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1.
BackgroundDNA and chromatin modifications are critical mediators in the establishment and maintenance of cell type-specific gene expression patterns that constitute cellular identities. One type of modification, the acetylation and deacetylation of histones, occurs reversibly on lysine ε-NH3+ groups of core histones via histone acetyl transferases (HAT) and histone deacetylases (HDAC). Hyperacetylated histones are associated with active chromatin domains, whereas hypoacetylated histones are enriched in non-transcribed loci.MethodsWe analyzed global histone H4 acetylation and HDAC activity levels in mature lineage marker-positive (Lin+) and progenitor lineage marker-negative (Lin?) hematopoietic cells from murine bone marrow (BM). In addition, we studied the effects of HDAC inhibition on hematopoietic progenitor/stem cell (HPSC) frequencies, cell survival, differentiation and HoxB4 dependence.ResultsWe observed that Lin? and Lin+ cells do not differ in global histone H4 acetylation but in HDAC activity levels. Further, we saw that augmented histone acetylation achieved by transient Trichostatin A (TSA) treatment increased the frequency of cells with HPSC immunophenotype and function in the heterogeneous pool of BM cells. Induction of histone hyperacetylation in differentiated BM cells was detrimental, as evidenced by preferential death of mature BM cells upon HDAC inhibition. Finally, TSA treatment of BM cells from HoxB4?/? mice revealed that the HDAC inhibitor-mediated increase in HPSC frequencies was independent of HoxB4.ConclusionsOverall, these data indicate the potential of chromatin modifications for the regulation of HPSC. Chromatin-modifying agents may provide potential strategies for ex vivo expansion of HPSC.  相似文献   

2.
BackgroundMost of the enzymes involved in the central carbon metabolism are acetylated in Lys residues. It has been claimed that this covalent modification represents a novel regulatory mechanism by which both enzyme/transporter activities and pathway fluxes can be modulated.MethodsTo establish which enzymes are regulated by acetylation, a systematic experimental analysis of activities and acetylation profile for several energy metabolism enzymes and pathway fluxes was undertaken in cells and mitochondria.ResultsThe majority of the glycolytic and neighbor enzymes as well as mitochondrial enzymes indeed showed Lys-acetylation, with GLUT1, HPI, CS, ATP synthase displaying comparatively lower acetylation patterns. The incubation of cytosolic and mitochondrial fractions with recombinant Sirt-3 produced lower acetylation signals, whereas incubation with acetyl-CoA promoted protein acetylation. Significant changes in acetylation levels of MDH and IDH-2 from rat liver mitochondria revealed no change in their activities. Similar observations were attained for the cytosolic enzymes from AS-30D and HeLa cells. A minor but significant (23%) increase in the AAT-MDH complex activity induced by acetylation was observed. To examine this question further, AS-30D and HeLa cells were treated with nicotinamide and valproic acid. These compounds promoted changes in the acetylation patterns of glycolytic proteins, although their activities and the glycolytic flux (as well as the OxPhos flux) revealed no clear correlation with acetylation.ConclusionAcetylation seems to play no predominant role in the control of energy metabolism enzyme activities and pathway fluxes.General significanceThe physiological function of protein acetylation on energy metabolism pathways remains to be elucidated.  相似文献   

3.
乙酰化修饰是由乙酰基转移酶、去乙酰化酶介导的可逆的蛋白质翻译后修饰。其中,乙酰基转移酶将乙酰辅酶A的乙酰基团转移至底物蛋白的氨基酸残基,而乙酰基团的去除由去乙酰化酶完成。乙酰化修饰参与许多基本生物学过程的调节作用,越来越多的研究表明,蛋白质乙酰化修饰在病原菌的致病过程中具有重要作用。病原菌,如引起非典型性肺炎的嗜肺军团菌,可以通过分泌具有乙酰基转移酶活性的效应蛋白靶向宿主细胞信号通路的关键蛋白质因子,干扰宿主细胞信号通路及免疫反应。本文主要从嗜肺军团菌的致病机制、乙酰化修饰及乙酰化修饰在病原体致病过程中的调控作用进行综述,突出已知的乙酰化毒力蛋白的例子,并讨论它们如何影响与宿主的相互作用,为理解乙酰化修饰在嗜肺军团菌致病过程中的作用机制提供参考。  相似文献   

