Aidan T. Pezacki

Cells continuously evaluate their nutrient status to make proliferation-quiescence decisions. Zinc is one such essential nutrient and zinc deprivation drives cells into quiescence. How other metal micronutrients are altered in quiescence has not been established. Here, we examined three quiescence triggers (zinc deficiency, serum starvation, and growth factor withdrawal) in MCF10A cells by modifying growth media, the primary source of metals. Fluorescence microscopy and elemental analysis reveal trigger-dependent changes in the labile and total transition metal pools of quiescent cells. RNA sequencing and differential expression analysis of metal associated, regulatory, and homeostasis genes shows that while core quiescence pathways are shared across triggers, quiescence states remain molecularly distinct. A significant percent of 2458 metal homeostasis genes are differentially expressed, including genes involved in antioxidant activity, oxidative phosphorylation, and heme metabolism. Overall, this work demonstrates that metal homeostasis is systematically rewired during cellular quiescence, with different signatures for different quiescence triggers.

Cuproptosis denotes cell death triggered by copper accumulation. However, its role in natural environmental responses remains unclear. Here, we demonstrate that cuproptosis contributes to cold-induced lethality in C. elegans. A mutant for the lysosomal copper transporter gene slcr-46.1 exhibited defective cold tolerance due to copper accumulation in the pharyngeal muscle upon cold exposure. The abnormal cold tolerance of the slcr-46.1 mutant was suppressed by interference with copper homeostasis and cuproptosis-signaling genes. Chelating copper ions in the slcr-46.1 mutant rescued the abnormal cold tolerance, while inhibiting cuproptosis signaling in wild-type worms inhibited cold-induced lethality. In addition, the cold lethality in the slcr-46.1 mutant was not caused by activation of the oxidative stress pathway observed in ferroptosis (iron-dependent cell death). Here, we show evidence that cuproptosis plays a critical role in cold-induced lethality in an animal.

Cells can enter a reversible non-proliferative state called quiescence either spontaneously or in response to nutrient deprivations. Metal ions are essential nutrients and play wide-ranging regulatory and signaling roles in biological processes. We previously showed that zinc is an essential nutrient for the mammalian cell cycle as zinc deprivation drives cells into quiescence, and this quiescent state is associated with changes in iron, copper, and manganese, suggesting broad remodeling of metal homeostasis. Here we examine whether metal remodeling is a general feature of quiescence by inducing quiescence via different triggers (zinc deficiency, serum starvation, and growth factor withdrawal) in MCF10A cells. Fluorescence microscopy and elemental analysis reveal significant trigger-dependent changes in the labile and total metal pools of quiescent cells. To gain insight into these differences, we carried out RNA sequencing and differential expression analysis, focusing on metal associated, metal regulatory, and metal homeostasis genes. While core quiescence pathways are shared across triggers, quiescence states remain molecularly distinct. A significant percent of 2458 metal homeostasis annotated genes are differentially expressed including 55% in starvation-induced quiescence, 50% in zinc deficiency-induced quiescence and 21% in growth factor withdrawal-induced quiescence. Our results also showed unique alteration of genes involved in major metal dependent processes including antioxidant activity, oxidative phosphorylation, heme metabolism, and chromatin accessibility in different quiescence states. Overall, this work demonstrates that metal homeostasis is systematically rewired during cellular quiescence with associated effects on genes that regulate critical biological processes. Significance Statement: Cells constantly evaluate their nutrient and energy status and integrate these signals into proliferation-quiescence decisions. Quiescence prevents cells from passing damage to daughter cells. Metals are essential micronutrients for biological processes. While limitation of zinc can drive cells into quiescence and alter other metals, how metals are remodeled and whether this is a common feature of quiescence was unknown. Here we examined three quiescence triggers by modifying growth media and serum, the primary source of metals. We report significant changes in labile and total Cu, Fe, Zn and Mn pools, and metal associated genes in response to distinct quiescence triggers. These changes converge on mitochondrial function, cellular antioxidant activity, and heme biosynthesis in a trigger specific manner.

Metabolic-associated steatotic liver disease (MASLD) is a prevalent liver disease driven by complex dysregulation of hepatic lipid metabolism. Here we show that copper deficiency is a nutrient vulnerability in steatotic liver disease and that selective liver-targeted copper supplementation can reduce excess lipid accumulation. Analysis of steatotic patient and mouse tissues identify widespread alterations in hepatic copper homeostasis markers. Integrated multi-omics analyses reveal that copper induces lipolysis of PLIN2-containing lipid droplets while lipid importer CD36 is downregulated. We show that copper inhibits cAMP hydrolase activity of PDE3B, thus activating PKA-mediated HSL and AMPK activation upstream of lipolysis. Fatty acids liberated through lipolysis are subsequently degraded via enhanced mitochondrial fatty acid oxidation, supported by energetic rewiring toward oxidative phosphorylation (OXPHOS) with increased copper-dependent complex IV and SOD1 activity. Our findings establish a multi-pronged mechanism by which hepatic copper supplementation coordinately regulates lipid metabolism in response to steatosis and unveils a therapeutic metallomedicine strategy to rewire lipid regulation. Summary: Liver-targeted copper supplementation reduces diet-induced liver steatosis by dual activation of lipolysis and fatty acid degradation pathways. Graphical

