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Respuesta al estrés catiónico en Aspergillus nidulans: studio del proceso de señalización del factor transcripcional SltA
|Authors:||Mellado, Laura CSIC|
|Advisor:||Espeso, Eduardo A. CSIC ORCID|
|Publisher:||CSIC - Centro de Investigaciones Biológicas Margarita Salas (CIB)|
Universidad Complutense de Madrid
|Abstract:||The ability of cells to sense and respond properly to environmental changes is essential for most metabolic processes of which life depends. Abiotic stresses although caused by natural elements these are not directly produced by organisms. Among them, and more interesting for this work, are high extracellular concentrations of mono and divalent cations, and environmental pH, which is a direct effect of the monovalent cation hydrogen .
Microorganisms have developed homeostatic systems which allow them to maintain intracellular ion concentration relatively constant in response to changes in external conditions. These organisms are classified according to their ability to tolerate different concentrations of ions, and among them, the model organism Aspergillus nidulans used in this thesis, can tolerate moderate amounts of a wide range of cations, from one molar concentrations of sodium or potassium up to millimolar concentrations of manganese or iron , as well as a wide range of environmental pH values, ranging from 2.5 to 10.5 values .
In A. nidulans tolerance to an alkaline pH requires the activities of three principal transcription factors: PacC, CrzA and SltA. In Saccharomyces cerevisiae Rim101p and Crz1p (PacC and CrzA, respectively), mediate the response to alkaline pH, and the latter is also involved in the regulation of cation homeostasis. However, in A. nidulans this regulatory function is shared between SltA and CrzA . SltA belongs to zinc-finger transcription factors family. Whereas SltA phylogenetic distribution is specific to filamentous fungi, CrzA and PacC are widely distributed among fungal kingdom.
Defects in SltA function, such as in the sltA1 mutant, cause sensitivity to NaCl, arginine, UV light and the alkylating agent N-methyl-N'-nitro-nitrosoguanidine . SltA shares significant similarity to Ace1p transcription factor from Hypocrea jecorina (~ 58%) but their functions are very different; while Ace1p controls cellulose utilization , SltA mediates the response to abiotic stress. More extensive studies carried out by the research group have shown that sltA deletant shows sensitivity to alkaline pH, Na+, K+, Mg2+, Li+ and Cs+, but not to calcium . Moreover, the deletant displayed reduced expression of a homologue ENA1, Na+-ATPase of S. cerevisiae, and also up-regulation of vacuolar Ca2+/H+ exchanger VcxA, VCX1 homologue in yeast.
The biological activity of many proteins, including transcription factors, is regulated by post-translational modifications, which are very varied and can be reversible or irreversible . These include phosphorylation and proteolysis, which are interesting for this work and can be modulated in response to extracellular signals.|
2. Objectives The main focus of this thesis is the study of the signaling process of SltA in the model organism Aspergillus nidulans, which mediates the response to abiotic stress. Additionally, we have discovered a new element of the Slt stress response pathway, SltB, through a previous work that showed a relationship between Slt and endocytic pathways in A. nidulans . This doctoral thesis is the starting point for the study of SltB protein and its function in the regulatory pathway after cation homeostasis and alkalinity tolerance. Three lines of research were proposed to pursue this main objective: 1) Study the signaling, activation and regulation mechanisms of the transcriptional factor SltA. 2) Characterize the signaling process of SltB and its cell function. 3) Determine the functional relationship between SltA and SltB, and other known elements involved in the response to cation and environmental pH. 3. Results The Results section is divided into five chapters. In the first chapter approaches a functional analysis of the SltB gene product. Deletion of sltB was not essential to the fungal development, but revealed a genetic interaction with sltA gene, because the sensitive phenotype to a number of salts and alkaline pH caused by deletion each gene was indistinguishable. This finding together with the similar phylogenetic distribution and its relationship with the endocytic pathway, led us to propose a initial working model where both proteins form part of the same signaling pathway. In this model, SltA