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DOI: 10.1002/jib.381

116 Old Yeasts - New Questions

**Provisional chapter**

#### *Saccharomyces cerevisiae* **Peroxiredoxins in Biological Processes: Antioxidant Defense, Signal Transduction, Circadian Rhythm, and More Processes: Antioxidant Defense, Signal Transduction, Circadian Rhythm, and More**

*Saccharomyces cerevisiae* **Peroxiredoxins in Biological**

DOI: 10.5772/intechopen.70401

Melina C. Santos, Carlos A. Breyer, Leonardo Schultz, Karen S. Romanello, Anderson F. Cunha, Carlos A. Tairum Jr and Marcos Antonio de Oliveira Schultz, Karen S. Romanello, Anderson F. Cunha, Carlos A. Tairum Jr and Marcos Antonio de Oliveira Additional information is available at the end of the chapter

Melina C. Santos, Carlos A. Breyer, Leonardo

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70401

#### **Abstract**

The yeast *Saccharomyces cerevisiae* is a model organism for biochemical and genetic studies, and several very important discoveries of fundamental biological processes have been conducted using this yeast as an experimental organism. An emerging concept, which is validated by several works using this organism, relies on the biological importance of oxidant species, specially the hydroperoxides. These molecules were formed during aerobic biological process and control several intracellular mechanisms such as a range of signaling pathways, cell cycle, programmed cell death, circadian rhythm, aging, and lifespan extension. Thereby, cellular homeostasis depends on a refined control of hydroperoxides levels and low-molecular-weight molecules in combination with antioxidant enzymes playing a role in this equilibrium. This proposal is focused on the *S. cerevisiae* peroxiredoxins and their role in peroxide decomposition, signal transduction, circadian clocks, and aging as model enzymes for the study and comprehension of these biological processes in living organisms, including humans.

**Keywords:** thiol-specific antioxidant protein, functional transitions, peroxidase, chaperone, overoxidation

#### **1. Introduction**

The use of *Saccharomyces cerevisiae* as a biological model in the field of oxidant species research represents a very important tool in an exciting area. Emerging concepts, validated by several

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

works, revealed the importance of oxidant molecules in biological processes, especially the hydroperoxides [1, 2]. These molecules are formed during several aerobic biological processes and, in adequate levels, are involved in a number of intracellular mechanisms, such as redox signaling pathways related to cell cycle progression, programmed cell death, circadian rhythm, aging, and lifespan extension [2–7]. However, the accumulation of these molecules can be harmful to the cells [3, 4]. In fact, highly deleterious radical species can be generated from hydroperoxides, such as hydroxyl radical (•OH), that is generated from hydrogen peroxide (H2 O2 ) through Fenton and Harber-Weiss reactions. The •OH is able to oxidize carbohydrates, lipids, proteins, and DNA, being extremely toxic to cells. Thereby, cellular homeostasis depends on a refined control of hydroperoxides levels, and this role is played by both low-molecular-weight molecules, such as glutathione and ascorbic acid, as well as by antioxidant enzymes such as glutathione peroxidases (Gpxs), catalases (Cats), and peroxiredoxins (Prxs) [2, 4]. The latter ones have been subject of intense studies since works involving kinetic approaches indicate that the Prxs decompose more than 90% of cellular hydroperoxides [8, 9]. Additionally, to exert their biological functions, several Prxs are able to perform amazing structural switches, revealing an intricate puzzle among protein structure and function [10–12].

The first Prx described was a cytosolic enzyme identified in *S. cerevisiae* and received the name of "thiol-specific antioxidant protein 1" (Tsa1) [13]. Subsequently, a second homologue cytosolic isoform, named Tsa2, was identified and characterized. Currently, there are five Prx isoforms identified in this yeast. In mammals, there are six isoforms described, and as in other organisms, they are located in several cellular environments as cytosol, nucleus, peroxisome, mitochondria, endoplasmic reticulum, and even in the nucleus [14, 15]. Furthermore, these proteins are very abundant. For example, in *S. cerevisiae*, they can reach ~0.9% of total soluble proteins and can represent one of ten most expressed enzymes in bacteria and in mammal cells [16]. In human erythrocytes, PrxII is the third most abundant protein, only losing in concentration for globins and carbonic anhydrase, and its level is modulated during cell differentiation [17].

Besides the widespread cellular distribution and abundance, Prx stands out due to their highly efficient ability to decompose a wide variety of hydroperoxides (H2 O2 , nitrite peroxide, lipid peroxides, among others), with second order rates reaching ~106 –108 M−1 s−1) [18–21]. These characteristics place the Prx as one of the main modulators of hydroperoxides levels and, consequently, of the cellular processes mediated by them. The Prx enzymes are able to decompose hydroperoxides without any prosthetic group, but using only a highly reactive cysteine residue named peroxidatic cysteine (CP) [5, 22]. All the Prxs described to date present a conserved motif containing the CP (PXXXT/SXXCP), which is oxidized to cysteine sulfenic acid (CP-SOH) after hydroperoxide reduction [10]. This enzyme family is very heterogeneous, and different classifications have been proposed; the most currently used one subdivides these proteins in three large subclasses,1-Cys Prx, typical 2-Cys Prx, and atypical 2-Cys Prx, based in the number of cysteines involved in catalytic cycle and structural aspects (**Figure 1**). The 1-Cys Prxs are homodimeric proteins that present only one cysteine residue, the CP , involved in hydroperoxide catalysis. 2-Cys Prxs may be monomeric (in the case of some atypical 2-Cys Prx) or homodimeric proteins and present a second cysteine residue, named resolving cysteine (CR), which condenses with CP forming a disulfide bond as final product during the catalytic cycle. In typical 2-Cys Prx, the disulfide is intermolecular (e.g., between different monomers), while in atypical 2-Cys Prx, the disulfide is intramolecular (in the same monomer) [23].

works, revealed the importance of oxidant molecules in biological processes, especially the hydroperoxides [1, 2]. These molecules are formed during several aerobic biological processes and, in adequate levels, are involved in a number of intracellular mechanisms, such as redox signaling pathways related to cell cycle progression, programmed cell death, circadian rhythm, aging, and lifespan extension [2–7]. However, the accumulation of these molecules can be harmful to the cells [3, 4]. In fact, highly deleterious radical species can be generated from hydroperoxides, such as hydroxyl radical (•OH), that is generated from hydrogen peroxide (H2

through Fenton and Harber-Weiss reactions. The •OH is able to oxidize carbohydrates, lipids, proteins, and DNA, being extremely toxic to cells. Thereby, cellular homeostasis depends on a refined control of hydroperoxides levels, and this role is played by both low-molecular-weight molecules, such as glutathione and ascorbic acid, as well as by antioxidant enzymes such as glutathione peroxidases (Gpxs), catalases (Cats), and peroxiredoxins (Prxs) [2, 4]. The latter ones have been subject of intense studies since works involving kinetic approaches indicate that the Prxs decompose more than 90% of cellular hydroperoxides [8, 9]. Additionally, to exert their biological functions, several Prxs are able to perform amazing structural switches,

The first Prx described was a cytosolic enzyme identified in *S. cerevisiae* and received the name of "thiol-specific antioxidant protein 1" (Tsa1) [13]. Subsequently, a second homologue cytosolic isoform, named Tsa2, was identified and characterized. Currently, there are five Prx isoforms identified in this yeast. In mammals, there are six isoforms described, and as in other organisms, they are located in several cellular environments as cytosol, nucleus, peroxisome, mitochondria, endoplasmic reticulum, and even in the nucleus [14, 15]. Furthermore, these proteins are very abundant. For example, in *S. cerevisiae*, they can reach ~0.9% of total soluble proteins and can represent one of ten most expressed enzymes in bacteria and in mammal cells [16]. In human erythrocytes, PrxII is the third most abundant protein, only losing in concentration for globins and carbonic anhydrase, and its level is modulated during cell differentiation [17].

Besides the widespread cellular distribution and abundance, Prx stands out due to their

These characteristics place the Prx as one of the main modulators of hydroperoxides levels and, consequently, of the cellular processes mediated by them. The Prx enzymes are able to decompose hydroperoxides without any prosthetic group, but using only a highly reactive cysteine residue named peroxidatic cysteine (CP) [5, 22]. All the Prxs described to date present a conserved motif containing the CP (PXXXT/SXXCP), which is oxidized to cysteine sulfenic acid (CP-SOH) after hydroperoxide reduction [10]. This enzyme family is very heterogeneous, and different classifications have been proposed; the most currently used one subdivides these proteins in three large subclasses,1-Cys Prx, typical 2-Cys Prx, and atypical 2-Cys Prx, based in the number of cysteines involved in catalytic cycle and structural aspects (**Figure 1**). The 1-Cys Prxs are homodimeric proteins that present only one cysteine residue,

, involved in hydroperoxide catalysis. 2-Cys Prxs may be monomeric (in the case of some atypical 2-Cys Prx) or homodimeric proteins and present a second cysteine residue,

revealing an intricate puzzle among protein structure and function [10–12].

highly efficient ability to decompose a wide variety of hydroperoxides (H2

ide, lipid peroxides, among others), with second order rates reaching ~106

the CP

120 Old Yeasts - New Questions

O2 )

O2

–108

, nitrite perox-

M−1 s−1) [18–21].

Among the different Prx subclasses, the typical 2-Cys Prxs are the best studied, and, from this point on, our focus will be on this Prx subclass. After oxidation, the disulfide bond of the typical 2-Cys Prx is frequently reduced by the low-molecular-weight (~11 kDa) enzyme thioredoxin (Trx). The oxidized Trx is reduced by thioredoxin reductase (TrxR), which uses electrons from nicotinamide adenine dinucleotide phosphate (NADPH) *via* a flavin adenine dinucleotide (FAD) molecule. Together, Trx, TrxR, and NADPH are named thioredoxin system (Trx system) [21]. It is important to mention that all electron exchanges between the proteins are performed using catalytic cysteines [25] (**Figure 2**).

**Figure 1.** Prx subclasses in reduced and oxidized states. For all enzymes, the first step of the catalytic cycle is represented by the attack of the CP-S− over the O─O from hydroperoxide forming cysteine sulfenic acid (CP-SOH) and releasing R─OH. (A) 1-Cys are dimeric enzymes containing only the peroxidatic cysteine, which is stable in oxidized state (CP-SOH). (B) In the atypical 2-Cys Prx, the oxidized cysteine (CP-SOH) formed after hydroperoxide decomposition condenses with the CR-SH from the same monomer forming an intramolecular disulfide bond. (C) In typical 2-Cys Prx, the CP-SOH condenses with CR from the adjacent monomer forming an intermolecular disulfide.

**Figure 2.** Hydroperoxide reduction steps by typical 2-Cys Prx and Trx system. The Prx CP in thiolate form (**1**) attacks the hydroperoxide (**2**), releasing a water molecule in the case of H2 O2 reduction, or an alcohol when the substrate is an organic hydroperoxide (the "R" represents the hydroperoxide radical). CP is oxidized to cysteine sulfenic acid (**3**), releases a water molecule (**4**) and condenses with CR forming an intermolecular disulfide (**5**), which is reduced by the enzyme Trx (**6**). Trx disulfide is reduced by the cysteines from TrxR enzyme (**7**) using electrons from NADPH (**9**) *via* a FAD molecule (**8**).

**Figure 3.** Quaternary structures of the typical 2-Cys Prx. (A) The yeast Tsa1 homodimer is represented in cartoon. (B) α2(5) decamer formed by the association of five homodimers. (C) Microenvironment of the CP in the active site. The Thr and Arg residues are involved in the thiolate (S−) stabilization. Additionally, the Thr residue is able to perform a CH-π interaction with the C atoms of a Tyr ring from the adjacent dimer. The proteins are represented in cartoon, and catalytic triad and the Tyr residue are represented in ball and stick. Figures were generated using the S. cerevisiae Tsa1 crystallographic coordinates (PDB: 3SBC) and Pymol software (http://www.pymol.org/).

Despite that the basic functional unit of the typical 2-Cys Prx is represented by a α(2) homodimer, studies using the Tsa1 and Tsa2 isoforms from *S. cerevisiae* revealed that this oligomeric state presents low peroxidase activity, and the highest reactivity of the typical 2-Cys Prx is reached when these proteins are found in a ring-shaped α2(5) decamers (association of five homodimers; **Figure 3A**). It is believed that the alternation between these two quaternary structures is responsible for the modulation of their peroxidase activity and may be involved in signal transduction (**Figure 3B**) [10]. Additionally, the typical 2-Cys Prx enzymes may also present other oligomeric states that will be discussed posteriorly.

The high reactivity of Prx over hydroperoxides is related to the maintenance of CP in thiolate form (CP-S− ), suitable for catalysis as a consequence of the microenvironment of the active site. The CP thiolate is stabilized by polar interactions with a threonine (or a serine, in some cases) and an arginine residue (**Figure 3C**). These three residues (Thr, CP , and Arg) are named catalytic triad and are widely conserved among all Prxs described to date [10]. During catalysis, a guanidine group of the Arg residue is able to perform a hydrogen bond with the proximal oxygen (O) of the hydroperoxide, allowing the nucleophilic attack of the CP over the hydroperoxide [24]. The Oγ from Thr, in turn, would act as an acceptor of the hydrogen bond with the distal O from hydroperoxide, aiding the positioning of the molecule in a productive way to catalysis [24].

