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1.
ACS Appl Mater Interfaces ; 12(3): 3854-3861, 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31874024

RESUMO

Radioactive contamination is a highly concerning global environmental issue along with the development of the nuclear industry. On account of sophisticated operations and high cost of instrument detection methods, numerous efforts have been focused on rapid and simple detection of pollution elements and uranium is the most common one. It is an enormous challenge to push the limit of determination as low as possible while carrying out ultrasensitive detection. Here, we report an intelligent platform based on functionalized solid nanochannels to monitor ultratrace uranyl ions. The platform has a detection limit of 1 fM, which is far below the value that traditional instrumental methods can reach. What is more, the system also exhibits uranyl removal property. The mesenchymal stem cells cultivated in media containing uranyl can achieve excellent viability in the presence of the membranes. This work provides a new choice for handling global radioactive contamination of water.

2.
PLoS Pathog ; 15(10): e1008079, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31603949

RESUMO

Interferon-inducible p200 family protein IFI204 was reported to be involved in DNA sensing, and subsequently induces the production of type I interferons and proinflammatory mediators. However, its function in the regulation of antiviral innate immune signaling pathway remains unclear. Here we reported a novel role of IFI204 that specifically inhibits the IRF7-mediated type I interferons response during viral infection. IFI204 and other p200 family proteins are highly expressed in mouse hepatitis coronavirus-infected bone marrow-derived dendritic cells. The abundant IFI204 could significantly interact with IRF7 in nucleus by its HIN domain and prevent the binding of IRF7 with its corresponding promoter. Moreover, other p200 family proteins that possess HIN domain could also inhibit the IRF7-mediated type I interferons. These results reveal that, besides the positive regulation function in type I interferon response at the early stage of DNA virus infection, the interferon-inducible p200 family proteins such as IFI204 could also negatively regulate the IRF7-mediated type I interferon response after RNA virus infection to avoid unnecessary host damage from hyper-inflammatory responses.


Assuntos
Infecções por Coronavirus/imunologia , Coronavirus/imunologia , Fator Regulador 7 de Interferon/metabolismo , Interferon Tipo I/imunologia , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Células 3T3 , Células A549 , Animais , Linhagem Celular , Infecções por Coronavirus/patologia , Células HEK293 , Humanos , Imunidade Inata/imunologia , Inflamação/imunologia , Inflamação/patologia , Fator Regulador 7 de Interferon/genética , Camundongos , Células RAW 264.7
3.
J Oral Rehabil ; 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31556140

RESUMO

BACKGROUND: Bone reconstruction of the maxillary bone defects is an urgent issue due to its functional and aesthetic influence. MicroRNAs (miRNAs) are a class of non-coding RNAs that function in diverse biological and pathological processes. Recently, microRNA-21 (miR-21) was reported to play significant roles in bone formation, suggesting that miR-21 can be novel biomarker and therapeutic target for bone remodelling and skeletal diseases. However, the role of miR-21 in maxillary bone defects remains unclear. OBJECTIVE AND METHODS: This study aimed to investigate the effect of miR-21 on the bone reconstruction by inducing maxillary bone defects in wild-type (WT) and miR-21 knockout (miR-21-KO) mice and explore these mice as maxillary bone defect models. RESULTS: Micro-computed tomography (micro-CT) and histochemistry showed that the miR-21-KO mice had reduced bone reformation ability compared with the WT mice. The expression levels of alkaline phosphatase (ALP) and osteocalcin (OCN) were dramatically decreased in the miR-21-KO mice. In addition, injection of miR-21 agomir intra-peritoneally into miR-21-KO mice (miR-21-KO+ agomir) following the maxillary bone defects surgery displayed a significantly enhanced bone formation -promoting ability, which indicated that miR-21 agomir could ameliorate maxillary bone defects in miR-21-KO mice in vivo. Furthermore, immunohistochemistry suggested that ALP and OCN expressions were prominently increased in miR-21-KO+ agomir mice. CONCLUSION: These findings demonstrated that miR-21 deficiency impaired bone reformation and miR-21 contributed to the bone reconstruction of the maxillary bone defects. The evidence also supported the use of WT and miR-21-KO mice as maxillary bone defect models for further research.

