Báo cáo khoa học: The yeast stress response Role of the Yap family of b-ZIP transcription factors The PABMB Lecture delivered on 30 June 2004 at the 29th FEBS Congress in Warsaw

The yeast stress response: Role of the Yap family of b-ZIP transcription factors

Tác giả: Claudina Rodrigues-Pousada, Tracy Nevitt, Regina Menezes

Lĩnh vực: Genomics and Stress Laboratory, Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Oeiras, Portugal

Nội dung tài liệu:
Bài viết này trình bày kiến thức hiện tại về vai trò của họ protein kích hoạt Yap, bao gồm tám yếu tố phiên mã b-ZIP, trong phản ứng với các tác nhân gây stress của nấm men Saccharomyces cerevisiae. Các yếu tố phiên mã này đóng vai trò quan trọng trong việc điều hòa biểu hiện gen, giúp tế bào nấm men thích nghi với nhiều loại áp lực môi trường khác nhau, bao gồm stress oxy hóa, stress kim loại nặng và stress thẩm thấu. Bài báo cũng thảo luận về cơ chế cảm nhận và truyền tín hiệu stress của các protein Yap, cũng như sự tương tác của chúng với các yếu tố khác trong tế bào để điều phối phản ứng của tế bào.

Mục lục chi tiết:

• The yeast stress response
• Role of the Yap family of b-ZIP transcription factors
• The PABMB Lecture delivered on 30 June 2004 at the 29th FEBS Congress in Warsaw
• Keywords
• Correspondence
• The budding yeast Saccharomyces cerevisiae possesses a very flexible and complex programme of gene expression when exposed to a plethora of environmental insults. Therefore, yeast cell homeostasis control is achieved through a highly coordinated mechanism of transcription regulation involving several factors, each performing specific functions. Here, we present our current knowledge of the function of the yeast activator protein family, formed by eight basic-leucine zipper trans-activators, which have been shown to play an important role in stress response.
• The capacity for adaptation to changes in intra- and extracellular conditions is a universal prerequisite for an organism’s survival and evolution. The existence of molecular mechanisms of response, repair and adaptation, many of which are greatly conserved across nature, endows the cell with the plasticity it requires to adjust to its ever-changing environment, a homeostatic event that is termed the stress response. Through the sensing and transduction of the stress signal into the nucleus, a genetic reprogramming occurs that leads, on the one hand, to a decrease in the expression of housekeeping genes and protein synthesis and, on the other hand, to an enhancement of the expression of genes encoding stress proteins. These include molecular chaperones responsible for maintaining protein folding, transcription factors that further modulate gene expression and a diverse network of players including membrane transporters and proteins involved in repair and detoxification pathways, nutrient metabolism, and osmolytes production, to name a few. Survival and growth resumption imply successful cellular adaptation to the new conditions as well as the repair of damage incurred to the cell. Although specific stress conditions elicit distinct cellular responses, underlying gene
• Abbreviations
• The Yap proteins and the yeast response to stress
• Oxidative stress
• The response to oxidative stress can be described as the phenomenon by which the cell responds to alterations in its redox state. As a consequence of aerobic growth, cells are continuously exposed to reactive oxygen species (ROS), potent oxidants capable of extensive cellular damage at the level of DNA, protein and membrane lipid content. As a result, organisms, from bacteria to humans, have developed mechanisms of maintaining cellular thiol redox homeostasis. This is achieved by limiting the accumulation of O2-derived oxidants, controlling iron and copper metabolism, the activation of thiol redox pathways and via damage repair [12].
