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1.
Se Pu ; 39(10): 1094-1101, 2021 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-34505431

RESUMO

Histone post-translational modifications (HPTMs) have been believed to play crucial roles in the regulation of gene transcription. Thus, aberrant modification of histone can contribute to the occurrence and development of diseases such as tumors. To date, formalin fixed paraffin-embedded (FFPE) clinical tissues are believed to be one of the most valuable sample resources in the study of human diseases. Therefore, it is of great significance to reveal the molecular mechanism of cancer and discover the markers of tumor. Proteomics, based on high performance liquid chromatograph-tandem mass spectrometry (HPLC-MS/MS), has become a powerful tool for HPTM identification. However, HPTM analysis of FFPE samples is yet to be explored; it has not been reported in China to our best knowledge. In this study, a new method based on HPLC-MS/MS was developed for the extraction and separation of histone proteins and analysis and quantification of HPTMs in FFPE tissues. First, the strategy for the extraction and separation of histone proteins from FFPE samples were optimized. After comparing the extraction efficiency of two different methods, which include the acid extraction of histone and extraction of total protein, a novel method was developed for histone extraction, separation, and HPTMs analysis by integrating dewaxed hydration treatment of FFPE tissues with protein extraction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation. Furthermore, the effects of operation parameters on histone extraction and HPTM identification were investigated, including number of paraffin embedded sections and chemical derivation of histone proteins. Thereafter, the identification and quantification of HPTMs were performed using reversed-phase HPLC-MS/MS in data independent acquisition (DIA) mode. Finally, the optimized methods were applied to quantitative analysis of HPTMs in FFPE tissues. A variety of HPTMs were identified; they included lysine methylation, acetylation, crotonylation, etc. Therefore, the spectrum of HPTMs on global level was set for human breast cancer and paracancerous tissues from two cases of FFPE clinical tissues. The sites holding differential HPTM level in cancer and paracancerous tissues were further obtained by quantifying the individual HPTMs. Thus, the relationship between HPTM level and tumor was discussed. Abnormal HPTM sites were characterized using cluster analysis, thus their similar tendency was found. For example, abnormal HPTM sites such as H3K9me3, H3K9ac, and H3K27me3 in cancers are associated with epigenetic regulation. It indicated that different epigenetic events might occur in cancer and paracancerous tissues. Interestingly, we found that the level of H4K20me3 were both significantly down-regulated in the two cancer tissues. HPTM had been thought to be a potential biomarker in breast cancer; therefore, these positive results indicated that our method is effective for HPTMs of clinical FFPE samples. Our study provides a useful tool for the isolation and analysis of HPTMs in clinical FFPE samples, showing the potential for the detection of epigenetic biomarker in cancer.


Assuntos
Histonas , Espectrometria de Massas em Tandem , Epigênese Genética , Formaldeído , Histonas/metabolismo , Humanos , Inclusão em Parafina , Processamento de Proteína Pós-Traducional
2.
Mol Psychiatry ; 2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34580416

RESUMO

Infectious diseases, including COVID-19, are crucial public health issues and may lead to considerable fear among the general public and stigmatization of, and discrimination against, specific populations. This meta-analysis aimed to estimate the pooled prevalence of stigma in infectious disease epidemics. We systematically searched PubMed, PsycINFO, Embase, MEDLINE, Web of Science, and Cochrane databases since inception to June 08, 2021, and reported the prevalence of stigma towards people with infectious diseases including SARS, H1N1, MERS, Zika, Ebola, and COVID-19. A total of 50 eligible articles were included that contributed 51 estimates of prevalence in 92722 participants. The overall pooled prevalence of stigma across all populations was 34% [95% CI: 28-40%], including enacted stigma (36% [95% CI: 28-44%]) and perceived stigma (31% [95% CI: 22-40%]). The prevalence of stigma in patients, community population, and health care workers, was 38% [95% CI: 12- 65%], 36% [95% CI: 28-45%], and 30% [95% CI: 20-40%], respectively. The prevalence of stigma in participants from low- and middle-income countries was 37% [95% CI: 29-45%], which is higher than that from high-income countries (27% [95% CI: 18-36%]) though this difference was not statistically significant. A similar trend of prevalence of stigma was also observed in individuals with lower education (47% [95% CI: 23-71%]) compared to higher education level (33% [95% CI: 23-4%]). These findings indicate that stigma is a significant public health concern, and effective and comprehensive interventions are needed to counteract the damaging effects of the infodemics during infectious disease epidemics, including COVID-19, and reduce infectious disease-related stigma.

