蛋白质组学研究中磷酸化蛋白质(肽)富集策略及展望

蛋白质磷酸化是细胞信号转导途径中重要的调控过程,但磷酸化蛋白质(肽)丰度低、化学计量低、易降解等限制了磷酸化蛋白质(肽)的研究.磷酸化蛋白质(肽)的制备是蛋白质磷酸化研究成功的关键步骤.笔者综述了磷酸化蛋白质(肽)的一些富集策略:基于抗体技术、亲和性标签磷酸化蛋白质富集策略,基于固相金属亲和层析、金属氧化亲和层析、IMAC连续洗脱、离子交换层析、亲水相互作用层析的磷酸化肽富集策略,熟练掌握及综合应用这些磷酸化蛋白质(肽)的富集策略,对于研究细胞信号转导途径中的调控因子具有重要作用.     

20℃和25℃条件下秀丽隐杆线虫衰老过程中磷酸化蛋白质组学的研究

衰老过程受到包括温度在内的众多环境因素的影响。蛋白质磷酸化是重要的翻译后修饰,调控多种生命活动,在衰老过程中也起着重要作用。秀丽隐杆线虫(Caenorhabditis elegans,C.elegans)是经典的衰老研究的模式生物。为了探究蛋白质磷酸化修饰在不同温度下衰老过程中的作用,我们对20℃(常温)和25℃(高温)培养条件下成年时期第1天、第5天、第10天的秀丽隐杆线虫样品进行了磷酸化组学分析工作。我们采用基于DIA的非标定量分析方法,系统地比较了不同样品间的磷酸化组学差异。共9 145条高可信度的磷酸化肽段被鉴定到,对应3 317个磷酸化蛋白质。经过筛选,最终6 624条磷酸化肽段被定量到,其中有1 093条显著变化的磷酸化肽段,来自于858个磷酸化蛋白质,包含1 426个磷酸化位点,通过进一步的生物信息学分析,揭示了衰老过程中的磷酸化变化规律。     

蛋白质磷酸化修饰研究进展

 蛋白质磷酸化是由蛋白质激酶催化的磷酸基转移反应,是最常见、最重要的蛋白质翻译后修饰方式之一,是一种普遍的生命活动调节方式,在细胞信号转导过程中起重要作用。本文介绍了蛋白质磷酸化修饰的主要类型与功能、磷酸化蛋白的鉴定及磷酸化位点的预测等方面研究进展,并着重介绍了一些灵敏度高、特异性强的以同位素标记、免疫印迹-化学发光法等作为核心的磷酸化蛋白质分析方案。Western blot方法被证明是鉴别磷蛋白的灵敏、特异方法,而NanoPro100/1000超微量蛋白分析系统等又在此基础上加以改善。蛋白磷酸化分析工具和软件的发展也很迅猛。     

禁食对小鼠下丘脑磷酸化蛋白质组的影响

本研究旨在通过干预小鼠进食,探讨对小鼠下丘脑中蛋白质磷酸化修饰和相关信号通路的影响,为完善蛋白磷酸化调控网络提供有价值的信息.将实验小鼠平均分为3组,分别为对照组(con)、禁食组(D2)和禁食后恢复组(DR),每组小鼠数量为3只.在48 h内,con组正常提供饲料,D2组不提供饲料,DR组前44 h不提供饲料、后4 h提供饲料.同时解剖3组小鼠并提取下丘脑组织,提取、纯化和酶解下丘脑组织的蛋白质,并采用二氧化钛富集分离技术富集磷酸化肽段,运用高通量液相色谱-质谱联用进行检测,以鉴定样品中的磷酸化蛋白质组.利用非标记定量方法筛选差异表达的磷酸化蛋白以及位点,对鉴定数据进行GO富集和KEGG通路分析,并探究磷酸化蛋白间的相互作用关系.结果显示,共鉴定到4 810个磷酸化蛋白质,对应于14 259个磷酸化位点发生了变化.采用数学统计方法分析,成功确认小鼠下丘脑中有681个磷酸化蛋白差异显著,观察到这些差异蛋白与蛋白结合、激酶行为、转运、轴突生成等分子活动和生物学过程有关,并富集到了MAPK、细胞内噬和环磷酸腺苷信号通路等11个主要的信号通路.这说明禁食会对小鼠下丘脑中多种蛋白质的磷酸化修饰...     

运用生物质谱分析磷酸化多肽合成中的主产物

蛋白质磷酸化是生物体内非常重要的翻译后修饰,磷酸化多肽及其类似物对研究和阐明蛋白质磷酸化修饰对生命活动的调节机制具有十分重要的作用,采用4-磺酸苯异硫腈酸酯修饰,源后衰减基质辅助激光解吸/电离飞行时间质谱(PSD-MALDI)模式分析合成磷酸化多肽,建立了一种简便易行的磷酸化多肽鉴定及定位方法.     

