Metallomics

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 StatementCells 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.

Cadmium, being a highly toxic metal, perturbs cellular homeostasis by forming stable complexes with numerous thiol-active proteins, ultimately leading to severe liver and lung damage. Despite its well-documented toxicity, the molecular mechanisms governing cadmium export remain poorly understood. Given the chemical similarity between cadmium and copper, we investigated whether the canonical copper-exporting ATPases, ATP7A and ATP7B participate in cadmium handling. Upon Cd treatment in hepatocytes, ATP7B undergoes trafficking to lysosomes via the retromer complex, as also observed in the case of elevated copper, accompanied by the upregulation of acidic lysosomal populations. In contrast, ATP7A expressed in lung adenocarcinoma cells, though exhibit vesicular redistribution upon Cd exposure, does not mediate lysosomal sequestration, suggesting distinct deployment of late secretory pathways by the two copper ATPases in response to cadmium. We have also observed that ATP7B-/- hepatocytes exhibit increased sensitivity to Cd exposure compared to wild-type cells. Whereas, overexpressing the ATP7B amino-terminal copper-binding domain in bacteria alleviates cadmium-induced stress, indicating its capacity to sequester Cd. Caenorhabditis elegans lacking copper-ATPase cua-1, displayed increased Cd sensitivity, while mutants (glo-1-/-), deficient in lysosome-related organelles (LRO), and (lmp-1-/-), deficient in lysosomal membrane glycoprotein, showed reduced resistance to cadmium toxicity. Treatment of the worm with cadmium increases the abundance of lysosomes marked by elevation in lysosomal biogenesis and functional genes, reinforcing the importance of lysosomal pathways in cadmium detoxification. To summarise, we delineated the non-canonical role of copper ATPases and lysosomes in cadmium-induced cellular toxicity.

Organic germanium, particularly carboxyethyl germanium sesquioxide (Ge-132), has been investigated for decades in relation to diverse biological effects, with a strong emphasis on its antioxidant properties. However, the available literature remains dispersed, encompassing heterogeneous experimental models and endpoints that limit mechanistic interpretation. While antiglycative activity has been described at the biochemical level, its downstream gene regulatory consequences under glycative stress remain inconsistently characterized. Here, we combined systematic review of the literature of experimental studies with targeted molecular analysis in a standardized cellular model. The literature mapping was used to guide pathway selection rather than to establish quantitative associations. Based on patterns emerging from literature, we focused on pathways associated with glycative stress responses, including carbonyl stress, inflammatory signaling, and autophagy regulation. Gene expression analysis revealed a limited and selective modulation of regulatory pathways under glycative stress conditions, consistent with a context-dependent effect rather than broad transcriptional reprogramming. In parallel, protein analysis showed reduced intracellular accumulation of advanced glycation end products (AGEs) in Ge-132-treated cells under glycative stress conditions. Importantly, these findings support a dissociation between glycative damage reduction and cellular stress-response pathways. This combined approach helps interpretation of previously fragmented observations across the literature and highlights gene regulation under glycative stress as a relevant but still unresolved aspect of organogermanium biology.

Chemotherapy treatment of colorectal cancers (CRC) using cisplatin (CDDP) encounters problems of drug resistance by the cancer cells and cytotoxicity to normal cells, highlighting the urgent need for joint therapeutical strategies. Selenium-enriched rapeseed extracts exhibit anti-cancer effects but the bioactive components and mechanisms remain unclear. Here, we applied different solvents to fractionate the extracts from Selenium-enriched rapeseed and found that the water extract (WE) fraction significantly enhanced the cytotoxic effect of CDDP on cancer cells but no damage on normal cells. HPLC-ICP-MS analysis revealed that methylselenocysteine (MSC) and selenocystine (SeCys2) were the main selenium speciation in WE. Through cell biology and integrative multi-omics analysis, we found a synergistic anti-CRC cell effect when combining CDDP with MSC, sulforaphane (SFN), celastrol (Cel), Indole-3-carbinol (I3C), -linolenic acid (ALA) or linoleic acid (LA). We propose that the CDDP-WE combination treatment holds the promise for improving curative efficacy for chemo-refractory CRC patients in the future.

