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.     

Regulation of cellular polyamines by antizyme

Proteins that are degraded by the proteasome are first modified by a set of enzymes that attach multiple copies of ubiquitin to substrate lysines, but a tiny minority, including the polyamine-synthesizing enzyme ornithine decarboxylase, is handled differently. This enzyme is targeted for destruction by another protein--antizyme. Why does ornithine decarboxylase have its own dedicated destruction mechanism, how does it work, and is it the only protein to be targeted to the proteasome in this way?     

FBXW7/hCDC4 controls glioma cell proliferation in vitro and is a prognostic marker for survival in glioblastoma patients

Ubiquitin is a highly versatile post-translational modification that controls virtually all types of cellular events. Over the past ten years we have learned that diverse forms of ubiquitin modifications and of ubiquitin binding modules co-exist in the cell, giving rise to complex networks of protein:protein interactions. A central problem that continues to puzzle ubiquitinologists is how cells translate this myriad of stimuli into highly specific responses. This is a classical signalling problem. Here, we draw parallels with the phosphorylation signalling pathway and we discuss the expanding repertoire of ubiquitin signals, signal tranducers and signalling-regulated E3 enzymes. We examine recent advances in the field, including a new mechanism of regulation of E3 ligases that relies on ubiquitination.     

Cul3-KLHL20 ubiquitin ligase: physiological functions,stress responses,and disease implications

Cullin-RING ubiquitin ligases are the largest Ubiquitin ligase family in eukaryotes and are multi-protein complexes. In these complexes, the Cullin protein serves as a scaffold to connect two functional modules of the ligases, the catalytic subunit and substrate-binding subunit. KLHL20 is a substrate-binding subunit of Cullin3 (Cul3) ubiquitin ligase. Recent studies have identified a number of substrates of KLHL20-based ubiquitin ligase. Through ubiquitination of these substrates, KLHL20 elicits diverse cellular functions, some of which are associated with human diseases. Furthermore, the functions, subcellular localizations, and expression of KLHL20 are regulated by several physiological and stressed signals, which allow KLHL20 to preferentially act on certain substrates to response to these signals. Here, we provide a summary of the functions and regulations of KLHL20 in several physiological processes and stress responses and its disease implications.     

Preparation of distinct ubiquitin chain reagents of high purity and yield

The complexity of protein ubiquitination signals derives largely from the variety of polyubiquitin linkage types that can modify a target protein, each imparting distinct functional consequences. Free ubiquitin chains of uniform linkages and length are important tools in understanding how ubiquitin-binding proteins specifically recognize these different polyubiquitin modifications. While some free ubiquitin chain species are commercially available, mutational analyses and labeling schemes are limited to select, marketed stocks. Furthermore, the multimilligram quantities of material required for detailed biophysical and/or structural studies often makes these reagents cost prohibitive. To address these limitations, we have optimized known methods for the synthesis and purification of linear, K11-, K48-, and K63-linked ubiquitin dimers, trimers, and tetramers on a preparative scale. The high purity and relatively high yield of these proteins readily enables material-intensive experiments and provides flexibility for engineering specialized ubiquitin chain reagents, such as fluorescently labeled chains of discrete lengths.     

Modulation of sensitivity and selectivity in plant signaling by proteasomal destabilization

The ubiquitin (Ub) system of intracellular protein degradation regulates the abundance of numerous proteins that control plant growth and development. Recent advances have begun to illustrate how environmental and endogenous signals affect plant responses through Ub-related proteolysis, the importance of combinatorial control in regulated protein destruction and how multiprotein complexes confer sensitivity and selectivity to ubiquitination. Further insight into the cell biology of Ub-chain assembly and proteasomal degradation, as well as into the relationship between proteolysis and other regulatory modifications, will be essential for understanding the mechanistic basis of the integration of diverse plant signals.     

