In Silico Biology 4, 0050 (2004); ©2004, Bioinformation Systems e.V.  


In silico reconstruction of nutrient-sensing signal transduction pathways in Aspergillus nidulans

Vignesh Muthuvijayan and Mark R. Marten*




University of Maryland, Baltimore County (UMBC)
Department of Chemical and Biochemical Engineering
1000 Hilltop Circle
Baltimore, MD 21250
USA



*  Corresponding author; Phone: +1-410-455 3439; Fax: +1-410-455 1049; Email: marten@umbc.edu





Edited by E. Wingender; received August 05, 2004; revised October 29, 2004; accepted October 31, 2004; published November 12, 2004


Abstract

We report here probable nutrient-sensing signal transduction pathways in Aspergillus nidulans, a model filamentous fungus, based on sequence homology studies with known Saccharomyces cerevisiae and Schizosaccharomyces pombe proteins. Specifically, we identified A. nidulans homologs for yeast proteins involved in (1) filamentation-invasion, (2) cAMP-PKA, (3) pheromone response, (4) cell integrity and (5) TOR signaling pathways. We have also studied autophagy, one of the most important cellular responses regulated by TOR signaling. The Basic Local Alignment Search Tool program "blastp" was used to assess the homology of proteins. We note that by using a highly conservative approach, 70% of the S. cerevisiae signal transduction proteins (107 proteins out of 153 proteins studied) have significant homologs in A. nidulans. Using a slightly less conservative approach, we are able to identify homologs for as high as 91% of the S. cerevisiae signal transduction proteins (139 proteins out of 153 proteins studied). The filamentation-invasion, cell integrity and TOR signaling pathways showed greatest similarity with S. cerevisiae, while the cAMP-PKA and pheromone response pathways showed greater similarity with S. pombe. Based on these results, probable pathways in A. nidulans were constructed using well-established S. cerevisiae and S. pombe models.

Key words: BLAST, signal transduction, bioinformatics, nutrient sensing, Aspergillus nidulans, comparative genomics


Introduction

Filamentous fungi are a diverse group of heterotrophic microorganisms that are agriculturally and medically important and are also widely used in the production of food and beverages [1], organic acids [2], enzymes [1, 3], antibiotics [4] and other pharmaceuticals [5]; as agents of biological control of pest insects and weeds, and in biomass conversion [6]. Hence filamentous fungi are currently responsible for about half the world’s pharmaceutical and biotechnology market [3]. Also, filamentous fungi have a significant medical importance as they are known to be serious human pathogens [7], especially to immuno-compromised patients [8] and have been reported to account for up to 40% of the deaths from hospital-acquired infections [9].

Aspergillus is a ubiquitous filamentous fungus found in nature [10]. It is commonly isolated from soil, plant debris, and indoor air environment [10]. The genus Aspergillus includes over 185 species, out of which approximately 20 have been identified as opportunistic pathogens [10]. The fungus also causes allergic diseases in asthmatics and patients suffering from cystic fibrosis [11]. Hence study of Aspergillus is of critical significance in the medical field. Aspergillus nidulans is a commonly used model filamentous fungus [1, 12]. It is closely related to a large number of other Aspergillus species of industrial and medical significance - e. g., A. niger, A. oryzae, A. flavus, and A. fumigatus [12]. Hence a better understanding of A. nidulans physiology will yield important information on critical mechanisms in other industrially and medically significant filamentous fungi.

Signal transduction is the cascade of processes by which an extra-cellular signal interacts with a receptor at the cell surface, causing a change in the level of a second messenger and ultimately effects a change in the cell’s function [13]. Recent studies show signal transduction pathways regulate fungal virulence in both human (Candida albicans and Cryptococcus neoformans [14]) and plant pathogens (Ustilago maydis, Magnaporthe grisea and Cryphonectria parasitica [14]). Also, many of these pathways have been identified as potential targets for new antifungal drugs [14]. Hence understanding fungal signal transduction pathways may aid in identifying targets for novel antifungal compounds.

The signal transduction pathways of model yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, have been studied extensively [15]. In contrast, information on signal transduction pathways in filamentous fungi is fragmentary [15]. For example, in S. cerevisiae about 2% of the proteins have been identified as kinases, classified under more than 30 families [16]. Whereas, in A. nidulans, a model filamentous fungus, only 18 kinases have been annotated. As the A. nidulans genome is approximately twice the size of S. cerevisiae genome, we predict the filamentous fungus contains at least as many, probably more, protein kinases as S. cerevisiae. This implies that many enzymes involved in the signal transduction pathways of the filamentous fungi have not yet been identified.