would be arrested in an inactive state but changing to an active state after the action of SltB, and then promoting a specific transcriptional response. In the second chapter a bioinformatic analysis of SltB protein sequence was conducted to identify the type and family to which SltB belongs. Multiple alignments performed among different Aspergillus orthologs, showed a prediction of two conserved functional domains in its sequence. The amino-terminal region displayed high similarity to non-functional protein kinases, and the carboxyl terminal region was identified as a trypsin-like family of serine/cysteine protease. Analysis of this domain using the MEROPS peptidase database classified SltB into the peptidase family S64 having a catalytic serine-type. The predicted active site residues for members of this family are His-Asp-Ser . Histidine 1008/1033, Aspartic 1036 and Serine 1142 residues were proposed as candidates to take part in the catalytic centre of SltB protease domain. Next detailed bioinformatic analyses of the putative protein kinase domain in SltB were carried out and determined that this region does not contain those essential catalytic residues defined in eukaryotic kinase families . We propose this region as a pseudokinase protein
. Chapter three describes classical genetics and molecular biology strategies to analyse this regulatory pathway. Mutant forms of SltA and SltB were constructed and experiments determined the existence of posttranslational regulatory mechanism for both proteins involving a proteolytic step. C-terminal labelling of SltA allowed the identification of three forms for this protein: SltA78kDa and SltA32kDa that, in turn, is in equilibrium with a modified form. A double-labelled SltB protein, both at the amino and carboxyl terminal regions, demonstrated the existence of two separate fractions which might contain those domains identified by bioinformatic analyses. Expression of sltB gene depends on the SltA transcriptional regulatory activity, since a SltB::GFP chimera was not detected in protein extracts of a null sltA strain. This fact was inconsistent with the proposed linear working model. A new model was presented to integrate this data showing cross-interactions between both proteins. Expression of SltB in a SltA-independent manner in a sltA null background demonstrated that SltA is the final effector of this regulatory pathway, and that proteolytic activation of SltB is independent of SltA activity. Through the generation and study of mutant strains, it was shown that SltB pseudokinase domain is enough for the process of SltA proteolysis. We identified mutations in the SltB protease domain blocking its proteolysis, suggesting a mechanism of self-catalysis. This proteolytic step in SltB is a prerequisite for the proteolytic cleavage of SltA. Generating diploid strains it has been proposed SltB proteolysis could occur in cis configuration, intramolecular, since failed to complement SltB activity in trans. We studied the catalytic triad of SltB through an in vitro mutagenesis process on the genomic sequence of sltB. Both histidine 1033 and serine 1142 amino acids were replaced by alanine residues, and these mutant versions of SltB were fused to the fluorescent protein GFP. The absence of serine 1142 resulted in the loss of the protein function, which corresponded with the absence of proteolysis of SltB and, consequently, of SltA. Conditional expression of the allele SltA32kDa with the thiamine promoter, gave rise a partial function of the protein. It has been studied the effect of the absence of SltA78kDa on sltB gene expression and the role of SltA32kDa in regulating sltB expression. It was determined that SltA32kDa is epistatic to the absence of sltB gene and, as a final effector, also promotes sltB expression. The posttranslational modification which occurs in SltA32kDa is not SltB-dependent and the main activity of SltB is likely to be exclusively restricted to the SltA proteolysis. In chapter four, the effects of stresses caused by cations and alkalinization in SltA and SltB proteins were studied. For this purpose, the presence of posttranslational changes in both SltA and SltB proteins was determined in mycelia cultured under different pH and osmotic stress conditions. No differences in the levels of each form were observed under those stress conditions analyzed. This result showed that signalling of SltA and SltB, as proteolytic cleavage, is independent of the abiotic stress present in the culture medium. These results suggest that Slt proteins are under a constitutive signalling system.
