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1.
Ecotoxicol Environ Saf ; 214: 112072, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33691243

RESUMO

The Green Revolution faced a great cost to meet ever-increasing demands for food, where indiscriminate use of agrochemicals resulted in non-friendly habitats. Therefore, the development of a sustainable approach to better crop production of onion seeds (Allium cepa L.) is very crucial. It is time to use organic waste as a replacement for agrochemicals by using arbuscular mycorrhizal fungi (AMF) and Trichoderma. Fish waste as representative of food waste acts as a leading cause of contamination of the environment. The interaction of AMF and Trichoderma viride on biomass, total soluble protein, mycorrhizal colonization, amino acids, phosphatases and phosphorus and nitrogen contents of onion plants grown in fish waste amended soil was studied. Fish waste has caused a slight increase in onions biomass, total free amino acids, and soluble protein content while with AMF and T. viride dual inoculation more increments were recorded; such increases were related to an increase in mycorrhizal colonization. T. viride application significantly increased the mycorrhizal colonization levels, but these were significantly reduced with waste addition. Analysis of amino acids in plants showed that their concentrations had changed as a result of waste addition combined with AMF and/or T. viride. The effectiveness of fish waste combined with low cost and health/environmental safety leads to a prediction that the introduction of fish waste coupled with fungi will become a more popular feature of agriculture in the future.


Assuntos
Micorrizas/fisiologia , Cebolas/fisiologia , Trichoderma/fisiologia , Agricultura , Aminoácidos/metabolismo , Biomassa , Alimentos , Fungos/metabolismo , Hypocreales , Micorrizas/metabolismo , Nitrogênio/metabolismo , Cebolas/química , Monoéster Fosfórico Hidrolases/metabolismo , Fósforo/metabolismo , Eliminação de Resíduos , Solo , Trichoderma/metabolismo
2.
Cells ; 10(2)2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33525562

RESUMO

Lithium salts have been in the therapeutic toolbox for better or worse since the 19th century, with purported benefit in gout, hangover, insomnia, and early suggestions that lithium improved psychiatric disorders. However, the remarkable effects of lithium reported by John Cade and subsequently by Mogens Schou revolutionized the treatment of bipolar disorder. The known molecular targets of lithium are surprisingly few and include the signaling kinase glycogen synthase kinase-3 (GSK-3), a group of structurally related phosphomonoesterases that includes inositol monophosphatases, and phosphoglucomutase. Here we present a brief history of the therapeutic uses of lithium and then focus on GSK-3 as a therapeutic target in diverse diseases, including bipolar disorder, cancer, and coronavirus infections.


Assuntos
Antimaníacos/uso terapêutico , Transtorno Bipolar/tratamento farmacológico , Compostos de Lítio/uso terapêutico , Neoplasias/tratamento farmacológico , Doenças Neurodegenerativas/tratamento farmacológico , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Animais , Antimaníacos/farmacologia , Transtorno Bipolar/metabolismo , Coronavirus/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Compostos de Lítio/farmacologia , Neoplasias/metabolismo , Doenças Neurodegenerativas/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Síndrome Respiratória Aguda Grave/metabolismo , Transdução de Sinais/efeitos dos fármacos
3.
Methods Mol Biol ; 2251: 39-53, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33481230

RESUMO

Our knowledge of the role and biology of the different phosphoinositides has greatly expanded over recent years. Reversible phosphorylation by specific kinases and phosphatases of positions 3, 4, and 5 on the inositol ring is a highly dynamic process playing a critical role in the regulation of the spatiotemporal recruitment and binding of effector proteins. The specific phosphoinositide kinases and phosphatases are key players in the control of many cellular functions, including proliferation, survival, intracellular trafficking, or cytoskeleton reorganization. Several of these enzymes are mutated in human diseases. The impact of the fatty acid composition of phosphoinositides in their function is much less understood. There is an important molecular diversity in the fatty acid side chains of PI. While stearic and arachidonic fatty acids are the major acyl species in PIP, PIP2, and PIP3, other fatty acid combinations are also found. The role of these different molecular species is still unknown, but it is important to quantify these different molecules and their potential changes during cell stimulation to better characterize this emerging field. Here, we describe a sensitive high-performance liquid chromatography-mass spectrometry method that we used for the first time to profile the changes in phosphoinositide molecular species (summed fatty acyl chain profiles) in human and mouse platelets under resting conditions and following stimulation. This method can be applied to other hematopoietic primary cells isolated from human or experimental animal models.


