Biology subjects

Backus, K.

Publications and source records attributed to Backus, K..

5 recordsLinked to original sources

bioRxiv2024

An activation-based high throughput screen identifies caspase-10 inhibitors

Caspases are a family of highly homologous cysteine proteases that play critical roles in inflammation and apoptosis. Small molecule inhibitors are useful tools for studying caspase biology, complementary to genetic approaches. However, achieving inhibitor selectivity for individual members of this highly homologous enzyme family remains a major challenge in developing such tool compounds. Prior studies have revealed that one strategy to tackle this selectivity gap is to target the precursor or zymogen forms of individual caspases, which share reduced structural homology when compared to active proteases. To establish a screening assay that favors the discovery of zymogen-directed caspase-10 selective inhibitors, we engineered a low-background and high-activity tobacco etch virus (TEV)--activated caspase-10 protein. We then subjected this turn-on protease to a high-throughput screen of approximately 100,000 compounds, with an average Z value of 0.58 across all plates analyzed. Counter screening, including against TEV protease, delineated bona fide procaspase-10 inhibitors. Confirmatory studies identified a class of low-pH activatable caspase-10 inhibitors. In parallel, mode-of-action studies revealed that pifithrin-{micro} (PFT{micro}), a reported TP53 inhibitor, also functions as a promiscuous caspase inhibitor. Both inhibitor classes showed preferential zymogen inhibition. Given the generalized utility of activation assays, we expect our screening platform to have widespread applications in identifying state-specific protease inhibitors.

biochemistry↗

bioRxiv2024

Cholesterol binding to VCAM-1 promotes vascular inflammation

Hypercholesterolemia has long been implicated in endothelial cell (EC) dysfunction, but the mechanisms by which excess cholesterol causes vascular pathology are incompletely understood. Here we used a cholesterol-mimetic probe to map cholesterol-protein interactions in primary human ECs and discovered that cholesterol binds to and stabilizes the adhesion molecule VCAM-1. We show that accessible plasma membrane (PM) cholesterol in ECs is acutely responsive to inflammatory stimuli and that the nonvesicular cholesterol transporter Aster-A regulates VCAM-1 stability in activated ECs by controlling the size of this pool. Deletion of Aster-A in ECs increases VCAM-1 protein, promotes immune cell recruitment to vessels, and impairs pulmonary immune homeostasis. Conversely, depleting cholesterol from the endothelium in vivo dampens VCAM-1 induction in response to inflammatory stimuli. These findings identify cholesterol binding to VCAM-1 as a key step during EC activation and provide a biochemical explanation for the ability of excess membrane cholesterol to promote immune cell recruitment to the endothelium.

physiology↗

bioRxiv2024

Lipid- and protein-directed photosensitizer proximity labeling captures the cholesterol interactome

The physical properties of cellular membranes, including fluidity and function, are influenced by protein and lipid interactions. In situ labeling chemistries, most notably proximity-labeling interactomics are well suited to characterize these dynamic and often fleeting interactions. Established methods require distinct chemistries for proteins and lipids, which limits the scope of such studies. Here we establish a singlet-oxygen-based photocatalytic proximity labeling platform (POCA) that reports intracellular interactomes for both proteins and lipids with tight spatiotemporal resolution using cell-penetrant photosensitizer reagents. Using both physiologically relevant lipoprotein-complexed probe delivery and genetic manipulation of cellular cholesterol handling machinery, cholesterol-directed POCA captured established and unprecedented cholesterol binding proteins, including protein complexes sensitive to intracellular cholesterol levels and proteins uniquely captured by lipoprotein uptake. Protein-directed POCA accurately mapped known intracellular membrane complexes, defined sterol-dependent changes to the non-vesicular cholesterol transport protein interactome, and captured state-dependent changes in the interactome of the cholesterol transport protein Aster-B. More broadly, we find that POCA is a versatile interactomics platform that is straightforward to implement, using the readily available HaloTag system, and fulfills unmet needs in intracellular singlet oxygen-based proximity labeling proteomics. Thus, we expect widespread utility for POCA across a range of interactome applications, spanning imaging to proteomics.

biochemistry↗

bioRxiv2023

Pervasive aggregation and depletion of host and viral proteins in response to cysteine-reactive electrophilic compounds

Protein homeostasis is tightly regulated, with damaged or misfolded proteins quickly eliminated by the proteasome and autophagosome pathways. By co-opting these processes, targeted protein degradation technologies enable pharmacological manipulation of protein abundance. Recently, cysteine-reactive molecules have been added to the degrader toolbox, which offer the benefit of unlocking the therapeutic potential of undruggable protein targets. The proteome-wide impact of these molecules remains to be fully understood and given the general reactivity of many classes of cysteine-reactive electrophiles, on- and off-target effects are likely. Using chemical proteomics, we identified a cysteine-reactive small molecule degrader of the SARS-CoV-2 non- structural protein 14 (nsp14), which effects degradation through direct modification of cysteines in both nsp14 and in host chaperones together with activation of global cell stress response pathways. We find that cysteine-reactive electrophiles increase global protein ubiquitylation, trigger proteasome activation, and result in widespread aggregation and depletion of host proteins, including components of the nuclear pore complex. Formation of stress granules was also found to be a remarkably ubiquitous cellular response to nearly all cysteine-reactive compounds and degraders. Collectively, our study sheds light on complexities of covalent target protein degradation and highlights untapped opportunities in manipulating and characterizing proteostasis processes via deciphering the cysteine-centric regulation of stress response pathways.

biochemistry↗

bioRxiv2023

Defining the Cell Surface Cysteinome using Two-step Enrichment Proteomics

The plasma membrane proteome is a rich resource of functional and therapeutically relevant protein targets. Distinguished by high hydrophobicity, heavy glycosylation, disulfide-rich sequences, and low overall abundance, the cell surface proteome remains undersampled in established proteomic pipelines, including our own cysteine chemoproteomics platforms. Here we paired cell surface glycoprotein capture with cysteine chemoproteomics to establish a two-stage enrichment method that enables chemoproteomic profiling of cell Surface Cysteinome. Our "Cys-Surf" platform captures >2,800 total membrane protein cysteines in 1,046 proteins, including 1,907 residues not previously captured by bulk proteomic analysis. By pairing Cys-Surf with an isotopic chemoproteomic readout, we uncovered 821 total ligandable cysteines, including known and novel sites. Cys-Surf also robustly delineates redox-sensitive cysteines, including cysteines prone to activation-dependent changes to cysteine oxidation state and residues sensitive to addition of exogenous reductants. Exemplifying the capacity of Cys-Surf to delineate functionally important cysteines, we identified a redox sensitive cysteine in the low-density lipoprotein receptor (LDLR) that impacts both the protein localization and uptake of LDL particles. Taken together, the Cys-Surf platform, distinguished by its two-stage enrichment paradigm, represents a tailored approach to delineate the functional and therapeutic potential of the plasma membrane cysteinome.

biochemistry↗