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1.
Nat Commun ; 14(1): 6248, 2023 10 06.
Article in English | MEDLINE | ID: mdl-37803021

ABSTRACT

YTHDF2 has been extensively studied and typified as an RNA-binding protein that specifically recognizes and destabilizes RNAs harboring N6-methyladenosine (m6A), the most prevalent internal modification found in eukaryotic RNAs. In this study, we unravel the m6A-independent role of YTHDF2 in the formation of an aggresome, where cytoplasmic protein aggregates are selectively sequestered upon failure of protein homeostasis mediated by the ubiquitin-proteasome system. Downregulation of YTHDF2 in HeLa cells reduces the circularity of aggresomes and the rate of movement of misfolded polypeptides, inhibits aggresome formation, and thereby promotes cellular apoptosis. Mechanistically, YTHDF2 is recruited to a misfolded polypeptide-associated complex composed of UPF1, CTIF, eEF1A1, and DCTN1 through its interaction with UPF1. Subsequently, YTHDF2 increases the interaction between the dynein motor protein and the misfolded polypeptide-associated complex, facilitating the diffusion dynamics of the movement of misfolded polypeptides toward aggresomes. Therefore, our data reveal that YTHDF2 is a cellular factor involved in protein quality control.


Subject(s)
Protein Folding , Proteolysis , Humans , Cytoplasm/metabolism , Dyneins/metabolism , HeLa Cells , Peptides/metabolism , RNA Helicases/genetics , RNA Helicases/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/metabolism , Organelles/metabolism
2.
Front Cell Dev Biol ; 10: 822026, 2022.
Article in English | MEDLINE | ID: mdl-35874812

ABSTRACT

Recent studies with single-particle tracking in live cells have revealed that chromatin dynamics are directly affected by transcription. However, how transcription alters the chromatin movements followed by changes in the physical properties of chromatin has not been elucidated. Here, we measured diffusion characteristics of chromatin by targeting telomeric DNA repeats with CRISPR-labeling. We found that transcription inhibitors that directly block transcription factors globally increased the movements of chromatin, while the other inhibitor that blocks transcription by DNA intercalating showed an opposite effect. We hypothesized that the increased mobility of chromatin by transcription inhibition and the decreased chromatin movement by a DNA intercalating inhibitor is due to alterations in chromatin rigidity. We also tested how volume confinement of nuclear space affects chromatin movements. We observed decreased chromatin movements under osmotic pressure and with overexpressed chromatin architectural proteins that compact chromatin.

3.
BMB Rep ; 54(10): 489-496, 2021 Oct.
Article in English | MEDLINE | ID: mdl-34488934

ABSTRACT

Chromatin has highly organized structures in the nucleus, and these higher-order structures are proposed to regulate gene activities and cellular processes. Sequencing-based techniques, such as Hi-C, and fluorescent in situ hybridization (FISH) have revealed a spatial segregation of active and inactive compartments of chromatin, as well as the non-random positioning of chromosomes in the nucleus, respectively. However, regardless of their efficiency in capturing target genomic sites, these techniques are limited to fixed cells. Since chromatin has dynamic structures, live cell imaging techniques are highlighted for their ability to detect conformational changes in chromatin at a specific time point, or to track various arrangements of chromatin through long-term imaging. Given that the imaging approaches to study live cells are dramatically advanced, we recapitulate methods that are widely used to visualize the dynamics of higher-order chromatin structures. [BMB Reports 2021; 54(10): 489-496].


Subject(s)
Chromatin/physiology , Imaging, Three-Dimensional/methods , Optical Imaging/methods , Cell Nucleus/metabolism , Chromatin/ultrastructure , Chromosomes/metabolism , Genome/genetics , Humans , Structure-Activity Relationship , Transcriptional Activation/genetics
5.
Oxid Med Cell Longev ; 2019: 8060962, 2019.
Article in English | MEDLINE | ID: mdl-31781352

ABSTRACT

Glaucoma is characterized by a progressive optic nerve degeneration and retinal ganglion cell loss, but the underlying biological basis for the accompanying neurodegeneration is not known. Accumulating evidence indicates that structural and functional abnormalities of astrocytes within the optic nerve head (ONH) have a role in glaucomatous neurodegeneration. Here, we investigate the impact of activation of cyclic adenosine 3',5'-monophosphate (cAMP)/protein kinase A (PKA) pathway on mitochondrial dynamics of ONH astrocytes exposed to oxidative stress. ONH astrocytes showed a significant loss of astrocytic processes in the glial lamina of glaucomatous DBA/2J mice, accompanied by basement membrane thickening and collagen deposition in blood vessels and axonal degeneration. Serial block-face scanning electron microscopy data analysis demonstrated that numbers of total and branched mitochondria were significantly increased in ONH astrocytes, while mitochondrial length and volume density were significantly decreased. We found that hydrogen peroxide- (H2O2-) induced oxidative stress compromised not only mitochondrial bioenergetics by reducing the basal and maximal respiration but also balance of mitochondrial dynamics by decreasing dynamin-related protein 1 (Drp1) protein expression in rat ONH astrocytes. In contrast, elevated cAMP by dibutyryl-cAMP (dbcAMP) or isobutylmethylxanthine treatment significantly increased Drp1 protein expression in ONH astrocytes. Elevated cAMP exacerbated the impairment of mitochondrial dynamics and reduction of cell viability to oxidative stress in ONH astrocytes by decreasing optic atrophy type 1 (OPA1), and mitofusin (Mfn)1/2 protein expression. Following combined treatment with H2O2 and dbcAMP, PKA inhibition restored mitochondrial dynamics by increasing mitochondrial length and decreasing mitochondrial number, and this promoted cell viability in ONH astrocytes. Also, PKA inhibition significantly promoted Akt/Bax phosphorylation and Mfn1/2 oligomerization in ONH astrocytes. These results suggest that modulation of the cAMP/PKA signaling pathway may have therapeutic potential by activating Akt/Bax phosphorylation and promoting Mfn1/2 oligomerization in glaucomatous ONH astrocytes.


