Biology subjects

Brady, D. C.

Publications and source records attributed to Brady, D. C..

10 recordsLinked to original sources

bioRxiv2026

Mitochondrial copper stabilizes lipoylated TCA cycle proteins to sustain metabolism and proliferation

Copper (Cu) is an essential cofactor for mitochondrial cytochrome c oxidase, yet whether it directly regulates mitochondrial metabolism beyond respiration remains unclear. Here we show that mitochondrial Cu, delivered by SLC25A3, is required to maintain the stability of lipoylated TCA cycle proteins. Loss of Slc25a3 or pharmacological Cu depletion selectively destabilized the lipoylated E2 subunits of mitochondrial dehydrogenases and the lipoylation enzymes LIPT1 and LIPT2, an effect not reproduced by acute electron transport chain inhibition. Mechanistically, we find that Cu directly engages the reduced lipoyl moiety using chemical probes and synthetic peptide approaches. Cu depletion impaired PDH and OGDH activity, rewired TCA cycle metabolism, and imposed a dependence on pyruvate carboxylase for anaplerosis. This metabolic defect depleted aspartate, suppressed mTORC1 signaling, and limited proliferation. Conversely, selective delivery of Cu to the mitochondria restored lipoylation, TCA cycle function, and cell growth. Together, these findings identify mitochondrial Cu as a structural regulator of the lipoylation machinery and reveal a direct link between Cu homeostasis and central carbon metabolism.

cell biology↗

bioRxiv2026

Reduction in Hepatic Phosphatidylcholine Biosynthesis Promotes MASH Through Copper Deficiency

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease for which the mechanisms linking lipid dysregulation to fibrosis remain poorly defined. Hepatic phosphatidylcholine (PC) content is reduced in MASH, but how this alteration drives disease progression is unclear. Here, we identify a role for copper (Cu) homeostasis as a downstream effector of impaired PC biosynthesis. Using single-nucleus RNA sequencing in complementary genetic and dietary mouse models, we found that reduced hepatic PC is associated with marked depletion of hepatic Cu and a concomitant increase in circulating Cu, indicating disrupted Cu distribution. Mechanistically, PC depletion impaired plasma membrane localization of the high-affinity Cu transporter CTR1 (SLC31A1) in hepatocytes, limiting Cu uptake. In human hepatic stellate cells, Cu promoted fibrogenic activation, whereas suppression of Cu import or pharmacologic inhibition of MAPK signaling attenuated fibronectin deposition. In vivo, liver-directed Cu supplementation restored hepatic Cu levels and reduced steatosis but failed to improve fibrosis. In contrast, pharmacologic Cu chelation with bathocuproinedisulfonic acid (BCS) reduced fibrosis without affecting inflammation. Together, these findings identify Cu redistribution as a consequence of impaired PC biosynthesis and implicate Cu-dependent signaling in stellate cell activation, fibrogenesis and MASH pathogenesis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/723926v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1c2bd52org.highwire.dtl.DTLVardef@1b8295org.highwire.dtl.DTLVardef@1a129dorg.highwire.dtl.DTLVardef@1bc2b87_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2025

MAPK Pathway Inhibition Reshapes Kinase Chemical Probe Reactivity Reflecting Cellular Activation States

Despite the pivotal role of oncogenic kinases in cancer initiation, progression, and therapeutic resistance, functionally profiling their activity and conformational dynamics in live cells remains challenging. Existing methods often fail to capture inhibitor-bound structural states of kinases, particularly in clinically relevant contexts such as treatment response and acquired resistance, where genomic data alone are insufficient. Here, we use activity-based protein profiling (ABPP) to monitor composite amino acid reactivity changes - across cysteine, lysine, and carboxylic acid residues - as a functional readout of kinase conformation and activation state. Using electrophilic probes, we show that treatment of BRAFV600E-mutant melanoma cells with vemurafenib or trametinib decreases overall cysteine and lysine reactivity in BRAFV600E and MEK1/2, likely reflecting composite changes in amino acid accessibility across multiple reactive residues associated with inhibitor binding. Changing the order of probe addition and inhibitor treatment altered labeling outcomes, consistent with competitive engagement and structural stabilization. Comparative analysis of ATP-competitive BRAFV600E inhibitors vemurafenib and dabrafenib revealed distinct impacts on aspartate and glutamate labeling patterns, suggesting that ABPP can distinguish inhibitor-dependent differences in residue accessibility that may reflect distinct inhibitor-bound conformations. In inhibitor-resistant melanoma models, ABPP detected differential residue reactivity relative to parental cells, consistent with BRAF overexpression and the MEK2 Q60P activation mutation, both established mechanisms of MAPK inhibitor resistance. Moreover, global proteome analyses of cysteine and lysine reactivity upon BRAFV600E inhibition, revealed probe-accessible cysteine labeling changes in labeling on KSR2, indicating broader MAPK pathway remodeling. Together, these findings establish ABPP as a powerful chemical biology approach for investigating inhibitor-dependent changes in kinase residue accessibility, providing a framework to explore how conformational dynamics and pathway adaptation shape therapeutic response and resistance in oncogenic signaling networks.

