Biology subjects

Conole, D.

Publications and source records attributed to Conole, D..

5 recordsLinked to original sources

bioRxiv2024

A spatiotemporally resolved GPCR interactome reveals novel mediators of receptor agonism

Cellular signaling by membrane G protein-coupled receptors (GPCRs) is orchestrated by a complex and diverse array of mechanisms. The dynamics of a GPCR interactome as it evolves over time and space in response to an agonist can offer a unique window on pleiotropic signaling decoding and functional selectivity at a cellular level. In this study, we employed proximity-based APEX2 proteomics to interrogate the interaction network of the GPCR for luteinizing hormone (LHR) on a minute-to-minute timescale. We developed an analytical approach integrating quantitative multiplexed proteomics and temporal reference profiles, providing a platform to identify the proteomic environment of APEX2-tagged LHR at the nanometer scale. LHR activity is exquisitely regulated at a spatial level, leading to identification of novel putative interactors including the Ras-related GTPase RAP2B that modulate both receptor signaling and post-endocytic trafficking, and providing a resource for spatiotemporal nanodomain mapping of LHR interactors across subcellular compartments. SignificanceG-protein coupled receptors (GPCRs) are the largest family of membrane proteins in the human body. GPCRs have established themselves as key drug targets, playing central roles in health and disease. The activity of this superfamily of receptors is highly dynamic, integrating diverse signaling pathways over time and across multiple subcellular compartments. Quantitative proximity proteomics using engineered ascorbate peroxidase (APEX2) has emerged as a powerful tool to map GPCR-protein interaction landscapes in both time and space. However, resolving these networks at a sub-minute timescale remains challenging, particularly for poorly characterized GPCRs with limited pharmacological tools or complex trafficking patterns. In this study, we applied APEX2 proximity proteomics to resolve at nanoscale the agonist (LH)-induced interactome of the luteinizing hormone receptor (LHR), a key GPCR in human reproduction. LHR undergoes rapid endocytosis upon LH stimulation, trafficking to a poorly understood compartment termed the very early endosome (VEE), where it engages in G protein signaling and post-endocytic sorting. Harnessing the temporal profile of GIPC1 protein, a key known interactor of LHR in the VEE pathway, we developed an analytical pipeline to identify novel endocytic interactors of LHR. Using our platform, we identified RAP2B and RAB38 as potential modulators of LHR signaling and trafficking. Overall, we present an APEX2 pipeline optimized for interrogating complex datasets, particularly for GPCRs, and potentially other membrane receptors, with limited known interactome data and minimal pharmacological tools. Our findings highlight both the complexity of GPCR interaction networks and an effective strategy to deconvolute them, providing a comprehensive resource to advance understanding of this large family of receptors. O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/599010v2_ufig1.gif" ALT="Figure 1"> View larger version (71K): org.highwire.dtl.DTLVardef@13badf7org.highwire.dtl.DTLVardef@11a5182org.highwire.dtl.DTLVardef@dd5453org.highwire.dtl.DTLVardef@cb2c27_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Chemical Tools for Profiling the Intracellular ADP-ribosylated Proteome

The post-translational modification (PTM) ADP-ribosylation plays an important role in cell signaling and regulating protein function and has been implicated in the development of multiple diseases, including breast and ovarian cancers. Studying the underlying mechanisms through which this PTM contributes towards disease development, however, has been hampered by the lack of appropriate tools for reliable identification of physiologically relevant ADP-ribosylated proteins in a live-cell environment. Herein, we explore the applications of an alkyne-tagged proprobe, 6Yn-ProTide-Ad (6Yn-Pro) as a chemical tool for the identification of intracellular ADP-ribosylated proteins through metabolic labelling. We applied targeted metabolomics and chemical proteomics in HEK293T cells treated with 6Yn-Pro to demonstrate intracellular metabolic conversion of the probe into ADP-ribosylation cofactor 6Yn-NAD+, and subsequent labelling and enrichment of PARP1 and multiple known ADP-ribosylated proteins in cells under hydrogen peroxide-induced stress. We anticipate that the approach and methodology described here will be useful for future identification of novel intracellular ADP-ribosylated proteins. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/555609v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@d50b3aorg.highwire.dtl.DTLVardef@d43023org.highwire.dtl.DTLVardef@a48ae2org.highwire.dtl.DTLVardef@9a0a1e_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2023

