to difference in their genome. Therefore, CYP450 genotyping especially for CYP2D6 might be helpful to refine HCQ dosage. Also, regular control of retina should be considered for patients under HCQ treatment. The major focus of the present review is to discuss about the pharmacokinetic and pharmacodynamic properties of CQ and HCQ that may be influenced by epigenetic mechanisms, and consequently cause several side effects especially retinopathy during SARS-CoV-2 therapy.


Assuntos
Betacoronavirus/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/genética , Epigênese Genética/efeitos dos fármacos , Hidroxicloroquina/efeitos adversos , Hidroxicloroquina/farmacologia , Doenças Retinianas/induzido quimicamente , Humanos , Isoenzimas/genética , Doenças Retinianas/genética
6.
Pestic Biochem Physiol ; 169: 104653, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32828371

RESUMO

Descurainia sophia L. is one of the most notorious broadleaf weeds in winter wheat fields of China. In this study, 95 out of 163 (58.3%) D. sophia populations which were collected from provinces of Hebei, Shandong, Henan, Shanxi, Shaanxi and Jiangsu, have evolved resistance to tribenuron-methyl. The als1 and als2 were cloned in all test D. sophia populations, while als3 and als4 were identified only in some of the populations. Resistant mutations of Pro-197-Ser/Thr/Leu/His/Ala/Arg, Asp-376-Glu and Trp-574-Leu were identified in tribenuron-methyl-resistant (TR) D. sophia plants, while the Pro-197-Arg was first identified in D. sophia in this study. These resistant mutations displayed no preference between ALS1 and ALS2. However, Pro-197-Ser/Thr and Trp-574-Leu were identified in all ALS isozymes, while the other mutations were not. In addition, some resistant mutations displayed regional differences, the frequency of Pro-197-Ser in Shandong and Trp-574-Leu in Shanxi province is much higher than that in other provinces.


Assuntos
Acetolactato Sintase/genética , Brassicaceae/efeitos dos fármacos , Herbicidas/farmacologia , Esclerose Amiotrófica Lateral , Sulfonatos de Arila , China , Resistência a Herbicidas , Isoenzimas/genética , Mutação
7.
Life Sci ; 259: 118168, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32739469

RESUMO

AIMS: Circular RNA PRKCI (circPRKCI) and poly ADP-ribose polymerase 9 (PARP9) are related to the development of cancers. In this study, we aimed to explore the regulatory mechanisms between circPRKCI and PARP9 in EC progression and radioresistance. MATERIALS AND METHODS: The levels of circPRKCI, PARP9 mRNA, and miR-186-5p were assessed by quantitative real time polymerase chain reaction (qRT-PCR). Western blot analysis was employed to examine the levels of several proteins. The viability, colony formation, cell cycle progression, and apoptosis of EC cells were determined with CCK-8, colony formation, or flow cytometry assays. The relationship between circPRKCI or PARP9 and miR-186-5p was verified with the dual-luciferase reporter and RIP assays. KEY FINDINGS: We observed that circPRKCI and PARP9 were upregulated while miR-186-5p was downregulated in EC tissues and cells. Furthermore, circPRKCI knockdown decreased tumor growth in vivo and constrained cell viability, colony formation, cell cycle progression, elevated cell radiosensitivity in EC cells in vitro. Importantly, circPRKCI modulated PARP9 expression through sponging miR-186-5p. Besides, PARP9 overexpression overturned circPRKCI silencing-mediated effects on the viability, colony formation, cell cycle progression, and radiosensitivity of EC cells. SIGNIFICANCE: CircPRKCI regulated cell malignancy and radioresistance through modulating the miR-186-5p /PARP9 axis in EC, which provided a might target for EC treatment.


