Biology subjects

Smith, J. W.

Publications and source records attributed to Smith, J. W..

8 recordsLinked to original sources

bioRxiv2026

Reversible, Orally Bioavailable Small-Molecule Inhibitors Occupy the Substrate Tunnel of the Thioesterase Domain of Fatty Acid Synthase

Fatty acid synthase (FASN) is the sole enzyme capable of lipogenesis in cells and is upregulated in many cancers. Herein we report the discovery and development of SBP-5917 (78S), a novel, reversible, small-molecule probe which targets the thioesterase (TE) domain of FASN. SBP-5917 and other compounds in the series possess IC50 < 1 M and < 5 M for inhibiting recombinantly purified FASN-TE and cellular FASN respectively. Co-crystal structures of our inhibitors bound to the FASN-TE domain show that they are positioned in the substrate tunnel of the enzyme. Biochemical and biophysical analyses quantify the strength of compound binding and demonstrate their selectivity for FASN-TE. Preliminary pharmacokinetic studies indicate promising in vivo potency for many of the compounds in the series. SBP-5917 is the first structurally validated, reversible inhibitor of FASN-TE with demonstrated cellular target engagement and can serve as a chemical probe to investigate FASN as a therapeutic target.

cell biology↗

bioRxiv2025

Optogenetic Clustering of Human IRE1 Reveals Differential Regulation of Transcription and mRNA Splice Isoform Abundance by the UPR

Inositol-requiring enzyme 1 (IRE1) is one of three known sensor proteins that respond to homeostatic perturbations in the metazoan endoplasmic reticulum. The three sensors collectively initiate an intertwined signaling network called the Unfolded Protein Response (UPR). Although IRE1 plays pivotal roles in human health and development, understanding its specific contributions to the UPR remains a challenge due to signaling crosstalk from the other two stress sensors. To overcome this problem, we engineered a light-activatable version of IRE1 and probed the transcriptomic effects of IRE1 activity in isolation from the other branches of the UPR. We demonstrate that 1) oligomerization alone is sufficient to activate IRE1 in human cells, 2) IRE1s transcriptional response evolves substantially under prolonged activation, and 3) the UPR induces major changes in mRNA splice isoform abundance in an IRE1-independent manner. Our data reveal previously unknown targets of IRE1 transcriptional regulation and direct degradation. Additionally, the tools developed here will be broadly applicable for precise dissection of signaling networks in diverse cell types, tissues, and organisms.

cell biology↗

bioRxiv2024

bayesTPC: Bayesian inference for Thermal Performance Curves in R

1O_LIReliable predictions of arthropod responses to climatic warming are important because many of these species perform important roles that can directly impact human society. C_LIO_LIThermal performance curves (TPCs) provide useful information on the physiological constraints that limit the capacity of temperature-sensitive organisms (like arthropods) to exist and grow. C_LIO_LINLS pipelines for fitting TPCs are widely available, but these approaches rely on assumptions that can yield unreliable parameter estimates. C_LIO_LIWe present bayesTPC, an R package for fitting TPCs to trait responses using the nimble language and machinery as the underlying engine for Markov Chain Monte Carlo. bayesTPC aims to support the adoption of Bayesian approaches in thermal physiology, and promote TPC fitting that adequately quantifies uncertainty. C_LI

ecology↗

bioRxiv2023

Molecular architecture and platelet-activating properties of small immune complexes assembled on intact heparin and their possible involvement in heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is an adverse reaction to heparin leading to a reduction in circulating platelets with an increased risk of thrombosis. It is precipitated by polymerized immune complexes consisting of pathogenic antibodies that recognize a small chemokine platelet factor 4 (PF4) bound to heparin, which trigger platelet activation and a hypercoagulable state. Characterization of these immune complexes is extremely challenging due to the enormous structural heterogeneity of such macromolecular assemblies and their constituents (especially heparin). We use native mass spectrometry to characterize small immune complexes formed by PF4, heparin and monoclonal HIT-specific antibodies. Up to three PF4 tetramers can be assembled on a heparin chain, consistent with the results of molecular modeling studies showing facile polyanion wrapping along the polycationic belt on the PF4 surface. Although these assemblies can accommodate a maximum of only two antibodies, the resulting immune complexes are capable of platelet activation despite their modest size. Taken together, these studies provide further insight into molecular mechanisms of HIT and other immune disorders where anti-PF4 antibodies play a central role.