4.
Curr Biol ; 29(14): R685-R688, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31336084

RESUMO

Mediator is a large, multi-module complex that plays a key role in transcription regulation in eukaryotes. A divergent Mediator from a unicellular eukaryote has been identified and characterized, revealing novel adaptations to mRNA and ncRNA transcription.

5.
Anal Chem ; 91(16): 10477-10483, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31318193

RESUMO

RNA molecules harbor diverse chemical modifications that play important regulatory roles in a variety of biological processes. Up to date, more than 150 modifications have been identified in various RNA species. Most of these modifications occurring in nucleic acids are the methylation of nucleic acids. It has been demonstrated that many of these methylation are reversible and undergo dynamic demethylation. Previous studies established that the demethylation of the two most important and prevalent modifications of 5-methylcytidine (m5C) and N6-methyladenosine (m6A) in nucleic acids is through the hydroxylation of m5C and m6A, forming 5-hydroxymethylcytidine (hm5C) and N6-hydroxymethyladenosine (hm6A), respectively. This indicates the hydroxylation of the methylated nucleosides may be a general pathway for the demethylation of nucleic acid methylation. However, few other hydroxylmethylation modifications have yet to be reported in existence in mammals. In the current study, we developed a neutral enzymatic digestion method for the mild digestion of nucleic acids, followed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. With the established method, we reported the existence of a new hydroxylmethylated nucleosides, N2-hydroxymethylguanosine (hm2G), in mammalian RNA. In addition, we found that the contents of hm2G, as well as N2-methylguanosine (m2G), showed significant differences between thyroid carcinoma tissues and tumor-adjacent normal tissues, indicating that m2G and hm2G in RNA may play certain roles in the carcinogenesis of thyroid carcinoma. Collectively, our study suggests that RNA hydroxylmethylation may be a new prevalent group of modifications existing in RNA, which expands the diversity of nucleic acid modifications and should exert regulatory functions in living organisms.

6.
J Proteomics ; 206: 103435, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31279926

RESUMO

The neurotoxins of venomous scorpion act on ion channels. Whether these neurotoxins are retained in processed Buthus martensii Karsch scorpions used in traditional Chinese medicine materials is unknown. Comprehensive mass spectrometry-based proteomic characterization of functionally active toxins in the processed medicinal scorpion material revealed 22 full-length and 44 truncated thermostable potassium channel-modulatory toxins that preserved six conserved cysteine residues capable of forming the three disulfide bonds necessary for toxicity. Additionally, a broad spectrum of degraded toxin fragments was found, indicating their relative thermal instability which enabled toxicity reduction. Furthermore, the suppression of interleukin-2 (IL-2) production in Jurkat cells and the reduced delayed-type hypersensitivity (DTH) response demonstrated that the extracts have immunoregulatory activity both in vitro and in vivo. Our work describes the first "map" of functionally active scorpion toxins in processed scorpion medicinal material, which is helpful to unveil the pharmaceutical basis of the processed scorpion medicinal material in traditional Chinese medicine. BIOLOGICAL SIGNIFICANCE: Scorpions have been used as medicinal materials in China for more than one thousand years. This is an example of the well-known "Combat poison with poison" strategy common to traditional Chinese medicine. In the past 30 years, extensive investigations of Chinese scorpions have indicated that the neurotoxins in the scorpion venom are the main toxic components and they target various ion channels in cell membranes. However, whether these neurotoxins are retained in processed Buthus martensii Karsch scorpions used for traditional Chinese medicine remains unknown. Our study described the thermal stability and instability of potassium channel-modulatory neurotoxins in processed scorpions and helps to understand the pharmaceutical basis underling the strategy of "combat poison with poison to cure diseases".

