Ubiquitin Ligation without a Ligase (original) (raw)
Related papers
Journal of Biological Chemistry, 2007
It is generally assumed that a specific ubiquitin ligase (E3) links protein substrates to polyubiquitin chains containing a single type of isopeptide linkage, and that chains composed of linkages through Lys 48 , but not through Lys 63 , target proteins for proteasomal degradation. However, when we carried out a systematic analysis of the types of ubiquitin (Ub) chains formed by different purified E3s and Ub-conjugating enzymes (E2s), we found, using Ub mutants and mass spectrometry, that the U-box E3, CHIP, and Ring finger E3s, MuRF1 and Mdm2, with the E2, UbcH5, form a novel type of Ub chain that contains all seven possible linkages, but predominantly Lys 48 , Lys 63 , and Lys 11 linkages. Also, these heterogeneous chains contain forks (bifurcations), where two Ub molecules are linked to the adjacent lysines at Lys 6 ؉ Lys 11 , Lys 27 ؉ Lys 29 , or Lys 29 ؉ Lys 33 on the preceding Ub molecule. However, the HECT domain E3s, E6AP and Nedd4, with the same E2, UbcH5, form homogeneous chains exclusively, either Lys 48 chains (E6AP) or Lys 63 chains (Nedd4). Furthermore, with other families of E2s, CHIP and MuRF1 synthesize homogeneous Ub chains on the substrates. Using the dimeric E2, UbcH13/Uev1a, they attach Lys 63 chains, but with UbcH1 (E2-25K), MuRF1 synthesizes Lys 48 chains on the substrate.
Regulation of E3 ubiquitin ligases by homotypic and heterotypic assembly
F1000Research, 2020
Protein ubiquitylation is essential for the maintenance of cellular homeostasis. E3 ubiquitin ligases are key components of the enzymatic machinery catalyzing the attachment of ubiquitin to substrate proteins. Consequently, enzymatic dysfunction has been associated with medical conditions including cancer, diabetes, and cardiovascular and neurodegenerative disorders. To safeguard substrate selection and ubiquitylation, the activity of E3 ligases is tightly regulated by post-translational modifications including phosphorylation, sumoylation, and ubiquitylation, as well as binding of alternative adaptor molecules and cofactors. Recent structural studies identified homotypic and heterotypic interactions between E3 ligases, adding another layer of control for rapid adaptation to changing environmental and physiological conditions. Here, we discuss the regulation of E3 ligase activity by combinatorial oligomerization and summarize examples of associated ubiquitylation pathways and mechan...
Isolation, Characterization, and Partial Purification of a Novel Ubiquitin-Protein Ligase, E3
Journal of Biological Chemistry, 1996
Degradation of a protein via the ubiquitin system involves two discrete steps, conjugation of ubiquitin to the substrate and degradation of the adduct. Conjugation follows a three-step mechanism. First, ubiquitin is activated by the ubiquitin-activating enzyme, E1. Following activation, one of several E2 enzymes (ubiquitin-carrier proteins or ubiquitin-conjugating enzymes, UBCs) transfers ubiquitin from E1 to the protein substrate that is bound to one of several ubiquitin-protein ligases, E3s. These enzymes catalyze the last step in the process, covalent attachment of ubiquitin to the protein substrate. The binding of the substrate to E3 is specific and implies that E3s play a major role in recognition and selection of proteins for conjugation and subsequent degradation. So far, only a few ligases have been identified, and it is clear that many more have not been discovered yet. Here, we describe a novel ligase that is involved in the conjugation and degradation of non "N-end rule" protein substrates such as actin, troponin T, and MyoD. This substrate specificity suggests that the enzyme may be involved in degradation of muscle proteins. The ligase acts in concert with E2-F1, a previously described non N-end rule UBC. Interestingly, it is also involved in targeting lysozyme, a bona fide N-end rule substrate that is recognized by E3␣ and E2-14 kDa. The novel ligase recognizes lysozyme via a signal(s) that is distinct from the N-terminal residue of the protein. Thus, it appears that certain proteins can be targeted via multiple recognition motifs and distinct pairs of conjugating enzymes. We have purified the ligase ϳ200-fold and demonstrated that it is different from other known E3s, including E3␣/UBR1, E3, and E6-AP. The native enzyme has an apparent molecular mass of ϳ550 kDa and appears to be a homodimer. Because of its unusual size, we designated this novel ligase E3L (large). E3L contains an-SH group that is essential for its activity. Like several recently described E3 enzymes, including E6-AP and the ligase involved in the processing of p105, the NF-B precursor, the novel ligase is found in mammalian tissues but not in wheat germ.
A ubiquitin-like protein unleashes ubiquitin ligases
Cell, 2008
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.
