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1.
Cell Rep ; 43(1): 113593, 2024 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-38113140

RESUMO

Nuclear mRNA export via nuclear pore complexes is an essential step in eukaryotic gene expression. Although factors involved in mRNA transport have been characterized, a comprehensive mechanistic understanding of this process and its regulation is lacking. Here, we use single-RNA imaging in yeast to show that cells use mRNA retention to control mRNA export during stress. We demonstrate that, upon glucose withdrawal, the essential RNA-binding factor Nab2 forms RNA-dependent condensate-like structures in the nucleus. This coincides with a reduced abundance of the DEAD-box ATPase Dbp5 at the nuclear pore. Depleting Dbp5, and consequently blocking mRNA export, is necessary and sufficient to trigger Nab2 condensation. The state of Nab2 condensation influences the extent of nuclear mRNA accumulation and can be recapitulated in vitro, where Nab2 forms RNA-dependent liquid droplets. We hypothesize that cells use condensation to regulate mRNA export and control gene expression during stress.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Proteínas de Saccharomyces cerevisiae , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
2.
Heart Lung Circ ; 32(12): 1434-1442, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38042639

RESUMO

OBJECTIVE: This study systematically assessed circulating proteins to identify new serum biomarkers and risk of hypertension using Mendelian randomisation. METHODS: The associations between 4,782 human circulating proteins and the risk of hypertension were evaluated using two-sample Mendelian randomisation. The FinnGen study demonstrated a link between genetic predisposition and hypertension in 85,438 cases and 223,663 controls. RESULTS: Inverse variance weighted and sensitivity analysis revealed nine proteins in circulation that have a causative effect on hypertension. SMOC1 and TIE1 were determined to be causative factors in the decreased likelihood of developing hypertension, with odds ratios of 0.86 (95% CI 0.81-0.91; p=1.06e-06) and 0.96 (95% CI 0.94-0.98; p=9.39e-05), respectively. NDUFB4, ETHE1, POFUT2, TRIL, ADAM23, GXYLT1, OXT, TPST2, and TMCC3 showed a possible connection to hypertension. CONCLUSIONS: This two-sample Mendelian randomisation study found that SMOC1 and TIE1 are causally linked to hypertension, making them a promising target for therapy.


Assuntos
Hipertensão , Humanos , Biomarcadores , Hipertensão/epidemiologia , Hipertensão/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Estudo de Associação Genômica Ampla , Proteínas Mitocondriais , Proteínas de Transporte Nucleocitoplasmático
3.
Sci Rep ; 13(1): 21723, 2023 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-38066085

RESUMO

The contact inhibition of proliferation (CIP) denotes the cell density-dependent inhibition of growth, and the loss of CIP represents a hallmark of cancer. However, the mechanism by which CIP regulates gene expression remains poorly understood. Chromatin is a highly complex structure consisting of DNA, histones, and trans-acting factors (TAFs). The binding of TAF proteins to specific chromosomal loci regulates gene expression. Therefore, profiling chromatin is crucial for gaining insight into the gene expression mechanism of CIP. In this study, using modified proteomics of TAFs bound to DNA, we identified a protein that shuttles between the nucleus and cytosol in a cell density-dependent manner. We identified TIPARP, PTGES3, CBFB, and SMAD4 as cell density-dependent nucleocytoplasmic shuttling proteins. In low-density cells, these proteins predominantly reside in the nucleus; however, upon reaching high density, they relocate to the cytosol. Given their established roles in gene regulation, our findings propose their involvement as CIP-dependent TAFs. We also identified and characterized potential open chromatin regions sensitive to changes in cell density. These findings provide insights into the modulation of chromatin structure by CIP.