7.
Virol Sin ; 34(5): 549-562, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31134586

RESUMO

Dengue virus (DENV) infection is a worldwide public health threat. To date, the knowledge about the pathogenesis and progression of DENV infection is still limited. Combining global profiling based on proteomic analysis together with functional verification analysis is a powerful strategy to investigate the interplay between the virus and host cells. In the present study, quantitative proteomics has been applied to evaluate host responses (as indicated by altered proteins and modifications) in human cells (using K562 cell line) upon DENV-2 infection, as DENV-2 spreads most widely among all DENV serotypes. Comparative analysis was performed to define differentially expressed proteins in the infected cells compared to the mock-control, and it revealed critical pathogen-induced changes covering a broad spectrum of host cellular compartments and processes. We also discovered more dramatic changes (> 20%, 160 regulated phosphoproteins) in protein phosphorylation compared to protein expression (14%, 321 regulated proteins). Most of these proteins/phosphoproteins were involved in transcription regulation, RNA splicing and processing, immune system, cellular response to stimulus, and macromolecule biosynthesis. Western blot analysis was also performed to confirm the proteomic data. Potential roles of these altered proteins were discussed. The present study provides valuable large-scale protein-related information for elucidating the functional emphasis of host cell proteins and their post-translational modifications in virus infection, and also provides insight and protein evidence for understanding the general pathogenesis and pathology of DENV.


Assuntos
Vírus da Dengue/patogenicidade , Interações Hospedeiro-Patógeno/genética , Proteômica , Replicação Viral , Biologia Computacional , Vírus da Dengue/fisiologia , Humanos , Células K562 , Fosforilação , Processamento de Proteína Pós-Traducional
8.
Methods Mol Biol ; 1977: 43-70, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30980322

RESUMO

Lysine acetylation is an important posttranslational modification (PTM) that regulates the function of proteins by affecting their localization, stability, binding, and enzymatic activity. Aberrant acetylation patterns have been observed in numerous diseases, most notably cancer, which has spurred the development of potential therapeutics that target acetylation pathways. Mass spectrometry (MS) has become the most adopted tool not only for the qualitative identification of acetylation sites but also for their large-scale quantification. By using heavy isotope labeling in cell culture combined with MS, it is now possible to accurately quantify newly synthesized acetyl groups and other PTMs, allowing differentiation between dynamically regulated and steady-state modifications. Here, we describe MS-based protocols to identify acetylation sites and quantify acetylation rates on both proteins in general and in the special case of histones. In the experimental approach for the former, 13C-glucose and D3-acetate are used to metabolically label protein acetylation in cells with stable isotopes, thus allowing isotope incorporation to be tracked over time. After protein extraction and digestion, acetylated peptides are enriched via immunoprecipitation and then analyzed by MS. For histones, a similar metabolic labeling approach is performed, followed by acid extraction, derivatization with propionic anhydride, and trypsin digestion prior to MS analysis. The procedures presented may be adapted to investigate acetylation dynamics in a broad range of experimental contexts, including different cell types and stimulation conditions.


Assuntos
Histonas/metabolismo , Marcação por Isótopo , Proteômica , Acetilação , Técnicas de Cultura de Células , Cromatografia Líquida , Interpretação Estatística de Dados , Bases de Dados de Proteínas , Humanos , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Espectrometria de Massas em Tandem
9.
Sci Adv ; 5(3): eaau7566, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30854428

RESUMO

Polo-like kinase 1 (Plk1) is a crucial regulator of cell cycle progression; but the mechanism of regulation of Plk1 activity is not well understood. We present evidence that Plk1 activity is controlled by a balanced methylation and phosphorylation switch. The methyltransferase G9a monomethylates Plk1 at Lys209, which antagonizes phosphorylation of T210 to inhibit Plk1 activity. We found that the methyl-deficient Plk1 mutant K209A affects DNA replication, whereas the methyl-mimetic Plk1 mutant K209M prolongs metaphase-to-anaphase duration through the inability of sister chromatids separation. We detected accumulation of Plk1 K209me1 when cells were challenged with DNA damage stresses. Ablation of K209me1 delays the timely removal of RPA2 and RAD51 from DNA damage sites, indicating the critical role of K209me1 in guiding the machinery of DNA damage repair. Thus, our study highlights the importance of a methylation-phosphorylation switch of Plk1 in determining its kinase activity and functioning in DNA damage repair.