Assuntos
Neoplasias Esofágicas/genética , Neoplasias Esofágicas/terapia , Isoenzimas/genética , MicroRNAs/genética , Proteínas de Neoplasias/genética , Poli(ADP-Ribose) Polimerases/genética , Proteína Quinase C/genética , RNA Circular/genética , Animais , Apoptose , Ciclo Celular , Linhagem Celular Tumoral , Progressão da Doença , Neoplasias Esofágicas/radioterapia , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Células-Tronco Neoplásicas , Interferência de RNA , Radiossensibilizantes/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
8.
PLoS One ; 15(8): e0236679, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32760087

RESUMO

The Drosophila shaggy gene (sgg, GSK-3) encodes multiple protein isoforms with serine/threonine kinase activity and is a key player in diverse developmental signalling pathways. Currently it is unclear whether different Sgg proteoforms are similarly involved in signalling or if different proteoforms have distinct functions. We used CRISPR/Cas9 genome engineering to tag eight different Sgg proteoform classes and determined their localization during embryonic development. We performed proteomic analysis of the two major proteoform classes and generated mutant lines for both of these for transcriptomic and phenotypic analysis. We uncovered distinct tissue-specific localization patterns for all of the tagged proteoforms we examined, most of which have not previously been characterised directly at the protein level, including one proteoform initiating with a non-standard codon. Collectively, this suggests complex developmentally regulated splicing of the sgg primary transcript. Further, affinity purification followed by mass spectrometric analyses indicate a different repertoire of interacting proteins for the two major proteoforms we examined, one with ubiquitous expression (Sgg-PB) and one with nervous system specific expression (Sgg-PA). Specific mutation of these proteoforms shows that Sgg-PB performs the well characterised maternal and zygotic segmentations functions of the sgg locus, while Sgg-PA mutants show adult lifespan and locomotor defects consistent with its nervous system localisation. Our findings provide new insights into the role of GSK-3 proteoforms and intriguing links with the GSK-3α and GSK-3ß proteins encoded by independent vertebrate genes. Our analysis suggests that different proteoforms generated by alternative splicing are likely to perform distinct functions.


Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/metabolismo , Quinase 3 da Glicogênio Sintase/fisiologia , Animais , Proteínas de Drosophila/genética , Quinase 3 da Glicogênio Sintase/genética , Isoenzimas/fisiologia , Proteômica/métodos
9.
Anticancer Res ; 40(7): 3857-3863, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32620625

RESUMO

BACKGROUND: The aim of this study was to define the alterations in the activity of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) in normal and cancerous lung cells. MATERIALS AND METHODS: Lung tissues were taken from 36 patients during surgical resection of cancer. The activities of tested enzymes were measured by spectrofluorometric method (ADH I, ADH II, total ALDH) and photometric method (ADH III, ADH IV, total ADH). RESULTS: The activities of class II and III ADH were significantly lower in lung cancer cells compared to histologically normal lung tissue. CONCLUSION: Reduced activity of isoenzyme class II ADH may affect disorders in retinoic acid biosynthesis, leading to its deficit. Lower ADH III activity may result in depletion of glutathione, and in initiation of oxidative stress, leading to cancer progression. These data suggest that alterations in ADH isoenzyme activities can contribute to carcinogenesis in human lungs.


Assuntos
Álcool Desidrogenase/metabolismo , Aldeído Desidrogenase/metabolismo , Neoplasias Pulmonares/enzimologia , Adenocarcinoma de Pulmão/enzimologia , Carcinoma de Células Escamosas/enzimologia , Feminino , Humanos , Isoenzimas/metabolismo , Masculino , Fatores Sexuais
10.
PLoS One ; 15(7): e0235747, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32658903

