Mecanismos de regulación de la enzima ribonucleótido reductasa de Saccharomyces cerevisiae en respuesta a la deficiencia de hierro

Mecanismos de regulación de la enzima ribonucleótido reductasa de Saccharomyces cerevisiae en respuesta a la deficiencia de hierro. Mantener un balance adecuado en los niveles de desoxirribonucleótidos trifosfato (dNTPs), precursores necesarios para la síntesis del DNA, es fundamental para preservar la estabilidad genómica y la viabilidad celular tanto durante la proliferación celular como en respuesta a daños en el DNA. La enzima ribonucléotido reductasa (RNR), responsable de la conversión de ribonucleótidos en desoxirribonucleótidos, es una de las dianas de regulación principales durante la síntesis de dNTPs, por lo que múltiples mecanismos controlan su actividad durante el progreso del ciclo celular y en respuesta a daños en el DNA. Las RNRs de clase Ia presentes en todas las células eucariotas son enzimas esenciales compuestas por una subunidad grande R1, en la que se encuentra el centro catalítico y los sitios de regulación alostérica, y una subunidad pequeña R2 en la que se sitúa un centro de oxo-dihierro y un radical tirosil esenciales para la actividad enzimática. Dado que la enzima RNR requiere de hierro como cofactor esencial para su función, en este trabajo se planteó utilizar la levadura Saccharomyces cerevisiae como modelo para estudiar los mecanismos moleculares que regulan la actividad RNR en respuesta a condiciones de deficiencia de hierro. En S. cerevisiae, la subunidad catalítica R1 se localiza en condiciones normales en el citoplasma, mientras que la mayor parte de la subunidad pequeña R2 formada por el heterodímero Rnr2-Rnr4 reside en el núcleo de la célula. Una proteína nuclear conocida como Wtm1, que interacciona con la subunidad R2, es la responsable de anclarla al núcleo. Durante la fase S del ciclo celular y en respuesta a daños en el DNA, la ruta de integridad del DNA formada por las quinasas Mec1-Rad53-Dun1 se activa y promueve la rotura de la interacción entre la subunidad R2 y Wtm1. Como consecuencia la subunidad R2 se relocaliza desde el núcleo hasta el citoplasma, donde se ensambla en un complejo RNR activo y permite la síntesis de dNTPs. En este trabajo hemos observado que en respuesta a la falta de hierro se produce la salida de la subunidad R2 del núcleo al citoplasma de una forma totalmente independiente de las quinasas Mec1 y Rad53, sugiriendo la existencia de un nuevo mecanismo de regulación de la actividad RNR en condiciones de deficiencia de hierro. Estudios previos han demostrado que la deficiencia de hierro induce la expresión de dos proteínas de unión a RNA denominadas Cth1 y Cth2. Estas proteínas desempeñan un papel clave en la remodelación del metabolismo dependiente de hierro mediante la unión y degradación selectiva de un gran número de mRNAs que codifican proteínas que contienen hierro o que participan en procesos dependientes de hierro. Una de las dianas principales de Cth1 y Cth2 es la respiración mitocondrial, que resulta reprimida en respuesta a la escasez de hierro. En este trabajo hemos podido demostrar que Cth1 y Cth2 controlan la localización subcelular de la subunidad R2 en condiciones de déficit de hierro mediante la degradación específica del mRNA WTM1. La consecuente bajada en los niveles de proteína Wtm1 permiten la relocalización de la subunidad R2 al citoplasma y la activación de la enzima RNR. Además, hemos descrito que, en condiciones de limitación de hierro, la quinasa Dun1 participa en la regulación de RNR. Por un lado, hemos observado que Dun1 promueve la relocalización al citoplasma de la subunidad pequeña R2, mecanismo que depende de su dominio quinasa, mientras que su domino FHA (del inglés, forkhead-associated) no es necesario. Por otro lado, nuestros resultados sugieren que cuando los niveles de hierro son bajos, Dun1 regula los niveles de proteína Sml1, inhibidor de la subunidad grande R1, a través de un mecanismo independiente de las quinasas Mec1 y Rad53. Análisis de la estructura de la proteína Dun1 muestran que tanto su dominio quinasa y como su dominio FHA se requieren para inducir la degradación de Sml1 en condiciones de escasez de hierro, mientras que el residuo T380, que es fosforilad pot la quinasa Rad53 en respuesta a daños en el DNA, no es necesario. Nuestros resultados indican que, en condiciones de deficiencia de hierro, el descenso de los niveles de Sml1 es mediado por el complejo de ubiquitín-ligasas Rad6-Urb2-Mub1 a través del proteasoma 26S y por la ruta vacuolar proteolítica. Estos resultados revelan un nuevo papel para la quinasa Dun1 en la degradación de Sml1 y en la relocalización de la subunidad pequeña R2 bajo condiciones de deficiencia de