Ubiquitin-based probes prepared by total synthesis to profile the activity of deubiquitinating enzymes

Epitope-tagged active-site-directed probes are widely used to visualize the activity of deubiquitinases (DUBs) in cell extracts, to investigate the specificity and potency of small-molecule DUB inhibitors, and to isolate and identify DUBs by mass spectrometry. With DUBs arising as novel potential drug targets, probes are required that can be produced in sufficient amounts and to meet the specific needs of a given experiment. The established method for the generation of DUB probes makes use of labor-intensive intein-based methods that have inherent limitations concerning the incorporation of unnatural amino acids and the amount of material that can be obtained. Here, we describe the total chemical synthesis of active-site-directed probes and their application to activity-based profiling and identification of functional DUBs. This synthetic methodology allowed the easy incorporation of desired tags for specific applications, for example, fluorescent reporters, handles for immunoprecipitation or affinity pull-down, and cleavable linkers. Additionally, the synthetic method can be scaled up to provide significant amounts of probe. Fluorescent ubiquitin probes allowed faster, in-gel detection of active DUBs, as compared to (immuno)blotting procedures. A biotinylated probe holding a photocleavable linker enabled the affinity pull-down and subsequent mild, photorelease of DUBs. Also, DUB activity levels were monitored in response to overexpression or knockdown, and to inhibition by small molecules. Furthermore, fluorescent probes revealed differential DUB activity profiles in a panel of lung and prostate cancer cells

Elucidating crosstalk mechanisms between phosphorylation and O-GlcNAcylation

Chemical Immunolog

Generation of the UFM1 Toolkit for Profiling UFM1-Specific Proteases and Ligases

Chemical Immunolog

Inhibition of transcription leads to rewiring of locus-specific chromatin proteomes

Transcription of a chromatin template involves the concerted interaction of many different proteins and protein complexes. Analyses of specific factors showed that these interactions change during stress and upon developmental switches. However, how the binding of multiple factors at any given locus is coordinated has been technically challenging to investigate. Here we used Epi-Decoder in yeast to systematically decode, at one transcribed locus, the chromatin binding changes of hundreds of proteins in parallel upon perturbation of transcription. By taking advantage of improved Epi-Decoder libraries, we observed broad rewiring of local chromatin proteomes following chemical inhibition of RNA polymerase. Rapid reduction of RNA polymerase II binding was accompanied by reduced binding of many other core transcription proteins and gain of chromatin remodelers. In quiescent cells, where strong transcriptional repression is induced by physiological signals, eviction of the core transcriptional machinery was accompanied by the appearance of quiescent cell-specific repressors and rewiring of the interactions of protein-folding factors and metabolic enzymes. These results show that Epi-Decoder provides a powerful strategy for capturing the temporal binding dynamics of multiple chromatin proteins under varying conditions and cell states. The systematic and comprehensive delineation of dynamic local chromatin proteomes will greatly aid in uncovering protein-protein relationships and protein functions at the chromatin template.Chemical Immunolog

Total Chemical Synthesis of SUMO and SUMO-Based Probes for Profiling the Activity of SUMO-Specific Proteases

Chemical Immunolog

SUMO targets the APC/C to regulate transition from metaphase to anaphase

Chemical Immunolog

A chemical switch for the modulation of the functional activity of higher homologues of histamine on the human histamine H3 receptor: effect of various substitutions at the primary amino function

In an effort to establish the structural requirements for agonism, neutral antagonism, and inverse agonism at the human histamine

High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4(+) T cells in human melanoma

Tumor-specific neo-antigens that arise as a consequence of mutations(1,2) are thought to be important for the therapeutic efficacy of cancer immunotherapies(3-5). Accumulating evidence suggests that neo-antigens may be commonly recognized by intratumoral CD8(+) T cells(3-7), but it is unclear whether neoantigen-specific CD4(+) T cells also frequently reside within human tumors. In view of the accepted role of tumor-specific CD4(+) T-cell responses in tumor control(8-10), we addressed whether neo-antigen-specific CD4(+) T-cell reactivity is a common property in human melanom