决策树算法预测人类病毒的蛋白质磷酸化位点

本文基于决策树分类算法构建人类病毒蛋白质磷酸化修饰位点的预测模型。采用氨基酸物理化学性质对蛋白质序列进行特征提取,并分析丝氨酸、苏氨酸和酪氨酸磷酸化位点邻近序列的氨基酸性质。同时考察了不同分类算法对预测结果的影响。通过10倍交叉验证,利用决策树算法预测丝氨酸、苏氨酸和酪氨酸磷酸化位点的MCC分别达到77.31%、75.91%和71.94%,表明本文提出的方法能有效地预测人类病毒的磷酸化修饰位点。     

基于 LibSVM 的 CKSAAP 蛋白特征提取预测水稻蛋白质磷酸化位点

本文从swiss-prot中选取经过试验验证的水稻蛋白质磷酸化位点数据作为训练集合，应用蛋白质序列特征提取方法Composition of k-spaced residues pairs （CKSAAP），为利用SVM算法构建专门针对水稻蛋白质磷酸化位点的预测工具做准备。 CKSAAP方法利用在序列片断中残基的K个间隔距离的组成，进一步反映了残基之间的相关性。本文利用LibSVM软件包对已通过改进过得CKSAAP方法特征提取出来的数值特征对磷酸化位点进行预测，从而为之后构建水稻蛋白质磷酸化位点的预测工具做准备。结果表明，本文基于SVM和CKSAAP方法的水稻蛋白质磷酸化位点预测在丝氨酸，苏氨酸和酪氨酸的平均预测准确性为80．638％，马修斯系数为0．611。与PlantPhos和Musite的预测性能的对比结果显示，在磷酸化各氨基酸位点的预测性能高于PlantPhos及Musite。     

抗三尖杉酯碱的HL60细胞蛋白质磷酸化

研究了抗三尖杉酯碱的ＨＬ６０细胞蛋白质磷酸化的变化。经差速离心得到纯膜蛋白，抗性细胞有一高度磷酸化的１１０ｋｕ的蛋白质存在，免疫沉淀ｃ－ＫＡＦ－１蛋白激酶，抗性细胞内ｃ－ＲＡＦ－１蛋白激酶磷酸化程度明显提高，其活性被蛋白激酶Ｃ抑制剂ＣａｌｐｈｏｓｔｉｎＣ明显地抑制。结果表明：ＨＬ６０细胞对三尖杉酯碱的抗药性与蛋白质高度磷酸化有关；抗性细胞内ｃ－ＲＡＦ－１蛋白激酶磷酸化程度明显提高，可能与多药抗药性和抗细胞调亡有关。     

小鼠Connexin31蛋白磷酸化定位

间隙连接蛋白31(Connexin31,Cx31)是间隙连接蛋(Connexin)家族的一员,目前对于Cx31的功能及其调节方式知之甚少.本文通过免疫沉淀、SDS-PAGE分离、蛋白质条带回收、蛋白质胶块酶解、4-磺酸苯异硫氰酸酯修饰、PSD-MALDI-TOF质谱分析、数据分析、确定小鼠Cx31磷酸化位点.     

人类病毒蛋白质磷酸化修饰位点的识别研究

蛋白质磷酸化翻译后修饰在病毒的复制和抑制宿主细胞功能方面发挥重要的作用。然而,利用实验的方法识别磷酸化位点既费时费力又耗财。因此基于蛋白质氨基酸序列发展一种机器学习方法对病毒蛋白磷酸化位点进行预测显得非常有必要。研究结合支持向量机提出识别病毒蛋白磷酸化位点的新方法。采用权重氨基酸成分和属性分组编码对病毒蛋白残基的氨基酸物理化学性质和序列信息进行特征提取,通过10倍交叉验证,丝氨酸、苏氨酸和酪氨酸磷酸化位点的预测准确率分别达到82.0%、85.8%和92.4%。运用该预测模型对丝氨酸残基磷酸化的激酶组进行分类评估,CMGC、AGC和CAMK激酶组的马氏相关系数分别达到69.3%、68.8%和68.2%。结果表明:构建的方法可以有效地预测激酶特异性的磷酸化位点。     

Calcium-dependent phosphorylation of histone H3 in butyrate-treated HeLa cells

Ca2+ is prominant in the control of cell proliferation and function. However, the biochemical mechanism(s) mediating its effects on nuclear events is unknown. We report here that Ca2+, at physiological concentrations, stimulates the phosphorylation of histone H3 by an endogenous protein kinase in HeLa cell nuclei. Also, pretreatment of cells with Na butyrate, which increases histone acetylation, selectively increases the susceptability of histone H3 to phosphorylation by the protein kinase. Our results reveal a potential link between histone H3 acetylation and phosphorylation, modifications which are thought to have important effects on chromatin structure and function and suggest a possible mechanism whereby stimuli at the cell surface (such as hormones, mitogens and drugs) may influence biochemical events at the nuclear level; changes in the intracellular Ca2+ concentration may influence the phosphorylation of chromosomal proteins, mediated by Ca2+ -dependent kinases in th nucleus.     