10.
Clin Epigenetics ; 11(1): 48, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30867030

RESUMO

BACKGROUND: Activation of transcription enhancers, especially super-enhancers, is one of the critical epigenetic features of tumorigenesis. However, very few studies have systematically identified the enhancers specific in cancer tissues. METHODS: Here, we studied the change of histone modifications in MMTV-PyVT breast cancer model, combining mass spectrometry-based proteomics and ChIP-seq-based epigenomics approaches. Some of the proteomic results were confirmed with western blotting and IHC staining. An inhibitor of H3K27ac was applied to study its effect on cancer development. RESULTS: H3K27ac and H4K8ac are elevated in cancer, which was confirmed in patient tissue chips. ChIP-seq revealed that H4K8ac is co-localized with H3K27ac on chromatin, especially on distal enhancers. Epigenomic studies further identified a subgroup of super-enhancers marked by H3K4me3 peaks in the intergenic regions. The H3K4me3-enriched regions enhancers are associated with higher level of H3K27ac and H4K8ac compared with the average level of conventional super-enhancers and are associated with higher transcription level of their adjacent genes. We identified 148 H3K4me3-enriched super-enhancers with higher gene expression in tumor, which may be critical for breast cancer. One inhibitor for p300 and H3K27ac, C646, repressed tumor formation probably through inhibiting Vegfa and other genes. CONCLUSIONS: Taken together, our work identifies novel regulators and provides important resource to the genome-wide enhancer studies in breast cancer and raises the possibility of cancer treatment through modulating enhancer activity.


Assuntos
Neoplasias da Mama/patologia , Elementos Facilitadores Genéticos , Histonas/genética , Histonas/metabolismo , Neoplasias Mamárias Experimentais/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Epigênese Genética , Epigenômica , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Código das Histonas , Humanos , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Proteômica , Ativação Transcricional , Regulação para Cima
11.
Genes Dev ; 33(5-6): 348-364, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30808657

RESUMO

RNAi and Polycomb repression play evolutionarily conserved and often coordinated roles in transcriptional silencing. Here, we show that, in the protozoan Tetrahymena thermophila, germline-specific internally eliminated sequences (IESs)-many related to transposable elements (TEs)-become transcriptionally activated in mutants deficient in the RNAi-dependent Polycomb repression pathway. Germline TE mobilization also dramatically increases in these mutants. The transition from noncoding RNA (ncRNA) to mRNA production accompanies transcriptional activation of TE-related sequences and vice versa for transcriptional silencing. The balance between ncRNA and mRNA production is potentially affected by cotranscriptional processing as well as RNAi and Polycomb repression. We posit that interplay between RNAi and Polycomb repression is a widely conserved phenomenon, whose ancestral role is epigenetic silencing of TEs.


Assuntos
Elementos de DNA Transponíveis/genética , Proteínas do Grupo Polycomb/genética , Proteínas de Protozoários/genética , Interferência de RNA , Tetrahymena thermophila/genética , Ativação Transcricional/genética , Epigênese Genética , Inativação Gênica , Mutação , RNA Mensageiro/genética , RNA não Traduzido/genética
12.
Nucleic Acids Res ; 47(D1): D1044-D1055, 2019 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-30445567

RESUMO

Whole-exome and whole-genome sequencing have revealed millions of somatic mutations associated with different human cancers, and the vast majority of them are located outside of coding sequences, making it challenging to directly interpret their functional effects. With the rapid advances in high-throughput sequencing technologies, genome-scale long-range chromatin interactions were detected, and distal target genes of regulatory elements were determined using three-dimensional (3D) chromatin looping. Herein, we present OncoBase (http://www.oncobase.biols.ac.cn/), an integrated database for annotating 81 385 242 somatic mutations in 68 cancer types from more than 120 cancer projects by exploring their roles in distal interactions between target genes and regulatory elements. OncoBase integrates local chromatin signatures, 3D chromatin interactions in different cell types and reconstruction of enhancer-target networks using state-of-the-art algorithms. It employs informative visualization tools to display the integrated local and 3D chromatin signatures and effects of somatic mutations on regulatory elements. Enhancer-promoter interactions estimated from chromatin interactions are integrated into a network diffusion system that quantitatively prioritizes somatic mutations and target genes from a large pool. Thus, OncoBase is a useful resource for the functional annotation of regulatory noncoding regions and systematically benchmarking the regulatory effects of embedded noncoding somatic mutations in human carcinogenesis.

