蛋白质亚磺酰化修饰及其在生理和病理过程中的作用

细胞正常生理或病理过程中均伴随着活性氧(ROS)和活性氮(RNS)的产生,引起蛋白质半胱氨酸发生氧化翻译后修饰。亚磺酰化是氧化翻译后修饰中的一种,指ROS将蛋白质的巯基氧化成亚磺酰基(R-SOH)的过程,广泛存在于多种物种中。亚磺酰化修饰蛋白质的捕获、富集和修饰位点的确定目前仍极具挑战性。半胱氨酸亚磺酰化的检测方法主要包括基于转录因子Yap1和基于小分子化合物dimedone或bicyclo[6.1.0]nonyne的分子探针。在此基础上,研究人员通过偶联生物素等标签分子又设计出了更多便于富集亚磺酰化蛋白质的衍生物探针。将亚磺酰化蛋白质捕获和富集后,与LC-MS/MS等质谱分析技术联用,则可确定发生亚磺酰化修饰的半胱氨酸位点。近几年的研究表明,细胞信号通路中的许多蛋白质或酶都会发生亚磺酰化修饰,调控蛋白质功能、稳定性或催化活性,从而引起下游信号通路或代谢过程的变化,进而影响机体生理或病理状态。随着对蛋白质亚磺酰化修饰的深入研究,越来越多疾病的发生发展新机制被发现,靶向该修饰有望为疾病治疗提供新的策略。本文从蛋白质氧化修饰的过程和亚磺酰化修饰检测的方法入手进行阐述,总结了近几年亚磺酰化修...     

斑马鱼蛋白酪氨酸硫酸化修饰的检测方法研究

蛋白酪氨酸硫酸化(protein tyrosine sulfation, PTS)是一种重要的翻译后修饰,调控生命活动中多种生理和病理过程,但由于PTS状态不稳定且目前缺乏有效的富集方法,因此在生物样品中难以进行有效地检测。本研究以模式动物斑马鱼(Danio rerio)为研究材料,利用Orbitrap Exploris 480高分辨质谱仪检测了斑马鱼胚胎发育早期总蛋白的酪氨酸硫酸化修饰水平,通过该方法共计检测到26种蛋白(包括膜蛋白、分泌蛋白、胞质蛋白和核蛋白等)存在潜在的29个酪氨酸硫酸化修饰位点。本研究建立了斑马鱼胚胎发育早期蛋白酪氨酸硫酸化修饰的检测方法,为探索生物体蛋白硫酸化修饰的作用机制奠定了技术基础。     

植物蛋白质翻译后修饰组学研究进展

蛋白质翻译后修饰(Protein post-translational modification,PTMs)是一种重要的细胞调控机制,通过在蛋白质的氨基酸侧链上共价结合一些化学小分子基团来调节蛋白质的活性、结构、定位和蛋白质间的互作关系,从而精细调控蛋白质生物学功能的动态变化。PTMs是植物对环境变化最快、最早的反应之一,是植物蛋白质组多样性的关键机制,在植物生长发育和对环境适应中起重要作用。主要介绍了近年来植物磷酸化、乙酰化、琥珀酰化、糖基化、泛素化、巴豆酰化、S-亚硝基化及2-羟基异丁酰化等PTMs研究进展,旨为认识植物PTMs的关键生物学功能和研究前景提供参考。     

蛋白质翻译后修饰研究进展

蛋白质是执行细胞功能的基本功能单元,其表达受基因组和表观遗传学的调控。通常,蛋白质在表达以后还需要经过不同程度的修饰才能发挥所需要的功能。这种翻译后修饰过程受到一系列修饰酶和去修饰酶的严格调控,使得在某一瞬间细胞中蛋白质表现出某种稳定或动态的特定功能。最新的研究表明,真核细胞中存在着各种各样的蛋白质修饰过程,其中大约70%目前还无法解释。有理由认为,这种经过了特定修饰的蛋白质,更客观地反映了细胞的各种生理以及病理过程。因此,除了基因组所编码的＂裸＂蛋白质组的表达以外,更需要对经过翻译后修饰的蛋白质及蛋白质组的调控过程进行深入的研究。该文对常见翻译后修饰以及研究方法进行了综述。     