Assuntos
Núcleo Celular , Cromatina , Cromatina/genética , Cromatina/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Transativadores/metabolismo , DNA/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Contagem de Células
4.
Int J Biol Sci ; 19(16): 5319-5336, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37928256

RESUMO

Transfer RNAs (tRNAs) impact the development and progression of various cancers, but how individual tRNAs are modulated during triple-negative breast cancer (TNBC) progression remains poorly understood. Here, we found that XPOT (Exportin-T), a nuclear export protein receptor of tRNAs, is associated with poor prognosis in breast cancer and directly orchestrates the nuclear export of a subset of tRNAs, subsequently promoting protein synthesis and proliferation of human TNBC cells. XPOT knockdown inhibited TNBC cell proliferation in vitro, and RNA-seq indicated that XPOT is involved in the completion of cytokinesis in TNBC cells. High-throughput sequencing of tRNA revealed that XPOT specifically influenced a subset of tRNA isodecoders involved in nucleocytoplasmic trafficking, including tRNA-Ala-AGC-10-1. Through codon preferential analysis and protein mass spectrometry, we found that XPOT preferentially transported nuclear tRNA-Ala-AGC-10-1 to the cytoplasm, driving the translation of TPR Repeat Protein 19 (TTC19). TTC19 is also indispensable for cytokinesis and proliferation of TNBC cells. Altogether, these findings provide a novel regulatory translation mechanism for preferential tRNA isodecoder nucleocytoplasmic transport through XPOT, which coordinates the spatial location of specific tRNA and the translation of mRNA to facilitate TNBC proliferation and progression. Targeting XPOT may be a novel therapeutic strategy for treating TNBC.


Assuntos
Citocinese , Neoplasias de Mama Triplo Negativas , Humanos , Citocinese/genética , Neoplasias de Mama Triplo Negativas/genética , Proliferação de Células/genética , Transporte Biológico , Citoplasma , RNA de Transferência/genética , Linhagem Celular Tumoral , Proteínas de Transporte Nucleocitoplasmático
5.
Int J Mol Sci ; 24(19)2023 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-37834012

RESUMO

Triple-negative breast cancer (TNBC) is the most fatal subtype of breast cancer; however, effective treatment strategies for TNBC are lacking. Therefore, it is important to explore the mechanism of TNBC metastasis and identify its therapeutic targets. Dysregulation of ETHE1 leads to ethylmalonic encephalopathy in humans; however, the role of ETHE1 in TNBC remains elusive. Stable cell lines with ETHE1 overexpression or knockdown were constructed to explore the biological functions of ETHE1 during TNBC progression in vitro and in vivo. Mass spectrometry was used to analyze the molecular mechanism through which ETHE1 functions in TNBC progression. ETHE1 had no impact on TNBC cell proliferation and xenograft tumor growth but promoted TNBC cell migration and invasion in vitro and lung metastasis in vivo. The effect of ETHE1 on TNBC cell migratory potential was independent of its enzymatic activity. Mechanistic investigations revealed that ETHE1 interacted with eIF2α and enhanced its phosphorylation by promoting the interaction between eIF2α and GCN2. Phosphorylated eIF2α in turn upregulated the expression of ATF4, a transcriptional activator of genes involved in cell migration and tumor metastasis. Notably, inhibition of eIF2α phosphorylation through ISRIB or ATF4 knockdown partially abolished the tumor-promoting effect of ETHE1 overexpression. ETHE1 has a functional and mechanistic role in TNBC metastasis and offers a new therapeutic strategy for targeting ETHE1-propelled TNBC using ISRIB.


Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/patologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Linhagem Celular Tumoral , Transdução de Sinais , Proliferação de Células/genética , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Mitocondriais/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo
6.
Adv Biol Regul ; 90: 100990, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37801910

RESUMO

Gle1 regulates gene expression at multiple steps from transcription to mRNA export to translation under stressed and non-stressed conditions. To better understand Gle1 function in stressed human cells, specific antibodies were generated that recognized the phosphorylation of threonine residue 102 (T102) in Gle1. A series of in vitro kinase assays indicated that T102 phosphorylation serves as a priming event for further phosphorylation in Gle1's N-terminal low complexity cluster. Indirect immunofluorescence microscopy with the anti-Gle1-pT102 antibodies revealed that basally phosphorylated Gle1 was pre-dominantly nuclear with punctate distribution; however, under sodium arsenite-induced stress, more cytoplasmic localization was detected. Immunoprecipitation with the anti-Gle1-pT102 antibody resulted in co-isolation of Gle1-pT102 with the DEAD-box protein DDX1 in a phosphatase sensitive manner. This suggested Gle1 phosphorylation might be linked to its role in regulating DDX1 during transcription termination. Notably, whereas the total Gle1-DDX1 association was decreased when Gle1 nucleocytoplasmic shuttling was disrupted, co-isolation of Gle1-pT102 and DDX1 increased under the same conditions. Taken together, these studies demonstrated that Gle1 phosphorylation impacts its cellular distribution and potentially drives nuclear Gle1 functions in transcription termination. We propose a model wherein phosphorylation of Gle1 either reduces its nucleocytoplasmic shuttling capacity or increases its binding affinity with nuclear interaction partners.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Humanos , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Fosforilação , Núcleo Celular/metabolismo
7.
RNA ; 29(12): 1870-1880, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37699651

