Biology subjects

Burslem, G. M.

Publications and source records attributed to Burslem, G. M..

10 recordsLinked to original sources

bioRxiv2026

Mitochondrial copper stabilizes lipoylated TCA cycle proteins to sustain metabolism and proliferation

Copper (Cu) is an essential cofactor for mitochondrial cytochrome c oxidase, yet whether it directly regulates mitochondrial metabolism beyond respiration remains unclear. Here we show that mitochondrial Cu, delivered by SLC25A3, is required to maintain the stability of lipoylated TCA cycle proteins. Loss of Slc25a3 or pharmacological Cu depletion selectively destabilized the lipoylated E2 subunits of mitochondrial dehydrogenases and the lipoylation enzymes LIPT1 and LIPT2, an effect not reproduced by acute electron transport chain inhibition. Mechanistically, we find that Cu directly engages the reduced lipoyl moiety using chemical probes and synthetic peptide approaches. Cu depletion impaired PDH and OGDH activity, rewired TCA cycle metabolism, and imposed a dependence on pyruvate carboxylase for anaplerosis. This metabolic defect depleted aspartate, suppressed mTORC1 signaling, and limited proliferation. Conversely, selective delivery of Cu to the mitochondria restored lipoylation, TCA cycle function, and cell growth. Together, these findings identify mitochondrial Cu as a structural regulator of the lipoylation machinery and reveal a direct link between Cu homeostasis and central carbon metabolism.

cell biology↗

bioRxiv2025

Quinazolinone and Phthalazinone Inhibitors of the HDAC6/Ubiquitin Protein-Protein Interaction

Histone deacetylase 6 (HDAC6) is a class IIb histone deacetylase that regulates diverse cytosolic acetylation through its two catalytic deacetylase domains and a C-terminal zinc finger ubiquitin-binding domain (ZnF-UBD). This ZnF-UBD mediates key protein-protein interactions (PPIs) that couple deacetylation and ubiquitin-dependent degradation. While most HDAC6 inhibitors target the catalytic domains, the ZnF-UBD represents an underexplored target. Here, we validate previously reported small-molecule inhibitors of the HDAC6 ZnF-UBD/ubiquitin interaction and describe novel N-alkyl moieties based on quinazolinone and phthalazinone scaffolds. Starting from known quinazolinone and phthalazinone scaffolds, a literature and modeling-guided scaffold hop revealed potential for an extended phthalazinone series. Results obtained both in fluorescence polarization (FP) and differential scanning fluorimetry (DSF) confirm this hypothesis. Additionally, late-stage diversification yields compounds with improved predicted physicochemical properties. Finally, machine-learning-based co-folding affinity predictions correlate with experimental IC{square}{square} rank order, highlighting their utility in PPI inhibitor design. These studies continue expanding the chemical space of HDAC6 ZnF-UBD inhibitors and build upon existing foundations for future therapeutic and mechanistic exploration of HDAC6- ubiquitin signaling.

biochemistry↗

bioRxiv2025

Coordination of nuclear RNA processing by speckle-localized kinase TAOK2

Nuclear speckles are membraneless organelles that act as active splicing hubs especially at sites of high transcription. Emerging views of this dynamic subnuclear structure place it as a hub of RNA processing, impacting steps from transcription to export. To manage this complex microcosm of RNA metabolism, nuclear speckles also require kinases and phosphorylation to execute their functions. The nuclear speckle-localized kinase, TAOK2, mediates the splicing and export of viral transcripts at the nuclear speckle, but its role in the processing of cellular transcripts was unknown. We used siRNA knockdown of TAOK2 and assessed RNA transcripts in both whole cell and nucleocytoplasmic fractions to characterize the complete endogenous effects of TAOK2. We found that TAOK2 knockdown impacts over 10% of the transcriptome, through changes in alternative splicing, export and transcript abundance. Cellular and biochemical phosphoproteomics further revealed nuclear speckle scaffolding proteins SRRM1 and SRRM2 as potential direct phosphorylation targets of TAOK2, mediating its large effects on speckle integrity and speckle-localized splicing. Indeed, knockdown of TAOK2 perturbs almost all speckle-resident serine/arginine (SR)-rich proteins while leaving heterogeneous ribonucleoproteins unperturbed. Altogether, we propose that phosphorylation of SRRM1/2 by TAOK2 plays a structural maintenance role that impacts SR protein-driven exon inclusion at the nuclear speckle.