Selenium is an essential trace element incorporated into selenoproteins as selenocysteine, yet the intestinal cellular programs associated with selenium utilization remain poorly defined. Here, we performed a large-scale single-cell transcriptomic analysis of 169,068 human enterocytes from 105 donors to systematically profile nine candidate selenium transporter systems and their relationship to selenoprotein expression. Among all transporters examined, the b0+ amino acid transporter system (SLC3A1/SLC7A9) showed the strongest and most maturation-independent association with selenoproteins, including SELENOP (Spearman r = 0.489) and GPX4 (r = 0.392), with enrichment across 21 of 24 detected selenoproteins in b0+-complete versus transporter-negative enterocytes. These associations were robust across individual donors and confirmed by pseudobulk validation, and were only partially explained by enterocyte maturation state and intestinal segment after covariate adjustment, indicating both differentiation-dependent and differentiation-independent components. Furthermore, LRP1 and LRP5, not the canonical SELENOP receptors LRP2 and LRP8, emerged as the predominant LRP family members in enterocytes and were preferentially enriched in b0+-complete cells, suggesting a distinct intestinal SELENOP receptor landscape. These findings provide the first single-cell transcriptomic evidence that b0+ transporter expression is associated with a selenoprotein-enriched enterocyte state and identify candidate receptor programs for intestinal SELENOP uptake.

The facultative methylotroph model organism Methylorubrum extorquens AM1 is a known lanthanide user, which has shed light on the role of rare-earth metals in biochemistry. The characterization of a methanol dehydrogenase (MDH) protein which requires lanthanides as an enzymatic cofactor outlined the question of how these metals are acquired from the environment. It has been proposed that mesophilic organisms as M. extorquens AM1 can produce siderophore-like molecules, which chelate, transport and traffic rare-earth elements into the microbial cell. Therefore, we performed the bioinformatic and chemical investigation of M. extorquens AM1 by using genome mining, the CAS and arsenazo assay, molecular networking and chemical analytical techniques. Our results showed that indeed Methylorubrum extorquens AM1 harbored a gene cluster to produce metal chelators. The chemical analysis confirmed the production of the known hybrid hydroxamate-citrate siderophores schizokinen A and N-deoxyschizokinen A, which are very likely the side products of the transformation of schizokinen and N-deoxyschizokinen. The determination of the lanthanide chelation activity of the schizokinen siderophores series against three different lanthanides (La, Eu and Lu) showed no coordination activity, thus ruling out the involvement of schizokinen siderophores in rare-earth metal transport.

Flavins are organic redox cofactors central to metabolism and uniquely capable of acting as extracellular electron shuttles. For life to have emerged, it must have disengaged itself from its stationary geochemical environment, a step requiring mobile redox-active components. The role of flavins at life's origin has been debated for decades, centered on their capacity for both one- and two-electron chemistry, distinguishing them from nicotinamides and iron-sulfur clusters. Here we chart the abiotic reduction of flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and riboflavin under hydrothermal conditions (40 {degrees}C, 1 bar N2 or 5 bar H2, pH 6, 8, and 10) by nickel (Ni) and iron (Fe). Flavins show greater environmental versatility than hydride carriers such as NAD and can harvest electrons from metals that would otherwise reduce water's protons to H2. Reduction is favoured under acidic conditions, while increasing molecular charge at higher pH impedes electron transfer. Ni acts as a hydrogenation catalyst, reducing deprotonated flavins via hydride transfer, suggesting mineral composition could have influenced geochemical selection of early electron carriers. Reduced FMNH2 and FADH2 were tested as electron shuttles toward Fe3+-containing minerals, revealing that FMNH2 enables faster mineral dissolution than FADH2. We further demonstrate complete redox cycling of FMN through Ni-assisted H2 reduction and subsequent oxidation by magnetite (Fe3O4) under inert atmosphere, releasing Fe2+. This study highlights the versatility, stability and redox chemical capabilities of flavins in prebiotic context.