3.
J Biol Chem ; 297(3): 101036, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34343566

RESUMO

Proteins containing breast cancer type 1 (BRCA1) C-terminal domains play crucial roles in response to and repair of DNA damage. Epithelial cell transforming factor (epithelial cell transforming sequence 2 [ECT2]) is a member of the BRCA1 C-terminal protein family, but it is not known if ECT2 directly contributes to DNA repair. In this study, we report that ECT2 is recruited to DNA lesions in a poly (ADP-ribose) polymerase 1-dependent manner. Using co-immunoprecipitation analysis, we showed that ECT2 physically associates with KU70-KU80 and BRCA1, proteins involved in nonhomologous end joining and homologous recombination, respectively. ECT2 deficiency impairs the recruitment of KU70 and BRCA1 to DNA damage sites, resulting in defective DNA double-strand break repair, an accumulation of damaged DNA, and hypersensitivity of cells to genotoxic insults. Interestingly, we demonstrated that ECT2 promotes DNA repair and genome integrity largely independently of its canonical guanine nucleotide exchange activity. Together, these results suggest that ECT2 is directly involved in DNA double-strand break repair and is an important genome caretaker.

4.
Talanta ; 234: 122656, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34364465

RESUMO

This work presents a new method of Cr speciation analysis based on micro sample pretreatment with a paper-based analytical device (PAD). By using electrokinetic separation and stacking on the PAD, Cr (VI) and Cr (III) can be separated and the recovered to achieve speciation analysis without have to be subjected to subtraction treatment. The separation and recovery properties of Cr (VI) and Cr (III) were characterized and optimized by UV-Vis spectrophotometry, with which the LOQ of 19.0 µg L-1 and 28.7 µg L-1, and the recoveries of 88-108% and 90-110%, were obtained for Cr (VI) and Cr (III), respectively. In addition, direct analysis of Cr (VI)/Cr (III) from an electroplating wastewater sample was also demonstrated with this method combined with atomic spectroscopy (GF-AAS and ICP-OES). This sample pretreatment method is fast, cheap and easy to be used. Combined with the high sensitivity and elemental selectivity of atomic spectroscopy and mass spectrometry, this PAD sample pretreatment method could be a compensation to their lack in speciation discrimination, and may play an important role in the speciation analysis of Cr.


Assuntos
Cromo , Espectrometria de Massas , Análise Espectral
5.
Food Funct ; 12(9): 4021-4033, 2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-33977946

RESUMO

Docosahexaenoic acid-enriched phosphatidylserine (DHA-PS) has attracted increasing attention because of its unique health benefits. In this study, DHA-PS was biosynthesized from DHA-enriched phosphatidylcholine (DHA-PC), which was extracted from herring roe, Clupea harengus. The ameliorating effect of DHA-PS on high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) was investigated using a mouse model. The DHA-PS treatment ameliorated NAFLD and effectively decreased the serum total cholesterol, triglyceride, non-esterified fatty acid, and low-density lipoprotein cholesterol levels and considerably increased the serum high-density lipoprotein cholesterol levels. Moreover, the DHA-PS treatment reduced the levels of liver-function enzymes and pro-inflammatory cytokines and also the oxidative stress indices. Furthermore, DHA-PS increased the diversity and richness of the beneficial intestinal microorganisms, suggesting its potential as a dietary supplement and functional food to combat HFD-induced NAFLD.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/administração & dosagem , Hepatopatia Gordurosa não Alcoólica/terapia , Fosfatidilserinas/administração & dosagem , Tecido Adiposo , Animais , Peso Corporal , Disbiose/terapia , Dislipidemias , Alimento Funcional , Microbioma Gastrointestinal , Metabolismo dos Lipídeos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia
6.
Sci Adv ; 7(19)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33952527

