N-乙酰葡萄糖胺糖基化修饰与糖尿病、阿尔茨海默病及心脏病的关系

蛋白质的O位N-乙酰葡萄糖胺(O-GlcNAc)糖基化作为一种区别于一般糖基化的翻译后修饰,会使蛋白质的功能发生多种改变。近年的研究发现,蛋白质O-GlcNAc糖基化修饰参与糖尿病、阿尔茨海默病与心脏病等多种疾病病理生理过程,因此对其研究具有积极意义。本文对蛋白质O-GlcNAc糖基化修饰与糖尿病、阿尔茨海默病和心脏病发生的关系作一综述。     

去泛素化酶的研究进展

<正>泛素蛋白酶体途径(UPP)将不需要的蛋白质降解,被降解的蛋白质大多都是调节蛋白质,比如转录因子、发生折叠错误的蛋白质等,以保证细胞周期的正常进行,它通过调控蛋白质水平来参与机体多数生命过程。该途径最主要的是调控蛋白质降解,但是并非所有泛素化修饰都会使蛋白质降解,有些泛素化会导致蛋白质活性和结构的改变,产生一系列生物效应,如介导膜蛋白内吞作用、DNA损伤修复、机体免疫应答等。蛋白质翻译后修饰较为常见的有     

马钱子生物碱在大鼠体内的组织分布

目的　研究马钱子生物碱在大鼠体内的组织分布。方法　大鼠静脉注射马钱子总碱后 ,定时用高效液相色谱法测定各组织中士的宁 (strychnine ,S)、马钱子碱 (brucine,B)、士的宁氮氧化物 (strychnineN oxide ,SNO)和马钱子碱氮氧化物(brucineN oxide ,BNO)的含量。结果　S、B、SNO和BNO在脑和脊髓中均有较多的分布。结论　S、B、SNO和BNO均可穿透血脑屏障 ,到达脑和脊髓 ,从而对中枢产生作用     

全面认识一氧化氮在心肌缺血再灌注损伤中的作用

90年代以来,人们对一氧化氮(Nitric Oxide,NO)在心肌缺血再灌注过程中的作用进行了大量研究,结论不甚一致,争论的焦点集中在以下两个方面:1、在心肌缺血再灌注过程中,NO的产生是增加还是减少?2、NO是参与了心肌缺血再灌注损伤,还是减轻心肌缺血再灌注损伤.本作者对近年来有关NO与心肌缺血再灌注损伤的实验进行了分析和总结,认为:NO在心肌缺血再灌注过程中起着双重作用.     

全面认识——氧化氮在心肌缺血再灌注损伤中的作用

90年代以来,人们对一氧化氮(Nitric Oxide,NO)在心肌缺血再灌注过程中的作用进行了大量研究,结论不甚一致,争论的焦点集中在以下两个方面:1、在心肌缺血再灌注过程中,NO的产生是增加还是减少?2、NO是参与了心肌缺血再灌注损伤,还是减轻心肌缺血再灌注损伤。本作者对近年来有关NO与心肌缺血再灌注损伤的实验进行了分析和总结,认为:NO在心肌缺血再灌注过程中起着双重作用。     

蛋白质药物聚乙二醇修饰方法的生物优化

聚乙二醇 (polyethyleneglycol,PEG)修饰又称分子的PEG化。是用来解决或缓解蛋白质和多肽类在药用过程中存在的诸多问题的有效途径 ,PEG修饰对具有药用潜能的蛋白来说具有 :(1)增加稳定性 ,延长血浆半衰期 ;(2 )降低免疫原性和抗原性 ;(3)降低毒副作用。但是 ,很重要的一点是蛋白质经PEG修饰后会引起生物学活性的降低。PEG化引起生物学活性降低的原因是多方面的 ,其主要原因为终产品中引进的基团 ,包括PEG以及PEG和修饰蛋白质之间的连接键所致 ;也与修饰反应的条件、产生的副产物有关。不同的蛋白质影响差异很大 ,这也是PEG化个体…     

蛋白质组分析在医学中的应用

随着人类基因组计划(HGP)的实施和完成,基因研究以近顶峰而进入后基因时代,后基因组研究以基因组学为中心,同时分化出代表生命科学不同侧面的多种"组学"研究,如蛋白质组学(proteome)、药物基因组学、比较基因组学等[1],在这些领域中蛋白质作为生命活动的"执行者",参与了DNA的合成、基因转录激活、蛋白质翻译、修饰和定位以及信息传导等重要的生物过程,已经成为新的研究焦点.     

