A Comparative Analysis of the Ubiquitination Kinetics of Multiple Degrons to Identify an Ideal Targeting Sequence for a Proteasome Reporter

The ubiquitin proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins. The conjugation of a polyubiquitin chain, or polyubiquitination, to a target protein requires an increasingly diverse cascade of enzymes culminating with the E3 ubiquitin ligases. Protein recognition by an E3 ligase occurs through a specific sequence of amino acids, termed a degradation sequence or degron. Recently, degrons have been incorporated into novel reporters to monitor proteasome activity; however only a limited few degrons have successfully been incorporated into such reporters. The goal of this work was to evaluate the ubiquitination kinetics of a small library of portable degrons that could eventually be incorporated into novel single cell reporters to assess proteasome activity. After an intensive literary search, eight degrons were identified from proteins recognized by a variety of E3 ubiquitin ligases and incorporated into a four component degron-based substrate to comparatively calculate ubiquitination kinetics. The mechanism of placement of multiple ubiquitins on the different degron-based substrates was assessed by comparing the data to computational models incorporating first order reaction kinetics using either multi-monoubiquitination or polyubiquitination of the degron-based substrates. A subset of three degrons was further characterized to determine the importance of the location and proximity of the ubiquitination site lysine with respect to the degron. Ultimately, this work identified three candidate portable degrons that exhibit a higher rate of ubiquitination compared to peptidase-dependent degradation, a desired trait for a proteasomal targeting motif.     

A new scheme to discover functional associations and regulatory networks of E3 ubiquitin ligases

Background

Protein ubiquitination catalyzed by E3 ubiquitin ligases play important modulatory roles in various biological processes. With the emergence of high-throughput mass spectrometry technology, the proteomics research community embraced the development of numerous experimental methods for the determination of ubiquitination sites. The result is an accumulation of ubiquitinome data, coupled with a lack of available resources for investigating the regulatory networks among E3 ligases and ubiquitinated proteins. In this study, by integrating existing ubiquitinome data, experimentally validated E3 ligases and established protein-protein interactions, we have devised a strategy to construct a comprehensive map of protein ubiquitination networks.

Results

In total, 41,392 experimentally verified ubiquitination sites from 12,786 ubiquitinated proteins of humans have been obtained for this study. Additional 494 E3 ligases along with 1220 functional annotations and 28588 protein domains were manually curated. To characterize the regulatory networks among E3 ligases and ubiquitinated proteins, a well-established network viewer was utilized for the exploration of ubiquitination networks from 40892 protein-protein interactions. The effectiveness of the proposed approach was demonstrated in a case study examining E3 ligases involved in the ubiquitination of tumor suppressor p53. In addition to Mdm2, a known regulator of p53, the investigation also revealed other potential E3 ligases that may participate in the ubiquitination of p53.

Conclusion

Aside from the ability to facilitate comprehensive investigations of protein ubiquitination networks, by integrating information regarding protein-protein interactions and substrate specificities, the proposed method could discover potential E3 ligases for ubiquitinated proteins. Our strategy presents an efficient means for the preliminary screen of ubiquitination networks and overcomes the challenge as a result of limited knowledge about E3 ligase-regulated ubiquitination.

     

A ubiquitin-like protein unleashes ubiquitin ligases

Modification of cullin-RING ubiquitin ligases by the ubiquitin-like molecule Nedd8 promotes substrate ubiquitination. A crystal structure of a cullin modified by Nedd8 recently reported in Cell (Duda et al., 2008) and a biochemical study in Molecular Cell (Saha and Deshaies, 2008) reveal the dramatic impact on the ligase machinery by conjugation of ubiquitin or ubiquitin-like proteins.     

Alternative Reading Frame Supports An Alternative Model for Retinoblastoma

The ubiqutin-proteasome system is the major pathway by which cells target proteins for degradation in a specific manner. The E3 ubiquitin ligase, which brings targeted proteins (substrates) and activated ubiquitin in close proximity, enabling covalent conjugation of ubiquitin to the substrate, is an essential component of this system. Of the E3 ligases, the cullin (CUL) ligases are of high interest because of their capacity to form multiple distinct E3 complexes to ubiquitinate a potentially large number of substrates. Of the six closely related cullins, very little is known about how specific substrates are recruited to CUL4-dependent ligases. A recent paper in Nature Cell Biology may shed some light on this issue as well as on the function of DDB1, a damaged-DNA binding protein that has long been associated with DNA repair.     

