The eukaryotic transcription factor NF-Y consists of three subunits （A, B, and C）, which are encoded in Ara- bidopsis thaliana in multigene families consisting of 10, 13, and 13 genes, respectively. In principle, al...The eukaryotic transcription factor NF-Y consists of three subunits （A, B, and C）, which are encoded in Ara- bidopsis thaliana in multigene families consisting of 10, 13, and 13 genes, respectively. In principle, all potential combi- nations of the subunits are possible for the assembly of the heterotrimeric complex. We aimed at assessing the probability of each subunit to participate in the assembly of NF-Y. The evaluation of physical interactions among all members of the NF-Y subunit families indicate a strong requirement for NF-YB/NF-YC heterodimerization before the entire complex can be accomplished. By means of a modified yeast two-hybrid system assembly of all three subunits to a heterotrimeric complex was demonstrated. Using GFP fusion constructs, NF-YA and NF-YC localization in the nucleus was demonstrated, while NF- YB is solely imported into the nucleus as a NF-YC-associated heterodimer NF-YC. This piggyback transport of the two Arabidopsis subunits differs from the import of the NF-Y heterotrimer of heterotrophic organisms. Based on a peptide structure model of the histone-fold-motifs, disulfide bonding among intramolecular conserved cysteine residues of NF-YB, which is responsible for the redox-regulated assembly of NF-YB and NF-YC in human and Aspergillus nidulans, can be excluded for Arabidopsis NF-YB.展开更多
Disrupted redox status primarily contributes to myocardial ischemia/reperfusion injury(MIRI).NRF2,the endogenous antioxidant regulator,might provide therapeutic benefits.Dihydrotanshinone-Ⅰ(DT)is an active component ...Disrupted redox status primarily contributes to myocardial ischemia/reperfusion injury(MIRI).NRF2,the endogenous antioxidant regulator,might provide therapeutic benefits.Dihydrotanshinone-Ⅰ(DT)is an active component in Salvia miltiorrhiza with NRF2 induction potency.This study seeks to validate functional links between NRF2 and cardioprotection of DT and to investigate the molecular mechanism particularly emphasizing on NRF2 cytoplasmic/nuclear translocation.DT potently induced NRF2 nuclear accumulation,ameliorating post-reperfusion injuries via redox alterations.Abrogated cardioprotection in NRF2-deficient mice and cardiomyocytes strongly supports NRF2-dependent cardioprotection of DT.Mechanistically,DT phosphorylated NRF2 at Ser40,rendering its nuclear-import by dissociating from KEAP1 and inhibiting degradation.Importantly,we identified PKC-δ-(Thr505)phosphorylation as primary upstream event triggering NRF2-(Ser40)phosphorylation.Knockdown of PKC-δdramatically retained NRF2 in cytoplasm,convincing its pivotal role in mediating NRF2 nuclear-import.NRF2 activity was further enhanced by activated PKB/GSK-3βsignaling via nuclear-export signal blockage independent of PKC-δactivation.By demonstrating independent modulation of PKC-δand PKB/GSK-3β/Fyn signaling,we highlight the ability of DT to exploit both nuclear import and export regulation of NRF2 in treating reperfusion injury harboring redox homeostasis alterations.Coactivation of PKC and PKB phenocopied cardioprotection of DT in vitro and in vivo,further supporting the potential applicability of this rationale.展开更多
A number of transmembrane receptors are targeted to the nucleus and convincingly localized therein. However, what remains a conundrum is how these cell-surface receptors end up in the nucleus. In this study, we report...A number of transmembrane receptors are targeted to the nucleus and convincingly localized therein. However, what remains a conundrum is how these cell-surface receptors end up in the nucleus. In this study, we reported that the transmembrane receptor phosphorylated TrkA was located in a series of carrier vesicles, including ring-like vesicles near the plasma membrane, large core vesicles and small dense core vesicles around the nuclei, as well as in the nucleus in human glioma cell line U251 using immunocytochemistry and immunofluorescence staining. Meanwhile, we also showed that small dense core vesicles budded from large core vesicles, and interacted with the nuclear envelope. Accordingly, our results suggested that such a series of membrane compartments might be involved in the pathway of nuclear translocation of the transmembrane receptor TrkA.展开更多
As shown in our previous study, two alternatively spliced androgen receptor(AR) variants, which are exclusively expressed in the granulosa cells of patients with polycystic ovary syndrome, exhibit retarded nuclear tra...As shown in our previous study, two alternatively spliced androgen receptor(AR) variants, which are exclusively expressed in the granulosa cells of patients with polycystic ovary syndrome, exhibit retarded nuclear translocation compared with wild-type AR. However, researchers have not yet determined whether these abnormalities correlate with heat shock protein 90(HSP90)and importin α(the former is a generally accepted co-chaperone of AR, and the latter is a component of classical nuclear import complexes). Here, these two variants were mainly retained in cytoplasm with HSP90 and importin α in the presence of dihydrotestosterone(DHT), and their levels in nucleus were significantly reduced, according to the immunofluorescence staining. The binding affinity of two AR variants for importin α was consistently decreased, while it was increased in WT-AR following DHT stimulation, leading to reduced nuclear import, particularly for the insertion-AR(Ins-AR). However, the binding affinities of two AR variants for HSP90 were increased in the absence of DHT compared with WT-AR, which functioned to maintain spatial structural stability, particularly for the deletion-AR(Del-AR). Therefore, the retarded nuclear translocation of two AR variants is associated with HSP90 and importin α, and the abnormal binding affinities for them play critical roles in this process.展开更多
