Use of the Baculovirus Expression System for the Generation of Virus-Like Particles

Protein ubiquitination is an important post-translational modification that regulates almost every aspect of cellular function and many cell signaling pathways in eukaryotes. Alterations of protein ubiquitination have been linked to many diseases, such as cancer, neurodegenerative diseases, cardiovascular diseases, immunological disorders and inflammatory diseases. To understand the roles of protein ubiquitination in these diseases and in cell signaling pathways, it is necessary to identify ubiquitinated proteins and their modification sites. However, owing to the nature of protein ubiquitination, it is challenging to identify the exact modification sites under physiological conditions. Recently, ubiquitin-remnant profiling, an immunoprecipitation approach, which uses monoclonal antibodies specifically to enrich for peptides derived from the ubiquitinated portion of proteins and mass spectrometry for their identification, was developed to determine ubiquitination events from cell lysates. This approach has now been widely applied to profile protein ubiquitination in several cellular contexts. In this review, we discuss mass-spectrometry-based methods for the identification of protein ubiquitination sites, analyze their advantages and disadvantages, and discuss their application for proteomic analysis of ubiquitination.     

Ubiquitination-mediated protein degradation and modification： an emerging theme in plant-microbe interactions

Post-translational modification is central to protein stability and to the naodulation of protein activity.Various types ofprotein modification,such as phosphorylation,methylation,acetylation,myristoylation,glycosylation,and ubiquitina-tion,have been reported.Among them,ubiquitination distinguishes itself from others in that most of the ubiquitinatedproteins are targeted to the 26S proteasome for degradation.The ubiquitin/26S proteasome system constitutes the majorprotein degradation pathway in the cell.In recent years,the importance of the ubiquitination machinery in the controlof numerous eukaryotic cellular functions has been increasingly appreciated.Increasing number of E3 ubiquitin ligasesand their substrates,including a variety of essential cellular regulators have been identified.Studies in the past severalyears have revealed that the ubiquitination system is important for a broad range of plant developmental processes andresponses to abiotic and biotic stresses.This review discusses recent advances in the functional analysis of ubiquitina-tion-associated proteins from plants and pathogens that play important roles in plant-microbe interactions.     

Non-degradative Ubiquitination of Protein Kinases

Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well.     

Gettin' down with ubiquitin: turning off cell-surface receptors, transporters and channels

G-protein-coupled receptors and transporters in Saccharomyces cerevisiae are modified with ubiquitin in response to ligand biding. In most cases, the proteasome does not recognize these ubiquitinated proteins. Instead, ubiquitination serves to trigger internalization and degradation of plasma membrane proteins in the lysosome-like vacuole. A number of mammalian receptors and at least one ion channel undergo ubiquitination at the plasma membrane, and this modification is required for their downregulation. Some of these cell-surface proteins appear to be degraded by both the proteasome and lysosomal proteases. Recent evidence indicates that other proteins required for receptor internalization might also be regulated by ubiquitination, suggesting that ubiquitin plays diverse roles in regulating plasma membrane protein activity.     

蛋白质的泛素化修饰在细胞应激反应中的作用

泛素是真核细胞内广泛存在的一种高度保守的蛋白质。在特定泛素化酶催化下实现的蛋白质泛素化修饰反应能够高选择性地降解细胞中的特定信号蛋白质,对维持细胞正常的生理功能具有非常重要的作用。另外,某些泛素化修饰反应也能够实现与蛋白质降解无关的功能调控作用。p53、NF-κB和GADD45α是在细胞应激损伤反应中具有广泛调控作用的信号蛋白,发生在这些分子上的泛素化修饰反应是它们发挥相关分子机制的重要基础。     

O-GlcNAc修饰对蛋白质泛素化降解途径的影响

O-GlcNAc修饰是一种特殊的糖基化修饰,几乎参与生物体内所有细胞过程的调控。该修饰与泛素化作为两种重要的蛋白质翻译后修饰形式,都与2型糖尿病、神经退行性疾病、癌症等疾病密切相关。O-GlcNAc修饰对蛋白质泛素化降解途径的影响主要体现在4个方面:(1)O-GlcNAc修饰能够抑制26S蛋白酶体的ATPase活性;(2)O-GlcNAc修饰会减少某些底物蛋白的泛素化降解;(3)O-GlcNAc修饰泛素化相关酶并调节其功能;(4)某些蛋白质(包括调控因子)发生O-GlcNAc修饰后间接影响蛋白质泛素化。     