Earlier, Han and Prade [17] reported the reconstruction of the A. nidulans salt stress-controlling pathway based on homology analysis with known S. cerevisiae genes. To accomplish this they identified sequence homologs of the S. cerevisiae High Osmolarity Glycerol (HOG) pathway genes in the A. nidulans EST database [17]. The results they obtained by computational techniques were validated by gene deletion studies [17]. But this study was limited by the EST information available. Now, with the availability of the entire A. nidulans genome [12], sequence homology studies can be conducted more effectively. A similar approach has been conducted with Aspergillus fumigatus to identify the existence of a sexual cycle [18] and sequence homologs of the S. cerevisiae pheromone response pathway were identified from an incomplete genome sequence of A. fumigatus [18].

Here we have studied various nutrient sensing signal transduction pathways in A. nidulans, using sequence homology studies to identify homologs for S. cerevisiae and S. pombe proteins involved in these pathways. Based on these observations, we have reconstructed probable A. nidulans signal transduction pathways.


Methods

We have used bioinformatics techniques for identifying new putative signal transduction proteins in the model fungus A. nidulans. As the genomes of A. nidulans [12], S. cerevisiae [19] and S. pombe [20] are now available, we performed sequence homology studies using the stand-alone BLAST program (Basic Local Alignment Search Tools) [21, 22]. We downloaded the A. nidulans genome [12] and re-formatted the FASTA database to a BLAST database using the "formatdb" program [23]. We compared the sequence homology of the signal transduction proteins in S. cerevisiae and S. pombe with the A. nidulans database created using "blastp" program [21, 22]. This method is very effective as it is fast and the reliability of the results is scored based on statistical techniques [21, 24].


Significance threshold

Percentage sequence identity and statistical score, such as expectation value (E-value) of BLAST or FASTA, are widely used measures for sequence comparison [25]. As it has been well-established that use of a statistical score such as E-value of BLAST is superior to percentage sequence identity in detecting remote homology [26], we have used the expectation value from the BLAST output as the measure for identifying the significance of the homology. Expectation value is defined as the number of different alignments with scores equivalent to or better than raw alignment score that are expected to occur in a database search by chance [23]. The statistical significance threshold for reporting matches against database sequences was E = 0.001. We have chosen stringent E-value thresholds for assigning homology based on the previous literature [25, 27, 28]. For example, Poggeler [18] had identified a probable pheromone response pathway in A. fumigatus using sequence homology with the S. cerevisiae pheromone response pathway. They had used an E-value of 10-6 as the threshold to assign homologs for all types of proteins. Other studies have used thresholds of E = 10-10 [29] and E = 10-15 [27]. We have taken a more conservative approach and we have used E = 10-15 as the threshold for assigning homology for only general proteins. Tian et al. used enzyme function conservation to derive the threshold of sequence identity necessary to transfer function from an enzyme of known function to an unknown protein to be E = 10-50. Hence, our significance threshold for enzymes in general was chosen to be E = 10-50 [25]. For identification of kinase homologs Wang et al. [28] showed an even lower E-value of 10-80 was required. Thus for kinase identifications we have used E = 10-80. We chose the threshold of E = 10-6 used by Poggeler [18] as a less conservative approach.


Results and discussion


Validation

For validation, we constructed a BLAST database using the S. cerevisiae FASTA genome. Using this database, we identified the S. cerevisiae homologs for the 18 annotated (4 experimentally and 14 electronically) A. nidulans kinases [30]. The results obtained are shown in Table 1. As can be seen, S. cerevisiae homologs of the A. nidulans kinases either have the same function or belong to the same family depending on the expectation value. For example, AnkA and its homolog Swe1p belong to the same family; KapR and its homolog Sra1p have the same function. We also observe that the homolog for NimA is Kin3p, a S. cerevisiae protein similar to NimA [31]. The only contradictory result is the high homology between Pho85p and PhoA(M1)/PhoA(M47). Even though they show high sequence homology, Pho85p is involved in phosphate and glycogen metabolism [32] while PhoA(M1)/PhoA(M47) are involved in phosphate limited growth [33]. Also, our results indicate that S. cerevisiae Hog1p shows significant homology with two A. nidulans kinases, namely, HogA and MpkC. This is probably because Hog1p in S. cerevisiae is involved in osmoregulation [34] as HogA of A. nidulans [17], and is induced under stress conditions [32]. As the function of MpkC is not yet clearly understood, we predict that MpkC may be involved in a stress response mechanism.

Based on the BLAST significance threshold, we have identified the proteins that are likely involved in A. nidulans signal transduction pathways. The results are shown in the Tables 2 through 9. Using this information, we have used the well-established models of S. cerevisiae [35] and S. pombe [14] to construct the A. nidulans signal transduction.

Table 1: Validation based on comparison of annotated kinases in A. nidulans with their S. cerevisiae homolog
(Click for viewing complete Table 1)



Filamentation-invasion pathway

Under specific culture conditions, diploid S. cerevisiae cells undergo a dimorphic switch and differentiate to form pseudohyphae, growing as extended and connected cells. Starvation of nitrogen has been identified to induce pseudohyphal differentiation [48]. In S. cerevisiae, the switch to pseudohyphal differentiation probably involves two signal transduction pathways [14, 35]. One of them is the filamentation-invasion pathway [14, 35] and the other is cAMP-Protein Kinase A pathway [14].