Copper and iron are redox-active micronutrients with tightly coupled homeostasis, yet how copper modulates iron-dependent stress responses remains unclear. Using Saccharomyces cerevisiae under nutrient-limited conditions, we uncoupled proliferative growth from long-term survival to dissect metal-dependent adaptation. Copper selectively preserved survival without affecting growth, whereas iron showed similar effects. Iron chelation impaired growth and suppressed electron transport chain gene expression; copper partially rescued these defects but required iron for its pro-survival activity. Despite this interdependence, copper and iron engaged distinct signaling programs. Iron-dependent survival required a Target of Rapamycin complex 1 (TORC1)-permissive state and was attenuated by rapamycin, whereas copper remained active under TORC1 inhibition. In contrast, copper promoted survival through AMP-activated protein kinase (AMPK) and antioxidant pathways, while iron exhibited context-dependent AMPK reliance. Together, these findings identify copper and iron as state-dependent regulators of cellular survival.

The rapid expansion of human genomic data has revealed a large number of naturally occurring variants, creating a major challenge for functional annotation. The human metal transporter SLC39A8 (ZIP8) is a clinically important, promiscuous divalent metal transporter, yet most of its documented variants remain uncharacterized. Here, we developed a workflow to functionally evaluate ZIP8 variants by integrating laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS) with scaled-up cell-based transport assays. Using this method, we systematically analyzed 33 naturally occurring missense variants located in the extracellular domain (ECD) of ZIP8. The assay enables direct quantification of intracellular metal accumulation with substantially improved throughput ([~]150 samples per hour). Functional screening identified 14 potential pathogenic variants with significantly reduced transport activity. Comparison with computational predictions revealed a moderate correlation between activity and AlphaMissense pathogenicity scores (R2 = 0.423), while an error rate of [~]20% underscores the need for experimental validation. Flow cytometry analysis showed that most loss-of-function variants exhibit impaired trafficking of the protein to the cell surface possibly due to mutation-caused protein misfolding or instability. Structural mapping of activity-compromised variants, together with functional assessment of the ZIP8-ECD, highlights the importance of this domain in ZIP8 expression and intracellular trafficking. Together, this work establishes a scalable approach for functional screening of metal transporter variants and provides new insights into the structure-function relationships of ZIP8.

Groundwater contamination presents challenges across world, yet remediation solutions in variably oxidized regions are limited and many co-interactions between contaminant metals and microbial reactions occur. Here we present a genomic and metabolic study into the biogeochemistry of a uranium-contaminated surficial aquifer site in Riverton, WY. We identified unique communities that varied based on geochemistry, geography, and compartment, matching microbial subsurface studies. Cross-site metabolism tests showed communities had functional capabilities of nitrogen respiration, manganese reduction, iron reduction, and sulfide oxidization. No sites showed evidence of microbial U-bioreduction nor ammonium oxidation. Only former tailings area groundwater and ditch surface water sites nearest a retention pond, and a downgradient oxbow lake exhibited sulfate reduction metabolisms. This was contrary to our hypothesis of near-river downgradient groundwater sites having U and S reduction capability. Most communities which showed S reduction capacity exhibited Fe oxidation capacity. Modeling demonstrated U as calcium uranyl carbonates. Based on our metabolism tests and known mineral and microbial metabolism reduction potentials, this suggests U reduction could only be achieved via abiotic reaction with biogenic sulfide. Of eleven sites tested, it is possible in four. This has impact on future site-specific remediation plans and understanding of microbial reactions in variably reduced zones. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/729369v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@1926923org.highwire.dtl.DTLVardef@13486f9org.highwire.dtl.DTLVardef@1895a91org.highwire.dtl.DTLVardef@990213_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical Abstract TextWe performed microbial membership and metabolism measurements across a uranium-contaminated sites surface and ground waters, then performed analyses relating these metrics to geochemistry at the site. Findings showed variations in the membership, yet mainly similar functional capabilities. Metabolic differences were explained in relationship to uranium cycling and remediation implications.

