Our group is interested in, using the fission yeast as a model system, studying the components and molecular mechanisms which regulate the responses to oxidative stress and the mitotic and meiotic cell cycle.
1) Imbalances between generation and scavenging of reactive oxygen species (ROS) give rise to an increase of their intracellular steady state concentrations and, as a consequence, to a complex situation so-called oxidative stress. A main goal of our laboratory is to study at the molecular level how does the cell sense oxidative stress and which mechanisms are triggered to allow adaptation to the stress. We, therefore, are trying to identify and characterize the components and mechanisms which activate signal transduction pathways to respond to oxidative stress in unicellular eukaryots (Schizosaccharomyces pombe). In particular, the fission yeast has two alternative pathways to respond to hydrogen peroxide (H2O2): the transcription factor Pap1 (section 1.1) and the MAP kinase Sty1 (section 1.2). Pap1 is specific for the response to oxidative stress, whereas Sty1 is able to sense multiple types of stresses, and to activate global responses.
We have recently identified the peroxiredoxin Tpx1 (section 1.3) as the real sensor of H2O2 in the Pap1 pathway. The work of many laboratories have reported the importance of peroxiredoxins in different cellular processes. In the fission yeast, our working hypothesis is that Tpx1 is the link between the physiological growth under aerobic conditions and a moderate extracellular oxidative stress, able to trigger the Pap1 pathway. A third quantitative degree of responses to H2O2 would lead to Sty1 activation. We believe that those three proteins protect the cell against a whole range of H2O2 concentrations.
The identification of Tpx1 as a regulator of both basal redox homeostasis and signal transduction in response to ROS has prompted us to study the sources of endogenous or intrinsic oxidative stress. Since intracellular ROS seem to be linked to pathological processes such as neurodegenerative diseases or aging, we have started to work on the global characterization of genes essential to control redox equilibrium, and the consequences at the proteomic level of redox imbalances (section 1.4).
1.1. In response to hydrogen peroxide and other oxidative agents, the transcription factor Pap1 of S. pombe translocates to the nucleus and activates transcription of antioxidant genes. We study the post-transcriptional modifications suffered by Pap1 in response to different inducers. In all cases, different cysteine residues of the protein are modified in response to the signals; the cysteine modifications include alkylation by diethylmaleate or disulfide bond formation in response to H2O2. In any case, oxidized Pap1 loses its ability to interact with the Crm1-dependent nuclear export machinery, and accumulates at the nucleus.
1.2. Sty1 is a MAP kinase from S. pombe which activates cellular defense responses versus a variety of extracellular signals, such as oxidative and osmotic stress, heat shock, nutrient starvation... One of the main substrates of Sty1 is the transcription factor Atf1, which in combination with Pcr1 induces global responses anti-stress. We study the participation of this kinase in each one of its cellular functions. Thus, we are interested in (i) identification and characterization of new substrates and upstream components of the MAP kinase Sty1; (ii) characterization of the role of Atf1 and/or Pcr1 in the responses to stress in S. pombe; (iii) role of the MAP kinase Sty1 in chronological aging (see section 2.2).
1.3. We study the peroxiredoxin Tpx1, and its role as a H2O2 scavenger and a H2O2 sensor. Our current working plans are: (i) elucidating why peroxiredoxins have evolved towards a protein sensitive to substrate inactivation; (ii) understanding why tpx1 is essential in S. pombe.
1.4. Regarding redox homeostasis and endogenous oxidative stress, we have already identified gene products essential for ROS scavenging and therefore for aerobic growth survival, as well as some genes which deletion enhances ROS production. A current goal of our lab is to characterize the oxyproteome of such mutant strains. Furthermore, we are currently performing a global screening of genes essential to overcome oxidative metabolism.
2) During the last two decades it has become possible to identify the molecular mechanisms that regulate the cell cycle and thereby cell division. The exact knowledge of how a healthy cell (wild type) divides will be very useful to understand what has gone wrong in a tumor cell. The use of a model organism, like S. pombe, has the advantage of a very simple and manipulative genetics. Furthermore, the fundamental mechanisms that govern the cell cycle are highly conserved through evolution and operate in the same manner in all eukaryotic organisms. In our laboratory, we are interested in characterizing a critical point in the cell cycle, START (Restriction point in mammalian cells) (section 2.1), where cells become committed to the mitotic cycle, stationary phase (section 2.2) or meiosis (section 2.3). We are currently also focused on the study of splicing of meiotic-specific genes.
2.1. From the early 80's it was shown that completion of START required the products of cdc2 and cdc10 genes. Cdc10 belongs to a transcription factor known as MBF, whose activity is required for the transcription of genes necessary for the completion of the S phase of the cell cycle. We are interested in determine how this transcription factor is regulated in parallel to cell cycle progression. We are using a proteomic approach (iTRAQ) to isolate proteins that interact with any component of the MBF complex (Cdc10, Res1 and/or Res2). Among those proteins we have found Max1/Yox1 as a strong interactor with intact MBF. Max1/Yox1 is the main repressor of MBF complex and also is a substrate of the kinase Cds1, which is the effector kinase of the DNA-synthesis checkpoint. Thus, Max1 is in charge of coupling the completion of DNA synthesis with the transcriptional activation of the MBF-dependent genes.
2.2. Most cells in a human body are quiescent. We are using S. pombe cells to determine which genes are required for survival in stationary phase, and are therefore important for chronological aging. Since cells deleted in the MAP kinase Sty1 show an inability to survive in stationary phase, we are determining the role of this MAP kinase during this quiescent growth phase. We are also interested in figuring out which is the role of Sty1 in cell cycle regulation after stress.
2.3. Meiosis is the only exception to a regular cell cycle since there are two nuclear divisions (Meiosis I and Meiosis II) without an intervening DNA synthesis. S. pombe cells require the activity of a meiotic-specific cyclin, Rem1, to progress along meiosis. In the laboratory we are characterizing the role of Rem1 during meiosis, as well as its transcriptional regulation. Since the rem1 gene has an intron, and we have recently demonstrated that transcription can occur without intron processing, we have become interested in studying splicing regulation in fission yeast. In fact, when rem1 is not spliced, translates into a truncated protein that brings about normal levels of recombination. This could account for a sophisticated alternative splicing scheme in a unicellular organism in which when the mRNA is not processed (during the pre-meiotic S phase) will render a 17 KDa protein that will have a role in recombination and when the mRNA is processed will produce a protein with a described function as a cyclin during meiosis I. This takes part of a regulatory mechanism which ensures the absence of the cyclin in mitotically growing cells, since even very low levels of Rem1 are toxic in non-meiotic cells. We have shown that the regulation of rem1 splicing is exclusively under the control of its own promoter. In this scenario, a transcription factor, Mei4, would recruit the active spliceosome to specific genes, and another transcription factor, Fkh2, would interfere with coupling splicing to transcription. Thus, a switch from Fkh2 to Mei4 could explain the effect on splicing regulation.