利用机器学习提高M. jannaschii酪氨酰tRNA合成酶底物特异性分子建模预测的准确度

设计结合不同化学结构底物的酶结合袋是一个巨大的挑战. 传统的湿实验要筛选成千上万甚至上百万个突变体来寻找对特定配体结合的突变体,此过程需要耗费大量的时间和资源. 为了加快筛选过程,我们提出了一种新的工作流程,将分子建模和数据驱动的机器学习方法相结合,生成具有高富集率的突变文库,用于高效筛选能识别特定底物的蛋白质突变体. M. jannaschii酪氨酰tRNA合成酶（Mj. TyrRS）能识别特定的非天然氨基酸并催化形成氨酰tRNA,其不同的突变体能够识别不同结构的非天然氨基酸,并且已经有了许多报道和数据的积累,因此我们使用TyrRS作为一个例子来进行此筛选流程的概念验证. 基于已知的多个Mj. TyrRS的晶体结构及分子建模的结果,我们发现D158G/P是影响残基158~163位α螺旋蛋白骨架变化的关键突变. 我们的模拟结果表明,在含有687个突变体的测试数据中,与随机突变相比,分子建模和打分函数计算排序可以将目标突变体的富集率提高2倍,而使用已知突变体和对应的非天然氨基酸数据训练的机器学习模型进行校准后,筛选富集率可提高11倍. 这种分子建模和机器学习相结合的计算和筛选流程非常有助于Mj.TyrRS的底物特异性设计,可以大大减少湿实验的时间和成本. 此外,这种新方法在蛋白质计算设计领域具有广泛的应用前景.     

应用IP-2D nano-HPLC-MALDI-TOF-TOF鉴定蛋白质泛素化修饰

近年来,在蛋白质研究中,特别是在蛋白质翻译后修饰(PTM)的研究中,生物质谱技术的应用越来越广泛,与纳升级HPLC的联合应用,使这一技术手段更加有效.针对泛素化在细胞功能调控中发挥关键作用的PTM的特点,将免疫沉淀、2D nano-HPLC和基质辅助激光解吸/电离串联飞行时间质谱(IP-2D nano-HPLC-MALDI-TOF-TOF)有机整合,建立了天然状态下蛋白质泛素化位点的鉴定方法,并应用这一方法确定出K562细胞内具有酪氨酸激酶活性的蛋白c-ABL的泛素化位点.为定性鉴定生理和病理状态下内源性蛋白的泛素化修饰提供了借鉴.     

新型蛋白质酰基化修饰与肿瘤的发生发展

基因的表观调控通常由化学基团对组蛋白和非组蛋白的动态调控共同决定,而蛋白质翻译后修饰(post-translational modification,PTM)作为表观调控的主要因素,以共价连接的方式在蛋白质特异位点添加小分子,进而对蛋白质结构、功能、稳定性以及活性产生一定影响,最终影响生命活动进程。而染色质中修饰语言的错误书写、拼读、删除是人类癌症中常见的、有时是早期和关键的事件,可通过诱导表观遗传、转录组和表型改变促进肿瘤的发生。其中赖氨酸作为一种两亲性氨基酸,具有疏水侧链,且赖氨酸具有正电荷可以被酰基化修饰所中和,因此赖氨酸是最常发生修饰的氨基酸。酰基化修饰不仅可以改变蛋白质的结构影响蛋白质的功能,而且在DNA转录、损伤修复、氧化应激、细胞代谢、细胞周期、衰老、血管生成等生命活动中起着至关重要的作用。接下来该文就最近发现的新型酰基化修饰展开综述,回顾新型酰基化修饰的发现过程、调控机制以及其在肿瘤发生发展中的重要作用。     

超氧化物歧化酶翻译后修饰的研究进展

超氧化物歧化酶(superoxide dismutase,SOD)是生物体内存在的一种抗氧化金属酶,它能够催化超氧阴离子自由基歧化生成氧(O₂)和过氧化氢(H₂O₂),在机体氧化与抗氧化平衡中起到至关重要的作用,且与很多疾病的发生、发展密不可分。对SOD的活性调节一直是研究热点,大多数研究都集中在转录水平(基因表达)和翻译水平(酶蛋白合成)两个方面。随着研究的深入,发现蛋白质翻译后修饰(PTM)对SOD的酶活性有重要影响。近年来,研究蛋白质翻译后修饰对SOD的酶活性的影响越来越受到重视。总结了硝基化、磷酸化、S-谷胱甘肽化、糖基化、乙酰化、次磺酸化、亚磺酸化、SUMO化等几种SOD翻译后的修饰方式,讨论了修饰后对SOD酶活性的影响和生理意义,并对SOD翻译后修饰的发展及面临的挑战进行了展望,为相关疾病的研究、治疗及靶向药物的研制提供了理论基础。     