molecular biology↗

bioRxiv2025

Repositioning of polyubiquitin alters the pathologic tau filament structure

Structurally diverse tau filaments form proteinaceous aggregates in a heterogeneous group of neurodegenerative diseases called tauopathies1. The factors extrinsic to the highly ordered core structure that influence tau filament stability are not well understood. Here, we found that polyubiquitinated tau filaments from Alzheimers disease and vacuolar tauopathy human brain tissue exhibit distinct seeding patterns in mice, in association with differences in tau filament ultrastructure determined by cryo-electron microscopy. Interestingly, chemical modulation of the polarity of polyubiquitin adjacent to the tau core with the small molecule ubistatin B resulted in the repositioning of poorly structured densities towards positively charged residues on the highly structured core filament, leading to shifting of the protofilament-protofilament interface of certain vacuolar tauopathy tau filaments. These results suggest that the structure of tau filaments that are associated with different seeding activities in vivo can be influenced by post-translational modifications.

neuroscience↗

bioRxiv2024

A selective S-acyltransferase inhibitor suppresses tumor growth

S-acyltransferases play integral roles in essential physiological processes including regulation of oncogenic signaling pathways. While discovered over 40 years ago the field still lacks specific S-acylation inhibitors thus the potential benefit of pharmacologically targeting S-acyltransferases for human disease is still unknown. Here we report the identification of an orally bioavailable acyltransferase inhibitor SD-066-4 that inhibits the acyltransferase ZDHHC20. We identified a specific alanine residue that accommodates the methyl group of SD-066-4, thus providing isoform selectivity. SD-066-4 stably reduces EGFR S-acylation in Kras mutant cells and blocks the growth of Kras mutant lung tumors extending overall survival. We find that lung cancer patients harboring deletions in ZDHHC20 or ZDHHC14 concurrent with Kras alterations have a significant survival benefit, underscoring the translational importance of these enzymes.

cancer biology↗

bioRxiv2023

Reversible histone deacetylase activity catalyzes lysine acylation

Starvation and low carbohydrate diets lead to the accumulation of the ketone body, {beta}-hydroxybutyrate (BHB), whose blood concentrations increase more than 10-fold into the millimolar range. In addition to providing a carbon source, BHB accumulation triggers lysine {beta}-hydroxybutyrylation (Kbhb) of proteins via unknown mechanisms. As with other lysine acylation events, Kbhb marks can be removed by histone deacetylases (HDACs). Here, we report that class I HDACs unexpectedly catalyze protein lysine modification with {beta}-hydroxybutyrate (BHB). Mutational analyses of the HDAC2 active site reveal a shared reliance on key amino acids for classical deacetylation and non-canonical HDAC-catalyzed {beta}-hydroxybutyrylation. Also consistent with reverse HDAC activity, Kbhb formation is driven by mass action and substrate availability. This reverse HDAC activity is not limited to BHB but also extends to multiple short-chain fatty acids. The reversible activity of class I HDACs described here represents a novel mechanism of PTM deposition relevant to metabolically-sensitive proteome modifications.

molecular biology↗

bioRxiv2023

Pooled tagging and hydrophobic targeting of endogenous proteins for unbiased mapping of unfolded protein responses

System-level understanding of proteome organization and function requires methods for direct visualization and manipulation of proteins at scale. We developed an approach enabled by high-throughput gene tagging for the generation and analysis of complex cell pools with endogenously tagged proteins. Proteins are tagged with HaloTag to enable visualization or direct perturbation. Fluorescent labeling followed by in situ sequencing and deep learning-based image analysis identifies the localization pattern of each tag, providing a birds-eye-view of cellular organization. Next, we use a hydrophobic HaloTag ligand to misfold tagged proteins, inducing spatially restricted proteotoxic stress that is read out by single cell RNA sequencing. By integrating optical and perturbation data, we map compartment-specific responses to protein misfolding, revealing inter-compartment organization and direct crosstalk, and assigning proteostasis functions to uncharacterized genes. Altogether, we present a powerful and efficient method for large-scale studies of proteome dynamics, function, and homeostasis.