A SltA and SltB expression analysis showed that sltB expression depends on the SltA activity. A notable reduction of SltB transcript levels were detected in RNA extracted from a null sltA strain. According to the amount and forms of protein detected in the mycelium cultured under different pH and osmotic stress conditions experiments, no variation in both genes expression levels in the different stress conditions tested was observed. In vitro dephosphorylation tests with Lambda protein phosphatase allowed to conclude that the intermediate band with a mobility between SltA78kDa and SltA32kDa detected in the protein extracts corresponded with a phosphorylated form of SltA32kDa, which coexists with another non-phosphorylated SltA32kDa form together with the native form SltA78kDa. The final part of this thesis is dedicated to the additional studies of SltA and SltB. Using a fluorescent-tagged version of SltB we showed a predominantly cytoplasmic localization for this protein. SltB is apparently excluded from the nuclei and organelles, and its distribution remains unaffected independently of which domain is expressed. In contrast, SltA cell distribution was studied by means of a subcellular fractionation experiment. Most notably, cytoplasmic levels of SltA are very low. SltA is mainly in the nuclear fraction but also associated with other large cellular elements. Finally, we have carried out genetic and functional studies between SltA, CrzA and PacC transcription factors. Mainly looking at the response to alkaline pH, primarily searching for a possible connection to PalB, the protease of PacC, and to the calcineurin signalling, the phosphatase of CrzA. The overall results suggested independent regulatory mechanisms between these transcription factors. 4. Conclusions 1) SltA and SltB are elements of the same signaling pathway by cation stress and environmental pH, where SltA is the final effector of the pathway and SltB is the signalling protein. 2) SltA and SltB have a restricted phylogenetic distribution to subphylum Pezizomycotina. 3) SltA is subjected to at least two post-translational modifications. A proteolysis process which determines the existence of two physiological forms of SltA, SltA78kDa and SltA32kDa, and a phosphorylation process of SltA32kDa form that determines the existence of the third SltA form characterized. 4) SltB is a protein with two putative functional domains identified in the sequence, a pseudokinase domain in the N-terminal region and a domain with protease activity at the C-terminus. 5) Also SltB is post-translationally modified by proteolysis, releasing the two domains found in its sequence. 6) The protease domain is required for proteolysis SltB itself, suggesting a mechanism of self-regulation by proteolysis. The Serine 1142 residue is a good candidate to be part of the catalytic center of this domain. The pseudokinase domain mediates the proteolysis of SltA.
A SltA and SltB expression analysis showed that sltB expression depends on the SltA activity. A notable reduction of SltB transcript levels were detected in RNA extracted from a null sltA strain. According to the amount and forms of protein detected in the mycelium cultured under different pH and osmotic stress conditions experiments, no variation in both genes expression levels in the different stress conditions tested was observed. In vitro dephosphorylation tests with Lambda protein phosphatase allowed to conclude that the intermediate band with a mobility between SltA78kDa and SltA32kDa detected in the protein extracts corresponded with a phosphorylated form of SltA32kDa, which coexists with another non-phosphorylated SltA32kDa form together with the native form SltA78kDa. The final part of this thesis is dedicated to the additional studies of SltA and SltB. Using a fluorescent-tagged version of SltB we showed a predominantly cytoplasmic localization for this protein. SltB is apparently excluded from the nuclei and organelles, and its distribution remains unaffected independently of which domain is expressed. In contrast, SltA cell distribution was studied by means of a subcellular fractionation experiment. Most notably, cytoplasmic levels of SltA are very low. SltA is mainly in the nuclear fraction but also associated with other large cellular elements. Finally, we have carried out genetic and functional studies between SltA, CrzA and PacC transcription factors. Mainly looking at the response to alkaline pH, primarily searching for a possible connection to PalB, the protease of PacC, and to the calcineurin signalling, the phosphatase of CrzA. The overall results suggested independent regulatory mechanisms between these transcription factors. 4. Conclusions 1) SltA and SltB are elements of the same signaling pathway by cation stress and environmental pH, where SltA is the final effector of the pathway and SltB is the signalling protein. 2) SltA and SltB have a restricted phylogenetic distribution to subphylum Pezizomycotina. 3) SltA is subjected to at least two post-translational modifications. A proteolysis process which determines the existence of two physiological forms of SltA, SltA78kDa and SltA32kDa, and a phosphorylation process of SltA32kDa form that determines the existence of the third SltA form characterized. 4) SltB is a protein with two putative functional domains identified in the sequence, a pseudokinase domain in the N-terminal region and a domain with protease activity at the C-terminus. 5) Also SltB is post-translationally modified by proteolysis, releasing the two domains found in its sequence. 6) The protease domain is required for proteolysis SltB itself, suggesting a mechanism of self-regulation by proteolysis. The Serine 1142 residue is a good candidate to be part of the catalytic center of this domain. The pseudokinase domain mediates the proteolysis of SltA. 7) SltB expression is dependent on the activity of SltA. 8) Signaling of SltA and SltB could be constitutive, independent of environmental stimulus. 9) SltB has a cytoplasmic cellular distribution, and the independent expression of its domains does not alter cellular localization. 10) SltA, CrzA and PacC mediate different pH-responsive signaling pathways.
