Biology subjects

Farley, S. E.

Publications and source records attributed to Farley, S. E..

8 recordsLinked to original sources

bioRxiv2026

Identifying Novel Estrogenic Mitochondrial Targets in Hypothalamic Proopiomelanocortin Neurons by Chemoproteomics

Loss of estrogens at menopause is linked to impaired brain metabolism and increased risk of Alzheimers disease (AD). However, estrogen replacement therapies are limited due to the deleterious effects of estrogen on peripheral organs and increased risk of vascular dementia. We have developed a non-steroidal estrogenic compound, STX, which does not bind to the classical estrogen receptors and {beta}, but mimics estrogenic signaling in the central nervous system (CNS) without the peripheral reproductive actions. STX is protective against neurodegeneration in stroke and AD models, but its molecular targets are unknown. Here, we identified and validated STX neural targets using chemoproteomic, molecular biological, electrophysiological and metabolic assays of hypothalamic proopiomelanocortin (POMC) neurons. Chemoproteomic profiling identified voltage dependent anion channels (VDAC1-3) as major intracellular binding partners in mHypo43 (POMC) cells. Based on quantitative single-cell PCR, Vdac2 was identified as the dominant isoform in female hypothalamic POMC neurons. Seahorse metabolic flux analyses showed that STX potently increased glycolysis, oxidative respiration and mitochondrial ATP production in mHypo43 cells. Nanomolar concentrations of STX enhanced VDAC2 voltage-dependent gating in reconstituted lipid membranes and shifted the low-conductance states toward anion selectivity, consistent with increased ATP flux. Together, these findings reveal a mechanism for the neuroprotective effects of STX through enhancing mitochondrial bioenergetics and modulating VDAC channel properties, potentially increasing cellular energy stores. Therefore, this work identifies previously unrecognized estrogenic mitochondrial targets and provides a mechanistic basis for the neuroprotective actions of STX relevant to menopause-associated brain vulnerability.

neuroscience↗

bioRxiv2026

An Integrated Method for Profiling Lipid-Protein Interactions Using Multifunctional Lipid Probes

Cellular lipids shape health and disease through specific protein interactions, yet lipid-protein networks remain poorly defined. Despite rapid advances in functional lipid probes, the field still lacks a practical, dedicated protocol for conducting lipid-protein interaction studies. We describe detailed methods for determining lipid interactomes from cells using multifunctionalized lipid derivatives. We provide protocols that detail 1) how to treat cells with lipid derivatives and perform photochemistry to obtain lipid-protein conjugates; 2) how to perform click chemistry with a fluorophore and observe lipid-protein conjugates by in-gel fluorescence; 3) how to perform click chemistry with azide beads and prepare lipid-protein conjugates for proteomic analysis. We provide context on important parameters for each step and include guidelines for controls, as well as suggestions for troubleshooting based on common problems encountered during the preparation of this protocol. This protocol enables mapping lipid interactomes across diverse biological systems. The entire workflow from cell treatment to complete proteomic sample preparation requires [~]15 hours over four days, depending on the type of experimental readout (in-gel fluorescence or proteomics), and the usage of pause points. Practitioners are expected to be familiar with standard biochemical techniques, such as sterile sample handling and tissue culture and gel electrophoresis. Additional skills are needed for mass spectrometric analysis, and collaboration with a proteomics core facility is recommended. The described procedures uniquely enable the identification of the protein interactors (the interactome) of select lipid species, providing for a major shift in the characterization of the biological roles of lipids in cellular systems.

biochemistry↗

bioRxiv2025

Multi-Omic Profiling Identifies Conserved Metabolic Pathways Critical for SARS-CoV-2 Variants Infection

AO_SCPLOWBSTRACTC_SCPLOWThe rapid evolution of SARS-CoV-2 has led to the emergence of numerous variants with enhanced transmissibility and immune evasion. Despite widespread vaccination, infections persist, and the mechanisms by which SARS-CoV-2 reprograms host metabolism remain incompletely understood. Here, we investigated whether virus-induced lipid remodeling is conserved across variants and whether changes in lipid abundance correlate with alterations in lipid biosynthetic enzymes. Using global untargeted lipidomics and quantitative proteomics, we analyzed A549-ACE2 cells infected with the Delta (B.1.617.2) or Omicron (B.1.1.529) variants and compared them to cells infected with the ancestral WA1 strain. In parallel, we conducted quantitative proteomics to assess virus-induced changes in the host proteome. Our results reveal that SARS-CoV-2 drives a remarkably consistent pattern of metabolic rewiring at both the lipidomic and proteomic levels across all three variants. We mapped changes in the expression of host metabolic enzymes and compared these to corresponding shifts in lipid abundance. This integrative analysis identified key host proteins involved in virus-mediated lipid remodeling, including fatty acid synthase (FASN), lysosomal acid lipase (LIPA), and ORM1-like protein 2 (ORMDL2). Together, these findings highlight conserved metabolic dependencies of SARS-CoV-2 variants and underscore host lipid metabolism as a potential target for broad-spectrum antiviral strategies.