Zinc is an essential transition metal that participates in many biological processes. In C. elegans , excess zinc is stored in lysosomes in intestinal cells; this process involves increasing the expression of the zinc transporter CDF-2 and remodeling of lysosomes characterized by an increase in the volume of the expansion compartment. To determine if this is a more general property, we investigated other metals. Here we report that lysosomes are remodeled in response to excess copper, manganese, and cadmium, with each metal causing an increase in the volume of the expansion compartment. Mutants with a reduced number of lysosomes were hypersensitive to growth retardation caused by excess copper and manganese, suggesting metal toxicity is prevented by metal sequestration in lysosomes. Using a novel method to analyze isolated lysosomes by X-ray Fluorescence Microscopy we demonstrated that zinc, copper and manganese are detectable in the lumen of lysosomes. To further analyze copper, we examined localization of CUA-1.1, a copper transporter that moves copper into the lumen of lysosomes. Like the zinc transporter CDF-2, CUA-1.1 localizes to both the acidified and expansion compartments in excess copper. These results indicate that the same intestinal lysosomes store zinc, copper and manganese. Lysosome remodeling characterized by an increase in volume of the expansion compartment is not specific to zinc but is a more general phenomenon during metal storage in lysosomes.

Chromatin remodeling by SWI/SNF complexes is essential for transcriptional regulation, yet how distinct SWI/SNF subcomplexes contribute to cellular stress responses remains incompletely understood. Here, we identify a specific role for the PBAF subunit Baf180 in regulating metal-responsive transcription and adaptation to metal stress in proliferating myoblasts. While knockdown (KD) of the BAF-specific subunit Baf250a or the ncBAF-specific subunit Brd9 significantly impairs myoblast proliferation, KD of the PBAF-specific subunit Baf180 has no effect under basal conditions. Notably, supplementation with copper (Cu) or zinc (Zn) restores proliferative capacity in Baf250a- and Brd9-deficient myoblasts. In contrast, Baf180-depleted myoblasts exhibit impaired proliferation upon metal exposure, accompanied by selective dysregulation of genes involved in Cu and Zn homeostasis. Transcriptomic and chromatin profiling further reveal that loss of Baf180 alters the activity of metal-regulatory transcription factor 1 (MTF1), including reduced chromatin occupancy at metal-responsive loci. Together, these findings support a model in which PBAF promotes metal-responsive gene regulation to maintain metal homeostasis and sustain myoblast proliferation, uncovering a previously unrecognized link between nucleosome remodeling and metal homeostasis during muscle cell proliferation.

Metal-based nanoparticles are a promising class of materials for diagnosis and treatment of cancer and other diseases. However, mechanisms of action of these nanomedicines remain insufficiently understood due in large part to our limited understanding of the dynamic equilibria between solid metal nanoparticles and labile metal ions generated from these nanoparticles within complex biological milieus. Here, we apply activity-based sensing to directly identify and investigate the fate of labile copper pools with metal and oxidation state-specificity generated by anticancer copper nanomedicines. We found that treatment of cells with copper-releasing nanoparticles alter labile Cu­(I)/Cu­(II) ratios through an increase in labile Cu­(II), while overall labile copper levels decrease. Labile copper release triggers compensatory responses in two major antioxidant pathways, glutathione (GSH) and nuclear factor erythroid 2-related factor 2 (NRF2), as well as in metal homeostasis to limit copper availability via regulation of copper export (ATP7B) and copper import (CTR1) proteins. These findings establish the value of activity-based sensing as a generalizable approach for labile metal imaging to help decipher molecular mechanisms of bioactive metal nanoparticles and guide the development of more effective nanomedicine diagnostics and therapies to target metal-dependent disease vulnerabilities.

