Elucidation of ubiquitin-conjugating enzymes that interact with RBR-type ubiquitin ligases using a liquid-liquid phase separation-based method.

RING-between RING (RBR)-type ubiquitin (Ub) ligases (E3s) such as Parkin receive Ub from Ub-conjugating enzymes (E2s) in response to ligase activation. However, the specific E2s that transfer Ub to each RBR-type ligase are largely unknown because of insufficient methods for monitoring their interaction. To address this problem, we have developed a method that detects intracellular interactions between E2s and activated Parkin. Fluorescent homotetramer Azami-Green fused with E2 and oligomeric Ash (Assembly helper) fused with Parkin form a liquid-liquid phase separation (LLPS) in cells only when E2 and Parkin interact. Using this method, we identified multiple E2s interacting with activated Parkin on damaged mitochondria during mitophagy. Combined with in vitro ubiquitination assays and bioinformatics, these findings revealed an underlying consensus sequence for E2 interactions with activated Parkin. Application of this method to other RBR-type E3s including HOIP, HHARI, and TRIAD1 revealed that HOIP forms an LLPS with its substrate NEMO in response to a proinflammatory cytokine and that HHARI and TRIAD1 form a cytosolic LLPS independent of Ub-like protein NEDD8. Since an E2-E3 interaction is a prerequisite for RBR-type E3 activation and subsequent substrate ubiquitination, the method we have established here can be an in-cell tool to elucidate the potentially novel mechanisms involved in RBR-type E3s.

Structural basis for UFM1 transfer from UBA5 to UFC1.

Ufmylation is a post-translational modification essential for regulating key cellular processes. A three-enzyme cascade involving E1, E2 and E3 is required for UFM1 attachment to target proteins. How UBA5 (E1) and UFC1 (E2) cooperatively activate and transfer UFM1 is still unclear. Here, we present the crystal structure of UFC1 bound to the C-terminus of UBA5, revealing how UBA5 interacts with UFC1 via a short linear sequence, not observed in other E1-E2 complexes. We find that UBA5 has a region outside the adenylation domain that is dispensable for UFC1 binding but critical for UFM1 transfer. This region moves next to UFC1's active site Cys and compensates for a missing loop in UFC1, which exists in other E2s and is needed for the transfer. Overall, our findings advance the understanding of UFM1's conjugation machinery and may serve as a basis for the development of ufmylation inhibitors.

Crystal structures of an E1-E2-ubiquitin thioester mimetic reveal molecular mechanisms of transthioesterification.

E1 enzymes function as gatekeepers of ubiquitin (Ub) signaling by catalyzing activation and transfer of Ub to tens of cognate E2 conjugating enzymes in a process called E1-E2 transthioesterification. The molecular mechanisms of transthioesterification and the overall architecture of the E1-E2-Ub complex during catalysis are unknown. Here, we determine the structure of a covalently trapped E1-E2-ubiquitin thioester mimetic. Two distinct architectures of the complex are observed, one in which the Ub thioester (Ub(t)) contacts E1 in an open conformation and another in which Ub(t) instead contacts E2 in a drastically different, closed conformation. Altogether our structural and biochemical data suggest that these two conformational states represent snapshots of the E1-E2-Ub complex pre- and post-thioester transfer, and are consistent with a model in which catalysis is enhanced by a Ub(t)-mediated affinity switch that drives the reaction forward by promoting productive complex formation or product release depending on the conformational state.

Legionella effector MavC targets the Ube2N~Ub conjugate for noncanonical ubiquitination.

The bacterial effector MavC modulates the host immune response by blocking Ube2N activity employing an E1-independent ubiquitin ligation, catalyzing formation of a γ-glutamyl-ε-Lys (Gln40Ub-Lys92Ube2N) isopeptide crosslink using a transglutaminase mechanism. Here we provide biochemical evidence in support of MavC targeting the activated, thioester-linked Ube2N~ubiquitin conjugate, catalyzing an intramolecular transglutamination reaction, covalently crosslinking the Ube2N and Ub subunits effectively inactivating the E2~Ub conjugate. Ubiquitin exhibits weak binding to MavC alone, but shows an increase in affinity when tethered to Ube2N in a disulfide-linked substrate that mimics the charged E2~Ub conjugate. Crystal structures of MavC in complex with the substrate mimic and crosslinked product provide insights into the reaction mechanism and underlying protein dynamics that favor transamidation over deamidation, while revealing a crucial role for the structurally unique insertion domain in substrate recognition. This work provides a structural basis of ubiquitination by transglutamination and identifies this enzyme's true physiological substrate.