On the packing density of the unbound protein-protein interaction interface and its implications in dynamics

Background

In eukaryotes, ubiquitin-conjugation is an important mechanism underlying proteasome-mediated degradation of proteins, and as such, plays an essential role in the regulation of many cellular processes. In the ubiquitin-proteasome pathway, E3 ligases play important roles by recognizing a specific protein substrate and catalyzing the attachment of ubiquitin to a lysine (K) residue. As more and more experimental data on ubiquitin conjugation sites become available, it becomes possible to develop prediction models that can be scaled to big data. However, no development that focuses on the investigation of ubiquitinated substrate specificities has existed. Herein, we present an approach that exploits an iteratively statistical method to identify ubiquitin conjugation sites with substrate site specificities.

Results

In this investigation, totally 6259 experimentally validated ubiquitinated proteins were obtained from dbPTM. After having filtered out homologous fragments with 40% sequence identity, the training data set contained 2658 ubiquitination sites (positive data) and 5532 non-ubiquitinated sites (negative data). Due to the difficulty in characterizing the substrate site specificities of E3 ligases by conventional sequence logo analysis, a recursively statistical method has been applied to obtain significant conserved motifs. The profile hidden Markov model (profile HMM) was adopted to construct the predictive models learned from the identified substrate motifs. A five-fold cross validation was then used to evaluate the predictive model, achieving sensitivity, specificity, and accuracy of 73.07%, 65.46%, and 67.93%, respectively. Additionally, an independent testing set, completely blind to the training data of the predictive model, was used to demonstrate that the proposed method could provide a promising accuracy (76.13%) and outperform other ubiquitination site prediction tool.

Conclusion

A case study demonstrated the effectiveness of the characterized substrate motifs for identifying ubiquitination sites. The proposed method presents a practical means of preliminary analysis and greatly diminishes the total number of potential targets required for further experimental confirmation. This method may help unravel their mechanisms and roles in E3 recognition and ubiquitin-mediated protein degradation.

     

A plant‐specific in vitro ubiquitination analysis system

Protein ubiquitination requires the concerted action of three enzymes: ubiquitin‐activating enzyme (E1), ubiquitin‐conjugating enzyme (E2) and ubiquitin ligase (E3). These ubiquitination enzymes belong to an abundant protein family that is encoded in all eukaryotic genomes. Describing their biochemical characteristics is an important part of their functional analysis. It has been recognized that various E2/E3 specificities exist, and that detection of E3 ubiquitination activity in vitro may depend on the recruitment of E2s. Here, we describe the development of an in vitro ubiquitination system based on proteins encoded by genes from Arabidopsis. It includes most varieties of Arabidopsis E2 proteins, which are tested with several RING‐finger type E3 ligases. This system permits determination of E3 activity in combination with most of the E2 sub‐groups that have been identified in the Arabidopsis genome. At the same time, E2/E3 specificities have also been explored. The components used in this system are all from plants, particularly Arabidopsis, making it very suitable for ubiquitination assays of plant proteins. Some E2 proteins that are not easily expressed in Escherichia coli were transiently expressed and purified from plants before use in ubiquitination assays. This system is also adaptable to proteins of species other than plants. In this system, we also analyzed two mutated forms of ubiquitin, K48R and K63R, to detect various types of ubiquitin conjugation.     

Ubiquitin and Ubiquitin-like Modifiers in Plants

Posttranslational modifications of proteins by small polypeptides including ubiquitination, neddylation (related to ubiquitin (RUB) conjugation), and sumoylation are implicated in plant growth and development, and they regulate protein degradation, location, and interaction with other proteins. Ubiquitination mediates the selective degradation of proteins by the ubiquitin (Ub)/proteasome pathway. The ubiquitin-like protein RUB is conjugated to cullins, which are part of a ubiquitin E3 ligase complex that is involved in auxin hormonal signaling. Sumoylation, by contrast, is known for its involvement in guiding protein interactions related to abiotic and biotic stresses and in the regulation of flowering time. ATG8/ATG12-mediated autophagy influences degradation and recycling of cellular components. Other ubiquitin-like modifiers (ULPs) such as homology to Ub-1, ubiquitin-fold modifier 1, and membrane-anchored Ub-fold are also found in Arabidopsis. ULPs share similar three-dimensional structures and a conjugation system, including E1 activating enzymes, E2 conjugation enzymes, and E3 ligases, as well as proteases for deconjugation and recycling of the tags. However, each of the ULP posttranslational modifications possesses its own specific enzymes and modifies its specific targets selectively. This review discusses recent findings on ubiquitination and ubiquitin-like modifier processes and their roles in the posttranslational modification of proteins in Arabidopsis.     