cancer biology↗

bioRxiv2024

A selective S-acyltransferase inhibitor suppresses tumor growth

S-acyltransferases play integral roles in essential physiological processes including regulation of oncogenic signaling pathways. While discovered over 40 years ago the field still lacks specific S-acylation inhibitors thus the potential benefit of pharmacologically targeting S-acyltransferases for human disease is still unknown. Here we report the identification of an orally bioavailable acyltransferase inhibitor SD-066-4 that inhibits the acyltransferase ZDHHC20. We identified a specific alanine residue that accommodates the methyl group of SD-066-4, thus providing isoform selectivity. SD-066-4 stably reduces EGFR S-acylation in Kras mutant cells and blocks the growth of Kras mutant lung tumors extending overall survival. We find that lung cancer patients harboring deletions in ZDHHC20 or ZDHHC14 concurrent with Kras alterations have a significant survival benefit, underscoring the translational importance of these enzymes.

cancer biology↗

bioRxiv2022

Copper as a Novel Regulator of PERK

PERK is a kinase that, in response to ER stress, mediates duplicitous homeostatic and pro-apoptotic signaling. Thus, intricate regulation is required for physiologic function. Attempts to modulate PERK activity have shown that the determinants of adaptive vs. mal-adaptive signaling remain ambiguous. Here, with purified protein, we provide direct evidence that PERK binds copper, identify the residues that coordinate binding, and demonstrate that binding is necessary for kinase activity. Furthermore cellular PERK activity can be modulated via copper availability, and this regulatory relationship can be manipulated to dictate ER stress tolerance. Critically, these phenomenon translate to phenotypes in vivo, as C. elegans harboring a "copper-binding mutant" of PERK exhibit enhanced ER-stress sensitivity. The copper-PERK paradigm adds onto a new class of dynamic copper-binding enzymes and implicates that copper homeostasis, as a regulator of PERK, may constitute a previously unknown variable to resolve long-standing ambiguity in endeavors to therapeutically target PERK.

biochemistry↗

bioRxiv2022

IGF2BP1/IMP1 contributes to autophagy modulation directly via MAP1LC3B

Homeostatic tissue maintenance requires coordinated regulation of metabolic processes including macroautophagy/autophagy. Autophagy dysregulation underlies numerous human diseases. Our prior work revealed that the RNA binding protein IGF2BP1/IMP1 binds transcripts encoding autophagy-related proteins. Furthermore, Imp1 deletion in gastrointestinal epithelial cells in mice was associated with enhanced autophagy flux and improved recovery from tissue injury. In the current study, we evaluated molecular mechanisms underlying IMP1 modulation of autophagy. We provide a mechanism of direct IMP1 regulation of MAP1LC3B that is dependent upon IMP1 phosphorylation or cell stress, suggesting dynamic modulation of Imp1-mediated autophagy repression that facilitates tissue regeneration. More broadly, our study supports a new mechanism by which tissue regeneration is modulated post-transcriptionally via cell state rather than changes in stem or other cell lineages. This new mechanism may be particularly important in gastrointestinal epithelial cells, where autophagy is essential for tissue recovery following injury, or in diseases such as inflammatory bowel disease where defective autophagy is implicated.

cell biology↗

bioRxiv2020

PAX8 orchestrates an angiogenic program through interaction with SOX17

Worldwide, the number of new ovarian cancer cases approaches 300,000 with more than 180,000 deaths every year. The low survival-rate reflects the limitations of current therapies and highlights the importance of identifying new therapeutic targets. Despite significant recent efforts to identify novel vulnerabilities in ovarian cancer, none have led to effective durable therapies with improvement in overall survival. PAX8, a lineage-transcription factor, whose expression is a major molecular feature of ovarian carcinomas, represents a novel therapeutic target. Herein, we have identified SOX17 as a bona fide PAX8-interacting partner and elucidated the impact of this interaction on the development of ovarian cancer. Importantly, we found that PAX8 and SOX17 regulate tumor angiogenesis in vitro and in vivo. The role of PAX8 and SOX17 in the regulation of angiogenesis reveals a novel function for these factors in regulating the tumor microenvironment and highlight this pathway as a viable therapeutic target.

cancer biology↗

bioRxiv2020

Structural and molecular determinants of CCS-mediated copper activation of MEK1/2