5. Discussion In this thesis has been studied the regulatory mechanisms of gene expression that allow the fungus tolerate high concentrations of cations and ambient pH alkalinization. In particular, the work has focused on determining how the transcriptional factor SltA is signalised through the activity of the SltB protein. This section is organized into five points which delves into the results from the point of view of its mechanism and biological function. I) From the genetic model to the molecular model. The initial working model was direct and simple. Since SltA is a transcriptional factor, this might be a final effector of Slt pathway. Thus, SltB would predictably be a signaling element that promotes a post-translational change in SltA transforming it into an active transcriptional form. This form would regulate the expression of the specific set of genes. The molecular work developed during this work has shown that the regulation of this system is more complex, there is a cross-link involving interactions of both proteins but SltA is the final regulatory element. These results allow us to propose two models of regulatory pathways to describe the role of SltA and SltB in cation homeostasis and environmental pH. The first would place both proteins as elements of a specific pathway, the Slt pathway; and the second would integrate into a more general pathway or network, which are the last effectors to the environmental stresses studied. II) Nature and dynamics of signaling Slt proteins. Various transcription factors are activated to mediate its action in the nucleus. Some are modified by proteolysis, leading to alternative forms where parts of the protein are eliminated to gain or lose a functional activity. Examples of transcription factors regulated by proteolysis are PacC and Stp1/2. This work shows that SltA is also a transcriptional factor regulated by limited proteolysis. The estimated size of SltA native form is 78kDa (SltA78kDa) and it is detectable in any culture condition. However, it is not the only form in the cell. Two forms, named as SltA32KDa, were identified after tagging this protein at the C-terminal end. One such forms is a phosphorylated SltA32KDa. The native form SltA78kDa is functional. We propose that proteolysis of SltA renders a new functional SltA form in the cell, since it retains the DNA binding domain. The SltA78KDa form is required for sltB gene expression. SltB protein is also subject to proteolytic cleavage. The analysis of the primary structure of SltB proposes at least two distinct domains, a pseudokinase domain and a protease domain. The pseudokinase domain is essential to accomplish the proteolysis of SltA. The protease domain is involved in at least the cleavage and release of the two domains in SltB. In this study has not been possible to determine the part of the cell where the signalling process occurs. However, all forms of SltB studied preferably maintain a cytoplasmic localisation. Labelling of SltA for determining its cell distribution resulted in non functional forms. Alternatively, an analysis was made of the cellular distribution of SltA by subcellular fractionation. This analysis places a portion of the cellular content of SltA (both forms) in the cytoplasm and partly in the nucleus. The SltA cytoplasmic fraction may be due to the formation of large protein complexes or association with membrane structures. This portion could receive the signalling or were in the process of it. The fact that pseudokinases may mediate formation of heterocomplexes and the need of SltBPsk activity for proteolysis of SltA, suggests SltA would be recruited by SltBPsk in a compartment/structure in which must contain the protease activity acting on the transcription factor.