RESUMO

The checkpoint kinase ATR [ATM (ataxia-telangiectasia mutated) and rad3-related] is a master regulator of DNA damage response. Yet, how ATR activity is regulated remains to be investigated. We report here that histone demethylase PHF8 (plant homeodomain finger protein 8) plays a key role in ATR activation and replication stress response. Mechanistically, PHF8 interacts with and demethylates TOPBP1 (DNA topoisomerase 2-binding protein 1), an essential allosteric activator of ATR, under unperturbed conditions, but replication stress results in PHF8 phosphorylation and dissociation from TOPBP1. Consequently, hypomethylated TOPBP1 facilitates RAD9 (RADiation sensitive 9) binding and chromatin loading of the TOPBP1-RAD9 complex to fully activate ATR and thus safeguard the genome and protect cells against replication stress. Our study uncovers a demethylation and phosphorylation code that controls the assembly of TOPBP1-scaffolded protein complex, and provides molecular insight into non-histone methylation switch in ATR activation.

7.
Cell Death Dis ; 12(2): 149, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542201

RESUMO

To support cellular homeostasis and mitigate chemotherapeutic stress, cancer cells must gain a series of adaptive intracellular processes. Here we identify that NUPR1, a tamoxifen (Tam)-induced transcriptional coregulator, is necessary for the maintenance of Tam resistance through physical interaction with ESR1 in breast cancers. Mechanistically, NUPR1 binds to the promoter regions of several genes involved in autophagy process and drug resistance such as BECN1, GREB1, RAB31, PGR, CYP1B1, and regulates their transcription. In Tam-resistant ESR1 breast cancer cells, NUPR1 depletion results in premature senescence in vitro and tumor suppression in vivo. Moreover, enforced-autophagic flux augments cytoplasmic vacuolization in NUPR1-depleted Tam resistant cells, which facilitates the transition from autophagic survival to premature senescence. Collectively, these findings suggest a critical role for NUPR1 as a transcriptional coregulator in enabling endocrine persistence of breast cancers, thus providing a vulnerable diagnostic and/or therapeutic target for endocrine resistance.


Assuntos
Antineoplásicos Hormonais/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Mama/tratamento farmacológico , Carcinoma Ductal de Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Tamoxifeno/farmacologia , Transcrição Genética , Animais , Autofagia/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Camundongos SCID , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas , Transcriptoma , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Mol Psychiatry ; 2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542468

RESUMO

Pandemics have become more frequent and more complex during the twenty-first century. Posttraumatic stress disorder (PTSD) following pandemics is a significant public health concern. We sought to provide a reliable estimate of the worldwide prevalence of PTSD after large-scale pandemics as well as associated risk factors, by a systematic review and meta-analysis. We systematically searched the MedLine, Embase, PsycINFO, Web of Science, CNKI, WanFang, medRxiv, and bioRxiv databases to identify studies that were published from the inception up to August 23, 2020, and reported the prevalence of PTSD after pandemics including sudden acute respiratory syndrome (SARS), H1N1, Poliomyelitis, Ebola, Zika, Nipah, Middle Eastern respiratory syndrome coronavirus (MERS-CoV), H5N1, and coronavirus disease 2019 (COVID-19). A total of 88 studies were included in the analysis, with 77 having prevalence information and 70 having risk factors information. The overall pooled prevalence of post-pandemic PTSD across all populations was 22.6% (95% confidence interval (CI): 19.9-25.4%, I2: 99.7%). Healthcare workers had the highest prevalence of PTSD (26.9%; 95% CI: 20.3-33.6%), followed by infected cases (23.8%: 16.6-31.0%), and the general public (19.3%: 15.3-23.2%). However, the heterogeneity of study findings indicates that results should be interpreted cautiously. Risk factors including individual, family, and societal factors, pandemic-related factors, and specific factors in healthcare workers and patients for post-pandemic PTSD were summarized and discussed in this systematic review. Long-term monitoring and early interventions should be implemented to improve post-pandemic mental health and long-term recovery.