13.
Proteomics ; 18(2)2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29251827

RESUMO

TBK1, STING, and MDA5 are important players within the antiviral innate immune response network. We mapped the interactome of endogenous TBK1, STING, and MDA5 by affinity enrichment MS in virally infected or uninfected THP-1 cells. Based on quantitative data of more than 2000 proteins and stringent statistical analysis, 58 proteins were identified as high-confidence interactors for at least one of three bait proteins. Our data indicated that TBK1 and MDA5 mostly interacted within preexisting protein networks, while STING interacted with different proteins with different viral infections. Functional analysis was performed on 17 interactors, and six were found to have functions in innate immune responses. We identified TTC4 as a TBK1 interactor and positive regulator of sendai virus-induced innate immunity.


Assuntos
Imunidade Inata , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica/métodos , Infecções por Respirovirus/imunologia , Vírus Sendai/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Células HEK293 , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Domínios e Motivos de Interação entre Proteínas , Infecções por Respirovirus/metabolismo , Infecções por Respirovirus/virologia , Vírus Sendai/isolamento & purificação , Células THP-1 , Replicação Viral
14.
J Proteome Res ; 17(1): 234-242, 2018 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-29121770

RESUMO

Histone post-translational modifications (PTMs) are fundamental players of chromatin regulation, as they contribute to editing histone chemical properties and recruiting proteins for gene transcription and DNA repair. Mass spectrometry (MS)-based proteomics is currently the most widely adopted strategy for high-throughput quantification of hundreds of histone PTMs. Samples such as primary tissues, complex model systems, and biofluids are hard to retrieve in large quantities. Because of this, it is critical to know whether the amount of sample available would lead to an exhaustive analysis if subjected to MS. In this work, we assessed the reproducibility in quantification of histone PTMs using a wide range of starting material, that is, from 5 000 000 to 50 000 cells. We performed the experiment using four different cell lines, that is, HeLa, 293T, human embryonic stem cells (hESCs), and myoblasts, and we quantified a list of 205 histone peptides using ion trap MS and our in-house software. Results highlighted that the relative abundance of some histone PTMs deviated as little as just 4% when comparing high starting material with histone samples extracted from 50 000 cells, for example, H3K9me2 (40% average abundance). Low abundance PTMs such as H3K4me2 (<3% average abundance) showed higher variability, but still ∼34%. This indicates that most PTMs, and especially abundant ones, are quantified with high precision starting from low cell counts. This study will help scientists to decide whether specific experiments are feasible and to plan how much sample should be reserved for histone analysis using MS.


Assuntos
Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Linhagem Celular , Humanos , Espectrometria de Massas , Reprodutibilidade dos Testes , Tamanho da Amostra
15.
Nucleic Acids Res ; 46(D1): D92-D99, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29040751

RESUMO

De novo mutations (DNMs) have been shown to be a major cause of severe early-onset genetic disorders such as autism spectrum disorder and intellectual disability. Over one million DNMs have been identified in developmental disorders by next generation sequencing, but linking these DNMs to the genes that they impact remains a challenge, as the majority of them are embedded in non-coding regions. As most developmental diseases occur in the early stages of development or during childhood, it is crucial to clarify the details of epigenetic regulation in early development in order to interpret the mechanisms underlying developmental disorders. Here, we develop EpiDenovo, a database that is freely available at http://www.epidenovo.biols.ac.cn/, and which provides the associations between embryonic epigenomes and DNMs in developmental disorders, including several neuropsychiatric disorders and congenital heart disease. EpiDenovo provides an easy-to-use web interface allowing users rapidly to find the epigenetic signatures of DNMs and the expression patterns of the genes that they regulate during embryonic development. In summary, EpiDenovo is a useful resource for selecting candidate genes for further functional studies in embryonic development, and for investigating regulatory DNMs as well as other genetic variants causing or underlying developmental disorders.


