Biology subjects

Burslem, G.

Publications and source records attributed to Burslem, G..

8 recordsLinked to original sources

bioRxiv2026

Sortase-mediated enrichment of ubiquitinated proteins from complex samples

Proteolytic processing is typically used to reveal post-translational modifications for mass spectrometric analysis. Here, we show that proteolysis of ubiquitinated proteins additionally generates a chemically addressable diglycine remnant that can be enzymatically remodeled. We exploit the ability of sortase to recognize ubiquitin-derived diglycine motifs as nucleophiles and ligate functionalized LPXTG peptides directly onto ubiquitin-remnant peptides. This reaction enables installation of a biotin affinity handle on defined ubiquitinated substrates and peptides derived from mammalian cell lysates, including lysine- and N-terminal ubiquitination. The resulting branched conjugates generate characteristic fragment ions that facilitate their identification. We further develop a bifunctional sortase donor containing an internal trypsin cleavage site, enabling affinity capture followed by conversion of enriched ubiquitin sites into a compact TGG mass-spectrometric signature. Alternatively, reversible sortase chemistry can regenerate the original diglycine-containing peptide. These studies establish ubiquitin-derived proteolytic remnants as latent chemical handles that can be converted into modular affinity and analytical tags, providing an antibody-independent approach to interrogating protein ubiquitination.

biochemistry↗

bioRxiv2026

A Modular Platform for Effector Discovery in Induced-Proximity Lysine Acetylation

The regulation of post-translational modifications (PTMs) is central to cellular biology and disease. Induced-proximity strategies enable manipulation of PTMs by recruiting modifying enzymes to proteins of interest, but identifying effective effector enzymes typically requires extensive heterobifunctional molecule synthesis before biological validation. Here we report a modular platform that enables rapid evaluation of PTM editing enzymes against defined protein substrates in living cells using compound-dependent or nanobody-mediated induced proximity. Using lysine acetylation as a model system, we demonstrate programmable acetylation of GFP, histone H3, and p53 through recruitment of diverse acetyltransferases. Effector identity dictates site-specific acetylation patterns, enabling selective PTM deposition across substrates and cellular compartments. This platform enables rapid identification of productive effector-substrate relationships prior to heterobifunctional molecule development, accelerating the design of induced-proximity chemical probes for targeted PTM editing.

biochemistry↗

bioRxiv2025

MAPK Pathway Inhibition Reshapes Kinase Chemical Probe Reactivity Reflecting Cellular Activation States

Despite the pivotal role of oncogenic kinases in cancer initiation, progression, and therapeutic resistance, functionally profiling their activity and conformational dynamics in live cells remains challenging. Existing methods often fail to capture inhibitor-bound structural states of kinases, particularly in clinically relevant contexts such as treatment response and acquired resistance, where genomic data alone are insufficient. Here, we use activity-based protein profiling (ABPP) to monitor composite amino acid reactivity changes - across cysteine, lysine, and carboxylic acid residues - as a functional readout of kinase conformation and activation state. Using electrophilic probes, we show that treatment of BRAFV600E-mutant melanoma cells with vemurafenib or trametinib decreases overall cysteine and lysine reactivity in BRAFV600E and MEK1/2, likely reflecting composite changes in amino acid accessibility across multiple reactive residues associated with inhibitor binding. Changing the order of probe addition and inhibitor treatment altered labeling outcomes, consistent with competitive engagement and structural stabilization. Comparative analysis of ATP-competitive BRAFV600E inhibitors vemurafenib and dabrafenib revealed distinct impacts on aspartate and glutamate labeling patterns, suggesting that ABPP can distinguish inhibitor-dependent differences in residue accessibility that may reflect distinct inhibitor-bound conformations. In inhibitor-resistant melanoma models, ABPP detected differential residue reactivity relative to parental cells, consistent with BRAF overexpression and the MEK2 Q60P activation mutation, both established mechanisms of MAPK inhibitor resistance. Moreover, global proteome analyses of cysteine and lysine reactivity upon BRAFV600E inhibition, revealed probe-accessible cysteine labeling changes in labeling on KSR2, indicating broader MAPK pathway remodeling. Together, these findings establish ABPP as a powerful chemical biology approach for investigating inhibitor-dependent changes in kinase residue accessibility, providing a framework to explore how conformational dynamics and pathway adaptation shape therapeutic response and resistance in oncogenic signaling networks.