Assuntos
Plaquetas/metabolismo , Fosfatidilinositóis/análise , Espectrometria de Massas em Tandem/métodos , 1-Fosfatidilinositol 4-Quinase/metabolismo , Animais , Fenômenos Bioquímicos , Linhagem Celular , Células Cultivadas , Cromatografia Líquida/métodos , Ácidos Graxos/metabolismo , Inositol/química , Camundongos , Fosfatidilinositol 3-Quinases/análise , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/análise , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transdução de Sinais/fisiologia
4.
Methods Mol Biol ; 2251: 73-89, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33481232

RESUMO

The dynamic phosphorylation of phosphatidylinositol produces seven distinct but interconvertible phosphatidylinositol phosphates (PIPs). Each PIP exhibits specific enrichment in a subset of membrane compartments as a result of dynamic phosphorylation and dephosphorylation by lipid kinases and phosphatases, and/or by vesicle-mediated transport. Several PIPs are found within the plasma membrane, such as phosphatidylinositol-4-phosphate [PI(4)P], phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2], and phosphatidylinositol-3,4,5-trisphosphate (PIP3), and these control many aspects of cell physiology, including receptor signaling and membrane traffic. As a result, measurement of the cell surface abundance of these PIPs is a valuable resource to allow understanding of the regulation and function of these cell surface lipids. Here, we describe methods based on quantification of the localization of genetically encoded fluorescent PIP probes to the cell surface by either spinning disc confocal microscopy or total internal reflection fluorescence microscopy that allow detection of changes in cell surface levels of PI(4,5)P2, PI(3,4)P2, and PIP3. These methods can also be applied to the measurement of other PIPs or lipid species at the cell surface, and thus represent a useful resource for the study of the cell biology of PIPs.


Assuntos
Técnicas Biossensoriais/métodos , Membrana Celular/química , Fosfatidilinositóis/análise , Animais , Técnicas de Cultura de Células/métodos , Membrana Celular/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Humanos , Microscopia de Fluorescência/métodos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases/metabolismo , Transporte Proteico/fisiologia
5.
Am J Physiol Gastrointest Liver Physiol ; 320(3): G351-G365, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33406007

RESUMO

Liver fibrosis, a major cause of morbidity and mortality worldwide, leads to liver damage, seriously threatening human health. In our previous study, we demonstrated that 14 kDa phosphohistidine phosphatase (PHP14) was upregulated in fibrotic liver tissue and involved in the migration and lamellipodia formation of hepatic stellate cells (HSCs). In this study, we evaluated PHP14 as a therapeutic target for liver fibrosis and investigated the mechanism by which it mediates liver fibrosis. AAV-shPhpt1 administration significantly attenuates CCl4-induced liver fibrosis in mice. In particular, fibrosis-associated inflammatory infiltration was significantly suppressed after PHP14 knockdown. Mechanistically, PHP14 regulated macrophage recruitment, infiltration, and migration by affecting podosome formation of macrophages. Inhibition of PHP14 decreased the expression of the fibrogenic signature at the early stage of liver fibrogenesis and the activation of HSCs in vivo. Thus, PHP14 can be considered a potential therapeutic target for liver fibrosis.NEW & NOTEWORTHY PHP14 inhibition via adeno-associated virus (AAV)-mediated gene silencing could potently attenuate carbon tetrachloride (CCl4)-induced liver fibrosis. PHP14 could regulate the migration of macrophages to the site of injury in vivo. PHP14 knockdown in vivo influenced the environment of fibrogenesis and relevant signaling pathways, subsequently affecting myofibroblast activation.