hierro. Así pues, los resultados obtenidos en este trabajo sugieren que, en respuesta a la falta de hierro, S. cerevisiae reorganiza el metabolismo celular priorizando procesos esenciales dependientes de hierro como la síntesis de dNTPs sobre vías metabólicas que consumen gran cantidad de hierro y que no esenciales para la levadura como la respiración mitocondrial.Regulatory mechanisms of the enzyme ribonucleotide reductase in Saccharomyces cerevisiae response to iron deficiency. Maintaining a proper balance in the levels of deoxyribonucleotide triphosphates (dNTPs), precursors required for DNA synthesis, is essential to maintain genomic stability and cell viability in both cell proliferation and during the response to DNA damage. The enzyme ribonucleotide reductase (RNR), responsible for the conversion of ribonucleotides to deoxyribonucleotides, one of the major targets of regulation during dNTPs synthesis, therefore multiple mechanisms control its activity during cell cycle progression and in response to damage in the DNA. The RNRs class Ia, present in all eukaryotic cells, are essential enzymes formed of a large subunit or R1, which is the catalytic center and allosteric regulation sites, and a small subunit or R2, which stands an oxo-diiron center and a tyrosyl radical, essential for enzymatic activity. Since RNR enzyme requires iron as an essential cofactor for their function, in this work we proposed to use the yeast Saccharomyces cerevisiae as a model to study the molecular mechanisms that regulate RNR activity in response to iron deficiency conditions. In S. cerevisiae, in normal conditions R1 catalytic subunit is located in the cytoplasm, whereas most of the small subunit R2 heterodimer formed by Rnr2-Rnr4 resides in the nucleus of cells. A nuclear protein knows as Wtm1, which interacts with the R2 subunit is responsible for anchoring the small subunit in the nucleus. During S-phase of the cell cycle and in response to DNA damage, the DNA damage checkpoint formed by Rad53-Mec1- Dun1 kinases is activated and promotes the lack of interacction between R2 subunit and Wtm1. As a consecuence, R2 subunit is relocated from the nucleus to the cytoplasm, where it is assembled into a complex active RNR and allows the synthesis of dNTPs. In this study, we observed that in response to iron deficiency R2 subunit relocalizes from the nucleus to the cytoplasm in a Mec1-Rad53 independent mechanim, suggesting the existence of a new mechanism of regulation of RNR activity under conditions of iron deficiency. Previous studies have shown that iron deficiency induces the expression of two RNA binding proteins called Cth1 and Cth2. These proteins play a key role in the remodeling of dependent metabolism by binding and selective degradation of many mRNAs encoding proteins that contain iron or are involved in iron-dependent processes. One of the main targets of Cth1 and Cth2 is the mitochondrial respiration, which is repressed in response to iron deficiency. In this work we have demonstrated that Cth1 and Cth2 control the subcellular localization of the R2 subunit in iron deficiency conditions by specific mRNA degradation WTM1. The resulting drop in Wtm1 protein levels allow relocation of the R2 subunit into the cytoplasm and the activation of the enzyme RNR. Furthermore, we reported that, under conditions of iron restriction, Dun1 kinase participates in the regulation of RNR. On one hand, we have observed that Dun1 promotes relocation to the cytoplasm of the small subunit R2, a mechanism that depends on the kinase domain, while its forkhead-associated domain or FHA is not necessary. Furthermore, our results suggest that when iron levels are low, Dun1 regulates Sml1 protein levels, R1 large subunit inhibitor, through a mechanism independent of Mec1 and Rad53 kinases. Analysis of protein structure Dun1 show that both its kinase domain as their FHA domain are required to induce Sml1 degradation in iron deficiency conditions, while the residue T380, which is phosphorylated by Rad53 kinases in response to DNA damage, it is not necessary. Our results indicate that, under conditions of iron deficiency, lthe ow levels of Sml1 are mediated by the ubiquitin-ligase complex Rad6-Urb2-Mub1 through 26S proteasome and vacuolar proteolytic pathway. These results reveal a new role for the Dun1 kinase in the Sml1 degradation and small subunit R2 relocation under iron deficiency conditions. Thus, the results obtained in this study suggest that, in response to lack of iron, S. cerevisiae reorganizes the metabolism prioritizing iron-essential dependent processes such dNTPs synthesis over metabolic pathways that consume large amounts of iron and are not essential for yeast cells as mitochondrial respiration

