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1.
Cell ; 183(2): 474-489.e17, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33035451

RESUMO

Mg2+ is the most abundant divalent cation in metazoans and an essential cofactor for ATP, nucleic acids, and countless metabolic enzymes. To understand how the spatio-temporal dynamics of intracellular Mg2+ (iMg2+) are integrated into cellular signaling, we implemented a comprehensive screen to discover regulators of iMg2+ dynamics. Lactate emerged as an activator of rapid release of Mg2+ from endoplasmic reticulum (ER) stores, which facilitates mitochondrial Mg2+ (mMg2+) uptake in multiple cell types. We demonstrate that this process is remarkably temperature sensitive and mediated through intracellular but not extracellular signals. The ER-mitochondrial Mg2+ dynamics is selectively stimulated by L-lactate. Further, we show that lactate-mediated mMg2+ entry is facilitated by Mrs2, and point mutations in the intermembrane space loop limits mMg2+ uptake. Intriguingly, suppression of mMg2+ surge alleviates inflammation-induced multi-organ failure. Together, these findings reveal that lactate mobilizes iMg2+ and links the mMg2+ transport machinery with major metabolic feedback circuits and mitochondrial bioenergetics.


Assuntos
Retículo Endoplasmático/metabolismo , Ácido Láctico/metabolismo , Magnésio/metabolismo , Animais , Células COS , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Chlorocebus aethiops , Retículo Endoplasmático/fisiologia , Feminino , Células HeLa , Células Hep G2 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo
2.
Proc Natl Acad Sci U S A ; 117(28): 16383-16390, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601238

RESUMO

Calcium uptake by the mitochondrial calcium uniporter coordinates cytosolic signaling events with mitochondrial bioenergetics. During the past decade all protein components of the mitochondrial calcium uniporter have been identified, including MCU, the pore-forming subunit. However, the specific lipid requirements, if any, for the function and formation of this channel complex are currently not known. Here we utilize yeast, which lacks the mitochondrial calcium uniporter, as a model system to address this problem. We use heterologous expression to functionally reconstitute human uniporter machinery both in wild-type yeast as well as in mutants defective in the biosynthesis of phosphatidylethanolamine, phosphatidylcholine, or cardiolipin (CL). We uncover a specific requirement of CL for in vivo reconstituted MCU stability and activity. The CL requirement of MCU is evolutionarily conserved with loss of CL triggering rapid turnover of MCU homologs and impaired calcium transport. Furthermore, we observe reduced abundance and activity of endogenous MCU in mammalian cellular models of Barth syndrome, which is characterized by a partial loss of CL. MCU abundance is also decreased in the cardiac tissue of Barth syndrome patients. Our work raises the hypothesis that impaired mitochondrial calcium transport contributes to the pathogenesis of Barth syndrome, and more generally, showcases the utility of yeast phospholipid mutants in dissecting the phospholipid requirements of ion channel complexes.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Síndrome de Barth/genética , Síndrome de Barth/metabolismo , Transporte Biológico , Canais de Cálcio/química , Canais de Cálcio/genética , Cardiolipinas/genética , Cardiolipinas/metabolismo , Humanos , Camundongos , Mitocôndrias/química , Mitocôndrias/genética , Mioblastos/metabolismo , Fosfolipídeos , Estabilidade Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
3.
EMBO Rep ; 21(10): e48483, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-32851774

RESUMO

MICU1 is a mitochondrial inner membrane protein that inhibits mitochondrial calcium entry; elevated MICU1 expression is characteristic of many cancers, including ovarian cancer. MICU1 induces both glycolysis and chemoresistance and is associated with poor clinical outcomes. However, there are currently no available interventions to normalize aberrant MICU1 expression. Here, we demonstrate that microRNA-195-5p (miR-195) directly targets the 3' UTR of the MICU1 mRNA and represses MICU1 expression. Additionally, miR-195 is under-expressed in ovarian cancer cell lines, and restoring miR-195 expression reestablishes native MICU1 levels and the associated phenotypes. Stable expression of miR-195 in a human xenograft model of ovarian cancer significantly reduces tumor growth, increases tumor doubling times, and enhances overall survival. In conclusion, miR-195 controls MICU1 levels in ovarian cancer and could be exploited to normalize aberrant MICU1 expression, thus reversing both glycolysis and chemoresistance and consequently improving patient outcomes.


