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1.
Proc Natl Acad Sci U S A ; 117(1): 415-425, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31871160

RESUMO

Transposable elements make up half of the mammalian genome. One of the most abundant is the short interspersed nuclear element (SINE). Among their million copies, B2 accounts for ∼350,000 in the mouse genome and has garnered special interest because of emerging roles in epigenetic regulation. Our recent work demonstrated that B2 RNA binds stress genes to retard transcription elongation. Although epigenetically silenced, B2s become massively up-regulated during thermal and other types of stress. Specifically, an interaction between B2 RNA and the Polycomb protein, EZH2, results in cleavage of B2 RNA, release of B2 RNA from chromatin, and activation of thermal stress genes. Although an established RNA-binding protein and histone methyltransferase, EZH2 is not known to be a nuclease. Here, we provide evidence for the surprising conclusion that B2 is a self-cleaving ribozyme. Ribozyme activity depends on Mg+2 and monovalent cations but is resistant to protease treatment. However, contact with EZH2 accelerates cleavage rate by >100-fold, suggesting that EZH2 promotes a cleavage-competent RNA conformation. B2 modification-interference analysis demonstrates that phosphorothioate changes at A and C nucleotides can substitute for EZH2. B2 nucleotides 45 to 55 and 100 to 101 are essential for activity. Finally, another family of SINEs, the human ALU element, also produces a self-cleaving RNA and is cleaved during T-cell activation as well as thermal and endoplasmic reticulum (ER) stress. Thus, B2/ALU SINEs may be classified as "epigenetic ribozymes" that function as transcriptional switches during stress. Given their high copy numbers, B2 and ALU may represent the predominant ribozyme activity in mammalian cells.


Assuntos
Elementos Alu/fisiologia , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Epigênese Genética , RNA Catalítico/metabolismo , Animais , Cromatina/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Proteína Potenciadora do Homólogo 2 de Zeste/isolamento & purificação , Células HeLa , Humanos , Células Jurkat , Camundongos , Conformação de Ácido Nucleico , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9 , Transcrição Gênica/fisiologia
3.
Blood ; 129(17): 2420-2428, 2017 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-28167662

RESUMO

The endoplasmic reticulum kinase inositol-requiring enzyme 1 (IRE1) and its downstream target X-box-binding protein 1 (XBP1) drive B-cell differentiation toward plasma cells and have been shown to contribute to multiple myeloma development; yet, little is known of the role of this pathway in diffuse large B-cell lymphoma (DLBCL). Here, we show that in the germinal center B-cell-like (GCB) DLBCL subtype, IRE1 expression is reduced to a level that prevents XBP1 activation. Gene expression profiles indicated that, in GCB DLBCL cancer samples, expression of IRE1 messenger RNA was inversely correlated with the levels and activity of the epigenetic repressor, histone methyltransferase enhancer of zeste homolog 2 (EZH2). Correspondingly, in GCB-derived cell lines, the IRE1 promoter carried increased levels of the repressive epigenetic mark histone 3 lysine 27 trimethylation. Pharmacological inhibition of EZH2 erased those marks and restored IRE1 expression and function in vitro and in vivo. Moreover, reconstitution of the IRE1-signaling pathway, by expression of the XBP1-active form, compromised GCB DLBCL tumor growth in a mouse xenograft cancer model. These findings indicate that IRE1-XBP1 downregulation distinguishes GCB DLBCL from other DLBCL subtypes and contributes to tumor growth.


Assuntos
Linfócitos B/imunologia , Endorribonucleases/genética , Regulação Neoplásica da Expressão Gênica , Centro Germinativo/imunologia , Linfoma Difuso de Grandes Células B/imunologia , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Proteína 1 de Ligação a X-Box/genética , Animais , Linfócitos B/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Endorribonucleases/antagonistas & inibidores , Endorribonucleases/imunologia , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/imunologia , Epigênese Genética , Centro Germinativo/patologia , Histonas/genética , Histonas/imunologia , Humanos , Indazóis/farmacologia , Leupeptinas/farmacologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Linfoma Difuso de Grandes Células B/terapia , Camundongos , Camundongos da Linhagem 129 , Plasmócitos/imunologia , Plasmócitos/patologia , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/imunologia , Piridonas/farmacologia , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/imunologia , Transdução de Sinais , Proteína 1 de Ligação a X-Box/antagonistas & inibidores , Proteína 1 de Ligação a X-Box/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Proc Natl Acad Sci U S A ; 113(32): E4671-80, 2016 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-27462105

RESUMO

Inflammasomes are critical sensors that convey cellular stress and pathogen presence to the immune system by activating inflammatory caspases and cytokines such as IL-1ß. The nature of endogenous stress signals that activate inflammasomes remains unclear. Here we show that an inhibitor of the HIV aspartyl protease, Nelfinavir, triggers inflammasome formation and elicits an IL-1R-dependent inflammation in mice. We found that Nelfinavir impaired the maturation of lamin A, a structural component of the nuclear envelope, thereby promoting the release of DNA in the cytosol. Moreover, deficiency of the cytosolic DNA-sensor AIM2 impaired Nelfinavir-mediated inflammasome activation. These findings identify a pharmacologic activator of inflammasome and demonstrate the role of AIM2 in detecting endogenous DNA release upon perturbation of nuclear envelope integrity.


Assuntos
Inflamassomos/efeitos dos fármacos , Nelfinavir/farmacologia , Membrana Nuclear/efeitos dos fármacos , Animais , Proteínas Adaptadoras de Sinalização CARD/fisiologia , Caspase 1/metabolismo , DNA/metabolismo , Inflamassomos/fisiologia , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Membrana Nuclear/fisiologia , Receptores de Interleucina-1/fisiologia
5.
Proc Natl Acad Sci U S A ; 113(2): E117-26, 2016 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-26715744

RESUMO

Inhibitors of the HIV aspartyl protease [HIV protease inhibitors (HIV-PIs)] are the cornerstone of treatment for HIV. Beyond their well-defined antiretroviral activity, these drugs have additional effects that modulate cell viability and homeostasis. However, little is known about the virus-independent pathways engaged by these molecules. Here we show that the HIV-PI Nelfinavir decreases translation rates and promotes a transcriptional program characteristic of the integrated stress response (ISR). Mice treated with Nelfinavir display hallmarks of this stress response in the liver, including α subunit of translation initiation factor 2 (eIF2α) phosphorylation, activating transcription factor-4 (ATF4) induction, and increased expression of known downstream targets. Mechanistically, Nelfinavir-mediated ISR bypassed direct activation of the eIF2α stress kinases and instead relied on the inhibition of the constitutive eIF2α dephosphorylation and down-regulation of the phophatase cofactor CReP (Constitutive Repressor of eIF2α Phosphorylation; also known as PPP1R15B). These findings demonstrate that the modulation of eIF2α-specific phosphatase cofactor activity can be a rheostat of cellular homeostasis that initiates a functional ISR and suggest that the HIV-PIs could be repositioned as therapeutics in human diseases to modulate translation rates and stress responses.


Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Inibidores da Protease de HIV/farmacologia , Protease de HIV/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Células HeLa , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Camundongos , Nelfinavir/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 1/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/metabolismo , Transcrição Gênica/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , eIF-2 Quinase/metabolismo
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