Typical 2-Cys Prxs, such as *S. cerevisiae* Tsa1 and Tsa2, are still able to perform additional structural and functional switches acting as peroxide sensors, molecular chaperones and are involved in several hydroperoxide-dependent signal transduction pathways, as it will be discussed further [20, 21]. Tsa1 and Tsa2 are also evolutionarily related to human PrxI and PrxII. In fact, Tsa1 presents 67% of identity and 77% of similarity with human PrxII, while Tsa2 presents 60% of identity and 76% of similarity with human PrxI, which places these proteins as important models to the study of the human Prx and the biological processes related to them.

#### **2. Redox cycle and structural transitions**

During the redox cycle, some typical 2-Cys Prxs are able to transit between different oligomeric species: α2(5) decamers (reduced enzyme) and α2 dimers (disulfide oxidized protein). Aiming to understand the details of the catalytic cycle and structural transitions, we have

*Saccharomyces cerevisiae* Peroxiredoxins in Biological Processes: Antioxidant Defense, Signal... http://dx.doi.org/10.5772/intechopen.70401 123

**Figure 4.** Tsa1 and Tsa2 interactions at dimer-dimer interface. Cartoon representations of quaternary structures of the Tsa1 and Tsa2. (A) Representation of theTsa1 decamer in cartoon. (B) Interaction between Thr44 and Tyr77 in Tsa1 decamer interface. (C) Tsa2 decamer in cartoon. (D) Interaction between Ser45 and Tyr78 in the Tsa2 decamer interface. In (B) and (D) the atoms are represented by spheres and colored as follow: O = red, C = yellow, N = blue. The gures were generated using the S. cerevisiae Tsa1 (PDB: 3SBC) and Tsa2 (PDB: 5DVB) coordinates and the Pymol software (http:// www.pymol.org/).

Despite that the basic functional unit of the typical 2-Cys Prx is represented by a α(2) homodimer, studies using the Tsa1 and Tsa2 isoforms from *S. cerevisiae* revealed that this oligomeric state presents low peroxidase activity, and the highest reactivity of the typical 2-Cys Prx is reached when these proteins are found in a ring-shaped α2(5) decamers (association of five homodimers; **Figure 3A**). It is believed that the alternation between these two quaternary structures is responsible for the modulation of their peroxidase activity and may be involved in signal transduction (**Figure 3B**) [10]. Additionally, the typical 2-Cys Prx enzymes may also

**Figure 3.** Quaternary structures of the typical 2-Cys Prx. (A) The yeast Tsa1 homodimer is represented in cartoon. (B) α2(5) decamer formed by the association of five homodimers. (C) Microenvironment of the CP in the active site. The Thr and Arg residues are involved in the thiolate (S−) stabilization. Additionally, the Thr residue is able to perform a CH-π interaction with the C atoms of a Tyr ring from the adjacent dimer. The proteins are represented in cartoon, and catalytic triad and the Tyr residue are represented in ball and stick. Figures were generated using the S. cerevisiae Tsa1

), suitable for catalysis as a consequence of the microenvironment of the active site.

thiolate is stabilized by polar interactions with a threonine (or a serine, in some cases)

triad and are widely conserved among all Prxs described to date [10]. During catalysis, a guanidine group of the Arg residue is able to perform a hydrogen bond with the proximal oxygen

The Oγ from Thr, in turn, would act as an acceptor of the hydrogen bond with the distal O from hydroperoxide, aiding the positioning of the molecule in a productive way to catalysis [24].

Typical 2-Cys Prxs, such as *S. cerevisiae* Tsa1 and Tsa2, are still able to perform additional structural and functional switches acting as peroxide sensors, molecular chaperones and are involved in several hydroperoxide-dependent signal transduction pathways, as it will be discussed further [20, 21]. Tsa1 and Tsa2 are also evolutionarily related to human PrxI and PrxII. In fact, Tsa1 presents 67% of identity and 77% of similarity with human PrxII, while Tsa2 presents 60% of identity and 76% of similarity with human PrxI, which places these proteins as important models to the study of the human Prx and the biological processes related

During the redox cycle, some typical 2-Cys Prxs are able to transit between different oligomeric species: α2(5) decamers (reduced enzyme) and α2 dimers (disulfide oxidized protein). Aiming to understand the details of the catalytic cycle and structural transitions, we have

in thiolate

, and Arg) are named catalytic

over the hydroperoxide [24].

present other oligomeric states that will be discussed posteriorly.

crystallographic coordinates (PDB: 3SBC) and Pymol software (http://www.pymol.org/).

and an arginine residue (**Figure 3C**). These three residues (Thr, CP

(O) of the hydroperoxide, allowing the nucleophilic attack of the CP

**2. Redox cycle and structural transitions**

form (CP-S−

122 Old Yeasts - New Questions

The CP

to them.

The high reactivity of Prx over hydroperoxides is related to the maintenance of CP

determined the crystallographic structure of Tsa1 [21]. In fact, the analysis of the structure revealed an interaction of the Thr from the active site motif, at the dimer-dimer interface of the decamer. Recently, using different methodological approaches as site-directed mutagenesis, biochemical approaches, size exclusion chromatography, and structural analysis, we have demonstrated that a slight difference in the PXXXT(S)XXCP is involved in decamers to dimers transitions [10]. While Tsa1 possess a Thr residue embedded in the conserved motif, in Tsa2, the Thr is naturally substituted by a Ser (**Figure 4**). In fact, the Tsa1 enzyme, containing Thr residue, transits between dimers (oxidized form) and decamers (reduced enzyme), but the Ser-containing enzyme Tsa2 is not able to dissociate in dimers. Indeed, the rearrangements as consequence of the redox states in the Tsa1 may cause hysteric hindrance of the Thr Oγ with the Tyr aromatic ring of the adjacent monomer, causing the decamer dissociation. Since Tsa2 presents a Ser residue, the hysteric clash probably is avoided. These characteristics may indicate an additional regulation of Prx quaternary structure, which may have implications in biological processes.

#### **3. Prx overoxidation: structural and functional implications**

During the typical 2-Cys Prx catalytic cycle under high levels of hydroperoxides, before disulfide formation, CP-SOH can be attacked by another hydroperoxide molecule and becomes overoxidized to cysteine sulfinic acid (CP-SO2 H) or sulfonic acid (CP-SO3 H). The CP overoxidation is related to spectacular functional and structural switch in typical 2-Cys Prx. As mentioned before, when the typical 2-Cys Prx are in reduced state (CP-S− ), these proteins are decamers, but when are oxidized in disulfide, they can be dimers and/or decamers and are able to act as peroxidases (**Figure 5A** and **B**) [9, 11]. However, when the CP is overoxidized, these enzymes are able to promote an intense oligomerization to form high-molecular-weight (HMW) spherical complexes (**Figure 5C**), with the concomitant inactivation of the peroxidase

**Figure 5.** Typical 2-Cys Prx overoxidation and high-molecular-weight complex formation. The typical 2-Cys Prx in reduced form are presented as α2(5) decamers (A). In low concentrations of hydroperoxides, the CP is oxidized in CP-SOH, and the intermolecular disulfide is formed with CR. The disulfide formation, in some cases, is able to destabilize the decamers, forming a mixture of decamers and dimers (B). The oxidized form is reduced by Trx system. When the typical 2-Cys Prx are challenged with high concentrations of hydroperoxides, the CP can be overoxidized to CP-SO2 H. The CP overoxidation promotes the HMW complexes formation which presents chaperone properties (C). The CP-SO2 H can be reduced by sulfiredoxin, in ATP and Mg2+ dependent manner (D).

activity. The HMW complexes formation was first reported in *S. cerevisiae* Tsa1 and Tsa2 by Jang and coworkers [11], and, posteriorly, very similar complexes were described to the human homologues typical 2-Cys Prxs (PrxI and PrxII) [26]. Using transmission electron microscopy (TEM), it was demonstrated that complexes are represented by heterogeneous spherical structures, which can reach 1 GDa, and biochemical approaches revealed that the complexes present an extraordinary chaperone holdase activity [9, 26]. Later on, similar spherical and another type of HWM complexes, represented by the stacking of several decamers (**Figure 5C**), were described for the plant chloroplastic 2-Cys Prxs, cyanobacterial Anabaena PCC7120 2-Cys Prx, among others [27–30]. The structural differences between the HMW complexes are not well understood to date.

A very important point relies on the fact that the Prx overoxidized species cannot be reduced by the Trx system, but some studies revealed that overoxidized typical 2-Cys Prx species could be regenerated to the reduced form *in vivo* [31]. Posteriorly, it was identified in *S. cerevisiae*, and after in human and other species, an enzyme named sulfiredoxin (Srx) which is able to reduce the CP-SO2 H in a ATP and Mg2+ dependent reaction, but not CP-SO3 H, suggesting that this oxidation state is refractive to reduction [31]. Curiously, the CP-SO2 H reduction rates by Srx are very slow when compared to the disulfide reduction by Trx (~2 M−1 s−1 and ~106 M−1 s−1, respectively) [32]. The biochemical steps of the CP-SO2 H reduction by Srx are represented in **Figure 5D**. It is important to highlight that the Srx was identified in several eukaryotes, but few prokaryotes possess this enzyme, which may be an evolutionary sophistication of the 2-Cys Prx redox cycle [33]. In fact, to the majority of the prokaryotes, no homologous Srx gene was detected in their genomes, and the typical 2-Cys Prxs are much more resistant to overoxidation. Moreover, an additional classification can be done based on the CP overoxidation susceptibility, and the 2-Cys Prx can be classified as sensitive or robust.

**Figure 6.** Structural comparison of the sensitive S. cerevisiae Tsa1 and the robust S. typhimurium AhpC. The comparison of Tsa1 (A) and AhpC (B) structures reveal the presence of the GGLG and YF motifs typically found in eukaryotes. The structures are represented in cartoon and the structural motifs as well the CP and CR are represented in ball and sticks. The figures were generated using the S. cerevisiae Tsa1 (PDB: 3SBC) and S. typhimurium AhpC (PDB: 4MA9) coordinates in Pymol software (http://www.pymol.org/).

The sensitive enzymes are present in eukaryotes and in some cyanobacteria, and the robust 2-Cys Prxs are exclusive to prokaryotes [34, 35]. The structural analyses of sensitive versus robust 2-Cys Prx revealed the presence of two motifs in the sensitive 2-Cys Prx. One is an insertion with conserved Gly-Gly-Leu-Gly, denominated GGLG motif (**Figure 6A**), and the other is an additional α-helix in C-terminal extension with a conserved Tyr-Phe sequence, the YF motif, both involved in CP overoxidation susceptibility. **Figure 6** shows the comparison of *S. cerevisiae* Tsa1, a sensitive typical 2-Cys Prx, and *Salmonella typhimurium* (AhpC), a robust enzyme [34]. This difference is associated with important effects in redox cell signaling transduction and will be detailed in the next topics.

#### **4. Typical 2-Cys Prx roles in redox signal transduction pathways**

activity. The HMW complexes formation was first reported in *S. cerevisiae* Tsa1 and Tsa2 by Jang and coworkers [11], and, posteriorly, very similar complexes were described to the human homologues typical 2-Cys Prxs (PrxI and PrxII) [26]. Using transmission electron microscopy (TEM), it was demonstrated that complexes are represented by heterogeneous spherical structures, which can reach 1 GDa, and biochemical approaches revealed that the complexes present an extraordinary chaperone holdase activity [9, 26]. Later on, similar spherical and another type of HWM complexes, represented by the stacking of several decamers (**Figure 5C**), were described for the plant chloroplastic 2-Cys Prxs, cyanobacterial Anabaena PCC7120 2-Cys Prx, among others [27–30]. The structural differences between the HMW complexes are not well

**Figure 5.** Typical 2-Cys Prx overoxidation and high-molecular-weight complex formation. The typical 2-Cys Prx in reduced form are presented as α2(5) decamers (A). In low concentrations of hydroperoxides, the CP is oxidized in CP-SOH, and the intermolecular disulfide is formed with CR. The disulfide formation, in some cases, is able to destabilize the decamers, forming a mixture of decamers and dimers (B). The oxidized form is reduced by Trx system. When the typical

2-Cys Prx are challenged with high concentrations of hydroperoxides, the CP can be overoxidized to CP-SO2

overoxidation promotes the HMW complexes formation which presents chaperone properties (C). The CP-SO2

A very important point relies on the fact that the Prx overoxidized species cannot be reduced by the Trx system, but some studies revealed that overoxidized typical 2-Cys Prx species could be regenerated to the reduced form *in vivo* [31]. Posteriorly, it was identified in *S. cerevisiae*, and after in human and other species, an enzyme named sulfiredoxin (Srx) which is able

by Srx are very slow when compared to the disulfide reduction by Trx (~2 M−1 s−1 and ~106

in **Figure 5D**. It is important to highlight that the Srx was identified in several eukaryotes, but few prokaryotes possess this enzyme, which may be an evolutionary sophistication of the 2-Cys Prx redox cycle [33]. In fact, to the majority of the prokaryotes, no homologous Srx gene was detected in their genomes, and the typical 2-Cys Prxs are much more resistant to overoxidation. Moreover, an additional classification can be done based on the CP overoxidation

that this oxidation state is refractive to reduction [31]. Curiously, the CP-SO2

susceptibility, and the 2-Cys Prx can be classified as sensitive or robust.

s−1, respectively) [32]. The biochemical steps of the CP-SO2

reduced by sulfiredoxin, in ATP and Mg2+ dependent manner (D).