Assuntos
Proteínas de Transporte de Cátions , MicroRNAs , Neoplasias Ovarianas , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Glicólise/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Neoplasias Ovarianas/genética
4.
Hum Mol Genet ; 27(10): 1794-1808, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29547888

RESUMO

The TMEM127 tumor suppressor gene encodes a transmembrane protein of unknown function mutated in pheochromocytomas and, rarely, in renal cancers. Tumors with inactivating TMEM127 mutations have increased mTORC1 signaling by undefined mechanisms. Here we report that TMEM127 interacts with the lysosome-anchored complex comprised of Rag GTPases, the LAMTOR pentamer (or 'ragulator') and vATPase, which controls amino acid-mediated mTORC1 activation. We found that under nutrient-rich conditions TMEM127 expression reduces mTORC1 recruitment to Rags. In addition, TMEM127 interacts with LAMTOR in an amino acid-dependent manner and decreases the LAMTOR1-vATPase association, while TMEM127-vATPase binding requires intact lysosomal acidification but is amino acid independent. Conversely, both murine and human cells lacking TMEM127 accumulate LAMTOR proteins in the lysosome. Consistent with these findings, pheochromocytomas with TMEM127 mutations have increased levels of LAMTOR proteins. These results suggest that TMEM127 interactions with ragulator and vATPase at the lysosome contribute to restrain mTORC1 signaling in response to amino acids, thus explaining the increased mTORC1 activation seen in TMEM127-deficient tumors.


Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Proteínas de Transporte/genética , Proteínas de Membrana/genética , Feocromocitoma/genética , Neoplasias das Glândulas Suprarrenais/metabolismo , Neoplasias das Glândulas Suprarrenais/patologia , Aminoácidos/genética , Animais , Regulação da Expressão Gênica , Genes Supressores de Tumor , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lisossomos/genética , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Complexos Multiproteicos/genética , Mutação , Feocromocitoma/metabolismo , Feocromocitoma/patologia , Transdução de Sinais
5.
Mol Cell ; 47(4): 648-55, 2012 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-22841487

RESUMO

Mammalian long intergenic noncoding RNAs (lincRNAs) are best known for modulating transcription. Here we report a posttranscriptional function for lincRNA-p21 as a modulator of translation. Association of the RNA-binding protein HuR with lincRNA-p21 favored the recruitment of let-7/Ago2 to lincRNA-p21, leading to lower lincRNA-p21 stability. Under reduced HuR levels, lincRNA-p21 accumulated in human cervical carcinoma HeLa cells, increasing its association with JUNB and CTNNB1 mRNAs and selectively lowering their translation. With elevated HuR, lincRNA-p21 levels declined, which in turn derepressed JunB and ß-catenin translation and increased the levels of these proteins. We propose that HuR controls translation of a subset of target mRNAs by influencing lincRNA-p21 levels. Our findings uncover a role for lincRNA as a posttranscriptional inhibitor of translation.


Assuntos
Biossíntese de Proteínas , Processamento Pós-Transcricional do RNA , RNA Longo não Codificante/genética , Sequência de Bases , Carboxipeptidases/genética , Carboxipeptidases/metabolismo , Proteínas ELAV/genética , Proteínas ELAV/metabolismo , Células HeLa , Humanos , MicroRNAs/genética , Dados de Sequência Molecular , Proteólise , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Células Tumorais Cultivadas , beta Catenina/genética , beta Catenina/metabolismo
6.
Mol Cell ; 43(3): 319-21, 2011 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-21816340

RESUMO

The RNA-binding protein HuR, while known to stabilize cytoplasmic mRNAs, is largely nuclear. In this issue of Molecular Cell, Mukherjee et al. (2011) and Lebedeva et al. (2011) identify transcriptome-wide HuR-RNA interactions using PAR-CLIP, unveiling HuR's nuclear role in pre-mRNA processing.

7.
Genes Dev ; 23(15): 1743-8, 2009 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-19574298

RESUMO

RNA-binding proteins (RBPs) and microRNAs (miRNAs) are potent post-transcriptional regulators of gene expression. Here, we show that the RBP HuR reduced c-Myc expression by associating with the c-Myc 3' untranslated region (UTR) next to a miRNA let-7-binding site. Lowering HuR or let-7 levels relieved the translational repression of c-Myc. Unexpectedly, HuR and let-7 repressed c-Myc through an interdependent mechanism, as let-7 required HuR to reduce c-Myc expression and HuR required let-7 to inhibit c-Myc expression. Our findings suggest a regulatory paradigm wherein HuR inhibits c-Myc expression by recruiting let-7-loaded RISC (RNA miRNA-induced silencing complex) to the c-Myc 3'UTR.