9.
Mol Cell Proteomics ; : 100053, 2021 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-33561546

RESUMO

Esophageal squamous cell cancer (ESCC) is an aggressive malignancy with poor therapeutic outcomes. However, the alterations in proteins and post-translational modifications (PTMs) leading to the pathogenesis of ESCC remains unclear. Here, we provide the comprehensive characterization of the proteome, phosphorylome, lysine acetylome and succinylome for ESCC and matched control cells using quantitative proteomic approach. We identify abnormal protein and post-translational modification (PTM) pathways, including significantly downregulated lysine succinylation sites in cancer cells. Focusing on hyposuccinylation, we reveal that this altered PTM was enriched on enzymes of metabolic pathways inextricably linked with cancer metabolism. Importantly, ESCC malignant behaviors such as cell migration are inhibited once the level of succinylation was restored in vitro or in vivo. This effect was further verified by mutations to disrupt succinylation sites in candidate proteins. Meanwhile, we found that succinylation has a negative regulatory effect on histone methylation to promote cancer migration. Finally, hyposuccinylation is confirmed in primary ESCC specimens. Our findings together demonstrate that lysine succinylation may alter ESCC metabolism and migration, providing new insights into the functional significance of PTM in cancer biology.

10.
Mol Cell Proteomics ; 2020 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-33077686

RESUMO

Esophageal squamous cell cancer (ESCC) is an aggressive malignancy with poor therapeutic outcomes. However, the alterations in proteins and post-translational modifications (PTMs) leading to the pathogenesis of ESCC remains unclear. Here, we provide the comprehensive characterization of the proteome, phosphorylome, lysine acetylome and succinylome for ESCC and matched control cells using quantitative proteomic approach. We identify abnormal protein and post-translational modification (PTM) pathways, including significantly downregulated lysine succinylation sites in cancer cells. Focusing on hyposuccinylation, we reveal that this altered PTM was enriched on enzymes of metabolic pathways inextricably linked with cancer metabolism. Importantly, ESCC malignant behaviors such as cell migration are inhibited once the level of succinylation was restored in vitro or in vivo This effect was further verified by mutations to disrupt succinylation sites in candidate proteins. Meanwhile, we found that succinylation has a negative regulatory effect on histone methylation to promote cancer migration. Finally, hyposuccinylation is confirmed in primary ESCC specimens. Our findings together demonstrate that lysine succinylation may alter ESCC metabolism and migration, providing new insights into the functional significance of PTM in cancer biology.

11.
Theranostics ; 10(23): 10769-10790, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32929379

RESUMO

Rationale: A number of guanine nucleotide exchange factors (GEFs) including epithelial cell transforming factor ECT2 are believed to drive carcinogenesis through activating distinct oncogenic GTPases. Yet, whether GEF-independent activity of ECT2 also plays a role in tumorigenesis remains unclear. Methods: Immunohistochemical (IHC) staining, colony formation and xenograft assays were used to examine the role of ECT2 in breast carcinogenesis. Co-immunoprecipitation, immunofluorescent stainings, in vivo deubiquitination and in vitro deubiquitination experiments were performed to examine the physical and functional interaction between ECT2 and ubiquitin-specific protease USP7. High-throughput RNA sequencing, quantitative reverse transcription-PCR and Western blotting were employed to investigate the biological significance of the interplay between ECT2 and USP7. Results: We report that ECT2 plays a tumor-promoting role in breast cancer, and GEF activity-deficient ECT2 is able to alleviate ECT2 depletion associated growth defects in breast cancer cells. Mechanistically, we demonstrated that ECT2 physically interacts with ubiquitin-specific protease USP7 and functionally facilitates USP7 intermolecular self-association, -deubiquitination and -stabilization in a GEF activity-independent manner. USP7 in turn, deubiquitinates and stabilizes ECT2, resulting in a feedforward regulatory circuit that ultimately sustains the expression of oncogenic protein MDM2. Conclusion: Our study uncovers a GEF-independent role of ECT2 in promoting survival of breast cancer cells, provides a molecular insight for the reciprocal regulation of ECT2 and USP7, and supports the pursuit of ECT2/USP7 as potential targets for breast cancer intervention.


