Neddylation修饰与病毒感染调控研究进展

Nedd8是一类结构上与泛素相似的分子,广泛参与蛋白质翻译后修饰,这一过程被称为Neddylation。Neddylation修饰已经被证实参与几乎所有细胞内的调控过程,并在病毒感染过程有着重要的调控作用,但对于其具体的调控机制研究还十分匮乏。深入开展Neddylation修饰与病毒感染之间的分子调控机制研究将有助于阐明病毒的致病机理。     

Neddylation及其在细胞中作用的研究进展

NEDD8是与泛素最相似的类泛素修饰蛋白,其参与翻译后可逆性修饰的过程被称为Neddylation,即NEDD化。近年来,继泛素化在生物细胞中的作用被不断探索后,NEDD化在细胞中的作用也被越来越多的人所研究。本文主要就NEDD化在信号转导以及转录因子调控等方面的作用作一综述。     

Deneddylation修饰酶NEDP1的作用机制及研究进展

神经前体表达发育下调蛋白8(neural precursor cell-expressed developmentally downregulated 8,Nedd8)是一种类泛素蛋白,其参与蛋白质修饰的作用机制与泛素高度相似,即通过与底物的赖氨酸残基共价结合,从而对底物进行Neddylation修饰。Neddylation修饰可调控多种重要的生命活动,如细胞周期和免疫应答等。Nedd8蛋白酶1(Nedd8 protease 1, NEDP1)是隶属于小类泛素修饰物特异性蛋白酶家族(small ubiquitin-like modifier specific protease family, Ulp/Senp family)家族的半胱氨酸蛋白酶,可以特异性去除底物与Nedd8的共价结合,例如,NEDP1能够去除p53、鼠双微体蛋白(murine double min-utes 2 protein, Mdm2)、smad泛素化调节因子1(smad ubiquitylation regulatory factor 1, Smurf1)等多个蛋白质的Neddylation修饰,进而调节Neddylation修饰蛋白的生物学功能。临床研究表明,NEDP1与肿瘤的发生发展密切相关,包括肺癌、结肠癌、胶质母细胞瘤等,另外,NEDP1还参与成骨发育异常、神经退行性疾病、血管炎症等病变过程。本文对NEDP1蛋白的结构,调控Neddylation通路的作用模式和NEDP1作用底物的进展进行总结,以期为肿瘤等相关疾病的诊断和治疗提供参考。     

非经典泛素化的研究进展

泛素化是真核生物最普遍最重要的翻译后修饰之一,控制基因转录表达、细胞生长死亡、分子运输、代谢、发育和免疫反应等大多数生理过程。经典泛素系统的通路和机制越来越明晰,同时非经典的泛素化也逐渐被发现。本文将对经典泛素系统进行简单回顾,并且对非蛋白底物泛素化、非赖氨酸位点泛素化、非经典E3泛素连接酶等最新非经典泛素化进行阐述。     

泛素连接酶E3与泛素链修饰类型特异性研究进展

泛素化修饰是真核细胞内广泛存在的一种修饰形式,受到该修饰的蛋白质分子遍及基因转录、蛋白质翻译、信号转导、细胞周期控制以及生长发育等几乎所有的生命活动过程,对生命体正常功能的发挥具有重要作用。泛素化修饰的失调会给生命体带来一系列负面影响,严重者将导致疾病,甚至危及生命。泛素连接酶E3是泛素化修饰反应中底物特异性的直接决定者,其机制研究不仅可揭示蛋白质质量控制和生命活动功能的奥秘,也将为疾病关联失调蛋白的精准调控和精准医学实践提供技术支撑。现结合当前对泛素连接酶E3研究的最新进展,阐述泛素连接酶E3发挥作用时与不同类型泛素链之间的特异性关系,旨在为蛋白质功能调控的分子机制、药物研制和疾病诊治提供新思路。     

泛素化研究专刊征稿启事北大核心CSCD

《生物化学与生物物理进展》拟于2023年1月出版泛素化研究专刊,诚邀海内外科研工作者来稿。生物大分子的共价修饰与生物大分子动态调控和细胞命运决定、机体组织稳态维持关系密切。在众多类型的蛋白质翻译后修饰中,泛素化修饰被广为关注。根据与底物结合的泛素分子的数量和拓扑结构,泛素化可分为单泛素化和多聚泛素化。     