H in a ATP and Mg2+ dependent reaction, but not CP-SO3

H, suggesting

H. The CP

H can be

M−1

H reduction rates

H reduction by Srx are represented

understood to date.

124 Old Yeasts - New Questions

to reduce the CP-SO2

Increasing evidence shows the involvement of the typical 2-Cys Prx with the redox signal transduction pathways. Several antioxidant coding genes are activated by the transcriptional regulator activator protein 1 (AP1) which is considered as the major transcriptional activator of the antioxidant proteins in eukaryotes. It has been shown that the translocation of the homologue factor in budding yeast (YAP1) from cytosol to the nucleus may be controlled by 2-Cys Prx indirectly by the modulation of the cytosolic hydroperoxide levels [36]. In mammals, the PrxII is able to perform a physical interaction with the transcription factor STAT 3 (signal transducer and activator of transcription 3), which is able to activate the transcription of several genes involved in cell growth and apoptosis [37]. The authors demonstrated that PrxII can form mixed disulfides through CP and cysteine residues of the DNA binding and trans-activating domains from STAT3, attenuating its transcriptional activity. Although the direct interaction of the typical 2-Cys Prx with target proteins is still an emerging area, this work reveals that the Prx may be an ultrasensitive hydroperoxide sensor that can form transient disulfides with unknown target proteins, which may have implications in biological processes. Additionally, the mammal PrxI can bind to several proteins including the tumor suppressor phosphatase and tensin homolog (PTEN), protecting it against suppression of its lipid phosphatase activity, which occurs under oxidative stress. On the other hand, PTEN deficiency causes decrease of PrxI, PrxII, PrxV, and PrxVI, suggesting that the Prxs and PTEN act together to maintain cellular antioxidant levels and suppress cancer-promoting pathways, such as the PI3K-Akt pathway [38].

Despite the importance of the physical interaction between typical 2-Cys Prx and biological targets, the indirect role in the regulation of the cell-signaling redox pathways is dependent of an intricate balance between peroxiredoxin, thioredoxin, and sulfiredoxin levels and their redox state. As an example, in yeast, the number of Tsa1 molecules per cell is estimated in 378,000 in aerobic conditions (log phase, SD medium), while its reductants represented by Trx and Srx molecules are much lower (~13,000 and 538 molecules/cell, respectively) [16]. In the case of Trx enzymes, additionally to the Prx reduction, these enzymes are involved in several biological processes as deoxyribonucleotide synthesis, repair of oxidatively damaged proteins, protein folding, sulfur metabolism, and activation of transcription factors among others [16]. The importance of Tsa1 reduction by Trx in redox signaling promoted by hydrogen peroxide may be significant in the cells since it produces oxidized Trx, and many signal transduction pathways are only activated by the reduced Trx enzyme [1, 39]. Because the oxidation of Trx by hydroperoxidesis is negligible, Prxs may act as a catalyst of this reaction in the cells [21].

The typical 2-Cys Prx inactivation by the CP overoxidation combined with the low rates of the reduction of the CP-SO2 H by sulfiredoxin (~2 M−1 s−1) [32] is able to enhance levels of the reduced Trx to participate of other biological processes. In fact, it has been shown that the CP overoxidation of the typical 2-Cys Prx from *Schizosaccharomyces pombe* (Tpx1) enhance the levels of the reduced Trx and allow the repair of damaged proteins increasing cell survival [40]. Accordingly, in mammals, only the reduced form of Trx is able to bind to the apoptosis signal regulating kinase (Ask-1), inhibiting the apoptosis, thus revealing a redox-dependent signal transduction pathway, which is induced by Trx oxidation [41]. Also in mammals, the activation of the nuclear factor kappa light chain enhancer of activated β cells (NF-kβ), a transcription factor that plays a central role regulating pathways of immune and inflammatory processes [42], is dependent on the reduction of a cysteine residue by Trx [43]. Additionally, Trx is involved in the reduction and activation of several transcription factors as the tumor-suppressor p53, the glucocorticoid and estrogen receptors, and c-Fos/c-Jun complexes [39].

Finally, the direct modulation of peroxides levels is an important role of the 2-Cys Prx enzymes in cell growth. It has been shown that PrxI and PrxII can eliminate the intracellular hydrogen peroxide generated by the receptors stimulation. Overexpression of PrxI and PrxII in culture cells dramatically reduces the intracellular hydrogen peroxide levels generated in response to platelet-derived growth factor (PDGF), epidermal growth factor (EGF), tirotropin (TSH), and TNF-related apoptosis inducing ligands (TRAIL). Furthermore, it has been shown that the expression of these proteins also led to a block of NF-κβ activity, which is induced by the extracellular addition of H2 O2 or tumor necrosis factor α (TNF-α) [44]. It has also been shown that PrxII regulates different MAP kinases. Under stimulation of TNF, in which the activity of PrxII was blocked or partially abolished (knockout and RNAi), the activity of JNK and P38 MAP kinase was increased [45]. Due to the involvement of PrxI and PrxII in cell growth events, several studies have demonstrated that these isoforms have elevated levels in distinct types of cancers in different organs and tissues such as esophagus, pancreas, thyroid, lung, and breast [44–46]. The high expression of PrxI/PrxII is also associated to a more aggressive phenotype of cancer cells resistant to chemotherapy and radiotherapy [44–46].

Some authors argued that the typical 2-Cys Prx enzymes maintain hydrogen peroxide in appropriate levels to cell growth but not to apoptosis. However, Liu et al. [47] showed that the neoplastic cells of acute myeloid leukemia treated with an inhibitor of the PrxI and PrxII peroxidase activity demonstrated that the accumulation of intracellular H2 O2 is related to the activation of the ERK1 and ERK2 (extracellular signal regulatory kinases). The kinases activation leads to an increase in the expression of the CCAAT-enhancer-binding proteins β (C/EBPβ). This condition resulted in cell differentiation and consequent tumor regression [47, 48], showing additional complexity of the neoplastic processes with the involvement of the Prx. Since there is a notable resemblance between human and yeasts typical 2-Cys Prx as also other proteins of these pathways, yeasts may be used to explore these mechanisms.

#### **5. Prx structural switch and circadian rhythm**

Despite the importance of the physical interaction between typical 2-Cys Prx and biological targets, the indirect role in the regulation of the cell-signaling redox pathways is dependent of an intricate balance between peroxiredoxin, thioredoxin, and sulfiredoxin levels and their redox state. As an example, in yeast, the number of Tsa1 molecules per cell is estimated in 378,000 in aerobic conditions (log phase, SD medium), while its reductants represented by Trx and Srx molecules are much lower (~13,000 and 538 molecules/cell, respectively) [16]. In the case of Trx enzymes, additionally to the Prx reduction, these enzymes are involved in several biological processes as deoxyribonucleotide synthesis, repair of oxidatively damaged proteins, protein folding, sulfur metabolism, and activation of transcription factors among others [16]. The importance of Tsa1 reduction by Trx in redox signaling promoted by hydrogen peroxide may be significant in the cells since it produces oxidized Trx, and many signal transduction pathways are only activated by the reduced Trx enzyme [1, 39]. Because the oxidation of Trx by hydroperoxidesis is negligible, Prxs may act as a catalyst of this reaction

The typical 2-Cys Prx inactivation by the CP overoxidation combined with the low rates of

reduced Trx to participate of other biological processes. In fact, it has been shown that the CP overoxidation of the typical 2-Cys Prx from *Schizosaccharomyces pombe* (Tpx1) enhance the levels of the reduced Trx and allow the repair of damaged proteins increasing cell survival [40]. Accordingly, in mammals, only the reduced form of Trx is able to bind to the apoptosis signal regulating kinase (Ask-1), inhibiting the apoptosis, thus revealing a redox-dependent signal transduction pathway, which is induced by Trx oxidation [41]. Also in mammals, the activation of the nuclear factor kappa light chain enhancer of activated β cells (NF-kβ), a transcription factor that plays a central role regulating pathways of immune and inflammatory processes [42], is dependent on the reduction of a cysteine residue by Trx [43]. Additionally, Trx is involved in the reduction and activation of several transcription factors as the tumor-suppressor p53, the

Finally, the direct modulation of peroxides levels is an important role of the 2-Cys Prx enzymes in cell growth. It has been shown that PrxI and PrxII can eliminate the intracellular hydrogen peroxide generated by the receptors stimulation. Overexpression of PrxI and PrxII in culture cells dramatically reduces the intracellular hydrogen peroxide levels generated in response to platelet-derived growth factor (PDGF), epidermal growth factor (EGF), tirotropin (TSH), and TNF-related apoptosis inducing ligands (TRAIL). Furthermore, it has been shown that the expression of these proteins also led to a block of NF-κβ activity, which is induced by the

that PrxII regulates different MAP kinases. Under stimulation of TNF, in which the activity of PrxII was blocked or partially abolished (knockout and RNAi), the activity of JNK and P38 MAP kinase was increased [45]. Due to the involvement of PrxI and PrxII in cell growth events, several studies have demonstrated that these isoforms have elevated levels in distinct types of cancers in different organs and tissues such as esophagus, pancreas, thyroid, lung, and breast [44–46]. The high expression of PrxI/PrxII is also associated to a more aggressive

phenotype of cancer cells resistant to chemotherapy and radiotherapy [44–46].

glucocorticoid and estrogen receptors, and c-Fos/c-Jun complexes [39].

O2

H by sulfiredoxin (~2 M−1 s−1) [32] is able to enhance levels of the

or tumor necrosis factor α (TNF-α) [44]. It has also been shown

in the cells [21].

126 Old Yeasts - New Questions

the reduction of the CP-SO2

extracellular addition of H2

The circadian rhythm is a fundamental process considered to be a feature of almost all living cells. The organisms are able to exhibit cycles in their metabolism, physiology, and behavior, even when isolated from external stimuli, maintaining a 24-h period [49]. However, the molecular mechanisms which drive the circadian rhythm are not simple to elucidate, since the already identified clock genes and proteins are not very conserved across phylogenetic kingdoms [49–53]. A common model for molecular mechanism has been described for all organisms which had their circadian rhythm investigated, named transcription-translation feedback loop (TTFL) [49]. However, the TTFL components are not shared between organisms, suggesting independent evolutionary processes. Additionally, it was showed that nontranscriptional mechanisms are sufficient to sustain circadian timekeeping in the eukaryotic lineage, although they normally function in conjunction with transcriptional components [51].

Recently, it has been demonstrated that in human erythrocytes, a cell type without transcriptional activity, the PrxI and PrxII exhibit an approximate 24-h rhythm according to the CP overoxidation. This characteristic is shared with several organisms, including *S. cerevisiae*, indicating that the typical 2-Cys Prxs constitute a universal rhythmic biomarker [52]. To reach this conclusion, the authors performed immunoblotting analyses using a Prx CP-SOH2/3 antibody and showed that 2-Cys Prx proteins from organisms of different domains have been oscillated to overoxidized Prx species, in constant conditions, exhibiting a circadian oscillation, probably reflecting an endogenous rhythm in the generation of reactive oxygen species (ROS; **Figure 7**) [51, 54–56]. Because all living organisms possess typical 2-Cys Prx enzymes that present remarkable conservation of the active site, the same antibody was able to detect overoxidized typical 2-Cys Prx in mice, fungi, plants, bacteria, and archaea. This indicates that the circadian clock mechanism is likely conserved across phylogenetic domains [54].

Yeast Tsa1 and Tsa2 isoforms exhibit relationship with the shorter period yeast respiratory oscillations, a cell-autonomous, temperature-compensated rhythm in oxygen consumption that synchronizes spontaneously when cells are grown at high density in aerobic, nutrientlimited, continuous culture [52]. Additionally, the yeast respiratory oscillation cycle shares

**Figure 7.** Redox circadian cycle of typical 2-Cys Prx. The circadian cycle of 2-Cys Prx could be detected by PRX-SOH2/3 immunoblot. Western blot representation shows that overoxidized Prx has a circadian rhythm (upper part of the figure), and, consequently, the oligomeric state follows the redox state from Prx, alternating between dimers and decamers, in oxidative and reduced states, respectively, and HMW formation in overoxidized species (represented in the right side of the figure).

key features with the clock in mammalian cells, which may contribute to the elucidation about the origins of biological timekeeping [52].