Assuntos
Bases de Dados Genéticas , Deficiências do Desenvolvimento/genética , Epigênese Genética , Mutação , Animais , Transtorno do Espectro Autista/genética , Criança , Imunoprecipitação da Cromatina , Desenvolvimento Embrionário/genética , Humanos , Deficiência Intelectual/genética , Internet , Camundongos , Interface Usuário-Computador
16.
Sci Rep ; 7(1): 10296, 2017 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-28860605

RESUMO

Protein acetylation plays a critical role in biological processes by regulating the functions and properties of proteins. Thus, the study of protein acetylation dynamics is critical for understanding of how this modification influences protein stability, localization, and function. Here we performed a comprehensive characterization of protein acetylation dynamics using mass spectrometry (MS) based proteomics through utilization of 13C-glucose or D3-acetate, which are metabolized into acetyl-coA, labeling acetyl groups through subsequent incorporation into proteins. Samples were collected at eight time points to monitor rates and trends of heavy acetyl incorporation. Through this platform, we characterized around 1,000 sites with significantly increasing acetylation trends, which we clustered based on their rates of acetylation. Faster rates were enriched on proteins associated with chromatin and RNA metabolism, while slower rates were more typical on proteins involved with lipid metabolism. Among others, we identified sites catalyzed at faster rates with potential critical roles in protein activation, including the histone acetyltransferase p300 acetylated in its activation loop, which could explain self-acetylation as an important feedback mechanism to regulate acetyltransferases. Overall, our studies highlight the dynamic nature of protein acetylation, and how metabolism plays a central role in this regulation.


Assuntos
Processamento de Proteína Pós-Traducional , Proteoma , Proteômica , Acetatos , Acetilcoenzima A/metabolismo , Acetilação , Biologia Computacional/métodos , Glucose/metabolismo , Células HeLa , Humanos , Peptídeos/metabolismo , Conformação Proteica , Proteômica/métodos , Relação Estrutura-Atividade
17.
Sci Rep ; 7(1): 8643, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28819100

RESUMO

Dysregulation of autophagy-mediated podocyte homeostasis is proposed to play a role in idiopathic membranous nephropathy (IMN). In the present study, autophagic activity and lysosomal alterations were investigated in podocytes of IMN patients and in cultured podocytes exposed to sublytic terminal complement complex, C5b-9. C5b-9 upregulated the number of LC3 positive puncta and the expression of p62 in patient podocytes and in C5b-9 injuried podocyte model. The lysosomal turnover of LC3-II was not influenced, although the BECN1 expression level was upregulated after exposure of podocytes to C5b-9. C5b-9 also caused a significant increase in the number of autophagosomes but not autolysosomes, suggesting that C5b-9 impairs the lysosomal degration of autophagosomes. Moreover, C5b-9 exacerbated the apoptosis of podocytes, which could be mimicked by chloroquine treatment, indicating that C5b-9 triggered podocyte injury, at least partially through inhibiting autophagy. Subsequent studies revealed that C5b-9 triggered lysosomal membrane permeabilization, which likely caused the decrease in enzymatic activity, defective acidification of lysosomes, and suppression of DQ-ovalbumin degradation. Taken together, our results suggest that the lysosomal-dependent autophagic pathway is blocked by C5b-9, which may play a key role in podocyte injury during the development of IMN.


Assuntos
Autofagia , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Glomerulonefrite Membranosa/metabolismo , Lisossomos/metabolismo , Podócitos/metabolismo , Transdução de Sinais , Adulto , Autofagossomos/metabolismo , Autofagia/imunologia , Permeabilidade da Membrana Celular , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Feminino , Glomerulonefrite Membranosa/tratamento farmacológico , Glomerulonefrite Membranosa/imunologia , Glomerulonefrite Membranosa/patologia , Humanos , Lisossomos/ultraestrutura , Masculino , Pessoa de Meia-Idade , Podócitos/patologia
18.
Nucleic Acids Res ; 45(9): 5183-5197, 2017 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-28334815