Enzymatic Assembly of Ubiquitin Chains.

The availability of different polyubiquitin chains of specific linkage types has changed the appreciation of the specificity in the ubiquitin (Ub) system. Numerous E2 Ub-conjugating enzymes and E3 Ub ligases, Ub-binding domains (UBDs), and deubiquitinases (DUBs) are now known to assemble, bind, or hydrolyze individual linkage types, respectively. Biochemical and structural studies of these processes require milligram quantities of pure polyUb. Here we describe protocols that allow the enzymatic synthesis and purification of six of the eight homotypic polyUb chains through the use of chain-specific Ub ligases and DUBs.

In Vitro Reconstitution of Atg8 Conjugation and Deconjugation.

Macroautophagy, hereafter autophagy, is a major degradation pathway in eukaryotic systems that allows the removal of large intracellular structures such as entire organelles or protein aggregates, thus contributing to the homeostasis of cells and tissues. Autophagy entails the de novo formation of an organelle termed autophagosome, where a cup-shaped structure called isolation membrane nucleates in proximity of a cytoplasmic cargo material. Upon elongation and closure of isolation membranes, the mature autophagosome delivers the sequestered cargo into the lysosomal system for degradation. Among the factors for autophagosome formation are the autophagy-related (Atg) proteins belonging to the Atg8 conjugation system. In this system, the ubiquitin-like Atg8 protein is conjugated to the membrane lipid phosphatidylethanolamine present in autophagosomal membranes. Atg8 can also be removed from membranes by Atg4-mediated deconjugation. Here, we describe in vitro systems that recapitulate the enzymatic reactions occurring in vivo by presenting expression and purification strategies for all the components of the Saccharomyces cerevisiae Atg8 conjugation system. We also present protocols for in vitro Atg8 conjugation and deconjugation reactions employing small and giant unilamellar vesicles.

Measuring APC/C-Dependent Ubiquitylation In Vitro.

The anaphase-promoting complex/cyclosome (APC/C) is a 1.2 MDa ubiquitin ligase complex with important functions in both proliferating and post-mitotic differentiated cells. In proliferating cells, APC/C controls cell cycle progression by targeting inhibitors of chromosome segregation and mitotic exit for degradation by the 26S proteasome. To understand how APC/C recruits and ubiquitylates its substrate proteins and how these processes are controlled, it is essential to analyze APC/C activity in vitro. In the past, such experiments have been limited by the fact that large quantities of purified APC/C were difficult to obtain and that mutated versions of the APC/C could not be easily generated. In this chapter we review recent advances in generating and purifying recombinant forms of the human APC/C and its co-activators, using methods that are scalable and compatible with mutagenesis. We also describe a method that allows the quantitative analysis of APC/C activity using fluorescently labeled substrate proteins.

The expression, purification and crystallization of a ubiquitin-conjugating enzyme E2 from Agrocybe aegerita underscore the impact of His-tag location on recombinant protein properties.

Ubiquitination is a post-translational modification involved in myriad cell regulation and disease pathways. The ubiquitin-conjugating (E2) enzyme is the central player in the ubiquitin-transfer pathway. Although a large array of E2 structures are available, not all E2 families have known structures and three-dimensional structures from fungal organisms other than yeast are lacking. Here, the expression, purification, crystallization and preliminary X-ray analysis of UbcA1, a novel ubiquitin-conjugating enzyme identified from the medicinal mushroom Agrocybe aegerita, which shows antitumour properties, are reported. As a potential anticancer drug candidate, the protein was expressed in either a C-terminally or an N-terminally His-tagged form. In the process of purification and crystallization, the location of the His tag seemed to play a crucial role in protein stability. In contrast to unsuccessful crystallization trials for the protein with a C-terminal tag, a crystal of N-terminally His-tagged UbcA1 grown under optimal conditions diffracted X-rays to 1.7 Å resolution. The crystal belonged to space group C2, with unit-cell parameters a = 84.93, b = 34.76, c = 128.10 Å, ß = 118.57°. An X-ray data set was collected that was suitable for structure determination, showing satisfactory completeness, and R factors. All of these results underscore the non-negligible impact of His-tag location on protein behaviour during the process of purification and crystallization.

