Elesclomol elevates cellular and mitochondrial iron levels by delivering copper to the iron import machinery.

Copper (Cu) and iron (Fe) are redox-active metals that serve as cofactors for many essential cellular enzymes. Disruption in the intracellular homeostasis of these metals results in debilitating and frequently fatal human disorders, such as Menkes disease and Friedreich's ataxia. Recently, we reported that an investigational anticancer drug, elesclomol (ES), can deliver Cu to critical mitochondrial cuproenzymes and has the potential to be repurposed for the treatment of Cu deficiency disorders. Here, we sought to determine the specificity of ES and the ES-Cu complex in delivering Cu to cuproenzymes in different intracellular compartments. Using a combination of yeast genetics, subcellular fractionation, and inductively coupled plasma-mass spectrometry-based metal measurements, we showed that ES and ES-Cu treatment results in an increase in cellular and mitochondrial Fe content, along with the expected increase in Cu. Using yeast mutants of Cu and Fe transporters, we demonstrate that ES-based elevation in cellular Fe levels is independent of the major cellular Cu importer but is dependent on the Fe importer Ftr1 and its partner Fet3, a multicopper oxidase. As Fet3 is metalated in the Golgi lumen, we sought to uncover the mechanism by which Fet3 receives Cu from ES. Using yeast knockouts of genes involved in Cu delivery to Fet3, we determined that ES can bypass Atx1, a metallochaperone involved in Cu delivery to the Golgi membrane Cu pump, Ccc2, but not Ccc2 itself. Taken together, our study provides a mechanism by which ES distributes Cu in cells and impacts cellular and mitochondrial Fe homeostasis.

Proteome plasticity in response to persistent environmental change.

Temperature is a variable component of the environment, and all organisms must deal with or adapt to temperature change. Acute temperature change activates cellular stress responses, resulting in refolding or removal of damaged proteins. However, how organisms adapt to long-term temperature change remains largely unexplored. Here we report that budding yeast responds to long-term high temperature challenge by switching from chaperone induction to reduction of temperature-sensitive proteins and re-localizing a portion of its proteome. Surprisingly, we also find that many proteins adopt an alternative conformation. Using Fet3p as an example, we find that the temperature-dependent conformational difference is accompanied by distinct thermostability, subcellular localization, and, importantly, cellular functions. We postulate that, in addition to the known mechanisms of adaptation, conformational plasticity allows some polypeptides to acquire new biophysical properties and functions when environmental change endures.

Targeting Copper Homeostasis Improves Functioning of vps13Δ Yeast Mutant Cells, a Model of VPS13-Related Diseases.

Ion homeostasis is crucial for organism functioning, and its alterations may cause diseases. For example, copper insufficiency and overload are associated with Menkes and Wilson's diseases, respectively, and iron imbalance is observed in Parkinson's and Alzheimer's diseases. To better understand human diseases, Saccharomyces cerevisiae yeast are used as a model organism. In our studies, we used the vps13Δ yeast strain as a model of rare neurological diseases caused by mutations in VPS13A-D genes. In this work, we show that overexpression of genes encoding copper transporters, CTR1, CTR3, and CCC2, or the addition of copper salt to the medium, improved functioning of the vps13Δ mutant. We show that their mechanism of action, at least partially, depends on increasing iron content in the cells by the copper-dependent iron uptake system. Finally, we present that treatment with copper ionophores, disulfiram, elesclomol, and sodium pyrithione, also resulted in alleviation of the defects observed in vps13Δ cells. Our study points at copper and iron homeostasis as a potential therapeutic target for further investigation in higher eukaryotic models of VPS13-related diseases.

Epigallocatechin-3-O-gallate, the main green tea component, is toxic to Saccharomyces cerevisiae cells lacking the Fet3/Ftr1.

Epigallocatechin-3-O-gallate (EGCG), the main green tea component, is intensively studied for its anti-oxidant, anti-inflammatory, anti-microbial and anti-cancer effects. In the present study, a screen on a Saccharomyces cerevisiae gene deletion library was performed to identify conditions under which EGCG had deleterious rather than beneficial effects. Two genes were identified whose deletion resulted in sensitivity to EGCG: FET3 and FTR1, encoding the components of the Fet3/Ftr1 high-affinity iron uptake system, also involved in Cu(I)/Cu(II) balance on the surface of yeast cells. The presence of EGCG in the growth medium induced the production of Cu(I), with deleterious effects on fet3Δ and ftr1Δ cells. Additionally, when combined, physiological surpluses of Cu(II) and EGCG acted in synergy not only against fet3Δ and ftr1Δ, but also against wild type cells, by generating surplus Cu(I) in the growth medium. The results imply that caution should be taken when combining EGCG-rich beverages/nutraceuticals with copper-rich foods.

Ace1 prevents intracellular copper accumulation by regulating Fet3 expression and thereby restricting Aft1 activity.