RESUMO

The conserved TREX complex has multiple functions in gene expression such as transcription elongation, 3' end processing, mRNP assembly and nuclear mRNA export as well as the maintenance of genomic stability. In Saccharomyces cerevisiae, TREX is composed of the pentameric THO complex, the DEAD-box RNA helicase Sub2, the nuclear mRNA export adaptor Yra1, and the SR-like proteins Gbp2 and Hrb1. Here, we present the structural analysis of the endogenous TREX complex of S. cerevisiae purified from its native environment. To this end, we used cross-linking mass spectrometry to gain structural information on regions of the complex that are not accessible to classical structural biology techniques. We also used negative-stain electron microscopy to investigate the organization of the cross-linked complex used for XL-MS by comparing our endogenous TREX complex with recently published structural models of recombinant THO-Sub2 complexes. According to our analysis, the endogenous yeast TREX complex preferentially assembles into a dimer.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , RNA Mensageiro/genética , Transporte de RNA , Transcrição Gênica , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Ligação a Poli(A)/genética
8.
Dev Biol ; 503: 43-52, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37597605

RESUMO

Transmembrane p24 trafficking protein 10 (TMED10) is a conserved vesicle trafficking protein. It is dysregulated in Alzheimer disease and plays a pivotal role in the pathogenesis of Alzheimer disease. In addition to the brain, TMED10 is highly expressed in the exocrine pancreas; however, its biological functions and underlying mechanisms remain largely unknown. We studied reduced Tmed10 in zebrafish embryos by morpholino oligonucleotide knockdown and CRISPR-Cas9 mutagenesis. Tmed10-deficient embryos showed extensive loss of acinar mass and impaired acinar differentiation. TMED10 has been reported to have an inhibitory effect on γ-secretase. As one of the substrates of γ-secretase, membrane-bound ß-catenin was significantly reduced in Tmed10-deficient embryos. Increased γ-secretase activity in wild-type embryos resulted in a phenotype similar to that of tmed10 mutants. And the mutant phenotype could be rescued by treatment with the γ-secretase inhibitor, N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-s-phenylglycinet-butyl ester (DAPT). In addition, the reduced membrane-bound ß-catenin was accompanied with up-regulated ß-catenin target genes in Tmed10-deficient embryos. Overexpression of ß-catenin signaling inhibitor Dickkopf-1 (DKK-1) could rescue the exocrine pancreas defects. Taken together, our study reveals that Tmed10 regulates exocrine pancreatic differentiation through γ-secretase. Reduced membrane-bound ß-catenin, accompanied with hyperactivation of ß-catenin signaling, is an important cause of exocrine pancreas defects in Tmed10-deficient embryos. Our study reaffirms the importance of appropriate ß-catenin signaling in exocrine pancreas development. These findings may provide a theoretical basis for the development of treatment strategies for TMED10-related diseases.


Assuntos
Doença de Alzheimer , Proteínas de Transporte Nucleocitoplasmático , Pâncreas Exócrino , Animais , Secretases da Proteína Precursora do Amiloide/genética , beta Catenina/genética , Larva , Pâncreas Exócrino/embriologia , Pâncreas Exócrino/metabolismo , Peixe-Zebra/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo
9.
Biol Chem ; 404(8-9): 845-850, 2023 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-37436777

RESUMO

Cell viability largely depends on the surveillance of mRNA export and translation. Upon pre-mRNA processing and nuclear quality control, mature mRNAs are exported into the cytoplasm via Mex67-Mtr2 attachment. At the cytoplasmic site of the nuclear pore complex, the export receptor is displaced by the action of the DEAD-box RNA helicase Dbp5. Subsequent quality control of the open reading frame requires translation. Our studies suggest an involvement of Dbp5 in cytoplasmic no-go-and non-stop decay. Most importantly, we have also identified a key function for Dbp5 in translation termination, which identifies this helicase as a master regulator of mRNA expression.