biochemistry↗

bioRxiv2023

Structure of alpha-synuclein fibrils derived from human Lewy body dementia tissue

The defining feature of Parkinson disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. We developed and validated a novel method to amplify Asyn fibrils extracted from LBD postmortem tissue samples and used solid state nuclear magnetic resonance (SSNMR) studies to determine atomic resolution structure. Amplified LBD Asyn fibrils comprise two protofilaments with pseudo-21 helical screw symmetry, very low twist and an interface formed by antiparallel beta strands of residues 85-93. The fold is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. These results expand the structural landscape of LBD Asyn fibrils and inform further studies of disease mechanisms, imaging agents and therapeutics targeting Asyn.

neuroscience↗

bioRxiv2022

Electron Videography of a Lipid-Protein Tango

Biological phenomena ranging from enzymatic catalysis to synaptic transmission find their origins in the morphological fluctuations of biomolecules and biomolecular assemblies in liquid water. However, directly imaging these nanoscopic dynamics without probes or labels has been a fundamental methodological challenge. Here we developed an approach for molecular videography--combining liquid-phase transmission electron microscopy with molecular modelling--with which we filmed the nanoscale morphological fluctuations of individual, freely suspended, and unlabelled membrane protein nanodiscs in liquid. These unprecedented observations enabled us to quantify fingerprints of nanoscale lipid-protein interactions directly, revealing that nanodisc shape undulations are defined by the molecular mechanics of encircling scaffold proteins, whereas lipid interactions with a membrane protein reach across surprisingly long ranges to delineate dynamically modified membrane domains and increase nanodisc stability. Overall, this work illustrates an experimental approach to image, quantify, and understand biomolecular dynamics at the nanometre scale.

biophysics↗

bioRxiv2021

Vascular proteome responses precede organ dysfunction in sepsis

Vascular dysfunction and organ failure are two distinct, albeit highly interconnected clinical outcomes linked to morbidity and mortality in human sepsis. The mechanisms driving vascular and parenchymal damage are dynamic and display significant molecular crosstalk between organs and tissues. Therefore, assessing their individual contribution to disease progression is technically challenging. Here, we hypothesize that dysregulated vascular responses predispose the organism to organ failure. To address this hypothesis, we have evaluated four major organs in a murine model of S. aureus sepsis by combining in vivo labeling of the endothelial proteome, data-independent acquisition (DIA) mass spectrometry, and an integrative computational pipeline. The data reveal, with unprecedented depth and throughput, that a septic insult evokes organ-specific proteome responses that are highly compartmentalized, synchronously coordinated, and significantly correlated with the progression of the disease. Vascular proteome changes were found to precede bacterial invasion and leukocyte infiltration into the organs, as well as to precede changes in various well-established cellular and biochemical correlates of systemic coagulopathy and tissue dysfunction. Importantly, our data suggests a potential role for the vascular proteome as a determinant of the susceptibility of the organs to undergo failure during sepsis.

immunology↗

bioRxiv2020

Quantitative profiling of protease specificity.

Proteases comprise an important class of enzymes, whose activity is central to many physiologic and pathologic processes. Detailed knowledge of protease specificity is key to understanding their function. Although many methodologies have been developed to profile specificities of proteases, few have the diversity and quantitative grasp necessary to fully define specificity of a protease, both in terms of substrate numbers and their catalytic efficiencies. We have developed a concept of “selectome”, which defines the set of substrates that uniquely represents specificity of a protease. We applied it to two closely related members of the Matrixin family – MMP-2 and MMP-9 by using substrate phage display coupled with Next Generation Sequencing and information theory-based data analysis. We have also derived a quantitative measure of substrate specificity, which accounts for both the numbers and relative catalytic efficiencies of substrates. Using these advances greatly facilitates uncovering selectivity between closely related members of protease families and provides insight into to the degree of contribution of catalytic cleft specificity to protein substrate recognition, thus providing basis to overcoming two of the major challenges in the field of proteolysis: 1) development of highly selective activity probes and inhibitors for studying proteases with overlapping specificities, and 2) distinguishing targeted proteolysis from bystander proteolytic events.View Full Text

biochemistry↗