Structure-guided design and optimization of covalent VHL-targeted sulfonyl fluoride PROTACs

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/545773v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@da22e5org.highwire.dtl.DTLVardef@1290278org.highwire.dtl.DTLVardef@1611c0eorg.highwire.dtl.DTLVardef@7902d2_HPS_FORMAT_FIGEXP M_FIG C_FIG Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules that have emerged as a therapeutic modality to induce targeted protein degradation (TPD) by harnessing cellular proteolytic degradation machinery. PROTACs which ligand the E3 ligase in a covalent manner have attracted intense interest, however, covalent PROTACs with a broad protein of interest (POI) scope have proven challenging to discover by design. Here, we report structure-guided design and optimization of Von Hippel-Lindau (VHL) protein-targeted sulfonyl fluorides which covalently bind Ser110 in the HIF1 binding site. We demonstrate that their incorporation in bifunctional degraders induces targeted protein degradation of BRD4 or androgen receptor (AR) without further linker optimization. Our study discloses the first covalent VHL ligands which can be implemented directly in bifunctional degrader design expanding the substrate scope of covalent E3 ligase PROTACs.

molecular biology↗

bioRxiv2021

A novel tubulin binding molecule drives differentiation of acute myeloid leukaemia cells

Acute Myeloid Leukaemia (AML) continues to have a poor prognosis, especially in the elderly. One reason for this is that many treatment regimens are not well tolerated by elderly patients. Much current focus is on the development of therapies that can target specific vulnerabilities of AML while having fewer toxic side effects. However, despite much recent progress in developing better drugs, many patients with AML still die within a year of diagnosis, partly due to the fact that it is difficult to identify therapeutic targets that are effective across multiple AML subtypes. One common factor across AML subtypes is the presence of a block in differentiation. Thus screening for compounds that can overcome this block in genetically diverse AML models should allow for the identification of agents that are not dependent on a specific mutation for their efficacy. Here, we used a phenotypic screen to identify novel compounds that stimulate differentiation in several AML cell lines. Lead compounds were shown to decrease tumour burden and to increase survival in vivo. Using multiple complementary target deconvolution approaches, these compounds were revealed to be anti-mitotic tubulin disruptors that cause differentiation by inducing a G2-M mitotic arrest. Together, these results reveal a novel function for tubulin disruptors in causing differentiation of AML cells.

cancer biology↗

bioRxiv2020

Targeting STAT3 signalling using stabilised sulforaphane (SFX-01) inhibits endocrine resistant stem-like cells in ER-positive breast cancer

PURPOSEEstrogen receptor (ER) positive breast cancer is frequently sensitive to endocrine therapy. Multiple mechanisms of endocrine therapy resistance have been identified, including cancer stem-like cell (CSC) activity. Sulforaphane (SFN) has previously been shown to target CSCs but its mechanism of action is unclear. Here we investigate SFX-01, a stabilised formulation of SFN, for its effects on breast CSC activity in ER+ preclinical models and to study its mechanism. EXPERIMENTAL DESIGNCSC activity was measured by mammosphere formation efficiency (MFE), aldehyde dehydrogenase (ALDH) activity, and tumor formation using patient samples and patient-derived xenograft (PDX) tumors treated with SFX-01 alone or in combination with tamoxifen or fulvestrant. Gene expression and SFN target proteins in treated samples were assessed. RESULTSSFX-01 reduced MFE of both ER+ primary and metastatic patient samples. Both tamoxifen and fulvestrant increased MFE and ALDH activity of PDX tumors, which was reversed by combination with SFX-01. SFX-01 significantly reduced tumor initiating cell frequency in secondary transplants at limiting dilution and reduced the formation of spontaneous lung micrometastases by PDX tumors in mice. Mechanistically, we establish that both tamoxifen and fulvestrant induce STAT3 phosphorylation. SFX-01 suppressed phospho-STAT3 and SFN directly bound STAT3 in patient and PDX samples. Analysis of ALDH+ cells from endocrine-resistant patient samples revealed activation of STAT3 target genes MUC1 and OSMR, which were inhibited by SFX-01 in patient samples. Increased expression of these genes after 3 months endocrine treatment of ER+ patients (n=68) predicted poor prognosis. CONCLUSIONSOur data establish the importance of STAT3 signaling in CSC-mediated resistance to endocrine therapy and the potential of SFX-01 for improving clinical outcomes in ER+ breast cancer.

cancer biology↗