Assuntos
Cirrose Hepática/induzido quimicamente , Monoéster Fosfórico Hidrolases/metabolismo , Transferência Adotiva , Animais , Intoxicação por Tetracloreto de Carbono , Técnicas de Cocultura , Sistemas de Liberação de Medicamentos , Técnicas de Silenciamento de Genes , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Monoéster Fosfórico Hidrolases/genética , Células RAW 264.7 , Regulação para Cima
6.
Mol Cell ; 81(3): 546-557.e5, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33378643

RESUMO

Eukaryotic cells regulate 5'-triphosphorylated RNAs (ppp-RNAs) to promote cellular functions and prevent recognition by antiviral RNA sensors. For example, RNA capping enzymes possess triphosphatase domains that remove the γ phosphates of ppp-RNAs during RNA capping. Members of the closely related PIR-1 (phosphatase that interacts with RNA and ribonucleoprotein particle 1) family of RNA polyphosphatases remove both the ß and γ phosphates from ppp-RNAs. Here, we show that C. elegans PIR-1 dephosphorylates ppp-RNAs made by cellular RNA-dependent RNA polymerases (RdRPs) and is required for the maturation of 26G-RNAs, Dicer-dependent small RNAs that regulate thousands of genes during spermatogenesis and embryogenesis. PIR-1 also regulates the CSR-1 22G-RNA pathway and has critical functions in both somatic and germline development. Our findings suggest that PIR-1 modulates both Dicer-dependent and Dicer-independent Argonaute pathways and provide insight into how cells and viruses use a conserved RNA phosphatase to regulate and respond to ppp-RNA species.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Processamento Pós-Transcricional do RNA , RNA/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Regulação da Expressão Gênica no Desenvolvimento , Monoéster Fosfórico Hidrolases/genética , Fosforilação , RNA/genética , Capuzes de RNA , /metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Espermatogênese , Especificidade por Substrato
7.
Elife ; 92020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33349335

RESUMO

Synaptojanin1 (Synj1) is a phosphoinositide phosphatase, important in clathrin uncoating during endocytosis of presynaptic vesicles. It was identified as a potential drug target for Alzheimer's disease, Down syndrome, and TBC1D24-associated epilepsy, while also loss-of-function mutations in Synj1 are associated with epilepsy and Parkinson's disease. Despite its involvement in a range of disorders, structural, and detailed mechanistic information regarding the enzyme is lacking. Here, we report the crystal structure of the 5-phosphatase domain of Synj1. Moreover, we also present a structure of this domain bound to the substrate diC8-PI(3,4,5)P3, providing the first image of a 5-phosphatase with a trapped substrate in its active site. Together with an analysis of the contribution of the different inositide phosphate groups to catalysis, these structures provide new insights in the Synj1 mechanism. Finally, we analysed the effect of three clinical missense mutations (Y793C, R800C, Y849C) on catalysis, unveiling the molecular mechanisms underlying Synj1-associated disease.


Assuntos
Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Humanos , Mutação de Sentido Incorreto , Conformação Proteica , Domínios Proteicos
8.
Int J Mol Sci ; 22(1)2020 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-33375448

RESUMO

Lithium is the prototype mood-stabilizer used for acute and long-term treatment of bipolar disorder. Cumulated translational research of lithium indicated the drug's neuroprotective characteristics and, thereby, has raised the option of repurposing it as a drug for neurodegenerative diseases. Lithium's neuroprotective properties rely on its modulation of homeostatic mechanisms such as inflammation, mitochondrial function, oxidative stress, autophagy, and apoptosis. This myriad of intracellular responses are, possibly, consequences of the drug's inhibition of the enzymes inositol-monophosphatase (IMPase) and glycogen-synthase-kinase (GSK)-3. Here we review lithium's neurobiological properties as evidenced by its neurotrophic and neuroprotective properties, as well as translational studies in cells in culture, in animal models of Alzheimer's disease (AD) and in patients, discussing the rationale for the drug's use in the treatment of AD.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Autofagia/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Lítio/uso terapêutico , Fármacos Neuroprotetores/farmacologia , Animais , Apoptose/efeitos dos fármacos , Transtorno Bipolar/tratamento farmacológico , Reposicionamento de Medicamentos , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Lítio/farmacologia , Lítio/toxicidade , Doenças Neurodegenerativas/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/metabolismo
9.
Anticancer Res ; 40(11): 6101-6113, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33109548