Assuntos
Neoplasias da Mama/genética , Carcinogênese/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Ensaios Enzimáticos , Retroalimentação Fisiológica , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Camundongos , Ligação Proteica/genética , Estabilidade Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/isolamento & purificação , RNA-Seq , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Peptidase 7 Específica de Ubiquitina/genética , Peptidase 7 Específica de Ubiquitina/isolamento & purificação , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Front Pharmacol ; 11: 614, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32595491

RESUMO

The incidence and mortality of cardiovascular disease in diabetic patients are 2-3 times higher than those in non-diabetic patients. Abnormal function of the L-type calcium channel in myocardial tissue might result in multiple cardiac disorders such as a prolonged QT interval. Therefore, QT prolongation is an independent risk factor of cardiovascular disease in patients with diabetes mellitus. Metformin, a hypoglycemic agent, is widely known to effectively reduce the occurrence of macrovascular diseases. The aim of the present study was to evaluate the effect of metformin on prolonged QT interval and to explore potential ionic mechanisms induced by diabetes. Diabetic mouse models were established with streptozotocin and an electrocardiogram was used to monitor the QT interval after 4 weeks of metformin treatment in each group. Action potential duration (APD) and L-type calcium current (I Ca-L) were detected by patch-clamp in isolated mice ventricular cardiomyocytes and neonatal cardiomyocytes of mice. The expression levels of CACNA1C mRNA and Cav1.2 were measured by real-time PCR, western blot and immunofluorescence. A shortened QT interval was observed after 4 weeks of metformin treatment in diabetic mice. Patch-clamp results revealed that both APD and I Ca-L were shortened in mouse cardiomyocytes. Furthermore, the expression levels of CACNA1C mRNA and Cav1.2 were decreased in the metformin group. The same results were also obtained in cultured neonatal mice cardiomyocytes. Overall, these results verify that metformin could shorten a prolonged QT interval by inhibiting the calcium current, suggesting that metformin may play a role in the electrophysiology underlying diabetic cardiopathy.

13.
Anal Chem ; 92(9): 6218-6223, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32243745

RESUMO

Histone post-translational modifications (HPTMs) serve as signal platforms for recruitment of binding proteins (readers) to regulate gene expression. Accumulated evidence suggests that the intensive distribution of HPTMs may result in crosstalk, which increases or inhibits the recruitment of reader proteins, further altering the functional outcome of HPTMs. Therefore, the comprehensive identification of multiple interactions between combinatorial HPTMs and reading domains is essential to understand the chromatin-templated processes. However, it is still a big challenge to profile these complicated interactions due to various limitations including rather weak, transient and multiple interactions between HPTMs and readers, the high dynamic property of HPTMs as well as the low abundance of reader proteins. Here we developed an integrated approach to profile the complicated interactions between combinatorial HPTMs and dual domains. Based on a combinatorial HPTM peptide library (trimethylation of histone H3 lysine 4 and its neighboring PTMs) and five affinity tag proteins containing tandem-domain probes, histone interactions can be profiled by pull-down assay combined with mass spectrometry analysis. The interactions were further verified by isothermal titration calorimetry and proximity ligation assay, as well as molecular docking. By use of combinatorial HPTMs, we demonstrated that this integrated approach can be successfully utilized for the characterization of multiple interactions between reading domains and combinatorial HPTMs including novel HPTMs with low stoichiometry. Thus, a novel chemical proteomics tool for profiling of multiple PTM-mediated protein-protein interactions was successfully developed and can be adapted for broad biomedical applications.


