RESUMO
RNA is an important target for chemical probes of function and lead therapeutics; however, it is difficult to target with small molecules. One approach to tackle this problem is to identify compounds that target RNA structures and utilize them to multivalently target RNA. Here we show that small molecules can be identified to selectively bind RNA base pairs by probing a library of RNA-focused small molecules. A small molecule that selectively binds AU base pairs informed design of a dimeric compound (2AU-2) that targets the pathogenic RNA, expanded r(AUUCU) repeats, that causes spinocerebellar ataxia type 10 (SCA10) in patient-derived cells. Indeed, 2AU-2 (50 nM) ameliorates various aspects of SCA10 pathology including improvement of mitochondrial dysfunction, reduced activation of caspase 3, and reduction of nuclear foci. These studies provide a first-in-class chemical probe to study SCA10 RNA toxicity and potentially define broadly applicable compounds targeting RNA AU base pairs in cells.
Assuntos
Ataxina-10/antagonistas & inibidores , Repetições de Microssatélites , Fármacos Neuroprotetores/síntese química , Splicing de RNA/efeitos dos fármacos , RNA Mensageiro/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/síntese química , Ataxina-10/genética , Ataxina-10/metabolismo , Pareamento de Bases , Caspase 3/genética , Caspase 3/metabolismo , Expansão das Repetições de DNA/genética , Desenho de Fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Células HeLa , Humanos , Mitocôndrias/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Cultura Primária de Células , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Ataxias Espinocerebelares/tratamento farmacológico , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/patologia , Relação Estrutura-AtividadeRESUMO
Chlamydia trachomatis is the leading causative agent of bacterial sexually transmitted infections worldwide which can lead to female pelvic inflammatory disease and infertility. A greater understanding of host response during chlamydial infection is essential to design intervention technique to reduce the increasing incidence rate of genital chlamydial infection. In this study, we investigated proteome changes in epithelial cells during C. trachomatis infection by using an isobaric tags for relative and absolute quantitation (iTRAQ) labeling technique coupled with a liquid chromatography-tandem mass spectrometry (LC-MS(3) ) analysis. C. trachomatis (serovar D, MOI 1)-infected HeLa-229 human cervical carcinoma epithelial cells (at 2, 4 and 8 h) showed profound modifications of proteome profile which involved 606 host proteins. MGST1, SUGP2 and ATXN10 were among the top in the list of the differentially upregulated protein. Through pathway analysis, we suggested the involvement of eukaryotic initiation factor 2 (eIF2) and mammalian target of rapamycin (mTOR) in host cells upon C. trachomatis infection. Network analysis underscored the participation of DNA repair mechanism during C. trachomatis infection. In summary, intense modifications of proteome profile in C. trachomatis-infected HeLa-229 cells indicate complex host-pathogen interactions at early phase of chlamydial infection.
Assuntos
Chlamydia trachomatis/crescimento & desenvolvimento , Fator de Iniciação 2 em Eucariotos/genética , Interações Hospedeiro-Patógeno , Serina-Treonina Quinases TOR/genética , Ataxina-10/genética , Ataxina-10/metabolismo , Chlamydia trachomatis/patogenicidade , Cromatografia Líquida , Fator de Iniciação 2 em Eucariotos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Redes e Vias Metabólicas/genética , Proteômica/métodos , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Transdução de Sinais , Coloração e Rotulagem/métodos , Serina-Treonina Quinases TOR/metabolismo , Espectrometria de Massas em Tandem , Fatores de TempoRESUMO
Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant neurologic disorder caused by ATTCT expansion in the ATXN10 gene. Previous investigations have identified that depletion of Ataxin-10, the gene product, leads to cellular apoptosis and cytokinesis failure. Herein we identify the mitotic kinase Aurora B as an Ataxin-10 interacting partner. Aurora B interacts with and phosphorylates Ataxin-10 at S12, as evidenced by in vitro kinase and mass spectrometry analysis. Both endogenous and S12-phosphorylated Ataxin-10 localizes to the midbody during cytokinesis, and cytokinetic defects induced by inhibition of ATXN10 expression is not rescued by the S12A mutant. Inhibition of Aurora B or expression of the S12A mutant renders reduced interaction between Ataxin-10 and polo-like kinase 1 (Plk1), a kinase previously identified to regulate Ataxin-10 in cytokinesis. Taken together, we propose a model that Aurora B phosphorylates Ataxin-10 at S12 to promote the interaction between Ataxin-10 and Plk1 in cytokinesis. These findings identify an Aurora B-dependent mechanism that implicates Ataxin-10 in cytokinesis.