Rescuing cells from stress damage emerges a potential therapeutic strategy to combat myocardial infarction.Protocatechuic aldehyde(PCA)is a major phenolic acid in Chinese herb Danshen(Salvia miltiorrhiza root).This st...Rescuing cells from stress damage emerges a potential therapeutic strategy to combat myocardial infarction.Protocatechuic aldehyde(PCA)is a major phenolic acid in Chinese herb Danshen(Salvia miltiorrhiza root).This study investigated whether PCA regulated nuclear pyruvate kinase isoform M2(PKM2)function to protect cardiomyocytes.In rats subjected to isoprenaline,PCA attenuated heart injury and protected cardiomyocytes from apoptosis.Through DARTS and CETSA assays,we identified that PCA bound and promoted PKM2 nuclear translocation in cardiomyocytes exposed to oxygen/glucose deprivation(OGD).In the nucleus,PCA increased the binding of PKM2 to β-Catenin via preserving PKM2 acetylation,and the complex,in cooperation with T-cell factor 4(TCF4),was required for transcriptional induction of genes encoding anti-apoptotic proteins,contributing to rescuing cardiomyocyte survival.In addition,PCA ameliorated mitochondrial dysfunction and prevented mitochondrial apoptosis dependent on PKM2.Consistently,PCA increased the binding of PKM2 to β-Catenin,improved heart contractive function,normalized heart structure and attenuated oxidative damage in mice subjected to artery ligation,but the protective effects were lost in Pkm2-deficient heart.Together,we showed that PCA regulated nuclear PKM2 function to rescue cardiomyocyte survival via β-Catenin/TCF4 signaling cascade,suggesting the potential of pharmacological intervention of PKM2 shuttle to protect the heart.展开更多
It has been shown clinically that continuous removal of ischemia/reperfusion-induced reactive oxygen species is not conducive to the recovery of late stroke.Indeed,previous studies have shown that excessive increases ...It has been shown clinically that continuous removal of ischemia/reperfusion-induced reactive oxygen species is not conducive to the recovery of late stroke.Indeed,previous studies have shown that excessive increases in hypochlorous acid after stroke can cause severe damage to brain tissue.Our previous studies have found that a small amount of hypochlorous acid still exists in the later stage of stroke,but its specific role and mechanism are currently unclear.To simulate stroke in vivo,a middle cerebral artery occlusion rat model was established,with an oxygen-glucose deprivation/reoxygenation model established in vitro to mimic stroke.We found that in the early stage(within 24 hours)of ischemic stroke,neutrophils produced a large amount of hypochlorous acid,while in the recovery phase(10 days after stroke),microglia were activated and produced a small amount of hypochlorous acid.Further,in acute stroke in rats,hypochlorous acid production was prevented using a hypochlorous acid scavenger,taurine,or myeloperoxidase inhibitor,4-aminobenzoic acid hydrazide.Our results showed that high levels of hypochlorous acid(200μM)induced neuronal apoptosis after oxygen/glucose deprivation/reoxygenation.However,in the recovery phase of the middle cerebral artery occlusion model,a moderate level of hypochlorous acid promoted the proliferation and differentiation of neural stem cells into neurons and astrocytes.This suggests that hypochlorous acid plays different roles at different phases of cerebral ischemia/reperfusion injury.Lower levels of hypochlorous acid(5 and 100μM)promoted nuclear translocation ofβ-catenin.By transfection of single-site mutation plasmids,we found that hypochlorous acid induced chlorination of theβ-catenin tyrosine 30 residue,which promoted nuclear translocation.Altogether,our study indicates that maintaining low levels of hypochlorous acid plays a key role in the recovery of neurological function.展开更多
文摘The eukaryotic transcription factor NF-Y consists of three subunits （A, B, and C）, which are encoded in Ara- bidopsis thaliana in multigene families consisting of 10, 13, and 13 genes, respectively. In principle, all potential combi- nations of the subunits are possible for the assembly of the heterotrimeric complex. We aimed at assessing the probability of each subunit to participate in the assembly of NF-Y. The evaluation of physical interactions among all members of the NF-Y subunit families indicate a strong requirement for NF-YB/NF-YC heterodimerization before the entire complex can be accomplished. By means of a modified yeast two-hybrid system assembly of all three subunits to a heterotrimeric complex was demonstrated. Using GFP fusion constructs, NF-YA and NF-YC localization in the nucleus was demonstrated, while NF- YB is solely imported into the nucleus as a NF-YC-associated heterodimer NF-YC. This piggyback transport of the two Arabidopsis subunits differs from the import of the NF-Y heterotrimer of heterotrophic organisms. Based on a peptide structure model of the histone-fold-motifs, disulfide bonding among intramolecular conserved cysteine residues of NF-YB, which is responsible for the redox-regulated assembly of NF-YB and NF-YC in human and Aspergillus nidulans, can be excluded for Arabidopsis NF-YB.
