亲和分离在蛋白质泛素化修饰研究中的应用进展

蛋白质泛素化是真核生物最普遍、最复杂的翻译后修饰方式之一,在细胞的信号转导、生长、发育、代谢等生命过程中发挥着重要作用。泛素化过程的失调则与神经退行性疾病、炎症反应、癌症等重大疾病的发生发展密切相关。分析和研究蛋白质泛素化的结构与功能,可望为认识生命、探索疾病调控内在规律和发现新的诊断策略提供重要信息。生命体系的高度复杂性,泛素化修饰位点、结构类型的多变和多样性,时空动态变化等特点给蛋白质泛素化分析研究带来了巨大的挑战。亲和分离以其高选择性成为泛素化蛋白质结构与功能研究的有力工具。免疫亲和分离法基于抗原-抗体相互作用,是最为经典的分离分析方法,已广泛应用于泛素化蛋白质或肽段的富集分离。源于天然泛素受体的泛素结合结构域(ubiquitin binding domains, UBDs)可与泛素或多聚泛素链相互作用。UBDs和基于此发展起来的串联泛素结合实体(tandem ubiquitin-binding entities, TUBEs)已成为蛋白质泛素化功能研究的热门识别分子。各种多肽类化合物的发展也为蛋白质泛素化的结构和功能解析提供新工具。此外,多种亲和识别配基的联合使用,在蛋白质泛素化修饰的高特异性、高灵敏度分析中展现了独特的优势,为认识生命体内的泛素化修饰提供了重要保障。该文对亲和分离方法在蛋白质泛素化修饰分析中的应用及进展进行了综述。

Advances in the application of affinity separation for analyzing protein ubiquitination

Protein ubiquitination is one of the most common yet complex post-translational modifications in eukaryotes that plays an important role in various biological processes including cell signal transduction,growth,and metabolism.Disorders in the ubiquitination process have been revealed to correlate with the occurrence and development of many diseases such as neurodegenerative disease,inflammation,and cancer.Investigation of protein ubiquitination is of great importance to uncover protein functions,understand the molecular mechanisms underlying biological processes,and develop novel strategies for disease treatment.Great advances have been made toward understanding protein ubiquitination;however,it remains a challenging task due to the high diversity of ubiquitination sites and structures,as well as the dynamic nature of ubiquitination in biological processes.Protein ubiquitination occurs through the formation of a covalent bond between the carboxyl terminus of ubiquitin and theε-amino group of a lysine residue in the substrate.As a small protein,ubiquitin itself can be further modified by another ubiquitin molecule to form homotypic or heterotypic polyubiquitin chains.There are eight sites,namely seven lysine residues(K6,K11,K27,K29,K33,K48,and K63)and one N-terminal methionine(M1),in one ubiquitin molecule that can be used to form a ubiquitin dimer.The variations in modification sites,ubiquitin chain lengths,and conformations result in differences in protein sorting,cell signaling,and function.To resolve the high complexity of protein ubiquitination,new separation approaches are required.Affinity separation based on the specific recognition between biomolecules offers high selectivity and has been employed to study the structures and functions of ubiquitination.In addition,affinity ligands are central to the separation performance.Different affinity ligands have been developed and employed for the capture and enrichment of ubiquitylated proteins.Immunoaffinity separation based on antigen-antibody interactions has been one of the most classical separation methods.Antibodies against ubiquitin or different ubiquitin linkages have been developed and widely applied for the enrichment of ubiquitylated proteins or peptides.The specific capture allows the downstream identification of endogenous ubiquitination sites via mass spectrometry and thus facilitates understanding of the roles and dynamics of polyubiquitin signals.Ubiquitin-binding domains(UBDs)are a collection of modular protein domains that can interact with ubiquitin or polyubiquitin chains.Ubiquitin-associated domains,ubiquitin-interacting motifs,and ubiquitin-binding zinc finger domains are the most frequently used UBDs.Due to the moderate affinity of UBDs toward ubiquitin or ubiquitin chains,tandem ubiquitin-binding entities(TUBEs)have been engineered with high affinities(Kd in the nanomolar range)and exhibit potential as powerful tools for ubiquitination analysis.Because of their affinity and selectivity,UBDs and TUBEs have been applied for the isolation and identification of ubiquitylated targets in cancer cells and yeasts.Compared with antibodies and UBDs,peptides are smaller in size and can be facilely synthesized via chemical approaches.The modular structure of peptides allows for de novo design and screening of artificial ubiquitin affinity ligands for targeted capture of ubiquitinated proteins.Furthermore,the polyhistidine tag at the N-terminus of ubiquitin facilitates the purification of ubiquitylated substrates using immobilized metal affinity chromatography.Considering the high complexity of biosystems,strategies combining multiple affinity ligands have emerged to further improve separation efficiency and reduce background interference.Several combinations of antibodies with UBDs,antibodies with peptidyl tags,and UBDs with peptidyl tags have been developed and proven to be effective for the analysis of protein ubiquitination.These affinity-based approaches serve as important solutions for studying the structure-activity relationship of protein ubiquitination.This review highlights the applications and recent advances in affinity separation techniques for analyzing protein ubiquitination,focusing on the methods using antibodies,UBDs,peptides,and their combinations as affinity ligands.Further,their applications in the enrichment of ubiquitin-modified substrates and the identification of ubiquitination structures are introduced.Additionally,remaining challenges in affinity separation of protein ubiquitination and perspectives are discussed.