RESUMO

BACKGROUND/AIM: Oral squamous cell carcinoma (OSCC) is a common malignancy with poor prognosis. Therefore, novel therapeutic options are needed to improve prognosis of OSCC. Recently, microRNAs (miRs) have received increasing attention as a potential therapeutic tool for carcinomas. However, no definitive miR-based drugs for patients with OSCC have been reported to date. The aim of this study was to identify new miRs potentially involved in cellular processes associated with OSCC malignancy, which could lead to novel therapeutic strategies. MATERIALS AND METHODS: We identified miRs that are modulated in OSCC and possibly regulate OSCC malignancy, using miR microarray on OSCC cell lines. RESULTS: miR-935 and miR-509-3p were down-regulated in OSCC cell lines and patient tissues. When miR-935 was overexpressed in HSC-3-M3 cells, proliferation, migration, and invasion of the cell line was suppressed, whereas apoptosis was increased. Moreover, we showed that the gene inositol polyphosphate-4-phosphatase type I A (INPP4A) is a potential target whose expression is positively regulated by miR-935. CONCLUSION: miR-935 may function as a tumor suppressor by inhibiting OSCC malignancy via INPP4A induction. Therefore, miR-935 can be a new therapeutic candidate for OSCC treatment.


Assuntos
Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/genética , MicroRNAs/metabolismo , Neoplasias Bucais/enzimologia , Neoplasias Bucais/genética , Monoéster Fosfórico Hidrolases/metabolismo , Apoptose/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Neoplasias Bucais/patologia , Invasividade Neoplásica , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
10.
J Pharmacol Sci ; 144(3): 139-146, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32921395

RESUMO

SHP2 is a non-receptor protein tyrosine phosphatase encoded by the PTPN11 gene in human. Clinically, SHP2 has been identified as a causal factor of several diseases, such as Noonan syndrome, LEOPARD syndrome as well as myeloid malignancies. Interestingly, both loss-of-function and gain-of-function mutations occur in the PTPN11 gene. Analyses by biochemical and cell biological means as well as probing with small molecule compounds have demonstrated that SHP2 has both phosphatase-dependent and independent functions. In comparison with its phosphatase activity, the non-phosphatase-like function of SHP2 has not been well introduced or summarized. This review mainly focuses on the phosphatase-independent functions and its regulation by small molecule compounds as well as their use for disease therapy.


Assuntos
Monoéster Fosfórico Hidrolases , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/fisiologia , Cerebrosídeos , Depsipeptídeos , Mutação com Ganho de Função , Humanos , Síndrome LEOPARD/genética , Mutação com Perda de Função , Terapia de Alvo Molecular , Síndrome de Noonan/genética , Monoéster Fosfórico Hidrolases/metabolismo , Piperidinas , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Pirimidinas , Transdução de Sinais/genética
11.
Oncogene ; 39(37): 5979-5994, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32753652

RESUMO

Non-small cell lung cancer (NSCLC) is the deadliest form of cancer worldwide, due in part to its proclivity to metastasize. Identifying novel drivers of invasion and metastasis holds therapeutic potential for the disease. We conducted a gain-of-function invasion screen, which identified two separate hits, IMPAD1 and KDELR2, as robust, independent drivers of lung cancer invasion and metastasis. Given that IMPAD1 and KDELR2 are known to be localized to the ER-Golgi pathway, we studied their common mechanism of driving in vitro invasion and in vivo metastasis and demonstrated that they enhance Golgi-mediated function and secretion. Therapeutically inhibiting matrix metalloproteases (MMPs) suppressed both IMPAD1- and KDELR2-mediated invasion. The hits from this unbiased screen and the mechanistic validation highlight Golgi function as one of the key cellular features altered during invasion and metastasis.


Assuntos
Complexo de Golgi/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Monoéster Fosfórico Hidrolases/genética , Proteínas de Transporte Vesicular/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Metaloproteinases da Matriz/metabolismo , Invasividade Neoplásica , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Transporte Vesicular/metabolismo
12.
Nat Commun ; 11(1): 4212, 2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32839469

RESUMO

Phosphatases, together with kinases and transcription factors, are key components in cellular signalling networks. Here, we present a systematic functional analysis of the phosphatases in Cryptococcus neoformans, a fungal pathogen that causes life-threatening fungal meningoencephalitis. We analyse 230 signature-tagged mutant strains for 114 putative phosphatases under 30 distinct in vitro growth conditions, revealing at least one function for 60 of these proteins. Large-scale virulence and infectivity assays using insect and mouse models indicate roles in pathogenicity for 31 phosphatases involved in various processes such as thermotolerance, melanin and capsule production, stress responses, O-mannosylation, or retromer function. Notably, phosphatases Xpp1, Ssu72, Siw14, and Sit4 promote blood-brain barrier adhesion and crossing by C. neoformans. Together with our previous systematic studies of transcription factors and kinases, our results provide comprehensive insight into the pathobiological signalling circuitry of C. neoformans.


