Resolving the Complexity of Ubiquitin Networks

Abstract

Ubiquitination regulates nearly all cellular processes by coordinated activity of ubiquitin writers (E1, E2, and E3 enzymes), erasers (deubiquitinating enzymes) and readers (proteins that recognize ubiquitinated proteins by their ubiquitin-binding domains). By differentially modifying cellular proteome and by recognizing these ubiquitin modifications, ubiquitination machinery tightly regulates execution of specific cellular events in space and time. Dynamic and complex ubiquitin architecture, ranging from monoubiquitination, multiple monoubiquitination, eight different modes of homotypic and numerous types of heterogeneous polyubiquitin linkages, enables highly dynamic and complex regulation of cellular processes. We discuss available tools and approaches to study ubiquitin networks, including methods for the identification and quantification of ubiquitin-modified substrates, as well as approaches to quantify the length, abundance, linkage type and architecture of different ubiquitin chains. Furthermore, we also summarize the available approaches for the discovery of novel ubiquitin readers and ubiquitin-binding domains, as well as approaches to monitor and visualize activity of ubiquitin conjugation and deconjugation machineries. We also discuss benefits, drawbacks and limitations of available techniques, as well as what is still needed for detailed spatiotemporal dissection of cellular ubiquitination networks.

Keywords: E3 ligase; affinity purification; deubiquitinating enzyme; mass spectrometry; ubiquitin; ubiquitin receptor.

FIGURE 1

Multicomponent enzymatic machineries assemble and disassemble ubiquitin modification. (A) Ub belongs to the β-grasp fold (β-GF) family (Vijay-Kumar et al., 1987), in which β-GF is formed by five-stranded β-sheet, a short 3₁₀ helix and a 3.5-turn α-helix. The C-terminal Ub tail is essential for Ub conjugation and hence, for all the Ub functions. Functionally relevant Ub residues are depicted in different colors. The figure was generated from the PDB entry 1UBQ by PyMOL v1.7.6.0 software. (B) Coordinated activity of Ub-activating (E1), Ub-conjugating (E2), and Ub-ligating enzyme (E3) is required for Ub attachment to substrate protein. The action modes of the three main groups of E3 ligases (RING, HECT and RBR) are also depicted. In mammals, Ub is encoded by four different genes: UBA52 and RPS27A genes encode a single Ub molecule fused to the ribosomal subunits L40 and S27a, respectively (depicted as RP-Ub), UBB and UBC genes encode 2 different polyUb precursor proteins (exemplified here as Ub₆ fusion). More than 100 cellular DUBs process newly translated Ub-containing polypeptides, remove Ub from modified substrates and disassemble unanchored Ub chains.

FIGURE 2

Cellular ubiquitin modification comes in different formats. Single Ub moieties can modify proteins at one (monoubiquitination) or several (multiple monoubiquitination) Lys residues. Ub can form eight distinctive homotypic linkages, either through Met1 (linear Ub chain) or 7 internal Lys residues (Lys6-, Lys11-, Lys27-, Lys29-, Lys33-, Lys48-, and Lys63-linked Ub chains). Additional complexity is achieved through the formation of heterotypic Ub chains, which contain multiple Ub linkages and adopt mixed or branched topology. Furthermore, heterologous polymers contain additional UBLs, such as SUMO or NEDD8, within Ub chains. Ub molecules undergo various PTMs, including phosphorylation and acetylation, which regulate their binding properties and abilities to generate Ub chains.

FIGURE 3

Ubiquitin-binding domains come in different shapes and forms. (A) Ub receptors contain single or multiple (identical or different) motifs or domains that non-covalently bind Ub or Ub chains. (B) UBDs differ in shape and Ub/Ub chain specificity. Several UBDs in complex with Ub or Ub chains are depicted: PLIC1 UBA (Ub-associated, PDB code: 2JY6), Hrs DUIM (double-sided Ub-interacting motif, PDB code: 2D3G), ZnF UBP/BUZ (zinc-finger Ub-binding, PDB code: 2G45), ABIN-1 UBAN (Ub-binding domain in ABINs and NEMO, PDB code: 5M6N) and RAP80 tandem UIMs (Ub-interacting motif, PDB code: 3A1Q). Ub and UBD structures are depicted in gray and blue, respectively. The figure was generated from PDB entries by PyMOL v1.7.6.0 software. (C) A subset of UBDs recognizes specific types of Ub modifications, such as specific Ub linkages. (D) Cooperative recognition of Ub modifications by two or more UBDs is one of many approaches to increase avidity of Ub:UBD interaction. (E) Monoubiquitinated proteins can bind their intrinsic UBDs to regulate their function. The interaction between Ub modification and UBD (on the same protein) provides an efficient switch between active and inactive Ub receptor conformation. (F) Ub:UBD interactions often lead to formation of large protein complexes. Most of the Ub:UBD interactions are relatively weak. Multiple UBDs, due to avidity, contribute to the strengthened interaction between Ub and UBDs. Such multiple UBDs and Ub modifications enable formation of highly dynamic protein complexes. (G) A subset of UBDs could potentially recognize specific PTM-modified Ub modifications.