Finally, it has been determined that the deregulation of the circadian rhythm is related to aging and genetic diseases [57]. Curiously, it has also been demonstrated that aging is related to the accumulation of the 2-Cys Prx overoxidized species in mammals [58]. Recently, a study involving the overoxidation of Tsa1 revealed that the chaperone activity detected in overoxidized species may be attributed to the association of this protein with the heat shock proteins Hsp70/Hsp104, revealing a pathway where the hydrogen peroxide is directly related to the aging process [12]. The authors also showed that the disaggregation process of the protein is dependent of Srx. Another study demonstrated that the presence of a mutant allele of Tsa1 resulted in accelerated aging in yeast [59]. One of the reasons for the involvement of these enzymes in the senescence process resides in the increase of the level of CP overoxidation in Prx over time, even in the absence of oxidative stress [6]. In fact, this process also involves the caloric restriction, a well-known intervention that extends life span [60]. The caloric restriction elevates the level of Srx, which is responsible to reduce the hyperoxidized Tsa1, the inhibition of Tsa1 causes a profound genome instability, like chromosomal rearrangements and recombination, therefore increasing aging process [6, 61].

Another situation in which Prxs are involved is in the telomere length homeostasis [62]. The telomere dysfunction causes cellular senescence due to DNA damage [63]. The yeast mutant with *tsa1* gene deleted displayed reduction of telomere lengthening, which was not observed in conditions of low-oxidative exposure, probably due to the role of Tsa1 in hydroperoxide decomposition, avoiding DNA damage [62]. The understanding of the aging process and its implications in yeast can be used to extrapolate to higher eukaryotes. In fact, even in erythrocytes, the 2-Cys Prxs are related to the aging process. PrxII also has the ability to associate with the erythrocyte cell membrane through the N-terminal cytoplasmic domain of band 3 protein, after which PrxII undergoes a conformational change that does not entail the loss of its peroxidase function. This association may indicate a potential role of this Prx in the protection of membrane lipids against oxidative damage increasing life span [64]. Accordingly, a study carried out using mouse erythrocytes showed that the levels of overoxidized PrxII are due to autoxidation of hemoglobin and to PrxII degradation by the 20S proteasome. Approximately 1% of PrxII-SO2 H is degraded daily, leading to progressive loss of this enzyme which is directly related with the erythrocyte senescence [65]. Additionally, the aging process is directly related the genome instability. This instability is maintained in part by Prx action, and it is also involved in some diseases like cancer. Together, the data presented here reveal a cross talk of the 2-Cys Prx CP overoxidation in circadian clocks, aging, and lifespan.

#### **6. Methodologies to detect different redox species of the typical 2-Cys Prx**

Several methodologies such as transmission electron microscopy (TEM), cryo-electron microscopy (Cryo-EM), size exclusion chromatography (SEC), mass spectrometry (MS), two-dimensional gel electrophoresis (2DGE), nonreducing SDS PAGE, and immunoblotting can be used to explore directly or indirectly the redox state of typical 2-Cys Prx [11, 66, 67]. However, for some experimental procedures, high-cost equipment and/or complex experimental procedures are necessary. Among these techniques, the nonreducing SDS PAGE, immunoblotting, and SEC are very good and cost-effective procedures, since no expensive equipment or complicated protocols are required. In this topic, these techniques and some experimental procedures will be discussed.

key features with the clock in mammalian cells, which may contribute to the elucidation

**Figure 7.** Redox circadian cycle of typical 2-Cys Prx. The circadian cycle of 2-Cys Prx could be detected by PRX-SOH2/3 immunoblot. Western blot representation shows that overoxidized Prx has a circadian rhythm (upper part of the figure), and, consequently, the oligomeric state follows the redox state from Prx, alternating between dimers and decamers, in oxidative and reduced states, respectively, and HMW formation in overoxidized species (represented in the right side

Finally, it has been determined that the deregulation of the circadian rhythm is related to aging and genetic diseases [57]. Curiously, it has also been demonstrated that aging is related to the accumulation of the 2-Cys Prx overoxidized species in mammals [58]. Recently, a study involving the overoxidation of Tsa1 revealed that the chaperone activity detected in overoxidized species may be attributed to the association of this protein with the heat shock proteins Hsp70/Hsp104, revealing a pathway where the hydrogen peroxide is directly related to the aging process [12]. The authors also showed that the disaggregation process of the protein is dependent of Srx. Another study demonstrated that the presence of a mutant allele of Tsa1 resulted in accelerated aging in yeast [59]. One of the reasons for the involvement of these enzymes in the senescence process resides in the increase of the level of CP overoxidation in Prx over time, even in the absence of oxidative stress [6]. In fact, this process also involves the caloric restriction, a well-known intervention that extends life span [60]. The caloric restriction elevates the level of Srx, which is responsible to reduce the hyperoxidized Tsa1, the inhibition of Tsa1 causes a profound genome instability, like chromosomal rearrangements and

Another situation in which Prxs are involved is in the telomere length homeostasis [62]. The telomere dysfunction causes cellular senescence due to DNA damage [63]. The yeast mutant with *tsa1* gene deleted displayed reduction of telomere lengthening, which was not observed in conditions of low-oxidative exposure, probably due to the role of Tsa1 in hydroperoxide decomposition, avoiding DNA damage [62]. The understanding of the aging process and its implications in yeast can be used to extrapolate to higher eukaryotes. In fact, even in erythrocytes, the 2-Cys Prxs are related to the aging process. PrxII also has the ability to associate with the erythrocyte cell membrane through the N-terminal cytoplasmic domain of band 3

about the origins of biological timekeeping [52].

of the figure).

128 Old Yeasts - New Questions

recombination, therefore increasing aging process [6, 61].

To access the formation of HMW complexes of purified 2-Cys Prx samples, the size exclusion chromatography (SEC) is the best choice. This methodology was used in the pioneer work performed by Jang and coworkers [11] using Tsa1 and Tsa2. In our lab, we performed a similar assay, using Tsa1, Trx system, and high concentration of cumene hydroperoxide (CHP) to promote the HMW complexes formation. Using SEC methodology, it is possible to separate several molecular species with mass range from ~45 kDa, correspondent to a dimer, followed by a ~200-kDa peak representing the decameric species, several oligomeric intermediates, and a prominent specie with more than 1000 kDa (**Figure 8**). These results are in accordance with structural analyses performed by transmission electron microscopy (TEM) by Jang and coworkers [11], using negative stain. These authors analyzed different fractions separated by SEC, and their results revealed three distinct oligomeric configurations: large spherical shaped particles, heterogeneous spherical particles, and ring-shaped structures, as represented in **Figure 8**. Currently, the cryo-electron microscopy development has provided pronounced advances to resolve complex protein structures in high resolution, such as the human Prx [67].

To reduced, oxidized, and overoxidized species from purified proteins samples *in vitro,* a simple nonreducing SDS PAGE (without DTT or another reductant) can be used to detect different redox species of the enzymes. In fact, the reduce Prx decamers and HMW complexes are held together by weak molecular forces as hydrophobic, van der Walls, and polar interactions, that are disrupted in SDS PAGE. As an example, on a nonreducing gel containing SDS,

**Figure 8.** Overoxidized Tsa1 complexes analyzed by SEC. Tsa1 HMW species formation was analyzed by size-exclusion chromatography. The assay was performed overnight at 4°C in Hepes-NaOH 50mM (pH 7.4), DTPA 100 μM, sodium azide 1 mM, NADPH 1 mM, S. cerevisiae Prx 43.6 μM; S. cerevisiae Trx1 1 μM; S. cerevisiae TrxR1 0.3 μM and CHP 10 mM. The reaction was injected into the system containing a BioSep-SEC-S3000 column, eluted at a ow rate of 1 ml min−1 and monitored by tryptophan uorescence (excitation, 280 nm; emission, 340 nm). The elution pro le of the molecular standards thyroglobulin (bovine) (670 kDa), γ-globulin (bovine) (158 kDa), and ovalbumin (chicken) (44 kDa) were used to identify the 2-Cys Prx oligomers.

the Tsa1 is detected as a monomer (~25 kDa). The oxidized form is detected as ~50 kDa bands (dimer) as a consequence of the intermolecular disulfide bond that is formed between the CP and CR that is not disrupted in the gel. The overoxidized forms (CysP-SO2 H or CysP-SO3 H) can also be visualized as monomers, since the disulfide bond formation is not achievable [68, 69] (**Figure 9A** and **B**).

**Figure 9B** shows the SDS-PAGE result of *in vitro* procedure to perform Tsa1 overoxidation using growing concentrations of organic hydroperoxide (*t*-BOOH) and the Trx system (see the legend for detail). In this example, it is possible to verify the presence of the Tsa1 overoxidized species when high concentrations of *t*-BOOH were used (**Figure 9B**, upper panel). In **Figure 9C**, it is represented the probable quaternary structure present in the correspondent lane of the gel. In low concentrations of hydroperoxides, there are, predominantly, reduced Tsa1 in decameric

**Figure 9.** Redox state analyses by nonreducing SDS-PAGE and immunoblotting of the typical 2-Cys Prx. Diagram of the different 2-Cys Prx redox species in SDS-PAGE in nonreducing conditions by monomer or dimer formation (A). The Tsa1 overoxidation can be followed by SDS-PAGE in nonreducing conditions using *in vitro* approaches with Trx system in growing concentrations of hydroperoxides (B). In the example, the reaction was performed in a final volume of 50 μl at 30°C in Hepes-NaOH 50 mM (pH 7.0), DTPA 100 μM, sodium azide 1 mM, NADPH 150 μM, *S. cerevisiae* Tsa1 9.3 μM; *S. cerevisiae* Trx1 1 μM; *S. cerevisiae* TrxR1 0.3 μM and growing concentrations of *t*-BOOH (0.01, 0.025, 0.05, 0.75, 0.125, 0.25, 0.5, 1, 5, and 10 mM).The CP overoxidized species can be observed in higher concentrations of *t*-BOOH. The oxidative state of Tsa1 induces the quaternary structural changes (C). The numbers in C demonstrate the possible structure of Tsa1 in different oxidative states (dimers, reduced decamers, oxidized decamers, and HMW complexes).

form, which is disrupted in SDS PAGE. At intermediate hydroperoxide concentrations, disulfide oxidized forms are detected, being represented by dimers and weak decamers (**Figure 9C**), that are detected as dimers in SDS PAGE. However, in high concentration of hydroperoxide, CP overoxidation and HMW structure formations that, in the gel, are detected as monomers occur [11] (**Figure 9B**). To confirm the redox state result, a western blot analysis can be performed using an anti-SO2/3 anti-body (**Figure 9B**, lower panel) [31, 70]. Additionally, the immunoblot technique can be used in nonpurified samples, as cell extracts, since antibodies to typical 2-Cys Prx are commercially available by several suppliers. Moreover, as mentioned before, the antibodies can be used in different species since the enzymes possess remarkable conservation [54].

#### **7. Conclusions**

the Tsa1 is detected as a monomer (~25 kDa). The oxidized form is detected as ~50 kDa bands (dimer) as a consequence of the intermolecular disulfide bond that is formed between the CP

**Figure 8.** Overoxidized Tsa1 complexes analyzed by SEC. Tsa1 HMW species formation was analyzed by size-exclusion chromatography. The assay was performed overnight at 4°C in Hepes-NaOH 50mM (pH 7.4), DTPA 100 μM, sodium azide 1 mM, NADPH 1 mM, S. cerevisiae Prx 43.6 μM; S. cerevisiae Trx1 1 μM; S. cerevisiae TrxR1 0.3 μM and CHP 10 mM. The reaction was injected into the system containing a BioSep-SEC-S3000 column, eluted at a ow rate of 1 ml min−1 and monitored by tryptophan uorescence (excitation, 280 nm; emission, 340 nm). The elution pro le of the molecular standards thyroglobulin (bovine) (670 kDa), γ-globulin (bovine) (158 kDa), and ovalbumin (chicken) (44 kDa) were used

also be visualized as monomers, since the disulfide bond formation is not achievable [68, 69]

**Figure 9B** shows the SDS-PAGE result of *in vitro* procedure to perform Tsa1 overoxidation using growing concentrations of organic hydroperoxide (*t*-BOOH) and the Trx system (see the legend for detail). In this example, it is possible to verify the presence of the Tsa1 overoxidized species when high concentrations of *t*-BOOH were used (**Figure 9B**, upper panel). In **Figure 9C**, it is represented the probable quaternary structure present in the correspondent lane of the gel. In low concentrations of hydroperoxides, there are, predominantly, reduced Tsa1 in decameric

**Figure 9.** Redox state analyses by nonreducing SDS-PAGE and immunoblotting of the typical 2-Cys Prx. Diagram of the different 2-Cys Prx redox species in SDS-PAGE in nonreducing conditions by monomer or dimer formation (A). The Tsa1 overoxidation can be followed by SDS-PAGE in nonreducing conditions using *in vitro* approaches with Trx system in growing concentrations of hydroperoxides (B). In the example, the reaction was performed in a final volume of 50 μl at 30°C in Hepes-NaOH 50 mM (pH 7.0), DTPA 100 μM, sodium azide 1 mM, NADPH 150 μM, *S. cerevisiae* Tsa1 9.3 μM; *S. cerevisiae* Trx1 1 μM; *S. cerevisiae* TrxR1 0.3 μM and growing concentrations of *t*-BOOH (0.01, 0.025, 0.05, 0.75, 0.125, 0.25, 0.5, 1, 5, and 10 mM).The CP overoxidized species can be observed in higher concentrations of *t*-BOOH. The oxidative state of Tsa1 induces the quaternary structural changes (C). The numbers in C demonstrate the possible structure of

Tsa1 in different oxidative states (dimers, reduced decamers, oxidized decamers, and HMW complexes).