Function and Regulation of Yeast Ribonucleotide Reductase: Cell Cycle, Genotoxic Stress, and Iron Bioavailability

All eukaryotic organisms require an adequate, balanced concentration of deoxyribonucleoside triphosphates (dNTPs) in order to assure accurate DNA replication and repair, and to maintain genomic integrity. The rate?limiting step in dNTP synthesis is catalyzed by ribonucleotide reductase (RNR), an essential enzyme mediating the reduction of ribonucleotides to desoxyribonucleotides, thereby providing the building blocks required for DNA synthesis. Consistent with its important role in cell proliferation, a significant increase in RNR activity has been associated with tumor cells and resistance to chemotherapy. Indeed, since the utilization of hydroxyurea in the 70s to the current development of sophisticated RNR inhibitors, RNR as been used as an important target for the chemotherapeutic treat? ment of numerous cancer types. [1] Therefore, understanding the molecular mechanisms that cells utilize to regulate RNR function in response to different stresses is critical for the development of new and efficient anticancer therapies. In this review, we focus on the yeast S. cerevisiae as a eukaryotic model to advance in our understanding of mechanisms regulating the function of eukaryotic RNRs during cell cycle progress and in response to environmental cues, including genotoxic stress and low iron bioavailability. RNR structure, assembly, and allosteric regulation In eukaryotes, class Ia RNRs are oxygen?dependent enzymes composed of a large R1 (? 2) and a small R2 (? 2 or ???) subunit. The R1 subunit contains the catalytic site and two allosteric effector binding sites that Review Article Ribonucleotide reductases (RNRs) are essential enzymes that catalyze the reduction of ribonucleotides to desoxyribonucleotides, thereby providing the building blocks required for de novo DNA biosynthesis. The RNR function is tightly regulated because an unbalanced or excessive supply of deoxyribonucleoside triphosphates (dNTPs) dramatically increases the mutation rates during DNA replication and repair that can lead to cell death or genetic anomalies. In this review, we focus on Saccharomyces cerevisiae class Ia RNR as a model to understand the different mechanisms controlling RNR function and regulation in eukaryotes. Many studies have contributed to our current understanding of RNR allosteric regulation and, more recently, to its link to RNR oligomerization. Cells have developed additional mechanisms that restrict RNR activity to particular periods when dNTPs are necessary, such as the S phase or upon genotoxic stress. These regulatory strategies include the transcriptional control of the RNR gene expression, inhibition of RNR catalytic activity, and the subcellular redistribution of RNR subunits. Despite class Ia RNRs requiring iron as an essential cofactor for catalysis, little is known about RNR function regulation depending on iron bioavailability. Recent studies into yeast have deciphered novel strategies for the delivery of iron to RNR and for its regulation in response to iron deficiency. Taken together, these studies open up new possibilities to explore in order to limit uncontrolled tumor cell proliferation via RNR