Assuntos
Calorimetria , Histonas/metabolismo , Simulação de Dinâmica Molecular , Corantes Fluorescentes/química , Células HEK293 , Histonas/química , Histonas/genética , Humanos , Microscopia de Fluorescência , Mutagênese , Biblioteca de Peptídeos , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
14.
J Adv Res ; 22: 35-46, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31956440

RESUMO

Histone posttranslational modifications (HPTMs) play important roles in eukaryotic transcriptional regulation. Recently, it has been suggested that combinatorial modification codes that comprise two or more HPTMs can recruit readers of HPTMs, performing complex regulation of gene expression. However, the characterization of the multiplex interactions remains challenging, especially for the molecular network of histone PTMs, readers and binding complexes. Here, we developed an integrated method that combines a peptide library, affinity enrichment, mass spectrometry (MS) and bioinformatics analysis for the identification of the interaction between HPTMs and their binding proteins. Five tandem-domain-reader proteins (BPTF, CBP, TAF1, TRIM24 and TRIM33) were designed and prepared as the enriched probes, and a group of histone peptides with multiple PTMs were synthesized as the target peptide library. First, the domain probes were used to pull down the PTM peptides from the library, and then the resulting product was characterized by MS. The binding interactions between PTM peptides and domains were further validated and measured by isothermal titration calorimetry analysis (ITC). Meanwhile, the binding proteins were enriched by domain probes and identified by HPLC-MS/MS. The interaction network of histone PTMs-readers-binding complexes was finally analyzed via informatics tools. Our results showed that the integrated approach combining MS analysis with ITC assay enables us to understand the interaction between the combinatorial HPTMs and reading domains. The identified network of "HPTMs-reader proteins-binding complexes" provided potential clues to reveal HPTM functions and their regulatory mechanisms.

15.
Anat Rec (Hoboken) ; 303(5): 1337-1345, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31502386

RESUMO

The NAD+ -dependent protein deacetylase silent information regulator 1 (SIRT1) targets multiple proteins for deacetylation, and it has been implicated in a variety of cellular pathways and human diseases. However, it remains unclear how the abundance of SIRT1 is regulated. Here, by mass spectrometry analysis of SIRT1-containing protein complexes, we revealed that SIRT1 is physically associated with the ubiquitin-specific protease USP7. Importantly, we found that USP7 cleaves K48-linked polyubiquitin chains of SIRT1 and promotes SIRT1 stabilization. Accordingly, we demonstrated that treatment of cells with an enzymatic inhibitor of USP7 led to a decreased level of SIRT1 expression and accumulation of SIRT1 polyubiquitination. Collectively, our findings indicate that USP7 is a critical regulator of SIRT1 and provide a new pathway for the maintenance of SIRT1 abundance in cells. Anat Rec, 303:1337-1345, 2020. © 2019 American Association for Anatomy.


Assuntos
Sirtuína 1/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Células HeLa , Humanos , Células MCF-7 , Espectrometria de Massas em Tandem
16.
Sci Adv ; 5(7): eaaw6703, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31328167

RESUMO

Lysine 2-hydroxyisobutyrylation (Khib) has recently been shown to be an evolutionarily conserved histone mark. Here, we report that CobB serves as a lysine de-2-hydroxyisobutyrylation enzyme that regulates glycolysis and cell growth in prokaryotes. We identified the specific binding of CobB to Khib using a novel self-assembled multivalent photocrosslinking peptide probe and demonstrated that CobB can catalyze lysine de-2-hydroxyisobutyrylation both in vivo and in vitro. R58 of CobB is a critical site for its de-2-hydroxyisobutyrylase activity. Using a quantitative proteomics approach, we identified 99 endogenous substrates that are targeted by CobB for de-2-hydroxyisobutyrylation. We further demonstrated that CobB can regulate the catalytic activities of enolase (ENO) by removing K343hib and K326ac of ENO simultaneously, which account for changes of bacterial growth. In brief, our study dissects a Khib-mediated molecular mechanism that is catalyzed by CobB for the regulation of the activity of metabolic enzymes as well as the cell growth of bacteria.