4.
Reversible lysine acetylation (RLA) is used by cells of all domains of life to modulate protein function. To date, bacterial acetylation/deacetylation systems have been studied in a few bacteria (e.g., Salmonella enterica, Bacillus subtilis, Escherichia coli, Erwinia amylovora, Mycobacterium tuberculosis, and Geobacillus kaustophilus), but little is known about RLA in antibiotic-producing actinomycetes. Here, we identify the Gcn5-like protein acetyltransferase AcuA of Saccharopolyspora erythraea (SacAcuA, SACE_5148) as the enzyme responsible for the acetylation of the AMP-forming acetyl coenzyme A synthetase (SacAcsA, SACE_2375). Acetylated SacAcsA was deacetylated by a sirtuin-type NAD+-dependent consuming deacetylase (SacSrtN, SACE_3798). In vitro acetylation/deacetylation of SacAcsA enzyme was studied by Western blotting, and acetylation of lysine residues Lys237, Lys380, Lys611, and Lys628 was confirmed by mass spectrometry. In a strain devoid of SacAcuA, none of the above-mentioned Lys residues of SacAcsA was acetylated. To our knowledge, the ability of SacAcuA to acetylate multiple Lys residues is unique among AcuA-type acetyltransferases. Results from site-specific mutagenesis experiments showed that the activity of SacAcsA was controlled by lysine acetylation. Lastly, immunoprecipitation data showed that in vivo acetylation of SacAcsA was influenced by glucose and acetate availability. These results suggested that reversible acetylation may also be a conserved regulatory posttranslational modification strategy in antibiotic-producing actinomycetes.  相似文献   

5.
6.
BackgroundThe damage of pancreatic β cells is a major pathogenesis of the development and progression of type 2 diabetes and there is still no effective therapy to protect pancreatic β cells clinically. In our previous study, we found that Quzhou Fructus Aurantii (QFA), which is rich in flavanones, had the protective effect of pancreatic β cells in diabetic mice. However, the underlying mechanism is still unclear.PurposeIn the current study, we administered naringenin and hesperetin, two major active components of QFA, to protect pancreatic β cells and to investigate the underlying molecular mechanism focusing on the epigenetic modifications.MethodsWe used diabetic db/db mouse and INS-1 pancreatic β cell line as in vivo and in vitro models to investigate the protective effect of naringenin and hesperetin on pancreatic β cells under high glucose environment and the related mechanism. The phenotypic changes were evaluatedby immunostaining and the measurement of biochemical indexes. The molecular mechanism was explored by biological techniques such as western blotting, qPCR, ChIP-seq and ChIP-qPCR, flow cytometry and lentivirus infection.ResultsWe found that naringenin and hesperetin had an inhibitory effect on histone acetylation. We showed that naringenin and hesperetin protected pancreatic β cells in vivo and in vitro, and this effect was independent of their direct antioxidant capacity. The further study found that the inhibition of thioredoxin-interacting protein (Txnip) expression regulated by histone acetylation was critical for the protective role of naringenin and hesperetin. Mechanistically, the histone acetylation inhibition by naringenin and hesperetin was achieved through regulating AMPK-mediated p300 inactivation.ConclusionThese findings highlight flavanones and the phytomedicine rich in flavanones as important dietary supplements in protecting pancreatic β cells in advanced diabetes. In addition, targeting histone acetylation by phytomedicine is a potential strategy to delay the development and progression of diabetes.  相似文献   