O2-tolerance is a desirable property for [FeFe] hydrogenases, which are highly efficient H2-producing catalysts. While most such enzymes are highly sensitive to aerobic environments, a small number of explored representatives exhibit exceptional stability and even H2-producing activity under oxygenic conditions. However, the genetic signatures of the O2-tolerance in this class of enzymes remain largely unknown. To address this knowledge gap, we explored a close homologue of a well-characterized O2-tolerant [FeFe] hydrogenase from Clostridium beijerinckii (CbHydA1) - a hydrogenase from Terrisporobacter glycolicus (TgHydA1). Our investigation indeed confirms that TgHydA1 can transition to the O2-stable Hinact state, a hallmark of O2 tolerance. The surprising outcome is that despite the high amino acid similarity, TgHydA1 shows a substantially higher propensity to remain in the Hinact state than CbHydA1. Using protein film electrochemical experiments, we demonstrate that the root of this behavior lies in roughly tenfold slower reactivation rates than those of CbHydA1 at any applied potential. This degree and direction of variation in reactivation kinetics have not been observed before for any other O2-tolerant [FeFe] hydrogenases or their variants to date, uncovering a yet-to-be-explored facet of reactivity alteration available to these enzymes. Overall, the results presented here highlight the importance of a holistic analysis of [FeFe] hydrogenase sequences in the context of their interaction with O2 that encompasses the protein environment and properties of the auxiliary metallocofactors.

Chemically complex pharmaceutical mixtures in wastewater and sludge can affect microbial adaptation; however, the responses to different co-occurring compounds have not been elucidated well. In this study, the adaptation of a strain derived from hospital sludge, Klebsiella pneumoniae SS02, to 17-ethinylestradiol (EE2), warfarin sodium, and their combination has been studied. The organism grows under all three conditions, and pre-exposure experiments show induction and cross-induction to substrates. UHPLC MS/MS analyses demonstrated that there is conditional depletion of the parent compound EE2 by [~]15% at 36 h post-treatment compared to initial concentrations, but not for the abiotic and non-adapted controls. The rate of warfarin sodium depletion was approximately [~]30% within 36 h and was in accordance with first order kinetics (k = 0.0102 /h; t{square}/{square}= 67.9 h). Under the combined treatment regime, there was a delay in warfarin sodium depletion, suggesting staged substrate consumption. Growth inhibition with efflux inhibitors confirmed transport-driven tolerance. A genome-based study revealed the coordinated response strategy that involved a proposed flavin-dependent monooxygenase (RutA), an oxidative entry into the pathway; redox processing linked to Hpa; aromatic metabolism through {beta}-ketoadipate pathway; and RND efflux system. The structural study additionally supported ligand-mediated decrease in DNA binding affinity of RutR, which is in agreement with de-repression of the substrate-activated regulatory mechanism. All these findings lead to the development of a dual-strategy for adaptation model in which oxidative modification and efflux-mediated protection work together under the influence of a mixture of pharmaceuticals.

Supersulfides are emerging sulfur-containing signaling molecules involved in redox regulation, mitochondrial function, and protein S-sulfhydration. However, their dynamic behavior in living mammalian systems remains poorly understood because existing analytical methods require destructive sample preparation or lack sufficient intracellular applicability. Here, we developed a genetically encoded supersulfide-dependent translocation reporter (SuTR) for mammalian cells and in vivo imaging. Although the previously reported probe psGFP failed to respond to supersulfides in mammalian cells, fusion of psGFP with the sulfide-responsive transcriptional repressor (SqrR) generated SuTR, a novel reporter that exhibited supersulfide-dependent translocation from the nucleus to the cytoplasm. Na2S2 and Na2S induced dose-dependent cytosolic translocation of SuTR, whereas Na2S showed no effect. Fluorescence recovery after photobleaching (FRAP) analysis revealed accelerated fluorescence recovery shortly after supersulfide stimulation, and overexpression of the endogenous supersulfide-producing enzyme Cysteinyl-tRNA Synthetase 2 (CARS2) similarly altered reporter dynamics. Mutational analyses demonstrated that reporter responsiveness depends on the DNA-binding activity of SqrR. Furthermore, SuTR successfully detected supersulfide induction in mouse liver in vivo following Na2S administration. These findings establish SuTR as a genetically encoded reporter for monitoring supersulfide dynamics in mammalian cells and tissues. HighlightsO_LIWe developed SuTR, a genetically encoded supersulfide-dependent translocation reporter. C_LIO_LISupersulfides induce nuclear-to-cytoplasmic translocation of SuTR C_LIO_LIFRAP enables rapid detection of endogenous and exogenous supersulfide responses C_LIO_LISuTR activity depends on the DNA-binding function of SqrR C_LIO_LISuTR enables visualization of supersulfide dynamics in mouse liver in vivo C_LI