Assuntos
Proteínas de Escherichia coli/metabolismo , Lisina/análogos & derivados , Células Procarióticas/metabolismo , Sirtuínas/metabolismo , Acetilação , Arginina/metabolismo , Biocatálise , Domínio Catalítico , Escherichia coli/metabolismo , Lisina/metabolismo , Proteus mirabilis/metabolismo , Especificidade por Substrato
17.
Br J Pharmacol ; 176(17): 3206-3219, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31162630

RESUMO

BACKGROUND AND PURPOSE: The increased proliferation and migration of vascular smooth muscle cells (VSMCs) after arterial injury contributes greatly to the pathogenesis of neointimal hyperplasia. As a major component of epigenetics, histone methylation plays an important role in several cardiovascular diseases. However, its role in restenosis is still unclear. EXPERIMENTAL APPROACH: Human aortic VSMCs were challenged with PDGF-BB, and total histones were extracted and analysed by HPLC/MS. For the in vivo study, rats were subjected to wire-guided common carotid injury. KEY RESULTS: PDGF-BB markedly increased the H3K27me3 level, as demonstrated by use of HPLC/MS and confirmed by western blot analysis. Enhancer of zeste homologue 2 (EZH2), the histone H3K27 methyltransferase component of polycomb repressive complex 2, was also up-regulated by PDGF-BB in VSMCs, and in the neointimal hyperplasia induced by wire injury of the rat carotid artery. Furthermore, inhibiting H3K27me3 by treatment with 3-µM UNC1999, an EZH2/1 inhibitor, significantly suppressed PDGF-BB-induced VSMC proliferation compared with the PDGF-BB-treated group. Consistently, neointimal formation was significantly attenuated by oral or perivascular administration of UNC1999 compared with the sham group. Mechanistically, the increase in H3K27me3 inhibited the transcription of the cyclin-dependent kinase inhibitor p16INK4A and thus promoted VSMC proliferation. CONCLUSIONS AND IMPLICATIONS: Vascular injury elevated the expression of EZH2 and the downstream target H3K27me3, which suppressed p16INK4A expression in VSMCs and promoted VSMC proliferation and neointimal hyperplasia. EZH2 inhibition might be a potential therapeutic target for restenosis.


Assuntos
Becaplermina/farmacologia , Histonas/antagonistas & inibidores , Hiperplasia/tratamento farmacológico , Músculo Liso Vascular/efeitos dos fármacos , Neointima/tratamento farmacológico , Complexo Repressor Polycomb 2/antagonistas & inibidores , Animais , Becaplermina/química , Lesões das Artérias Carótidas/tratamento farmacológico , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Histonas/metabolismo , Humanos , Hiperplasia/patologia , Masculino , Metilação/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Neointima/patologia , Complexo Repressor Polycomb 2/metabolismo , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade
18.
Sensors (Basel) ; 19(11)2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31163585

RESUMO

Hemiparesis is one of the common sequelae of neurological diseases such as strokes, which can significantly change the gait behavior of patients and restrict their activities in daily life. The results of gait characteristic analysis can provide a reference for disease diagnosis and rehabilitation; however, gait correlation as a gait characteristic is less utilized currently. In this study, a new non-contact electrostatic field sensing method was used to obtain the electrostatic gait signals of hemiplegic patients and healthy control subjects, and an improved Detrended Cross-Correlation Analysis cross-correlation coefficient method was proposed to analyze the obtained electrostatic gait signals. The results show that the improved method can better obtain the dynamic changes of the scaling index under the multi-scale structure, which makes up for the shortcomings of the traditional Detrended Cross-Correlation Analysis cross-correlation coefficient method when calculating the electrostatic gait signal of the same kind of subjects, such as random and incomplete similarity in the trend of the scaling index spectrum change. At the same time, it can effectively quantify the correlation of electrostatic gait signals in subjects. The proposed method has the potential to be a powerful tool for extracting the gait correlation features and identifying the electrostatic gait of hemiplegic patients.