线性泛素化修饰研究进展

蛋白质泛素化修饰过程在调节各种细胞生物学功能的过程中发挥了非常重要的作用,如细胞周期进程、DNA损伤修复、信号转导和各种蛋白质膜定位等。泛素化修饰可分为多聚泛素化修饰和单泛素化修饰。多聚泛素化修饰系统可以通过对底物连接不同类型的多泛素化链调节蛋白质的功能。多聚泛素化修饰中已知7种泛素链连接方式均为泛素内赖氨酸连接方式。近几年发现了第8种类型的泛素链连接形式即线性泛素化,其泛素链的连接方式是由泛素甲硫氨酸的氨基基团与另一泛素甘氨酸的羧基基团相连形成泛素链标记。目前研究表明线性泛素化修饰在先天性免疫和炎症反应等多个过程中发挥着非常重要的作用。募集线性泛素链的泛素连接酶E3被称为LUBAC复合体,其组成底物以及其活性调控机制和功能所知甚少。本文综述了募集线性泛素化链的泛素连接酶、去泛素化酶、底物等活性调控机制及其在先天性免疫等多个领域中的功能,分析了后续研究方向,以期为相关研究提供参考。     

K27泛素化修饰研究进展

泛素化修饰作为最普遍存在的翻译后修饰形式之一,介导了生物体内蛋白质稳态调控等功能。泛素分子的7个赖氨酸和N端甲硫氨酸可以继续被泛素分子修饰,进而形成8种类型的泛素链。其中,K48和K63泛素链由于丰度高且功能研究相对清楚被称为经典泛素链,而其他6种泛素链被称为非经典泛素链。在非经典泛素链中,K27泛素链是在泛素分子的Lys27 (K27)位点上继续发生泛素化形成的,具有紧密的空间结构。近些年,K27泛素链在固有免疫、蛋白稳态和DNA损伤修复等方面的功能逐渐被报道,但K27泛素链的合成、修剪过程及其下游招募特定蛋白质的分子调控机制还所知甚少。文中结合实验室研究,综述了K27泛素链结构特征、结合方式和生物学功能,为未来K27泛素链结构和生物学功能的深入研究提供参考。     

NEDD化修饰系统及其在肿瘤发展中的作用

目前已经鉴定出17种类泛素蛋白（ubiquitin like proteins,UBLs）,这些蛋白与底物的结合方式与泛素相似．根据进化特征,可将UBLs分为9类,分别为：NEDD8、SUMO、ISG15、FUB1、FAT10、Atg8、Atg12、Urm1和UFM1．NEDD8是目前研究最多的UBLs之一,与泛素的氨基酸序列具有高度相似性．NEDD化修饰是一种动态的可逆蛋白质翻译后修饰方式,可以将NEDD8共价结合到靶蛋白之上,也可以将NEDD8从靶蛋白上去除．NEDD化修饰对蛋白功能具有重要的调节作用,如改变蛋白质的空间构象、阻碍底物与其它蛋白质的相互作用和招募与NEDD8相互作用的蛋白等．最新研究表明,NEDD化与肿瘤的发生发展密切相关,但具体的机制还不清楚．本文将就NEDD化修饰在肿瘤发展过程中的作用机制做一综述．     

泛素链修饰类型研究进展

泛素化修饰的蛋白质底物广泛参与蛋白质降解、胞内蛋白质转运、细胞信号转导、自噬和DNA损伤修复等重要的生物学过程。泛素化修饰包括单泛素化修饰和多泛素化修饰。因泛素分子含有7个赖氨酸残基和1个N端甲硫氨酸残基,多泛素化修饰又可分为同型或异型的多聚泛素化修饰。此外,泛素分子的乙酰化修饰和磷酸化修饰大大增加了泛素链的复杂性。不同泛素链的形成往往依赖泛素连接酶或者去泛素化酶。现综述不同类型的泛素链修饰类型的编辑、识别、去除机制及其生物学功能,并讨论泛素分子自身的乙酰化和磷酸化修饰。     

Rbx1 Flexible Linker Facilitates Cullin-RING Ligase Function Before Neddylation and After Deneddylation