诱导型一氧化氮合酶对心血管疾病的调节作用

一氧化氮(NO)是心血管系统中重要的信号传递分子,人体内的NO是由三种不同的一氧化氮合酶合成。其中诱导型一氧化氮合酶(iNOS)一经诱导生成,产生大量NO,参与了多种心血管疾病的发生发展过程。现对iNOS在心血管疾病中的调节作用进行综述。     

Neddylation修饰及其抑制剂MLN4924研究进展

蛋白质的Neddylation修饰是一种类泛素化修饰,是将类泛素蛋白NEDD8标记到底物蛋白的可逆修饰过程。Neddylation修饰对蛋白质功能具有重要调节作用,可诱导底物蛋白发生构象变化、改变其亚细胞定位、激活或抑制其酶活性,还可通过与其它泛素分子竞争结合改变其蛋白稳定性等。研究表明,Neddylation修饰的异常与包括肿瘤在内的多种疾病相关,提示Neddylation修饰是疾病治疗的新靶标。Neddylation修饰的小分子抑制剂MLN4924的发现极大地促进了Neddylation修饰领域的发展,为多种疾病的治疗开辟了新思路。研究表明,MLN4924具有抗病毒、抗炎及广泛的抗肿瘤活性,对多种疾病具有良好潜在治疗作用,作为癌症治疗药物的多项临床试验研究正在进行中。文章就蛋白质Neddylation修饰及其抑制剂MLN4924与疾病的最新研究进展进行综述。     

聚乙二醇定点修饰蛋白质药物中巯基的研究进展

聚乙二醇(PEG)修饰是解决蛋白质药物溶解度低、稳定性差、半衰期短、具有免疫原性等问题的有效方法,通常在氨基上进行修饰,但在巯基上进行定点修饰更有利于获得结构明确、组成稳定的修饰产物。综述了聚乙二醇定点修饰蛋白质药物中巯基,包括在游离巯基、二硫键和引入的巯基上进行修饰的研究进展。     

Vitamin D deficiency-induced hypertension is associated with vascular oxidative stress and altered heart gene expression

S-nitrosylation is a ubiquitous protein modification in redox-based signaling and forms S-nitrosothiol from nitric oxide (NO) on cysteine residues. Dysregulation of (S)NO signaling (nitrosative stress) leads to impairment of cellular function. Protein kinase C (PKC) is an important signaling protein that plays a role in the regulation of vascular function, and it is not known whether (S)NO affects PKC's role in vascular reactivity. We hypothesized that S-nitrosylation of PKC in vascular smooth muscle would inhibit its contractile activity. Aortic rings from male C57BL/6 mice were treated with auranofin or 1-chloro-2,4-dinitrobenzene (DNCB) as pharmacological tools, which lead to stabilize S-nitrosylation, and propylamine propylamine NONOate (PANOate) or S-nitrosocysteine (CysNO) as NO donors. Contractile responses of aorta to phorbol-12,13-dibutyrate, a PKC activator, were attenuated by auranofin, DNCB, PANOate, and CysNO. S-nitrosylation of PKCα was increased by auranofin or DNCB and CysNO as compared with control protein. Augmented S-nitrosylation inhibited PKCα activity and subsequently downstream signal transduction. These data suggest that PKC is inactivated by S-nitrosylation, and this modification inhibits PKC-dependent contractile responses. Because S-nitrosylation of PKC inhibits phosphorylation and activation of target proteins related to contraction, this posttranslational modification may be a key player in conditions of decreased vascular reactivity.     

