Here we demonstrate that quantitation of stimuli-induced proteome dynamics in primary cells is feasible by combining the power of Bio-Orthogonal Non Canonical Amino acid Tagging (BONCAT) and Stable Isotope Labelling of Amino acids in Cell culture (SILAC). In conjunction with nanoLC-MS/MS QuaNCAT allowed us to monitor the early expression changes of > 600 proteins in primary resting T cells subjected to activation stimuli.
The JmjC histone demethylases (KDMs) are linked to tumour cell proliferation and are current cancer targets; however, very few highly selective inhibitors for these are available. Here we report cyclic peptide inhibitors of the KDM4A-C with selectivity over other KDMs/2OG oxygenases, including closely related KDM4D/E isoforms. Crystal structures and biochemical analyses of one of the inhibitors (CP2) with KDM4A reveals that CP2 binds differently to, but competes with, histone substrates in the active site. Substitution of the active site binding arginine of CP2 to N-ɛ-trimethyl-lysine or methylated arginine results in cyclic peptide substrates, indicating that KDM4s may act on non-histone substrates. Targeted modifications to CP2 based on crystallographic and mass spectrometry analyses results in variants with greater proteolytic robustness. Peptide dosing in cells manifests KDM4A target stabilization. Although further development is required to optimize cellular activity, the results reveal the feasibility of highly selective non-metal chelating, substrate-competitive inhibitors of the JmjC KDMs.
Cross-metathesis (CM) has recently emerged as a viable strategy for protein modification. Here, efficient protein CM has been demonstrated through biomimetic chemical access to Se-allyl-selenocysteine (Seac), a metathesis-reactive amino acid substrate, via dehydroalanine. On-protein reaction kinetics reveal a rapid reaction with rate constants of Seac-mediated-CM comparable or superior to off-protein rates of many current bioconjugations. This use of Se-relayed Seac CM on proteins has now enabled reactions with substrates (allyl GlcNAc, N-allyl acetamide) that were previously not possible for the corresponding sulfur analogue. This CM strategy was applied to histone proteins to install a mimic of acetylated lysine (KAc, an epigenetic marker). The resulting synthetic H3 was successfully recognized by antibody that binds natural H3-K9Ac. Moreover, Cope-type selenoxide elimination allowed this putative marker (and function) to be chemically expunged, regenerating an H3 that can be rewritten to complete a chemically enabled “write (CM)–erase (ox)–rewrite (CM)” cycle.
Olefin cross-metathesis (CM) is a viable reaction for the modification of alkene-containing proteins. Although allyl sulfide or selenide side-chain motifs in proteins can critically enhance the rate of CM reactions, no efficient method for their site-selective genetic incorporation into proteins has yet been reported. Here, through the systematic evaluation of olefin-bearing unnatural amino acids for their metabolic incorporation, we have discovered S-allylhomocysteine (Ahc) as a genetically encode-able Met analogue that is both processed by translational cellular machinery and is also a privileged CM substrate residue in proteins. In this way, Ahc was used for efficient Met-codon reassignment in a Met-auxotrophic strain of E. coli (B834 (DE3)), as well as metabolic labeling of protein in human cells and was reactive towards CM in several representative proteins. This expands the use of CM in the tool kit for 'tagand-modify' functionalization of proteins. Alkenes can be installed into proteins by incorporation of some unnatural amino acids. 1-4 However, although aliphatic alkene-containing amino acids such as homoallylglycine (Hag) 4 are reactive in both self-metathesis and crossmetathesis reactions as monomeric, protected amino acids in organic solvents 5 and can be metabolically incorporated into proteins, they are unreactive in CM reactions in aqueous media. 6 On the other hand, chemically-installed 7 unnatural amino acids such as S-allylcysteine (Sac) 6 ASSOCIATED CONTENT Supporting Information Full procedures and protein ESI-MS. Material is available free of charge via the Internet at http://pubs.acs.org.
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