H or CysP-SO3

H) can

and CR that is not disrupted in the gel. The overoxidized forms (CysP-SO2

(**Figure 9A** and **B**).

130 Old Yeasts - New Questions

to identify the 2-Cys Prx oligomers.

*S. cerevisiae* is continuously used as a model organism by several researchers, being associated with significant advances in life sciences. In this chapter, we exposed several discoveries related with the role of the yeast Prx as a model in several studies related to hydroperoxide detoxification and signaling, and how these characteristics influence physiological processes like circadian rhythm and aging and diseases like cancer. All these features are related to the redox state of Prx and amazing functional and structural switches and the cross talk with different pathways that are regulated by hydroperoxide levels. Additionally, we present some practical approaches which can be easily implemented to Prx studies, like nonredox SDS-PAGE, size exclusion chromatography, and transmission electron microscopy. We believe that the use of these techniques may facilitate the study of these intricate enzymes for those interested in joining to this exciting research area.

#### **Acknowledgements**

We acknowledge the financial support from the Fundação de Amparo à Pesquisa do Estado de São Paulo grants 07/50930-3, 16/10130-7, and 13/07937-8 (Redox Processes in Biomedicine [REDOXOMA]).

#### **Author details**

Melina C. Santos1,2, Carlos A. Breyer1 , Leonardo Schultz1 , Karen S. Romanello3 , Anderson F. Cunha3 , Carlos A. Tairum Jr1,4 and Marcos Antonio de Oliveira1 \*

\*Address all correspondence to<EMAIL_ADDRESS>

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200006.112

#### **Chapter 7**

**Provisional chapter**

#### **HMGB Proteins from Yeast to Human. Gene Regulation, DNA Repair and Beyond DNA Repair and Beyond**

DOI: 10.5772/intechopen.70126

**HMGB Proteins from Yeast to Human. Gene Regulation,**

Vizoso-Vázquez Ángel, Barreiro-Alonso Aida, Rico-Díaz Agustín, Lamas-Maceiras Mónica, Rodríguez-Belmonte Esther, Becerra Manuel, González-Siso María Isabel and Cerdán María Esperanza Rico-Díaz Agustín, Lamas-Maceiras Mónica, Rodríguez-Belmonte Esther, Becerra Manuel, González-Siso María Isabel and Cerdán María Esperanza Additional information is available at the end of the chapter

Vizoso-Vázquez Ángel, Barreiro-Alonso Aida,

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70126

#### **Abstract**

HMGB proteins are characterized for containing one or more HMG-box domains and are well conserved from yeasts to higher eukaryotes. The HMG-box domain is formed by three α-helices with an L-shaped fold. Although HMGB proteins also have cytoplasmic and extracellular functions, they bind to nuclear or mitochondrial DNA in a highly dynamic process that affects chromatin organization. In this review, we mainly focus on HMGB proteins from yeast and their human homologs as functionally involved in DNA repair and transcriptional regulation. Recent research reveals that these proteins participate in epigenetic control of gene expression, aging, disease, or stem-cell biology.

**Keywords:** nonhistone proteins, epigenetics, transcriptional regulation

#### **1. Introduction**

Nucleosomes are fairly stable basic units of DNA packaging. Nevertheless, nucleosomal chromatin is surrounded by a highly dynamic protein pool that allows chromatin remodeling and favors replication, DNA repair, and gene transcription. Among proteins that transiently associate with chromatin are variants of the linker histone H1 family [1–3] and members of the high mobility group (HMG) protein superfamily [4–6]. Although HMG motifs are present in many nuclear proteins, the classification and nomenclature of the considered "canonical"

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

HMG proteins is organized in three families named HMGA, HMGB, and HMGN, each one having a specific functional domain: the "AT hook" in HMGA, the "HMG-box" in HMGB, and the "nucleosomal binding domain" in HMGN proteins [7].

Some HMGB proteins have been related to nuclear, extranuclear, and extracellular functions during inflammation, cell differentiation, cell migration, and tumor metastasis [8, 9]. Their HMG-box domain contains 65–85 amino acids and has a characteristic L-shaped fold formed by three α-helices with an angle of ≈80° between the two arms. The long arm, or minor wing, is composed by the extended N-terminal strand and third α-helix, while first and second α-helices form the short arm, or major wing (**Figure 1(a)**). Because of protein interaction in the minor groove, DNA-bending and widening of the double helix is produced (**Figure 1(b)**).

There are two broad subfamilies of HMGB-containing proteins, based on structural and phylogenetic studies. One class includes those that bind to distorted DNA with low or without sequence specificity (nonsequence specificity (NSS), HMG-box domains) and have, in general, two or more *in tandem* arranged HMG-box domains [10, 11]. Examples of proteins without sequence specificity are the mammalian Hmgb1-4 and Ubf proteins, Hmgd from *Drosophila*, or Nhp6a and Nhp6b from *Saccharomyces cerevisiae*. Their role is related to chromatin modification, participating in many different functions such as co-activation or silencing of transcription and V(D)J junction recombination. A second class of HMGB-containing proteins binds to DNA by recognizing a specific DNA sequence (sequence specificity (SS), HMG-box domains), and they usually contain a single HMG-box domain [10, 11]. They generally function as transcription factors, only expressed in a few cell types, and they also contain other regulatory associated domain. The determinants for DNA sequence specificity lie mainly in the minor wing of the HMG-box. Examples of this kind of HMGB proteins are the mammalian lymphoid enhancer factor (Lef-1), the sex determining factor (Sry), and the Sry-related HMG-box (SOX) family, or the hypoxic gene repressor (Rox1) from *S. cerevisiae*.

In this review, we focus on HMGB proteins from yeast, as functionally involved in DNA repair and transcriptional regulation, but also in their homologs from multicellular eukaryotes, with special reference to human proteins. Their functions may be modulated by nucleosome positioning and stability [12]. Interestingly, recent findings support that HMGB proteins may also

**Figure 1.** (a) Characteristic HMG-box fold (based on Sox17 protein structure; PDBID: 3F27). (b) Bending and widening produced in the double strand of DNA after protein binding.

play diverse roles in epigenetic control, since their interaction with chromatin affects the level of histone modifications [13]. In the light of recently opened research areas, in which HMGB proteins are involved, available knowledge is also discussed.

#### **2. HMGB proteins from** *Saccharomyces cerevisiae*

HMG proteins is organized in three families named HMGA, HMGB, and HMGN, each one having a specific functional domain: the "AT hook" in HMGA, the "HMG-box" in HMGB, and

Some HMGB proteins have been related to nuclear, extranuclear, and extracellular functions during inflammation, cell differentiation, cell migration, and tumor metastasis [8, 9]. Their HMG-box domain contains 65–85 amino acids and has a characteristic L-shaped fold formed by three α-helices with an angle of ≈80° between the two arms. The long arm, or minor wing, is composed by the extended N-terminal strand and third α-helix, while first and second α-helices form the short arm, or major wing (**Figure 1(a)**). Because of protein interaction in the minor groove, DNA-bending and widening of the double helix is produced (**Figure 1(b)**). There are two broad subfamilies of HMGB-containing proteins, based on structural and phylogenetic studies. One class includes those that bind to distorted DNA with low or without sequence specificity (nonsequence specificity (NSS), HMG-box domains) and have, in general, two or more *in tandem* arranged HMG-box domains [10, 11]. Examples of proteins without sequence specificity are the mammalian Hmgb1-4 and Ubf proteins, Hmgd from *Drosophila*, or Nhp6a and Nhp6b from *Saccharomyces cerevisiae*. Their role is related to chromatin modification, participating in many different functions such as co-activation or silencing of transcription and V(D)J junction recombination. A second class of HMGB-containing proteins binds to DNA by recognizing a specific DNA sequence (sequence specificity (SS), HMG-box domains), and they usually contain a single HMG-box domain [10, 11]. They generally function as transcription factors, only expressed in a few cell types, and they also contain other regulatory associated domain. The determinants for DNA sequence specificity lie mainly in the minor wing of the HMG-box. Examples of this kind of HMGB proteins are the mammalian lymphoid enhancer factor (Lef-1), the sex determining factor (Sry), and the Sry-related

HMG-box (SOX) family, or the hypoxic gene repressor (Rox1) from *S. cerevisiae*.

In this review, we focus on HMGB proteins from yeast, as functionally involved in DNA repair and transcriptional regulation, but also in their homologs from multicellular eukaryotes, with special reference to human proteins. Their functions may be modulated by nucleosome positioning and stability [12]. Interestingly, recent findings support that HMGB proteins may also

**Figure 1.** (a) Characteristic HMG-box fold (based on Sox17 protein structure; PDBID: 3F27). (b) Bending and widening

produced in the double strand of DNA after protein binding.

the "nucleosomal binding domain" in HMGN proteins [7].

140 Old Yeasts - New Questions

In *S. cerevisiae*, the genes *ABF2, HMO1, IXR1, NHP6A, NHP6B, NHP10*, and *ROX1* encode HMGB proteins [7]. The protein Spp41 also contains a HMG-like motif although homology searches reveal that it is far related to the others. The structural characteristics and functions of these yeast proteins are shown in **Table 1**. Only one HMG-box domain is present in most of them, but Abf2 and Ixr1 have two in tandem "HMG-box" motifs.

With the exception of Rox1 that behaves as a specific transcriptional regulator of the hypoxic yeast regulon [14] and Ixr1 that has a dual function as specific transcription factor and DNAbinding protein without sequence specificity, also participating in DNA repair [15], the other

**Table 1.** Characteristics of HMGB proteins in *Saccharomyces cerevisiae.*

HMGB proteins from *S. cerevisiae* might be considered as chromatin architectural proteins, but with wide influence on gene expression [16]. This is not a HMGB-exclusive mechanism since, in eukaryotes, many other chromatin components, such as histones [17], histone chaperones and modifiers [18], chromatin remodel complexes [19], and long noncoding RNAs [20], affect gene expression by different mechanisms.

Although Abf2 and Ixr1 are considered paralogs, resulting from the whole genome duplication in an ancestor of *Saccharomyces*, the function of Abf2 is not related to transcriptional regulation of hypoxic regulons. Abf2 is a mitochondrial DNA-binding protein involved in mitochondrial DNA replication and recombination [21, 22]. *In vivo*, PKA-mediated phosphorylation of Abf2 during glucose repression may regulate its functions on maintaining mitochondrial DNA content during the shift from gluconeogenic to fermentative growth [21].

Hmo1 is not considered a specific transcriptional factor either, although it regulates rDNA transcription from RNA polymerase I promoters and also regulates start site selection of ribosomal protein genes by RNA polymerase II [23–25].

Nhp10 (alias Hmo2) is a nonessential subunit of the INO80 chromatin remodeling complex, and it affects telomere maintenance via recombination [26, 27].

Nhp6a and Nhp6b are also paralogs and functionally redundant [28], they bind to and remodel nucleosomes [29, 30], and both are required for transcriptional initiation fidelity of some tRNA genes [31]. Their protein levels increase in response to DNA replication stress [32]. Besides, Nhp6a and Nhp6b acting on chromatin tightly repress histone expression; paradoxically, histone gene overexpression in the double *nhp6a∆ nhp6b∆* mutant is compensated by downregulation of translation, finally determining a histone-decreased phenotype to avoid the toxic effect of histone overproduction [33].

Although few data are available about Ssp41 functions, it has been associated with chromatin remodeling [34], transcription, and RNA processing [35, 36]. Besides, overexpression causes chromosomal instability [36] and under hypoxia, it is rapidly exported to the cytosol [34].

An intriguing question is whether the *S. cerevisiae* HMGB proteins contribute altogether to regulate specific cell functions. An interesting perspective comes from the terms "environmental stress response" (ESR) or "common environmental response" (CER). These terms refer to adaptive yeast responses against acute changes in diverse environmental parameters (e.g., O2 , osmolarity, nutrients, pH, UV, etc.), which evoke a common transcriptional response, initially devoted to mitigate the deleterious effect of the specific stressor, but principally to balance cell energetics and to coordinate progression through the cell cycle [37]. We have summarized the information available in SGD about protein interactants of HMGB proteins from *S. cerevisiae* (http://www.yeastgenome.org/ as accessed date February 22, 2017) and used this information to construct a interactome network using STRING facilities (http://string-db. org/). **Figure 2** shows that this network statistically has significantly more interactions among HMG-box proteins and their previously reported partners than randomly expected, with a *p*-value < 0.01 according to STRING analysis. This result suggests that yeast HMGB proteins are related, not only structurally but also as a functional group. **Table 2** summarizes GOTerm enrichment analysis among the components of this network and their statistical significances evaluated by false discovery rate (FDR) according to STRING analysis [120].