Cardiac capsaicin-sensitive sensory nerves regulate myocardial relaxation via S-nitrosylation of SERCA: role of peroxynitrite

BACKGROUND AND PURPOSE: Sensory neuropathy develops in the presence of cardiovascular risk factors (e.g. diabetes, dyslipidemia), but its pathological consequences in the heart are unclear. We have previously shown that systemic sensory chemodenervation by capsaicin leads to impaired myocardial relaxation and diminished cardiac nitric oxide (NO) content. Here we examined the mechanism of diminished NO formation and if it may lead to a reduction of peroxynitrite (ONOO(-))-induced S-nitrosylation of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA2a). EXPERIMENTAL APPROACH: Male Wistar rats were treated with capsaicin for 3 days to induce sensory chemodenervation. Seven days later, myocardial function and biochemical parameters were measured. KEY RESULTS: Capsaicin pretreatment significantly increased left ventricular end-diastolic pressure (LVEDP) decreased cardiac NO level, Ca(2+)-dependent NO synthase (NOS) activity, and NOS-3 mRNA. Myocardial superoxide content, xanthine oxidoreductase and NADPH oxidase activities did not change, although superoxide dismutase (SOD) activity increased. Myocardial and serum ONOO(-) concentration and S-nitrosylation of SERCA2a were significantly decreased. CONCLUSIONS AND IMPLICATIONS: Our results show that sensory chemodenervation decreases cardiac NO via decreased expression and activity of Ca(2+)-dependent NOS and increases SOD activity, thereby leading to decreased basal ONOO(-) formation and reduction of S-nitrosylation of SERCA2a, which causes impaired myocardial relaxation characterized by increased left ventricular end-diastolic pressure (LVEDP). This suggests that capsaicin sensitive sensory neurons regulate myocardial relaxation via maintaining basal ONOO(-) formation and SERCA S-nitrosylation.     

Regulation of cardiovascular TRP channel functions along the NO–cGMP–PKG axis

There is growing body of evidence that nitric oxide (NO)–cGMP–PKG signaling plays a central role in negative regulation of cardiovascular (CV) responses and its disorders through suppressed Ca²⁺ dynamics. Other lines of evidence also reveal the stimulatory effects of this signaling on some CV functions. Recently, transient receptor potential (TRP) channels have received much attention as non-voltage-gated Ca²⁺ channels involved in CV physiology and pathophysiology. Available information suggests that these channels undergo both inhibition and activation by NO via PKG-mediated phosphorylation and S-nitrosylation, respectively, and also act as upstream regulators to promote endothelial NO production. This review summarizes the roles of NO–cGMP–PKG signaling pathway, particularly in regulating TRP channel functions with their associated physiology and pathophysiology.     

Breast cancer drugs perturb fundamental vascular functions of endothelial cells by attenuating protein S-nitrosylation

Cardiovascular side effects of broadly used chemotherapeutic drugs such as Tamoxifen citrate (TC), Capecitabine (CP) and Epirubicin (EP) among cancer survivors are well established. Nitric oxide (NO) is known to protect cardiovascular tissues under conditions of stress. NO can act through cyclic guanosine monophosphate (cGMP)-dependent and -independent pathways. Particularly, the S-nitrosylation of SH-groups in a protein by NO falls under cGMP-independent effects of NO. TC, CP, and EP are hypothesized as interfering with cellular protein S-nitrosylation, which, in turn, may lead to endothelial dysfunctions. The results show that all three drugs attenuate nitrosylated proteins in endothelial cells. A significant reduction in endogenous S-nitrosylated proteins was revealed by Saville–Griess assay, immunofluorescence and western blot. Incubation with the drugs causes a reduction in endothelial migration, vasodilation and tube formation, while the addition of S-nitrosoglutathione (GSNO) has a reversal of this effect. In conclusion, results indicate the possibility of decreased cellular nitrosothiols as being one of the reasons for endothelial dysfunctions under TC, CP and EP treatment. Identification of the down-regulated S-nitrosylated proteins so as to correlate their implications on fundamental vascular functions could be an interesting phenomenon.     