RESUMO

Despite development of markers for identification of cancer stem cells, the mechanism underlying the survival and division of cancer stem cells in breast cancer remains unclear. Here we report that PKCλ expression was enriched in basal-like breast cancer, among breast cancer subtypes, and was correlated with ALDH1A3 expression (p = 0.016, χ2-test). Late stage breast cancer patients expressing PKCλhigh and ALDH1A3high had poorer disease-specific survival than those expressing PKCλlow and ALDH1A3low (p = 0.018, log rank test for Kaplan-Meier survival curves: hazard ratio 2.58, 95% CI 1.24-5.37, p = 0.011, multivariate Cox regression analysis). Functional inhibition of PKCλ through siRNA-mediated knockdown or CRISPR-Cas9-mediated knockout in ALDH1high MDA-MB 157 and MDA-MB 468 basal-like breast cancer cells led to increases in the numbers of trypan blue-positive and active-caspase 3-positive cells, as well as suppression of tumor-sphere formation and cell migration. Furthermore, the amount of CASP3 and PARP mRNA and the level of cleaved caspase-3 protein were enhanced in PKCλ-deficient ALDH1high cells. An Apoptosis inhibitor (z-VAD-FMK) suppressed the enhancement of cell death as well as the levels of cleaved caspase-3 protein in PKCλ deficient ALDH1high cells. It also altered the asymmetric/symmetric distribution ratio of ALDH1A3 protein. In addition, PKCλ knockdown led to increases in cellular ROS levels in ALDH1high cells. These results suggest that PKCλ is essential for cancer cell survival and migration, tumorigenesis, the asymmetric distribution of ALDH1A3 protein among cancer cells, and the maintenance of low ROS levels in ALDH1-positive breast cancer stem cells. This makes it a key contributor to the poorer prognosis seen in late-stage breast cancer patients.


Assuntos
Aldeído Oxirredutases/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/mortalidade , Regulação Neoplásica da Expressão Gênica , Isoenzimas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteína Quinase C/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Movimento Celular , Proliferação de Células , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/metabolismo , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas
11.
Int J Occup Environ Med ; 11(3): 140-147, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32683426

RESUMO

BACKGROUND: The major portion of lead in the body resides in skeletal system. The bone turnover affects the release of lead into the circulation from bones. The bone turnover biomarkers (BTM) in lead-battery workers with long-term exposure to lead have not been explored yet. OBJECTIVE: To evaluate the BTM (formation and resorption) in lead-battery workers with long-term exposure to lead in lead-battery manufacturing plant. METHODS: 176 male lead-exposed workers and 80 matched comparison group were studied. All participants were examined for blood lead levels (BLLs), bone formation biomarkers- serum osteocalcin (OC), alkaline phosphatase (ALP), bone-specific alkaline phosphatase (BALP)-and bone resorption biomarkers-serum pyridinoline (PYD), deoxypyridinoline (DPYD), tartarate-resistant acid phosphatase-5b (TRACP-5b), and urinary hydroxyproline (UHYP). RESULTS: We found a significantly higher bone formation biomarkers such as BALP (p=0.007) and bone resorption biomarkers, eg, PYD (p=0.048), TRCAP-5b (p=0.001), and UHYP (p=0.001) in lead-exposed workers. A significant (p=0.041) negative correlation (ρ ­0.128) was noted between BLLs and OC. A significant positive correlation was noted between BLLs and TRACP-5b (ρ 0.176, p=0.005) and UHYP (ρ 0.258, p=0.004). Serum OC (p=0.040) and UHYP (p=0.015) levels changed significantly with BLL level. Bone resorption biomarkers levels- PYD, TRACP-5b, and BALP-were higher among those with higher BLLs levels. The duration of exposure was significantly associated with BALP (p=0.037), DPYD (p=0.016), TRACP-5b (p=0.001), and UHYP (p=0.002) levels. CONCLUSION: Long-term lead exposure affects the bone turnover.