Assuntos
Cryptococcus neoformans/genética , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica/métodos , Genoma Fúngico/genética , Estudo de Associação Genômica Ampla/métodos , Monoéster Fosfórico Hidrolases/genética , Animais , Análise por Conglomerados , Criptococose/microbiologia , Cryptococcus neoformans/patogenicidade , Feminino , Proteínas Fúngicas/classificação , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Camundongos Endogâmicos , Monoéster Fosfórico Hidrolases/classificação , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases/classificação , Fosfotransferases/genética , Fosfotransferases/metabolismo , Transdução de Sinais/genética , Termotolerância/genética , Fatores de Transcrição/classificação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Virulência/genética
13.
Mol Cell ; 79(2): 342-358.e12, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32645368

RESUMO

Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.


Assuntos
Calcineurina/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Biotinilação , Centrossomo/metabolismo , Simulação por Computador , Células HEK293 , Células HeLa , Humanos , Espectrometria de Massas , Monoéster Fosfórico Hidrolases/química , Fosforilação , Mapas de Interação de Proteínas , Proteoma/metabolismo , Receptor Notch1/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais
14.
Life Sci ; 256: 117924, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32522568

RESUMO

AIMS: Osteoarthritis (OA) is a common degenerative joint disease characterized by cartilage degeneration and joint inflammation. As its pathogenesis remains unclear, there are no effective treatments established. Circular RNA (circRNA), microRNA (miRNA), and other noncoding RNAs participate in OA development; however, the effects and mechanisms of circRNA and miRNA in OA remain unknown. MAIN METHODS: Cartilage miRNA was examined in patients with and without OA. KEY FINDINGS: CircRNA-9119 and phosphatase and tensin homolog (PTEN) expression decreased in OA-affected cartilage and interleukin (IL)-1ß-induced chondrocytes, and miR-26a expression significantly decreased in normal cells and tissues. CircRNA-9119 overexpression restored chondrocyte growth, whereas IL-1ß treatment impaired chondrocyte growth. Annexin V-FITC & PI flow cytometry and Bcl-2/Bax ratio measurement indicated that the apoptosis of IL-1ß-treated articular chondrocytes was decreased by circRNA-9119 upregulation. Bioinformatic prediction and the dual-luciferase reporter assay indicated that circRNA-9119 served as a miR-26a sponge and that miR-26a targeted the 3'-UTR of PTEN. Transfection of chondrocytes with a circRNA-9119-overexpressing vector revealed downregulation of miR-26a expression. Furthermore, circRNA-9119 overexpression induced PTEN expression. In addition, a miR-26a mimic induced IL-1ß-induced chondrocyte apoptosis, and circRNA-9119 overexpression inhibited IL-1ß-induced chondrocyte apoptosis. SIGNIFICANCE: CircRNA-9119 is an important regulator of IL-1ß-treated chondrocytes through the miR-26a/PTEN axis, possibly contributing to OA development.


Assuntos
Condrócitos/metabolismo , Interleucina-1beta/metabolismo , Osteoartrite/tratamento farmacológico , Substâncias Protetoras/metabolismo , RNA Circular/metabolismo , Regiões 3' não Traduzidas , Apoptose/efeitos dos fármacos , Cartilagem/metabolismo , Cartilagem Articular/metabolismo , Linhagem Celular , Condrócitos/citologia , Regulação para Baixo , Matriz Extracelular/metabolismo , Humanos , MicroRNAs/genética , PTEN Fosfo-Hidrolase/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Regulação para Cima , Proteína X Associada a bcl-2/metabolismo
15.
Proc Natl Acad Sci U S A ; 117(21): 11715-11726, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32398371

