Chemical Tools for Probing the Ub/Ubl Conjugation Cascades

Conjugation of ubiquitin (Ub) and structurally related ubiquitin‐like proteins (Ubls), essential for many cellular processes, employs multi‐step reactions orchestrated by specific E1, E2 and E3 enzymes. The E1 enzyme activates the Ub/Ubl C‐terminus in an ATP‐dependent process that results in the formation of a thioester linkage with the E1 active site cysteine. The thioester‐activated Ub/Ubl is transferred to the active site of an E2 enzyme which then interacts with an E3 enzyme to promote conjugation to the target substrate. The E1‐E2‐E3 enzymatic cascades utilize labile intermediates, extensive conformational changes, and vast combinatorial diversity of short‐lived protein‐protein complexes to conjugate Ub/Ubl to various substrates in a regulated manner. In this review, we discuss various chemical tools and methods used to study the consecutive steps of Ub/Ubl activation and conjugation, which are often too elusive for direct studies. We focus on methods developed to probe enzymatic activities and capture and characterize stable mimics of the transient intermediates and transition states, thereby providing insights into fundamental mechanisms in the Ub/Ubl conjugation pathways.

• Publication year

  2024

• Venue

  ChemBioChem

• Publication date

  2024-09-23

• Fields of study

  Medicine, Chemistry

• Identifiers
  DOI 10.1002/cbic.202400659PMID 39313481PMCID 11727022
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Chemical Tools for Probing the Ub/Ubl Conjugation Cascades

  2024cited by this paper
• Capturing the catalytic intermediates of parkin ubiquitination

  2024cited by this paper
• Structural basis for transthiolation intermediates in the ubiquitin pathway

  2024cited by this paper
• Design and Semisynthesis of Biselectrophile-Functionalized Ubiquitin Probes To Investigate Transthioesterification Reactions

  2024cited by this paper
• Insights into the ISG15 transfer cascade by the UBE1L activating enzyme

  2023cited by this paper
• Structural snapshots along K48-linked ubiquitin chain formation by the HECT E3 UBR5

  2023cited by this paper
• Cryo-EM structures of Uba7 reveal the molecular basis for ISG15 activation and E1-E2 thioester transfer

  2023cited by this paper
• Structural insights into the regulation of the human E2∼SUMO conjugate through analysis of its stable mimetic

  2023cited by this paper
• Biotin‐Based Strategies to Explore the World of Ubiquitin and Ubiquitin‐Like Modifiers

  2023cited by this paper
• The unifying catalytic mechanism of the RING-between-RING E3 ubiquitin ligase family

  2023cited by this paper
• Simultaneous capture of ISG15 conjugating and deconjugating enzymes using a semi-synthetic ISG15-Dha probe

  2023cited by this paper
• Bivalent binding of p14ARF to MDM2 RING and acidic domains inhibits E3 ligase function

  2022cited by this paper
• MUL1‐RING recruits the substrate, p53‐TAD as a complex with UBE2D2–UB conjugate

  2022cited by this paper
• Ubiquitin and a charged loop regulate the ubiquitin E3 ligase activity of Ark2C

  2022cited by this paper
• Divergent self-association properties of paralogous proteins TRIM2 and TRIM3 regulate their E3 ligase activity

  2022cited by this paper
• Cullin-independent recognition of HHARI substrates by a dynamic RBR catalytic domain.

  2022cited by this paper
• A bifunctional molecule-assisted synthesis of mimics for use in probing the ubiquitination system

  2022cited by this paper
• Production and characterisation of modularly deuterated UBE2D1–Ub conjugate by small angle neutron and X-ray scattering

  2022cited by this paper
• An expanded lexicon for the ubiquitin code

  2022cited by this paper
• A non‐canonical scaffold‐type E3 ligase complex mediates protein UFMylation

  2022cited by this paper
• Unveiling the Essential Role of Arkadia’s Non-RING Elements in the Ubiquitination Process

  2022cited by this paper
• Activity-based probe profiling of RNF12 E3 ubiquitin ligase function in Tonne-Kalscheuer syndrome

  2022cited by this paper
• Structure of UBE2K–Ub/E3/polyUb reveals mechanisms of K48-linked Ub chain extension

  2022cited by this paper
• Functional conservation and divergence of the helix‐turn‐helix motif of E2 ubiquitin‐conjugating enzymes

  2021cited by this paper
• Structural basis for the E3 ligase activity enhancement of yeast Nse2 by SUMO-interacting motifs

  2021cited by this paper
• Structural insights into Ubr1-mediated N-degron polyubiquitination

  2021cited by this paper
• Ubiquitin ligation to F-box protein targets by SCF–RBR E3–E3 super-assembly

  2021cited by this paper
• Reconstitution and Structural Analysis of a HECT Ligase-Ubiquitin Complex via an Activity-Based Probe

  2021cited by this paper
• Chemical synthesis of activity-based E2-ubiquitin probes for the structural analysis of E3 ligase-catalyzed transthiolation.

