Ubiquitin‐fold modifier 1 (UFM1) is a reversible post‐translational modifier that is covalently attached to target proteins through an enzymatic cascade and removed by designated proteases. ...Abnormalities in this process, referred to as Ufmylation, have been associated with a variety of human diseases. Given this, the UFM1‐specific enzymes represent potential therapeutic targets; however, understanding of their biological function has been hampered by the lack of chemical tools for activity profiling. To address this unmet need, a diversifiable platform for UFM1 activity‐based probes (ABPs) utilizing a native chemical ligation (NCL) strategy was developed, enabling the generation of a variety of tools to profile both UFM1 conjugating and deconjugating enzymes. The use of the probes is demonstrated in vitro and in vivo for monitoring UFM1 enzyme reactivity, opening new research avenues.
Introducing the UFM1 toolkit: A facile native chemical ligation strategy to enable the development of UFM1 reagents and activity‐based probes is presented. This toolkit permits the study of both UFM1‐specific proteases and conjugating enzymes in vitro and in cells, thereby opening new research avenues for the discovery of specific UFM1 inhibitors.
Major histocompatibility complex (MHC) class I molecules present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. MHC-bound peptides are critical for the ...stability of the MHC complex, and standard strategies for the production of recombinant MHC complexes are based on in vitro refolding reactions with specific peptides. This strategy is not amenable to high-throughput production of vast collections of MHC molecules. We have developed conditional MHC ligands that form stable complexes with MHC molecules but can be cleaved upon UV irradiation. The resulting empty, peptide-receptive MHC molecules can be charged with epitopes of choice under native conditions. Here we describe in-depth procedures for the high-throughput production of peptide-MHC (pMHC) complexes by MHC exchange, the analysis of peptide exchange efficiency by ELISA and the parallel production of MHC tetramers for T-cell detection. The production of the conditional pMHC complex by an in vitro refolding reaction can be achieved within 2 weeks, and the actual high-throughput MHC peptide exchange and subsequent MHC tetramer formation require less than a day.
Plasmodium parasites are the causative agents of malaria, a disease with wide public health repercussions. Increasing drug resistance and the absence of a vaccine make finding new chemotherapeutic ...strategies imperative. Components of the ubiquitin and ubiquitin-like pathways have garnered increased attention as novel targets given their necessity to parasite survival. Understanding how these pathways are regulated in Plasmodium and identifying differences to the host is paramount to selectively interfering with parasites. Here, we focus on Nedd8 modification in Plasmodium falciparum, given its central role to cell division and DNA repair, processes critical to Plasmodium parasites given their unusual cell cycle and requirement for refined repair mechanisms. By applying a functional chemical approach, we show that deNeddylation is controlled by a different set of enzymes in the parasite versus the human host. We elucidate the molecular determinants of the unusual dual ubiquitin/Nedd8 recognition by the essential PfUCH37 enzyme and, through parasite transgenics and drug assays, determine that only its ubiquitin activity is critical to parasite survival. Our experiments reveal interesting evolutionary differences in how neddylation is controlled in higher versus lower eukaryotes, and highlight the Nedd8 pathway as worthy of further exploration for therapeutic targeting in antimalarial drug design.
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.
Sirtuin 1 (Sirt1) is a NAD
dependent lysine deacetylase associated with the pathogenesis of various diseases including cancer. In many cancer types Sirt1 expression is increased and higher levels ...have been associated with metastasis and poor prognosis. However, it was also shown, that Sirt1 can have tumor suppressing properties and in some instances even a dual role for the same cancer type has been reported. Increased Sirt1 activity has been linked to extension of the life span of cells, respectively, organisms by promoting DNA repair processes and downregulation of tumor suppressor proteins. This may have the downside of enhancing tumor growth and metastasis. In mice embryonic fibroblasts depletion of Sirt1 was shown to decrease levels of the DNA damage sensor histone H2AX. Impairment of DNA repair mechanisms by Sirt1 can promote tumorigenesis but also lower chemoresistance toward DNA targeting therapies. Despite many biological studies, there is currently just one small molecule Sirt1 inhibitor in clinical trials. Selisistat (EX-527) reached phase III clinical trials for treatment of Huntington's Disease. New small molecule Sirt1 modulators are crucial for further investigation of the contradicting roles of Sirt1 in cancer. We tested a small library of commercially available compounds that were proposed by virtual screening and docking studies against Sirt1, 2 and 3. A thienopyrimidone featuring a phenyl thiocyanate moiety was found to selectively inhibit Sirt1 with an IC
of 13 μM. Structural analogs lacking the thiocyanate function did not show inhibition of Sirt1 revealing this group as key for the selectivity and affinity toward Sirt1. Further analogs with higher solubility were identified through iterative docking studies and
testing. The most active compounds (down to 5 μM IC
) were further studied in cells. The ratio of phosphorylated γH2AX to unmodified H2AX is lower when Sirt1 is depleted or inhibited. Our new Sirtuin 1 inhibiting thiocyanates (S1th) lead to similarly lowered γH2AX/H2AX ratios in mouse embryonic fibroblasts as Sirt1 knockout and treatment with the reference inhibitor EX-527. In addition to that we were able to show antiproliferative activity, inhibition of migration and colony forming as well as hyperacetylation of Sirt1 targets p53 and H3 by the S1th in cervical cancer cells (HeLa). These results reveal thiocyanates as a promising new class of selective Sirt1 inhibitors.