RESUMO

Campylobacter jejuni monitors intestinal metabolites produced by the host and microbiota to initiate intestinal colonization of avian and animal hosts for commensalism and infection of humans for diarrheal disease. We previously discovered that C. jejuni has the capacity to spatially discern different intestinal regions by sensing lactate and the short-chain fatty acids acetate and butyrate and then alter transcription of colonization factors appropriately for in vivo growth. In this study, we identified the C. jejuni butyrate-modulated regulon and discovered that the BumSR two-component signal transduction system (TCS) directs a response to butyrate by identifying mutants in a genetic screen defective for butyrate-modulated transcription. The BumSR TCS, which is important for infection of humans and optimal colonization of avian hosts, senses butyrate likely by indirect means to alter transcription of genes encoding important colonization determinants. Unlike many canonical TCSs, the predicted cytoplasmic sensor kinase BumS lacked in vitro autokinase activity, which would normally lead to phosphorylation of the cognate BumR response regulator. Instead, BumS has likely evolved mutations to naturally function as a phosphatase whose activity is influenced by exogenous butyrate to control the level of endogenous phosphorylation of BumR and its ability to alter transcription of target genes. To our knowledge, the BumSR TCS is the only bacterial signal transduction system identified so far that mediates responses to the microbiota-generated intestinal metabolite butyrate, an important factor for host intestinal health and homeostasis. Our findings suggest that butyrate sensing by this system is vital for C. jejuni colonization of multiple hosts.


Assuntos
Proteínas de Bactérias , Butiratos/metabolismo , Campylobacter jejuni , Regulação Bacteriana da Expressão Gênica/genética , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Infecções por Campylobacter/microbiologia , Galinhas , Humanos , Monoéster Fosfórico Hidrolases/genética , Transdução de Sinais/genética
16.
mSphere ; 5(3)2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32434842

RESUMO

The control of the virulence response regulator and sensor (CovR-CovS) two-component regulatory system in group A Streptococcus (GAS) strains regulates more than 15% of gene expression and has critical roles in invasive GAS infection. The membrane-embedded CovS has kinase and phosphatase activities, and both are required for modulating the phosphorylation level of CovR. Regulator of Cov (RocA) is a positive regulator of covR and also been shown to be a pseudokinase that interacts with CovS to enhance the phosphorylation level of CovR; however, how RocA modulates the activity of CovS has not been determined conclusively. Although the phosphorylation level of CovR was decreased in the rocA mutant in the exponential phase, the present study shows that phosphorylated CovR in the rocA mutant increased to levels similar to those in the wild-type strain in the stationary phase of growth. In addition, acidic stress, which is generally present in the stationary phase, enhanced the phosphorylation level of CovR in the rocA mutant. The phosphorylation levels of CovR in the CovS phosphatase-inactivated mutant and its rocA mutant were similar under acidic stress and Mg2+ (the signal that inhibits CovS phosphatase activity) treatments, suggesting that the phosphatase activity, but not the kinase activity, of CovS is required for RocA to modulate CovR phosphorylation. The phosphorylation level of CovR is crucial for GAS strains to regulate virulence factor expression; therefore, the growth phase- and pH-dependent RocA activity would contribute significantly to GAS pathogenesis.IMPORTANCE The emergence of invasive group A streptococcal infections has been reported worldwide. Clinical isolates that have spontaneous mutations or a truncated allele of the rocA gene (e.g., emm3-type isolates) are considered to be more virulent than isolates with the intact rocA gene (e.g., emm1-type isolates). RocA is a positive regulator of covR and has been shown to enhance the phosphorylation level of intracellular CovR regulator through the functional CovS protein. CovS is the membrane-embedded sensor and modulates the phosphorylation level of CovR by its kinase and phosphatase activities. The present study shows that the enhancement of CovR phosphorylation is mediated via the repression of CovS's phosphatase activity by RocA. In addition, we found that RocA acts dominantly on modulating CovR phosphorylation under neutral pH conditions and in the exponential phase of growth. The phosphorylation level of CovR is crucial for group A Streptococcus species to regulate virulence factor expression and is highly related to bacterial invasiveness; therefore, growth phase- and pH-dependent RocA activity and the sequence polymorphisms of rocA gene would contribute significantly to bacterial phenotype variations and pathogenesis.