7.
A member of the sirtuin family of NAD+-dependent deacetylases, SIRT3, is located in mammalian mitochondria and is important for regulation of mitochondrial metabolism, cell survival, and longevity. In this study, MRPL10 (mitochondrial ribosomal protein L10) was identified as the major acetylated protein in the mitochondrial ribosome. Ribosome-associated SIRT3 was found to be responsible for deacetylation of MRPL10 in an NAD+-dependent manner. We mapped the acetylated Lys residues by tandem mass spectrometry and determined the role of these residues in acetylation of MRPL10 by site-directed mutagenesis. Furthermore, we observed that the increased acetylation of MRPL10 led to an increase in translational activity of mitochondrial ribosomes in Sirt3−/− mice. In a similar manner, ectopic expression and knockdown of SIRT3 in C2C12 cells resulted in the suppression and enhancement of mitochondrial protein synthesis, respectively. Our findings constitute the first evidence for the regulation of mitochondrial protein synthesis by the reversible acetylation of the mitochondrial ribosome and characterize MRPL10 as a novel substrate of the NAD+-dependent deacetylase, SIRT3.  相似文献   

8.
周盈  毕利军 《微生物学报》2018,58(7):1233-1244
【目的】研究乙酰化修饰对Ku蛋白活性的影响。【方法】利用耻垢分枝杆菌为表达菌株,转入Ku蛋白表达质粒,纯化具有乙酰化修饰的Ku蛋白和无乙酰化的Ku蛋白突变体,比较两类蛋白的生化活性;分析氧化压力和酸性环境下耻垢分枝杆菌细胞内Ku蛋白乙酰化水平的变化。【结果】Ku蛋白过量表达的耻垢分枝杆菌比转入空质粒的对照菌株生长缓慢;乙酰化Ku蛋白比未发生乙酰化Ku蛋白修复断裂DNA的活性降低、DNA结合活性降低;氧化压力和酸性压力环境下,耻垢分枝杆菌细胞内Ku蛋白数量降低,乙酰化Ku蛋白数量变化不大。【结论】乙酰化修饰能够调节Ku蛋白的DNA结合活性,从而调节非同源末端连接修复系统的活性;Ku蛋白乙酰化程度升高是耻垢分枝杆菌对不良生长环境的反应。  相似文献   

9.
【背景】正常生理状况下核糖体蛋白SA (ribosomal protein SA, RPSA)主要在细胞内表达,参与多种细胞功能。在发生感染性疾病时,RPSA往往会异位于胞膜,介导微生物的感染。【目的】全面揭示RPSA在猪链球菌2型(Streptococcus suis serotype 2, SS2)感染宿主过程中的作用。【方法】首先利用本课题组已有的脑脊液和血清蛋白组学数据库(SS2脑膜炎感染模型的仔猪和健康仔猪),借助生物信息学手段分别筛选脑脊液和血清中的差异表达蛋白(differentially expressed proteins, DEPs),并对其涉及的信号通路进行分析。通过体外烯醇化酶(enolase, ENO)刺激宿主细胞,检测宿主细胞线粒体膜电位、钙离子含量和活性氧(reactive oxygen species, ROS)等指标变化,揭示RPSA介导SS2-ENO对宿主细胞主要能量细胞器——线粒体功能的影响。【结果】生物信息学揭示SS2感染宿主后,RPSA和相关蛋白显著富集在代谢和糖酵解/糖异生等能量有关通路。SS2-ENO刺激导致宿主细胞线粒体膜电位下降、钙离子和ROS水平升高。封闭RPSA后缓解了ENO对线粒体膜电位、细胞活性氧和细胞内钙离子含量的影响。【结论】RPSA介导SS2毒力因子ENO损伤宿主细胞线粒体功能。本研究丰富了SS2感染时RPSA的作用机制,为SS2脑膜炎疾病的防治提供了理论基础。  相似文献   

