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1.
RNA ; 24(12): 1785-1802, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30232101

RESUMO

While a large number of long noncoding RNAs (lncRNAs) are transcribed from the genome of higher eukaryotes, systematic prediction of their functionality has been challenging due to the lack of conserved sequence motifs or structures. Assuming that some lncRNAs function as large ribonucleoprotein complexes and thus are easily crosslinked to proteins upon UV irradiation, we performed RNA-seq analyses of RNAs recovered from the aqueous phase after UV irradiation and phenol-chloroform extraction (UPA-seq). As expected, the numbers of UPA-seq reads mapped to known functional lncRNAs were remarkably reduced upon UV irradiation. Comparison with ENCODE eCLIP data revealed that lncRNAs that exhibited greater decreases upon UV irradiation preferentially associated with proteins containing prion-like domains (PrLDs). Fluorescent in situ hybridization (FISH) analyses revealed the nuclear localization of novel functional lncRNA candidates, including one that accumulated at the site of transcription. We propose that UPA-seq provides a useful tool for the selection of lncRNA candidates to be analyzed in depth in subsequent functional studies.


Assuntos
Complexos Multiproteicos/genética , RNA Longo não Codificante/genética , Ribonucleoproteínas/genética , Proteínas Ligadas por GPI/síntese química , Proteínas Ligadas por GPI/genética , Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Complexos Multiproteicos/química , Complexos Multiproteicos/efeitos da radiação , Príons/síntese química , Príons/genética , RNA Longo não Codificante/química , RNA Longo não Codificante/efeitos da radiação , Ribonucleoproteínas/química , Ribonucleoproteínas/efeitos da radiação , Raios Ultravioleta
2.
Biochem J ; 475(15): 2559-2576, 2018 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-30045873

RESUMO

Hsp90 is an ATP-dependent molecular chaperone that assists folding and conformational maturation/maintenance of many proteins. It is a potential cancer drug target because it chaperones oncoproteins. A prokaryotic homolog of Hsp90 (HtpG) is essential for thermo-tolerance in some bacteria and virulence of zoonotic pathogens. To identify a new class of small molecules which target prokaryotic and eukaryotic Hsp90s, we studied the effects of a naturally occurring cyclic sesquiterpene, zerumbone, which inhibits proliferation of a wide variety of tumor cells, on the activity of Hsp90. Zerumbone enhanced the ATPase activity of cyanobacterial Hsp90 (Hsp90SE), yeast Hsp90, and human Hsp90α. It also enhanced the catalytic efficiency of Hsp90SE by greatly increasing kcat Mass analysis showed that zerumbone binds to cysteine side chains of Hsp90SE covalently. Mutational studies identified 3 cysteine residues (one per each domain of Hsp90SE) that are involved in the enhancement, suggesting the presence of allosteric sites in the middle and C-terminal domains of Hsp90SE Treatment of cyanobacterial cells with zerumbone caused them to become very temperature-sensitive, a phenotype reminiscent of cyanobacterial Hsp90 mutants, and also decreased the cellular level of linker polypeptides that are clients for Hsp90SE Zerumbone showed cellular toxicity on cancer-derived mammalian cells by inducing apoptosis. In addition, zerumbone inhibited the binding of Hsp90/Cdc37 to client kinases. Altogether, we conclude that modification of cysteine residues of Hsp90 by zerumbone enhances its ATPase activity and inhibits physiological Hsp90 function. The activation of Hsp90 may provide new strategies to inhibit its chaperone function in cells.


Assuntos
Adenosina Trifosfatases , Proteínas de Bactérias , Cianobactérias/enzimologia , Citotoxinas , Proteínas de Choque Térmico HSP90 , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Sesquiterpenos , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cisteína/química , Cisteína/metabolismo , Estabilidade Enzimática/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Sesquiterpenos/química , Sesquiterpenos/farmacologia
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