蛋白质翻译后修饰在肝癌免疫治疗中的作用机制

蛋白质翻译后修饰（PTM）是蛋白质活性调节、定位、表达以及与其他细胞分子相互作用的调节机制,能引起蛋白质性质和功能的变化,其传统形式包括磷酸化、糖基化、甲基化、泛素化等。越来越多的研究表明,PTMs不仅调节肝癌的发生和发展,而且在抗癌免疫反应中起着至关重要的作用。本文综述了目前几种传统类型的PTMs在肝癌免疫治疗中的作用机制,为肝癌治疗提供新的见解和未来研究方向。     

蛋白质棕榈酰化修饰在病毒感染过程中的作用

蛋白质棕榈酰化修饰是脂质修饰的一种,赋予了底物蛋白更加多样化的生物学功能.在哺乳动物细胞中,棕榈酰化修饰主要是由ZDHHC家族介导的.病毒入侵细胞后,可利用宿主的棕榈酰化修饰促进自身的复制和感染.宿主通过模式识别受体识别病原体相关分子模式诱发天然免疫应答以保护自身免受病毒的伤害并达到清除病原体的目的.天然免疫是宿主抵抗...     

Analysis of sequence requirements for protein tyrosine sulfation.

We analyzed sequences surrounding known tyrosine sulfation sites to determine the characteristics that distinguish these sites from those that do not undergo sulfation. Tests evaluated the number and position of acidic, basic, hydrophobic, and small amino acids, as well as disulfide and N-glycosylation (sugar) sites. We determined that composition-based tests that select close to 100% of known tyrosine sulfation sites reject 97% of the non-sulfated tyrosines. The acidic test, by far the most selective, eliminated 95% of the non-sulfated tyrosine residues and none of the sulfated tyrosines. Including the basic, hydrophobic, and disulfide tests increased the elimination rate to 97%. Whereas no position flanking the tyrosine residues had the same amino acid always present, imperfectly conserved amino acids found in some positions will improve the specificity of the tests.     

Mapping of Post-translational Modifications of Transition Proteins,TP1 and TP2, and Identification of Protein Arginine Methyltransferase 4 and Lysine Methyltransferase 7 as Methyltransferase for TP2

In a unique global chromatin remodeling process during mammalian spermiogenesis, 90% of the nucleosomal histones are replaced by testis-specific transition proteins, TP1, TP2, and TP4. These proteins are further substituted by sperm-specific protamines, P1 and P2, to form a highly condensed sperm chromatin. In spermatozoa, a small proportion of chromatin, which ranges from 1 to 10% in mammals, retains the nucleosomal architecture and is implicated to play a role in transgenerational inheritance. However, there is still no mechanistic understanding of the interaction of chromatin machinery with histones and transition proteins, which facilitate this selective histone replacement from chromatin. Here, we report the identification of 16 and 19 novel post-translational modifications on rat endogenous transition proteins, TP1 and TP2, respectively, by mass spectrometry. By in vitro assays and mutational analysis, we demonstrate that protein arginine methyltransferase PRMT4 (CARM1) methylates TP2 at Arg⁷¹, Arg⁷⁵, and Arg⁹² residues, and lysine methyltransferase KMT7 (Set9) methylates TP2 at Lys⁸⁸ and Lys⁹¹ residues. Further studies with modification-specific antibodies that recognize TP2K88me1 and TP2R92me1 modifications showed that they appear in elongating to condensing spermatids and predominantly associated with the chromatin-bound TP2. This work establishes the repertoire of post-translational modifications that occur on TP1 and TP2, which may play a significant role in various chromatin-templated events during spermiogenesis and in the establishment of the sperm epigenome.     

