Biology subjects

Pezacki, A. T.

Publications and source records attributed to Pezacki, A. T..

7 recordsLinked to original sources

bioRxiv2026

Lysosomal Expansion Compartments Mediate Zinc and Copper Homeostasis in Caenorhabditis elegans

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.

molecular biology↗

bioRxiv2025

Repurposing Melatonin in dual-mode for Wilson disease therapy as a Copper Chelator and an antioxidant agent

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 Cu2+ affinity (K ITC=4.54 x 103 M-{superscript 1}), rationalized by MD-simulations showing an interaction energy of 18.5 x 10-3 kcal{middle dot}mol-{superscript 1} via amide-Cu{superscript 2} 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 melatonins 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.

cell biology↗

bioRxiv2025

The PBAF chromatin remodeling complex contributes to metal homeostasis through Mtf1 regulation

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.

molecular biology↗

bioRxiv2025

Endothelial Cu Uptake Transporter CTR1 Senses Disturbed Flow to Promote Atherosclerosis through Cuproptosis

Endothelial cells (ECs) lining blood vessels sense disturbed blood flow (D-flow), which drives mitochondrial dysfunction and atherosclerosis. Copper (Cu) is an essential micronutrient, and its disruption of homeostasis has been implicated in atherosclerosis. Cellular Cu levels are tightly controlled by Cu transport proteins including the Cu importer CTR1. Cuproptosis is a recently discovered form of regulated cell death triggered by mitochondrial Cu accumulation, but its endogenous stimulants and role in atherosclerosis remain unknown. Using EC-specific CTR1-deficient mice and cultured ECs, we show that endothelial CTR1 responds to D-flow by increasing mitochondrial Cu levels through its interaction with the mitochondrial Cu transporter SLC25A3 at caveolae/lipid rafts. This leads to the aggregation of lipoylated mitochondrial proteins, mitochondrial dysfunction, and cuproptosis, thereby exacerbating atherosclerosis. Importantly, mitochondria-targeted Cu-chelating nanoparticles effectively mitigate D-flow-induced cuproptosis and atherosclerosis, highlighting the endothelial CTR1-SLC25A3-mitochondrial Cu axis as a potential therapeutic target.

cell biology↗

bioRxiv2024

Cysteine Rich Intestinal Protein 2 is a copper-Responsive regulator of skeletal muscle differentiation.

Copper (Cu) is an essential trace element required for respiration, neurotransmitter synthesis, oxidative stress response, and transcriptional regulation. Imbalance in Cu homeostasis can lead to several pathological conditions, affecting neuronal, cognitive, and muscular development. Mechanistically, Cu and Cu-binding proteins (Cu-BPs) have an important but underappreciated role in transcription regulation in mammalian cells. In this context, our lab investigates the contributions of novel Cu-BPs in skeletal muscle differentiation using murine primary myoblasts. Through an unbiased synchrotron X-ray fluorescence-mass spectrometry (XRF/MS) metalloproteomic approach, we identified the murine cysteine rich intestinal protein 2 (mCrip2) in a sample that showed enriched Cu signal, which was isolated from differentiating primary myoblasts derived from mouse satellite cells. Immunolocalization analyses showed that mCrip2 is abundant in both nuclear and cytosolic fractions. Thus, we hypothesized that mCrip2 might have differential roles depending on its cellular localization in the skeletal muscle lineage. mCrip2 is a LIM-family protein with 4 conserved Zn2+-binding sites. Homology and phylogenetic analyses showed that mammalian Crip2 possesses histidine residues near two of the Zn2+-binding sites (CX2C-HX2C) which are potentially implicated in Cu+-binding and competition with Zn2+. Biochemical characterization of recombinant human hsCRIP2 revealed a high Cu+-binding affinity for two and four Cu+ ions and limited redox potential. Functional characterization using CRISPR/Cas9-mediated deletion of mCrip2 in primary myoblasts did not impact proliferation, but impaired myogenesis by decreasing the expression of differentiation markers, possibly attributed to Cu accumulation. Transcriptome analyses of proliferating and differentiating mCrip2 KO myoblasts showed alterations in mRNA processing, protein translation, ribosome synthesis, and chromatin organization. CUT&RUN analyses showed that mCrip2 associates with a select set of gene promoters, including MyoD1 and metallothioneins, acting as a novel Cu-responsive or Cu-regulating protein. Our work demonstrates novel regulatory functions of mCrip2 that mediate skeletal muscle differentiation, presenting new features of the Cu-network in myoblasts.

