Enzymes that bind and process ubiquitin, a small 76-amino-acid protein, have been recognized as pharmacological targets in oncology, immunological disorders, and neurodegeneration. Mass spectrometry ...technology has now reached the capacity to cover the proteome with enough depth to interrogate entire biochemical pathways including those that contain DUBs and E3 ligase substrates. We have recently characterized the breast cancer cell (MCF7) deep proteome by detecting and quantifying ~10,000 proteins, and within this data set, we can detect endogenous expression of 65 deubiquitylating enzymes (DUBs), whereas matching transcriptomics detected 78 DUB mRNAs. Since enzyme activity provides another meaningful layer of information in addition to the expression levels, we have combined advanced mass spectrometry technology, pre-fractionation, and more potent/selective ubiquitin active-site probes with propargylic-based electrophiles to profile 74 DUBs including distinguishable isoforms for 5 DUBs in MCF7 crude extract material. Competition experiments with cysteine alkylating agents and pan-DUB inhibitors combined with probe labeling revealed the proportion of active cellular DUBs directly engaged with probes by label-free quantitative (LFQ) mass spectrometry. This demonstrated that USP13, 39, and 40 are non-reactive to probe, indicating restricted enzymatic activity under these cellular conditions. Our extended chemoproteomics workflow increases depth of covering the active DUBome, including isoform-specific resolution, and provides the framework for more comprehensive cell-based small-molecule DUB selectivity profiling.
Jumonji domain-containing demethylases (JmjC-KDMs) catalyse demethylation of Nε-methylated lysines on histones and play important roles in gene regulation. We report selectivity studies on KDM6B ...(JMJD3), a disease-relevant JmjC-KDM, using synthetic lysine analogues. The results unexpectedly reveal that KDM6B accepts multiple Nε-alkylated lysine analogues, forming alcohol, aldehyde and carboxylic acid products.
Formaldehyde (HCHO) is a potent electrophile that is toxic above threshold levels, but which is also produced in the nuclei of eukaryotic cells by demethylases. We report studies with the four ...canonical human histones revealing that histone H2B reacts with HCHO, including as generated by a histone demethylase, to give a stable product. NMR studies show that HCHO reacts with the N-terminal proline and associated amide of H2B to give a 5,5-bicyclic aminal that is relatively stable to competition with HCHO scavengers. While the roles of histone modification by this reaction require further investigation, we demonstrated the potential of N-terminal aminal formation to modulate protein function by conducting biochemical and cellular studies on the effects of HCHO on catalysis by 4-oxalocrotonate tautomerase, which employs a nucleophilic N-terminal proline. The results suggest that reactions of N-terminal residues with HCHO and other aldehydes have potential to alter protein function.
The main protease (Mpro) of SARS-CoV-2 is central to viral maturation and is a promising drug target, but little is known about structural aspects of how it binds to its 11 natural cleavage sites. We ...used biophysical and crystallographic data and an array of biomolecular simulation techniques, including automated docking, molecular dynamics (MD) and interactive MD in virtual reality, QM/MM, and linear-scaling DFT, to investigate the molecular features underlying recognition of the natural Mpro substrates. We extensively analysed the subsite interactions of modelled 11-residue cleavage site peptides, crystallographic ligands, and docked COVID Moonshot-designed covalent inhibitors. Our modelling studies reveal remarkable consistency in the hydrogen bonding patterns of the natural Mpro substrates, particularly on the N-terminal side of the scissile bond. They highlight the critical role of interactions beyond the immediate active site in recognition and catalysis, in particular plasticity at the S2 site. Building on our initial Mpro-substrate models, we used predictive saturation variation scanning (PreSaVS) to design peptides with improved affinity. Non-denaturing mass spectrometry and other biophysical analyses confirm these new and effective ‘peptibitors’ inhibit Mpro competitively. Our combined results provide new insights and highlight opportunities for the development of Mpro inhibitors as anti-COVID-19 drugs.
Apoptosis signal-regulating kinase 1 (ASK1) plays an essential role in stress and immune response and has been linked to the development of several diseases. Here, we present the structure of the ...human ASK1 catalytic domain in complex with staurosporine. Analytical ultracentrifugation (AUC) and crystallographic analysis showed that ASK1 forms a tight dimer (Kd ∼ 0.2 μM) interacting in a head-to-tail fashion. We found that the ASK1 phosphorylation motifs differ from known ASK1 phosphorylation sites but correspond well to autophosphorylation sites identified by mass spectrometry. Reporter gene assays showed that all three identified in vitro autophosphorylation sites (Thr813, Thr838, Thr842) regulate ASK1 signaling, but site-directed mutants showed catalytic activities similar to wild-type ASK1, suggesting a regulatory mechanism independent of ASK1 kinase activity. The determined high-resolution structure of ASK1 and identified ATP mimetic inhibitors will provide a first starting point for the further development of selective inhibitors.