Calreticulin and Arginylated Calreticulin Have Different Susceptibilities to Proteasomal Degradation

Post-translational arginylation has been suggested to target proteins for proteasomal degradation. The degradation mechanism for arginylated calreticulin (R-CRT) localized in the cytoplasm is unknown. To evaluate the effect of arginylation on CRT stability, we examined the metabolic fates and degradation mechanisms of cytoplasmic CRT and R-CRT in NIH 3T3 and CHO cells. Both CRT isoforms were found to be proteasomal substrates, but the half-life of R-CRT (2 h) was longer than that of cytoplasmic CRT (0.7 h). Arginylation was not required for proteasomal degradation of CRT, although R-CRT displays ubiquitin modification. A CRT mutant incapable of dimerization showed reduced metabolic stability of R-CRT, indicating that R-CRT dimerization may protect it from proteasomal degradation. Our findings, taken together, demonstrate a novel function of arginylation: increasing the half-life of CRT in cytoplasm.     

Effects of Serine 129 Phosphorylation on α-Synuclein Aggregation,Membrane Association,and Internalization

Although trace levels of phosphorylated α-synuclein (α-syn) are detectable in normal brains, nearly all α-syn accumulated within Lewy bodies in Parkinson disease brains is phosphorylated on serine 129 (Ser-129). The role of the phosphoserine residue and its effects on α-syn structure, function, and intracellular accumulation are poorly understood. Here, co-expression of α-syn and polo-like kinase 2 (PLK2), a kinase that targets Ser-129, was used to generate phosphorylated α-syn for biophysical and biological characterization. Misfolding and fibril formation of phosphorylated α-syn isoforms were detected earlier, although the fibrils remained phosphatase- and protease-sensitive. Membrane binding of α-syn monomers was differentially affected by phosphorylation depending on the Parkinson disease-linked mutation. WT α-syn binding to presynaptic membranes was not affected by phosphorylation, whereas A30P α-syn binding was greatly increased, and A53T α-syn was slightly lower, implicating distal effects of the carboxyl- on amino-terminal membrane binding. Endocytic vesicle-mediated internalization of pre-formed fibrils into non-neuronal cells and dopaminergic neurons matched the efficacy of α-syn membrane binding. Finally, the disruption of internalized vesicle membranes was enhanced by the phosphorylated α-syn isoforms, a potential means for misfolded extracellular or lumenal α-syn to access cytosolic α-syn. Our results suggest that the threshold for vesicle permeabilization is evident even at low levels of α-syn internalization and are relevant to therapeutic strategies to reduce intercellular propagation of α-syn misfolding.     

An Unprecedented Combination of Serine and Cysteine Nucleophiles in a Split Intein with an Atypical Split Site

Protein splicing mediated by inteins is a self-processive reaction leading to the excision of the internal intein domain from a precursor protein and the concomitant ligation of the flanking sequences, the extein-N and extein-C parts, thereby reconstituting the host protein. Most inteins employ a splicing pathway in which the upstream scissile peptide bond is consecutively rearranged into two thioester or oxoester intermediates before intein excision and rearrangement into the new peptide bond occurs. The catalytically critical amino acids involved at the two splice junctions are cysteine, serine, or threonine. Notably, the only potential combination not observed so far in any of the known or engineered inteins corresponds to the transesterification from an oxoester to a thioester, which suggested that this formal uphill reaction with regard to the thermodynamic stability might be incompatible with intein-mediated catalysis. We show that corresponding mutations also led to inactive gp41-1 and AceL-TerL inteins. We report the novel GOS-TerL split intein identified from metagenomic databases as the first intein harboring the combination of Ser1 and Cys+1 residues. Mutational analysis showed that its efficient splicing reaction indeed follows the shift from oxoester to thioester and thus represents a rare diversion from the canonical pathway. Furthermore, the GOS-TerL intein has an atypical split site close to the N terminus. The Int^N fragment could be shortened from 37 to 28 amino acids and exchanged with the 25-amino acid Int^N fragment from the AceL-TerL intein, indicating a high degree of promiscuity of the Int^C fragment of the GOS-TerL intein.     

Nitro- and Amino-polysaccharide Formation from Dextran

Dextran was subjected to oxidative scission by periodate, followed by ring closure with nitromethane to form nitrodextran. The nitro group attached to the ring was reduced by LiAlH₄ to yield amino-polysaccharide of which the molecular weight was about 10,000. It became clear that nitrodextran consisted of 3-deoxy-3-nitro-mannopyranoside, -glucopyranoside, -galactopyranoside and -talopyranoside and their molar ratio was 6: 5: 1: 2 as determined by column Chromatographic separation and gas Chromatographic analysis of the methanolyzate of nitro-dextran.