Biology subjects

Backus, K. M.

Publications and source records attributed to Backus, K. M..

8 recordsLinked to original sources

bioRxiv2026

A unified photosensitizer platform for in situ DNA, RNA, and protein directed proximity labeling

Cells depend on the spatial organization of proteins, RNA, and DNA into discrete subcellular compartments. Previous methods have largely centered on measuring spatial organization based on only one of these biomolecular classes at a time. Here, we demonstrate that POCA photocatalytic proximity labeling can serve as a unified photosensitizer-based platform for profiling the proximal proteomes of protein, RNA, and DNA targets within a single experimental framework. We show that POCA can harness standard immunofluorescence or in situ hybridization workflows to specifically target organic fluorophore photosensitizers to intracellular targets for proximity labeling in fixed cells. POCA-targeted proximity labeling requires minimal cellular input and does not require genetic engineering. Additionally, POCA photosensitizers are selected to also be fluorescent, enabling direct confirmation of on-target localization by imaging prior to proteomic analysis. To demonstrate broad utility, we apply POCA across multiple molecular targets spanning protein, RNA, and genomic DNA, including components of the nuclear pore complex, nucleolus, nuclear speckles, telomeres, and pericentromeric heterochromatin. By anchoring proximity labeling to both a protein and an RNA within the same nuclear compartment, we resolve shared and distinct proximal proteomes from orthogonal molecular perspectives.

cell biology↗

bioRxiv2026

A Scalable Design for Proximity-Inducing Molecules

Chimeric molecules, which bring together an effector enzyme and a protein-of-interest (POI) to add/remove post-translational modifications (PTMs), are furnishing transformative modalities (e.g., PROTACs). However, these chimeras scalability is limited as they employ rare, non-inhibitory binders of effectors. We report GRoup-transfer chimeras for Inducing Proximity (GRIPs) that employ abundantly available effectors inhibitors to append POI binder on the effector using group-transfer handles. To demonstrate scalability, we develop 6 GRIPs classes for 3 PTMs utilizing diverse inhibitor, spanning 16 effector-POI pairs. Furthermore, we report a toolbox of 42 tunable group-transfer handles for Cys/Lys residues and [~]5000 inhibitor-residue pairs for diverse effectors. Using global proteomics, we confirm the specificity for group transfer and PTM editing. GRIPs endowed new functionalities to POI drugs, including preventing rebound signaling upon drug withdrawal, a more potent/persistent inhibition, and inhibitor-induced pathway activation in 4 fully-endogenous systems. In diverse hemi-endogenous systems (tagged POI), GRIPs induced condensate formation with reduced off-targets, cleared pathogenic PTMs, and initiated PTM crosstalk. Overall, GRIPs provide a scalable and versatile platform for PTM editing. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/706349v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@132f70dorg.highwire.dtl.DTLVardef@f9aa62org.highwire.dtl.DTLVardef@12143edorg.highwire.dtl.DTLVardef@f296d4_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Chemoproteomics identifies proteoform-selective caspase-2 inhibitors

Caspases are a highly conserved family of cysteine-aspartyl proteases known for their essential roles in regulating apoptosis, inflammation, cell differentiation, and proliferation. Complementary to genetic approaches, small-molecule probes have emerged as useful tools for modulating caspase activity. However, due to the high sequence and structure homology of all twelve human caspases, achieving selectivity remains a central challenge for caspase-directed small-molecule inhibitor development efforts. Here, using mass spectrometry-based chemoproteomics, we first identify a highly reactive non-catalytic cysteine that is unique to caspase-2. By combining both gel-based activity-based protein profiling (ABPP) and a tobacco etch virus (TEV) protease activation assay, we then identify covalent lead compounds that react preferentially with this cysteine and afford a complete blockade of caspase-2 activity. Inhibitory activity is restricted to the zymogen or precursor form of monomeric caspase-2. Focused analogue synthesis combined with chemoproteomic target engagement analysis in cellular lysates and in cells yielded both pan-caspase reactive molecules and caspase-2 selective lead compounds together with a structurally matched inactive control. Application of this focused set of tool compounds to stratify caspase contributions to initiation of intrinsic apoptosis, supports compensatory caspase-9 activity in the context of caspase-2 inactivation. More broadly, our study highlights future opportunities for the development of proteoform-selective caspase inhibitors that target non-conserved and non-catalytic cysteine residues.

biochemistry↗

bioRxiv2023

Multi-omic stratification of the missense variant cysteinome

Cancer genomes are rife with genetic variants; one key outcome of this variation is gain-of-cysteine, which is the most frequently acquired amino acid due to missense variants in COSMIC. Acquired cysteines are both driver mutations and sites targeted by precision therapies. However, despite their ubiquity, nearly all acquired cysteines remain uncharacterized. Here, we pair cysteine chemoproteomics--a technique that enables proteome-wide pinpointing of functional, redox sensitive, and potentially druggable residues--with genomics to reveal the hidden landscape of cysteine acquisition. For both cancer and healthy genomes, we find that cysteine acquisition is a ubiquitous consequence of genetic variation that is further elevated in the context of decreased DNA repair. Our chemoproteogenomics platform integrates chemoproteomic, whole exome, and RNA-seq data, with a customized 2-stage false discovery rate (FDR) error controlled proteomic search, further enhanced with a user-friendly FragPipe interface. Integration of CADD predictions of deleteriousness revealed marked enrichment for likely damaging variants that result in acquisition of cysteine. By deploying chemoproteogenomics across eleven cell lines, we identify 116 gain-of-cysteines, of which 10 were liganded by electrophilic druglike molecules. Reference cysteines proximal to missense variants were also found to be pervasive, 791 in total, supporting heretofore untapped opportunities for proteoform-specific chemical probe development campaigns. As chemoproteogenomics is further distinguished by sample-matched combinatorial variant databases and compatible with redox proteomics and small molecule screening, we expect widespread utility in guiding proteoform-specific biology and therapeutic discovery.