In ubiquitination, cullin-RING E3 ubiquitin ligases (CRLs) assist in ubiquitin transfer from ubiquitin-conjugating enzyme E2 to the substrate. Neddylation, which involves NEDD8 transfer from E2 to E3-cullin, stimulates ubiquitination by inducing conformational change in CRLs. However, deneddylation, which removes NEDD8 from cullin, does not suppress ubiquitination in vivo, raising the question of how neddylation/deneddylation exerts its effects. Using molecular-dynamics simulations, we demonstrate that before neddylation occurs, the linker flexibility of Rbx1, a CRL component, leads to conformational changes in CRLs that allow neddylation and initiation of ubiquitination. These large NEDD8-induced conformational changes are retained after deneddylation, allowing both initiation of the ubiquitination process and ubiquitin chain elongation after deneddylation. Furthermore, mutation of lysine, the cullin residue to which NEDD8 covalently attaches, dramatically reduces CRL conformational changes, suggesting that the acceptor lysine allosterically regulates CRLs. Thus, our results imply that neddylation stimulates ubiquitination by CRL conformational control via lysine modification.     

Protection of cullin-RING E3 ligases by CSN-UBP12

Neddylation, a process that conjugates the ubiquitin-like polypeptide NEDD8 to cullin proteins, activates cullin-RING ubiquitin ligases (CRLs). Deneddylation, in which the COP9 signalosome (CSN) removes NEDD8 from cullins, inactivates CRLs. However, genetic studies of CSN function conclude that deneddylation also promotes CRL activity. It has been proposed that a cyclic transition through neddylation and deneddylation is required for the regulation of CRL activity in vivo. Recent discoveries suggest that an additional level of complexity exists, whereby CRL components are targets for degradation, mediated either by autocatalytic ubiquitination or by unknown mechanisms. Deneddylation by CSN and deubiquitylation by CSN-associated ubiquitin-specific protease 12 protect CRL components from cellular depletion, thus maintaining the physiological CRL activities.     

Cutting edge: bacterial modulation of epithelial signaling via changes in neddylation of cullin-1

The human enteric flora plays a significant role in intestinal health and disease. Certain enteric bacteria can inhibit the NF-kappaB pathway by blockade of IkappaB-alpha ubiquitination. IkappaB-alpha ubiquitination is catalyzed by the E3-SCF(betaTrCP) ubiquitin ligase, which is itself regulated via covalent modification of the cullin-1 subunit by the ubiquitin-like protein NEDD8. Neddylation is a biochemical event associated with diverse cellular processes related to cell signaling, however, physiological regulation of cullin neddylation has not been described in mammalian systems. We report that interaction of nonpathogenic bacteria with epithelial cells resulted in a rapid loss of neddylated Cul-1 and consequent repression of the NF-kappaB pathway. This observation may explain the ability of intestinal bacterial communities to influence diverse eukaryotic processes in general and inflammatory tolerance of the mammalian intestinal epithelia specifically.     

Upregulation of SIRT1 by 17β-estradiol depends on ubiquitin-proteasome degradation of PPAR-γ mediated by NEDD4-1

17β-estradiol (E2) treatment of cells results in an upregulation of SIRT1 and a down-regulation of PPARγ. The decrease in PPARγ expression is mediated by increased degradation of PPARγ. Here we report that PPARγ is ubiquitinated by HECT E3 ubiquitin ligase NEDD4-1 and degraded, along with PPARγ, in response to E2 stimulation. The PPARγ interacts with ubiquitin ligase NEDD4-1 through a conserved PPXY-WW binding motif. The WW3 domain in NEDD4-1 is critical for binding to PPARγ. NEDD4-1 overexpression leads to PPARγ ubiquitination and reduced expression of PPARγ. Conversely, knockdown of NEDD4-1 by specific siRNAs abolishes PPARγ ubiquitination. These data indicate that NEDD4-1 is the E3 ubiquitin ligase responsible for PPARγ ubiquitination. Here, we show that NEDD4-1 delays cellular senescence by degrading PPARγ expression. Taken together, our data show that E2 could upregulate SIRT1 expression via promoting the PPARγ ubiquitination-proteasome degradation pathway to delay the process of cell senescence.     