Assuntos
Biomarcadores/sangue , Remodelação Óssea/fisiologia , Fontes de Energia Elétrica , Chumbo/toxicidade , Exposição Ocupacional/análise , Fosfatase Ácida/sangue , Fosfatase Ácida/metabolismo , Adulto , Fosfatase Alcalina/sangue , Biomarcadores/análise , Reabsorção Óssea/sangue , Estudos de Casos e Controles , Estudos Transversais , Fontes de Energia Elétrica/efeitos adversos , Humanos , Isoenzimas/sangue , Isoenzimas/metabolismo , Chumbo/química , Intoxicação por Chumbo/sangue , Intoxicação por Chumbo/diagnóstico , Masculino , Instalações Industriais e de Manufatura , Pessoa de Meia-Idade , Exposição Ocupacional/efeitos adversos , Exposição Ocupacional/estatística & dados numéricos , Osteocalcina/sangue , Local de Trabalho
12.
Int J Mol Sci ; 21(13)2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32629995

RESUMO

Peptidylarginine deiminases (PADs) are a family of calcium-regulated enzymes that are phylogenetically conserved and cause post-translational deimination/citrullination, contributing to protein moonlighting in health and disease. PADs are implicated in a range of inflammatory and autoimmune conditions, in the regulation of extracellular vesicle (EV) release, and their roles in infection and immunomodulation are known to some extent, including in viral infections. In the current study we describe putative roles for PADs in COVID-19, based on in silico analysis of BioProject transcriptome data (PRJNA615032 BioProject), including lung biopsies from healthy volunteers and SARS-CoV-2-infected patients, as well as SARS-CoV-2-infected, and mock human bronchial epithelial NHBE and adenocarcinoma alveolar basal epithelial A549 cell lines. In addition, BioProject Data PRJNA631753, analysing patients tissue biopsy data (n = 5), was utilised. We report a high individual variation observed for all PADI isozymes in the patients' tissue biopsies, including lung, in response to SARS-CoV-2 infection, while PADI2 and PADI4 mRNA showed most variability in lung tissue specifically. The other tissues assessed were heart, kidney, marrow, bowel, jejunum, skin and fat, which all varied with respect to mRNA levels for the different PADI isozymes. In vitro lung epithelial and adenocarcinoma alveolar cell models revealed that PADI1, PADI2 and PADI4 mRNA levels were elevated, but PADI3 and PADI6 mRNA levels were reduced in SARS-CoV-2-infected NHBE cells. In A549 cells, PADI2 mRNA was elevated, PADI3 and PADI6 mRNA was downregulated, and no effect was observed on the PADI4 or PADI6 mRNA levels in infected cells, compared with control mock cells. Our findings indicate a link between PADI expression changes, including modulation of PADI2 and PADI4, particularly in lung tissue, in response to SARS-CoV-2 infection. PADI isozyme 1-6 expression in other organ biopsies also reveals putative links to COVID-19 symptoms, including vascular, cardiac and cutaneous responses, kidney injury and stroke. KEGG and GO pathway analysis furthermore identified links between PADs and inflammatory pathways, in particular between PAD4 and viral infections, as well as identifying links for PADs with a range of comorbidities. The analysis presented here highlights roles for PADs in-host responses to SARS-CoV-2, and their potential as therapeutic targets in COVID-19.


Assuntos
Infecções por Coronavirus/patologia , Pneumonia Viral/patologia , Desiminases de Arginina em Proteínas/metabolismo , Betacoronavirus/isolamento & purificação , Estudos de Casos e Controles , Linhagem Celular , Infecções por Coronavirus/metabolismo , Citocinas/metabolismo , Bases de Dados Factuais , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Vesículas Extracelulares/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Pulmão/enzimologia , Pulmão/patologia , Pulmão/virologia , Pandemias , Pneumonia Viral/metabolismo , Mapas de Interação de Proteínas , Desiminases de Arginina em Proteínas/genética , RNA Mensageiro/metabolismo
13.
Environ Sci Pollut Res Int ; 27(32): 40652-40663, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32671708