  2021cited by this paper
• Site-Specific Conversion of Cysteine in a Protein to Dehydroalanine Using 2-Nitro-5-thiocyanatobenzoic Acid

  2021cited by this paper
• Crystal structures of an E1–E2–ubiquitin thioester mimetic reveal molecular mechanisms of transthioesterification

  2021cited by this paper
• Structure of the FA core ubiquitin ligase closing the ID clamp on DNA

  2021cited by this paper
• RING domains act as both substrate and enzyme in a catalytic arrangement to drive self-anchored ubiquitination

  2021cited by this paper
• Structural basis for RING-Cys-Relay E3 Ligase Activity and its Role in Axon Integrity

  2020cited by this paper
• Linkage-specific ubiquitin chain formation depends on a lysine hydrocarbon ruler

  2020cited by this paper
• Who with whom: functional coordination of E2 enzymes by RING E3 ligases during poly‐ubiquitylation

  2020cited by this paper
• Structural insights into ADP-ribosylation of ubiquitin by Deltex family E3 ubiquitin ligases

  2020cited by this paper
• Molecular basis for the bifunctional Uba4–Urm1 sulfur‐relay system in tRNA thiolation and ubiquitin‐like conjugation

  2020cited by this paper
• Chain reactions: molecular mechanisms of RBR ubiquitin ligases

  2020cited by this paper
• Structural basis for DNA damage-induced phosphoregulation of MDM2 RING domain

  2020cited by this paper
• HECT E3 ubiquitin ligases – emerging insights into their biological roles and disease relevance

  2020cited by this paper
• NEDD8 nucleates a multivalent cullin-RING-UBE2D ubiquitin ligation assembly

  2020cited by this paper
• Hybrid Chains: A Collaboration of Ubiquitin and Ubiquitin-Like Modifiers Introducing Cross-Functionality to the Ubiquitin Code

  2020cited by this paper
• An activity-based probe developed by a sequential dehydroalanine formation strategy targets HECT E3 ubiquitin ligases.

  2019cited by this paper
• Photocrosslinking Activity-Based Probes for Ubiquitin RING E3 Ligases

  2019cited by this paper
• Chemical Synthesis of Atomically Tailored SUMO E2 Conjugating Enzymes for the Formation of Covalently Linked SUMO-E2-E3 Ligase Ternary Complexes.

  2019cited by this paper
• A tri-ionic anchor mechanism drives Ube2N-specific recruitment and K63-chain ubiquitination in TRIM ligases

  2019cited by this paper
• E3 ubiquitin-protein ligase TRIM21-mediated lysine capture by UBE2E1 reveals substrate-targeting mode of a ubiquitin-conjugating E2

  2019cited by this paper
• Structural insights into E1 recognition and the ubiquitin-conjugating activity of the E2 enzyme Cdc34

  2019cited by this paper
• Structural basis for adenylation and thioester bond formation in the ubiquitin E1

  2019cited by this paper
• Analysis of ubiquitin recognition by the HECT ligase E6AP provides insight into its linkage specificity

  2019cited by this paper
• Characterisation of class VI TRIM RING domains: linking RING activity to C-terminal domain identity

  2019cited by this paper
• A Bifunctional Role for the UHRF1 UBL Domain in the Control of Hemi-methylated DNA-Dependent Histone Ubiquitylation.

  2018cited by this paper
• Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation

  2018cited by this paper
• A small-molecule inhibitor of the ubiquitin activating enzyme for cancer treatment

  2018cited by this paper
• Mechanistic insights revealed by a UBE2A mutation linked to intellectual disability

  2018cited by this paper
• Structural insights into non-covalent ubiquitin activation of the cIAP1-UbcH5B∼ubiquitin complex

  2018cited by this paper
• Determinants of E2-ubiquitin conjugate recognition by RBR E3 ligases

  2018cited by this paper
• Insights into the ubiquitin transfer cascade catalyzed by the Legionella effector SidC

  2018cited by this paper
• Activity-based E3 ligase profiling uncovers an E3 ligase with esterification activity

  2018cited by this paper
• Generation of the UFM1 Toolkit for Profiling UFM1‐Specific Proteases and Ligases

  2018cited by this paper
• UbMES and UbFluor: Novel probes for ring-between-ring (RBR) E3 ubiquitin ligase PARKIN

  2017cited by this paper
• Mind the Metal: A Fragment Library-Derived Zinc Impurity Binds the E2 Ubiquitin-Conjugating Enzyme Ube2T and Induces Structural Rearrangements

  2017cited by this paper
• Activity‐Based Probes for HECT E3 Ubiquitin Ligases

  2017cited by this paper
• Ubiquitin-like Protein Conjugation: Structures, Chemistry, and Mechanism

  2017cited by this paper
• SUMO chain formation relies on the amino-terminal region of SUMO-conjugating enzyme and has dedicated substrates in plants

  2017cited by this paper
• S. pombe Uba1-Ubc15 Structure Reveals a Novel Regulatory Mechanism of Ubiquitin E2 Activity.