Molecular Cell, 2016
The Doa10 quality control ubiquitin (Ub) ligase labels proteins with uniform lysine 48linked poly-Ub (K48-pUB) chains for proteasomal degradation. Processing of Doa10 substrates requires the activity of two Ub conjugating enzymes. Here we show that the non-canonical conjugating enzyme Ubc6 attaches single Ub molecules not only to lysines but also to hydroxylated amino acids. These Ub moieties serve as primers for subsequent poly-ubiquitylation by Ubc7. We propose that the evolutionary conserved propensity of Ubc6 to mount Ub on diverse amino acids augments the ubiquitylation sites within a substrate and thereby increases the target range of Doa10. Our work provides new insights on how the consecutive activity of two specialized conjugating enzymes facilitates the attachment of poly-Ub to very heterogeneous client molecules. Such stepwise ubiquitylation reactions most likely represent a more general cellular phenomenon that extends the versatility, yet sustains the specificity of the Ub conjugation system.
BioE3 identifies specific substrates of ubiquitin E3 ligases
Nature Communications, 2023
Hundreds of E3 ligases play a critical role in recognizing specific substrates for modification by ubiquitin (Ub). Separating genuine targets of E3s from E3-interactors remains a challenge. We present BioE3, a powerful approach for matching substrates to Ub E3 ligases of interest. Using BirA-E3 ligase fusions and bioUb, site-specific biotinylation of Ub-modified substrates of particular E3s facilitates proteomic identification. We show that BioE3 identifies both known and new targets of two RING-type E3 ligases: RNF4 (DNA damage response, PML bodies), and MIB1 (endocytosis, autophagy, centrosome dynamics). Versatile BioE3 identifies targets of an organellespecific E3 (MARCH5) and a relatively uncharacterized E3 (RNF214). Furthermore, BioE3 works with NEDD4, a HECT-type E3, identifying new targets linked to vesicular trafficking. BioE3 detects altered specificity in response to chemicals, opening avenues for targeted protein degradation, and may be applicable for other Ub-likes (UbLs, e.g., SUMO) and E3 types. BioE3 applications shed light on cellular regulation by the complex UbL network. Protein ubiquitination is conserved in all eukaryotes and plays crucial roles in almost all cellular processes. Ubiquitin (Ub) conjugation is coordinated by a three-step enzymatic cascade, which can be reversed by the action of deubiquitinating enzymes (DUBs). This cycle is conserved among the different ubiquitin-like proteins (UbLs), each using their own set of enzymes, often depicted as E1 (activating), E2 (conjugating), E3 (ligating), and DUBs. Specificity of ubiquitin toward particular targets is achieved as the cycle progresses. In humans, two Ub E1 enzymes, around 40 E2s and about 700 E3 ligases cooperate to selectively target thousands of substrates 1. The question of how substrate specificity is achieved might benefit from a compendium of targets for specific E3 ligases. Ub E3 ligases are subdivided into categories, according to shared domains and modes of action for substrate modification 2. The main family covers more than 600 RING (Really Interesting New Gene) type Ub E3 ligases. The RING domain allows the direct transfer of Ub from the E2 to the target protein by placing them in close proximity 3. To function, some RING E3 ligases (e.g., RNF4; RING Finger protein 4), dimerize through their RING domain 4 , or create multi-subunit complexes, (e.g., Cullin RING Ligases). CRLs can recognize diverse targets with specificity by forming complexes with >300 different substrate receptors 2. In the case of HECT (Homology to E6AP C Terminus) and RBR (RING-Between-RING) E3 ligases, a covalent E3~Ub thioester
A Human Ubiquitin Conjugating Enzyme (E2)-HECT E3 Ligase Structure-function Screen
Molecular & Cellular Proteomics, 2012
Here we describe a systematic structure-function analysis of the human ubiquitin (Ub) E2 conjugating proteins, consisting of the determination of 15 new high-resolution three-dimensional structures of E2 catalytic domains, and autoubiquitylation assays for 26 Ub-loading E2s screened against a panel of nine different HECT (homologous to E6-AP carboxyl terminus) E3 ligase domains. Integration of our structural and biochemical data revealed several E2 surface properties associated with Ub chain building activity; (1) net positive or neutral E2 charge, (2) an "acidic trough" located near the catalytic Cys, surrounded by an extensive basic region, and (3) similarity to the previously described HECT binding signature in UBE2L3 (UbcH7).
Nature protocols, 2016
Ubiquitination is an essential protein modification that influences eukaryotic processes ranging from substrate degradation to nonproteolytic pathway alterations, including DNA repair and endocytosis. Previous attempts to analyze substrates via physical association with their respective ubiquitin ligases have had some success. However, because of the transient nature of enzyme-substrate interactions and rapid protein degradation, detection of substrates remains a challenge. Ligase trapping is an affinity purification approach in which ubiquitin ligases are fused to a polyubiquitin-binding domain, which allows the isolation of ubiquitinated substrates. Immunoprecipitation is first used to enrich for proteins that are bound to the ligase trap. Subsequently, affinity purification is used under denaturing conditions to capture proteins conjugated with hexahistidine-tagged ubiquitin. By using this protocol, ubiquitinated substrates that are specific for a given ligase can be isolated for...