Assuntos
Antígenos de Superfície/metabolismo , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas de Ligação a RNA/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Regiões 3' não Traduzidas , Proteínas Argonautas , Sequência de Bases , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Fator de Iniciação 2 em Eucariotos/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/metabolismo
8.
Nucleic Acids Res ; 42(2): 1196-208, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24106086

RESUMO

In response to stress conditions, many mammalian mRNAs accumulate in stress granules (SGs) together with numerous RNA-binding proteins that control mRNA turnover and translation. However, the signaling cascades that modulate the presence of ribonucleoprotein (RNP) complexes in SGs are poorly understood. Here, we investigated the localization of human antigen R (HuR), an mRNA-stabilizing RNA-binding protein, in SGs following exposure to the stress agent arsenite. Unexpectedly, the mobilization of HuR to SGs was prevented through the activation of Janus kinase 3 (JAK3) by the vitamin K3 analog menadione. JAK3 phosphorylated HuR at tyrosine 200, in turn inhibiting HuR localization in SGs, reducing HuR interaction with targets SIRT1 and VHL mRNAs, and accelerating target mRNA decay. Our findings indicate that HuR is tyrosine-phosphorylated by JAK3, and link this modification to HuR subcytoplasmic localization and to the fate of HuR target mRNAs.


Assuntos
Proteínas ELAV/metabolismo , Janus Quinase 3/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Tirosina/metabolismo , Grânulos Citoplasmáticos/metabolismo , Proteínas ELAV/química , Células HeLa , Humanos , Fosforilação
9.
EMBO J ; 30(6): 1040-53, 2011 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-21317874

RESUMO

Ionizing radiation (IR) triggers adaptive changes in gene expression. Here, we show that survival after IR strongly depends on the checkpoint kinase Chk2 acting upon its substrate HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. Microarray analysis showed that in human HCT116 colorectal carcinoma cells (WT), IR-activated Chk2 triggered the dissociation of virtually all of HuR-bound mRNAs, since IR did not dissociate HuR target mRNAs in Chk2-null (CHK2-/-) HCT116 cells. Accordingly, several HuR-interacting mRNAs encoding apoptosis- and proliferation-related proteins (TJP1, Mdm2, TP53BP2, Bax, K-Ras) dissociated from HuR in WT cells, but remained bound and showed altered post-transcriptional regulation in CHK2-/- cells. Use of HuR mutants that were not phosphorylatable by Chk2 (HuR(3A)) and HuR mutants mimicking constitutive phosphorylation by Chk2 (HuR(3D)) revealed that dissociation of HuR target transcripts enhanced cell survival. We propose that the release of HuR-bound mRNAs via an IR-Chk2-HuR regulatory axis improves cell outcome following IR.


Assuntos
Antígenos de Superfície/metabolismo , Células Epiteliais/efeitos da radiação , Regulação da Expressão Gênica , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Radiação Ionizante , Antígenos de Superfície/genética , Linhagem Celular , Sobrevivência Celular , Quinase do Ponto de Checagem 2 , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Técnicas de Inativação de Genes , Humanos , Proteínas de Ligação a RNA/genética
10.
Nucleic Acids Res ; 40(22): 11531-44, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23066106

RESUMO

MicroRNA (miRNA) biogenesis is tightly regulated by numerous proteins. Among them, Dicer is required for the processing of the precursor (pre-)miRNAs into the mature miRNA. Despite its critical function, the mechanisms that regulate Dicer expression are not well understood. Here we report that the RNA-binding protein (RBP) AUF1 (AU-binding factor 1) associates with the endogenous DICER1 mRNA and can interact with several segments of DICER1 mRNA within the coding region (CR) and the 3'-untranslated region (UTR). Through these interactions, AUF1 lowered DICER1 mRNA stability, since silencing AUF1 lengthened DICER1 mRNA half-life and increased Dicer expression, while overexpressing AUF1 lowered DICER1 mRNA and Dicer protein levels. Given that Dicer is necessary for the synthesis of mature miRNAs, the lowering of Dicer levels by AUF1 diminished the levels of miRNAs tested, but not the levels of the corresponding pre-miRNAs. In summary, AUF1 suppresses miRNA production by reducing Dicer production.