-Methylation of lysine residues in histones plays an essential role in the regulation of eukaryotic transcription. The 'highest' methylation mark,
-trimethyllysine, is specifically recognised by
...-trimethyllysine binding 'reader' domains, and undergoes demethylation, as catalysed by 2-oxoglutarate dependent JmjC oxygenases. We report studies on the recognition of the closest positively charged
-trimethyllysine analogue, i.e. its trimethylphosphonium derivative (K
me
), by
-trimethyllysine histone binding proteins and
trimethyllysine demethylases. Calorimetric and computational studies with histone binding proteins reveal that H3K
4me
binds more tightly than the natural H3K4me
substrate, though the relative differences in binding affinity vary. Studies with JmjC demethylases show that some, but not all, of them can accept the phosphonium analogue of their natural substrates and that the methylation state selectivity can be changed by substitution of nitrogen for phosphorus. The combined results reveal that very subtle changes,
. substitution of nitrogen for phosphorus, can substantially affect interactions between ligand and reader domains / demethylases, knowledge that we hope will inspire the development of highly selective small molecules modulating their activity.
The demethylation of Nε‐methyllysine residues on histones by Jumonji‐C lysine demethylases (JmjC‐KDMs) has been established. A subset of JmjC‐KDMs has also been reported to have Nω‐methylarginine ...residue demethylase (RDM) activity. Here, we describe biochemical screening studies, showing that the catalytic domains of all human KDM5s (KDM5A‐KDM5D), KDM4E and, to a lesser extent, KDM4A/D, have both KDM and RDM activities with histone peptides. Ras GTPase‐activating protein‐binding protein 1 peptides were shown to be RDM substrates for KDM5C/D. No RDM activity was observed with KDM1A and the other JmjC‐KDMs tested. The results highlight the potential of JmjC‐KDMs to catalyse reactions other than Nε‐methyllysine demethylation. Although our study is limited to peptide fragments, the results should help guide biological studies investigating JmjC functions.
Methylation of lysines and arginines in histones is a key epigenetic modification that regulates eukaryotic transcription. A peptide library screen against an extended set of purified human lysine demethylases (KDMs) by mass spectrometry reveals that all KDM5 subfamily members and some KDM4 subfamily members catalyse demethylation of both Nε‐methyllysines and Nω‐methylarginines. Not all KDMs tested show arginine demethylase activity.
Enzyme inhibitors working by
O
-acylation of nucleophilic serine residues are of immense medicinal importance, as exemplified by the β-lactam antibiotics. By contrast, inhibition of nucleophilic ...cysteine enzymes by
S
-acylation has not been widely exploited for medicinal applications. The SARS-CoV-2 main protease (M
pro
) is a nucleophilic cysteine protease and a validated therapeutic target for COVID-19 treatment using small-molecule inhibitors. The clinically used M
pro
inhibitors nirmatrelvir and simnotrelvir work
via
reversible covalent reaction of their electrophilic nitrile with the M
pro
nucleophilic cysteine (Cys145). We report combined structure activity relationship and mass spectrometric studies revealing that appropriately functionalized γ-lactams can potently inhibit M
pro
by reversible covalent reaction with Cys145 of M
pro
. The results suggest that γ-lactams have potential as electrophilic warheads for development of covalently reacting small-molecule inhibitors of M
pro
and, by implication, other nucleophilic cysteine enzymes.
Thiophene-fused γ-lactams are reversible covalent inhibitors of the SARS-CoV-2 main protease, a nucleophilic cysteine enzyme. γ-Lactams can inhibit nucleophilic cysteine enzymes by
S
-acylation as well as nucleophilic serine enzymes by
O
-acylation.
Protein kinases are major targets for the development of new medicines and play key roles in cellular signaling. The flexible nature of these proteins, posttranslational modifications, and the large ...size of some protein kinases pose a particular challenge obtaining homogeneous, active recombinant protein kinases suitable for functional or structural studies. Here we describe our expertise expressing protein kinases in two frequently used host systems: E. coli and insect cells using the baculovirus expression vector system. In particular, we will discuss and provide detailed methods on construct design, high-throughput cloning, parallel expression testing and scale up as well as purification and co-expression strategies leading to stable and homogeneous recombinant protein samples.
Long-term potentiation (LTP), a long-lasting enhancement in communication between neurons, is considered to be the major cellular mechanism underlying learning and memory. LTP triggers high-frequency ...calcium pulses that result in the activation of Calcium/Calmodulin (CaM)-dependent kinase II (CaMKII). CaMKII acts as a molecular switch because it remains active for a long time after the return to basal calcium levels, which is a unique property required for CaMKII function. Here we describe the crystal structure of the human CaMKIIdelta/Ca2+/CaM complex, structures of all four human CaMKII catalytic domains in their autoinhibited states, as well as structures of human CaMKII oligomerization domains in their tetradecameric and physiological dodecameric states. All four autoinhibited human CaMKIIs were monomeric in the determined crystal structures but associated weakly in solution. In the CaMKIIdelta/Ca2+/CaM complex, the inhibitory region adopted an extended conformation and interacted with an adjacent catalytic domain positioning T287 into the active site of the interacting protomer. Comparisons with autoinhibited CaMKII structures showed that binding of calmodulin leads to the rearrangement of residues in the active site to a conformation suitable for ATP binding and to the closure of the binding groove for the autoinhibitory helix by helix alphaD. The structural data, together with biophysical interaction studies, reveals the mechanism of CaMKII activation by calmodulin and explains many of the unique regulatory properties of these two essential signaling molecules.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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