泛素化和SUMO化对DEC1的拮抗调控作用

分化的胚软骨表达蛋白1(differentiated embryo-chondrocyte expressed gene 1,DEC1)作为一种时钟蛋白,除了在周期节律的调控中发挥转录抑制作用外,还在能量代谢以及多种肿瘤相关的信号通路的调控中发挥重要作用。此外,蛋白质的翻译后修饰是实现蛋白质功能精细调控的一种重要方式。目前发现,DEC1主要可被两种翻译后修饰,即泛素化和SUMO化修饰。尽管泛素化和SUMO化是两种过程非常类似的蛋白质翻译后修饰方式,但是它们对目的蛋白功能的调控却截然不同。由于泛素化和SUMO化与底物的作用靶点都是赖氨酸(Lys),因此在多数情况下,泛素化和SUMO化以拮抗性的方式调控底物蛋白的功能。鉴于此,该文旨在阐述泛素化和SUMO化修饰对DEC1功能的拮抗调节过程,为了解时钟蛋白DEC1对多种信号通路的调控过程中的分子机制提供新的思路。     

Site-specific ubiquitination: Deconstructing the degradation tag

Ubiquitin is a small eukaryotic protein so named for its cellular abundance and originally recognized for its role as the posttranslational modification (PTM) “tag” condemning substrates to degradation by the 26S proteasome. Since its discovery in the 1970s, protein ubiquitination has also been identified as a key regulatory feature in dozens of non-degradative cellular processes. This myriad of roles illustrates the versatility of ubiquitin as a PTM; however, understanding the cellular and molecular factors that enable discrimination between degradative versus non-degradative ubiquitination events has been a persistent challenge. Here, we discuss recent advances in uncovering how site-specificity — the exact residue that gets modified — modulates distinct protein fates and cellular outcomes with an emphasis on how ubiquitination site specificity regulates proteasomal degradation. We explore recent advances in structural biology, biophysics, and cell biology that have enabled a broader understanding of the role of ubiquitination in altering the dynamics of the target protein, including implications for the design of targeted protein degradation therapeutics.     

Ubiquitin Transfer from the E2 Perspective: Why is UbcH5 So Promiscuous?

Protein ubiquitination is a regulatory process that influences nearly every aspect of eukaryotic cell biology. Pathways that range from cell-cycle progression and differentiation to DNA repair to vesicle budding all rely on regulated modification of target proteins by ubiquitin. Target proteins can be tagged by a single molecule of ubiquitin or modified by ubiquitin polymers that can vary in length and linkage specificity, and these variations influence how ubiquitination signals are interpreted. Surprisingly, little is understood regarding mechanisms of protein ubiquitination and how poly-ubiquitin chains are synthesized. Simple models to explain ubiquitin transfer have dominated the literature, but recent work suggests basic assumptions as to how proteins assemble to facilitate protein ubiquitination and poly-ubiquitin chain synthesis should be re-examined. This is particularly necessary for understanding the roles played by E2 ubiquitin-conjugating enzymes, a central protein component in all ubiquitin transfer reactions. In particular, UbcH5, a canonical E2 protein that is active in a broad number of in vitro ubiquitin transfer reactions, is capable of binding ubiquitin non-covalently on a surface distinct from its active site. This unique property allows activated UbcH5~Ub complexes to self-assemble and has a profound influence on poly-ubiquitin chain synthesis.     

RUBI: rapid proteomic-scale prediction of lysine ubiquitination and factors influencing predictor performance

Post-translational modification of protein lysines was recently shown to be a common feature of eukaryotic organisms. The ubiquitin modification is regarded as a versatile regulatory mechanism with many important cellular roles. Large-scale datasets are becoming available for H. sapiens ubiquitination. However, using current experimental techniques the vast majority of their sites remain unidentified and in silico tools may offer an alternative. Here, we introduce Rapid UBIquitination (RUBI) a sequence-based ubiquitination predictor designed for rapid application on a genome scale. RUBI was constructed using an iterative approach. At each iteration, important factors which influenced performance and its usability were investigated. The final RUBI model has an AUC of 0.868 on a large cross-validation set and is shown to outperform other available methods on independent sets. Predicted intrinsic disorder is shown to be weakly anti-correlated to ubiquitination for the H. sapiens dataset and improves performance slightly. RUBI predicts the number of ubiquitination sites correctly within three sites for ca. 80 % of the tested proteins. The average potentially ubiquitinated proteome fraction is predicted to be at least 25 % across a variety of model organisms, including several thousand possible H. sapiens proteins awaiting experimental characterization. RUBI can accurately predict ubiquitination on unseen examples and has a signal across different eukaryotic organisms. The factors which influenced the construction of RUBI could also be tested in other post-translational modification predictors. One of the more interesting factors is the influence of intrinsic protein disorder on ubiquitinated lysines where residues with low disorder probability are preferred.     