HMGB Proteins from Yeast to Human. Gene Regulation, DNA Repair and Beyond http://dx.doi.org/10.5772/intechopen.70126 143

**Figure 2.** Network of yeast HMGB interactants according to STRING analysis.

HMGB proteins from *S. cerevisiae* might be considered as chromatin architectural proteins, but with wide influence on gene expression [16]. This is not a HMGB-exclusive mechanism since, in eukaryotes, many other chromatin components, such as histones [17], histone chaperones and modifiers [18], chromatin remodel complexes [19], and long noncoding RNAs

Although Abf2 and Ixr1 are considered paralogs, resulting from the whole genome duplication in an ancestor of *Saccharomyces*, the function of Abf2 is not related to transcriptional regulation of hypoxic regulons. Abf2 is a mitochondrial DNA-binding protein involved in mitochondrial DNA replication and recombination [21, 22]. *In vivo*, PKA-mediated phosphorylation of Abf2 during glucose repression may regulate its functions on maintaining mitochondrial DNA content during the shift from gluconeogenic to fermentative growth [21].

Hmo1 is not considered a specific transcriptional factor either, although it regulates rDNA transcription from RNA polymerase I promoters and also regulates start site selection of ribo-

Nhp10 (alias Hmo2) is a nonessential subunit of the INO80 chromatin remodeling complex,

Nhp6a and Nhp6b are also paralogs and functionally redundant [28], they bind to and remodel nucleosomes [29, 30], and both are required for transcriptional initiation fidelity of some tRNA genes [31]. Their protein levels increase in response to DNA replication stress [32]. Besides, Nhp6a and Nhp6b acting on chromatin tightly repress histone expression; paradoxically, histone gene overexpression in the double *nhp6a∆ nhp6b∆* mutant is compensated by downregulation of translation, finally determining a histone-decreased phenotype to avoid

Although few data are available about Ssp41 functions, it has been associated with chromatin remodeling [34], transcription, and RNA processing [35, 36]. Besides, overexpression causes chromosomal instability [36] and under hypoxia, it is rapidly exported to the cytosol [34].

An intriguing question is whether the *S. cerevisiae* HMGB proteins contribute altogether to regulate specific cell functions. An interesting perspective comes from the terms "environmental stress response" (ESR) or "common environmental response" (CER). These terms refer to adaptive yeast responses against acute changes in diverse environmental parameters

initially devoted to mitigate the deleterious effect of the specific stressor, but principally to balance cell energetics and to coordinate progression through the cell cycle [37]. We have summarized the information available in SGD about protein interactants of HMGB proteins from *S. cerevisiae* (http://www.yeastgenome.org/ as accessed date February 22, 2017) and used this information to construct a interactome network using STRING facilities (http://string-db. org/). **Figure 2** shows that this network statistically has significantly more interactions among HMG-box proteins and their previously reported partners than randomly expected, with a *p*-value < 0.01 according to STRING analysis. This result suggests that yeast HMGB proteins are related, not only structurally but also as a functional group. **Table 2** summarizes GOTerm enrichment analysis among the components of this network and their statistical significances

evaluated by false discovery rate (FDR) according to STRING analysis [120].

, osmolarity, nutrients, pH, UV, etc.), which evoke a common transcriptional response,

[20], affect gene expression by different mechanisms.

142 Old Yeasts - New Questions

somal protein genes by RNA polymerase II [23–25].

the toxic effect of histone overproduction [33].

(e.g., O2

and it affects telomere maintenance via recombination [26, 27].

References to the existence of interplay between the response to hypoxia, oxidative stress, and mitochondrial function have been reported, i.e., it is known that when cells experience hypoxia, up- or downregulation of an important number of oxygen-regulated genes in yeast depends on an active mitochondrial respiratory chain [38]. Treatment with antimycin A (respiration inhibitor) or oxygen deprivation cause downregulation of networks involved in the G1/S transition of the cell cycle as well as of those involved in energetically costly programs of ribosomal biogenesis and protein synthesis [37]. Similar regulation occurs in the response to DNA stress [39–41], and therefore, a wide gene-regulatory response might engage the functions of the HMGB proteins coordinately. **Figure 3** summarizes the participation of HMGB proteins from *S. cerevisiae* in functional responses against external (nutrient availability, oxidants, oxygen levels, DNA damaging agents) or internal (replicative stress) stressors.

**Table 2.** GOTerm enrichment in the interactome network depicted in **Figure 2**.

**Pathway ID Biological function; pathway description**

144 Old Yeasts - New Questions

GO.0051171 Regulation of nitrogen compound metabolic process

DNA-templated

GO.0006355 Regulation of transcription,

GO.0051252 Regulation of RNA metabolic process

GO.0071824 Protein-DNA complex subunit organization

GO.0043933 Macromolecular complex subunit organization

GO.0006974 Cellular response to DNA damage stimulus

GO.0006357 Regulation of transcription from

GO.0006366 Transcription from RNA

GO.0043044 ATP-dependent chromatin remodeling

RNA polymerase II promoter

polymerase II promoter

GO.0006325 Chromatin organization 50 4.50E-27 GO.0010468 Regulation of gene expression 81 5.85E-25

GO.0051276 Chromosome organization 63 5.85E-25

GO.0090304 Nucleic acid metabolic process 95 1.53E-21 GO.0034728 Nucleosome organization 27 3.55E-21 GO.0006338 Chromatin remodeling 26 3.00E-20 GO.0006351 Transcription, DNA-templated 61 3.69E-19

GO.0006333 Chromatin assembly or disassembly 24 7.13E-19 GO.0006281 DNA repair 39 2.21E-18 GO.0016568 Chromatin modification 36 3.02E-18 GO.0006259 DNA metabolic process 47 1.11E-17 GO.0010467 Gene expression 82 1.39E-15 GO.0016070 RNA metabolic process 78 1.73E-15

GO.0006323 DNA packaging 16 1.73E-11

GO.0006950 Response to stress 54 6.10E-10 GO.0016458 Gene silencing 22 1.12E-09

**Observed gene count**

84 5.85E-25

71 3.30E-24

72 3.30E-24

39 1.27E-22

78 1.24E-21

43 5.34E-19

45 8.94E-14

29 1.99E-11

14 5.72E-11

**False discovery rate**

**Figure 3.** Orchestrated action of *S. cerevisiae* HMGB proteins in cellular responses to stress.

#### **3. HMGB proteins from other yeasts**

Although the complete sequences of a huge number of genomes from yeast and fungi are available, functional studies of HMGB proteins are not very frequent and only a few HMGB homologs have been characterized so far.

In *Yarrowia lipolytica*, YlMhb1, the homologous of Abf2 from *S. cerevisiae*, compacts mitochondrial DNA *in vitro*. Phenotypic analysis of a *mhb1∆* strain reveals a large decrease in the mitochondrial DNA copy number and also shows that the protein protects the mitochondrial genome against mutagenic events. Like Abf2, YlMhb1 has two HMG-box domains [42]. In *Candida parapsilosis*, the homologous of Abf2 has been named Gcf1 and diverse experimental data support its role in the maintenance of the *C. parapsilosis* mitochondrial genome; in contrast to Abf2 and YlMhb1, Gcf1contains a coiled-coil domain and a single high-mobility HMG-box domain [43]. A similar structure is observed in *Candida albicans* [44].

In *C. albicans*, proteins with DNA-binding activity and high similarity to Nhp6 promote changes in chromatin structure, which are involved in hypha-specific gene regulation [45].

Regarding the Rox1 homolog in *Kluyveromyces lactis*, its molecular function, synteny, and HMG-box structural features were shown to be different from that of *S. cerevisiae* [46, 47]. The *KlROX1* gene from *K. lactis* does not regulate the hypoxic response in this yeast neither interacts with the components of the general corepressor factor (Tup1-Ssn6) that mediates the transcriptional repression exerted by Rox1 in *S. cerevisiae*. However, KlRox1 mediates the response to metals [47].

Although a low number of functional data is available, we may speculate that in yeasts the functions of "architectural" HMGB proteins are probably more conserved than those with functions as specific transcriptional factors. This is also predictable considering that transcriptional factors are among the proteins more strongly diverged between yeasts [48].

#### **4. HMGB proteins in multicellular organisms**

**Figure 3.** Orchestrated action of *S. cerevisiae* HMGB proteins in cellular responses to stress.

Although the complete sequences of a huge number of genomes from yeast and fungi are available, functional studies of HMGB proteins are not very frequent and only a few HMGB

In *Yarrowia lipolytica*, YlMhb1, the homologous of Abf2 from *S. cerevisiae*, compacts mitochondrial DNA *in vitro*. Phenotypic analysis of a *mhb1∆* strain reveals a large decrease in the mitochondrial DNA copy number and also shows that the protein protects the mitochondrial genome against mutagenic events. Like Abf2, YlMhb1 has two HMG-box domains [42]. In *Candida parapsilosis*, the homologous of Abf2 has been named Gcf1 and diverse experimental data support its role in the maintenance of the *C. parapsilosis* mitochondrial genome; in contrast to Abf2 and YlMhb1, Gcf1contains a coiled-coil domain and a single high-mobility HMG-box domain [43]. A similar structure is observed in *Candida*

**3. HMGB proteins from other yeasts**

homologs have been characterized so far.

*albicans* [44].

146 Old Yeasts - New Questions

In multicellular eukaryotes, a large number of proteins contain HMG boxes, most of which are transcription factors that contain a single HMG-box [49], although some may have up to 6 HMG-box domains, like Ubf1 [50]. According to the classification from Bustin [7], "canonical" chromatin HMGB proteins represent a subgroup that invariably contains two in tandem HMG boxes. A model for the phylogenesis of HMGB genes in metazoan suggests that these two HMG boxes have their origin in the duplication of an ancient single HMG-box; even those which are part of HMG-box transcription factors might evolve from this ancestral ProtoBox [51].

Transcription factors (including SOX factors) are the most divergent group of HMG-box proteins in humans, whereas in plants the chromosomal HMGB-type proteins are most variable [52]. In plants, HMG-box proteins classify into four groups: HMGB-type proteins, structure-specific recognition protein 1 (SSRP1), proteins containing 3 HMG-box domains (3xHMG-box), and proteins that contain both an AT-rich interaction domain (ARID) and an HMG-box domain (ARID/HMG). These latter two groups are apparently specific for plants [52]. Conversely, HMG-box containing transcription factors such as Sry, a sex-determining factor that is necessary for testes development [53], Lef-1, which regulates gene expression during cell differentiation [54], and the SOX family are presumably not present in plants [52].

**Table 3** resumes the homologies found between *S. cerevisiae* and human HMGB groups using the YeastMine facility "Yeast gene-human homolog(s)-Disease" (http://yeastmine.yeastgenome.org/yeastmine/begin.do accessed on date February 25, 2017) and completed with functional data from SGD (http://www.yeastgenome.org/) and associated human diseases. **Figure 4** summarizes the structural and phylogenetic relationships between several HMGB proteins from *S. cerevisiae* and their human homologs.

**Table 3.** Human homologs to HMGB yeast proteins and associated diseases.

**Figure 4.** Molecular phylogenetic analysis of HMG-box domains by maximum likelihood method. (a) Characteristic HMG-box conservation. The evolutionary history was inferred by using the maximum likelihood method based on the JTT matrix-based model [118]. (b) The tree with the highest log likelihood (−3421.5683) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 39 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 63 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 [119].