Microbial ammonia oxidation, the first and rate-limiting step of nitrification, plays a central role in soil nitrogen cycling. It is most relevant in agricultural soils as nitrifiers compete with crops for ammonia-based fertilizers. Therefore, synthetic nitrification inhibitors are widely used alongside fertilizers to reduce the activities of dominant drivers of this process, i.e. ammonia-oxidizing archaea (AOA) and bacteria (AOB). However, the physiological responses of ammonia oxidizers remain poorly resolved. Here the response of the AOA Nitrososphaera viennensis to the nitrification inhibitors 3,4-dimethylpyrazole phosphate (DMPP) and allylthiourea (ATU) were investigated using a combination of functional genomics, physiological assays, and relief experiments. The results overturn earlier assumptions that DMPP and ATU act by chelating free copper. Both compounds affected ammonia oxidation and triggered broader shifts in energy metabolism and stress-response pathways, which diverged markedly between the two inhibitors. We propose a competitive inhibition of the ammonia monooxygenase complex with DMPP as it can be alleviated by additional ammonia and elicits activation of urea acquisition, while ATU acted as a non-competitive inhibitor generally inducing quiescence. Both modes of inhibition were associated with clear transcriptomic and proteomic signals that will be advantageous for the identification of mechanisms of other nitrification inhibitors in the future. Key word: Ammonia-oxidizing archaea, nitrification, nitrification inhibitors, archaea, nitrogen cycle

The tight regulation of iron homeostasis is of great importance for cellular health. An increase in intracellular iron levels results in the formation of free radicals, which damages macromolecules and membranes, eventually resulting in cell death by Ferroptosis. Recently, we showed that patients with mutations in VPS41 display a severe neurodegenerative phenotype with iron deposition in the brain. VPS41 is well known as subunit of the HOPS complex required for fusion of late endosomes and autophagosomes with lysosomes. However, VPS41 has also been identified as inhibitor of Ferroptosis and regulator of redox homeostasis. How VPS41 exerts these functions and if these are dependent on the HOPS complex is unknown. Here we show that depletion of VPS41 results in increased intracellular iron levels, ROS formation and mitochondrial fission. Our findings indicate an important role for VPS41 in the regulation of iron homeostasis and mitochondrial fission and suggest Ferroptosis as a possible cause for neurodegeneration in VPS41 patients.

Peroxiredoxin 2 (Prdx2) mediates redox signaling by transferring oxidative equivalents to target proteins such as STAT3, a redox-sensitive transcription factor implicated in inflammation and cancer. Although this interaction has been demonstrated in cells, reconstituting the Prdx2:STAT3 complex in vitro remains challenging due to its transient and redox-dependent nature. Here we test various conditions to stabilize the complex between taggless Prdx2 and the core fragment of STAT3 (CF-STAT3), including oxidants, detergents, the facilitator Annexin A2, anaerobic environments, and CovalX crosslinking. Complex formation was assessed via mass photometry, analytical size-exclusion chromatography (SEC), SEC-MALS, and electron microscopy (EM). No stable complex was observed under standard conditions. Anaerobic environments briefly stabilized the interaction, but cryo-EM could not resolve the structure. CovalX crosslinking yielded short-lived but homogeneous complexes. We found that Prdx2 is highly susceptible to hyperoxidation at its peroxidatic cysteine, particularly in the presence of DTT or excess H2O2, resulting in loss of function. Maintaining non-reducing conditions during purification preserved Prdx2 in an oxidation-competent state, promoting formation of the disulfide bond between the peroxidatic and resolving cysteines and thereby enabling reproducible detection of a weak complex with CF-STAT3. Our findings establish a framework for studying redox-relay protein complexes in vitro and highlight the importance of oxidation state management during protein handling.