Research progress in protein post-translational modification

A. Ciechanover and A. Hershko, Israel scientists, and American scientist O. Rose have received the Nobel Prize of 2004 in chemistry because they have discovered themechanism of the ubiquitin-regulated proteolysis. Their work indicates the research directi…     

Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation

Tri-methylation of histone H3 lysine 9 is important for recruiting heterochromatin protein 1 (HP1) to discrete regions of the genome, thereby regulating gene expression, chromatin packaging and heterochromatin formation. Here we show that HP1alpha, -beta, and -gamma are released from chromatin during the M phase of the cell cycle, even though tri-methylation levels of histone H3 lysine 9 remain unchanged. However, the additional, transient modification of histone H3 by phosphorylation of serine 10 next to the more stable methyl-lysine 9 mark is sufficient to eject HP1 proteins from their binding sites. Inhibition or depletion of the mitotic kinase Aurora B, which phosphorylates serine 10 on histone H3, causes retention of HP1 proteins on mitotic chromosomes, suggesting that H3 serine 10 phosphorylation is necessary for the dissociation of HP1 from chromatin in M phase. These findings establish a regulatory mechanism of protein-protein interactions, through a combinatorial readout of two adjacent post-translational modifications: a stable methylation and a dynamic phosphorylation mark.     

Photosystem II core phosphorylation and photosynthetic acclimation require two different protein kinases

Illumination changes elicit modifications of thylakoid proteins and reorganization of the photosynthetic machinery. This involves, in the short term, phosphorylation of photosystem II (PSII) and light-harvesting (LHCII) proteins. PSII phosphorylation is thought to be relevant for PSII turnover, whereas LHCII phosphorylation is associated with the relocation of LHCII and the redistribution of excitation energy (state transitions) between photosystems. In the long term, imbalances in energy distribution between photosystems are counteracted by adjusting photosystem stoichiometry. In the green alga Chlamydomonas and the plant Arabidopsis, state transitions require the orthologous protein kinases STT7 and STN7, respectively. Here we show that in Arabidopsis a second protein kinase, STN8, is required for the quantitative phosphorylation of PSII core proteins. However, PSII activity under high-intensity light is affected only slightly in stn8 mutants, and D1 turnover is indistinguishable from the wild type, implying that reversible protein phosphorylation is not essential for PSII repair. Acclimation to changes in light quality is defective in stn7 but not in stn8 mutants, indicating that short-term and long-term photosynthetic adaptations are coupled. Therefore the phosphorylation of LHCII, or of an unknown substrate of STN7, is also crucial for the control of photosynthetic gene expression.     

ATP-dependent reduction of cysteine-sulphinic acid by S. cerevisiae sulphiredoxin

Proteins contain thiol-bearing cysteine residues that are sensitive to oxidation, and this may interfere with biological function either as 'damage' or in the context of oxidant-dependent signal transduction. Cysteine thiols oxidized to sulphenic acid are generally unstable, either forming a disulphide with a nearby thiol or being further oxidized to a stable sulphinic acid. Cysteine-sulphenic acids and disulphides are known to be reduced by glutathione or thioredoxin in biological systems, but cysteine-sulphinic acid derivatives have been viewed as irreversible protein modifications. Here we identify a yeast protein of relative molecular mass M(r) = 13,000, which we have named sulphiredoxin (identified by the US spelling 'sulfiredoxin', in the Saccharomyces Genome Database), that is conserved in higher eukaryotes and reduces cysteine-sulphinic acid in the yeast peroxiredoxin Tsa1. Peroxiredoxins are ubiquitous thiol-containing antioxidants that reduce hydroperoxides and control hydroperoxide-mediated signalling in mammals. The reduction reaction catalysed by sulphiredoxin requires ATP hydrolysis and magnesium, involving a conserved active-site cysteine residue which forms a transient disulphide linkage with Tsa1. We propose that reduction of cysteine-sulphinic acids by sulphiredoxin involves activation by phosphorylation followed by a thiol-mediated reduction step. Sulphiredoxin is important for the antioxidant function of peroxiredoxins, and is likely to be involved in the repair of proteins containing cysteine-sulphinic acid modifications, and in signalling pathways involving protein oxidation.     