biochemistry↗

bioRxiv2023

Distributable, Metabolic PET Reporting of Tuberculosis

Tuberculosis remains a large global disease burden for which treatment regimens are protracted and monitoring of disease activity difficult. Existing detection methods rely almost exclusively on bacterial culture from sputum which limits sampling to organisms on the pulmonary surface. Advances in monitoring tuberculous lesions have utilized the common glucoside [18F]FDG, yet lack specificity to the causative pathogen Mycobacterium tuberculosis (Mtb) and so do not directly correlate with pathogen viability. Here we show that a close mimic that is also positron-emitting of the non-mammalian Mtb disaccharide trehalose - 2-[18F]fluoro-2-deoxytrehalose ([18F]FDT) - can act as a mechanism-based enzyme reporter in vivo. Use of [18F]FDT in the imaging of Mtb in diverse models of disease, including non-human primates, successfully co-opts Mtb-specific processing of trehalose to allow the specific imaging of TB-associated lesions and to monitor the effects of treatment. A pyrogen-free, direct enzyme-catalyzed process for its radiochemical synthesis allows the ready production of [18F]FDT from the most globally-abundant organic 18F-containing molecule, [18F]FDG. The full, pre-clinical validation of both production method and [18F]FDT now creates a new, bacterium-specific, clinical diagnostic candidate. We anticipate that this distributable technology to generate clinical-grade [18F]FDT directly from the widely-available clinical reagent [18F]FDG, without need for either bespoke radioisotope generation or specialist chemical methods and/or facilities, could now usher in global, democratized access to a TB-specific PET tracer.

biochemistry↗

bioRxiv2023

Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome

Proteinaceous cysteines function as essential sensors of cellular redox state. Consequently, defining the cysteine redoxome is a key challenge for functional proteomic studies. While proteome-wide inventories of cysteine oxidation state are readily achieved using established, widely adopted proteomic methods such as OxiCat, Biotin Switch, and SP3-Rox, they typically assay bulk proteomes and therefore fail to capture protein localization-dependent oxidative modifications. To obviate requirements for laborious biochemical fractionation, here, we develop and apply an unprecedented two step cysteine capture method to establish the Local Cysteine Capture (Cys-LoC), and Local Cysteine Oxidation (Cys-LOx) methods, which together yield compartment-specific cysteine capture and quantitation of cysteine oxidation state. Benchmarking of the Cys-LoC method across a panel of subcellular compartments revealed more than 3,500 cysteines not previously captured by whole cell proteomic analysis. Application of the Cys-LOx method to LPS stimulated murine immortalized bone marrow-derived macrophages (iBMDM), revealed previously unidentified mitochondria-specific inflammation-induced cysteine oxidative modifications including those associated with oxidative phosphorylation. These findings shed light on post-translational mechanisms regulating mitochondrial function during the cellular innate immune response.

biochemistry↗

bioRxiv2020

From Chemoproteomic-Detected Amino Acids to Genomic Coordinates: Insights into Precise Multi-omic Data Integration

The integration of proteomic, transcriptomic, and genetic-variant annotation data will improve our understanding genotype-phenotype associations. Due, in part, to challenges associated with accurate inter-database mapping, such multi-omic studies have not extended to chemoproteomics, a method that measure the intrinsic reactivity and potential druggability of nucleophilic amino acid side chains. Here, we evaluated two mapping approaches to match chemoproteomic-detected cysteine and lysine residues with their genetic coordinates. Our analysis reveals that databases update cycles and reliance on stable identifiers can lead to pervasive misidentification of labeled residues. Enabled by this examination of mapping strategies, we then integrated our chemoproteomic data with in silico generated predictions of genetic variant pathogenicity, which revealed that codons of highly reactive cysteines are enriched for genetic variants that are predicted to be more deleterious. Our study provides a roadmap for more precise inter-database comparisons and points to untapped opportunities to improve the predictive power of pathogenicity scores and to advance prioritization of putative druggable sites through integration of predictions of pathogenicity with chemoproteomic datasets.

systems biology↗

bioRxiv2019

Integrative x-ray structure and molecular modeling for the rationalization of procaspase-8 inhibitor potency and selectivity

Caspases are a critical class of proteases involved in regulating programmed cell death and other biological processes. Selective inhibitors of individual caspases, however, are lacking, due in large part to the high structural similarity found in the active sites of these enzymes. We recently discovered a small-molecule inhibitor, 63-R, that covalently binds the zymogen, or inactive precursor (pro-form), of caspase-8, but not other caspases, pointing to an untapped potential of procaspases as targets for chemical probes. Realizing this goal would benefit from a structural understanding of how small molecules bind to and inhibit caspase zymogens. There have, however, been very few reported procaspase structures. Here, we employ x-ray crystallography to elucidate a procaspase-8 crystal structure in complex with 63-R, which reveals large conformational changes in active-site loops that accommodate the intramolecular cleavage events required for protease activation. Combining these structural insights with molecular modeling and mutagenesis-based biochemical assays, we elucidate key interactions required for 63-R inhibition of procaspase-8. Our findings inform the mechanism of caspase activation and its disruption by small molecules, and, more generally, have implications for the development of small molecule inhibitors and/or activators that target alternative (e.g., inactive precursor) protein states to ultimately expand the druggable proteome.

biochemistry↗