Degradation for better survival? Role of ubiquitination in epithelial morphogenesis

As a prevalent post‐translational modification, ubiquitination is essential for many developmental processes. Once covalently attached to the small and conserved polypeptide ubiquitin (Ub), a substrate protein can be directed to perform specific biological functions via its Ub‐modified form. Three sequential catalytic reactions contribute to this process, among which E3 ligases serve to identify target substrates and promote the activated Ub to conjugate to substrate proteins. Ubiquitination has great plasticity, with diverse numbers, topologies and modifications of Ub chains conjugated at different substrate residues adding a layer of complexity that facilitates a huge range of cellular functions. Herein, we highlight key advances in the understanding of ubiquitination in epithelial morphogenesis, with an emphasis on the latest insights into its roles in cellular events involved in polarized epithelial tissue, including cell adhesion, asymmetric localization of polarity determinants and cytoskeletal organization. In addition, the physiological roles of ubiquitination are discussed for typical examples of epithelial morphogenesis, such as lung branching, vascular development and synaptic formation and plasticity. Our increased understanding of ubiquitination in epithelial morphogenesis may provide novel insights into the molecular mechanisms underlying epithelial regeneration and maintenance.     

An efficient system to detect protein ubiquitination by agroinfiltration in Nicotiana benthamiana

The ubiquitination proteasome pathway has been demonstrated to regulate all plant developmental and signaling processes. E3 ligase/substrate‐specific interactions and ubiquitination play important roles in this pathway. However, due to technical limitations only a few instances of E3 ligase–substrate binding and protein ubiquitination in plants have been directly evidenced. An efficient in vivo and in vitro ubiquitination assay was developed for analysis of protein ubiquitination reactions by agroinfiltration expression of both substrates and E3 ligases in Nicotiana benthamiana. Using a detailed analysis of the well‐known E3 ligase COP1 and its substrate HY5, we demonstrated that this assay allows for fast and reliable detection of the specific interaction between the substrate and the E3 ligase, as well as the effects of MG132 and substrate ubiquitination and degradation. We were able to differentiate between the original and ubiquitinated forms of the substrate in vivo with antibodies to ubiquitin or to the target protein. We also demonstrated that the substrate and E3 ligase proteins expressed by agroinfiltration can be applied to analyze ubiquitination in in vivo or in vitro reactions. In addition, we optimized the conditions for different types of substrate and E3 ligase expression by supplementation with the gene‐silencing suppressor p19 and by time‐courses of sample collection. Finally, by testing different protein extraction buffers, we found that different types of buffer should be used for different ubiquitination analyses. This method should be adaptable to other protein modification studies.     

SUMO modification of proteins other than transcription factors

A wide range of eukaryotic proteins has been shown to be sumoylated. Most, but not all of these proteins are nuclear. In all cases documented so far, sumoylation has been shown to occur on lysine residues. In general these are located within the consensus sequence psiKxE, although there are some exceptions to this. The role of sumoylation has been investigated for a number of identified targets. Unlike the situation with ubiquitination, sumoylation does not appear to target proteins for proteasome-mediated degradation. In contrast, the effect of SUMO modification appears to depend on the target protein and includes roles in altering protein activity, protein-protein interactions or protein localisation.     

Direct identification of ubiquitination sites on ubiquitin-conjugated CHIP using MALDI mass spectrometry

The study of protein ubiquitination, a post-translational modification by ubiquitin, has emerged as one of the most active areas in biology because of the important role of this type of modification on the regulation of various cellular proteins. Advances in techniques for the determination and site mapping of protein ubiquitination can facilitate the elucidation of molecular mechanisms of this modification. We have recently described a novel method for identifying peptides containing ubiquitinated amino acid residues, based on the MALDI-MS/MS analysis of tryptic peptide derivatives. In particular, we have utilized N-terminal sulfonation of these peptides to provide a unique fragmentation pattern that leads to the direct identification and sequencing of ubiquitin modified peptides. Here we present an application of this new method on the characterization of ubiquitin conjugated C-terminal Hsc70-interacting protein (CHIP), a recently identified U-box containing E3 enzyme. Three peptides bearing ubiquitination sites have been identified from the digest of ubiquitinated CHIP; one of these was a site on CHIP, while the other two were found on the ubiquitin molecules, demonstrating that sulfonation of tryptic peptides is a general and efficient method for characterizing protein ubiquitination.     