Histidine phosphorylation and phosphoryl group transfer in bacterial chemotaxis

A cascade of protein phosphorylation, initiated by autophosphorylation of the CheA protein, may be important in the signal transduction pathway of bacterial chemotaxis. A proteolytic fragment of CheA cannot autophosphorylate, but can still transfer phosphate to proteins that generate excitation and adaptation signals. The site of CheA phosphorylation is His 48; mutants altered at this position are non-chemotactic. Similar mechanisms of transient protein phosphorylation and phosphoryl group transfer seem to be involved in processing sensory data and in activating specific gene expression.     

FMRFamide reverses protein phosphorylation produced by 5-HT and cAMP in Aplysia sensory neurons

Neurotransmitter can modulate neuronal activity through a variety of second messengers that act on ion channels and other substrate proteins. The most commonly described effector mechanism for second messengers in neurons depends on protein phosphorylation mediated by one of three sets of kinases: the cyclic AMP-dependent protein kinases, the Ca2+-calmodulin-dependent protein kinases, and the Ca2+-phospholipid-dependent protein kinases. In addition, some neurotransmitters and second messengers can also inhibit protein phosphorylation by lowering cAMP levels (either by inhibiting adenylyl cyclase or activating phosphodiesterases). This raises the question: can neurotransmitters also modulate neuronal activity by decreasing protein phosphorylation that is independent of cAMP? Various biochemical experiments show that a decrease in protein phosphorylation can arise through activation of a phosphatase or inhibition of kinases. In none of these cases, however, is the physiological role for the decrease in protein phosphorylation known. Here we report that in Aplysia sensory neurons, the presynaptic inhibitory transmitter FMRFamide decreases the resting levels of protein phosphorylation without altering the level of cAMP. Furthermore, FMRFamide overrides the cAMP-mediated enhancement of transmitter release produced by 5-hydroxytryptamine (5-HT), and concomitantly reverses the cAMP-dependent increase in protein phosphorylation produced by 5-HT. These findings indicate that a receptor-mediated decrease in protein phosphorylation may play an important part in the modulation of neurotransmitter release.     

Phosphorylation of the nicotinic acetylcholine receptor regulates its rate of desensitization

Recent studies have provided evidence for a role of protein phosphorylation in the regulation of the function of various potassium and calcium channels (for reviews, see refs 1, 2). As these ion channels have not yet been isolated and characterized, it has not been possible to determine whether phosphorylation of the ion channels themselves alters their properties or whether some indirect mechanism is involved. In contrast, the nicotinic acetylcholine receptor, a neurotransmitter-dependent ion channel, has been extensively characterized biochemically and has been shown to be directly phosphorylated. The phosphorylation of this receptor is catalysed by at least three different protein kinases (cyclic AMP-dependent protein kinase, protein kinase C and a tyrosine-specific protein kinase) on seven different phosphorylation sites. However, the functional significance of phosphorylation of the receptor has been unclear. We have now examined the functional effects of phosphorylation of the nicotinic acetylcholine receptor by cAMP-dependent protein kinase. We investigated the ion transport properties of the purified and reconstituted acetylcholine receptor before and after phosphorylation. We report here that phosphorylation of the nicotinic acetylcholine receptor on the gamma- and delta-subunits by cAMP-dependent protein kinase increases the rate of the rapid desensitization of the receptor, a process by which the receptor is inactivated in the presence of acetylcholine (ACh). These results provide the first direct evidence that phosphorylation of an ion channel protein modulates its function and suggest that phosphorylation of postsynaptic receptors in general may play an important role in synaptic plasticity.     

Regulation of Arabidopsis cryptochrome 2 by blue-light-dependent phosphorylation

Cryptochromes are blue/ultraviolet-A light receptors that mediate various light responses in plants and animals. But the initial photochemical reaction of cryptochrome is still unclear. For example, although most photoreceptors are known to undergo light-dependent protein modification such as phosphorylation, no blue-light dependent phosphorylation has been reported for a cryptochrome. Arabidopsis cryptochrome 2 (cry2) mediates light regulation of seedling development and photoperiodic flowering. The physiological activity and cellular level of cry2 protein are light-dependent, and protein protein interactions are important for cry2 function. Here we report that cry2 undergoes a blue-light-dependent phosphorylation, and that cry2 phosphorylation is associated with its function and regulation. Our results suggest that, in the absence of light, cry2 remains unphosphorylated, inactive and stable; absorption of blue light induces the phosphorylation of cry2, triggering photomorphogenic responses and eventually degradation of the photoreceptor.