#### **5. Mechanisms of transcriptional regulation mediated by HMGB proteins**

#### **5.1. Direct binding to target promoters**

**Yeast** *H. sapiens* **Associated human diseases**

Sox12 Sox13 Sox14 Sox15

Sox21

Sox30 Sox4 Sox5 Sox6 Sox7 Sox8

Hmg20b

Sp140 Tfam Ubtf Ubtfl1

Hmg20b

Sp140 Tfam Ubtf Ubtfl1

Hmg20b Hmgb1

Sp140 Tfam Ubtf Ubtfl1

**Table 3.** Human homologs to HMGB yeast proteins and associated diseases.

Sox10 Peripheral demyelinating neuropathy, central dysmyelination,

Sox18 Hypotrichosis-lymphedema-telangiectasia-renal defect syndrome

Sox11 Mental retardation, autosomal dominant 27

Sox17 Vesicoureteral reflux

Sox2 Microphthalmia, syndromic 3

Sox3 Mental retardation, X-linked

Sox9 Campomelic dysplasia Sry 46,Xx sex reversal 1

Smarce1 Susceptibility to familial meningioma Sp110 Susceptibility to *Mycobacterium tuberculosis*

Smarce1 Susceptibility to familial meningioma Sp110 Susceptibility to *Mycobacterium tuberculosis*

Hmgb3 Microphthalmia, syndromic 13 Smarce1 Susceptibility to familial meningioma Sp110 Susceptibility to *Mycobacterium tuberculosis*

Waardenburg syndrome, and Hirschsprung disease

Rox1 Sox1

148 Old Yeasts - New Questions

Ixr1 Hmg20a

Abf2 Tfam Hmo1 Hmg20a

Nhp6a/b Hmg20a

In *S. cerevisiae*, Rox1 is a DNA-binding protein with an HMG-box domain that binds to the consensus sequence YYYATTGTTCTC present in the promoter regions of genes related to hypoxia, causing a DNA bending of 90° in the double strand [55, 56]. Up to one-third of the *S. cerevisiae* hypoxic genes are transcriptionally repressed during aerobic growth by Rox1, through the recruitment of the general corepressor complex Ssn6/Tup1 [14, 57]. In several promoters, this repression is synergic with the caused by regulator Mot3 [58]. *ROX1* expression is dependent of oxygen and heme levels in the cell, since its transcription is under the control of Hap1 [59], and therefore, it is induced aerobically [60]. In addition to aerobic upregulation produced by Hap1, the *ROX1* expression is counterbalanced by self-repression, to avoid cell toxic effects produced by an eventual overexpression. At low oxygen levels, the Rox1 protein levels rapidly decay by degradation, since it is labile in these conditions, and because the *ROX1* gene is no longer transcribed. Under normoxic (aerobic) conditions, the heme-activated Hap1 complex increases *ROX1* expression, allowing in turn Rox1 repression of hypoxic genes. In hypoxia, the situation is reversed, since the low levels of Rox1 allow derepression. The genes that are under the control of Rox1, either directly by the protein binding to their promoter regions, or indirectly through signal transduction pathways, are those related to efficient metabolism under low oxygen levels, ergosterol and heme synthesis, cell wall maintenance, or electron chain transport [61]. The genes repressed simultaneously by Mot3 and Rox1 preferentially encode proteins of the cell wall and plasma membrane; cell conjugationrelated genes are negatively regulated by both factors and by osmotic stress [62]. During anaerobiosis, the histone deacetylase and global repressor complex Rpd3 act at the promoter of the anaerobic gene *DAN1* to antagonize the chromatin-mediated repression caused by Mot3 and Rox1 and chromatin remodeling by Swi/Snf is necessary for expression [63].

The first report about the participation of Ixr1 in the yeast hypoxic response was the aerobic repression of the *COX5B* gene, which encodes the hypoxic isoform of the subunit Vb of the mitochondrial complex cytochrome c oxidase [64]. Ixr1 also regulates other hypoxic genes like *TIR1*, a cell wall mannoprotein of the serine-alanine-rich protein family [65] and *HEM13*, which encodes the enzyme coproporphyrinogen III oxidase in the heme biosynthetic pathway [66]. The whole set of genes that are regulated by Ixr1 during the hypoxic response was determined in a genome-wide approach [67]. Hypoxic genes are also regulated by oxidative stress. Indeed, reactive oxygen species (ROS) induce expression of *CYC7* and *COX5B* through an Ixr1-independent mechanism that diminishes the access of Rox1 to its promoter targets [68].

A cross-regulation between Rox1 and Ixr1 in the yeast hypoxic response has been reported [66]. In aerobiosis, low levels of *IXR1* expression are maintained by Rox1 repression and during hypoxia Ixr1 auto-enhances *IXR1* expression [66]. Ixr1 is also required for hypoxic repression of *ROX1*. Binding to specific regions of the *ROX1, IXR1, HEM13*, and *TIR1* promoters were probed *in vitro* and *in vivo* [66, 69]. Ixr1 is also known by binding to cisplatin-DNA adducts with high affinity [70]. We have recently evidenced that functional specialization of the 2 HMG boxes, which are present in Ixr1, may explain its dual function. Regulation of transcription and DNA repair is achieved through differential recognition of specific regulatory sequences in the target promoters, or DNA disturbances caused by cisplatin treatment [15].

Rox1 from *S. cerevisiae* is homologous to the SOX family of transcriptional factors from human (**Table 2**) and other metazoan, from which SRY was the founding member. In vertebrates, there are more than 20 SOX genes characterized, which originate through a process of duplication and divergence [71], and they play important roles in tissue homeostasis, organogenesis, and cell fate decision during developmental processes (thoroughly reviewed by Ref. [72]). For most mammals, SRY is the only member of the SOXA group [73]. SOXB1 group (SOX1, SOX2, and SOX3) participates in neural, lens, and ear development; SOXB2 group (SOX14 and SOX21) in neuronal differentiation SOXC group (SOX4, SOX11, and SOX12) in nervous system development and retinal differentiation; and SOXD group (SOX5, SOX6, and SOX13) in chondrocyte differentiation, cartilage formation, and neural development. SOXE group (SOX8, SOX9, and SOX10) is involved in primary sex determination and neural development, and SOXF group (SOX7, SOX17, and SOX18) in cardiac, vascular, and lymphatic development [72]. The SOXG group has only one member in mammals, and SOX15 involved in skeletal muscle regeneration [72, 74]. Besides, SOX4 and SOX11 are involved in tumorigenesis, and SOX7, SOX17, and SOX18 in endoderm development [72]. **Figure 5** summarizes the functions of these human SOX factors.

**Figure 5.** Functional groups of human SOX factors.

through the recruitment of the general corepressor complex Ssn6/Tup1 [14, 57]. In several promoters, this repression is synergic with the caused by regulator Mot3 [58]. *ROX1* expression is dependent of oxygen and heme levels in the cell, since its transcription is under the control of Hap1 [59], and therefore, it is induced aerobically [60]. In addition to aerobic upregulation produced by Hap1, the *ROX1* expression is counterbalanced by self-repression, to avoid cell toxic effects produced by an eventual overexpression. At low oxygen levels, the Rox1 protein levels rapidly decay by degradation, since it is labile in these conditions, and because the *ROX1* gene is no longer transcribed. Under normoxic (aerobic) conditions, the heme-activated Hap1 complex increases *ROX1* expression, allowing in turn Rox1 repression of hypoxic genes. In hypoxia, the situation is reversed, since the low levels of Rox1 allow derepression. The genes that are under the control of Rox1, either directly by the protein binding to their promoter regions, or indirectly through signal transduction pathways, are those related to efficient metabolism under low oxygen levels, ergosterol and heme synthesis, cell wall maintenance, or electron chain transport [61]. The genes repressed simultaneously by Mot3 and Rox1 preferentially encode proteins of the cell wall and plasma membrane; cell conjugationrelated genes are negatively regulated by both factors and by osmotic stress [62]. During anaerobiosis, the histone deacetylase and global repressor complex Rpd3 act at the promoter of the anaerobic gene *DAN1* to antagonize the chromatin-mediated repression caused by Mot3 and Rox1 and chromatin remodeling by Swi/Snf is necessary for expression [63].

150 Old Yeasts - New Questions

The first report about the participation of Ixr1 in the yeast hypoxic response was the aerobic repression of the *COX5B* gene, which encodes the hypoxic isoform of the subunit Vb of the mitochondrial complex cytochrome c oxidase [64]. Ixr1 also regulates other hypoxic genes like *TIR1*, a cell wall mannoprotein of the serine-alanine-rich protein family [65] and *HEM13*, which encodes the enzyme coproporphyrinogen III oxidase in the heme biosynthetic pathway [66]. The whole set of genes that are regulated by Ixr1 during the hypoxic response was determined in a genome-wide approach [67]. Hypoxic genes are also regulated by oxidative stress. Indeed, reactive oxygen species (ROS) induce expression of *CYC7* and *COX5B* through an Ixr1-independent mechanism that diminishes the access of Rox1 to its promoter targets [68]. A cross-regulation between Rox1 and Ixr1 in the yeast hypoxic response has been reported [66]. In aerobiosis, low levels of *IXR1* expression are maintained by Rox1 repression and during hypoxia Ixr1 auto-enhances *IXR1* expression [66]. Ixr1 is also required for hypoxic repression of *ROX1*. Binding to specific regions of the *ROX1, IXR1, HEM13*, and *TIR1* promoters were probed *in vitro* and *in vivo* [66, 69]. Ixr1 is also known by binding to cisplatin-DNA adducts with high affinity [70]. We have recently evidenced that functional specialization of the 2 HMG boxes, which are present in Ixr1, may explain its dual function. Regulation of transcription and DNA repair is achieved through differential recognition of specific regulatory sequences in the target promoters, or DNA disturbances caused by cisplatin treatment [15].

Rox1 from *S. cerevisiae* is homologous to the SOX family of transcriptional factors from human (**Table 2**) and other metazoan, from which SRY was the founding member. In vertebrates, there are more than 20 SOX genes characterized, which originate through a process of duplication and divergence [71], and they play important roles in tissue homeostasis, organogenesis, and cell fate decision during developmental processes (thoroughly reviewed by Ref. [72]). SOX proteins are highly dynamic regulators of cell functions due to their nucleocytoplasmic shuttling properties [75]. However, because of their low affinity for DNA binding, and despite SOX proteins usually have their own C-terminal activation/repression domain, they are committed to recruit partner proteins to fulfill their transcriptional regulatory task [76]. Homo- and heterodimerization of SOX proteins is also a mechanism used for the formation of these regulatory complexes [77]. SOX proteins also interact with signaling effectors, Wnt/β-catenin being one of the most studied signaling pathways [78]. Different molecular complexes of SOX factors and their partner proteins are formed along developmental processes. Besides, these specific interactions are usually dependent on posttranslational modifications of SOX proteins like phosphorylation, acetylation, SUMOylation, and ubiquitination [72].

#### **5.2. Other mechanisms for transcriptional regulation**

The HMGB proteins that are not classified as transcriptional factors also influence transcription by different mechanisms, which affect chromatin. Since these HMGB proteins are very dynamic in their interactions and have no DNA sequence specificity, they usually help transcription factors or cofactors to bind to their cognate sites by bending the DNA molecule, but are rarely retained within the formed complexes [79].

In plants, HMGB proteins contribute to transcriptional regulation by functional interaction with certain transcription factors like Dof2 [80]. In mammals, Hmgb1 alters the structure and stability of the canonical nucleosome in a nonenzymatic, ATP-independent way to facilitate strong binding of estrogen receptor to their regulatory elements [81].

HMGB proteins also interact with nucleosomes to promote their sliding or other chromatin remodeling processes [79]. Yeast Nhp6a, Nhp6b, and Hmo1 proteins stimulate the sliding activity of the yeast remodeler complex SWI/SNF, while octamer transfer and transient exposure of nucleosomal DNA catalyzed by this complex are only stimulated by Hmo1. Hmo1 also favors the sliding activity of the ISW1a complex [82].

Hmo1 in yeasts and the upstream binding factor (Ubf) in mammals function as cofactors in RNA polymerase I transcription and therefore are essential for transcription of the rRNA genes *in vivo*, but also have more generalized roles in chromatin structure. Binding of Ubf to human rRNA genes is accompanied by a reduction in core histone binding at the same sequences [83, 84], and a similar mechanism has been described for its ortholog Hmo1 in yeast [25]. Similarly, mammalian cells lacking Hmgb1 and yeast *nhp6* mutants contain a reduced amount of core, linker, and variant histones [85]. Consequently, the reduced number of nucleosomes produces a global increment of transcription and affects the relative expression of about 10% of genes [85].

Finally, HMGB proteins have been involved in the selection of modified histone variants. Studies carried out in mouse showed that conditional inactivation of Ubf is also accompanied by recruitment of H3K9me3, which reveals its function in the epigenetic control of gene expression [86].

#### **6. Mechanisms of DNA repair mediated by HMGB proteins**

SOX proteins are highly dynamic regulators of cell functions due to their nucleocytoplasmic shuttling properties [75]. However, because of their low affinity for DNA binding, and despite SOX proteins usually have their own C-terminal activation/repression domain, they are committed to recruit partner proteins to fulfill their transcriptional regulatory task [76]. Homo- and heterodimerization of SOX proteins is also a mechanism used for the formation of these regulatory complexes [77]. SOX proteins also interact with signaling effectors, Wnt/β-catenin being one of the most studied signaling pathways [78]. Different molecular complexes of SOX factors and their partner proteins are formed along developmental processes. Besides, these specific interactions are usually dependent on posttranslational modifications of SOX proteins like phosphorylation, acetylation, SUMOylation,

The HMGB proteins that are not classified as transcriptional factors also influence transcription by different mechanisms, which affect chromatin. Since these HMGB proteins are very dynamic in their interactions and have no DNA sequence specificity, they usually help transcription factors or cofactors to bind to their cognate sites by bending the DNA molecule, but

In plants, HMGB proteins contribute to transcriptional regulation by functional interaction with certain transcription factors like Dof2 [80]. In mammals, Hmgb1 alters the structure and stability of the canonical nucleosome in a nonenzymatic, ATP-independent way to facilitate

HMGB proteins also interact with nucleosomes to promote their sliding or other chromatin remodeling processes [79]. Yeast Nhp6a, Nhp6b, and Hmo1 proteins stimulate the sliding activity of the yeast remodeler complex SWI/SNF, while octamer transfer and transient exposure of nucleosomal DNA catalyzed by this complex are only stimulated by Hmo1. Hmo1

Hmo1 in yeasts and the upstream binding factor (Ubf) in mammals function as cofactors in RNA polymerase I transcription and therefore are essential for transcription of the rRNA genes *in vivo*, but also have more generalized roles in chromatin structure. Binding of Ubf to human rRNA genes is accompanied by a reduction in core histone binding at the same sequences [83, 84], and a similar mechanism has been described for its ortholog Hmo1 in yeast [25]. Similarly, mammalian cells lacking Hmgb1 and yeast *nhp6* mutants contain a reduced amount of core, linker, and variant histones [85]. Consequently, the reduced number of nucleosomes produces a global increment of transcription and affects the relative expres-

Finally, HMGB proteins have been involved in the selection of modified histone variants. Studies carried out in mouse showed that conditional inactivation of Ubf is also accompanied by recruitment of H3K9me3, which reveals its function in the epigenetic control of gene

and ubiquitination [72].