cancer biology↗

bioRxiv2025

Class I histone deacetylases catalyze lysine lactylation

Metabolism and post-translational modifications (PTMs) are intrinsically linked and the number of identified metabolites that can covalently modify proteins continues to increase. This metabolism/PTM crosstalk is especially true for lactate, the product of anaerobic metabolism following glycolysis. Lactate forms an amide bond with the {varepsilon}-amino group of lysine, a modification known as lysine lactylation, or Kla. Multiple independent mechanisms have been proposed in the formation of Kla, including p300/CBP-dependent transfer from lactyl-CoA, via a high-energy intermediate lactoylglutathione species that non-enzymatically lactylates proteins, and several enzymes are reported to have lactyl transferase capability. We recently discovered that class I histone deacetylases (HDACs) 1, 2, and 3 can all reverse their canonical chemical reaction to catalyze lysine {beta}-hydroxybutyrylation. Here we tested the hypothesis that HDACs can also catalyze Kla formation. Using biochemical, pharmacological, and genetic approaches, we found that HDAC-catalyzed lysine lactylation accounts for the majority of Kla formation in cells. Dialysis experiments confirm this is a reversible reaction that depends on lactate concentration. We also directly quantified intracellular lactyl-CoA and found that Kla abundance can be uncoupled from lactyl-CoA levels. Therefore, we propose a model in which the majority of Kla is formed through enzymatic addition of lactate by HDACs 1, 2, and 3.

biochemistry↗

bioRxiv2025

Chemical Tools Based on the Tetrapeptide Sequence of IL-18 Reveals Shared Specificities between Inflammatory and Apoptotic Initiator Caspases

Caspases are a family of cysteine proteases that act as molecular scissors to cleave substrates and regulate biological processes such as programmed cell death and inflammation. Extensive efforts have been made to identify caspase substrates and to determine factors that dictate substrate specificity. We recently discovered that that the human inflammatory caspases (caspases-1, -4, and -5) cleave the cytokines IL-1{beta} and IL-18 in a sequence-dependent manner. Here, we report the development of a new peptide-based probe and inhibitor based on the tetrapeptide sequence of IL-18 (LESD). We found that this inhibitor was most selective and potent at inhibiting caspase-8 activity (IC50 = 50 nM). We also discovered that our LESD-based inhibitor is more potent than the currently used z-IETD-FMK inhibitor that is thought to be the most selective and potent inhibitor of caspase-8. Accordingly, we demonstrate that the LESD based inhibitor prevents caspase-8 activation during Yersinia pseudotuberculosis infection in primary bone-marrow derived macrophages. Furthermore, we characterize the selectivity and potency of currently known substrates and inhibitors for the apoptotic and inflammatory caspases using the same activity units of each caspase. Our findings reveal that VX-765, a known caspase-1 inhibitor, also inhibits caspase-8 (IC50 = 1 {micro}M) and even when specificities are shared, the caspases have different efficiencies and potencies for shared substrates and inhibitors. Altogether, we report the development of new tools that will facilitate the study of caspases and their roles in biology.

biochemistry↗

bioRxiv2024

Intracellular Protein Editing to Enable Incorporation of Non-Canonical Residues into Endogenous Proteins