Assuntos
RNA Helicases DEAD-box/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Ribonuclease III/genética , Regiões 3' não Traduzidas , Linhagem Celular , RNA Helicases DEAD-box/metabolismo , Regulação da Expressão Gênica , Ribonucleoproteína Nuclear Heterogênea D0 , Humanos , MicroRNAs/metabolismo , Neoplasias/enzimologia , Neoplasias/metabolismo , Ribonuclease III/metabolismo
11.
EMBO J ; 28(9): 1271-82, 2009 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-19322201

RESUMO

The RNA-binding protein HuR regulates the stability and translation of numerous mRNAs encoding stress-response and proliferative proteins. Although its post-transcriptional influence has been linked primarily to its cytoplasmic translocation, here we report that moderate heat shock (HS) potently reduces HuR levels, thereby altering the expression of HuR target mRNAs. HS did not change HuR mRNA levels or de novo translation, but instead reduced HuR protein stability. Supporting the involvement of the ubiquitin-proteasome system in this process were results showing that (1) HuR was ubiquitinated in vitro and in intact cells, (2) proteasome inhibition increased HuR abundance after HS, and (3) the HuR kinase checkpoint kinase 2 protected against the loss of HuR by HS. Within a central, HS-labile approximately 110-amino-acid region, K182 was found to be essential for HuR ubiquitination and proteolysis as mutant HuR(K182R) was left virtually unubiquitinated and was refractory to HS-triggered degradation. Our findings reveal that HS transiently lowers HuR by proteolysis linked to K182 ubiquitination and that HuR reduction enhances cell survival following HS.


Assuntos
Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Temperatura Alta , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ubiquitina/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Quinase do Ponto de Checagem 2 , Dactinomicina/farmacologia , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Lisina/metabolismo , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade Proteica , Ubiquitina/química , Ubiquitinação/genética , Ubiquitinação/fisiologia
12.
Methods ; 58(2): 81-7, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22813890

RESUMO

Cellular transcripts of all types, including coding messenger (m)RNAs and noncoding (nc)RNAs, are subject to extensive post-transcriptional regulation. Among the factors that elicit post-transcriptional control, microRNAs (miRNAs) have emerged as a major class of small regulatory RNAs. Since RNA-RNA interactions can be modeled computationally, several excellent programs have been developed to predict the interaction of miRNAs with target transcripts. However, many such predictions are not realized for different reasons, including absent or low-abundance expression of the miRNA in the cell, the existence of competing factors or conformational changes masking the microRNA site, and the possibility that target transcripts are not present in the prediction databases, as is the case for long ncRNAs. Here, we provide a systematic approach termed MS2-TRAP (tagged RNA affinity purification) for identifying miRNAs associated with a target transcript in the cellular context. We illustrate the use of this methodology by identifying microRNAs that associate with a long intergenic (li)ncRNA, based on the expression of the lincRNA tagged with MS2 RNA hairpins (lincRNA-p21-MS2) and the concomitant expression of a fusion protein recognizing the MS2 RNA hairpins, MS2-GST. After affinity pulldown of the ribonucleoprotein (RNP) complex comprising [MS2-GST/lincRNA-p21-MS2], the RNA in the pulldown material was isolated and reverse transcribed (RT). Subsequent assessment of the microRNAs present in the pulldown complex by using real-time quantitative (q)PCR analysis led to the identification of bona fide miRNAs that interact with and control the abundance of lincRNA-p21. We describe alternative designs and applications of this approach, and discuss its implications in deciphering post-transcriptional gene regulatory schemes.


Assuntos
Biologia Computacional/métodos , MicroRNAs , RNA Longo não Codificante , RNA Mensageiro , RNA , Cromatografia de Afinidade , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , MicroRNAs/química , MicroRNAs/genética , RNA/química , RNA/genética , RNA Longo não Codificante/química , RNA Longo não Codificante/genética , RNA Mensageiro/química , RNA Mensageiro/genética
13.
Gen Comp Endocrinol ; 186: 25-32, 2013 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-23453961