10.
BackgroundAlanine and proline-rich protein (Apa) is a secreted antigen of Mycobacterium spp. which involves in stimulating immune responses and adhering to host cells by binding to fibronectin (Fn). Here, we report the crystal structure of Apa from Mycobacterium tuberculosis (Mtb) and its Fn-binding characteristics.MethodsThe crystal structure of Mtb Apa was determined at resolutions of 1.54 Å. The dissociation constants (KD) of Apa and individual modules of Fn were determined by surface plasmon resonance and enzyme-linked immunosorbent assay. Site-directed mutagenesis was performed to investigate the putative Fn-binding motif of Apa.ResultsMtb Apa folds into a large seven-stranded anti-parallel β-sheet which is flanked by three α-helices. The binding affinity of Mtb Apa to individual Fn modules was assessed and the results indicated that the Mtb Apa binds to FnIII-4 and FnIII-5 of Fn CBD segment. Notably, structure analysis suggested that the previously proposed Fn-binding motif 258RWFV261 is buried within the protein and may not be accessible to the binding counterpart.ConclusionsThe structural and Fn-binding characteristics we reported here provide molecular insights into the multifunctional protein Mtb Apa. FnIII-4 and FnIII-5 of CBD are the only two modules contributing to Apa-Fn interaction.General significanceThis is the first study to report the structure and Fn-binding characteristics of mycobacterial Apa. Since Apa plays a central role in stimulating immune responses and host cells adhesion, these results are of great importance in understanding the pathogenesis of mycobacterium. This information shall provide a guidance for the development of anti-mycobacteria regimen.  相似文献   

11.
Apurinic/apyrimidinic endonuclease 1 (APE1) is the main abasic endonuclease in the base excision repair (BER) pathway of DNA lesions caused by oxidation/alkylation in mammalian cells; within nucleoli it interacts with nucleophosmin and rRNA through N-terminal Lys residues, some of which (K27/K31/K32/K35) may undergo acetylation in vivo. Here we study the functional role of these modifications during genotoxic damage and their in vivo relevance. We demonstrate that cells expressing a specific K-to-A multiple mutant are APE1 nucleolar deficient and are more resistant to genotoxic treatment than those expressing the wild type, although they show impaired proliferation. Of interest, we find that genotoxic treatment induces acetylation at these K residues. We also find that the charged status of K27/K31/K32/K35 modulates acetylation at K6/K7 residues that are known to be involved in the coordination of BER activity through a mechanism regulated by the sirtuin 1 deacetylase. Of note, structural studies show that acetylation at K27/K31/K32/K35 may account for local conformational changes on APE1 protein structure. These results highlight the emerging role of acetylation of critical Lys residues in regulating APE1 functions. They also suggest the existence of cross-talk between different Lys residues of APE1 occurring upon genotoxic damage, which may modulate APE1 subnuclear distribution and enzymatic activity in vivo.  相似文献   

12.
Lysine acetylation is a reversible and dynamic post-translational modification that plays vital roles in regulating multiple cellular processes including aging. However, acetylome-wide analysis in the aging process remains poorly studied in mammalian tissues. Nicotinamide adenine dinucleotide (NAD+), a hub metabolite, benefits health span at least in part due to the activation of Sirtuins, a family of NAD+-consuming deacetylases, indicating changes in acetylome. Here, we combine two antibodies for the enrichment of acetylated peptides and perform label-free quantitative acetylomic analysis of mouse livers during natural aging and upon the treatment of beta-nicotinamide mononucleotide (NMN), a NAD+ booster. Our study describes previously unknown acetylation sites and reveals the acetylome-wide dynamics with age as well as upon the treatment of NMN. We discover protein acetylation events as potential aging biomarkers. We demonstrate that the life-beneficial effect of NMN could be partially reflected by the changes in age-related protein acetylation. Our quantitative assessment indicates that NMN has mild effects on acetylation sites previously reported as substrates of Sirtuins. Collectively, our data analyze protein acetylation with age, laying critical foundation for the functional study of protein post-translational modification essential for healthy aging and perhaps disease conditions.  相似文献   