Insights into links between autophagy and the ubiquitin system from the structure of LC3B bound to the LIR motif from the E3 ligase NEDD4

Members of the LC3/GABARAP family of ubiquitin‐like proteins function during autophagy by serving as membrane linked protein‐binding platforms. Their C‐termini are physically attached to membranes through covalent linkage to primary amines on lipids such as phosphatidylethanolamine, while their ubiquitin‐like fold domains bind “LIR” (LC3‐Interacting Region) sequences found within an extraordinarily diverse array of proteins including regulators of autophagy, adaptors that recruit ubiquitinated cargoes to be degraded, and even proteins controlling processes at membranes that are not associated with autophagy. Recently, LC3/GABARAP proteins were found to bind the ubiquitin E3 ligase NEDD4 to influence ubiquitination associated with autophagy in human cell lines. Here, we use purified recombinant proteins to define LC3B interactions with a specific LIR sequence from NEDD4, present a crystal structure showing atomic details of the interaction, and show that LC3B‐binding can steer intrinsic NEDD4 E3 ligase activity. The data provide detailed molecular insights underlying recruitment of an E3 ubiquitin ligase to phagophores during autophagy.     

NEDD8 overexpression results in neddylation of ubiquitin substrates by the ubiquitin pathway

Ubiquitin and ubiquitin-like proteins use unique E1, E2, and E3 enzymes for conjugation to their substrates. We and others have recently reported that increases in the relative concentration of the ubiquitin-like protein NEDD8 over ubiquitin lead to activation of NEDD8 by the ubiquitin E1 enzyme. We now show that this results in erroneous conjugation of NEDD8 to ubiquitin substrates, such as p53, Caspase 7, and Hif1α, demonstrating that overexpression of NEDD8 is not appropriate for identification of substrates of the NEDD8 pathway.     

Protein neddylation and its alterations in human cancers for targeted therapy

Neddylation, a post-translational modification that conjugates an ubiquitin-like protein NEDD8 to substrate proteins, is an important biochemical process that regulates protein function. The best-characterized substrates of neddylation are the cullin subunits of Cullin-RING ligases (CRLs), which, as the largest family of E3 ubiquitin ligases, control many important biological processes, including tumorigenesis, through promoting ubiquitylation and subsequent degradation of a variety of key regulatory proteins. Recently, increasing pieces of experimental evidence strongly indicate that the process of protein neddylation modification is elevated in multiple human cancers, providing sound rationale for its targeting as an attractive anticancer therapeutic strategy. Indeed, neddylation inactivation by MLN4924 (also known as pevonedistat), a small molecule inhibitor of E1 NEDD8-activating enzyme currently in phase I/II clinical trials, exerts significant anticancer effects by inducing cell cycle arrest, apoptosis, senescence and autophagy in a cell-type and context dependent manner. Here, we summarize the latest progresses in the field with a major focus on preclinical studies in validation of neddylation modification as a promising anticancer target.     

Changes in the ratio of free NEDD8 to ubiquitin triggers NEDDylation by ubiquitin enzymes

Ubiquitin and UBL (ubiquitin-like) modifiers are small proteins that covalently modify other proteins to alter their properties or behaviours. Ubiquitin modification (ubiquitylation) targets many substrates, often leading to their proteasomal degradation. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is the UBL most closely related to ubiquitin, and its best-studied role is the activation of CRLs (cullin-RING ubiquitin ligases) by its conjugation to a conserved C-terminal lysine residue on cullin proteins. The attachment of UBLs requires three UBL-specific enzymes, termed E1, E2 and E3, which are usually well insulated from parallel UBL pathways. In the present study, we report a new mode of NEDD8 conjugation (NEDDylation) whereby the UBL NEDD8 is linked to proteins by ubiquitin enzymes in vivo. We found that this atypical NEDDylation is independent of classical NEDD8 enzymes, conserved from yeast to mammals, and triggered by an increase in the NEDD8 to ubiquitin ratio. In cells, NEDD8 overexpression leads to this type of NEDDylation by increasing the concentration of NEDD8, whereas proteasome inhibition has the same effect by depleting free ubiquitin. We show that bortezomib, a proteasome inhibitor used in cancer therapy, triggers atypical NEDDylation in tissue culture, which suggests that a similar process may occur in patients receiving this treatment.