III) SltB protease activity. The initial role proposed for SltB protein has been continuously modified throughout the progress of this thesis. Firstly, the genetic model visualized SltB activity as a trigger of the activation of SltA. The need of SltB activity for signaling SltA defined a direct relationship between the proposed activity for its protease domain and the proteolysis of the transcriptional factor. However, classical genetics provided a new molecular model to characterize the mutant protein of allele sltB56. The protein encoded by the sltB56 allele is truncated at amino acid 477, containing largely of the pseudokinase domain. The phenotype of this mutation is a partial loss of function, which we explain later determining existence of proteolysis of SltA. This result discarded SltB as the protease of SltA. Classical genetics again provided a loss of function mutant, sltB53. The mutant protein StlB53 is visualized in its native form, and in this genetic background, SltA is not proteolysed to SltA32kDa. Therefore, SltB must be self-proteolysed and this process should render the separation of the two functional domains in SltB. The pseudokinase domain is needed to transmit SltA signalling. Although there are examples described in pseudokinases proteins such as Titin or VNK, which maintain a remainder catalytic activity , it could not been attributed to SltBPsk an involvement in the phosphorylation of SltA32kDa, since this is phosphorylated in a sltB null genetic background. As already mentioned, a possible role for SltB pseudokinase domain could be to recruit the necessary elements for SltA proteolysis, as it has been described a scaffold function for pseudokinase proteins. Sequence analysis of the protease domain suggests a certain similarity between SltB and Ssy5p protein from S. cerevisiae. This protein has been classified as a possible member of the family of trypsin-like serine proteases, pointing to the H465, D545 and S640 residues as elements of the catalytic triad . Alignments made between SltB and Ssy5 sequences identified the possible catalytic triad in SltB composed of H1008/1033, D1036 and S1142 residues. Using reverse genetics we have demonstrated that substitution of H1033 and Ser 1142 to alanine residues causes partial or total loss of function of SltB, respectively. Both phenotypes are due to the reduction, in the first case, or the absence of proteolysis of SltB and consequently altering the proteolysis of SltA. Thus, we define Ser 1142 of SltB sequence as a functional and essential residue of the putative catalytic triad of SltB protease domain and therefore, we classify SltB within the serine protease group.
IV) Regulation of the SltA activity. Functional regions. There are regulatory interactions of the expression and signaling of Slt proteins, indicating that the form SltA78kDa must be functional to promote the transcription of sltB gene. In this regard, it has been shown how the form SltA78kDa mediates the expression of seveeral non-functional forms of SltB. Accordingly, analysis SltB promoter shows putative target sequences for SltA factor. It has also been shown that the conditional expression of the form that mimics SltA32kDa (thiA::sltAMet400) allows equivalent levels of SltB than in a wild type background. These results show a positive role for SltA regulating the expression of sltB, which is activated by the SltB protein itself. Analysis of mutant forms obtained by classical genetics, and those generated in this study, allow us to define two functional regions in SltA protein. The similar phenotype displayed by sltA and sltA1 mutants highlighted the importance of the last 116 amino acids of the protein sequence of SltA. However, the degree of conservation of this region is very divergent between SltA homologs in different species of the genus Aspergillus identified to date. This C- terminal region of SltA might have been acquired as an evolutionary advantage over the different orthologs described, acquiring additional regulatory function in cis. In this regard, modification determined for SltA32KDa by phosphorylation and truncated forms generated, SltAMet400 and SltAArg331, indicate C- terminal region as a candidate to contain the elements involved in the phosphorylation, thus supporting this hypothesis The amino terminal region of SltA (SltA46kDa) could have a regulatory role on its activity, as described for many eukaryotic transcription factors (eg Stp1 or NRF1, Nuclear factor-erythroid 2-related factor 1). V) Finding new components of the Slt pathway. One of the difficulties in understanding this regulatory system has been the lack of additional elements to SltA and SltB. It has been demonstrated that at least two proteins are involved in this signalling process, a protease and a kinase, both of them mediating its action on SltA. These two proteins could be or not specific of Slt pathway. Has been performed in silico searches for candidate genes that could encode these two elements of the Slt pathway. Thus, we identified in the A. nidulans protein database the protein sequence called PskA, a pseudokinase with high similarity to the SltB pseudokinase domain, and specific to filamentous fungi too. However, after generating a strain carrying the null allele of this gene, we could not prove their relationship to Slt pathway. The identification of the kinase and protease of SltA could be done by searching for supressor mutants of the lethal phenotype caused by the deletion of vps genes . So far, no mutations have been found in different loci from those corresponding to SltA or SltB, so it is necessary
Since only two effectors of the response to salt stress and alkaline pH in A. nidulans are known, SltA and SltB, we cannot discard that these were trans modulators of a general stress response pathway. As SltA is involved in different cellular processes (cation homeostasis, environmental pH, conidiation and secondary metabolism), the proposal on the connection between different signaling cascades could occur by posttranslational modifications that would regulate the overall SltA activity. This idea is described for other transcription factors metazoans, such as FOXO1, belongs to the Forkhead family of transcription factors . This TF can modulate the specificity of cellular process that is involved in response to different signals by their posttranslational modifications. A similar role could have the modifications by proteolysis and phosphorylation determined for SltA. In this regard, it should be studied the state of post-translational modification of SltA in other study conditions for which function has been determined.
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