[FeFe]-hydrogenases are very active biocatalysts for H2 conversion. However, their active site is vulnerable to irreversible degradation initiated by O2 binding at the catalytic iron ion (Fed) of the active center. CbA5H, the [FeFe]-hydrogenases from Clostridium beijerinckii exhibits stability towards oxygen (O2) due to its ability to reversibly enter an inactive state termed Hinact upon contact with O2. We previously proposed that the close distance of approximately 3.1 [A] between the thiol of a nearby cysteine (C367) and the Fed, based on a 2.9 [A] crystal structure of CbA5H in the Hinact state, enables their binding to each other. This binding therefore was suggested to shield the Fed from O2 damage. However, there is currently a lack of evidence to support this hypothesis. Furthermore, density functional theory (DFT) calculations based on a homologous model favored hydroxide as the binding ligand of the Fed over the thiol of C367. In this study, we present the crystal structure of CbA5H in the Hinact state at an improved resolution of 2.15 [A]. The structure reveals a direct binding between the thiol of C367 and the Fed with a distance of approximated 2.77 [A] which is well supported by our DFT calculations based on the new crystallographic data. It is noteworthy that the 2.77 [A] bond distance is strikingly long when compared with other iron-sulfur bonds. This finding may provide a crucial foundation for understanding the rapid reversibility of the Hinact state.

BackgroundWhile -synuclein (-syn) accumulation and iron dysregulation are hallmarks of Parkinsons Disease, the adaptations that enable neuronal survival under chronic protein stress remain unclear. Here, we investigated how -syn overexpression and ceruloplasmin (Cp)-mediated iron modulation alters iron and redox homeostasis. MethodsWe utilized human BE(2)-M17 neuroblastoma cell lines stably expressing different levels of -syn to examine the interplay between -syn, Ceruloplasmin (Cp)-mediated iron modulation, and the cellular response to oxidative stress. Analyses included Western blotting, immunofluorescence staining, soluble/insoluble fractionation, glutathione, reactive oxygen species (ROS) and hydrogen peroxide (H2O2) quantification, lipid peroxidation, ferrous iron, and cell viability. ResultsOur data suggest an unexpected relationship between chronic -syn expression and cellular redox regulation. Despite carrying a greater -syn burden, cells with higher -syn expression exhibit lower basal ROS, H2O2, and lipid peroxidation compared to control cells. These changes are not accompanied by activation of canonical antioxidant pathways suggesting that the reduced oxidative profile arises through alternative mechanisms. Besides, -syn over-expressing cells display significant remodeling of iron-handling pathways, including altered expression of ferritin heavy chain, transferrin receptor, and ferroportin, suggesting that chronically high -syn levels are associated with changes in iron homeostasis. In addition, this phenotype is not maintained following Cp overexpression. Although Cp reduces Fe{superscript 2} levels, it also induces substantial increases in ROS and H2O2 without corresponding changes in GPX4, glutathione, or related antioxidant systems. Thus, the reduced basal oxidative profile observed in -syn-over-expressing cells does not reflect enhanced canonical antioxidant capacity. Instead, chronically high -syn levels appear to be associated with adaptive remodeling of iron and redox pathways that become sensitive to oxidative imbalance. ConclusionChronic -syn over-expression promotes adaptive remodeling of iron and redox homeostasis, associated with reduced basal oxidative stress but increased sensitivity to Cp-mediated perturbation. These data link -syn burden to iron metabolism and stress-dependent vulnerability in synucleinopathies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/732494v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@9ab785org.highwire.dtl.DTLVardef@186a200org.highwire.dtl.DTLVardef@1f5c13forg.highwire.dtl.DTLVardef@162249e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Ferrosomes are recently discovered lipid-bound bacterial organelles that store iron as iron-phosphate biominerals, yet the chemical nature and physiological consequences of ferrosome-stored iron remain poorly understood. Here, we combined X-ray absorption spectroscopy (XAS), electron microscopy, inductively coupled plasma mass spectrometry (ICP-MS), and physiological analyses to characterize ferrosome iron in Clostridioides difficile. XAS analysis of isolated ferrosomes revealed an amorphous iron-phosphate biomineral containing mixed Fe(II)/Fe(III), consistent with partial oxidation during aerobic isolation. In contrast, whole-cell XAS of intact anaerobically maintained cells demonstrated that ferrosomes predominantly contain a structurally disordered ferrous phosphate biomineral with local Fe-O-P coordination features similar to those of vivianite. Upon air exposure, this ferrous biomineral rapidly oxidized to a ferric phosphate-like state, revealing a highly oxygen-sensitive iron-storage phase. Despite containing abundant redox-active Fe(II), ferrosome-stored iron contributed minimally to the cytosolic labile iron pool. Consistent with this observation, isolated ferrosomes exhibited little ROS-generating activity, and ferrosome-overproducing cells displayed no substantial increase in sensitivity to oxygen, peroxide, or paraquat stress relative to ferrosome-deficient controls. Together, these results establish ferrosomes as iron-storage organelles that sequester redox-active Fe(II) in a mineralized ferrous phosphate phase, limiting its participation in cytosolic ROS chemistry and providing a mechanism for the safe storage of reactive iron. Significance StatementIron is essential for life but can also damage cells because ferrous iron drives oxidative stress. How cells store large amounts of ferrous iron while limiting toxicity therefore remains a fundamental biological question. Ferrosomes are recently discovered bacterial organelles that store iron as iron-phosphate biominerals, but the chemical nature and physiological consequences of ferrosome-associated iron remained unknown. Using Fe K-edge X-ray absorption spectroscopy, we show that ferrosomes in Clostridioides difficile contain a redox-sensitive ferrous phosphate biomineral. Physiological analyses demonstrate that this iron is largely inaccessible to cytosolic reactive oxygen species (ROS) chemistry. These findings reveal that bacteria can combine biomineralization and subcellular compartmentalization to maintain large intracellular iron reservoirs while limiting iron-dependent oxidative damage.