Copper is an essential element for sustaining life. However, disruptions in copper homeostasis underpin disease, as illustrated by cuproptosis, an emerging form of cell death resulting from aberrant accumulation of copper pools in loosely bound, labile forms. Along these lines, activity-based sensing (ABS) offers a powerful strategy for tracking labile copper fluxes with metal and oxidation state selectivity by exploiting analyte reactivity for analyte detection. Traditional ABS probes for Cu(I), the major oxidation state of copper in cells, are selective but require O₂ as a coadditive, thus limiting their temporal resolution and sensitivity. Here, we present the design, synthesis, and biological evaluation of a first-generation ABS strategy for direct Cu(I) sensing by leveraging alkyne-directed cleavage reactivity. Copper Alkyne Probe-1 (CAP-1) features a rapid response to changes in intracellular Cu(I) pools with over 400-fold selectivity for Cu(I) over competing biological metals. We apply this probe to identify novel metal-metal crosstalk in cuproptosis, where we observe that Mn(II) exposure sensitizes cells to cuproptosis through upregulating the mitochondrial reductase FDX1 and depleting reduced glutathione, thus synergistically elevating labile Cu(I) levels. By revealing an interplay between copper and manganese in regulating cell death, this work provides a starting point for broader investigations of metal-metal nutrient crosstalk in biology and medicine.

Loss-of-function mutations in copper-ATPase ATP7B underlie Wilson disease (WD), a disorder characterized by hepatic copper accumulation and severe hepato-neuropathology. Existing chelation therapeutics remove excess copper but lack intrinsic antioxidant capacity and frequently cause systemic toxicity. Here we evaluate melatonin, an FDA-approved indoleamine with antioxidant and putative metal-chelating activity, as a candidate therapeutic for WD. In ATP7B⁻ ^/ ⁻ hepatocytes, melatonin restored copper-induced reactive oxygen species (ROS) to basal levels, reduced apoptosis twofold, and attenuated Nrf2 nuclear translocation leading to reduction of Hemoxygenase-1 abundance. Live-cell ratiometric analysis of GSSG/GSH using GRX1-roGFP2 expressed in melatonin-treated ATP7B⁻ ^/ ⁻ hepatocytes revealed a significant reduction in intensity-ratio, indicating an effective mitigation of copper-induced glutathione oxidation. Isothermal calorimetric titration revealed moderate Cu ²⁺ affinity (K ^ITC =4.54 × 10³ M⁻¹), rationalized by MD-simulations showing an interaction energy of 18.5 × 10⁻³ kcal·mol⁻¹ via amide–Cu²⁺ coordination. In-cellulo studies also revealed that copper-induced vesicularized ATP7B reinstates to Golgi in melatonin-treated hepatocytes. In vivo , melatonin treatment reduced copper-induced oxidative stress in zebrafish embryos and lowered copper burden in Caenorhabditis elegans WD model. Our studies revealed that encapsulation of melatonin within an engineered polymeric nanocapsules having dithiol linkers, susceptible to cleavage by GSH, extended melatonin’s circulatory half-life ten-fold and enhanced its ROS-scavenging efficacy three-fold relative to free melatonin. This work introduces a unique dual-function therapeutic strategy that integrates antioxidant activity with copper chelation, simultaneously addressing copper overload and redox imbalance. Repurposing melatonin, with its established clinical safety, offers rapid and cost-effective translational pathway toward WD-therapy while providing a generalizable platform for redox- and metal-associated disorders.

SWI/SNF chromatin remodeling complexes regulate gene expression by modulating nucleosome positioning, yet their roles in metal homeostasis during skeletal muscle development remain unclear. Here, we uncover distinct functions of the BAF, PBAF, and ncBAF complexes in myoblast proliferation under metal stress. While knockdown (KD) of Baf250a (BAF-specific) or Brd9 (ncBAF-specific) reduces myoblast proliferation, Baf180 (PBAF-specific) KD does not impair cell proliferation under basal conditions. Interestingly, supplementation with copper (Cu) or zinc (Zn) rescues proliferation in Baf250a - and Brd9 -deficient myoblasts but paradoxically inhibits growth in Baf180 KD cells. Mechanistically, Baf180 KD disrupts Cu and Zn homeostasis, leading to intracellular Cu accumulation without labile Cu⁺ pools and impaired expression of Atp7a , a key Cu exporter. Transcriptomic analyses reveal widespread gene dysregulation in metal-treated Baf180 -deficient cells, while metal supplementation promotes pro-proliferative gene expression in Baf250a - and Brd9 -KD myoblasts. CUT&RUN assays demonstrate that metal-responsive transcription factor Mtf1 exhibits increased chromatin binding upon Cu treatment, targeting genes involved in stress response and myogenesis. Notably, Mtf1 colocalizes with Baf180 in the nucleus and co-immunoprecipitates with both conserved SWI/SNF subunits and Baf180, suggesting a functional interplay between PBAF and Mtf1 in regulating metal-dependent gene expression. Our findings establish the PBAF complex as a crucial regulator of Cu/Zn homeostasis in myoblast proliferation via Mtf1, while metal supplementation compensates for BAF and ncBAF dysfunction but exacerbates defects in PBAF-deficient cells. This study reveals a novel link between chromatin remodeling, metal signaling, and muscle development, with implications for stress adaptation and metabolic regulation in myogenesis.