Assuntos
Proteínas de Transporte Nucleocitoplasmático , Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas de Saccharomyces cerevisiae/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Expressão Gênica
10.
Commun Biol ; 6(1): 664, 2023 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-37353594

RESUMO

Self-renewing somatic tissues rely on progenitors to support the continuous tissue regeneration. The gene regulatory network maintaining progenitor function remains incompletely understood. Here we show that NUP98 and RAE1 are highly expressed in epidermal progenitors, forming a separate complex in the nucleoplasm. Reduction of NUP98 or RAE1 abolishes progenitors' regenerative capacity, inhibiting proliferation and inducing premature terminal differentiation. Mechanistically, NUP98 binds on chromatin near the transcription start sites of key epigenetic regulators (such as DNMT1, UHRF1 and EZH2) and sustains their expression in progenitors. NUP98's chromatin binding sites are co-occupied by HDAC1. HDAC inhibition diminishes NUP98's chromatin binding and dysregulates NUP98 and RAE1's target gene expression. Interestingly, HDAC inhibition further induces NUP98 and RAE1 to localize interdependently to the nucleolus. These findings identified a pathway in progenitor maintenance, where HDAC activity directs the high levels of NUP98 and RAE1 to directly control key epigenetic regulators, escaping from nucleolar aggregation.


Assuntos
Cromatina , Proteínas de Transporte Nucleocitoplasmático , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Cromatina/genética , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Sítios de Ligação
11.
Methods Mol Biol ; 2666: 115-136, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37166661

RESUMO

tRNAs are small noncoding RNAs that are predominantly known for their roles in protein synthesis and also participate in numerous other functions ranging from retroviral replication to apoptosis. In eukaryotic cells, all tRNAs move bidirectionally, shuttling between the nucleus and the cytoplasm. Bidirectional nuclear-cytoplasmic tRNA trafficking requires a complex set of conserved proteins. Here, we describe an in vivo biochemical methodology in Saccharomyces cerevisiae to assess the ability of proteins implicated in tRNA nuclear export to form nuclear export complexes with tRNAs. This method employs tagged putative tRNA nuclear exporter proteins and co-immunoprecipitation of tRNA-exporter complexes using antibody-conjugated magnetic beads. Because the interaction between nuclear exporters and tRNAs may be transient, this methodology employs strategies to effectively trap tRNA-protein complexes in vivo. This pull-down method can be used to verify and characterize candidate proteins and their potential interactors implicated in tRNA nuclear-cytoplasmic trafficking.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transporte Ativo do Núcleo Celular/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , RNA de Transferência/genética , Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo
12.
Cell Rep ; 42(3): 112242, 2023 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-36924490

RESUMO

Here, we ask how developing precursors maintain the balance between cell genesis for tissue growth and establishment of adult stem cell pools, focusing on postnatal forebrain neural precursor cells (NPCs). We show that these NPCs are transcriptionally primed to differentiate and that the primed mRNAs are associated with the translational repressor 4E-T. 4E-T also broadly associates with other NPC mRNAs encoding transcriptional regulators, and these are preferentially depleted from ribosomes, consistent with repression. By contrast, a second translational regulator, Cpeb4, associates with diverse target mRNAs that are largely ribosome associated. The 4E-T-dependent mRNA association is functionally important because 4E-T knockdown or conditional knockout derepresses proneurogenic mRNA translation and perturbs maintenance versus differentiation of early postnatal NPCs in culture and in vivo. Thus, early postnatal NPCs are primed to differentiate, and 4E-T regulates the balance between cell genesis and stem cell expansion by sequestering and repressing mRNAs encoding transcriptional regulators.


Assuntos
Células-Tronco Neurais , Diferenciação Celular/fisiologia , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Corpos de Processamento , Biossíntese de Proteínas , Proteínas Repressoras/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo
13.
Am J Med Genet A ; 191(6): 1614-1618, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36891747

RESUMO

Ethylmalonic encephalopathy (EE) is a rare, severe, autosomal recessive condition caused by pathogenic variants in ETHE1 leading to progressive encephalopathy, hypotonia evolving to dystonia, petechiae, orthostatic acrocyanosis, diarrhea, and elevated ethylmalonic acid in urine. In this case report, we describe a patient with only mild speech and gross motor delays, subtle biochemical abnormalities, and normal brain imaging found to be homozygous for a pathogenic ETHE1 variant (c.586G>A) via whole exome sequencing. This case highlights the clinical heterogeneity of ETHE1 mutations and the utility of whole-exome sequencing in diagnosing mild cases of EE.