FIGURE 4

Tools to study ubiquitin system. Overview of different approaches to study features of Ub signaling: E3 ligase and DUB enzymes (enzyme abundance, activity, cellular localization), Ub chains (type, architecture, length, quantity, cellular localization, PTMs), ubiquitinated substrates (identity, modification site, type of modification) and Ub receptors (identity, Ub linkage preference).

FIGURE 5

Various approaches to study ubiquitination targets. (A) Various N-terminally tagged Ub variants can be exogenously added to cells to enable affinity purifications of ubiquitinated substrates upon denaturing lysis. Short and relatively linear tags (such as 6xHIS, STREP, HA and FLAG), combined with their respective affinity resins (Ni-NTA, strep-tactin, HA agarose and FLAG agarose) are often used in such experiments. Besides N-terminally tagged wild-type Ub, additional Ub variants are often used, such as specific Lys mutants (single or multiple Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 and Lys63 residues mutated to either Arg or Ala). Additionally, residues relevant for Ub binding properties, such as Ile44 and Ile36, can also be mutated to either Arg or Ala. (B) Dual BirA system contains synthetic hexaUb sequence fused C-terminally to E. coli BirA gene (Ub₆-BirA). Each Ub in the construct contains 16 amino acids sequence at the N terminus that can be biotinylated by BirA. Once expressed in cells (or organisms), linear hexaUb is processed by cellular DUBs and undergoes biotinylation by BirA. When used by cellular ubiquitination machinery, biotin-containing Ub conjugates can be efficiently affinity purified with neutravidin resins and subsequently analyzed by Western blot and mass spectrometry. Due to the N-terminal tagging of Ub, such approach cannot be used to enrich linear ubiquitination targets. (C) DUB-resistant Ub variant Leu73Pro increases the half-life and stability of the formed Ub linkages and facilitates their subsequent identification. (D) Two MS-coupled approaches rely on the use of internally tagged Ub variants. Unlike N-terminally tagged Ub, INT-Ub and INT-Ub.7KR variants enable affinity purification of ubiquitinated proteins and Met1-Ub-modified substrates, respectively, due to the existence of internal affinity purification STREP-tag between Ub residues Lys48 and Lys63 that keeps Ub Met1 free to interact with Gly76 of another Ub molecule. (E) INT-Ub approach is based on inducible expression of INT-Ub variants in SILAC-treated cells, followed by denaturing lysis, strep-tactin pull-down and subsequent MS analysis. The presence of the internal tag does not affect the overall behavior of Ub. Similar to that, 6xHIS insertion near C-terminus of Ub in StUbEx PLUS approach enables enrichment of HIS-Ub-modified substrates under denaturing conditions. The latter approach can be combined with Ub remnant profiling to identify Ub-modified substrate sites.

FIGURE 6

Methods for mapping ubiquitination sites in proteins. (A) Ub remnant profiling is based on trypsin digestion of the proteome (cells are previously lysed in urea-containing buffer) combined with immunoprecipitation with monoclonal antibody raised against Lys-ε-Gly-Gly motif that remains on ubiquitinated substrate after trypsin cleavage. Samples are further processed and analyzed by LC-MS/MS. Such approach does not distinguish between modifications by Ub and other UBLs (such as NEDD8 and ISG15), and cannot be applied for Met1- and N-terminally Ub-modified proteins. (B) UbiSite antibody recognizes the last 13 amino acids of Ub that remain attached to ubiquitinated proteins upon LysC cleavage. Enriched ubiquitinated proteins are further analyzed by MS. Even though UbiSite approach distinguishes between modifications by Ub and other UBLs, it cannot be used for studying linear ubiquitination, as it does not recognize the signature peptide of linear ubiquitination after tryptic cleavage: Gly-Gly-Met-Gln-Ile-Phe-Val-Lys. (C) Ub-COFRADIC approach distinguishes between free (α or ε) and modified primary amines to enable identification of ubiquitinated Lys residues. Initial acetylation by NHS-acetate is only possible on free amines, leaving ubiquitinated Lys residues non-acetylated. Subsequent addition of USP2cc removes all the Ub moieties from Lys residues and enables the attachment of Gly linked to a hydrophobic tert-butyloxycarbonyl (Gly-BOC tag) to previously non-acetylated Lys residues. Trypsin then cleaves C-terminally of Arg residues (but not C-terminally of acetylated Lys). Peptides collected after the first reversed phase (RP)-HPLC run are treated with TFA to remove BOC groups, followed by additional RP-HPLC and MS. In enzyme setting during MS data analysis, ArgC (and not trypsin) should be selected, as cleavage after Lys residues is blocked.