Ubiquitin modifications

Protein ubiquitination is a dynamic multifaceted post-translational modification involved in nearly all aspects of eukaryotic biology. Once attached to a substrate, the 76-amino acid protein ubiquitin is subjected to further modifications, creating a multitude of distinct signals with distinct cellular outcomes, referred to as the ''ubiquitin code''. Ubiquitin can be ubiquitinated on seven lysine (Lys) residues or on the N-terminus, leading to polyubiquitin chains that can encompass complex topologies. Alternatively or in addition, ubiquitin Lys residues can be modified by ubiquitin-like molecules (such as SUMO or NEDD8). Finally, ubiquitin can also be acetylated on Lys, or phosphorylated on Ser, Thr or Tyr residues, and each modification has the potential to dramatically alter the signaling outcome. While the number of distinctly modified ubiquitin species in cells is mind-boggling, much progress has been made to characterize the roles of distinct ubiquitin modifications, and many enzymes and receptors have been identified that create, recognize or remove these ubiquitin modifications. We here provide an overview of the various ubiquitin modifications present in cells, and highlight recent progress on ubiquitin chain biology. We then discuss the recent findings in the field of ubiquitin acetylation and phosphorylation, with a focus on Ser65-phosphorylation and its role in mitophagy and Parkin activation.     

Ubiquitin Signaling Regulates RNA Biogenesis,Processing, and Metabolism

The fate of eukaryotic proteins, from their synthesis to destruction, is supervised by the ubiquitin–proteasome system (UPS). The UPS is the primary pathway responsible for selective proteolysis of intracellular proteins, which is guided by covalent attachment of ubiquitin to target proteins by E1 (activating), E2 (conjugating), and E3 (ligating) enzymes in a process known as ubiquitylation. The UPS can also regulate protein synthesis by influencing multiple steps of RNA (ribonucleic acid) metabolism. Here, recent publications concerning the interplay between the UPS and different types of RNA are reviewed. This interplay mainly involves specific RNA-binding E3 ligases that link RNA-dependent processes with protein ubiquitylation. The emerging understanding of their modes of RNA binding, their RNA targets, and their molecular and cellular functions are primarily focused on. It is discussed how the UPS adapted to interact with different types of RNA and how RNA molecules influence the ubiquitin signaling components.     

Chromatin and cell death

HMGB1, a very mobile chromatin protein, leaks out from necrotic cells and signals to neighbouring cells that tissue damage has occurred. At least one receptor for extracellular HMGB1 exists, and signals to different cells to divide, migrate, activate inflammation or start an immune response. Remarkably, apoptotic chromatin binds HMGB1 irreversibly, thereby ensuring that it will not diffuse away to activate responses from neighbouring cells. Thus, dying cells use their own chromatin to signal how they have died. We argue that the nuclear events in apoptosis serve to control the molecular signals that dying cells send out.     

The Ubiquitin System in Higher and Lower Plants — Pathways in Protein Metabolism

The multiple biological functions of the small polypeptide ubiquitin are mirrored by its unparalleled conservation on the amino acid and gene organization level. During the last years, it has become widely accepted that ubiquitin is an essential component in the ATP-dependent nonlysosomal protein degradation pathway occurring in all eukaryotic organisms. As turnover, consisting of protein synthesis and disassembly, is a central and vital process for each living cell, ubiquitin-mediated proteolysis is of enormous physiological value. The components of the ubiquitin ligation system have been characterized skillfully in plant and animal cells, but at the moment many questions remain as to how the high degree of specificity that is necessary for the regulation of intracellular breakdown is ensured. The recent hypotheses and models proposed for the basic mechanisms of protein recognition, conjugation and degradation will be discussed in detail. The existence of ubiquitin-protein conjugates which are not rapidly degraded clearly suggested that the role of ubiquitin is not restricted in its implication for protein turnover. Alterations of DNA structure, specific cell recognition mechanisms and cytoskeletal variations were observed as further ubiquitin-dependent processes which are not directly coupled to protein degradation.     