• Yap1 was initially characterized through the observation that the deletion mutant is hypersensitive to the oxidants H2O2 and t-BOOH, and to chemicals that generate superoxide anions, including menadione, plumbagine and methylviologen as well as to cadmium, methylglyoxal and cycloheximide. Recent genome-wide studies have focused on modulation of the gene expression programmes that occur following exposure to an oxidative insult in S. cerevisiae. Indeed, Gasch et al. [7] and Causton et al. [13] have demonstrated that the response to mild doses of H2O2 leads to the immediate and transient modulation of ~ 24% of the genome. Although approximately half of this response can be attributed to the ESR, there is an H2O2-specific response comprising genes encoding most cellular antioxidants and components of thiol redox pathways, heat shock proteins, drug transporters and enzymes involved in carbohydrate metabolism. Among these genes are TRX2 [14] and GSH1 [15], two of the first Yap1 targets to be characterized and induced under oxidative stress imposed by H2O2, diamide and t-BOOH. Since then, several Yap1 targets involved in ROS detoxification have been identified, including those involved in the thioredoxin and glutathione systems, and other antioxidants such as catalase and superoxide dismutase, among others. Yap1 is therefore central to the adaptive response to oxidative stress, regulating not only the response to H2O2-induced stress, but also that to chemical oxidants (redox cycling chemicals, thiol oxidants and alkylating agents), cadmium and drug stress. Purified as a 90 kDa protein [16], Yap1 has a basal expression and, in unstressed cells, shifts to and from the cytoplasm
• Yap proteins and the yeast response to stress
• The Yap protein family
• The Yap family of b-ZIP proteins comprises eight members with a significant sequence similarity to the true yeast AP-1 factor Gen4 at the DNA-binding domain [8]. However, in addition and common to all family members, are several key residues that impart distinct binding properties to these transcription factors. It has been determined that Yap1 through to Yap5 preferentially bind to the consensus site TTAG/CTAA, which differs from the true AP-1 recognition element bound by Gcn4 (TGAG/CTCA). It is unclear how many YAP sites are required for target gene regulation. Work performed by Cohen et al. [9] indicates that gene clusters enriched for Yap1- and Yap2-dependent genes have, on average, 1.9 (P-value 8.0 × 10-4) and 1.8 (P-value 2.0 × 10-3) consensus Yap sites, respectively. We cannot, however, exclude the possibility that flanking bases around this core consensus are also required. In the case of Yap8, it has been shown that this protein binds the sequence TTAATAA on target gene promoters [10] (and our own results). The Yap family has been found to be implicated in a variety of stress responses including oxidative, osmotic,
• arsenic, drug and heat stress, among others [11]. Although much is currently understood about Yap1, the major regulator of the oxidative stress response, comparatively less is known about the remaining family members.
• Oxidative stress
• The response to oxidative stress can be described as the phenomenon by which the cell responds to alterations in its redox state. As a consequence of aerobic growth, cells are continuously exposed to reactive oxygen species (ROS), potent oxidants capable of extensive cellular damage at the level of DNA, protein and membrane lipid content. As a result, organisms, from bacteria to humans, have developed mechanisms of maintaining cellular thiol redox homeostasis. This is achieved by limiting the accumulation of O2-derived oxidants, controlling iron and copper metabolism, the activation of thiol redox pathways and via damage repair [12].
• Yap1 was initially characterized through the observation that the deletion mutant is hypersensitive to the oxidants H2O2 and t-BOOH, and to chemicals that generate superoxide anions, including menadione, plumbagine and methylviologen as well as to cadmium, methylglyoxal and cycloheximide. Recent genome-wide studies have focused on modulation of the gene expression programmes that occur following exposure to an oxidative insult in S. cerevisiae. Indeed, Gasch et al. [7] and Causton et al. [13] have demonstrated that the response to mild doses of H2O2 leads to the immediate and transient modulation of ~ 24% of the genome. Although approximately half of this response can be attributed to the ESR, there is an H2O2-specific response comprising genes encoding most cellular