Comprehensive approach to study branched ubiquitin chains reveals roles for K48-K63 branches in VCP/p97-related processes

Lange, Sven M.; McFarland, Matthew R.; Lamoliatte, Frederic; Kwaśna, Dominika; Shen, Linnan; Wallace, Iona; Cole, Isobel; Armstrong,; Knebel, Axel; Johnson, Clare; Cesare, Virginia De; Kulathu, Yogesh

doi:10.1101/2023.01.10.523363

Cited by 13 publications

(30 citation statements)

References 107 publications

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“…Several tens of microgram of polyubiquitin is generally sufficient for biochemical experiments including the gel-based evaluation of DUB-mediated cleavage and the pull-down assay for studies on protein−ubiquitin interactions. 13,20,53,54 The synthesized K63/K48 Ub 4 with a K48-linked branch at the middle site was supplied to partial trypsin digestion under nondenaturing conditions to validate the branch structure. Trypsin treatment under nondenaturing conditions leads to the cleavage of peptide bonds between the 74th arginine and 75th glycine in the polyubiquitin chain, thereby allowing the characterization of distal and branched ubiquitins as ubiquitin units lacking C-terminal GG (Ub 1−74 ) and harboring multiple GG remnants on lysine residues engaged in the linkage formation, respectively (Figure 4e, left).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…The target K63/K48 Ub 4 synthesized on a larger scale was isolated by cation-exchange chromatography (52 μg, 4.3% isolated yield) (Figure S10) and characterized by MALDI-TOF MS (Figure d). Several tens of microgram of polyubiquitin is generally sufficient for biochemical experiments including the gel-based evaluation of DUB-mediated cleavage and the pull-down assay for studies on protein–ubiquitin interactions. ,,, …”

Section: Resultsmentioning

confidence: 99%

“…The time traces of biotinmodified ubiquitin species during AMSH processing of synthesized K63/K48 Ub 4 indicated the preference of AMSH for the distal side over the proximal side of K63-linked isopeptide bonds at the K63/K48 branching position, as suggested recently (Figure S12). 54 The DUB-mediated trimming of polyubiquitin is one of the essential aspects of the structural regulation of polyubiquitin chains underlying the precise control of ubiquitin functions. 59 Thus, the structuredefined heterotypic chains would offer a robust platform for evaluating how the structure of heterotypic polyubiquitin chains is controlled by enzymes including ubiquitin ligases and DUBs for precise modulation of ubiquitin signaling.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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One-Pot, Photocontrolled Enzymatic Assembly of the Structure-Defined Heterotypic Polyubiquitin Chain

Furuhata,

Devadasan Racheal,

Murayama

et al. 2023

J. Am. Chem. Soc.

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Heterotypic polyubiquitins are an emerging class of polyubiquitins that have attracted interest because of their potential diversity of structures and physiological functions. There is an increasing demand for structure-defined synthesis of heterotypic chains to investigate the topological factors underlying the intracellular signals that are characteristically mediated by the heterotypic chain. However, the applicability of chemical and enzymatic polyubiquitin synthesis developed to date has been limited by laborious rounds of ligation and purification or by a lack of modularity of the chain structure with respect to the length and the branch position. Here, we established a one-pot, photocontrolled synthesis of structurally defined heterotypic polyubiquitin chains. We designed ubiquitin derivatives with a photolabile protecting group at a lysine residue used for polymerization. Repetitive cycles of linkage-specific enzymatic elongation and photoinduced deprotection of the protected ubiquitin units enabled stepwise addition of ubiquitins with appropriate functionalities to control the length and branching positions. The positional control of branching was achieved without isolation of intermediates, allowing one-pot synthesis of K63 triubiqutin chains and a K63/K48 heterotypic tetraubiquitin chain with defined branching positions. The present study provides a chemical platform for the efficient construction of long polyubiquitin chains with defined branch structures that will facilitate the understanding of the essential relationships between functions and structures of the heterotypic chain that have hitherto been overlooked.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

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One-Pot, Photocontrolled Enzymatic Assembly of the Structure-Defined Heterotypic Polyubiquitin Chain

Furuhata,

Devadasan Racheal,

Murayama

et al. 2023

J. Am. Chem. Soc.

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“…The synthesis of Ub chains requires sequential conjugation and deprotection as illustrated in Figure A. We adopted the recently proposed nomenclatures for branched ubiquitin from Kulath et al, which provides a method of describing heterotypic ubiquitin chains . In our synthesis, the removal of excess Ub D and the E2-conjugating enzymes after the conjugation as well as buffer exchange before each conjugation/deprotection are inevitable.…”

