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1.
Cell Death Dis ; 14(1): 44, 2023 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-36658121

RESUMO

The alteration of cellular energy metabolism is a hallmark of colorectal cancer (CRC). Accumulating evidence has suggested oxidative phosphorylation (OXPHOS) is upregulated to meet the demand for energy in tumor initiation and development. However, the role of OXPHOS and its regulatory mechanism in CRC tumorigenesis and progression remain unclear. Here, we reveal that Prohibitin 2 (PHB2) expression is elevated in precancerous adenomas and CRC, which promotes cell proliferation and tumorigenesis of CRC. Additionally, knockdown of PHB2 significantly reduces mitochondrial OXPHOS levels in CRC cells. Meanwhile, NADH:ubiquinone oxidoreductase core subunit S1 (NDUFS1), as a PHB2 binding partner, is screened and identified by co-immunoprecipitation and mass spectrometry. Furthermore, PHB2 directly interacts with NDUFS1 and they co-localize in mitochondria, which facilitates NDUFS1 binding to NADH:ubiquinone oxidoreductase core subunit V1 (NDUFV1), regulating the activity of complex I. Consistently, partial inhibition of complex I activity also abrogates the increased cell proliferation induced by overexpression of PHB2 in normal human intestinal epithelial cells and CRC cells. Collectively, these results indicate that increased PHB2 directly interacts with NDUFS1 to stabilize mitochondrial complex I and enhance its activity, leading to upregulated OXPHOS levels, thereby promoting cell proliferation and tumorigenesis of CRC. Our findings provide a new perspective for understanding CRC energy metabolism, as well as novel intervention strategies for CRC therapeutics.


Assuntos
Neoplasias Colorretais , NADH Desidrogenase , Fosforilação Oxidativa , Proibitinas , Humanos , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Neoplasias Colorretais/genética , NAD/metabolismo , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Oxirredutases/metabolismo , Proteínas Repressoras/metabolismo , Ubiquinona/metabolismo , Proibitinas/genética
2.
Front Cell Dev Biol ; 9: 669379, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34055802

RESUMO

Mitochondria are double membrane organelles in eukaryotic cells that provide energy by generating adenosine triphosphate (ATP) through oxidative phosphorylation. They are crucial to many aspects of cellular metabolism. Mitochondria contain their own DNA that encodes for essential proteins involved in the execution of normal mitochondrial functions. Compared with nuclear DNA, the mitochondrial DNA (mtDNA) is more prone to be affected by DNA damaging agents, and accumulated DNA damages may cause mitochondrial dysfunction and drive the pathogenesis of a variety of human diseases, including neurodegenerative disorders and cancer. Therefore, understanding better how mtDNA damages are repaired will facilitate developing therapeutic strategies. In this review, we focus on our current understanding of the mtDNA repair system. We also discuss other mitochondrial events promoted by excessive DNA damages and inefficient DNA repair, such as mitochondrial fusion, fission, and mitophagy, which serve as quality control events for clearing damaged mtDNA.

3.
Transl Vis Sci Technol ; 9(9): 47, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32934897

RESUMO

Purpose: Fuchs' endothelial corneal dystrophy (FECD) is the leading indication for corneal transplantation. Seventy percent of cases are caused by an intronic CTG triplet repeat expansion in the TCF4 gene that results in accumulation of pathogenic expanded CUG repeat RNA (CUGexp) as nuclear foci in corneal endothelium. A catalytically dead Cas9 (dCas9) can serve as an effective guide to target genomic DNA or RNA transcripts. Here, we examined the utility of the clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 system to effectively target and reduce CUGexp. Methods: We delivered dCas9 and repeat-targeting single guide RNA (sgRNA) expression plasmids to patient-derived endothelial cells using lipofection or lentiviral transduction. We used fluorescence in situ hybridization (FISH) and RNA dot-blot hybridization to quantify CUGexp foci and repeat RNA levels, respectively. TCF4 expression levels were assessed using quantitative PCR (qPCR). Results: Using FISH, we found that expression of both dCas9 and a (CAG)n sgRNA complementary to CUGexp are necessary to reduce foci. We observed a reduction in percentage of cells with foci from 59% to 5.6% and number of foci per 100 cells from 73.4 to 7.45 (P < 0.001) in cells stably expressing dCas9-(CAG)n sgRNA but saw no decrease in cells expressing dCas9-(CUG)n sgRNA or nontargeting control sgRNA. In cells with dCas9-(CAG)n sgRNA, we detected a reduction in CUGexp RNA by dot-blot without any reduction in TCF4 mRNA levels using qPCR. Conclusions: Using CRISPR-dCas9 to target the trinucleotide repeat is a promising treatment for FECD contingent on effective in vivo delivery. Translational Relevance: This work advances a gene therapy for a common age-related degenerative disorder.