FIGURE 7

Tandems of ubiquitin entities can be utilized in various ubiquitin tools. (A) Tandems of Ub chain-specific or promiscuous UBDs, additionally equipped with affinity tags (such as FLAG-, HA-, GST-, or 6xHIS) and bound to appropriate resins, can be used for affinity purification of ubiquitinated proteins. The use of tandem UBDs increases affinity toward Ub due to avidity, as well as protects ubiquitinated proteins from endogenous DUBs during purification steps. Purified ubiquitinated proteins can be further analyzed by either Western blotting or MS. (B) Ub chain enrichment middle-down MS (UbiChEM-MS) approach is based on the enrichment of the specific Ub linkages by linkage-specific UBDs or antibodies, combined with minimal trypsinolysis of Ub that induces a single Ub cleavage after Arg74 and leaves the rest of the Ub molecule intact. By using that approach, Ub molecules within chain, as well as capping and branched Ub conjugates can be detected and quantified by MS. (C) Ub-ProT (Ub chain protection from trypsinization) method determines the length of Ub chains bound to target proteins. Trypsin-resistant (TR)-TUBE (i.e., PLIC1 UBA domain lacking Arg residues) is used for the enrichment of ubiquitinated proteins that can be analyzed by Western blotting. Furthermore, since TR-TUBE-protected sample is resistant to trypsin digestion, it can be applied for the determination of the length of Ub chains by quantitative MS.

FIGURE 8

Quantification of ubiquitination in vivo. (A) Synthetic peptide absolute quantification (Ub-AQUA) MS approach is based on synthetic peptides as quantification standards for both mono- and polyubiquitination. (B) Ub-PSAQ approach is based on the use of stable isotope-labeled free Ub and Ub conjugates as protein standards. They are added to lysates and captured with UBD BUZ that is selective for free Ub and UBD PLIC2 UBA (or similar) that recognizes Ub chains. Half of the sample is treated with USP2cc and total free Ub captured by BUZ affinity reagent. Sample is quantified by MS, relative to the peptide standard.

FIGURE 9

Different approaches to identify E3 ligase substrates. (A) Ligase trapping approach “stabilizes” E3 ligase:substrate non-covalent interactions by UBDs (such as UBA domain) fused to E3 ligase substrate-interacting domains (such as F-box of the multi-protein E3 ligase complex SCF). When such ligase traps and 6xHIS-tagged Ub are overexpressed in cells, the UBA interacts with the nascent Ub chain on endogenous SCF substrates, thereby delaying their release. Cells are then lysed and subjected to an anti-FLAG coimmunoprecipitation under native conditions, to isolate ligase trap complexes (FLAG tag is inserted between F-box and UBA). FLAG eluates are then used in denaturing Ni-NTA agarose pull-down to exclusively enrich ubiquitinated substrates (and to remove any non-covalently interacting proteins). (B) UBAITs, similar to ligase traps, enable identification of E3 ligase substrates (for both HECT and RING E3 ligases), as well as their adaptors and regulators. Unlike ligase traps, UBAITs are fusions of N-terminal affinity-tagged E3 and C-terminal Ub molecule. With the help of cellular E1 and E2, UBAIT E3 component transfers UBAIT Ub component (by forming amide bond) to proteins that interact with the E3, such as E3 ligase substrates. Formed complex is easily affinity purified and analyzed by mass spectrometry. For HECT E3s, both E3 and E2 thioester-linked interacting proteins can be captured by UBAITs. (C) NEDDylator is a catalytic tagging tool, in which Ubc12, an E2 enzyme for NEDD8, is fused to an E3 ligase substrate-binding domain, allowing for the transfer of NEDD8 to the E3 substrate, and MS-based identification of E3 ligase-target pairs.

FIGURE 10

Approaches to identify ubiquitin receptors. (A) A very common type of Y2H approach is based on the use of Ub sequence (lacking C-terminal Gly-Gly motif) as N-terminal fusion with the GAL4 binding domain (BD). The cDNA library containing putative UBDs consists of cDNAs cloned under the control of the lacZ promoter downstream of the DNA sequence encoding the activating domain (AD) of the yeast GAL4 transcription factor. Protein interaction induces the close proximity of GAL4AD and GAL4BD, forming the active transcription factor, which binds to the GAL1 upstream activating sequence (UAS) and activates the transcription of several GAL4-responsive genes, which are used as reporters. For example, yeast strain S. cerevisiae YTHGold (Clontech) enables very stringent quadruple selection, since it contains 4 reporters. In that way background growth and detection of false positive interacting proteins is significantly decreased, simplifying further evaluation steps. (B) UbIA-MS method relies on the use of 8 chemically synthesized non-hydrolyzable biotinylated diUbs that can be used for in vitro affinity purification of Ub interactors. These diUb linkages mimic native diUb, and have advantage of not being cleaved by cellular DUBs, which prevents the loss of captured material and decrease in Ub chain specificity. Upon purification, samples are digested on beads with trypsin, followed by liquid chromatography (LC)-MS/MS analysis. (C) Ub-PT is a synthetic Ub variant that contains a photo-activatable crosslinking Leu mimic photoleucine (pLeu) at positions 8 or 73 in Ub molecule. Importantly, these modifications do not affect Ub functionality, including its ability to bind UBDs. Enzymatic polymerization of Ub-PT into Ub chains of defined lengths and linkage types allows the use of Ub-PT as UV-activatable crosslinking reagent (phototrap) for irreversibly capturing Ub receptors. Furthermore, the existence of 6xHIS tag in Ub-PT-containing reagents allows stringent isolation of Ub interactors, without co-purification of their binding proteins.