RESUMO

The main objective of this study was to characterize the Giardia duodenalis isolates from Iranian patients in Fars Province, south of Iran by biochemical and molecular methods. Fifteen mass cultivated of G. duodenalis isolates in modified TYI-S-33 medium were analyzed using isoenzyme electrophoresis and PCR genotyping. Polyacrylamide gel electrophoresis (PAGE) of five different enzyme systems was used to characterize isolates: (i) glucose-6-phosphate dehydrogenase, (ii) glucose phosphate isomerase, (iii) malate dehydrogenase, (iv) malic enzyme, and (v) phosphoglucomutase. As well, a fragment of the SSU-rDNA (292 bp) gene was amplified by PCR using the primers RH11 and RH4. The sequencing of the PCR products and phylogenetic tree were performed. The isoenzyme electrophoretic profiles divided fifteen G. duodenalis isolates into four zymodemes. G6PD, GPI, MDH, ME, and PGM enzyme systems showed 1, 2, 2, 3, and 3 enzyme pattern, respectively. G6PD isoenzyme pattern had the most homogeneity, while isoenzyme patterns of ME and PGM had the most heterogeneity in our study. Genotyping results indicated that the zymodemes 1-4 were categorized in assemblage A based on the SSU-rDNA gene. Phylogenetic analysis showed that all four zymodemes were distributed within the cluster of assemblage A. Our results indicated that both isoenzyme and DNA analyses were useful to characterize the isolates of Giardia and distinguishing various zymodemes and assemblages. It could be suggested that the genetic diversity among isoenzymes profiles of G. duodenalis may explain the variable clinical manifestations, pathogenicity, host response, drug susceptibility, and treatment efficacy of human giardiasis.


Assuntos
Giardia lamblia , Giardíase , Fezes , Genótipo , Giardia lamblia/genética , Humanos , Irã (Geográfico) , Isoenzimas/genética , Filogenia
14.
Eur J Endocrinol ; 183(4): 369-379, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32621582

RESUMO

Background: The '3PAs' syndrome, associating pituitary adenoma (PA) and pheochromocytoma/paraganglioma (PPGL), is sometimes associated with mutations in PPGL-predisposing genes, such as SDHx or MAX. In '3PAs' syndrome, PAs can occur before PPGL, suggesting a new gateway into SDHx/MAX-related diseases. Objective: To determine the SDHx/MAX mutation prevalence in patients with isolated PAs and characterize PAs of patients with SDHx/MAX mutations. Design: Genes involved in PAs (AIP/MEN1/CDKN1B) or PPGLs (SDHx/MAX) were sequenced in patients with isolated PAs. We then conducted a review of cases of PA in the setting of '3PAs' syndrome. Results: A total of 263 patients were recruited. Seven (likely) pathogenic variants were found in AIP, two in MEN1, two in SDHA, and one in SDHC. The prevalence of SDHx mutations reached 1.1% (3/263). Of 31 reported patients with PAs harboring SDHx/MAX mutations (28 published cases and 3 cases reported here), 6/31 (19%) developed PA before PPGL and 8/31 (26%) had isolated PA. The age of onset was later than in patients with AIP/MEN1 mutations. PAs were mainly macroprolactinomas and showed intracytoplasmic vacuoles seen on histopathology. Conclusions: We discovered SDHx mutations in patients bearing PA who had no familial or personal history of PPGL. However, the question of incidental association remains unresolved and data to determine the benefit of SDHx/MAX screening in these patients are lacking. We recommend that patients with isolated PA should be carefully examined for a family history of PPGLs. A family history of PPGL, as well as the presence of intracytoplasmic vacuoles in PA, requires SDHx/MAX genetic testing of patients.


Assuntos
Adenoma/genética , Mutação em Linhagem Germinativa , Neoplasias Hipofisárias/genética , Succinato Desidrogenase/genética , Adenoma/epidemiologia , Adenoma/patologia , Adolescente , Neoplasias das Glândulas Suprarrenais/epidemiologia , Neoplasias das Glândulas Suprarrenais/genética , Neoplasias das Glândulas Suprarrenais/patologia , Adulto , Idade de Início , Idoso , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Criança , Análise Mutacional de DNA/métodos , Feminino , França/epidemiologia , Predisposição Genética para Doença , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Isoenzimas/genética , Masculino , Pessoa de Meia-Idade , Paraganglioma/epidemiologia , Paraganglioma/genética , Paraganglioma/patologia , Feocromocitoma/epidemiologia , Feocromocitoma/genética , Feocromocitoma/patologia , Neoplasias Hipofisárias/epidemiologia , Neoplasias Hipofisárias/patologia , Prolactinoma/epidemiologia , Prolactinoma/genética , Prolactinoma/patologia , Subunidades Proteicas/genética , Estudos Retrospectivos , Adulto Jovem
15.
Mol Cell ; 79(3): 376-389.e8, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32640193

