Biology subjects

Bradshaw, G. A.

Publications and source records attributed to Bradshaw, G. A..

8 recordsLinked to original sources

bioRxiv2026

K63-linked ubiquitin chains mark inactive Smoothened for packaging into ciliary extracellular vesicles

Signaling receptors can exit cilia either by retrieval back into the cell or by secretion in extracellular vesicles (EVs), a process known as ectocytosis. The mechanisms that govern ectocytosis remain poorly understood. Here, we leverage quantitative proteomic profiling to identify the Hedgehog signaling receptor Smoothened (SMO) as a major cargo of cilia-derived EVs. Surprisingly, ligands that promote ciliary accumulation of SMO strongly suppressed its packaging into EVs, indicating that ectocytosis selectively packages specific conformational states of SMO. We further find that SMO packaged into EVs is extensively modified with K63-linked ubiquitin chains. Preventing SMO ubiquitination or selectively removing K63-linked ubiquitin chains from SMO markedly reduced its secretion into EVs and caused SMO accumulation within cilia. Together, these results identify K63-linked ubiquitin chains as a sorting signal for ciliary ectocytosis and indicate that ubiquitin-dependent packaging of inactive SMO contributes to the dynamic redistribution of SMO during Hedgehog signaling.

cell biology↗

bioRxiv2025

BBSome-Mediated Clearance of Ubiquitinated IMPG2 Defines a Constitutive Ciliary Retrieval Pathway in Photoreceptors

The BBSome mediates the retrieval of ubiquitinated membrane proteins from cilia, but its physiological cargoes in photoreceptors remain largely unidentified. Here, we find that K63-linked ubiquitin (UbK63) chains accumulate in the outer segment (OS, equivalent of cilia) of Bbs4-/- photoreceptors from the onset of OS formation. Through quantitative profiling of the UbK63-associated OS proteome, we identify the transmembrane fragment of interphotoreceptor matrix proteoglycan 2 (IMPG2m) as a principal cargo of the BBSome. In Bbs4-/- mice, ubiquitinated IMPG2m aberrantly accumulates in OSs, and disruption of IMPG2m ubiquitination impairs its retrieval and clearance. Because full-length IMPG2 traffics to the OS to deliver its extracellular domain to the matrix, our data support a model in which IMPG2m undergoes constitutive cycling between the inner and outer segments. These findings redefine the BBSomes role in photoreceptors from quality control to constitutive membrane protein turnover.

cell biology↗

bioRxiv2025

Probing the CaV1.2 interactome in heart failure identifies a positive modulator of inotropy and lusitropy

BackgroundMaladaptive changes in the function, expression and localization of proteins involved in calcium-handling worsen the impaired contractility of systolic heart failure (HF). Standard proteomics techniques require cell lysis and so are unable to characterize changes specific to the critical sub-cellular domain bounded by the T-tubule and the sarcoplasmic reticulum, known as the cardiac dyad. Traditional approaches are also less likely to capture low-affinity protein-protein interactions on lipid membranes. To improve our understanding of heart failure pathophysiology, we applied proximity proteomics to the cardiac dyad of mice with ischemic cardiomyopathy. MethodsUsing two lines of transgenic mice expressing fusion proteins of the engineered ascorbate peroxidase APEX2 with subunits of the cardiac voltage gated calcium channel, CaV1.2, we labeled the dyad proteome of live, intact myocytes from healthy mice (N=6) and mice with coronary artery ligation HF (N=5) by peroxidase-catalyzed biotinylation. Quantitative mass spectrometry with isobaric tandem mass tags (TMT) was used to assess alterations in the local dyad proteome in myocytes from mice with chronic, remodeled HF. We subsequently generated a mouse with inducible cardiac overexpression of Galectin-1 to examine the effects of this protein on cardiac function and more specifically on the calcium-handling properties of mature myocytes, using cellular electrophysiology, calcium imaging, and echocardiography. ResultsFrom mice with HF, we found significant enrichment of 43 proteins defined by their abundance and proximity to transgenic CaV1.2 1C channels, and a significant reduction in 22 proteins, out a of a total of 2326 proteins quantified. We also significantly enriched 286 proteins, and saw a reduction in 13 proteins, defined by proximity and abundance to CaV1.2 {beta}2B-subunits, out of a total 2236 proteins identified. Pathway analysis revealed HF is associated with increased abundance of components of the 26s proteasome and microtubules in the dyad, as well as the dimerizing, carbohydrate binding protein Galectin-1. Cardiac specific overexpression of Galectin-1 in healthy mice increases activation of CaV1.2 and the Ryanodine receptor and accelerates myocyte relaxation through phosphorylation of Phospholamban. ConclusionsUsing proximity proteomics to examine the effects of HF in vivo, we find increased localization of Galectin-1 to the cardiac dyad, and that overexpression of Galectin-1 accelerates calcium kinetics in the heart.

cell biology↗

bioRxiv2024

Acute BRCAness Induction and AR Signaling Blockage through CDK12/7/9 Degradation Enhances PARP Inhibitor Sensitivity in Prostate Cancer