Assuntos
Proteínas de Bactérias/metabolismo , Histidina Quinase/metabolismo , Proteínas Repressoras/metabolismo , Streptococcus pyogenes/patogenicidade , Transativadores/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação/genética , Infecções Estreptocócicas/patologia , Streptococcus pyogenes/genética , Transativadores/genética
17.
PLoS One ; 15(5): e0233163, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32413098

RESUMO

Inositol polyphosphate-4-phosphatase type II (INPP4B) is a dual-specificity phosphatase that acts as a tumor suppressor in multiple cancers. INPP4B dephosphorylates phospholipids at the 4th position of the inositol ring and inhibits AKT and PKC signaling by hydrolyzing of PI(3,4)P2 and PI(4,5)P2, respectively. INPP4B protein phosphatase targets include phospho-tyrosines on Akt and phospho-serine and phospho-threonine on PTEN. INPP4B is highly expressed in testes, suggesting its role in testes development and physiology. The objective of this study was to determine whether Inpp4b deletion impacts testicular function in mice. In testis, Inpp4b expression was the highest in postmeiotic germ cells in both mice and men. The testes of Inpp4b knockout male mice were significantly smaller compared to the testes of wild-type (WT) males. Inpp4b-/- males produced fewer mature sperm cells compared to WT, and this difference increased with age and high fat diet (HFD). Reduction in early steroidogenic enzymes and luteinizing hormone (LH) receptor gene expression was detected, although androgen receptor (AR) protein level was similar in WT and Inpp4b-/- testes. Germ cell apoptosis was significantly increased in the knockout mice, while expression of meiotic marker γH2A.X was decreased. Our data demonstrate that INPP4B plays a role in maintenance of male germ cell differentiation and protects testis functions against deleterious effects of aging and high fat diet.


Assuntos
Monoéster Fosfórico Hidrolases/metabolismo , Espermatogênese/genética , Espermatozoides/metabolismo , Testículo/metabolismo , Animais , Apoptose/genética , Citocinas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Regulação da Expressão Gênica no Desenvolvimento/genética , Histonas/metabolismo , Humanos , Masculino , Meiose/genética , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Monoéster Fosfórico Hidrolases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA-Seq , Receptores Androgênicos/metabolismo , Receptores do LH/genética , Receptores do LH/metabolismo , Análise de Célula Única , Contagem de Espermatozoides , Testículo/crescimento & desenvolvimento
18.
PLoS One ; 15(3): e0230819, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32231398

RESUMO

STAT3 mediates signalling downstream of cytokine and growth factor receptors where it acts as a transcription factor for its target genes, including oncogenes and cell survival regulating genes. STAT3 has been found to be persistently activated in many types of cancers, primarily through its tyrosine phosphorylation (Y705). Here, we show that constitutive STAT3 activation protects cells from cytotoxic drug responses of several drug classes. To find novel and potentially targetable STAT3 regulators we performed a kinase and phosphatase siRNA screen with cells expressing either a hyperactive STAT3 mutant or IL6-induced wild type STAT3. The screen identified cell division cycle 7-related protein kinase (CDC7), casein kinase 2, alpha 1 (CSNK2), discoidin domain-containing receptor 2 (DDR2), cyclin-dependent kinase 8 (CDK8), phosphatidylinositol 4-kinase 2-alpha (PI4KII), C-terminal Src kinase (CSK) and receptor-type tyrosine-protein phosphatase H (PTPRH) as potential STAT3 regulators. Using small molecule inhibitors targeting these proteins, we confirmed dose and time dependent inhibition of STAT3-mediated transcription, suggesting that inhibition of these kinases may provide strategies for dampening STAT3 activity in cancers.


Assuntos
Antineoplásicos/farmacologia , Biologia Computacional , Fator de Transcrição STAT3/metabolismo , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Monoéster Fosfórico Hidrolases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/genética , Fator de Transcrição STAT3/deficiência , Fator de Transcrição STAT3/genética , Fatores de Tempo
19.
Int J Med Microbiol ; 310(4): 151418, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32245626