  2017cited by this paper
• Structural analysis of MDM2 RING separates degradation from regulation of p53 transcription activity

  2017cited by this paper
• UbFluor: A Fluorescent Thioester to Monitor HECT E3 Ligase Catalysis

  2017cited by this paper
• Ubiquitin Ligases: Structure, Function, and Regulation.

  2017cited by this paper
• The activity of TRAF RING homo- and heterodimers is regulated by zinc finger 1

  2017cited by this paper
• Structure and catalytic activation of the TRIM23 RING E3 ubiquitin ligase

  2017cited by this paper
• Label-Free and Real-Time Detection of Protein Ubiquitination with a Biological Nanopore

  2017cited by this paper
• Structural Studies of HHARI/UbcH7∼Ub Reveal Unique E2∼Ub Conformational Restriction by RBR RING1

  2017cited by this paper
• A General Strategy for Discovery of Inhibitors and Activators of RING and U-box E3 Ligases with Ubiquitin Variants

  2017cited by this paper
• Mechanism of catalysis, E2 recognition, and autoinhibition for the IpaH family of bacterial E3 ubiquitin ligases

  2017cited by this paper
• Allosteric auto‐inhibition and activation of the Nedd4 family E3 ligase Itch

  2017cited by this paper
• Probing the roles of SUMOylation in cancer cell biology by using a selective SAE inhibitor.

  2017cited by this paper
• Structural insights into the mechanism and E2 specificity of the RBR E3 ubiquitin ligase HHARI

  2017cited by this paper
• Dissecting the Specificity of Adenosyl Sulfamate Inhibitors Targeting the Ubiquitin-Activating Enzyme.

  2017cited by this paper
• RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment*

  2016cited by this paper
• Facile synthesis of covalent probes to capture enzymatic intermediates during E1 enzyme catalysis.

  2016cited by this paper
• A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes.

  2016cited by this paper
• Capturing a substrate in an activated RING E3/E2-SUMO complex

  2016cited by this paper
• Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

  2016cited by this paper
• Systematic approaches to identify E3 ligase substrates

  2016cited by this paper
• Crystal Structure of a Ube2S-Ubiquitin Conjugate

  2016cited by this paper
• UbFluor: a mechanism-based probe for HECT E3 ligases

  2016cited by this paper
• Substrate specificity of the ubiquitin and Ubl proteases

  2016cited by this paper
• Two Distinct Types of E3 Ligases Work in Unison to Regulate Substrate Ubiquitylation.

  2016cited by this paper
• Structural insights into the catalysis and regulation of E3 ubiquitin ligases

  2016cited by this paper
• The increasing complexity of the ubiquitin code

  2016cited by this paper
• Mechanism of TRIM25 Catalytic Activation in the Antiviral RIG-I Pathway

  2016cited by this paper
• Probes of Ubiquitin E3 ligases distinguish different stages of Parkin activation

  2016cited by this paper
• Cryo-EM of Mitotic Checkpoint Complex-Bound APC/C Reveals Reciprocal and Conformational Regulation of Ubiquitin Ligation.

  2016cited by this paper
• The molecular basis of lysine 48 ubiquitin chain synthesis by Ube2K

  2015cited by this paper
• Mechanism of phospho-ubiquitin induced PARKIN activation

  2015cited by this paper
• Orthogonal thiol functionalization at a single atomic center for profiling transthiolation activity of E1 activating enzymes.

  2015cited by this paper
• RING E3 mechanism for ubiquitin ligation to a disordered substrate visualized for human anaphase-promoting complex

  2015cited by this paper
• Itch WW Domains Inhibit Its E3 Ubiquitin Ligase Activity by Blocking E2-E3 Ligase Trans-thiolation*

  2015cited by this paper

• Lock, relax, load, and shoot: a molecular perspective on Nedd4 regulation.

  2026cites this paper
• From Covalent Traps to Fluorescent Beacons: The Expanding Arsenal of Chemical Probes for Studying Ubiquitin and Ubiquitin-Like Proteins.

  2026cites this paper
• Internal Ubiquitin Electrophiles for Covalent Trapping and Inhibition of Deubiquitinases

  2025cites this paper
• A Simple, Quick, and Scalable Route to Fluorogenic Ubiquitin and Ubiquitin-Like Protein Substrates for Assessing Activities of Deubiquitinases and Ubiquitin-Like Protein-Specific Proteases

  2025cites this paper
• Investigating the Interaction Profile of Unconjugated Ubiquitin: Chemical Biology and Affinity Enrichment Mass Spectrometric Approaches

  2025cites this paper
• Chemical Tools for Probing the Ub/Ubl Conjugation Cascades

  2024cites this paper