Section: Resultsmentioning

confidence: 99%

Biocompatible Lysine Protecting Groups for the Chemoenzymatic Synthesis of K48/K63 Heterotypic and Branched Ubiquitin Chains

et al. 2023

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The elucidation of emerging biological functions of heterotypic and branched ubiquitin (Ub) chains requires new strategies for their preparation with defined lengths and connectivity. While in vitro enzymatic assembly using expressed E1-activating and E2-conjugating enzymes can deliver homotypic chains, the synthesis of branched chains typically requires extensive mutations of lysines or other sequence modifications. The combination of K48- and K63-biased E2-conjugating enzymes and two new carbamate protecting groupspyridoxal 5′-phosphate (PLP)-cleavable aminobutanamide carbamate (Abac group) and periodate-cleavable aminobutanol carbamate (Aboc group)provides a strategy for the synthesis of heterotypic and branched Ub trimers, tetramers, and pentamers. The Abac- and Aboc-protected lysines are readily prepared and incorporated into synthetic ubiquitin monomers. As these masking groups contain a basic amine, they preserve the overall charge and properties of the Ub structure, facilitating folding and enzymatic conjugations. These protecting groups can be chemoselectively removed from folded Ub chains and monomers by buffered solutions of PLP or NaIO4. Through the incorporation of a cleavable C-terminal His-tag on the Ub acceptor, the entire process of chain building, iterative Abac deprotections, and global Aboc cleavage can be conducted on a resin support, obviating the need for handling and purification of the intermediate oligomers. Simple modulation of the Ub monomers affords various K48/K63 branched chains, including tetramers and pentamers not previously accessible by synthetic or biochemical methods.

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“…We adopted the recently proposed nomenclatures for branched ubiquitin from Kulath et al, which provides a method of describing heterotypic ubiquitin chains. 50 In our synthesis, the removal of excess Ub D and the E2 conjugating enzymes after the conjugation, as well as buffer exchange before each conjugation/deprotection, is inevitable. To facilitate the synthesis and avoid chromatographic purification after each conjugation or deprotection, we implemented an on-resin synthesis of ubiquitin This facilitated the removal of non-tagged Ub D and E2s from the mixture and enabled buffer exchange by simple washing of the resin, analogous to SPPS.…”

Section: Lysine Deprotections On Folded Ubiquitinmentioning

confidence: 99%

Biocompatible lysine protecting groups for the chemoenzymatic synthesis of K48/K63 heterotypic and branched ubiquitin chains

Mikami

Majima

Song

et al. 2023

Preprint

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The elucidation of emerging biological functions of heterotypic and branched ubiquitin (Ub) chains requires new strategies for their preparation with defined length and connectivity. While in vitro enzymatic assembly using expressed E1 activating and E2 conjugating enzymes can deliver homotypic chains, the synthesis of branched chains typically requires extensive mutations of lysines or other sequence modifications. The combination of K48 and K63 biased E2 conjugating enzymes and two new carbamate protecting groups – pyridoxal 5’-phosphate (PLP) cleavable aminobutanamide carbamate (Abac group) and periodate cleavable aminobutanol carbamate (Aboc group) – provides a strategy for the synthesis of heterotypic and branched Ub trimers, tetramers, and pentamers. The Abac- and Aboc-protected lysines are readily prepared and incorporated into synthetic ubiquitin monomers. As these masking groups contain a basic amine, they preserve the overall charge and properties of the Ub structure, facilitating folding and enzymatic conjugations. These protecting groups can be chemoselectively removed from folded Ub chains and monomers with buffered solutions of PLP or NaIO4. Through the incorporation of a cleavable C-terminal His tag on the Ub acceptor, the entire process of chain building, iterative Abac deprotections, and global Aboc cleavage can be conducted on a resin-support, obviating the need for handling and purification of the intermediate oligomers. Simple modulation of the Ub monomers affords various K48/K63 branched chains, including tetramers and pentamers not previously accessible by synthetic or biochemical methods.

show abstract

Comprehensive approach to study branched ubiquitin chains reveals roles for K48-K63 branches in VCP/p97-related processes

Cited by 13 publications

References 107 publications

One-Pot, Photocontrolled Enzymatic Assembly of the Structure-Defined Heterotypic Polyubiquitin Chain

One-Pot, Photocontrolled Enzymatic Assembly of the Structure-Defined Heterotypic Polyubiquitin Chain

Biocompatible Lysine Protecting Groups for the Chemoenzymatic Synthesis of K48/K63 Heterotypic and Branched Ubiquitin Chains

Biocompatible lysine protecting groups for the chemoenzymatic synthesis of K48/K63 heterotypic and branched ubiquitin chains

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