molecular biology↗

bioRxiv2023

Glial swip-10 expression controls systemic mitochondrial function, oxidative stress, and neuronal viability via copper ion homeostasis

Cuprous copper (Cu(I)) is an essential cofactor for enzymes supporting many cellular functions including mitochondrial respiration and suppression of oxidative stress. Neurons are particularly dependent on these pathways, with multiple neurodegenerative diseases, including Alzheimers disease (AD), Parkinsons disease, associated with their dysfunction. Key features of Cu(I) contributions to neuronal health in vivo remain to be defined, owing largely to the complex processes involved in Cu(I) production, intracellular transport, and systemic redistribution. Here, we provide genetic and pharmacological evidence that swip-10 is a critical determinant of systemic Cu(I) levels in C. elegans, with deletion leading to systemic deficits in mitochondrial respiration, production of oxidative stress, and neurodegeneration. These phenotypes can be reproduced in wild-type worms by Cu(I)-specific chelation and offset in swip-10 mutants by growth on the Cu(I) enhancing molecule elesclomol, as well as by glial expression of wildtype swip-10. MBLAC1, the most closely related mammalian ortholog to swip-10, encodes for a pre-mRNA processing enzyme for H3 histone, a protein whose actions surprisingly include an enzymatic capacity to produce Cu(I) via the reduction of Cu(II). Moreover, genome-wide association studies and post-mortem molecular studies implicate reductions of MBLAC1 expression in risk for AD with cardiovascular disease comorbidity. Consistent with these studies, we demonstrate that the deposition of {beta}-amyloid plaques, an AD pathological hallmark, in worms engineered to express human A{beta}1-42, is greatly exaggerated by mutation of swip-10. Together, these studies identify a novel glial-expressed, and pathway for Cu(I) production that may be targeted for the treatment of AD and other neurodegenerative diseases. Significance StatementDevastating neurodegenerative diseases such as Alzheimers disease, and Parkinsons disease are associated with disruptions in copper (Cu) homeostasis. Alterations in Cu(I) give rise to increased oxidative stress burden, mitochondrial and metabolic dysfunction, and can accelerate production and/or potentiate toxicity of disease-associated protein aggregates. Here, using the model system Caenorhabditis elegans, we establish a role for the gene swip-10 in systemic Cu(I) homeostasis. Perturbation of this pathway in worms recapitulates biochemical, histological, and pathological features seen in human neurodegenerative disease. We reveal that these changes can be suppressed pharmacologically and arise when swip-10 expression is eliminated from glial cells. Our work implicates swip-10 and orthologs as key players in Cu(I) homeostasis that may be exploitable to treat multiple neurodegenerative diseases.

neuroscience↗

bioRxiv2023

Leishmania major-induced alteration of host cellular and systemic copper homeostasis drives the fate of infection

Copper plays a key role in host-pathogen interaction. We found that during Leishmania major infection, the parasite-harboring macrophage regulates its copper homeostasis pathway in a way to facilitate copper-mediated neutralization of the pathogen. Copper-ATPase ATP7A transports copper to amastigote-harboring phagolysosomes to induce stress on parasites. Leishmania in order to evade the copper stress, utilizes a variety of manipulative measures to lower the host-induced copper stress. It induces deglycosylation and degradation of host-ATP7A and downregulation of copper importer, CTR1 by cysteine oxidation. Additionally, Leishmania induces CTR1 endocytosis that arrests copper uptake. In mouse model of infection, we report an increase in systemic bioavailable copper in infected animals. Heart acts as the major organ for diverting its copper reserves to systemic circulation to fight-off infection by downregulating its CTR1. Our study explores reciprocal mechanism of manipulation of host copper homeostasis pathway by macrophage and Leishmania to gain respective advantages in host-pathogen interaction.

cell biology↗