Assuntos
Células Endoteliais , Distrofia Endotelial de Fuchs , Distrofia Endotelial de Fuchs/genética , Humanos , Hibridização in Situ Fluorescente , Fator de Transcrição 4/genética , Expansão das Repetições de Trinucleotídeos/genética
4.
Invest Ophthalmol Vis Sci ; 60(12): 3980-3991, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31560764

RESUMO

Purpose: Fuchs' endothelial corneal dystrophy (FECD) is a major cause of vision loss and the most common nucleotide repeat disorder, affecting 4% of United States population greater than 40 years of age. Seventy percent of FECD cases are due to an intronic CTG expansion within the TCF4 gene, resulting in accumulation of CUG repeat RNA nuclear foci in corneal endothelium. Each endothelial cell has approximately two sense foci, and each focus is a single RNA molecule. This study aimed to obtain a better understanding of how rare repeat RNA species lead to disease. Methods: We quantitatively examined muscleblind-like (MBNL) proteins and their interaction with foci in both patient-derived corneal endothelial cell lines and human corneal endothelial tissue. Results: Using fluorescent in situ hybridization and immunofluorescence, we found that depletion of both MBNL1 and MBNL2 reduces nuclear RNA foci formed by the repeat, suggesting that both are necessary for foci. Quantitative studies of RNA and protein copy number revealed MBNLs to be abundant in the total cellular pool in endothelial cell lines but are much lower in human corneal endothelial tissue. Studies using human tissue nuclear and cytoplasmic fractions indicate that most MBNL proteins are localized to the cytoplasm. Conclusions: The low levels of MBNL1/2 in corneal tissue, in combination with the small fraction of protein in the nucleus, may make corneal endothelial cells especially susceptible to sequestration of MBNL1/2 by CUG repeat RNA. These observations may explain how a limited number of RNA molecules can cause widespread alteration of splicing and late-onset degenerative FECD.


Assuntos
Endotélio Corneano/metabolismo , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/genética , Fator de Transcrição 4/genética , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Imunofluorescência , Humanos , Immunoblotting , Hibridização in Situ Fluorescente , Mapas de Interação de Proteínas , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Transfecção , Expansão das Repetições de Trinucleotídeos
5.
Hum Mol Genet ; 27(6): 1015-1026, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29325021

RESUMO

Fuchs' endothelial corneal dystrophy (FECD) is the most common repeat expansion disorder. FECD impacts 4% of U.S. population and is the leading indication for corneal transplantation. Most cases are caused by an expanded intronic CUG tract in the TCF4 gene that forms nuclear foci, sequesters splicing factors and impairs splicing. We investigated the sense and antisense RNA landscape at the FECD gene and find that the sense-expanded repeat transcript is the predominant species in patient corneas. In patient tissue, sense foci number were negatively correlated with age and showed no correlation with sex. Each endothelial cell has ∼2 sense foci and each foci is single RNA molecule. We designed antisense oligonucleotides (ASOs) to target the mutant-repetitive RNA and demonstrated potent inhibition of foci in patient-derived cells. Ex vivo treatment of FECD human corneas effectively inhibits foci and reverses pathological changes in splicing. FECD has the potential to be a model for treating many trinucleotide repeat diseases and targeting the TCF4 expansion with ASOs represents a promising therapeutic strategy to prevent and treat FECD.


Assuntos
Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/metabolismo , Fator de Transcrição 4/genética , Idoso , Idoso de 80 Anos ou mais , Alelos , Endotélio Corneano/metabolismo , Feminino , Predisposição Genética para Doença , Humanos , Íntrons , Masculino , Pessoa de Meia-Idade , Oligorribonucleotídeos Antissenso/genética , Oligorribonucleotídeos Antissenso/uso terapêutico , RNA/metabolismo , Splicing de RNA , Fator de Transcrição 4/metabolismo , Fatores de Transcrição/genética , Expansão das Repetições de Trinucleotídeos
6.
Invest Ophthalmol Vis Sci ; 58(11): 4579-4585, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28886202