Nucleosomal H2B ubiquitylation with purified factors.

Diverse histone modifications play important roles in transcriptional regulation throughout eukaryotes, and recent studies have implicated histone H2B ubiquitylation in active transcription. The necessity of at least three enzymes (E1-E3), as well as ongoing transcription events, for efficient H2B ubiquitylation complicates mechanistic studies of H2B ubiquitylation relative to other histone modifications. Here we describe experimental protocols for preparation of human H2B ubiquitylation factors, ubiquitylation substrates and transcription factors, as well as the use of these factors to establish H2B ubiquitylation mechanisms during transcription. The methods include reliable protein interaction and E3 ubiquitylation assays that can be widely applied to confirm cognate E2-E3 pairs in other protein ubiquitylation systems, optimized in vitro ubiquitylation assays for various histone substrates, and a transcription-coupled H2B ubiquitylation assay in a highly purified transcription system. These comprehensive analyses have revealed (i) that RAD6 serves as the cognate E2 for the BRE1 complex in human cells, as previously established in yeast, (ii) that RAD6, through direct interaction with the BRE1 complex, ubiquitylates chromatinized H2B at lysine 120 and (iii) that PAF1 complex-mediated transcription is required for efficient H2B ubiquitylation. This experimental system permits detailed mechanistic analyses of H2B ubiquitylation during transcription by providing information concerning both precise enzyme functions and physical interactions between the transcription and histone modification machineries.

Cell-free-based protein microarray technology using agarose/DNA microplate.

Protein microarray is considered to be one of the key analytical tools for high-throughput protein function analysis. We found that Arabidopsis HY5 protein functions as a novel DNA-binding tag (DBtag), and DBtagged proteins are immobilized and purified on a newly designed agarose/DNA microplate. In this chapter, we demonstrate a protocol for making the DBtag-based protein microarray and will provide protocols for two applications using the microarray: (1) detection of autophosphorylation activity of DBtagged human protein kinases and inhibition of their activity by staurosporine, and (2) detection of a protein-protein interaction between the DBtagged UBE2N and UBE2v1.

Insights into the conformational dynamics of the E3 ubiquitin ligase CHIP in complex with chaperones and E2 enzymes.

The dimeric E3 ubiquitin ligase CHIP binds with its tetratricopeptide repeat (TPR) domain the C-terminus of molecular chaperones Hsp70 and Hsp90 and with its U-box region E2 ubiquitin-conjugating enzymes. By ubiquitinating chaperone-bound polypeptides, CHIP thus links the chaperone machinery to the proteasomal degradation pathway. The molecular mechanism of how CHIP discriminates between folding and destruction of chaperone substrates is not yet understood. Two recently published crystal structures of mouse and zebrafish CHIP truncation constructs differ substantially, showing either an asymmetric assembly or a symmetric assembly with a highly ordered middle domain. To characterize the conformational properties of the intact full-length protein in solution, we performed amide hydrogen exchange mass spectrometry (HX-MS) with human CHIP. In addition, we monitored conformational changes in CHIP upon binding of Hsp70, Hsp90, and their respective C-terminal EEVD peptides, and in complex with the different E2 ubiquitin-conjugating enzymes UbcH5a and Ubc13. Solution HX-MS data suggest a symmetric dimer assembly with highly flexible parts in the middle domain contrasting both the asymmetric and the symmetric crystal structure. CHIP exhibited an extraordinary flexibility with a largely unprotected N-terminal TPR domain. Formation of a complex with intact Hsp70 and Hsp90 or their respective C-terminal octapeptides induced folding of the TPR domain to a defined, highly stabilized structure with protected amide hydrogens. Interaction of CHIP with two different E2 ubiquitin-conjugating enzymes, UbcH5a and Ubc13, had distinct effects on the conformational dynamics of CHIP, suggesting different roles of the CHIP-E2 interaction in the ubiquitination of substrates and interaction with chaperones.