antioxidants and components of thiol redox pathways, heat shock proteins, drug transporters and enzymes involved in carbohydrate metabolism. Among these genes are TRX2 [14] and GSH1 [15], two of the first Yap1 targets to be characterized and induced under oxidative stress imposed by H2O2, diamide and t-BOOH. Since then, several Yap1 targets involved in ROS detoxification have been identified, including those involved in the thioredoxin and glutathione systems, and other antioxidants such as catalase and superoxide dismutase, among others. Yap1 is therefore central to the adaptive response to oxidative stress, regulating not only the response to H2O2-induced stress, but also that to chemical oxidants (redox cycling chemicals, thiol oxidants and alkylating agents), cadmium and drug stress. Purified as a 90 kDa protein [16], Yap1 has a basal expression and, in unstressed cells, shifts to and from the cytoplasm
• via interaction of the Crm1 nuclear exportin with the Yap1 nuclear export signal (NES) [17,18]. Although YAP1 mRNA basal levels are enhanced upon exposure to an oxidative stimulus, the control of Yap1 activity is primarily regulated through subcellular localization. Indeed, Kuge et al. [14] demonstrated that Yap1 nuclear retention is mediated by the cysteine-rich domain (CRD) located at the C-terminus of the protein which contains two cysteine-rich regions designated as the n-CRD (C303, C310 and C315) and c-CRD (C598, C620 and C629) (Fig. 1A). In response to diamide, the c-CRD is sufficient to mediate a response. However, in the case of H2O2, both n- and c-CRD regions are required [17,19]. How does Yap1 sense oxidative stress? It has been shown that the oxidant receptor peroxidase Orp1 (also designated Hyrl and Gpx3), is the main signal sensor and that a third component of this signal relay, Yap1 binding protein (Ybp1) is associated with Yap1 [20]. Orp1 carries a conserved peroxi-dase-active site cysteine residue (Cys36) of the Gpx family, whose catalytic cycle is first oxidized to a sulf-enic acid (Cys-SOH), and then reduced by GSH [18]. Orpl, however, contributes towards H2O2 resistance not as a peroxidase, but as a sensor of oxidative stress. Orpl activates Yap1 by forming an intermole-cular disulfide bond between its Cys36 and the Yap1 Cys598, which is then converted into the
• Yapl intramolecular Cys303-Cys598 disulfide bond (Fig. 1B). Veal et al. [20] have shown that Ybpl is required for the signal transduction from Orpl to Yap1 because in its absence the intermolecular disul-fide bond does not form. It has been suggested that Ybpl could act as chaperoning the formation of disul-fide bonds through the guiding of Orp1 Cys36SOH to Yap1 Cys598, and/or preventing the formation of the competing Orp1 Cys36-Cys82 disulfide bond. Once activated, the Yap1 NES that lies within the c-CRD is masked leading to its retention in the nucleus and the up-regulation of target genes. Ybp2/Ybhl, a protein homologous to Ybpl, was found in the genome of S. cerevisiae and described as having an effect on H2O2 tolerance, through different mechanisms [21]. However, these data should be regarded with caution because most of the conclusions are derived from indirect results. These sensing mechanisms appear con-served in Schizosaccharomyces pombe in which a two-cysteine-based peroxidase functions in a similar way to Orpl in the activation of Papl, the Yap1 orthologue [22]. In addition, a second Yap1 redox centre involved in the direct binding of N-ethylmaleimide (NEM), the quinone menadione, both an electrophile and super-oxide anion generator, was shown to operate. Under conditions favouring superoxide anion generation, Yap1 is activated by H2O2 formed by the dismutation of the
• Fig. 1. (A) Comparison of the CRD of Yap1, Yap2 and Yap8, NES is underlined. (B) The two Yap1 redox centres. Under nonoxidizing conditions, Yap1 is cytoplasmic owing to Crm1-dependent nuclear export. Upon H2O2 exposure, the formation of an intermolecular bond occurs between the Orp1 Cys36 and the Cys598 of Yap1 leading to its activation. The subsequent formation of the Yap1 Cys303-Cys598 disulfide bond masks the NES retaining it in the nucleus where it activates target genes. Under thiol-reactive agents, and possibly the metalloids, a second redox centre operates involving the Cys598, Cys620 and Cys629 of Yap1, to which the drug binds directly.
• superoxide. In contrast, menadione acts as an electro-phile in the absence of oxygen and in this case binds directly to the c-CRD Cys598, Cys620 and Cys629 in a manner independent of the Orp1 pathway [23].