The ability to study proteins in a cellular context is crucial to our understanding of biology. Here, we report a new technology for "intracellular protein editing", drawing from intein- mediated protein splicing, genetic code expansion, and endogenous protein tagging. This protein editing approach enables us to rapidly and site specifically install residues and chemical handles into a protein of interest. We demonstrate the power of this protein editing platform to edit cellular proteins, inserting epitope peptides, protein-specific sequences, and non-canonical amino acids (ncAAs). Importantly, we employ an endogenous tagging approach to apply our protein editing technology to endogenous proteins with minimal perturbation. We anticipate that the protein editing technology presented here will be applied to a diverse set of problems, enabling novel experiments in live mammalian cells and therefore provide unique biological insights. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/602493v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@8957b8org.highwire.dtl.DTLVardef@de02beorg.highwire.dtl.DTLVardef@1863cb4org.highwire.dtl.DTLVardef@1fb8bc0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

The Interplay of Acetylation and Ubiquitination Controls PRMT1 Homeostasis

PRMT1 plays many important roles in both normal and disease biology, thus understanding its regulation is crucial. Herein, we report the role of p300-mediated acetylation at K228 in triggering PRMT1 degradation through FBXL17-mediated ubiquitination. Utilizing mass-spectrometry, cellular biochemistry, and genetic code-expansion technologies, we elucidate a crucial mechanism independent of PRMT1 transcript levels. These results underscore the significance of acetylation in governing protein stability and expand our understanding of PRMT1 homeostasis. By detailing the molecular interplay between acetylation and ubiquitination involved in PRMT1 degradation, this work contributes to broader efforts in deciphering post-translational mechanisms that influence protein homeostasis.

biochemistry↗

bioRxiv2023

Universal open MHC-I molecules for rapid peptide loading and enhanced complex stability across HLA allotypes

The polymorphic nature and intrinsic instability of class I major histocompatibility complex (MHC-I) and MHC-like molecules loaded with suboptimal peptides, metabolites, or glycolipids presents a fundamental challenge for identifying disease-relevant antigens and antigen-specific T cell receptors (TCRs), hindering the development of autologous therapeutics. Here, we leverage the positive allosteric coupling between the peptide and light chain ({beta}2 microglobulin, {beta}2m) subunits for binding to the MHC-I heavy chain (HC) through an engineered disulfide bond bridging conserved epitopes across the HC/{beta}2m interface, to generate conformationally stable, open MHC-I molecules. Biophysical characterization shows that open MHC-I molecules are properly folded protein complexes of enhanced thermal stability compared to the wild type, when loaded with low- to intermediate-affinity peptides. Using solution NMR, we characterize the effects of the disulfide bond on the conformation and dynamics of the MHC-I structure, ranging from local changes in {beta}2m interacting sites of the peptide binding groove to long-range effects on the 2-1 helix and 3 domain. The interchain disulfide bond stabilizes empty MHC-I molecules in a peptide-receptive, open conformation to promote peptide exchange across multiple human leucocyte antigen (HLA) allotypes, covering representatives from five HLA-A, six HLA-B supertypes, and oligomorphic HLA-Ib molecules. Our structural design, combined with conditional {beta}-peptide ligands, provides a universal platform for generating ready-to-load MHC-I systems of enhanced stability, enabling a range of approaches to screen antigenic epitope libraries and probe polyclonal TCR repertoires in the context of highly polymorphic HLA-I allotypes, as well as oligomorphic nonclassical molecules. Significance StatementWe outline a structure-guided approach for generating conformationally stable, open MHC-I molecules with enhanced ligand exchange kinetics spanning five HLA-A, all HLA-B supertypes, and oligomorphic HLA-Ib allotypes. We present direct evidence of positive allosteric cooperativity between peptide binding and {beta}2m association with the heavy chain by solution NMR and HDX-MS spectroscopy. We demonstrate that covalently linked {beta}2m serves as a conformational chaperone to stabilize empty MHC-I molecules in a peptide-receptive state, by inducing an open conformation and preventing intrinsically unstable heterodimers from irreversible aggregation. Our study provides structural and biophysical insights into the conformational properties of MHC-I ternary complexes, which can be further applied to improve the design of ultra-stable, universal ligand exchange systems in a pan-HLA allelic setting.

biochemistry↗