Purification and characterization of a ubiquitin-like system for autophagosome formation.

Autophagy refers to the bulk degradation of cellular proteins and organelles through an autophagosome and plays a pivotal role in the development, cellular differentiation, aging, and elimination of aberrant structures. A failure of autophagy has been implicated in a growing list of mammalian disease states, including cancer and cardiomyopathy. Two ubiquitin-like systems are highly involved in autophagy, especially in the formation of autophagosomes. Here, we purified and characterized Atg7 (an E1-like enzyme), and Atg3 and Atg10 (E2-like enzymes) in order to gain an insight into the role played by ubiquitin-like systems in the formation of autophagosomes. Interestingly, we observed that Atg7 forms a homodimer to construct an active conformation, unlike other E1-like enzymes. Although Atg3 was detected as a monomer under physiological conditions, Atg10 existed in an oligomeric form, indicating that the mechanism by which Atg10 functions may differ from that of Atg3.

Identification of SUMO-conjugated proteins and their SUMO attachment sites using proteomic mass spectrometry.

The covalent modification of cellular factors by the small ubiquitin-like modifier (SUMO) has emerged as a key regulatory pathway for many biological processes. One recent advance in the field of SUMO modification that has provided important insights into SUMO-mediated regulatory networks is the ability to use proteomic mass spectrometry to identify the substrates of SUMO modification as well as their sites of conjugation (1-10). In this chapter, we describe a global strategy for affinity purifying and identifying a broad spectrum of SUMO-conjugated proteins and a focused approach for purifying a selected SUMO target and mapping its SUMO attachment site(s). Although both methods were initially developed for use in S. cerevisiae, they can be readily adapted to study the SUMO pathway in higher eukaryotes.

Performing in vitro sumoylation reactions using recombinant enzymes.

Sumoylation of proteins in vitro has evolved as an indispensable tool for the functional analysis of this post-translational modification. In this article we present detailed protocols for bacterial production of mammalian proteins necessary to perform in vitro sumoylation reactions, namely the E1 activating enzyme Aos1/Uba2 (SAE1/SAE2), the E2 conjugating enzyme Ubc9, SUMO-1 (identical protocols can be used for SUMO-2/3), and the catalytic domain of the E3 ligase RanBP2/Nup358. Two alternative procedures are described for the E1 enzyme, one depending on co-expression of His-Aos1 and untagged Uba2, and a second protocol for separate expression of His-Aos1 and Uba2-His and subsequent reconstitution of the active dimer. Two example conditions for in vitro sumoylation of RanGAP1 and Sp100 in the absence or presence of the SUMO E3 ligase RanBP2, respectively, are provided. Both protocols can be adapted easily to test in vitro conjugation of other target proteins and/or E3 ligases.

Preparation of sumoylated substrates for biochemical analysis.

Covalent modification of proteins with SUMO (small ubiquitin related modifier) affects many cellular processes like transcription, nuclear transport, DNA repair and cell cycle progression. Although hundreds of SUMO targets have been identified, for several of them the function remains obscure. In the majority of cases sumoylation is investigated via "loss of modification" analysis by mutating the relevant target lysine. However, in other cases this approach is not successful since mapping of the modification site is problematic or mutation does not cause an obvious phenotype. These latter cases ask for different approaches to investigate the target modification. One possibility is to choose the opposite approach, a "gain in modification" analysis by producing both SUMO modified and unmodified protein in vitro and comparing them in functional assays. Here, we describe the purification of the ubiquitin conjugating enzyme E2-25K, its in vitro sumoylation with recombinant enzymes and the subsequent separation and purification of the modified and the unmodified forms.

Identification of human cytomegalovirus UL84 virus- and cell-encoded binding partners by using proteomics analysis.