Current treatments for advanced prostate cancer (PCa) primarily target the androgen receptor (AR) pathway. However, the emergence of castration-resistant prostate cancer (CRPC) and resistance to AR pathway inhibitors (APSIs) remains ongoing challenges. Here, we present BSJ-5-63, a novel proteolysis-targeting chimera (PROTAC) targeting cyclin-dependent kinases (CDKs) CDK12, CDK7, and CDK9, offering a multi-pronged approach to CRPC therapy. BSJ-5-63 degrades CDK12, diminishing BRCA1 and BRCA2 expression and inducing a sustained "BRCAness" state. This sensitizes cancer cells to PARP inhibitors (PARPis) regardless of their homologous recombination repair (HRR) status. Furthermore, CDK7 and CDK9 degradation attenuates AR signaling, enhancing its therapeutic efficacy. Preclinical studies, including both in vitro and in vivo CRPC models, demonstrate that BSJ-5-63 exerts potent anti-tumor activity in both AR-positive and AR-negative setting. This study introduces BSJ-5-63 as a promising therapeutic agent that addresses both DNA repair and AR signaling mechanisms, with potential benefits for a board patient population.

cancer biology↗

bioRxiv2023

Cell wall fluidization by mechano-endopeptidases sets up a turgor-mediated volumetric pacemaker

The cell wall is an essential cellular component of bacteria and the target of many antibiotics. However, how bacteria regulate the rate of cell wall biosynthesis as growth rates change remains unresolved. In E. coli, cell wall growth was thought to proceed independently from turgor pressure1, the osmotic pressure that the cytoplasm exerts on the cell wall. Here, we uncover a striking increase of turgor pressure with growth rate. Modulating turgor pressure and measuring cell wall biosynthesis, we find that turgor pressure is directly controls the rate of cell wall biosynthesis. The picture that emerges is that turgor pressure is largely generated by counterions of negatively charged cellular biomass. The increase in turgor pressure with growth rates results from more ribosomes and therefore higher concentrations of negatively charged ribosomal RNA. Elegantly, the coupling between biomass composition, turgor pressure and cell wall biosynthesis simultaneously explains how bacteria achieve homeostasis of cytoplasmic crowding and how they regulate the rate of cell wall biosynthesis across growth rates.

microbiology↗

bioRxiv2023

A Spatiotemporal Map of Co-Receptor Signaling Networks Underlying B Cell Activation

The B cell receptor (BCR) signals together with a multi-component co-receptor complex to initiate B cell activation in response to antigen binding. This process underlies nearly every aspect of proper B cell function. Here, we take advantage of peroxidase-catalyzed proximity labeling combined with quantitative mass spectrometry to track B cell co-receptor signaling dynamics from 10 seconds to 2 hours after BCR stimulation. This approach enables tracking of 2,814 proximity-labeled proteins and 1,394 quantified phosphosites and provides an unbiased and quantitative molecular map of proteins recruited to the vicinity of CD19, the key signaling subunit of the co-receptor complex. We detail the recruitment kinetics of essential signaling effectors to CD19 following activation, and then identify new mediators of B cell activation. In particular, we show that the glutamate transporter SLC1A1 is responsible for mediating rapid metabolic reprogramming immediately downstream of BCR stimulation and for maintaining redox homeostasis during B cell activation. This study provides a comprehensive map of the BCR signaling pathway and a rich resource for uncovering the complex signaling networks that regulate B cell activation.

immunology↗

bioRxiv2022

A Spatiotemporal Notch Interaction Map from Membrane to Nucleus

Notch signaling relies on ligand-induced proteolysis to liberate a nuclear effector that drives cell fate decisions. The location and timing of individual steps required for proteolysis and movement of Notch from membrane to nucleus, however, remain unclear. Here, we use proximity labeling with quantitative multiplexed mass spectrometry to monitor the microenvironment of endogenous Notch2 after ligand stimulation in the presence of a gamma secretase inhibitor and then as a function of time after inhibitor removal. Our studies show that gamma secretase cleavage of Notch2 occurs in an intracellular compartment and that formation of nuclear complexes and recruitment of chromatin-modifying enzymes occurs within 45 minutes of inhibitor washout. This work provides a spatiotemporal map of unprecedented detail tracking the itinerary of Notch from membrane to nucleus after activation and identifies molecular events in signal transmission that are potential targets for modulating Notch signaling activity.

molecular biology↗

bioRxiv2020

Multiplexed proteomics and imaging of resolving and lethal SARS-CoV-2 infection in the lung

Normal tissue physiology and repair depends on communication with the immune system. Understanding this communication at the molecular level in intact tissue requires new methods. The consequences of SARS-CoV-2 infection, which can result in acute respiratory distress, thrombosis and death, has been studied primarily in accessible liquid specimens such as blood, sputum and bronchoalveolar lavage, all of which are peripheral to the primary site of infection in the lung. Here, we describe the combined use of multiplexed deep proteomics with multiplexed imaging to profile infection and its sequelae directly in fixed lung tissue specimens obtained from necropsy of infected animals and autopsy of human decedents. We characterize multiple steps in disease response from cytokine accumulation and protein phosphorylation to activation of receptors, changes in signaling pathways, and crosslinking of fibrin to form clots. Our data reveal significant differences between naturally resolving SARS-CoV-2 infection in rhesus macaques and lethal COVID-19 in humans. The approach we describe is broadly applicable to other tissues and diseases. SummaryProteomics of infected tissue reveals differences in inflammatory and thrombotic responses between resolving and lethal COVID-19.

pathology↗