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1.
Front Immunol ; 12: 705232, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34295340

RESUMO

Increasing evidence supports that N6-methyladenosine (m6A) mRNA modification may play an important role in regulating immune responses. Intestinal epithelial cells orchestrate gastrointestinal mucosal innate defense to microbial infection, but underlying mechanisms are still not fully understood. In this study, we present data demonstrating significant alterations in the topology of host m6A mRNA methylome in intestinal epithelial cells following infection by Cryptosporidium parvum, a coccidian parasite that infects the gastrointestinal epithelium and causes a self-limited disease in immunocompetent individuals but a life-threatening diarrheal disease in AIDS patients. Altered m6A methylation in mRNAs in intestinal epithelial cells following C. parvum infection is associated with downregulation of alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 and the fat mass and obesity-associated protein with the involvement of NF-кB signaling. Functionally, m6A methylation statuses influence intestinal epithelial innate defense against C. parvum infection. Specifically, expression levels of immune-related genes, such as the immunity-related GTPase family M member 2 and interferon gamma induced GTPase, are increased in infected cells with a decreased m6A mRNA methylation. Our data support that intestinal epithelial cells display significant alterations in the topology of their m6A mRNA methylome in response to C. parvum infection with the involvement of activation of the NF-кB signaling pathway, a process that modulates expression of specific immune-related genes and contributes to fine regulation of epithelial antimicrobial defense.


Assuntos
Adenosina/análogos & derivados , Criptosporidiose/imunologia , Cryptosporidium parvum/imunologia , Epitélio/imunologia , Imunidade Inata , Mucosa Intestinal/imunologia , Processamento Pós-Transcricional do RNA , RNA Mensageiro/imunologia , Adenosina/fisiologia , Homólogo AlkB 5 da RNA Desmetilase/antagonistas & inibidores , Homólogo AlkB 5 da RNA Desmetilase/biossíntese , Homólogo AlkB 5 da RNA Desmetilase/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/biossíntese , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Animais , Sistemas CRISPR-Cas , GTP Fosfo-Hidrolases/biossíntese , GTP Fosfo-Hidrolases/genética , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica/imunologia , Humanos , Mucosa Intestinal/citologia , Metilação , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética
2.
Nat Chem Biol ; 16(12): 1394-1402, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32719557

RESUMO

Metabolism is often regulated by the transcription and translation of RNA. In turn, it is likely that some metabolites regulate enzymes controlling reversible RNA modification, such as N6-methyladenosine (m6A), to modulate RNA. This hypothesis is at least partially supported by the findings that multiple metabolic diseases are highly associated with fat mass and obesity-associated protein (FTO), an m6A demethylase. However, knowledge about whether and which metabolites directly regulate m6A remains elusive. Here, we show that NADP directly binds FTO, independently increases FTO activity, and promotes RNA m6A demethylation and adipogenesis. We screened a set of metabolites using a fluorescence quenching assay and NADP was identified to remarkably bind FTO. In vitro demethylation assays indicated that NADP enhances FTO activity. Furthermore, NADP regulated mRNA m6A via FTO in vivo, and deletion of FTO blocked NADP-enhanced adipogenesis in 3T3-L1 preadipocytes. These results build a direct link between metabolism and RNA m6A demethylation.


Assuntos
Adenosina/análogos & derivados , Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , NADP/farmacologia , RNA Mensageiro/genética , Células 3T3-L1 , Adenosina/metabolismo , Adipócitos/citologia , Adipócitos/enzimologia , Adipogenia/genética , Homólogo AlkB 5 da RNA Desmetilase/antagonistas & inibidores , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Animais , Sítios de Ligação , Diferenciação Celular/efeitos dos fármacos , Desmetilação , Ensaios Enzimáticos , Deleção de Genes , Regulação da Expressão Gênica , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Cinética , Metiltransferases/antagonistas & inibidores , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , NADP/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
3.
Bioorg Med Chem ; 28(4): 115300, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31937477