cell biology↗

bioRxiv2023

Pooled endogenous protein tagging and recruitment for scalable discovery of effectors for induced proximity therapeutics

The field of induced proximity therapeutics is in its ascendancy but is limited by a lack of scalable tools to systematically explore effector-target protein pairs in an unbiased manner. Here, we combined Scalable POoled Targeting with a LIgandable Tag at Endogenous Sites (SPOTLITES) for the high-throughput tagging of endogenous proteins, with generic small molecule-based protein recruitment to screen for novel proximity-based effectors. We apply this methodology in two orthogonal screens for targeted protein degradation: the first using fluorescence to monitor target protein levels directly, and the second using a cellular growth phenotype that depends on the degradation of an essential protein. Our screens revealed a multitude of potential new effector proteins for degradation and converged on members of the CTLH complex which we demonstrate potently induce degradation. Altogether, we introduce a platform for pooled induction of endogenous protein-protein interactions that can be used to expand our toolset of effector proteins for targeted protein degradation and other forms of induced proximity.

cell biology↗

bioRxiv2023

A Chicken Tapasin ortholog can chaperone empty HLA molecules independently of other peptide-loading components

Human Tapasin (hTapasin) is the main chaperone of MHC-I molecules, enabling peptide loading and antigen repertoire optimization across HLA allotypes. However, it is restricted to the endoplasmic reticulum (ER) lumen as part of the protein loading complex (PLC) and therefore is highly unstable when expressed in recombinant form. Additional stabilizing co-factors such as ERp57 are required to catalyze peptide exchange in vitro, limiting uses for the generation of pMHC-I molecules of desired antigen specificities. Here, we show that the chicken Tapasin (chTapasin) ortholog can be expressed recombinantly at high yields in stable form, independently of co-chaperones. chTapasin can bind the human HLA-B*37:01 with low micromolar-range affinity to form a stable tertiary complex. Biophysical characterization by methyl-based NMR methods reveals that chTapasin recognizes a conserved {beta}2m epitope on HLA-B*37:01, consistent with previously solved X-ray structures of hTapasin. Finally, we provide evidence that the B*37:01/chTapasin complex is peptide-receptive and can be dissociated upon binding of high-affinity peptides. Our results highlight the use of chTapasin as a stable scaffold for future protein engineering applications aiming to expand the ligand exchange function on human MHC-I and MHC-like molecules.

immunology↗

bioRxiv2020

Structural and molecular determinants of CCS-mediated copper activation of MEK1/2

SummaryNormal physiology relies on the precise coordination of intracellular signal transduction pathways that respond to nutrient availability to balance cell growth and cell death. We recently established a critical mechanistic function for the redox-active micronutrient copper (Cu) in the canonical mitogen activated protein kinase (MAPK) pathway at the level of MEK1 and MEK2. Here we report the X-ray crystal structure of Cu-MEK1 and reveal active site chemical ligands and oxidation state specificity for MEK1 Cu coordination. Mechanistically, the Cu chaperone CCS selectively bound to and facilitated Cu transfer to MEK1. Mutations in MEK1 that disrupt Cu(I) affinity or a CCS small molecule inhibitor reduced Cu-stimulated MEK1 kinase activity. These atomic and molecular level data provide the first mechanistic insights of Cu kinase signaling and could be exploited for the development of novel MEK1/2 inhibitors that either target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS.Competing Interest StatementD.C.B holds ownership in Merlon Inc. D.C.B. is an inventor on the patent application 20150017261 entitled Methods of treating and preventing cancer by disrupting the binding of copper in the MAP kinase pathway. No potential conflicts of interest were disclosed by the other authors.View Full Text

biochemistry↗