基金supported by the National Key R&D Program of China(No.2019YFC1711000)National Natural Science Foundation of China(81421005 and 81722048)+2 种基金111 Project(B16046,China)“Double First-Class”University Project(CPU2018GF04,China)the Qing Lan Project of Jiangsu Province(China)
文摘Disrupted redox status primarily contributes to myocardial ischemia/reperfusion injury(MIRI).NRF2,the endogenous antioxidant regulator,might provide therapeutic benefits.Dihydrotanshinone-Ⅰ(DT)is an active component in Salvia miltiorrhiza with NRF2 induction potency.This study seeks to validate functional links between NRF2 and cardioprotection of DT and to investigate the molecular mechanism particularly emphasizing on NRF2 cytoplasmic/nuclear translocation.DT potently induced NRF2 nuclear accumulation,ameliorating post-reperfusion injuries via redox alterations.Abrogated cardioprotection in NRF2-deficient mice and cardiomyocytes strongly supports NRF2-dependent cardioprotection of DT.Mechanistically,DT phosphorylated NRF2 at Ser40,rendering its nuclear-import by dissociating from KEAP1 and inhibiting degradation.Importantly,we identified PKC-δ-(Thr505)phosphorylation as primary upstream event triggering NRF2-(Ser40)phosphorylation.Knockdown of PKC-δdramatically retained NRF2 in cytoplasm,convincing its pivotal role in mediating NRF2 nuclear-import.NRF2 activity was further enhanced by activated PKB/GSK-3βsignaling via nuclear-export signal blockage independent of PKC-δactivation.By demonstrating independent modulation of PKC-δand PKB/GSK-3β/Fyn signaling,we highlight the ability of DT to exploit both nuclear import and export regulation of NRF2 in treating reperfusion injury harboring redox homeostasis alterations.Coactivation of PKC and PKB phenocopied cardioprotection of DT in vitro and in vivo,further supporting the potential applicability of this rationale.
基金the National Natural Science Foundation of China (Grant Nos. 30270639, 30570894 and 30570981)the Doctoral Research Foundation of the Jiangsu University, China (Grant No. 02JDG028)
文摘A number of transmembrane receptors are targeted to the nucleus and convincingly localized therein. However, what remains a conundrum is how these cell-surface receptors end up in the nucleus. In this study, we reported that the transmembrane receptor phosphorylated TrkA was located in a series of carrier vesicles, including ring-like vesicles near the plasma membrane, large core vesicles and small dense core vesicles around the nuclei, as well as in the nucleus in human glioma cell line U251 using immunocytochemistry and immunofluorescence staining. Meanwhile, we also showed that small dense core vesicles budded from large core vesicles, and interacted with the nuclear envelope. Accordingly, our results suggested that such a series of membrane compartments might be involved in the pathway of nuclear translocation of the transmembrane receptor TrkA.