systems biology↗

bioRxiv2024

Trifunctional fatty acid derivatives demonstrate the impact of diazirine placement

Functionalized lipid probes are a critical new tool to interrogate the function of individual lipid species, but the structural parameters that constrain their utility have not been thoroughly described. Here, we synthesize three palmitic acid derivatives with a diazirine at different positions on the acyl chain and examine their metabolism, subcellular localization, and protein interactions. We demonstrate that while they produce very similar metabolites and subcellular distributions, probes with the diazirine at either end pulldown distinct subsets of proteins after photo-crosslinking. This highlights the importance of thoughtful diazirine placement when developing probes based on biological molecules.

biochemistry↗

bioRxiv2023

Trifunctional sphinganine: a new tool to dissect sphingolipid function

Functions of the sphingolipids sphingosine and sphinganine in cells are not well established. While some signaling roles for sphingosine have been elucidated, the closely related sphinganine has been described only insofar as it does not elicit many of the same signaling responses. The underlying mechanisms behind the cell biological differences between these lipids are not well understood. Here, we prepared multifunctionalized derivatives of the two lipid species that only differ in a single double bond of the carbon backbone. Using these novel probes, we were able to define their spatiotemporal distribution within cells. Furthermore, we used these tools to systematically map the protein interactomes of both lipids. The lipid-protein conjugates, prepared through photo-crosslinking in live cells and extraction via click chemistry to azide beads, revealed significant differences in the captured proteins, highlighting their distinct roles in various cellular processes. This work elucidates mechanistic differences between these critical lipids and sets the foundation for further studies on the functions of sphingosine and sphinganine.

biochemistry↗

bioRxiv2023

Systematic analysis of the sphingomyelin synthase family in C. elegans

Sphingomyelin (SM) is a major component of mammalian cell membranes and particularly abundant in the myelin sheath that surrounds nerve fibers. Its production is catalyzed by SM synthases SMS1 and SMS2, which interconvert phosphatidylcholine and ceramide to diacylglycerol and SM in the Golgi and at the plasma membrane, respectively. As the lipids participating in this reaction fulfill both structural and signaling functions, SMS enzymes have considerable potential to influence diverse important cellular processes. The nematode Caenorhabditis elegans is an attractive model for studying both animal development and human disease. The organism contains five SMS homologues but none of these have been characterized in any detail. Here, we carried out the first systematic analysis of SMS family members in C. elegans. Using heterologous expression systems, genetic ablation, metabolic labeling and lipidome analyses, we show that C. elegans harbors at least three distinct SM synthases and one ceramide phosphoethanolamine (CPE) synthase. Moreover, C. elegans SMS family members have partially overlapping but also unique subcellular distributions and together occupy all principal compartments of the secretory pathway. Our findings shed light on crucial aspects of sphingolipid metabolism in a valuable animal model and opens avenues for exploring the role of SM and its metabolic intermediates in organismal development.

molecular biology↗

bioRxiv2022

A global lipid map reveals host dependency factors conserved across SARS-CoV-2 variants

A comprehensive understanding of host dependency factors for SARS-CoV-2 remains elusive. We mapped alterations in host lipids following SARS-CoV-2 infection using nontargeted lipidomics. We found that SARS-CoV-2 rewires host lipid metabolism, altering 409 lipid species up to 64-fold relative to controls. We correlated these changes with viral protein activity by transfecting human cells with each viral protein and performing lipidomics. We found that lipid droplet plasticity is a key feature of infection and that viral propagation can be blocked by small-molecule glycerolipid biosynthesis inhibitors. We found that this inhibition was effective against the main variants of concern (alpha, beta, gamma, and delta), indicating that glycerolipid biosynthesis is a conserved host dependency factor that supports this evolving virus.

microbiology↗

bioRxiv2020

Cross-reactivity of SARS-CoV structural protein antibodies against SARS-CoV-2

There is currently a lack of biological tools to study the replication cycle and pathogenesis of SARS-CoV-2, the etiological agent of COVID-19. Repurposing the existing tools, including antibodies of SARS-CoV, is an effective way to accelerate the development of therapeutics for COVID-19. Here, we extensively characterized antibodies of the SARS-CoV structural proteins for their cross-reactivity, experimental utility, and neutralization of SARS-CoV-2. We assessed a total of 10 antibodies (six for Spike, two for Membrane, and one for Nucleocapsid and Envelope viral protein). We evaluated the utility of these antibodies against SARS-CoV-2 in a variety of assays, including immunofluorescence, ELISA, biolayer interferometry, western blots, and micro-neutralization. Remarkably, a high proportion of the antibodies we tested showed cross-reactivity, indicating a potentially generalizable theme of cross-reactivity between SARS-CoV and SARS-CoV-2 antibodies. These antibodies should help facilitate further research into SARS-CoV-2 basic biology. Moreover, our study provides critical information about the propensity of SARS-CoV antibodies to cross-react with SARS-CoV-2 and highlights its relevance in defining the clinical significance of such antibodies to improve testing and guide the development of novel vaccines and therapeutics.

immunology↗