Dataset Information

Nonenzymatic acetylation of ubiquitin Lys side chains is modulated by their neighboring residues.

ABSTRACT: Nonenzymatic acetylation of Lys side chains (Lys-SCs) by various in vivo reactive molecules has been suggested to play novel regulatory roles. Ubiquitin (UB) has seven Lys residues that are utilized for synthesis of specific poly-UB chains. To understand the nature of these Lys-SC modifications, the chemical acetylation rate and pKa and Hill coefficient of each UB-Lys-SC were measured. Mutagenesis studies combined with the determination of activation energy indicated that specific neighboring residues of the Lys-SCs have a potential catalytic activity during nonenzymatic acetylation. Based on the shared chemistry between nonenzymatic Lys acetylation and ubiquitylation, the characterized chemical properties of the UB-Lys-SCs could be a reference for deciphering both mechanisms. Our NMR approaches could be useful for studying general nonenzymatic Lys acylations of various proteins.

SUBMITTER: Lee SY

PROVIDER: S-EPMC5947880 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Nonenzymatic acetylation of ubiquitin Lys side chains is modulated by their neighboring residues.

Lee Seo-Yeon SY Choi Yun-Seok YS Kim Eun-Hee EH Cheong Hae-Kap HK Lee Yun-Ju YJ Lee Jin-Gu JG Ye Yihong Y Ryu Kyoung-Seok KS

The FEBS journal 20180304 7

Nonenzymatic acetylation of Lys side chains (Lys-SCs) by various in vivo reactive molecules has been suggested to play novel regulatory roles. Ubiquitin (UB) has seven Lys residues that are utilized for synthesis of specific poly-UB chains. To understand the nature of these Lys-SC modifications, the chemical acetylation rate and pK<sub>a</sub> and Hill coefficient of each UB-Lys-SC were measured. Mutagenesis studies combined with the determination of activation energy indicated that specific nei ...[more]

PMID: 29430834

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Project description:Polymeric chains of a small protein ubiquitin are involved in regulation of nearly all vital processes in eukaryotic cells. Elucidating the signaling properties of polyubiquitin requires the ability to make these chains in vitro. In recent years, chemical and chemical-biology tools have been developed that produce fully natural isopeptide-linked polyUb chains with no need for linkage-specific ubiquitin-conjugating enzymes. These methods produced unbranched chains (in which no more than one lysine per ubiquitin is conjugated to another ubiquitin). Here we report a nonenzymatic method for the assembly of fully natural isopeptide-linked branched polyubiquitin chains. This method is based on the use of mutually orthogonal removable protecting groups (e.g., Boc- and Alloc-) on lysines combined with an Ag-catalyzed condensation reaction between a C-terminal thioester on one ubiquitin and a specific ε-amine on another ubiquitin, and involves genetic incorporation of more than one Lys(Boc) at the desired linkage positions in the ubiquitin sequence. We demonstrate our method by making a fully natural branched tri-ubiquitin containing isopeptide linkages via Lys11 and Lys33, and a (15)N-enriched proximal ubiquitin, which enabled monomer-specific structural and dynamical studies by NMR. Furthermore, we assayed disassembly of branched and unbranched tri-ubiquitins as well as control di-ubiquitins by the yeast proteasome-associated deubiquitinase Ubp6. Our results show that Ubp6 can recognize and disassemble a branched polyubiquitin, wherein cleavage preferences for individual linkages are retained. Our spectroscopic and functional data suggest that, at least for the chains studied here, the isopeptide linkages are effectively independent of each other. Together with our method for nonenzymatic assembly of unbranched polyubiquitin, these developments now provide tools for making fully natural polyubiquitin chains of essentially any type of linkage and length.

Dataset Information

Nonenzymatic acetylation of ubiquitin Lys side chains is modulated by their neighboring residues.

Publications

Nonenzymatic acetylation of ubiquitin Lys side chains is modulated by their neighboring residues.

Similar Datasets

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