Assuntos
Fenômenos Biomecânicos , Transtornos Neurológicos da Marcha/fisiopatologia , Eletricidade Estática , Caminhada/fisiologia , Feminino , Marcha/fisiologia , Humanos , Masculino , Acidente Vascular Cerebral/fisiopatologia
19.
J Biol Chem ; 294(25): 9844-9857, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31073027

RESUMO

Hyperactivation of the canonical Wnt-signaling pathway is a prominent feature of a number of human malignancies. Transcriptional activation of this signaling cascade depends on the formation of the ß-catenin-B-cell CLL/lymphoma 9 (BCL9)-pygopus (PYGO) family plant homeodomain finger 1 complex, yet how the assembly of this complex is regulated remains to be investigated. Here, using MCF-7, HeLa, HEK293T, MDA-MB-231, and Sf9 cells, along with immunoblotting and immunofluorescence, nano-HPLC-MS/MS, deubiquitination, immunoprecipitation, and chromatin immunoprecipitation (ChIP) assays, we report that BCL9 physically associates with a protein deubiquitinase, ubiquitin-specific peptidase 9, X-linked (USP9X), and that USP9X removes Lys-63-linked polyubiquitin on Lys-212 of BCL9. Importantly, the USP9X-mediated BCL9 deubiquitination facilitated the formation of the ß-catenin-BCL9-PYGO complex, thereby potentiating the transcriptional activation of Wnt/ß-catenin target genes. We also show that USP9X-mediated BCL9 deubiquitination promotes the proliferation and invasion of breast cancer cells. Together, these results uncover USP9X as a deubiquitinase of BCL9, implicating USP9X in Wnt/ß-catenin signaling and breast carcinogenesis.


Assuntos
Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina/metabolismo , Proteína Wnt1/metabolismo , beta Catenina/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Movimento Celular , Proliferação de Células , Feminino , Humanos , Proteólise , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Ubiquitina Tiolesterase/genética , Proteína Wnt1/genética , beta Catenina/genética
20.
Sensors (Basel) ; 19(7)2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30978981

RESUMO

Walking is a basic requirement for participating in daily activities. Neurological diseases such as stroke can significantly affect one's gait and thereby restrict one's activities that are a part of daily living. Previous studies have demonstrated that gait temporal parameters are useful for characterizing post-stroke hemiparetic gait. However, no previous studies have investigated the symmetry, regularity and stability of post-stroke hemiparetic gaits. In this study, the dynamic time warping (DTW) algorithm, sample entropy method and empirical mode decomposition-based stability index were utilized to obtain the three aforementioned types of gait features, respectively. Studies were conducted with 15 healthy control subjects and 15 post-stroke survivors. Experimental results revealed that the proposed features could significantly differentiate hemiparetic patients from healthy control subjects by a Mann-Whitney test (with a p-value of less than 0.05). Finally, four representative classifiers were utilized in order to evaluate the possible capabilities of these features to distinguish patients with hemiparetic gaits from the healthy control subjects. The maximum area under the curve values were shown to be 0.94 by the k-nearest-neighbor (kNN) classifier. These promising results have illustrated that the proposed features have considerable potential to promote the future design of automatic gait analysis systems for clinical practice.


Assuntos
Marcha/fisiologia , Paresia/fisiopatologia , Reabilitação do Acidente Vascular Cerebral/métodos , Acidente Vascular Cerebral/fisiopatologia , Adulto , Algoritmos , Feminino , Análise da Marcha/métodos , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Paresia/terapia , Estatísticas não Paramétricas , Acidente Vascular Cerebral/terapia , Caminhada/fisiologia
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