基金supported by the National Key Research and Development Program of China (2017YFC1001303)International Cooperation Project of China and Canada NSFC (81661128010)+2 种基金National Natural Science Foundation of China (31471405, 81671456, 81671412)the National Key Basic Research Program (2013CB967404) the Doctoral Innovation Fund of School of Medicine, Shanghai Jiao Tong University (BXJ201640)
文摘As shown in our previous study, two alternatively spliced androgen receptor(AR) variants, which are exclusively expressed in the granulosa cells of patients with polycystic ovary syndrome, exhibit retarded nuclear translocation compared with wild-type AR. However, researchers have not yet determined whether these abnormalities correlate with heat shock protein 90(HSP90)and importin α(the former is a generally accepted co-chaperone of AR, and the latter is a component of classical nuclear import complexes). Here, these two variants were mainly retained in cytoplasm with HSP90 and importin α in the presence of dihydrotestosterone(DHT), and their levels in nucleus were significantly reduced, according to the immunofluorescence staining. The binding affinity of two AR variants for importin α was consistently decreased, while it was increased in WT-AR following DHT stimulation, leading to reduced nuclear import, particularly for the insertion-AR(Ins-AR). However, the binding affinities of two AR variants for HSP90 were increased in the absence of DHT compared with WT-AR, which functioned to maintain spatial structural stability, particularly for the deletion-AR(Del-AR). Therefore, the retarded nuclear translocation of two AR variants is associated with HSP90 and importin α, and the abnormal binding affinities for them play critical roles in this process.
基金supported by the National Key R&D Program of China(2019YFC1711000)the National Natural Science Foundation of China(81421005 and 81722048)“Double First-Class”University project(CPU2018GF04 and CPU2018GY09,China)。
文摘Rescuing cells from stress damage emerges a potential therapeutic strategy to combat myocardial infarction.Protocatechuic aldehyde(PCA)is a major phenolic acid in Chinese herb Danshen(Salvia miltiorrhiza root).This study investigated whether PCA regulated nuclear pyruvate kinase isoform M2(PKM2)function to protect cardiomyocytes.In rats subjected to isoprenaline,PCA attenuated heart injury and protected cardiomyocytes from apoptosis.Through DARTS and CETSA assays,we identified that PCA bound and promoted PKM2 nuclear translocation in cardiomyocytes exposed to oxygen/glucose deprivation(OGD).In the nucleus,PCA increased the binding of PKM2 to β-Catenin via preserving PKM2 acetylation,and the complex,in cooperation with T-cell factor 4(TCF4),was required for transcriptional induction of genes encoding anti-apoptotic proteins,contributing to rescuing cardiomyocyte survival.In addition,PCA ameliorated mitochondrial dysfunction and prevented mitochondrial apoptosis dependent on PKM2.Consistently,PCA increased the binding of PKM2 to β-Catenin,improved heart contractive function,normalized heart structure and attenuated oxidative damage in mice subjected to artery ligation,but the protective effects were lost in Pkm2-deficient heart.Together,we showed that PCA regulated nuclear PKM2 function to rescue cardiomyocyte survival via β-Catenin/TCF4 signaling cascade,suggesting the potential of pharmacological intervention of PKM2 shuttle to protect the heart.
基金supported by the Natural Science Foundation of Jiangsu Province of China,No.BK20211348(to SHQ)Xuzhou Basic Research Program,No.KC21030(to LYH)+1 种基金Leadership Program of Xuzhou Medical University,No.JBGS202203(to SHQ)Research Grant Council GRF of Hong Kong Special Administrative Region of China,No.17105220(to JGS)。
文摘It has been shown clinically that continuous removal of ischemia/reperfusion-induced reactive oxygen species is not conducive to the recovery of late stroke.Indeed,previous studies have shown that excessive increases in hypochlorous acid after stroke can cause severe damage to brain tissue.Our previous studies have found that a small amount of hypochlorous acid still exists in the later stage of stroke,but its specific role and mechanism are currently unclear.To simulate stroke in vivo,a middle cerebral artery occlusion rat model was established,with an oxygen-glucose deprivation/reoxygenation model established in vitro to mimic stroke.We found that in the early stage(within 24 hours)of ischemic stroke,neutrophils produced a large amount of hypochlorous acid,while in the recovery phase(10 days after stroke),microglia were activated and produced a small amount of hypochlorous acid.Further,in acute stroke in rats,hypochlorous acid production was prevented using a hypochlorous acid scavenger,taurine,or myeloperoxidase inhibitor,4-aminobenzoic acid hydrazide.Our results showed that high levels of hypochlorous acid(200μM)induced neuronal apoptosis after oxygen/glucose deprivation/reoxygenation.However,in the recovery phase of the middle cerebral artery occlusion model,a moderate level of hypochlorous acid promoted the proliferation and differentiation of neural stem cells into neurons and astrocytes.This suggests that hypochlorous acid plays different roles at different phases of cerebral ischemia/reperfusion injury.Lower levels of hypochlorous acid(5 and 100μM)promoted nuclear translocation ofβ-catenin.By transfection of single-site mutation plasmids,we found that hypochlorous acid induced chlorination of theβ-catenin tyrosine 30 residue,which promoted nuclear translocation.Altogether,our study indicates that maintaining low levels of hypochlorous acid plays a key role in the recovery of neurological function.