Subject(s)
Astrocytes/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , GTP Phosphohydrolases/metabolism , Glaucoma/metabolism , Membrane Proteins/metabolism , Mitochondrial Proteins/metabolism , Optic Nerve/metabolism , Oxidative Stress , Protein Multimerization , Signal Transduction , bcl-2-Associated X Protein/metabolism , Animals , Astrocytes/pathology , Female , Glaucoma/pathology , Mice , Optic Nerve/pathology , Rats , Rats, Sprague-Dawley
6.
Biochem Biophys Res Commun ; 503(4): 2639-2645, 2018 09 18.
Article in English | MEDLINE | ID: mdl-30107910

ABSTRACT

Coenzyme Q10 (CoQ10) protects retinal ganglion cells (RGCs) in experimental retinal ischemia and glaucoma by scavenging reactive oxygen species. We tested whether a diet supplemented with ubiquinol, the reduced form of CoQ10, promotes RGC survival and blocks the apoptotic pathway in ischemic mouse retina induced by acute high intraocular pressure (IOP) elevation. Ubiquinol (1%) treatment significantly promoted RGC survival at 2 weeks after ischemia/reperfusion. The ubiquinol treatment significantly blocked activation of astroglial and microglial cells in the ischemic retina at 2 weeks. While the ubiquinol treatment significantly decreased active Bax protein expression in the ischemic retina, phosphorylation of Bad at serine 112 and Bcl-xL protein expression were preserved in the ubiquinol-treated ischemic retina at 12 h. Consistently, the ubiquinol treatment prevented apoptotic cell death by blocking caspase-3 cleavage. These results suggest that the ubiquinol enhances RGC survival by modulating the Bax/Bad/Bcl-xL-mediated apoptotic pathway in the ischemic retina. Ubiquinol has therapeutic potential for ameliorating elevated IOP-induced ischemic retinal degeneration.


Subject(s)
Antioxidants/pharmacology , Gene Expression Regulation/drug effects , Reperfusion Injury/drug therapy , Retinal Degeneration/prevention & control , Retinal Ganglion Cells/drug effects , Ubiquinone/analogs & derivatives , Animals , Apoptosis , Astrocytes/drug effects , Astrocytes/metabolism , Astrocytes/pathology , Caspase 3/genetics , Caspase 3/metabolism , Cell Survival/drug effects , Disease Models, Animal , Female , Intraocular Pressure , Mice , Mice, Inbred C57BL , Microglia/drug effects , Microglia/metabolism , Microglia/pathology , Oxidative Stress/drug effects , Phosphorylation/drug effects , Reactive Oxygen Species/antagonists & inhibitors , Reactive Oxygen Species/metabolism , Reperfusion Injury/genetics , Reperfusion Injury/metabolism , Reperfusion Injury/pathology , Retinal Degeneration/genetics , Retinal Degeneration/metabolism , Retinal Degeneration/pathology , Retinal Ganglion Cells/metabolism , Retinal Ganglion Cells/pathology , Signal Transduction , Ubiquinone/pharmacology , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolism , bcl-Associated Death Protein/genetics , bcl-Associated Death Protein/metabolism , bcl-X Protein/genetics , bcl-X Protein/metabolism
7.
Cell Death Dis ; 9(3): 285, 2018 02 19.
Article in English | MEDLINE | ID: mdl-29459737

ABSTRACT

Glaucoma is characterized by a progressive loss of retinal ganglion cells and their axons, but the underlying biological basis for the accompanying neurodegeneration is not known. Accumulating evidence indicates that structural and functional abnormalities of astrocytes within the optic nerve head (ONH) have a role. However, whether the activation of cyclic adenosine 3',5'-monophosphate (cAMP) signaling pathway is associated with astrocyte dysfunction in the ONH remains unknown. We report here that the cAMP/protein kinase A (PKA) pathway is critical to ONH astrocyte dysfunction, leading to caspase-3 activation and cell death via the AKT/Bim/Bax signaling pathway. Furthermore, elevated intracellular cAMP exacerbates vulnerability to oxidative stress in ONH astrocytes, and this may contribute to axonal damage in glaucomatous neurodegeneration. Inhibition of intracellular cAMP/PKA signaling activation protects ONH astrocytes by increasing AKT phosphorylation against oxidative stress. These results strongly indicate that activation of cAMP/PKA pathway has an important role in astrocyte dysfunction, and suggest that modulating cAMP/PKA pathway has therapeutic potential for glaucomatous ONH degeneration.


Subject(s)
Astrocytes/metabolism , Cyclic AMP/metabolism , Glaucoma/metabolism , Optic Nerve/metabolism , Oxidative Stress , Animals , Astrocytes/cytology , Axons/metabolism , Cell Death , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Female , Glaucoma/genetics , Glaucoma/physiopathology , Humans , Mice , Mice, Inbred DBA , Optic Nerve/cytology , Phosphorylation , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Rats , Rats, Sprague-Dawley , Signal Transduction
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