152 Old Yeasts - New Questions

**5.2. Other mechanisms for transcriptional regulation**

are rarely retained within the formed complexes [79].

also favors the sliding activity of the ISW1a complex [82].

sion of about 10% of genes [85].

expression [86].

strong binding of estrogen receptor to their regulatory elements [81].

The three HMG families (A, B, N) are involved in the four major DNA repair pathways. HMGB proteins contribute to nucleotide excision repair (NER), base excision repair (BER), doublestrand break repair (DSBR), and mismatch repair (MMR), but with specific particularities (reviewed in Ref. [87]). The first report about participation of HMGB proteins in DNA repair was the identification of Hmgb1 binding to the major DNA lesions formed in cells treated with cisplatin, which are repaired by the NER pathway [88]. In general, the effects of HMGB proteins on DNA repair are achieved by different mechanisms. First, they contribute to modulate chromatin compaction and nucleosome occupancy; through interactions with chromatin-modifying enzymes and energy-dependent remodeling complexes, HMGB proteins favor or avoid the access of the repair machinery to altered DNA. Second, HMGB proteins can also regulate repair by direct modulation of the enzymatic activities and/or mechanistic steps implied in the diverse repair pathways. Third, acting as transcriptional regulators, HMGB proteins may change the expression levels of genes involved in DNA repair processes.

Hmgb1 and many other HMGB proteins (e.g., Ubf, Lef-1, Sry, and human mtTFA) inhibit NER [87]. If Hmgb1 binds first to a cisplatin adduct, the replication protein A (hRPA), necessary for NER repair, cannot displace it, thus potentially inhibiting repair [89]. On the contrary, Hmgb1 stimulates *in vitro* NER of triplex DNA interstrand crosslinks, caused by psoralen, by facilitating the interaction with components of this pathway [83, 90].

Hmgb1 coimmunoprecipitates with proteins from the BER pathway, including Ape1, Fen-1, and Pol-beta, and *in vitro*, modulates the deoxyribose phosphate lyase activity of Pol-beta [91].

Also *in vitro*, purified Hmgb1 binds to the ends of the double-strand breaks, similarly to the Ku proteins, and stimulates kinase and ligase activities required for DBSR of these lesions [92, 93]. Oppositely, in yeast, the HMGB protein Hmo1 must be evicted, along with core histones, for efficient DSBR [94].

Hmgb1 and Hmgb2 form part of a pentameric "damage-sensing" complex (also including heat shock protein 70, protein disulfide-isomerase Erp60, and glyceraldehyde3-phosphate dehydrogenase) specifically recruited to nonnatural nucleosides *in vivo* as part of the MMR pathway [95]. *In vitro*, Hmgb1 also interacts with the MMR proteins Msh2 and Mlh1 and cooperates with the replication protein A to mediate the exonuclease I activity that creates a gap, which is filled in by DNA polymerase, and finally, the broken strands are sealed by DNA ligase [96]. In yeast, following Nhp6a interaction to DNA, the mismatch repair complex Msh2-Msh6 is excluded from binding, unless a mismatch is present. *In vitro* the complex Msh2-Msh6-Nhp6a is stable and responsive to ATP on mismatched substrates [97].

Other important connection between Hmgb1 and DNA repair comes from the observation that this protein interacts with p53 *in vitro* and *in vivo*, stimulating p53 binding to sequencespecific recognition sites as well as to cisplatin-modified DNA [98, 99]. p53 directly impacts the activity of various DNA-repair systems, and besides, it halts cell cycle, thus allowing the repair machineries to restore genome stability [100].

#### **7. HMGB proteins at the forefront of cutting-edge research**

Recent publications on HMGB proteins reveal that these proteins are becoming a focus of interest due to their participation in cellular processes of great importance for humankind like epigenetic control of gene expression, aging, disease, or regenerative cellular therapies.

An interesting research field concerning HMGB proteins is their function replacing histones under specific conditions. In eukaryotic chromatin, histone H1 associates with the linker DNA in the nucleosome core particle to stabilize the higher-order chromatin structure and to modulate the ability of specific regulatory factors to access their final targets. It has been demonstrated that in *S. cerevisiae* Hmo1 might replace histone H1 and protect linker DNA from nuclease digestion, creating a less dynamic chromatin environment that depends on its lysine-rich domain. This lysine-rich extension is unusual in other HMGB proteins, which have an acidic domain instead [101, 102].

Environmental changes, sensed through signaling cascades, regulate chromatin organization, thus contributing to gene expression and, ultimately, cell adaptation to external stimulus. These responses are related to cell fate and aging. In yeast, the nutrient-dependent target-ofrapamycin complex 1 (TORC1) pathway and histone H3 collaborate to retain HMGB proteins within the nucleus, and in this way, they increase longevity [103].

The role of HMGB proteins remodeling chromatin on a genome-wide scale relates to the onset of several human diseases. Two chromatin structural proteins, CCCTC-binding factor (Ctcf) and high mobility group protein B2 (Hmgb2), regulate pathologic transcription in myocytes during heart disease [104]. The response of macrophages to inflammation starts by nucleosome loss and cell lacking Hmgb1 contains 20% less nucleosomes and has a specific transcription pattern. In a mouse model, unstimulated Hmgb1-/- macrophages activate transcriptional pathways associated with cell migration and chemotaxis. Wild-type macrophages, under lipopolysaccharide (LPS)/interferon (IFN)-γ exposure, rapidly secrete Hmgb1 and reduce their histone content [105].

Hmgb1 is overexpressed in many types of cancer, including those of etiology based on oxidative damage [8], and frequently, Hmgb1 expression increases with tumor stage and metastasis. In the pediatric acute lymphoblastic leukemia, autophagy is regarded as a mechanism that underlies chemoresistance. Since autophagy depends on the Hmgb1 translocation from nucleus to cytoplasm, this protein is a good target of study in order to overcome the problem [106]. It has been found that Hmgb1 expression is inversely correlated with semaphorin 3A expression, a suppressor of angiogenesis and cell migration. The epigenetic mechanism causing semaphorin 3A repression by Hmgb1 implies that it promotes heterochromatin formation and decreased occupancy of acetylated histones at the semaphorin 3A locus [107].

Other remarkable function of HMGB proteins, yet not fully understood, is their participation in telomere maintenance, studied in yeast [108] plants [109] and notoriously in animals [110], because of their implications in cancer development. The telomerase that conserves telomere structures is formed by a catalytic protein subunit (telomerase reverse transcriptase (TERT)) and an RNA subunit (telomerase RNA, TR), and both physically interact with Hmgb1 *in vitro*. Knockout of the HMGB1 gene in mouse embryonic fibroblasts (MEFs) causes chromosomal abnormalities, enhanced localization of γ-H2AX at telomeres, moderate shortening of telomere lengths, and lower telomerase activity compared to the wild-type cells. Oppositely, knockout of the HMGB2 gene elevates telomerase activity, which reveals the intricate interplay of these proteins in chromosome stability and cancer [110].

Evidences linking HMGB proteins with stem cell biology and cellular reprograming are also found. Sox factors participate in embryonic pluripotent cell differentiation; Oct4 interacts with Sox2 to maintain pluripotency or with Sox17 to promote endoderm commitment [111]. Expression of Hmgb2 changes notably at different time points during embryogenesis [112] and controls the differentiation of neural stem cells into neurons, astrocytes, and oligodendrocytes. Besides, several Sox factors [113, 114] and also chromatin HMGB proteins [115] are involved in back-reprograming differentiated cells into stem cells. Hmgb1 was also proposed as an efficient stem cell recruiter with tissue-regenerating roles; it was able to induce stem cell transmigration through an endothelial barrier or to capture in muscle the stem cells injected into the general circulation [116]. In murine and human mammary cancer stem cells, Hmgb1 promotes self-renewal of these cells [117], which are responsible for tumor progression, metastases, resistance to therapy, and tumor recurrence. Therefore, HMGB proteins are clues in the search of more effective cancer therapies and cellular regenerative treatments.

#### **Acknowledgements**

**7. HMGB proteins at the forefront of cutting-edge research**

within the nucleus, and in this way, they increase longevity [103].

an acidic domain instead [101, 102].

154 Old Yeasts - New Questions

their histone content [105].

Recent publications on HMGB proteins reveal that these proteins are becoming a focus of interest due to their participation in cellular processes of great importance for humankind like epigenetic control of gene expression, aging, disease, or regenerative cellular therapies.

An interesting research field concerning HMGB proteins is their function replacing histones under specific conditions. In eukaryotic chromatin, histone H1 associates with the linker DNA in the nucleosome core particle to stabilize the higher-order chromatin structure and to modulate the ability of specific regulatory factors to access their final targets. It has been demonstrated that in *S. cerevisiae* Hmo1 might replace histone H1 and protect linker DNA from nuclease digestion, creating a less dynamic chromatin environment that depends on its lysine-rich domain. This lysine-rich extension is unusual in other HMGB proteins, which have

Environmental changes, sensed through signaling cascades, regulate chromatin organization, thus contributing to gene expression and, ultimately, cell adaptation to external stimulus. These responses are related to cell fate and aging. In yeast, the nutrient-dependent target-ofrapamycin complex 1 (TORC1) pathway and histone H3 collaborate to retain HMGB proteins

The role of HMGB proteins remodeling chromatin on a genome-wide scale relates to the onset of several human diseases. Two chromatin structural proteins, CCCTC-binding factor (Ctcf) and high mobility group protein B2 (Hmgb2), regulate pathologic transcription in myocytes during heart disease [104]. The response of macrophages to inflammation starts by nucleosome loss and cell lacking Hmgb1 contains 20% less nucleosomes and has a specific transcription pattern. In a mouse model, unstimulated Hmgb1-/- macrophages activate transcriptional pathways associated with cell migration and chemotaxis. Wild-type macrophages, under lipopolysaccharide (LPS)/interferon (IFN)-γ exposure, rapidly secrete Hmgb1 and reduce

Hmgb1 is overexpressed in many types of cancer, including those of etiology based on oxidative damage [8], and frequently, Hmgb1 expression increases with tumor stage and metastasis. In the pediatric acute lymphoblastic leukemia, autophagy is regarded as a mechanism that underlies chemoresistance. Since autophagy depends on the Hmgb1 translocation from nucleus to cytoplasm, this protein is a good target of study in order to overcome the problem [106]. It has been found that Hmgb1 expression is inversely correlated with semaphorin 3A expression, a suppressor of angiogenesis and cell migration. The epigenetic mechanism causing semaphorin 3A repression by Hmgb1 implies that it promotes heterochromatin formation

Other remarkable function of HMGB proteins, yet not fully understood, is their participation in telomere maintenance, studied in yeast [108] plants [109] and notoriously in animals [110], because of their implications in cancer development. The telomerase that conserves telomere structures is formed by a catalytic protein subunit (telomerase reverse transcriptase (TERT))

and decreased occupancy of acetylated histones at the semaphorin 3A locus [107].

Funding is acknowledged both from the Instituto de Salud Carlos III under Grant Agreement no. PI14/01031 cofinanced by FEDER and from Xunta de Galicia (Consolidación D.O.G. X-12- 2016. Contract Number: 2016/012). Aida Barreiro-Alonso was funded by a predoctoral fellowship from Plan I2C Xunta de Galicia-2013 (Spain). Agustín Rico-Diaz was funded by a predoctoral fellowship from Plan I2C Xunta de Galicia-2012 (Spain). We thank STRING facilities and development.

#### **Author details**

Vizoso-Vázquez Ángel, Barreiro-Alonso Aida, Rico-Díaz Agustín, Lamas-Maceiras Mónica, Rodríguez-Belmonte Esther, Becerra Manuel, González-Siso María Isabel and Cerdán María Esperanza\*

\*Address all correspondence to<EMAIL_ADDRESS>
Facultade de Ciencias, Centro de Investigacións Científicas Avanzadas (CICA) e Instituto de Investigacións Biomédicas Coruña (INIBIC), Universidade da Coruña, Grupo EXPRELA, A Coruña, Spain

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