13.
Protein acetylation is a reversible posttranslational modification, which is regulated by lysine acetyltransferase (KAT) and lysine deacetyltransferase (KDAC). Although protein acetylation has been shown to regulate synaptic plasticity, this was mainly for histone protein acetylation. The function and regulation of nonhistone protein acetylation in synaptic plasticity and learning remain largely unknown. Calmodulin (CaM), a ubiquitous Ca2+ sensor, plays critical roles in synaptic plasticity such as long-term potentiation (LTP). During LTP induction, activation of NMDA receptor triggers Ca2+ influx, and the Ca2+ binds with CaM and activates calcium/calmodulin-dependent protein kinase IIα (CaMKIIα). In our previous study, we demonstrated that acetylation of CaM was important for synaptic plasticity and fear learning in mice. However, the KAT responsible for CaM acetylation is currently unknown. Here, following an HEK293 cell-based screen of candidate KATs, steroid receptor coactivator 3 (SRC3) is identified as the most active KAT for CaM. We further demonstrate that SRC3 interacts with and acetylates CaM in a Ca2+ and NMDA receptor-dependent manner. We also show that pharmacological inhibition or genetic downregulation of SRC3 impairs CaM acetylation, synaptic plasticity, and contextual fear learning in mice. Moreover, the effects of SRC3 inhibition on synaptic plasticity and fear learning could be rescued by 3KQ-CaM, a mutant form of CaM, which mimics acetylation. Together, these observations demonstrate that SRC3 acetylates CaM and regulates synaptic plasticity and learning in mice.  相似文献   

14.
Protein acetylation is a rapid mechanism for control of protein function. Acetyl‐CoA synthetase (AMP‐forming, Acs) is the paradigm for the control of metabolic enzymes by lysine acetylation. In many bacteria, type I or II protein acetyltransferases acetylate Acs, however, in actinomycetes type III protein acetyltransferases control the activity of Acs. We measured changes in the activity of the Streptomyces lividans Acs (SlAcs) enzyme upon acetylation by PatB using in vitro and in vivo analyses. In addition to the acetylation of residue K610, residue S608 within the acetylation motif of SlAcs was also acetylated (PKTRSGK610). S608 acetylation rendered SlAcs inactive and non‐acetylatable by PatB. It is unclear whether acetylation of S608 is enzymatic, but it was clear that this modification occurred in vivo in Streptomyces. In S. lividans, an NAD+‐dependent sirtuin deacetylase from Streptomyces, SrtA (a homologue of the human SIRT4 protein) was needed to maintain SlAcs function in vivo. We have characterized a sirtuin‐dependent reversible lysine acetylation system in Streptomyces lividans that targets and controls the Acs enzyme of this bacterium. These studies raise questions about acetyltransferase specificity, and describe the first Acs enzyme in any organism whose activity is modulated by O‐Ser and N?Lys acetylation.  相似文献   

15.
目的 研究严重急性呼吸综合征冠状病毒2(SARS-CoV-2)膜蛋白对宿主细胞mRNA前体(pre-mRNA)3"非翻译区(UTR)加工的影响。方法 本研究以人肺上皮细胞系A549为模型,利用瞬时转染在细胞内过表达SARS-CoV-2膜蛋白;利用RNA-Seq测序技术及生物信息学分析方法,系统性描绘宿主细胞选择性多聚腺苷酸化(alternative polyadenylation,APA)事件;Metascape数据库对发生显著APA变化的基因进行功能富集分析;RT-qPCR验证靶基因3"UTR长度变化;蛋白质免疫印迹(Western blot)检测目的蛋白表达水平。结果 SARS-CoV-2膜蛋白外源表达后宿主细胞内共813个基因发生显著APA变化。GO和KEGG分析显示,差异APA基因广泛参与有丝分裂细胞周期、调节细胞应激等生物过程,涉及病毒感染和蛋白质加工等。从中进一步筛选出AKT1基因,在IGV软件中显示3"UTR延长;RT-qPCR验证AKT1基因的3"UTR长度变化趋势;Western blot结果显示AKT1蛋白磷酸化水平增加。结论 SARS-CoV-2膜蛋白潜在影响宿主pre-mRNA的3"UTR加工,其中参与多种病毒性生物过程的AKT1基因 3"UTR延长,且其编码的蛋白质功能在细胞内被激活。  相似文献   