RESUMO

Salivary glands, although widely considered as typically exocrine, may also release specific proteins in an endocrine manner. However, endocrine release of salivary gland proteins is not generally acknowledged since the evidences are not easily demonstrable. Submandibular salivary glands (SMG) of male Syrian hamsters express male-specific secretory proteins (MSP; which are lipocalins) visible in SDS-PAGE of SMG extracts, as major bands and also detectable in immunoblots of whole-saliva and urine as low MSP crossreactions. We report here that MSP is localized in acinar cells of SMG and acute treatment with isoproterenol (IPR; non-specific ß1/ß2-adrenergic agonist) results in considerable release of MSP in SMG-saliva. Moreover, acute IPR treatment markedly depletes SMG-MSP in a dose- and time-dependent manner. However, MSP depleted from SMG, far exceeds that recovered in SMG-saliva. Blood, submandibular lymph nodes and kidney of IPR-treated males showed MSP crossreactions and SDS-PAGE of their urine revealed profuse MSP excretion; this was undetectable in IPR-treated-SMG-ablated males, confirming that a substantial amount of MSP depleted from SMG after IPR treatment enters circulation and is excreted in urine. Treatments with specific ß1- or ß2-adrenergic agonists also reduced SMG-MSP levels and resulted in copious urinary excretion of MSP. Co-treatments with specific ß1/ß2-blockers indicated that above effects of IPR, ß1- and even ß2-agonists are very likely mediated by ß1-adrenoceptors. MSP's detection by SDS-PAGE in urine after ß-agonist treatment is a compelling and easily demonstrable evidence of release into circulation of a salivary gland protein. The possible means (endocrine-like or otherwise) of MSP's release into circulation and significance of its presence in saliva, blood and urine of male hamsters are discussed.


Assuntos
Proteínas e Peptídeos Salivares/urina , Células Acinares/efeitos dos fármacos , Células Acinares/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Cricetinae , Eletroforese em Gel de Poliacrilamida , Isoproterenol/farmacologia , Rim/efeitos dos fármacos , Rim/metabolismo , Lipocalinas/urina , Masculino , Mesocricetus
14.
Nucleic Acids Res ; 39(19): 8513-30, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21737422

RESUMO

RNA-binding proteins (RBPs) regulate gene expression at many post-transcriptional levels, including mRNA stability and translation. The RBP nucleolin, with four RNA-recognition motifs, has been implicated in cell proliferation, carcinogenesis and viral infection. However, the subset of nucleolin target mRNAs and the influence of nucleolin on their expression had not been studied at a transcriptome-wide level. Here, we globally identified nucleolin target transcripts, many of which encoded cell growth- and cancer-related proteins, and used them to find a signature motif on nucleolin target mRNAs. Surprisingly, this motif was very rich in G residues and was not only found in the 3'-untranslated region (UTR), but also in the coding region (CR) and 5'-UTR. Nucleolin enhanced the translation of mRNAs bearing the G-rich motif, since silencing nucleolin did not change target mRNA stability, but decreased the size of polysomes forming on target transcripts and lowered the abundance of the encoded proteins. In summary, nucleolin binds G-rich sequences in the CR and UTRs of target mRNAs, many of which encode cancer proteins, and enhances their translation.


Assuntos
Fosfoproteínas/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Genes Reporter , Guanina/análise , Células HeLa , Humanos , Proteínas de Neoplasias/genética , Motivos de Nucleotídeos , Estabilidade de RNA , RNA Mensageiro/química , Transcriptoma , Regiões não Traduzidas , Nucleolina
15.
iScience ; 25(1): 103722, 2022 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-35005527

RESUMO

SARS-CoV-2 is a newly identified coronavirus that causes the respiratory disease called coronavirus disease 2019 (COVID-19). With an urgent need for therapeutics, we lack a full understanding of the molecular basis of SARS-CoV-2-induced cellular damage and disease progression. Here, we conducted transcriptomic analysis of human PBMCs, identified significant changes in mitochondrial, ion channel, and protein quality-control gene products. SARS-CoV-2 proteins selectively target cellular organelle compartments, including the endoplasmic reticulum and mitochondria. M-protein, NSP6, ORF3A, ORF9C, and ORF10 bind to mitochondrial PTP complex components cyclophilin D, SPG-7, ANT, ATP synthase, and a previously undescribed CCDC58 (coiled-coil domain containing protein 58). Knockdown of CCDC58 or mPTP blocker cyclosporin A pretreatment enhances mitochondrial Ca2+ retention capacity and bioenergetics. SARS-CoV-2 infection exacerbates cardiomyocyte autophagy and promotes cell death that was suppressed by cyclosporin A treatment. Our findings reveal that SARS-CoV-2 viral proteins suppress cardiomyocyte mitochondrial function that disrupts cardiomyocyte Ca2+ cycling and cell viability.