The Ubiquitin CODE constitutes a unique post-translational modification language relying on the covalent attachment of Ubiquitin (Ub) to substrates, with Ub serving as the minimum entity to generate ...a message that is translated into different cellular pathways. The creation of this message is brought about by the dedicated action of writers, erasers, and readers of the Ubiquitin CODE. This CODE is greatly expanded through the generation of polyUb chains of different architectures on substrates thus regulating their fate. Through additional post-translational modification by Ub-like proteins (UbL), hybrid Ub/UbL chains, which either alter the originally encrypted message or encode a completely new one, are formed. Hybrid Ub/UbL chains are generated under both stress or physiological conditions and seem to confer improved specificity and affinity toward their cognate receptors. In such a manner, their formation must play a specific, yet still undefined role in cellular signaling and thus understanding the UbCODE message is crucial. Here, we discuss the evidence for the existence of hybrid Ub/UbL chains in addition to the current understanding of its biology. The modification of Ub by another UbL complicates the deciphering of the spatial and temporal order of events warranting the development of a hybrid chain toolbox. We discuss this unmet need and expand upon the creation of tailored tools adapted from our previously established toolkit for the Ubiquitin Proteasome System to specifically target these hybrid Ub/UbL chains.
Protein modification by interferon‐stimulated gene 15 (ISG15), an ubiquitin‐like modifier, affects multiple cellular functions and represents one of the major antiviral effector systems. Covalent ...linkage of ISG15 to proteins was previously reported to be counteracted by ubiquitin‐specific protease 18 (USP18). To date, analysis of the molecular properties of USP18 was hampered by low expression yields and impaired solubility. We established high‐yield expression of USP18 in insect cells and purified the protease to homogeneity. USP18 binds with high affinity to ISG15, as shown by microscale thermophoresis with a Kd of 1.3 ± 0.2 μm. The catalytic properties of USP18 were characterized by a novel assay using ISG15 fused to a fluorophore via an isopeptide bond, giving a Km of 4.6 ± 0.2 μm and a kcat of 0.23 ± 0.004 s−1, respectively, at pH 7.5. Furthermore, the recombinant enzyme cleaves efficiently ISG15 but not ubiquitin from endogenous cellular substrates. In line with these data, USP18 exhibited neither cross‐reactivity with an ubiquitin isopeptide fluorophore substrate, nor with a ubiquitin vinyl sulfone, showing that the enzyme is specific for ISG15.
Structured digital
●ISG15 and USP18 bind by microscale thermophoresis (View interaction)●USP18 cleaves ISG15 by enzymatic study (View interaction)
Protein modification by the interferon‐stimulated gene 15 (ISG15), an ubiquitin‐like modifier, represents one of the major antiviral effector systems. Covalent linkage of ISG15 to proteins is counteracted by the protease USP18. We established expression and purification of USP18 from insect cells and show that the enzyme is highly specific for ISG15, cleaving ISG15 but not ubiquitin from different substrates.
The ubiquitination of NEMO with linear ubiquitin chains by the E3-ligase LUBAC is important for the activation of the canonical NF-κB pathway. NEMO ubiquitination requires a dual target specificity ...of LUBAC, priming on a lysine on NEMO and chain elongation on the N terminus of the priming ubiquitin. Here we explore the minimal requirements for these specificities. Effective linear chain formation requires a precise positioning of the ubiquitin N-terminal amine in a negatively charged environment on the top of ubiquitin. Whereas the RBR-LDD region on HOIP is sufficient for targeting the ubiquitin N terminus, the priming lysine modification on NEMO requires catalysis by the RBR domain of HOIL-1L as well as the catalytic machinery of the RBR-LDD domains of HOIP. Consequently, target specificity toward NEMO is determined by multiple LUBAC components, whereas linear ubiquitin chain elongation is realized by a specific interplay between HOIP and ubiquitin.
Background: Linear ubiquitination of NEMO by LUBAC is important for NF-κB activation.
Results: HOIP and the “top” of ubiquitin are essential for linear ubiquitination, whereas NEMO ubiquitination additionally requires HOIL-1L.
Conclusion: NEMO priming and ubiquitin chain elongation rely on different LUBAC contributions.
Significance: Novel insights in the requirements for linear ubiquitin chain formation and target selection.
Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for ...proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and protein degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world's most devastating pathogens.
Autotaxin (ATX) is a secreted phosphodiesterase that hydrolyzes the abundant phospholipid lysophosphatidylcholine (LPC) to produce lysophosphatidic acid (LPA). The ATX-LPA signaling axis has been ...implicated in inflammation, fibrosis, and tumor progression, rendering ATX an attractive drug target. We recently described a boronic acid-based inhibitor of ATX, named HA155 (1). Here, we report the design of new inhibitors based on the crystal structure of ATX in complex with inhibitor 1. Furthermore, we describe the syntheses and activities of these new inhibitors, whose potencies can be explained by structural data. To understand the difference in activity between two different isomers with nanomolar potencies, we performed molecular docking experiments. Intriguingly, molecular docking suggested a remarkable binding pose for one of the isomers, which differs from the original binding pose of inhibitor 1 for ATX, opening further options for inhibitor design.
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