ABSTRACT
Deubiquitinating enzymes are key regulators in the ubiquitin system and an emerging class of drug targets. These proteases disassemble polyubiquitin chains and many deubiquitinases show selectivity for specific polyubiquitin linkages. However, most biochemical insights originate from studies of single diubiquitin linkages in isolation, whereas in cells all linkages coexist. To better mimick this diubiquitin substrate competition, we develop a multiplexed mass spectrometry-based deubiquitinase assay that can probe all ubiquitin linkage types simultaneously to quantify deubiquitinase activity in the presence of all potential diubiquitin substrates. For this, all eight native diubiquitins are generated and each linkage type is designed with a distinct molecular weight by incorporating neutron-encoded amino acids. Overall, 22 deubiquitinases are profiled, providing a three-dimensional overview of deubiquitinase linkage selectivity over time and enzyme concentration.
Subject(s)
Deubiquitinating Enzymes , Polyubiquitin , Ubiquitination , Polyubiquitin/metabolism , Deubiquitinating Enzymes/metabolism , Ubiquitin/metabolism , Ubiquitins/metabolismABSTRACT
In this issue of Cell Chemical Biology,De Cesare et al. (2018) report the development of a high-throughput assay that measures E2/E3 enzyme activity by MALDI-TOF mass spectrometry and apply this to screen for small molecule E3 inhibitors. This assay potentially accelerates the drug discovery for the ubiquitin ligation pathway.
Subject(s)
Ubiquitin-Protein Ligases , Ubiquitin , Drug Discovery , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , UbiquitinationABSTRACT
The development of γ-thionorleucine (ThioNle) as a handle for native chemical ligation-desulfurization is reported here. ThioNle is a new addition to the expanding thiolated amino acid toolbox and serves as a methionine substitute in NCL with the advantage that it lacks the undesirable oxidation-prone thioether moiety. Its usefulness for N-terminal ubiquitination is demonstrated by efficient preparation of fully synthetic linear diubiquitin with preserved protein folding compared to the expressed material. Interestingly, gel-based deubiquitinating assays revealed that the methionine to norleucine substitution did affect diubiquitin cleavage, which may indicate a more profound role for methionine in the interaction between ubiquitin and the deubiquitinating enzymes than has been known so far.
Subject(s)
Methionine/chemistry , Norleucine/chemistry , Ubiquitins/chemistry , Ubiquitins/metabolismABSTRACT
The post-translational modification of proteins with polyubiquitin regulates virtually all aspects of cell biology. Eight distinct chain linkage types co-exist in polyubiquitin and are independently regulated in cells. This 'ubiquitin code' determines the fate of the modified protein. Deubiquitinating enzymes of the ovarian tumour (OTU) family regulate cellular signalling by targeting distinct linkage types within polyubiquitin, and understanding their mechanisms of linkage specificity gives fundamental insights into the ubiquitin system. Here we reveal how the deubiquitinase Cezanne (also known as OTUD7B) specifically targets Lys11-linked polyubiquitin. Crystal structures of Cezanne alone and in complex with monoubiquitin and Lys11-linked diubiquitin, in combination with hydrogen-deuterium exchange mass spectrometry, enable us to reconstruct the enzymatic cycle in great detail. An intricate mechanism of ubiquitin-assisted conformational changes activates the enzyme, and while all chain types interact with the enzymatic S1 site, only Lys11-linked chains can bind productively across the active site and stimulate catalytic turnover. Our work highlights the plasticity of deubiquitinases and indicates that new conformational states can occur when a true substrate, such as diubiquitin, is bound at the active site.
Subject(s)
Deubiquitinating Enzymes/metabolism , Endopeptidases/metabolism , Lysine/metabolism , Polyubiquitin/metabolism , Biocatalysis , Catalytic Domain , Crystallography, X-Ray , Deubiquitinating Enzymes/chemistry , Deubiquitinating Enzymes/genetics , Deuterium Exchange Measurement , Endopeptidases/chemistry , Endopeptidases/genetics , Enzyme Activation , Humans , Mass Spectrometry , Models, Molecular , Protein Binding , Protein Conformation , Substrate Specificity , Ubiquitination , Ubiquitins/metabolismABSTRACT
Deubiquitinating enzymes (DUBs) are proteases that fulfill crucial roles in the ubiquitin (Ub) system, by deconjugation of Ub from its targets and disassembly of polyUb chains. The specificity of a DUB towards one of the polyUb chain linkages largely determines the ultimate signaling function. We present a novel set of diubiquitin FRET probes, comprising all seven isopeptide linkages, for the absolute quantification of chain cleavage specificity of DUBs by means of Michaelis-Menten kinetics. Each probe is equipped with a FRET pair consisting of Rhodamine110 and tetramethylrhodamine to allow the fully synthetic preparation of the probes by SPPS and NCL. Our synthetic strategy includes the introduction of N,N'-Boc-protected 5-carboxyrhodamine as a convenient building block in peptide chemistry. We demonstrate the value of our probes by quantifying the linkage specificities of a panel of nine DUBs in a high-throughput manner.