16.
Nucleic Acids Res ; 37(8): 2658-71, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19270063

RESUMO

The molecular basis underlying the aberrant DNA-methylation patterns in human cancer is largely unknown. Altered DNA methyltransferase (DNMT) activity is believed to contribute, as DNMT expression levels increase during tumorigenesis. Here, we present evidence that the expression of DNMT3b is post-transcriptionally regulated by HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. The presence of a putative HuR-recognition motif in the DNMT3b 3'UTR prompted studies to investigate if this transcript associated with HuR. The interaction between HuR and DNMT3b mRNA was studied by immunoprecipitation of endogenous HuR ribonucleoprotein complexes followed by RT-qPCR detection of DNMT3b mRNA, and by in vitro pulldown of biotinylated DNMT3b RNAs followed by western blotting detection of HuR. These studies revealed that binding of HuR stabilized the DNMT3b mRNA and increased DNMT3b expression. Unexpectedly, cisplatin treatment triggered the dissociation of the [HuR-DNMT3b mRNA] complex, in turn promoting DNMT3b mRNA decay, decreasing DNMT3b abundance, and lowering the methylation of repeated sequences and global DNA methylation. In summary, our data identify DNMT3b mRNA as a novel HuR target, present evidence that HuR affects DNMT3b expression levels post-transcriptionally, and reveal the functional consequences of the HuR-regulated DNMT3b upon DNA methylation patterns.


Assuntos
Antígenos de Superfície/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Antígenos de Superfície/análise , Antineoplásicos/farmacologia , Sequência de Bases , Linhagem Celular Tumoral , Cisplatino/farmacologia , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Humanos , Dados de Sequência Molecular , Proteínas de Ligação a RNA/análise , DNA Metiltransferase 3B
17.
Proc Natl Acad Sci U S A ; 105(51): 20297-302, 2008 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-19088191

RESUMO

Gene expression is potently regulated through the action of RNA-binding proteins (RBPs) and microRNAs (miRNAs). Here, we present evidence of a miRNA regulating an RBP. The RBP HuR can stabilize and modulate the translation of numerous target mRNAs involved in cell proliferation, but little is known about the mechanisms that regulate HuR abundance. We identified two putative sites of miR-519 interaction on the HuR mRNA, one in its coding region (CR), one in its 3'-untranslated region (UTR). In several human carcinoma cell lines tested, HeLa (cervical), HCT116 and RKO (colon), and A2780 (ovarian), overexpression of a miR-519 precursor [(Pre)miR-519] reduced HuR abundance, while inhibiting miR-519 by using an antisense RNA [(AS)miR-519] elevated HuR levels. The influence of miR-519 was recapitulated using heterologous reporter constructs that revealed a greater repressive effect on the HuR CR than the HuR 3'-UTR target sequences. miR-519 did not alter HuR mRNA abundance, but reduced HuR biosynthesis, as determined by measuring nascent HuR translation and HuR mRNA association with polysomes. Modulation of miR-519 leading to altered HuR levels in turn affected the levels of proteins encoded by HuR target mRNAs. In keeping with HuR's proliferative influence, (AS)miR-519 significantly increased cell number and [(3)H]-thymidine incorporation, while (Pre)miR-519 reduced these parameters. Importantly, the growth-promoting effects of (AS)miR-519 required the presence of HuR, because downregulation of HuR by RNAi dramatically suppressed its proliferative action. In sum, miR-519 represses HuR translation, in turn reducing HuR-regulated gene expression and cell division.


Assuntos
Antígenos de Superfície/genética , Proliferação de Células , Regulação da Expressão Gênica , MicroRNAs/fisiologia , Proteínas de Ligação a RNA/genética , Regiões 3' não Traduzidas , Antígenos de Superfície/biossíntese , Linhagem Celular Tumoral , Regulação para Baixo , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Humanos , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/biossíntese
18.
Artigo em Inglês | MEDLINE | ID: mdl-20445246