RESUMO

Purpose: The most common cause of Fuchs' endothelial corneal dystrophy (FECD) is an intronic CTG repeat expansion in TCF4. Expanded CUG repeat RNA colocalize with splicing factor, muscleblind-like 1 (MBNL1), in nuclear foci in endothelium as a molecular hallmark. Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a CTG repeat expansion in the 3'-untranslated region (UTR) of DMPK. In this study, we examine for RNA-MBNL1 foci in endothelial cells of FECD subjects with DM1, test the hypothesis that DM1 patients are at risk for FECD, and determine prevalence of TCF4 and DMPK expansions in a FECD cohort. Methods: Using FISH, we examined for nuclear RNA-MBNL1 foci in endothelial cells from FECD subjects with DM1. We examined 13 consecutive unrelated DM1 patients for FECD using slit-lamp and specular microscopy. We genotyped TCF4 and DMPK repeat polymorphisms in a FECD cohort of 317 probands using short-tandem repeat and triplet repeat-primed PCR assays. Results: We detected abundant nuclear RNA foci colocalizing with MBNL1 in endothelial cells of FECD subjects with DM1. Six of thirteen DM1 patients (46%) had slit-lamp and specular microscopic findings of FECD, compared to 4% disease prevalence (P = 5.5 × 10-6). As expected, 222 out of 317 (70%) FECD probands harbored TCF4 expansion, while one subject harbored DMPK expansion without prior diagnosis of DM1. Conclusions: Our work suggests that DM1 patients are at risk for FECD. DMPK mutations contribute to the genetic burden of FECD but are uncommon. We establish a connection between two repeat expansion disorders converging upon RNA-MBNL1 foci and FECD.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Distrofia Endotelial de Fuchs/genética , Distrofia Miotônica/genética , Miotonina Proteína Quinase/genética , RNA Nuclear , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Adulto , Idoso , Endotélio Corneano/metabolismo , Endotélio Corneano/patologia , Feminino , Distrofia Endotelial de Fuchs/patologia , Técnicas de Genotipagem , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Distrofia Miotônica/patologia , Reação em Cadeia da Polimerase , Splicing de RNA , Lâmpada de Fenda , Fator de Transcrição 4 , Expansão das Repetições de Trinucleotídeos
7.
J Biol Chem ; 291(36): 19079-91, 2016 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-27422821

RESUMO

During the cell cycle, sister-chromatid cohesion tethers sister chromatids together from S phase to the metaphase-anaphase transition and ensures accurate segregation of chromatids into daughter cells. N-terminal acetylation is one of the most prevalent protein covalent modifications in eukaryotes and is mediated by a family of N-terminal acetyltransferases (NAT). Naa50 (also called San) has previously been shown to play a role in sister-chromatid cohesion in metazoans. The mechanism by which Naa50 contributes to cohesion is not understood however. Here, we show that depletion of Naa50 in HeLa cells weakens the interaction between cohesin and its positive regulator sororin and causes cohesion defects in S phase, consistent with a role of Naa50 in cohesion establishment. Strikingly, co-depletion of NatA, a heterodimeric NAT complex that physically interacts with Naa50, rescues the sister-chromatid cohesion defects and the resulting mitotic arrest caused by Naa50 depletion, indicating that NatA and Naa50 play antagonistic roles in cohesion. Purified recombinant NatA and Naa50 do not affect each other's NAT activity in vitro Because NatA and Naa50 exhibit distinct substrate specificity, we propose that they modify different effectors and regulate sister-chromatid cohesion in opposing ways.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromátides/enzimologia , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Humanos/enzimologia , Mitose/fisiologia , Acetiltransferase N-Terminal A/metabolismo , Acetiltransferase N-Terminal E/metabolismo , Fase S/fisiologia , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromossômicas não Histona/genética , Cromossomos Humanos/genética , Células HeLa , Humanos , Acetiltransferase N-Terminal A/genética , Acetiltransferase N-Terminal E/genética , Coesinas
8.
Sheng Wu Gong Cheng Xue Bao ; 24(11): 1918-23, 2008 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-19256339

RESUMO

We studied the aggregation of a recombinant engineering antibody (chA21). Anti-ErbB2 antibody chA21 was produced by fusing single-chain Fv (scFv) with human IgG1 Fc fragment, and it was proved to be a drug candidate for cancer therapy. We characterized the aggregation of chA21 by high performance sized-exclusive chromatography (HPSEC), dynamic light scattering (DLS), SDS-PAGE, indirect ELISA assay, and compared the influence of temperature and additive on the level of aggregation and binding activity. Conformation changes of different levels of aggregation were also analyzed via circular dichroism (CD). Finally, we analyzed which part of chA21 was involved in aggregation by cleaving it into scFv and Fc fragments. The results showed that chA21 could form aggregates in the storage solution. The aggregates interacted through non-covalent bonds and remained binding activity. Temperature and additive could slightly affect the level of aggregation and binding activity, while the conformations of chA21 were stable. Aggregation propensity of scFv fragment was almost same as chA21, indicating that scFv may be the major part to form the aggregates. The research on aggregation may be helpful to develop a suitable formulation for chA21 clinical application as well as provide direction for future antibody design and reconstruction.


Assuntos
Anticorpos/química , Fragmentos Fc das Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Agregação de Receptores/imunologia , Receptor ErbB-2/imunologia , Anticorpos/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/metabolismo , Conformação Proteica , Engenharia de Proteínas/métodos , Receptor ErbB-2/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia
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