Animal HECT ubiquitin ligases: evolution and functional implications

Background  

The patterns of emergence and diversification of the families of ubiquitin ligases provide insights about the evolution of the eukaryotic ubiquitination system. U-box ubiquitin ligases (UULs) are proteins characterized by containing a peculiar protein domain known as U box. In this study, the origin of the animal UUL genes is described.     

泛素化和磷酸化协同作用调控蛋白质降解

在真核细胞中,泛素化和磷酸化是2种常见的蛋白质修饰方式。泛素在蛋白酶体降解途径中发挥重要的靶向作用,细胞外信号严格调控着目的蛋白的泛素化。在很多情况下,这种调控依赖于蛋白质的磷酸化。由磷酸化影响的调控步骤可能与E3泛素连接酶对底物的识别有关,也可能与实际的交联反应有关。这种调控是通过对底物或E3连接酶本身的磷酸化实现的。     

类泛素化修饰Neddylation的功能和调控机制研究进展

NEDD8 (neural precursor cell-expressed developmentally downregulated 8) 分子是一类结构上与泛素相似的分子,参与蛋白质翻译后修饰,这一过程被称为Neddylation．Neddylation的发生机制与泛素化相似,需要E1、E2、E3介导的一系列酶促反应．Neddylation修饰在Cullin-Roc类泛素连接酶的活性调控中具有至关重要的作用,与泛素化研究相比,在真核细胞内仅发现了很少的能被Neddylation修饰的底物,Neddylation的生理功能也有待深入研究．     

Ubiquitin-protein ligases in muscle wasting

Muscle wasting occurs when rates of protein degradation outstrip rates of protein synthesis. Accelerated rates of protein degradation develop in atrophying muscle largely through activation of the ubiquitin-proteasome pathway. The complexity of the ubiquitination process, however, has hampered our understanding of how this pathway is activated in atrophying muscles and which enzymes of the ubiquitin conjugation system are responsible. Recent studies demonstrate that two ubiquitin-protein ligases (E3s), atrogin-1/MAFbx and MuRF1 are critical in the development of muscle atrophy. Other experiments implicate E2(14k) and E3alpha, of the N-end rule pathway, as important players in the process. It seems likely that multiple pathways of ubiquitin conjugation are activated in parallel in atrophying muscle, perhaps to target for degradation specific classes of muscle proteins. The emerging challenge will be to define the protein targets for, as well as to develop inhibitors of, these E3s.     

U-box泛素连接酶调控植物抗逆和生长发育

泛素化是真核生物蛋白质转录后修饰的重要方式之一。泛素连接酶决定了泛素化过程底物的特异性, 在植物抗病、抗旱、耐盐、抗寒和生长发育各个阶段都发挥重要作用。泛素连接酶包括RING、U-box、HECT和F-box四大类。该文对U-box泛素连接酶在植物抗逆和生长发育过程中的作用进行了总结, 并对今后的研究提出了建议, 以期为进一步了解植物泛素化调控通路提供依据。     

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.     

Ubiquitin-mediated modulation of the cytoplasmic viral RNA sensor RIG-I

RIG-I-like receptors, including RIG-I, MDA5 and LGP2, recognize cytoplasmic viral RNA. The RIG-I protein consists of N-terminal CARDs, central RNA helicase and C-terminal domains. RIG-I activation is regulated by ubiquitination. Three ubiquitin ligases target the RIG-I protein. TRIM25 and Riplet ubiquitin ligases are positive regulators of RIG-I and deliver the K63-linked polyubiquitin moiety to RIG-I CARDs and the C-terminal domain. RNF125, another ubiquitin ligase, is a negative regulator of RIG-I and mediates K48-linked polyubiquitination of RIG-I, leading to the degradation of the RIG-I protein by proteasomes. The K63-linked polyubiquitin chains of RIG-I are removed by a deubiquitin enzyme, CYLD. Thus, CYLD is a negative regulator of RIG-I. Furthermore, TRIM25 itself is regulated by ubiquitination. HOIP and HOIL proteins are ubiquitin ligases and are also known as linear ubiquitin assembly complexes (LUBACs). The TRIM25 protein is ubiquitinated by LUBAC and then degraded by proteasomes. The splice variant of RIG-I encodes a protein that lacks the first CARD of RIG-I, and the variant RIG-I protein is not ubiquitinated by TRIM25. Therefore, ubiquitin is the key regulator of the cytoplasmic viral RNA sensor RIG-I.     