RESUMO

Activation of dual-specificity tyrosine-phosphorylation-regulated kinases 1A and 1B (DYRK1A and DYRK1B) requires prolyl hydroxylation by PHD1 prolyl hydroxylase. Prolyl hydroxylation of DYRK1 initiates a cascade of events leading to the release of molecular constraints on von Hippel-Lindau (VHL) ubiquitin ligase tumor suppressor function. However, the proline residue of DYRK1 targeted by hydroxylation and the role of prolyl hydroxylation in tyrosine autophosphorylation of DYRK1 are unknown. We found that a highly conserved proline in the CMGC insert of the DYRK1 kinase domain is hydroxylated by PHD1, and this event precedes tyrosine autophosphorylation. Mutation of the hydroxylation acceptor proline precludes tyrosine autophosphorylation and folding of DYRK1, resulting in a kinase unable to preserve VHL function and lacking glioma suppression activity. The consensus proline sequence is shared by most CMGC kinases, and prolyl hydroxylation is essential for catalytic activation. Thus, formation of prolyl-hydroxylated intermediates is a novel mechanism of kinase maturation and likely a general mechanism of regulation of CMGC kinases in eukaryotes.


Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Isoenzimas/genética , Prolina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Glioma/patologia , Células HEK293 , Xenoenxertos , Humanos , Hidroxilação , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Camundongos , Camundongos Nus , Proteína Quinase 14 Ativada por Mitógeno/química , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Mutação , Neuroglia/metabolismo , Neuroglia/patologia , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
16.
Mol Genet Metab ; 130(4): 227-229, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32561366

RESUMO

Fabry disease is an X-linked disease due to a deficiency of the lysosomal enzyme alpha-galactosidase A. Clinical symptoms in classically affected males include acroparesthesia, anhydrosis and angiokeratoma, which may present during childhood followed by cardiac, cerebral and renal complications. Even though pulmonary involvement is not widely appreciated by clinicians, an obstructive lung disease is another recognized component of Fabry disease. Coronavirus Disease-19 (COVID-19), caused by the SARS-CoV-2 virus was labeled as a global pandemic and patients with Fabry disease can be considered at high risk of developing severe complications. The impact of COVID-19 on patients with Fabry disease receiving enzyme replacement therapy is still unknown. Many patients who receive treatment in the hospital experienced infusion disruptions due to fear of infection. Effects of temporary treatment interruption was described in more detail in other lysosomal storage diseases, but the recommencement of therapy does not fully reverse clinical decline due to the temporary discontinuation. When possible, home-therapy seems to be the most efficient way to maintain enzyme replacement therapy access during pandemic. Sentence take-home message: Home-therapy, when possible, seems to be the most efficient way to maintain enzyme replacement therapy access during pandemic in patients with Fabry disease.