RESUMO

Proteins belonging to the lipocalin superfamily are usually secretory proteins of molecular mass approximately 20 kDa with a hydrophobic pocket for the binding and transport of diverse small ligands. Various lipocalins have been associated with many biological processes, e.g. immunomodulation, odorant transport, pheromonal activity, retinoid transport, cancer-cell interactions etc. However, the exact functions of many lipocalins and the ligands bound by them are unclear. Previously, the cDNA of a 20 kDa lipocalin (FLP) which is female-specifically expressed in the lacrimal glands of Syrian (golden) hamsters and secreted in the tears of females has been identified and cloned. His-tagged recombinant FLP (rFLP) has now been cloned, overexpressed in Escherichia coli as a soluble protein and purified to homogeneity using Ni-affinity followed by size-exclusion chromatography. Purified rFLP was crystallized using the sitting-drop vapour-diffusion method. The crystals tested belonged to space group P2(1)2(1)2(1) and diffracted to beyond 1.86 A resolution. Solvent-content analysis indicated the presence of one monomer in the asymmetric unit.


Assuntos
Aparelho Lacrimal/química , Lipocalinas/química , Mesocricetus , Animais , Clonagem Molecular , Cricetinae , Cristalização , Cristalografia por Raios X , Feminino , Expressão Gênica , Lipocalinas/genética , Lipocalinas/isolamento & purificação
19.
Cell Chem Biol ; 27(5): 538-550.e7, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32101699

RESUMO

Mitochondrial D2HGDH and L2HGDH catalyze the oxidation of D-2-HG and L-2-HG, respectively, into αKG. This contributes to cellular homeostasis in part by modulating the activity of αKG-dependent dioxygenases. Signals that control the expression/activity of D2HGDH/L2HGDH are presumed to broadly influence physiology and pathology. Using cell and mouse models, we discovered that MYC directly induces D2HGDH and L2HGDH transcription. Furthermore, in a manner suggestive of D2HGDH, L2HGDH, and αKG dependency, MYC activates TET enzymes and RNA demethylases, and promotes their nuclear localization. Consistent with these observations, in primary B cell lymphomas MYC expression positively correlated with enhancer hypomethylation and overexpression of lymphomagenic genes. Together, these data provide additional evidence for the role of mitochondria metabolism in influencing the epigenome and epitranscriptome, and imply that in specific contexts wild-type TET enzymes could demethylate and activate oncogenic enhancers.


Assuntos
Oxirredutases do Álcool/genética , Epigenoma , Linfoma de Células B/genética , Proteínas Proto-Oncogênicas c-myc/genética , Ativação Transcricional , Animais , Linhagem Celular , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Transcriptoma , Células Tumorais Cultivadas
20.
Sci Signal ; 13(628)2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32317369

RESUMO

The tricarboxylic acid (TCA) cycle converts the end products of glycolysis and fatty acid ß-oxidation into the reducing equivalents NADH and FADH2 Although mitochondrial matrix uptake of Ca2+ enhances ATP production, it remains unclear whether deprivation of mitochondrial TCA substrates alters mitochondrial Ca2+ flux. We investigated the effect of TCA cycle substrates on MCU-mediated mitochondrial matrix uptake of Ca2+, mitochondrial bioenergetics, and autophagic flux. Inhibition of glycolysis, mitochondrial pyruvate transport, or mitochondrial fatty acid transport triggered expression of the MCU gatekeeper MICU1 but not the MCU core subunit. Knockdown of mitochondrial pyruvate carrier (MPC) isoforms or expression of the dominant negative mutant MPC1R97W resulted in increased MICU1 protein abundance and inhibition of MCU-mediated mitochondrial matrix uptake of Ca2+ We also found that genetic ablation of MPC1 in hepatocytes and mouse embryonic fibroblasts resulted in reduced resting matrix Ca2+, likely because of increased MICU1 expression, but resulted in changes in mitochondrial morphology. TCA cycle substrate-dependent MICU1 expression was mediated by the transcription factor early growth response 1 (EGR1). Blocking mitochondrial pyruvate or fatty acid flux was linked to increased autophagy marker abundance. These studies reveal a mechanism that controls the MCU-mediated Ca2+ flux machinery and that depends on TCA cycle substrate availability. This mechanism generates a metabolic homeostatic circuit that protects cells from bioenergetic crisis and mitochondrial Ca2+ overload during periods of nutrient stress.


Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Ácidos Graxos/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Ácido Pirúvico/metabolismo , Animais , Transporte Biológico Ativo/genética , Canais de Cálcio/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte de Cátions/genética , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Camundongos Knockout , Mitocôndrias Hepáticas/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas Mitocondriais/genética
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