Colletotrichum sublineola (Cs), the hemibiotrophic fungus that causes sorghum anthracnose, impacts sorghum grain and biomass crop production worldwide. Although nutrient availability is known to influence development in filamentous fungi, including Colletotrichum species, how in vitro nutrient limitation reprograms the Cs cellular state remains unclear. We cultured Cs on full-strength, half-strength, and one-tenth-strength potato dextrose agar (PDA) to define responses across a nutrient gradient. Nutrient limitation induced a pronounced high-sporulation phenotype, with one-tenth-strength PDA producing the strongest conidiation response, followed by half-strength PDA. To study the underlying molecular programs in each condition, we employed a multiplexed metabolite, protein, and lipid extraction (MPLEx) protocol for global proteomics and metabolomics. Global proteomics resulted in 4,590 protein identifications, including 204 unique to one-tenth-strength PDA. Among them are proteins linked to sporulation, vesicular transport, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, and common in fungal extracellular membrane (CFEM)-domain proteins. Differential abundance and pathway analyses revealed a broad reduction of central carbon and energy metabolism, including glycolysis/gluconeogenesis, pentose phosphate, pyruvate metabolism, and glyoxylate pathways, together with increased ribosome-related processes, cAMP signaling, and cell-surface remodeling in one-tenth-strength PDA conditions. In addition, correlative metabolomics supported selective metabolic depletion and resource reallocation toward stress adaptation, membrane remodeling, and conidiation, supporting proteomics findings. Together, these data support a starvation-adapted Cs developmental state associated with enhanced sporulation, cellular pathway reprogramming, and potential virulence linked preparedness under nutrient-limited growth conditions in vitro. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/724728v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@f6ceb2org.highwire.dtl.DTLVardef@17c4836org.highwire.dtl.DTLVardef@68e995org.highwire.dtl.DTLVardef@1bf3983_HPS_FORMAT_FIGEXP M_FIG C_FIG