SummaryNormal physiology relies on the precise coordination of intracellular signal transduction pathways that respond to nutrient availability to balance cell growth and cell death. We recently established a critical mechanistic function for the redox-active micronutrient copper (Cu) in the canonical mitogen activated protein kinase (MAPK) pathway at the level of MEK1 and MEK2. Here we report the X-ray crystal structure of Cu-MEK1 and reveal active site chemical ligands and oxidation state specificity for MEK1 Cu coordination. Mechanistically, the Cu chaperone CCS selectively bound to and facilitated Cu transfer to MEK1. Mutations in MEK1 that disrupt Cu(I) affinity or a CCS small molecule inhibitor reduced Cu-stimulated MEK1 kinase activity. These atomic and molecular level data provide the first mechanistic insights of Cu kinase signaling and could be exploited for the development of novel MEK1/2 inhibitors that either target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS.Competing Interest StatementD.C.B holds ownership in Merlon Inc. D.C.B. is an inventor on the patent application 20150017261 entitled Methods of treating and preventing cancer by disrupting the binding of copper in the MAP kinase pathway. No potential conflicts of interest were disclosed by the other authors.View Full Text

biochemistry↗

bioRxiv2019

Copper is an essential regulator of the autophagic kinases ULK1/2 to drive lung adenocarcinoma

Targeting aberrant kinase activity in cancer relies on unmasking cellular inputs such as growth factors, nutrients, and metabolites that contribute to cancer initiation and progression1. While the transition metal copper (Cu) is an essential nutrient that is traditionally viewed as a static cofactor within enzyme active sites2, a newfound role for Cu as a modulator of kinase signaling is emerging3, 4. We discovered that Cu is required for the activity of the autophagic kinases ULK1/2 through a direct Cu-ULK1/2 interaction. Genetic loss of the Cu transporter Ctr1 or mutations in ULK1 that disrupt Cu-binding reduced ULK1/2-dependent signaling and autophagosome complex formation. Elevated intracellular Cu levels are associated with starvation induced autophagy and sufficient to enhance ULK1 kinase activity and in turn autophagic flux. Targeting autophagy machinery is a promising therapeutic strategy in cancers5, but is limited by the absence of potent inhibitors and the emergence of resistance. The growth and survival of lung tumors driven by KRASG12D is diminished in the absence of Ctr1, depends on ULK1 Cu-binding, and is associated with reduced autophagy levels and signaling. These findings suggest a new molecular basis for exploiting Cu-chelation therapy to forestall autophagy signaling to limit proliferation and survival in cancer.

cancer biology↗

bioRxiv2019

Activity-Based Ratiometric FRET Probe Reveals Oncogene-Driven Changes in Labile Copper Pools Induced by Altered Glutathione Metabolism

Copper is essential for life, and beyond its well-established ability to serve as a tightly-bound, redox-active active site cofactor for enzyme function, emerging data suggest that cellular copper also exists in labile pools, defined as loosely bound to low molecular weight ligands, which can regulate diverse transition metal signaling processes spanning neural communication and olfaction, lipolysis, rest-activity cycles, and kinase pathways critical for oncogenic signaling. To help decipher this growing biology, we report a first-generation ratiometric fluorescence resonance energy transfer (FRET) copper probe, FCP-1, for activity-based sensing of labile Cu(I) pools in live cells. FCP-1 links fluorescein and rhodamine dyes through a tris[(2-pyridyl)methyl]amine (TPA) bridge. Bioinspired Cu(I)-induced oxidative cleavage decreases FRET between fluorescein donor and rhodamine acceptor. FCP-1 responds to Cu(I) with high metal selectivity and oxidation-state specificity and facilitates ratiometric measurements that minimize potential interferences arising from variations in sample thickness, dye concentration, and light intensity. FCP-1 enables imaging of dynamic changes in labile Cu(I) pools in live cells in response to copper supplementation/depletion, differential expression of the copper importer CTR1, and redox stress induced by manipulating intracellular glutathione levels and GSH/GSSG ratios. FCP-1 imaging reveals a labile Cu(I) deficiency induced by oncogene-driven cellular transformation that promotes fluctuations in glutathione metabolism, where lower GSH/GSSG ratios decrease labile Cu(I) availability without affecting total copper levels. By connecting copper dysregulation and glutathione stress in cancer, this work provides a valuable starting point to study broader crosstalk between metal and redox pathways in health and disease with activity-based probes.\n\nSignificanceCopper is a required metal nutrient for life, yet its altered homeostasis is associated with many diseases. Thus, to develop new methods to help decipher copper biology, we present an activity-based ratiometric FRET probe that exploits a biomimetic, copper(I)-dependent cleavage reaction to enable imaging of loosely-bound, labile copper pools in cells with metal and oxidation state selectivity and a self-calibrating ratiometric response. Application of this technology to cellular models of cancer reveals that oncogene-driven changes in the metabolism of glutathione, a major cellular redox buffer, leads to a labile copper(I) deficiency. This work establishes the relevance of copper dysregulation to cancer metabolism and presages further opportunities for activity-based sensing in studies of metal biology.

biochemistry↗