RESUMO

The imidazobenzoxazin-5-thione MV1035, synthesized as a new sodium channel blocker, has been tested on tumoral cells that differ for origin and for expressed NaV pool (U87-MG, H460 and A549). In this paper we focus on the effect of MV1035 in reducing U87 glioblastoma cell line migration and invasiveness. Since the effect of this compound on U87-MG cells seemed not dependent on its sodium channel blocking capability, alternative off-target interaction for MV1035 have been identified using SPILLO-PBSS software. This software performs a structure-based in silico screening on a proteome-wide scale, that allows to identify off-target interactions. Among the top-ranked off-targets of MV1035, we focused on the RNA demethylase ALKBH5 enzyme, known for playing a key role in cancer. In order to prove the effect of MV1035 on ALKBH5 in vitro coincubation of MV1035 and ALKBH5 has been performed demonstrating a consequent increase of N6-methyladenosine (m6A) RNA. To further validate the pathway involving ALKBH5 inhibition by MV1035 in U87-MG reduced migration and invasiveness, we evaluated CD73 as possible downstream protein. CD73 is an extrinsic protein involved in the generation of adenosine and is overexpressed in several tumors including glioblastoma. We have demonstrated that treating U87-MG with MV1035, CD73 protein expression was reduced without altering CD73 transcription. Our results show that MV1035 is able to significantly reduce U87 cell line migration and invasiveness inhibiting ALKBH5, an RNA demethylase that can be considered an interesting target in fighting glioblastoma aggressiveness. Our data encourage to further investigate the MV1035 inhibitory effect on glioblastoma.


Assuntos
Homólogo AlkB 5 da RNA Desmetilase/antagonistas & inibidores , Benzoxazinas/farmacologia , Inibidores Enzimáticos/farmacologia , Proteoma/efeitos dos fármacos , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Benzoxazinas/síntese química , Benzoxazinas/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Relação Estrutura-Atividade
4.
J Pharm Biomed Anal ; 162: 9-15, 2019 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-30219599

RESUMO

N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic messenger RNA (mRNA). Until now, two RNA demethylases have been identified, including FTO (fat mass and obesity-associated protein) and ALKBH5 (α-ketoglutarate-dependent dioxygenase alkB homologue 5). As a mammalian m6A demethylase, ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles, and ALKBH5 may play a significant role in these biological processes. Nevertheless, no modulator of ALKBH5 has been reported. The reason for that may be the lack of in vitro assays for ALKBH5 inhibitor screening. Herein, we describe the development of two homogeneous assays for ALKBH5 using N6-methyladenosine as substrate with different principles. Using ALKBH5 recombinant, we developed a formaldehyde dehydrogenase coupled fluorescence based assay and an antibody based assay for the activity evaluation of ALKBH5. These robust coupled assays are suitable for screening ALKBH5 inhibitors in 384-well format (Z' factors of 0.74), facilitating the discovery of modulators in the quest for the regulation of biological processes.


Assuntos
Adenosina/análogos & derivados , Aldeído Oxirredutases/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Descoberta de Drogas/métodos , Imunofluorescência , Formaldeído/metabolismo , Ensaios de Triagem em Larga Escala/métodos , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Aldeído Oxirredutases/antagonistas & inibidores , Homólogo AlkB 5 da RNA Desmetilase/antagonistas & inibidores , Desmetilação , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Reprodutibilidade dos Testes , Espectrometria de Fluorescência
5.
Cell Physiol Biochem ; 48(2): 838-846, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30032148