Yeast Dun1 Kinase Regulates Ribonucleotide Reductase Small Subunit Localization in Response to Iron Deficiency

Ribonucleotide reductase (RNR) is an essential iron-dependent enzyme that catalyzes deoxyribonucleotide synthesis in eukaryotes. Living organisms have developed multiple strategies to tightly modulate RNR function to avoid inadequate or unbalanced deoxyribonucleotide pools that cause DNA damage and genome instability. Yeast cells activate RNR in response to genotoxic stress and iron deficiency by facilitating redistribution of its small heterodimeric subunit Rnr2-Rnr4 from the nucleus to the cytoplasm, where it forms an active holoenzyme with large Rnr1 subunit. Dif1 protein inhibits RNR by promoting nuclear import of Rnr2-Rnr4. Upon DNA damage, Dif1 phosphorylation by the Dun1 checkpoint kinase and its subsequent degradation enhances RNR function. In this report, we demonstrate that Dun1 kinase triggers Rnr2-Rnr4 redistribution to the cytoplasm in response to iron deficiency. We show that Rnr2-Rnr4 relocalization by low iron requires Dun1 kinase activity and phosphorylation site Thr-380 in the Dun1 activation loop, but not the Dun1 forkhead-associated domain. By using different Dif1 mutant proteins, we uncover that Dun1 phosphorylates Dif1 Ser-104 and Thr-105 residues upon iron scarcity. We observe that the Dif1 phosphorylation pattern differs depending on the stimuli, which suggests different Dun1 activating pathways. Importantly, the Dif1-S104A/T105A mutant exhibits defects in nucleus-to-cytoplasm redistribution of Rnr2-Rnr4 by iron limitation. Taken together, these results reveal that, in response to iron starvation, Dun1 kinase phosphorylates Dif1 to stimulate Rnr2-Rnr4 relocalization to the cytoplasm and promote RNR function.This work has been supported by a predoctoral fellowship from “Conselleria d'Educació de la Generalitat Valenciana” (to N. S.), a predoctoral fellowship from the Spanish Ministry of Economy and Competitiveness (to A. M. R.), Spanish Ministry of Economy and Competitiveness Grants AGL2011-29099 and BIO2014-56298-P (to S. P.), and National Institutes of Health Grant CA125574 (to M. H.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.Peer reviewe

Yeast Dun1 Kinase Regulates Ribonucleotide Reductase Inhibitor Sml1 in Response to Iron Deficiency

Iron is an essential micronutrient for all eukaryotic organisms because it participates as a redox-active cofactor in many biological processes, including DNA replication and repair. Eukaryotic ribonucleotide reductases (RNRs) are Fe-dependent enzymes that catalyze deoxyribonucleoside diphosphate (dNDP) synthesis. We show here that the levels of the Sml1 protein, a yeast RNR large-subunit inhibitor, specifically decrease in response to both nutritional and genetic Fe deficiencies in a Dun1-dependent but Mec1/Rad53- and Aft1-independent manner. The decline of Sml1 protein levels upon Fe starvation depends on Dun1 forkhead-associated and kinase domains, the 26S proteasome, and the vacuolar proteolytic pathway. Depletion of core components of the mitochondrial iron-sulfur cluster assembly leads to a Dun1-dependent diminution of Sml1 protein levels. The physiological relevance of Sml1 downregulation by Dun1 under low-Fe conditions is highlighted by the synthetic growth defect observed between dun1? and fet3? fet4? mutants, which is rescued by SML1 deletion. Consistent with an increase in RNR function, Rnr1 protein levels are upregulated upon Fe deficiency. Finally, dun1? mutants display defects in deoxyribonucleoside triphosphate (dNTP) biosynthesis under low-Fe conditions. Taken together, these results reveal that the Dun1 checkpoint kinase promotes RNR function in response to Fe starvation by stimulating Sml1 protein degradation