UPL1 and 2, two 405 kDa ubiquitin-protein ligases from Arabidopsis thaliana related to the HECT-domain protein family

The ubiquitin/26S proteasome pathway is a major route for degrading abnormal and important short-lived regulatory proteins in eukaryotes. Covalent attachment of ubiquitin, which triggers entry of target proteins into the pathway, is accomplished by an ATP-dependent reaction cascade involving the sequential action of three enzymes, E1s, E2s and E3s. Although much of the substrate specificity of the pathway is determined by E3s (or ubiquitin-protein ligases, UPLs), little is known about these enzymes in plants and how they choose appropriate targets for ubiquitination. Here, we describe two 405 kDa E3s (UPL1 and 2) from Arabidopsis thaliana related to the HECT-E3 family that is essential in yeast and animals. UPL1 and 2 are encoded by 13 kbp genes 26 cM apart on chromosome I, that are over 95% identical within both the introns and exons, suggesting that the two loci arose from a recent gene duplication. The C-terminal HECT domain of UPL1 is necessary and sufficient to conjugate ubiquitin in vitro in a reaction that requires the positionally conserved cysteine within the HECT domain, E1, and an E2 of the UBC8 family. Given that HECT E3s help define target specificity of the ubiquitin conjugation, a continued characterization of UPL1 and 2 should be instrumental in understanding the functions of ubiquitin-dependent protein turnover in plants and for identifying pathway substrates.     

Multidimensional protein identification technology (MudPIT) analysis of ubiquitinated proteins in plants

Protein conjugation with ubiquitin, known as ubiquitination, is a key regulatory mechanism to control protein abundance, localization, and activity in eukaryotic cells. To identify ubiquitin-dependent regulatory steps in plants, we developed a robust affinity purification/identification system for ubiquitinated proteins. Using GST-tagged ubiquitin binding domains, we performed a large scale affinity purification of ubiquitinated proteins from Arabidopsis cell suspension culture. High molecular weight ubiquitinated proteins were separated by SDS-PAGE, and the trypsin-digested samples were then analyzed by a multidimensional protein identification technology (MudPIT) system. A total of 294 proteins specifically bound by the GST-tagged ubiquitin binding domains were identified. From these we determined 85 ubiquitinated lysine residues in 56 proteins, confirming the enrichment of the target class of proteins. Our data provide the first view of the ubiquitinated proteome in plants. We also provide evidence that this technique can be broadly applied to the study of protein ubiquitination in diverse plant species.     

Immunolocalization of ubiquitin conjugates at Z-bands and intercalated discs of rat cardiomyocytes in vitro and in vivo.

Ubiquitin, a highly conserved 76-residue protein found in all eukaryotic cells, can be covalently bound to a wide variety of proteins in the nucleus, cytosol, cytoskeleton, and plasmalemma. This diversity of target proteins reflects a diversity of functions for ubiquitin conjugation. Previous studies have showed enhanced localization of ubiquitin conjugates to Z-bands of normal skeletal muscle and increased ubiquitination in atrophic muscles. These results have implicated a ubiquitin-mediated pathway in protein turnover and degradation in striated muscle. To investigate whether such a pathway might also exist in cardiac striated muscle, we used an affinity-purified polyclonal antibody (conjugate specific) and indirect immunofluorescence to localize ubiquitin conjugates in neonatal and adult rat cardiac myocytes both in vitro and in vivo. In both cultured myocytes and heart tissue, fluorescent ubiquitin conjugates were found in the nucleus as aggregates, in the cytoplasm in a striated pattern indicative of Z-bands, and in intercellular junctions at the intercalated discs between myocytes. Although the acceptor proteins and the physiological significance of ubiquitination at these locations are unknown, the targeting of ubiquitin to specific sites within the nucleus, myofibrils, and sarcolemma could provide a means for selective processing of individual components within these larger macromolecular assemblies, thus implying a regulatory role for ubiquitin conjugation in turnover or stability of proteins in the heart.