RESUMO

Phosphoinositides (PIPs) are one kind of membrane components functioning in many intracellular processes, especially in signaling transduction and membrane transport. Phosphatidylinositide phosphatases (PIPases) are specifically important for the PIP homeostasis in cell. In our previous study, we have identified the actin-related protein CaSac1 in Candida albicans, while its functional mechanisms in regulating membrane homeostasis has not been identified. Here, we show that the PIPase CaSac1 is a main membrane-related protein and regulates hyphal polarization by governing phosphoinositide dynamic and plasma membrane (PM) electrostatic field. Deletion of CaSAC1 resulted in large-scale abnormal redistribution of phosphatidylinositide 4-phosphate (PI4P) from the endomembrane to the PM. This abnormality further led to disturbance of the PM's negative electrostatic field and abnormally spotted distribution of phosphatidylinositide 4,5-bisphosphate (PI(4,5)P2). These changes led to a severe defect in polarized hyphal growth, which could be diminished with recovery of the PM's negative electrostatic field by the anionic polymer polyacrylic acid (PAA). This study revealed that the PIPase CaSac1 plays an essential role in regulating membrane homeostasis and membrane traffic, contributing to establishment of polarized hyphal growth.


Assuntos
Candida albicans/enzimologia , Candida albicans/crescimento & desenvolvimento , Homeostase , Proteínas de Membrana/metabolismo , Fosfatidilinositóis/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Candida albicans/genética , Membrana Celular , Hifas/crescimento & desenvolvimento , Proteínas de Membrana/genética , Monoéster Fosfórico Hidrolases/genética
20.
J Virol ; 94(12)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32269127

RESUMO

Positive-strand RNA [(+)RNA] viruses assemble numerous membrane-bound viral replicase complexes (VRCs) with the help of viral replication proteins and co-opted host proteins within large viral replication compartments in the cytosol of infected cells. In this study, we found that deletion or depletion of Sac1 phosphatidylinositol 4-phosphate [PI(4)P] phosphatase reduced tomato bushy stunt virus (TBSV) replication in yeast (Saccharomyces cerevisiae) and plants. We demonstrate a critical role for Sac1 in TBSV replicase assembly in a cell-free replicase reconstitution assay. The effect of Sac1 seems to be direct, based on its interaction with the TBSV p33 replication protein, its copurification with the tombusvirus replicase, and its presence in the virus-induced membrane contact sites and within the TBSV replication compartment. The proviral functions of Sac1 include manipulation of lipid composition, sterol enrichment within the VRCs, and recruitment of additional host factors into VRCs. Depletion of Sac1 inhibited the recruitment of Rab5 GTPase-positive endosomes and enrichment of phosphatidylethanolamine in the viral replication compartment. We propose that Sac1 might be a component of the assembly hub for VRCs, likely in collaboration with the co-opted the syntaxin18-like Ufe1 SNARE protein within the TBSV replication compartments. This work also led to demonstration of the enrichment of PI(4)P phosphoinositide within the replication compartment. Reduction in the PI(4)P level due to chemical inhibition in plant protoplasts; depletion of two PI(4)P kinases, Stt4p and Pik1p; or sequestration of free PI(4)P via expression of a PI(4)P-binding protein in yeast strongly inhibited TBSV replication. Altogether, Sac1 and PI(4)P play important proviral roles during TBSV replication.IMPORTANCE Replication of positive-strand RNA viruses depends on recruitment of host components into viral replication compartments or organelles. Using TBSV, we uncovered the critical roles of Sac1 PI(4)P phosphatase and its substrate, PI(4)P phosphoinositide, in promoting viral replication. Both Sac1 and PI(4)P are recruited to the site of viral replication to facilitate the assembly of the viral replicase complexes, which perform viral RNA replication. We found that Sac1 affects the recruitment of other host factors and enrichment of phosphatidylethanolamine and sterol lipids within the subverted host membranes to promote optimal viral replication. In summary, this work demonstrates the novel functions of Sac1 and PI(4)P in TBSV replication in the model host yeast and in plants.


Assuntos
Interações Hospedeiro-Patógeno/genética , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Tombusvirus/genética , Replicação Viral/genética , 1-Fosfatidilinositol 4-Quinase/genética , 1-Fosfatidilinositol 4-Quinase/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Endossomos/metabolismo , Regulação da Expressão Gênica , Fosfatidiletanolaminas/genética , Fosfatidiletanolaminas/metabolismo , Monoéster Fosfórico Hidrolases/deficiência , Monoéster Fosfórico Hidrolases/metabolismo , Células Vegetais/metabolismo , Células Vegetais/virologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Protoplastos/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , /metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/virologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Esteróis/metabolismo , Tabaco/genética , Tabaco/metabolismo , Tabaco/virologia , Tombusvirus/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo
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