RESUMO

Histone modifiers regulate proper cellular activities in response to various environmental stress by modulating gene expression. In budding yeast, Rph1 transcriptionally represses many DNA damage or autophagy-related gene expression. However, little is known how Rph1 is regulated during these stress conditions. Here, we report that Rph1 is degraded upon DNA damage stress conditions. Notably, this degradation occurs via the autophagy pathway rather than through 26S proteasome proteolysis. Deletion of ATG genes or inhibition of vacuole protease activity compromises Rph1 turnover. We also determine that Rph1 and nuclear export protein Crm1 interact, which is required for Rph1 translocation from the nucleus to the cytoplasm. More importantly, Gcn5 directly acetylates Rph1 in vitro and in vivo, and Gcn5-containing complex, SAGA, is required for autophagic degradation of Rph1. Gcn5-mediated Rph1 acetylation is essential for the association of Rph1 with the nuclear pore protein Nup1. Finally, we show that sustaining high levels of Rph1 during DNA damage stress results in cell growth defects. Thus, we propose that Gcn5-mediated acetylation finely regulates Rph1 protein level and that autophagic degradation of Rph1 is important for cell homeostasis. Our findings may provide a general connection between DNA damage, protein acetylation and autophagy.


Assuntos
Autofagia , Dano ao DNA , Histona Acetiltransferases/metabolismo , Histona Desmetilases/metabolismo , Proteólise , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico , Acetilação/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Homeostase/efeitos dos fármacos , Carioferinas/metabolismo , Metanossulfonato de Metila/toxicidade , Modelos Biológicos , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo
19.
Sci Rep ; 7: 45103, 2017 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-28338045

RESUMO

In this study we quantified the alterations of retinal histone post-translational modifications (PTMs) in diabetic rats using a liquid chromatography - tandem mass spectrometry (LC-MS/MS) approach. Some diabetic rats were subsequently treated with minocycline, a tetracycline antibiotic, which has been shown to inhibit the diabetes-induced chronic inflammation in the retinas of rodents. We quantified 266 differentially modified histone peptides, including 48 out of 83 methylation marks with significantly different abundancein retinas of diabetic rats as compared to non-diabetic controls. About 67% of these marks had their relative abundance restored to non-diabetic levels after minocycline treatment. Mono- and di-methylation states of histone H4 lysine 20 (H4K20me1/me2), markers related to DNA damage response, were found to be up-regulated in the retinas of diabetic rats and restored to control levels upon minocycline treatment. DNA damage response biomarkers showed the same pattern once quantified by western blotting. Collectively, this study indicates that alteration of some histone methylation levels is associated with the development of diabetic retinopathy in rodents, and the beneficial effect of minocycline on the retinas of diabetic rodents is partially through its ability to normalize the altered histone methylation levels.


Assuntos
Antibacterianos/uso terapêutico , Retinopatia Diabética/tratamento farmacológico , Código das Histonas , Histonas/metabolismo , Minociclina/uso terapêutico , Retina/metabolismo , Animais , Antibacterianos/farmacologia , Técnicas de Cultura de Células , Dano ao DNA , Retinopatia Diabética/metabolismo , Metilação , Minociclina/farmacologia , Processamento de Proteína Pós-Traducional , Ratos , Ratos Sprague-Dawley , Retina/efeitos dos fármacos
20.
Sci China Life Sci ; 60(3): 264-270, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27761696

RESUMO

DNA replication elongation is tightly controlled by histone-modifying enzymes. Our previous studies showed that the histone methytransferase TXR1 (Tetrahymena Trithorax related protein 1) specifically catalyzes H3K27 monomethylation and affects DNA replication elongation in Tetrahymena thermophila. In this study, we investigated whether TXR1 has a substrate preference to the canonical H3 over the replacement variant H3.3. We demonstrated by histone mutagenesis that K27Q mutation in H3.3 further aggravated the replication stress phenotype of K27Q mutation in canonical H3, supporting H3.3 as a physiologically relevant substrate of TXR1. This result is in apparent contrast to the strong preference for canonical H3 recently reported in Arabidopsis homologues ATXR5 and ATXR6, and further corroborates the role of TXR1 in DNA replication.


Assuntos
Replicação do DNA , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Lisina/química , Tetrahymena thermophila/enzimologia , Sequência de Aminoácidos , Histona Metiltransferases , Metilação , Mutação , Proteínas de Protozoários/metabolismo , Especificidade por Substrato
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