Human cytomegalovirus (HCMV) UL84 is a phosphoprotein that shuttles from the nucleus to the cytoplasm and is required for oriLyt-dependent DNA replication and viral growth. UL84 was previously shown to interact with IE2 (IE86) in infected cells, and this interaction down-regulates IE2-mediated transcriptional activation in transient assays. UL84 and IE2 were also shown to cooperatively activate a promoter within HCMV oriLyt. UL84 alone can interact with an RNA stem-loop within oriLyt and is bound to this structure within the virion. In an effort to investigate the binding partners for UL84 in infected cells, we pulled down UL84 from protein lysates prepared from HCMV-infected human fibroblasts by using a UL84-specific antibody and resolved the immunoprecipitated protein complexes by two-dimensional gel electrophoresis. We subsequently identified individual proteins by matrix-assisted laser desorption ionization-tandem time of flight analysis. This analysis revealed that UL84 interacts with viral proteins UL44, pp65, and IE2. In addition, a number of cell-encoded proteins were identified, including ubiquitin-conjugating enzyme E2, casein kinase II (CKII), and the multifunctional protein p32. We also confirmed the interaction between UL84 and IE2 as well as the interaction of UL84 with importin alpha. UL44, pp65, and CKII interactions were confirmed to occur in infected and cotransfected cells by coimmunoprecipitation assays followed by Western blotting. Ubiquitination of UL84 occurred in the presence and absence of the proteasome activity inhibitor MG132 in infected cells. The identification of UL84 binding partners is a significant step toward the understanding of the function of this significant replication protein.

Expression, purification, and structural analysis of (HIS)UBE2G2 (human ubiquitin-conjugating enzyme).

The ubiquitin system represents a selective mechanism for intracellular proteolysis in eukaryotic cells that involves the sequential activity of three enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3). The identification of these proteins and their cellular targets, as well as structural data, are essential to understanding how this system operates in the eukaryotic cell. In the present study, the open reading frame of the human ubiquitin-conjugating enzyme UBE2G2 was isolated from a human brain cDNA panel, cloned into pET28a vector and expressed in Escherichia coli. The His-tagged protein was then purified through nickel-affinity chromatography and subjected to structural and functional studies using circular dichroism (CD) and an in vitro ubiquitin-binding assay, respectively. Our results showed that the production of the HISUBE2G2 protein in bacteria, carried out with 0.1 mM of IPTG at 30 degrees C, was successfully achieved, rendering high concentrations of soluble, pure and stable enzyme after a single purification step. The recombinant protein was able to bind ubiquitin molecules when exposed to a HeLa cell extract during the ubiquitin assay. Moreover, the fact that HISUBE2G2 was expressed in its active form is supported by the typical alpha/beta secondary structure specific to other class I E2 enzymes displayed during the CD assay.

High-level expression and purification of recombinant E1 enzyme.

The ubiquitin E1 enzyme is an ATP-dependent enzyme that activates ubiquitin for use in all ubiquitin conjugation pathways. This chapter describes the expression and purification of human E1 enzyme for use in in vitro ubiquitination reactions.

Expression, purification, and characterization of the E1 for human NEDD8, the heterodimeric APPBP1-UBA3 complex.

The NEDD8 pathway is important for numerous biological processes, including cell proliferation, signal transduction, and development. The heterodimeric activating enzyme of NEDD8, APPBP1-UBA3, plays an essential role in NEDD8 conjugation. Not surprisingly, mutations in APPBP1 and UBA3 lead to defects in many cellular functions. The APPBP1-UBA3 complex initiates NEDD8 conjugation by first catalyzing adenylation of the C terminus of NEDD8 and ultimately catalyzing transfer of NEDD8 to the downstream enzyme in the pathway, Ubc12. This chapter describes methods for expressing and purifying APPBP1-UBA3 for in vitro studies of NEDD8 conjugation.

Purification and properties of the ubiquitin-conjugating enzymes Cdc34 and Ubc13.Mms2.

A prerequisite for structure/function studies on the ubiquitin-conjugating enzymes (Ubc) Cdc34 and Ubc13.Mms2 has been the ability to express and purify recombinant derivatives of each. This chapter describes the methods used in the expression and purification of these proteins from Escherichia coli, including variations of these protocols used to generate (35)S, (15)N, (13)C/(15)N, and seleno-L-methionine derivatives. Assays used to measure the Ub thiolester and Ub conjugation activities of these Ubcs are also described.