A proteomic study of S-nitrosylation in the rat cardiac proteins in vitro

Protein S-nitrosylation in the heart tissue has been implicated in several patho (physiological) processes. However, specific protein targets for S-nitrosylation remain largely unknown. In this study, the rat cardiac proteins were incubated in vitro with S-nitrosoglutathione (GSNO), a biologically existing nitric oxide (NO) donor and S-nitrosating agent, to induce protein S-nitrosylation, and the resulting S-nitrosylated proteins were purified by the biotin switch method, followed by two-dimensional gel electrophoresis (2-DE) separation and matrix-assisted laser desorption ionization/time of flight tandem mass spectrometry (MALDI-TOF-MS/MS) identification. Candidate Western blot analysis was also used to identify potential S-nitrosylated proteins. A total of ten proteins including triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, creatine kinase, adenylate kinase 1 (AK1), enolase 1, destrin, actin, myosin, albumin and Hsp27 were unambiguously identified, among which AK1 was found as a novel target of S-nitrosylation. Further studies showed that AK1 activity in the rat heart extracts was significantly inhibited by GSNO but not oxidized glutathione (GSSG), and the inhibition was completely reversed by dithiothreitol (DTT) post-treatment, demonstrating that S-nitrosylation might serve as a new regulatory mechanism in controlling AK1 activity. This study represents an initial attempt to characterize the S-nitrosoproteome in the heart and highlights the importance of protein S-nitrosylation in cardio function regulation.     

S-nitrosylation of cysteine 289 of the AT1 receptor decreases its binding affinity for angiotensin II

1. Nitric oxide (NO) is known to affect the properties of various proteins via the S-nitrosylation of cysteine residues. This study evaluated the direct effects of the NO donor sodium nitroprusside (SNP) on the pharmacological properties of the AT1 receptor for angiotensin II expressed in HEK-293 cells. 2. SNP dose-dependently decreased the binding affinity of the AT1 receptor without affecting its total binding capacity. This modulatory effect was reversed within 5 min of removing SNP. 3. The effect of SNP was not modified in the presence of the G protein uncoupling agent GTPgammaS or the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. 4. The binding properties of a mutant AT1 receptor in which all five cysteine residues within the transmembrane domains had been replaced by serine was not affected by SNP. Systematic analysis of mutant AT1 receptors revealed that cysteine 289 conferred the sensitivity to SNP. 5. These results suggest that NO decreased the binding affinity of the AT1 receptor by S-nitrosylation of cysteine 289. This modulatory mechanism may be particularly relevant in pathophysiological situations where the beneficial effects of NO oppose the deleterious effects of angiotensin II.     

Involvement of constitutive nitric oxide synthase in ghrelin-induced cytosolic phospholipase A2 activation in gastric mucosal cell protection against ethanol cytotoxicity

Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, is an important regulator of nitric oxide synthase (NOS) and cyclooxygenase (COX) enzyme systems, the products of which are of major significance to the processes of gastric mucosal defense and repair. Here, using primary culture of rat gastric mucosal cells, we report on the mechanism of ghrelin protection against ethanol cytotoxicity. We show that the protective effect of ghrelin was associated with the increase in NO and PGE2 production, and characterized by a marked up-regulation in cytosolic phospholipase A₂ (cPLA₂) activity and arachidonic acid (AA) release. The loss in countering effect of ghrelin on the ethanol cytotoxicity was attained with constitutive NOS (cNOS) inhibitor, L-NAME, as well as indomethacin and a specific COX-1 inhibitor, SC-560, while specific COX-2 inhibitor, NS-398, and a selective inducible NOS (iNOS) inhibitor, 1400W, had no effect. The effect of L-NAME was reflected in the inhibition of ghrelin-induced mucosal cell capacity for NO production, cPLA₂ activation, and PGE2 generation, whereas indomethacin caused only the inhibition in PGE2 generation. Moreover, the ghrelin-induced up-regulation in AA release was reflected in the cPLA₂ enzyme protein phosphorylation and S-nitrosylation. Preincubation with L-NAME resulted in the inhibition of the ghrelin-induced S-nitrosylation, whereas the ERK inhibitor, PD98059, caused the blockage in cPLA₂ protein phosphorylation as well as S-nitrosylation. The findings demonstrate that ghrelin protection of gastric mucosa against ethanol cytotoxicity involves cNOS-derived NO induction of cPLA₂ activation for the increase in PGE2 synthesis. This activation process apparently includes the cPLA₂ phosphorylation followed by S-nitrosylation.     