Assuntos
Betacoronavirus/patogenicidade , Continuidade da Assistência ao Paciente/normas , Infecções por Coronavirus/prevenção & controle , Terapia de Reposição de Enzimas/normas , Doença de Fabry/terapia , Terapia por Infusões no Domicílio/normas , Pneumopatias Obstrutivas/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Adulto , Continuidade da Assistência ao Paciente/organização & administração , Continuidade da Assistência ao Paciente/estatística & dados numéricos , Infecções por Coronavirus/complicações , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Terapia de Reposição de Enzimas/estatística & dados numéricos , Doença de Fabry/complicações , Doença de Fabry/diagnóstico , Feminino , Terapia por Infusões no Domicílio/estatística & dados numéricos , Humanos , Controle de Infecções/normas , Infusões Intravenosas , Isoenzimas/administração & dosagem , Pneumopatias Obstrutivas/diagnóstico , Pneumopatias Obstrutivas/etiologia , Masculino , Pessoa de Meia-Idade , Pneumonia Viral/complicações , Pneumonia Viral/transmissão , Pneumonia Viral/virologia , Proteínas Recombinantes/administração & dosagem , Índice de Gravidade de Doença , Fatores de Tempo , alfa-Galactosidase/administração & dosagem
17.
Proc Natl Acad Sci U S A ; 117(25): 14280-14291, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513715

RESUMO

In utero mammalian development relies on the establishment of the maternal-fetal exchange interface, which ensures transportation of nutrients and gases between the mother and the fetus. This exchange interface is established via development of multinucleated syncytiotrophoblast cells (SynTs) during placentation. In mice, SynTs develop via differentiation of the trophoblast stem cell-like progenitor cells (TSPCs) of the placenta primordium, and in humans, SynTs are developed via differentiation of villous cytotrophoblast (CTB) progenitors. Despite the critical need in pregnancy progression, conserved signaling mechanisms that ensure SynT development are poorly understood. Herein, we show that atypical protein kinase C iota (PKCλ/ι) plays an essential role in establishing the SynT differentiation program in trophoblast progenitors. Loss of PKCλ/ι in the mouse TSPCs abrogates SynT development, leading to embryonic death at approximately embryonic day 9.0 (E9.0). We also show that PKCλ/ι-mediated priming of trophoblast progenitors for SynT differentiation is a conserved event during human placentation. PKCλ/ι is selectively expressed in the first-trimester CTBs of a developing human placenta. Furthermore, loss of PKCλ/ι in CTB-derived human trophoblast stem cells (human TSCs) impairs their SynT differentiation potential both in vitro and after transplantation in immunocompromised mice. Our mechanistic analyses indicate that PKCλ/ι signaling maintains expression of GCM1, GATA2, and PPARγ, which are key transcription factors to instigate SynT differentiation programs in both mouse and human trophoblast progenitors. Our study uncovers a conserved molecular mechanism, in which PKCλ/ι signaling regulates establishment of the maternal-fetal exchange surface by promoting trophoblast progenitor-to-SynT transition during placentation.


Assuntos
Diferenciação Celular/fisiologia , Isoenzimas/metabolismo , Troca Materno-Fetal/fisiologia , Placenta/metabolismo , Proteína Quinase C/metabolismo , Trofoblastos/fisiologia , Animais , Proteínas de Ligação a DNA/metabolismo , Feminino , Fator de Transcrição GATA2/metabolismo , Humanos , Isoenzimas/genética , Masculino , Camundongos , Camundongos Knockout , Modelos Animais , PPAR gama/metabolismo , Placenta/citologia , Placentação/fisiologia , Gravidez , Proteína Quinase C/genética , Transdução de Sinais , Células-Tronco/citologia , Fatores de Transcrição/metabolismo , Trofoblastos/citologia
18.
Nat Commun ; 11(1): 2738, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483131

RESUMO

Almost half of all enzymes utilize a metal cofactor. However, the features that dictate the metal utilized by metalloenzymes are poorly understood, limiting our ability to manipulate these enzymes for industrial and health-associated applications. The ubiquitous iron/manganese superoxide dismutase (SOD) family exemplifies this deficit, as the specific metal used by any family member cannot be predicted. Biochemical, structural and paramagnetic analysis of two evolutionarily related SODs with different metal specificity produced by the pathogenic bacterium Staphylococcus aureus identifies two positions that control metal specificity. These residues make no direct contacts with the metal-coordinating ligands but control the metal's redox properties, demonstrating that subtle architectural changes can dramatically alter metal utilization. Introducing these mutations into S. aureus alters the ability of the bacterium to resist superoxide stress when metal starved by the host, revealing that small changes in metal-dependent activity can drive the evolution of metalloenzymes with new cofactor specificity.