In the yeast Saccharomyces cerevisiae Aft1, the low iron-sensing transcription factor is known to regulate the expression of the FET3 gene. However, we found that a strain-lacking FET3 is more sensitive to copper excess than a strain-lacking AFT1, and accordingly, FET3 expression is not fully compromised in the latter. These findings suggest that, under such conditions, another regulator comes into play and controls FET3 expression. In this work, we identify Ace1, the regulator of copper detoxification genes, as a regulator of FET3. We suggest that the activation of FET3 by Ace1 prevents the hyper activation of Aft1, possibly by assuring the adequate functioning of mitochondrial iron-sulfur cluster biogenesis. While reinforcing the link between iron and copper homeostasis, this work unveils a novel protection mechanism against copper toxicity mediated by Ace1, which relies in the activation of FET3 and results in the restriction of Aft1 activity as a means to prevent excessive copper accumulation.

Synergistic Effects of Copper Sites on Apparent Stability of Multicopper Oxidase, Fet3p.

Saccharomyces cerevisiae Fet3p is a multicopper oxidase that contains three cupredoxin-like domains and four copper ions located in three distinct metal sites (T1 in domain 3; T2 and the binuclear T3 at the interface between domains 1 and 3). To probe the role of the copper sites in Fet3p thermodynamic stability, we performed urea-induced unfolding experiments with holo-, apo- and three partially-metallated (T1, T2 and T1/T2 sites depleted of copper) forms of Fet3p. Using a combination of spectroscopic probes (circular dichroism, fluorescence intensity and maximum, 8-anilinonaphthalene-1-sulfonic acid (ANS) emission, oxidase activity and blue color), we reveal that all forms of Fet3p unfold in a four-state reaction with two partially-folded intermediates. Using phase diagrams, it emerged that Fet3p with all copper sites filled had a significantly higher stability as compared to the combined contributions of the individual copper sites. Hence, there is long-range inter-domain communication between distal copper sites that contribute to overall Fet3p stability.

The Phosphoinositide 3-Kinase Regulates Retrograde Trafficking of the Iron Permease CgFtr1 and Iron Homeostasis in Candida glabrata.

The phosphoinositide 3-kinase (PI3K), which phosphorylates phosphatidylinositol and produces PI3P, has been implicated in protein trafficking, intracellular survival, and virulence in the pathogenic yeast Candida glabrata Here, we demonstrate PI3-kinase (CgVps34) to be essential for maintenance of cellular iron homeostasis. We examine how CgVps34 regulates the fundamental process of iron acquisition, and underscore its function in vesicular trafficking as a central determinant. RNA sequencing analysis revealed iron homeostasis genes to be differentially expressed upon CgVps34 disruption. Consistently, the Cgvps34Δ mutant displayed growth attenuation in low- and high-iron media, increased intracellular iron content, elevated mitochondrial aconitase activity, impaired biofilm formation, and extenuated mouse organ colonization potential. Furthermore, we demonstrate for the first time that C. glabrata cells respond to iron limitation by expressing the iron permease CgFtr1 primarily on the cell membrane, and to iron excess via internalization of the plasma membrane-localized CgFtr1 to the vacuole. Our data show that CgVps34 is essential for the latter process. We also report that macrophage-internalized C. glabrata cells express CgFtr1 on the cell membrane indicative of an iron-restricted macrophage internal milieu, and Cgvps34Δ cells display better survival in iron-enriched medium-cultured macrophages. Overall, our data reveal the centrality of PI3K signaling in iron metabolism and host colonization.

Expression in Arabidopsis and cellular localization reveal involvement of rice NRAMP, OsNRAMP1, in arsenic transport and tolerance.

Irrigation of paddy fields to arsenic (As) containing groundwater leads to As accumulation in rice grains and causes serious health risk to the people worldwide. To reduce As intake via consumption of contaminated rice grain, identification of the mechanisms for As accumulation and detoxification in rice is a prerequisite. Herein, we report involvement of a member of rice NRAMP (Natural Resistance-Associated Macrophage Protein) transporter, OsNRAMP1, in As, in addition to cadmium (Cd), accumulation through expression in yeast and Arabidopsis. Expression of OsNRAMP1 in yeast mutant (fet3fet4) rescued iron (Fe) uptake and exhibited enhanced accumulation of As and Cd. Expression of OsNRAMP1 in Arabidopsis provided tolerance with enhanced As and Cd accumulation in root and shoot. Cellular localization revealed that OsNRAMP1 resides on plasma membrane of endodermis and pericycle cells and may assist in xylem loading for root to shoot mobilization. This is the first report demonstrating role of NRAMP in xylem mediated loading and enhanced accumulation of As and Cd in plants. We propose that genetic modification of OsNRAMP1 in rice might be helpful in developing rice with low As and Cd content in grain and minimize the risk of food chain contamination to these toxic metals.