Structural basis for ubiquitin recognition by SH3 domains

The SH3 domain is a protein-protein interaction module commonly found in intracellular signaling and adaptor proteins. The SH3 domains of multiple endocytic proteins have been recently implicated in binding ubiquitin, which serves as a signal for diverse cellular processes including gene regulation, endosomal sorting, and protein destruction. Here we describe the solution NMR structure of ubiquitin in complex with an SH3 domain belonging to the yeast endocytic protein Sla1. The ubiquitin binding surface of the Sla1 SH3 domain overlaps substantially with the canonical binding surface for proline-rich ligands. Like many other ubiquitin-binding motifs, the SH3 domain engages the Ile44 hydrophobic patch of ubiquitin. A phenylalanine residue located at the heart of the ubiquitin-binding surface of the SH3 domain serves as a key specificity determinant. The structure of the SH3-ubiquitin complex explains how a subset of SH3 domains has acquired this non-traditional function.     

Selective recruitment of an E2~ubiquitin complex by an E3 ubiquitin ligase

RING E3 ligases are proteins that must selectively recruit an E2-conjugating enzyme and facilitate ubiquitin transfer to a substrate. It is not clear how a RING E3 ligase differentiates a naked E2 enzyme from the E2∼ubiquitin-conjugated form or how this is altered upon ubiquitin transfer. RING-box protein 1 (Rbx1/ROC1) is a key protein found in the Skp1/Cullin-1/F-box (SCF) E3 ubiquitin ligase complex that functions with the E2 ubiquitin conjugating enzyme CDC34. The solution structure of Rbx1/ROC1 revealed a globular RING domain (residues 40–108) stabilized by three structural zinc ions (root mean square deviation 0.30 ± 0.04 Å) along with a disordered N terminus (residues 12–39). Titration data showed that Rbx1/ROC1 preferentially recruits CDC34 in its ubiquitin-conjugated form and favors this interaction by 50-fold compared with unconjugated CDC34. Furthermore, NMR and biochemical assays identified residues in helix α2 of Rbx1/ROC1 that are essential for binding and activating CDC34∼ubiquitin for ubiquitylation. Taken together, this work provides the first direct structural and biochemical evidence showing that polyubiquitylation by the RING E3 ligase Rbx1/ROC1 requires the preferential recruitment of an E2∼ubiquitin complex and subsequent release of the unconjugated E2 protein upon ubiquitin transfer to a substrate or ubiquitin chain.     

UMI, a novel RNF168 ubiquitin binding domain involved in the DNA damage signaling pathway

Ubiquitination regulates important cellular processes, including the DNA damage response (DDR) and DNA repair. The complexity of the ubiquitin-mediated signals is decoded by ubiquitin receptors, which contain protein modules named ubiquitin binding domains (UBDs). We previously identified a new ubiquitin ligase, RNF168, involved in DDR and endowed with two UBDs named MIU (motif interacting with ubiquitin). Here we have provided the identification of a novel UBD, the UMI (UIM- and MIU-related UBD), present in RNF168, and characterized the interaction surface with ubiquitin, centered on two Leu residues. We have demonstrated that integrity of the UMI, in addition to the MIUs, is necessary for the proper localization of RNF168 and for ubiquitination of nuclear proteins, including histone H2A. Finally, we have shown that simultaneous inactivation of UMI and MIUs prevents the recruitment to DDR foci of the crucial downstream mediator 53BP1.     

Modes of regulation of ubiquitin-mediated protein degradation

The ubiquitin-proteasome pathway is responsible for the major portion of specific cellular protein degradation. Ubiquitin-mediated degradation is involved in physiological regulation of many cellular processes, including cell cycle progression, differentiation, and signal transduction. Here, we review the basic mechanisms of the ubiquitin system and the various ways in which ubiquitin-mediated degradation can be modulated by physiological signals.     