Roles of ubiquitination in pattern-recognition receptors and type I interferon receptor signaling

Post-translational protein modifications are involved in all functions of living cells. This includes the ability of cells to recognize pathogens and regulate genes involved in their clearance, a concept known as innate immunity. While phosphorylation mechanisms play essential roles in regulating different aspects of the innate immune response, ubiquitination is now recognized as another post-translational modification that works in parallel with phosphorylation to orchestrate the final proper innate immune response against invading pathogens. More precisely, this review will discuss the most recent advances that address the role of ubiquitination in pattern-recognition receptors and type I interferon receptor signaling.     

It’s Time for Some “Site”-Seeing: Novel Tools to Monitor the Ubiquitin Landscape in Arabidopsis thaliana

Ubiquitination, the covalent binding of the small protein modifier ubiquitin to a target protein, is an important and frequently studied posttranslational protein modification. Multiple reports provide useful insights into the plant ubiquitinome, but mostly at the protein level without comprehensive site identification. Here, we implemented ubiquitin combined fractional diagonal chromatography (COFRADIC) for proteome-wide ubiquitination site mapping on Arabidopsis thaliana cell cultures. We identified 3009 sites on 1607 proteins, thereby greatly increasing the number of known ubiquitination sites in this model plant. Finally, The Ubiquitination Site tool (http://bioinformatics.psb.ugent.be/webtools/ubiquitin_viewer/) gives access to the obtained ubiquitination sites, not only to consult the ubiquitination status of a given protein, but also to conduct intricate experiments aiming to study the roles of specific ubiquitination events. Together with the antibodies recognizing the ubiquitin remnant motif, ubiquitin COFRADIC represents a powerful tool to resolve the ubiquitination maps of numerous cellular processes in plants.     

A Conserved Deubiquitinating Enzyme Uses Intrinsically Disordered Regions to Scaffold Multiple Protein Interaction Sites

In the canonical view of protein function, it is generally accepted that the three-dimensional structure of a protein determines its function. However, the past decade has seen a dramatic growth in the identification of proteins with extensive intrinsically disordered regions (IDRs), which are conformationally plastic and do not appear to adopt single three-dimensional structures. One current paradigm for IDR function is that disorder enables IDRs to adopt multiple conformations, expanding the ability of a protein to interact with a wide variety of disparate proteins. The capacity for many interactions is an important feature of proteins that occupy the hubs of protein networks, in particular protein-modifying enzymes that usually have a broad spectrum of substrates. One such protein modification is ubiquitination, where ubiquitin is attached to proteins through ubiquitin ligases (E3s) and removed through deubiquitinating enzymes. Numerous proteomic studies have found that thousands of proteins are dynamically regulated by cycles of ubiquitination and deubiquitination. Thus, how these enzymes target their wide array of substrates is of considerable importance for understanding the function of the cell''s diverse ubiquitination networks. Here, we characterize a yeast deubiquitinating enzyme, Ubp10, that possesses IDRs flanking its catalytic protease domain. We show that Ubp10 possesses multiple, distinct binding modules within its IDRs that are necessary and sufficient for directing protein interactions important for Ubp10''s known roles in gene silencing and ribosome biogenesis. The human homolog of Ubp10, USP36, also has IDRs flanking its catalytic domain, and these IDRs similarly contain binding modules important for protein interactions. This work highlights the significant protein interaction scaffolding abilities of IDRs in the regulation of dynamic protein ubiquitination.     

Ubiquitination and filamentous structure of cytidine triphosphate synthase

Living organisms respond to nutrient availability by regulating the activity of metabolic enzymes. Therefore, the reversible post-translational modification of an enzyme is a common regulatory mechanism for energy conservation. Recently, cytidine-5′-triphosphate (CTP) synthase was discovered to form a filamentous structure that is evolutionarily conserved from flies to humans. Interestingly, induction of the formation of CTP synthase filament is responsive to starvation or glutamine depletion. However, the biological roles of this structure remain elusive. We have recently shown that ubiquitination regulates CTP synthase activity by promoting filament formation in Drosophila ovaries during endocycles. Intriguingly, although the ubiquitination process was required for filament formation induced by glutamine depletion, CTP synthase ubiquitination was found to be inversely correlated with filament formation in Drosophila and human cell lines. In this article, we discuss the putative dual roles of ubiquitination, as well as its physiological implications, in the regulation of CTP synthase structure.