Regulation of programmed cell death in neuronal cells by nitric oxide

Nitric oxide (NO), produced from L-arginine and molecular oxygen in a reaction catalyzed by one of three NO synthase isoenzymes, can prevent or induce neuronal apoptosis depending on its concentration and cellular redox state. This molecule affords neuroprotection by post-translational S-nitrosylation of NMDA receptor, caspases and p21ras, and increases the expression of cytoprotective genes such as HSP70, heme oxygenase and Bcl-2. Moreover, the NO/cGMP pathway activates the anti-apoptotic serine/threonine kinase Akt by protein kinase G-dependent activation of phosphatidylinositol 3-kinase. A high concentration of NO and peroxynitrite, a reaction product of NO with superoxide anion, can promote apoptotic pathways in neuronal cells through the indirect activation of caspases. We review the molecular mechanism by which NO exerts both pro- and anti-apoptotic actions in neuronal cells and the clinical implications for regulating neuronal apoptosis.     

Regulation of cardiovascular TRP channel functions along the NO-cGMP-PKG axis

There is growing body of evidence that nitric oxide (NO)-cGMP-PKG signaling plays a central role in negative regulation of cardiovascular (CV) responses and its disorders through suppressed Ca(2+) dynamics. Other lines of evidence also reveal the stimulatory effects of this signaling on some CV functions. Recently, transient receptor potential (TRP) channels have received much attention as non-voltage-gated Ca(2+) channels involved in CV physiology and pathophysiology. Available information suggests that these channels undergo both inhibition and activation by NO via PKG-mediated phosphorylation and S-nitrosylation, respectively, and also act as upstream regulators to promote endothelial NO production. This review summarizes the roles of NO-cGMP-PKG signaling pathway, particularly in regulating TRP channel functions with their associated physiology and pathophysiology.     

Aberrant regulation and function of Src family tyrosine kinases: their potential contributions to glutamate-induced neurotoxicity

Excitotoxicity, a major cause of neuronal death in acute and chronic neurodegenerative diseases and conditions such as stroke and Parkinson's disease, is initiated by overstimulation of glutamate receptors, leading to calcium overload in affected neurons. The sustained high concentration of intracellular calcium constitutively activates a host of enzymes, notably the calcium-activated proteases calpains, neuronal nitric oxide synthase (nNOS) and NADPH oxidase (NOX), to antagonise the cell survival signalling pathways and induce cell death. Upon overactivation by calcium, calpains catalyse limited proteolysis of specific cellular proteins to modulate their functions; nNOS produces excessive amounts of nitric oxide (NO), which, in turn, covalently modifies specific enzymes by S-nitrosylation; and NOX produces excessive amounts of reactive oxygen species (ROS) to inflict damage to key metabolic enzymes. Presumably, key regulatory enzymes governing cell survival and cell death are aberrantly modified and regulated by calpains, NO and ROS in affected neurons; these aberrantly modified enzymes then cooperate to induce the death of affected neurons. c-Src, an Src family kinase (SFK) member, is one of the aberrantly regulated enzymes involved in excitotoxic neuronal death. Herein we review how SFKs are functionally linked to the glutamate receptors and the biochemical and structural basis of the aberrant regulation of SFKs. Results in the literature suggest that SFKs are aberrantly activated by calpain-mediated truncation and S-nitrosylation. Thus, the aberrantly activated SFKs are targets for therapeutic intervention to reduce the extent of brain damage caused by stroke.