Assuntos
Encefalopatias Metabólicas Congênitas , Encefalopatias , Púrpura , Humanos , Encefalopatias Metabólicas Congênitas/diagnóstico , Encefalopatias Metabólicas Congênitas/genética , Púrpura/diagnóstico , Púrpura/genética , Encéfalo/patologia , Encefalopatias/diagnóstico , Encefalopatias/genética , Encefalopatias/patologia , Proteínas Mitocondriais/genética , Proteínas de Transporte Nucleocitoplasmático/genética
14.
Mol Cell ; 83(5): 759-769.e7, 2023 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-36736315

RESUMO

The AAA+ ATPase Cdc48 utilizes the cofactor Ufd1/Npl4 to bind and thread polyubiquitinated substrates for their extraction from complexes or membranes and often for subsequent proteasomal degradation. Previous studies indicated that Cdc48 engages polyubiquitin chains through the Npl4-mediated unfolding of an initiator ubiquitin; yet, the underlying principles remain largely unknown. Using FRET-based assays, we revealed the mechanisms and kinetics of ubiquitin unfolding, insertion into the ATPase, and unfolding of the ubiquitin-attached substrate. We found that Cdc48 uses Ufd1's UT3 domain to bind a K48-linked ubiquitin on the initiator's proximal side of the chain, thereby directing the initiator toward rapid unfolding by Npl4 and engagement by Cdc48. Ubiquitins on the initiator's distal side increase substrate affinity and facilitate unfolding but impede substrate release from Cdc48-Ufd1/Npl4 in the absence of additional cofactors. Our findings explain how Cdc48-UN efficiently processes substrates with K48-linked chains of 4-6 ubiquitins, which represent most cellular polyubiquitinated proteins.


Assuntos
Poliubiquitina , Proteínas de Saccharomyces cerevisiae , Poliubiquitina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteína com Valosina/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Proteínas de Ciclo Celular/metabolismo
15.
Redox Biol ; 60: 102629, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36780769

RESUMO

Hydrogen sulfide (H2S) was previously revealed to inhibit osteoblastic differentiation of valvular interstitial cells (VICs), a pathological feature in calcific aortic valve disease (CAVD). This study aimed to explore the metabolic control of H2S levels in human aortic valves. Lower levels of bioavailable H2S and higher levels of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) were detected in aortic valves of CAVD patients compared to healthy individuals, accompanied by higher expression of cystathionine γ-lyase (CSE) and same expression of cystathionine ß-synthase (CBS). Increased biogenesis of H2S by CSE was found in the aortic valves of CAVD patients which is supported by increased production of lanthionine. In accordance, healthy human aortic VICs mimic human pathology under calcifying conditions, as elevated CSE expression is associated with low levels of H2S. The expression of mitochondrial enzymes involved in H2S catabolism including sulfide quinone oxidoreductase (SQR), the key enzyme in mitochondrial H2S oxidation, persulfide dioxygenase (ETHE1), sulfite oxidase (SO) and thiosulfate sulfurtransferase (TST) were up-regulated in calcific aortic valve tissues, and a similar expression pattern was observed in response to high phosphate levels in VICs. AP39, a mitochondria-targeting H2S donor, rescued VICs from an osteoblastic phenotype switch and reduced the expression of IL-1ß and TNF-α in VICs. Both pro-inflammatory cytokines aggravated calcification and osteoblastic differentiation of VICs derived from the calcific aortic valves. In contrast, IL-1ß and TNF-α provided an early and transient inhibition of VICs calcification and osteoblastic differentiation in healthy cells and that effect was lost as H2S levels decreased. The benefit was mediated via CSE induction and H2S generation. We conclude that decreased levels of bioavailable H2S in human calcific aortic valves result from an increased H2S metabolism that facilitates the development of CAVD. CSE/H2S represent a pathway that reverses the action of calcifying stimuli.