16.
Post-translational modification of proteins is an efficient way cells use to control the activity of structural proteins, gene expression regulatory proteins, and enzymes. In eukaryotes, the Sir2-dependent system of protein acetylation/deacetylation controls a number of processes that affect cell longevity. Sir2 proteins have NAD+-dependent protein deacetylase activity and are found in all forms of life. Although the identity of the acetyltransferases that partner with Sir2 enzymes is known in eukaryotes, the identity of the prokaryotic acetyltransferases is not. We report the identification of the gene of Salmonella enterica serovar Typhimurium LT2 encoding the major protein acetyltransferase (Pat) enzyme that, in concert with the CobB sirtuin of this bacterium, regulates the activity of the central metabolic enzyme acetyl-coenzyme A synthetase (Acs). The Pat enzyme uses acetyl-CoA as substrate to modify residue Lys609 of Acs. The Pat/CobB system of S. enterica should serve as the paradigm to further investigate the contributions of this system to the physiology of prokaryotes.  相似文献   

17.
《Genomics》2019,111(4):786-792
BackgroundRenal fibrosis is a final common pathway of chronic kidney disease. SIRT1, a NAD+-dependent protein deacetylase, deacetylates the p65 of NF-κB and shows protective effects in kidney disorders. miR-373 directly targets the 3′UTR of SIRT1. However, roles of miR-373 in renal fibrosis are unclear.MethodsTGF-β1, a critical regulator of fibrosis, was used to stimulate human kidney-2 cells to establish cell model for renal fibrosis. Unilateral ureteral obstruction (UUO) was performed as an in vivo model.ResultsTGF-β1 induced the level of miR-373, reduced level of SIRT1, and promoted p65 acetylation and MMP-9 expression. These effects were reversed by the miR-373 inhibitor. In the animal model, UUO caused a consistent pattern as demonstrated in vitro.ConclusionThese results indicated an undesired effect of miR-373 in the regulation of renal injury and fibrosis by targeting SIRT1-mediated NF-κB/MMP-9 signaling, which might provide a potential therapeutic strategy for renal fibrosis.  相似文献   

18.
19.
Host cell protein synthesis continues when cultured cells are infected by Toxoplasma gondii. In order to determine if this host function is necessary for the parasite we used two independent methods that specifically block cellular protein synthesis. In the first, we infected a temperature-sensitive Chinese hamster ovary cell mutant that has a thermolabile leucyl tRNA synthetase. At the restrictive temperature of 40 C, the mutant cells showed only negligible protein synthesis that was probably mitochondrial. At this temperature, the growth and nucleic acid synthesis of T. gondii proceeded normally and [3H]leucine was specifically incorporated into the parasite as demonstrated by autoradiography. A secpnd method for blocking protein synthesis by the host cell employed treatment of uninfected human fibroblast cells with muconomycin A, an inhibitor of initiation. Repeated washing of monolayer cultures reduced the free muconomycin A to an insignificant level while the cells remained incapable of protein synthesis. T. gondii infected and grew normally in the inhibited cells. Autoradiographic localization of the incorporation of [3H]leucine showed that it was almost exclusively in the intracellular parasites in the cells pretreated with muconomycin A. In the untreated control most of the [3H]leucine was incorporated by the host cell rather than the parasite. We conclude that de novo protein synthesis by the host cell is not required to support the growth of intracellular T. gondii.  相似文献   

20.
目的:探讨NDRG2对胶质瘤U87-MG细胞组蛋白乙酰化的影响,从代谢组学角度明确其抑癌机制,为胶质瘤治疗提供新思路。方法:利用慢病毒介导的外源性NDRG2基因在胶质瘤U87-MG细胞株中过表达,并采用MTT检测其对胶质瘤U87-MG细胞增殖的影响,采用Western blot技术研究其对胶质瘤U87-MG细胞组蛋白乙酰化及AKT-ACLY通路磷酸化状态的影响,并使用酶联反应检测胞内乙酰辅酶A的水平。结果:NDRG2在胶质瘤U87-MG细胞中外源过表达可降低AKT及下游分子ACLY的磷酸化水平,减少胞内乙酰辅酶A的合成,抑制组蛋白乙酰化。结论:NDRG2可能通过抑制AKT通路,减少组蛋白乙酰化,进而抑制胶质瘤U87-MG细胞增殖。  相似文献   

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