RESUMO

BACKGROUND/AIMS: Mounting evidence suggests that epitranscriptional modifications regulate multiple cellular processes. N6-Methyladenosine (m6A), the most abundant reversible methylation of mRNA, has critical roles in cancer pathogenesis. However, the mechanisms and functions of long non-coding RNA (lncRNA) methylation remain unclear. Pancreatic cancer resulted in 411,600 deaths globally in 2015. By the time of pancreatic cancer diagnosis, metastasis has often occurred in other parts of the body. The present study sought to investigate lncRNA m6A modification and its roles in pancreatic cancer. METHODS: Differential expression between cancer cells and matched normal cells was evaluated to identify candidate lncRNAs. The lncRNA KCNK15-AS1 was detected in cancer tissues and various pancreatic cells using RT-qPCR. KCNK15-AS1 was transfected into cells to explore its role in migration and invasion. Then, m6A RNA immunoprecipitation was performed to detect methylated KCNK15-AS1 in tissues and cells. Epithelial-mesenchymal transition (EMT) markers were used to evaluate KCNK15-AS1-mediated EMT processes. RESULTS: KCNK15-AS1 was downregulated in pancreatic cancer tissues compared with paired adjacent normal tissues. KCNK15-AS1 inhibited migration and invasion in MIA PaCa-2 and BxPC-3 cells. Furthermore, total RNA methylation in cancer cells was significantly enriched relative to that in immortalized human pancreatic duct epithelial (HPDE6-C7) cells. In addition, the m6A eraser ALKBH5 was downregulated in cancer cells, which can demethylate KCNK15-AS1 and regulate KCNK15-AS1-mediated cell motility. CONCLUSION: Our results have revealed a novel mechanism by which ALKBH5 inhibits pancreatic cancer motility by demethylating lncRNA KCNK15-AS1, identifying a potential therapeutic target for pancreatic cancer.


Assuntos
Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Neoplasias Pancreáticas/patologia , RNA Longo não Codificante/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/antagonistas & inibidores , Homólogo AlkB 5 da RNA Desmetilase/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Metilação de DNA , Regulação para Baixo , Transição Epitelial-Mesenquimal/genética , Humanos , Neoplasias Pancreáticas/metabolismo , Interferência de RNA , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , Vimentina/metabolismo
6.
Chem Asian J ; 13(19): 2854-2867, 2018 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-29917331

RESUMO

Dynamic combinatorial chemistry (DCC) is a powerful supramolecular approach for discovering ligands for biomolecules. To date, most, if not all, biologically templated DCC systems employ only a single biomolecule to direct the self-assembly process. To expand the scope of DCC, herein, a novel multiprotein DCC strategy has been developed that combines the discriminatory power of a zwitterionic "thermal tag" with the sensitivity of differential scanning fluorimetry. This strategy is highly sensitive and could differentiate the binding of ligands to structurally similar subfamily members. Through this strategy, it was possible to simultaneously identify subfamily-selective probes against two clinically important epigenetic enzymes: FTO (7; IC50 =2.6 µm) and ALKBH3 (8; IC50 =3.7 µm). To date, this is the first report of a subfamily-selective ALKBH3 inhibitor. The developed strategy could, in principle, be adapted to a broad range of proteins; thus it is of broad scientific interest.


Assuntos
Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/antagonistas & inibidores , Dioxigenase FTO Dependente de alfa-Cetoglutarato/antagonistas & inibidores , Técnicas de Química Combinatória/métodos , Inibidores Enzimáticos/química , Oxirredutases O-Desmetilantes/antagonistas & inibidores , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/química , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/genética , Homólogo AlkB 5 da RNA Desmetilase/antagonistas & inibidores , Homólogo AlkB 5 da RNA Desmetilase/química , Homólogo AlkB 5 da RNA Desmetilase/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/química , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Catálise , Fluorometria/métodos , Humanos , Hidrazonas/química , Cinética , Ligantes , Estrutura Molecular , Oxirredutases O-Desmetilantes/química , Oxirredutases O-Desmetilantes/genética , Peptídeos/química , Peptídeos/genética , Desnaturação Proteica , Engenharia de Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Temperatura de Transição
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