Assuntos
Estenose da Valva Aórtica , Calcinose , Sulfeto de Hidrogênio , Humanos , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Sulfeto de Hidrogênio/metabolismo , Calcinose/metabolismo , Calcinose/patologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Células Cultivadas , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo
17.
Nucleic Acids Res ; 51(3): 1393-1408, 2023 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-36620872

RESUMO

In eukaryotic cells, various classes of RNAs are exported to the cytoplasm by class-specific factors. Accumulating evidence has shown that export factors affect the fate of RNA, demonstrating the importance of proper RNA classification upon export. We previously reported that RNA polymerase II transcripts were classified after synthesis depending on their length, and identified heterogeneous nuclear ribonucleoprotein (hnRNP) C as the key classification factor. HnRNP C inhibits the recruitment of PHAX, an adapter protein for spliceosomal U snRNA export, to long transcripts, navigating these RNAs to the mRNA export pathway. However, the mechanisms by which hnRNP C inhibits PHAX recruitment to mRNA remain unknown. We showed that the cap-binding complex, a bridging factor between m7G-capped RNA and PHAX, directly interacted with hnRNP C on mRNA. Additionally, we revealed that the tetramer-forming activity of hnRNP C and its strong RNA-binding activity were crucial for the inhibition of PHAX binding to longer RNAs. These results suggest that mRNA is wrapped around the hnRNP C tetramer without a gap from the cap, thereby impeding the recruitment of PHAX. The results obtained on the mode of length-specific RNA classification by the hnRNP C tetramer will provide mechanistic insights into hnRNP C-mediated RNA biogenesis.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo C , RNA Polimerase II , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/genética , Células Eucarióticas/metabolismo
18.
Life Sci Alliance ; 6(3)2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36599624

RESUMO

Replication licensing, a prerequisite of DNA replication, helps to ensure once-per-cell-cycle genome duplication. Some DNA replication-initiation proteins are sequentially loaded onto replication origins to form pre-replicative complexes (pre-RCs). ORC and Noc3p bind replication origins throughout the cell cycle, providing a platform for pre-RC assembly. We previously reported that cell cycle-dependent ORC dimerization is essential for the chromatin loading of the symmetric MCM double-hexamers. Here, we used Saccharomyces cerevisiae separation-of-function NOC3 mutants to confirm the separable roles of Noc3p in DNA replication and ribosome biogenesis. We also show that an essential and cell cycle-dependent Noc3p dimerization cycle regulates the ORC dimerization cycle. Noc3p dimerizes at the M-to-G1 transition and de-dimerizes in S-phase. The Noc3p dimerization cycle coupled with the ORC dimerization cycle enables replication licensing, protects nascent sister replication origins after replication initiation, and prevents re-replication. This study has revealed a new mechanism of replication licensing and elucidated the molecular mechanism of Noc3p as a mediator of ORC dimerization in pre-RC formation.


Assuntos
Multimerização Proteica , Proteínas de Saccharomyces cerevisiae , Ciclo Celular/genética , Dimerização , Replicação do DNA/genética , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Multimerização Proteica/genética , Multimerização Proteica/fisiologia , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia
20.
Nat Commun ; 13(1): 5881, 2022 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-36202822

RESUMO

The changes occurring in mRNA organization during nucleo-cytoplasmic transport and export, are not well understood. Moreover, directionality of mRNA passage through the nuclear pore complex (NPC) has not been examined within individual NPCs. Here we find that an mRNP is compact during nucleoplasmic travels compared to a more open structure after transcription and at the nuclear periphery. Compaction levels of nuclear transcripts can be modulated by varying levels of SR proteins and by changing genome organization. Nuclear mRNPs are mostly rod-shaped with distant 5'/3'-ends, although for some, the ends are in proximity. The latter is more abundant in the cytoplasm and can be modified by translation inhibition. mRNAs and lncRNAs exiting the NPC exhibit predominant 5'-first export. In some cases, several adjacent NPCs are engaged in export of the same mRNA suggesting 'gene gating'. Altogether, we show that the mRNP is a flexible structure during travels, with 5'-directionality during export.


Assuntos
Poro Nuclear , RNA Longo não Codificante , Transporte Ativo do Núcleo Celular/genética , Núcleo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Transporte de RNA , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
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