Assuntos
Proteínas de Bactérias/metabolismo , Ferro/metabolismo , Manganês/metabolismo , Metaloproteínas/metabolismo , Staphylococcus aureus/enzimologia , Superóxido Dismutase/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico , Evolução Molecular , Ferro/química , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , Manganês/química , Metaloproteínas/química , Metaloproteínas/genética , Mutação , Oxirredução , Filogenia , Homologia de Sequência de Aminoácidos , Staphylococcus aureus/genética , Superóxido Dismutase/química , Superóxido Dismutase/genética , Superóxidos/metabolismo
19.
Mol Cell ; 78(5): 803-805, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32502418

RESUMO

In this issue of Molecular Cell, Schumann et al. (2020) present a novel strategy to dissect the regulation of protein O-glycosylation by a large family of isoenzymes in cells. They employ a bump-and-hole engineering approach to capture the specific contribution of individual isoenzymes to O-glycosylation of proteins.


Assuntos
Glicosiltransferases , Proteínas , Glicosilação , Isoenzimas
20.
Mol Pharmacol ; 98(2): 88-95, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32487734

RESUMO

Arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic-metabolizing enzyme that also has a role in cancer cell growth and metabolism. Recently, it was reported that NAT1 undergoes lysine acetylation, an important post-translational modification that can regulate protein function. In the current study, we use site-directed mutagenesis to identify K100 and K188 as major sites of lysine acetylation in the NAT1 protein. Acetylation of ectopically expressed NAT1 in HeLa cells was decreased by C646, an inhibitor of the protein acetyltransferases p300/CREB-binding protein (CBP). Recombinant p300 directly acetylated NAT1 in vitro. Acetylation of NAT1 was enhanced by the sirtuin (SIRT) inhibitor nicotinamide but not by the histone deacetylase inhibitor trichostatin A. Cotransfection of cells with NAT1 and either SIRT 1 or 2, but not SIRT3, significantly decreased NAT1 acetylation. NAT1 activity was evaluated in cells after nicotinamide treatment to enhance acetylation or cotransfection with SIRT1 to inhibit acetylation. The results indicated that NAT1 acetylation impaired its enzyme kinetics, suggesting decreased acetyl coenzyme A binding. In addition, acetylation attenuated the allosteric effects of ATP on NAT1. Taken together, this study shows that NAT1 is acetylated by p300/CBP in situ and is deacetylated by the sirtuins SIRT1 and 2. It is hypothesized that post-translational modification of NAT1 by acetylation at K100 and K188 may modulate NAT1 effects in cells. SIGNIFICANCE STATEMENT: There is growing evidence that arylamine N-acetyltransferase 1 has an important cellular role in addition to xenobiotic metabolism. Here, we show that NAT1 is acetylated at K100 and K188 and that changes in protein acetylation equilibrium can modulate its activity in cells.


Assuntos
Arilamina N-Acetiltransferase/química , Arilamina N-Acetiltransferase/metabolismo , Proteína de Ligação a CREB/genética , Proteína p300 Associada a E1A/genética , Isoenzimas/química , Isoenzimas/metabolismo , Sirtuína 1/genética , Sirtuína 2/genética , Acetilcoenzima A/metabolismo , Acetilação/efeitos dos fármacos , Arilamina N-Acetiltransferase/genética , Benzoatos/farmacologia , Proteína de Ligação a CREB/metabolismo , Cristalografia por Raios X , Proteína p300 Associada a E1A/metabolismo , Células HeLa , Humanos , Ácidos Hidroxâmicos/farmacologia , Isoenzimas/genética , Lisina/química , Lisina/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Niacinamida/farmacologia , Conformação Proteica , Pirazóis/farmacologia , Sirtuína 1/metabolismo , Sirtuína 2/metabolismo , Transfecção
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