Stress,specificity and the NEDD8 proteome

Comment on: Leidecker O, et al. Cell Cycle 2012; 1142–50In an exciting and surprising paper in a recent issue of Cell Cycle, Leidecker et al. show that the balance between protein modification by ubiquitin or the ubiquitin like protein NEDD8 is dramatically altered by cellular stress. In a variety of conditions that reduce the concentration of free ubiquitin, a very dramatic increase in protein modification by neddylation is revealed. Importantly, this process is shown to arise as NEDD8 is activated under these conditions by the ubiquitin-activating enzyme Ube1 and not by the typical NEDD8 specific EI enzyme, NAE. This results in many proteins in stressed cells being modified by mixed ubiquitin NEDD8 chains, which is highly relevant in the development of novel cancer therapeutics, as the NAE specific inhibitor MLN4924²does not block this new pathway despite its promising anticancer activity.Initial comparative studies on the ubiquitin and ubiquitin-like (Ubl) protein pathways have established that each pathway has separate and specific enzymes both for activating the Ubl and for removing it.³ In the case of NEDD8, the E1 is NAE; the E2s are Ubc12 and Ube2F, and the E3s include the Rbx1 and Rbx2 RING finger proteins as well as members of the DCN family of proteins. The first studies of the NEDD8 system suggested that there were very few substrates for this modification, with most emphasis placed on the cullin proteins. The cullins are components of the cullin-RING ligases (CRLs) that are responsible for the ubiquitylation of many critical substrates, for example, oncoproteins such as cyclin E and c-myc. The cullins are modified by neddylation, which increases the E3 activity of the CRLs, probably through structural alterations that free the Ring domain of the E3 and/or by blocking the binding of inhibitory proteins such as CAND 1.^4,5 Recently, many new substrates and E3 ligases for NEDD8 have been uncovered, with initial studies identifying p53 and Mdm2 as substrates for neddylation, and Mdm2 as a E3 ligase for both NEDD8 and ubiquitin.⁶ Proteomic approaches have now identified many more substrates, notable among them being the ribosomal proteins involved in signaling to p53.^7,8 In the current study, the authors found that a high level of NEDD8-conjugated proteins were rapidly induced by proteasome inhibition with MG132, but that this reaction was not inhibited by MLN4924, even while the same compound was blocking cullin neddylation. This meant that another E1 had to be in play for the neddylation of these new substrates, and knockdown of Ube1 (which was known to be able to activate NEDD8 in vitro)⁹ showed that it was, indeed, responsible. Exploring further stress signals showed that this increased neddylation response was induced by heat shock and by elevated levels of reactive oxygen species (ROS). Since all of these stress pathways reduce free ubiquitin levels, the authors asked if NAE-independent neddylation could be triggered simply by reducing free ubiquitin levels. The clearly positive results of this study suggested that competition with ubiquitin for Ube1 may normally limit Ube1 activation of NEDD8 and the neddylation of non-cullin substrates (Fig. 1). Open in a separate windowFigure 1. Nedd8 pathway and stress. (A) In unstressed cells, two parallel and non-overlapping pathways are in play. Nedd8 activation is through the action of NAE, while ubiquitin is activated by Ube1. Substrate selectivity of the E2 and E3 results in many proteins being ubiquitinated, but few are Nedd8-modified, notably, the cullins. (B) Low free ubiquitin levels in stress conditions results in Nedd8 being activated by the ubiquitin Ube1 as well as NAE1. This, in turn, results in a large increase in the variety of protein substrates that are NEDD8-modified, in addition to the cullins.In stress conditions then, when free ubiquitin levels fall, Ube1 acts as a sensor of this state and neddylation increases. Why would this be useful? The speculation is that the modification of substrate proteins by NEDD8 may help the cell to cope with stress signals, for example, by promoting cell survival through inhibition of the degradation of very labile pro-survival proteins, such as Mcl-1. After the stress signal abates, the many effective de-ubiquitinating and de-neddylating enzymes can come into play to restore homeostasis. Improved mass spectrometry methods developed in this paper using Lys-C to digest neddylated proteins allow one to distinguish NEDD8 modification from ubiquitination. This helps to further refine our knowledge of this fascinating system, but, meanwhile, protein neddylation may provide a new biomarker for cellular stress. Many critical issues remain to be resolved: are there proteins with ubiquitin/NEDD8 binding domains that specifically recognize the ubiquitin NEDD8 hybrid chains that result from these stress signals? Which E2s and E3s are responsible for stress-induced neddylation? Should Ube1 inhibitors be developed to complement the NAE inhibitor in cancer treatments, or would they prove too toxic? The next few years promise to reveal critical insights into the crosstalk between the different Ubl pathways.