• Metalloid and metal stress
• The widespread distribution of the toxic metalloid arsenic in nature leads to the acquisition of its resist-ance in almost all living organisms [24,25]. In S. cere-visiae, resistance to arsenic is achieved through the activation of the arsenic compounds-resistance (ACR) cluster [26], which is composed by the positive regula-tor Acrl (Yap8), the arsenate-reductase Acr2 and the plasma membrane arsenite efflux protein Acr3 [27]. The yeast cadmium factor (YCFI) gene encodes an independent detoxification system that operates by sequestering As(GS)3 into the vacuole [28-30]. Induc-tion of the expression of ACR2, ACR3 and also YCF1 by the transcription factor Yap8 is essential to arsenic stress response. Like Yap1, Yap8 is constitutively expressed, and under physiological conditions shuttles to and from the nucleus [31]. This is in contrast to the results obtained by Wysocki et al. [10] and may be due to the fact that the latter use a multicopy vector, whereas the former look at the green fluorescent pro-tein construct within a normal chromosomal context. Under arsenic stress conditions, Yap8 is activated at the level of its transactivation potential as well as its nuclear accumulation, which is triggered by the loss of interaction with Crm1 [31]. Yap8 cysteine residues Cys132, Cys137 and Cys274 are essential to both pro-cesses (Fig. 1A). Work by Haugen et al. [32] on the inte-gration of phenotypic and expression profiles involved in arsenic response has revealed the array of genes whose transcription is enriched, including those involved in methionine metabolism and sulfur assimil-ation, protein degradation and transcriptional regula-tion, and by proteins that form a stress response network, including Fhll, Msn2 Msn4, Yapl, Cad1 (Yap2), Hsfl and Rpn4 among others. Furthermore, results obtained in microarray analyses point towards the existence of further Yap8-mediated arsenic detoxifi-cation pathways (C Amaral, F Devause, R Menezes, C Facq & C Rodrigues-Pousada, unpublished observa-tions), highlighting the relevance of multiple mecha-nisms of arsenic management. A distinct detoxification strategy employed by S. pombe, nematodes and plants makes use of phytochelatins (PCs) for metalloid che-lation. The observation that overexpression of the S. cerevisiae ACR3-encoded arsenite transporter not only complements the lack of phytochelatins in S. pombe, but also confers hyper-resistance to arsenic
• compounds to the levels observed in the budding yeast and prokaryotes [33] further accentuates the effective-ness of this pathway in arsenic detoxification. Yapl activation by arsenic compounds is similar to its acti-vation by thiol-reactive chemicals [23] because it is unaffected by the absence of the sensor Orpl/Gpx3 and does not depend on the n-CRD cysteines (Fig. 1B). In contrast to Yap8, under arsenic stress conditions YAPI basal expression is slightly enhanced and the presence of this metalloid does not signifi-cantly modulate Yap1 transactivation function [11]. Heavy metals including copper, zinc, iron and man-ganese play an important role in cellular biochemistry and physiology [34]. However, when the concentration of these metals is elevated, toxicity arises for the organ-isms. Although cadmium and mercury are not essential metals they cause severe damage even in low amounts. Organisms therefore possess cellular detoxification mechanisms that maintain homeostasis through the con-trol of intracellular ion levels. One of these involves the activation of Yap1 and Yap2 (Cad1) [9,35,36]. Yap2 over-expression confers resistance to a plethora of stress agents such as cadmium, cerulenin and 1,10-phenanthro-line among others, suggesting a role in the response to drug stress. Indeed, several target genes encoding a set of proteins involved in the stabilization and folding of pro-teins in an oxidative environment have been identified by microarray analyses [9]. Induced upon exposure to cad-mium stress [8], Yap2 re-localizes to the nucleus via a Crm1-dependent mechanism, where it activates the tran-scription of its target gene FRM2, encoding a protein homologous to nitroreductase, whose precise role in the metal stress response remains unclear. The strong sequence homology between Yap2 and Yap1 in the C-terminal CRD (residues 570-650 in Yap1 and 330-409 in Yap2) was used to further provide an insight into the function Yap2. Domain swapping of the Yap1 c-CRD by that of Yap2 has shown that the fusion protein is regulated by cadmium but not by H2O2 (D Azevedo & C Rodrigues-Pousada, unpublished data). Nuclear localization of the fusion protein correlates not only with activation of FRM2 transcription, but also with growth