Filamentation-invasion pathway (Fig. 1) contains a mitogen-activated protein kinase (MAPK) cascade. These cascades generally contain three protein kinases that act in series, namely mitogen-activated protein kinase kinase kinase (MAPKKK or MEKK), mitogen-activated protein kinase kinase (MAPKK or MEK) and mitogen-activated protein kinase (MAPK). When the cascade is activated, MEKK phosphorylates the MEK, which in turn phosphorylates the MAPK [35]. These MAPK cascades regulate transcription factors by MAPK-mediated phosphorylation [14, 15, 35]. The components of the MAPK cascade of the S. cerevisiae filamentation-invasion pathway are Ste11p (MEKK), Ste7p (MEK) and Kss1p (MAPK). Ste20p acts as the upstream kinase. The MAPK cascade mediates signal transduction from two small GTP binding proteins, Ras2p and Cdc42p [35].

Our results show that 11 of the 20 S. cerevisiae proteins involved in filamentation-invasion pathway show significant A. nidulans homologs (Table 2, Fig. 1). Furthermore, an additional 6 S. cerevisiae filamentation-invasion proteins have homologs in A. nidulans under less conservative thresholds (Table 2, Fig. 1). These results imply A. nidulans is likely to have a pathway similar to S. cerevisiae filamentation-invasion pathway.

As A. nidulans is a filamentous fungus, it seems likely that the pathway homologous to the S. cerevisiae filamentation-invasion pathway would trigger a different cellular response. We predict that the A. nidulans pathway may be involved in the regulation of asexual sporulation. In support of this prediction we note a number of consistent observations from the literature. Asexual sporulation in A. nidulans is known to be regulated by two different mechanisms similar to the pseudohyphal differentiation in S. cerevisiae [14]. Initiation of conidiation is contingent upon cells having undergone a defined period of growth to reach developmental competence [49]. This is an intrinsically programmed process that is independent of external changes in growth medium [50]. Although conidiation largely occurs in a medium-independent manner, it can be induced in hyphae grown in submerged culture by nutrient (carbon or nitrogen)-limiting condition [51]. Conidiation in confluent cultures of A. nidulans depends upon inoculum density; and higher initial cell densities promote sporulation [33]. Induction of morphogenesis involves temporal and spatial regulation of several hundred genes, many of which function in conidiophore assembly or conidiospore differentiation [14]. The sequential expression of bristle (brlA), abacus (abaA), and wet-white conidia (wetA) establishes a central regulatory pathway (brlA --> abaA --> wetA) required for the transition from vegetative growth to asexual reproduction [52, 53]. We observe that the S. cerevisiae transcription factor Tec1p, which is involved in the regulation of filamentation-invasion proteins is homologous to AbaA in A. nidulans (Table 2). AbaA controls the temporal and spatial specificity of A. nidulans [34]. Expression of abaA leads to activation of brlA and wetA and cessation of vegetative growth [34]. AbaA also regulates the expression of numerous sporulation specific genes [34]. This implies that the signal transduction pathway homologous to filamentation-invasion pathway may regulate the A. nidulans asexual sporulation.

Table 2: S. cerevisiae filamentation-invasion pathway proteins and their A. nidulans homologs
(Click for viewing complete Table 2)



Figure 1: In silico reconstruction of the S. cerevisiae filamentation-invasion pathway [35] in A. nidulans. A. nidulans sequence homologs of S. cerevisiae proteins are listed in square brackets. The proteins listed in bold are homologous to more than one S. cerevisiae protein. Homology of the underlined proteins is less than the significance threshold. Proteins with just the annotation numbers are hypothetical proteins. --> activation, --| inhibition, ? No homologs identified.



cAMP-PKA Pathway

The S. cerevisiae cAMP-PKA pathway plays a vital role in regulating carbon metabolism and cell cycle progression and also functions in parallel with the filamentation-invasion pathway to regulate pseudohyphal growth [14]. This nutrient-sensing pathway involves a novel G protein-coupled receptor (GPCR), G-proteins, Ga protein Gpa2p, adenylyl cyclase, cAMP, and cAMP-dependent protein kinase A (PKA) [54, 55]. In S. cerevisiae, Gpa2p regulates cAMP levels [56, 57], which then modulate the activity of cAMP-dependent protein kinase (PKA); Tpk2p, one of the three catalytic subunits of PKA, specifically controls the filamentation response [58, 59].

Except for the S. cerevisiae GPCR, all other cAMP-PKA pathway proteins had homologs in A. nidulans (Table 3, Fig. 2). The absence of an A. nidulans homolog for the GPCR is not surprising as this gene shows no sequence homology with a functional homolog from S. pombe. Hence, A. nidulans may also have a GPCR functional homolog that shows little sequence homology to Gpr1.

It has been established that in A. nidulans, a pathway involving a heterotrimeric G protein a subunit encoded by fadA regulates mycelium proliferation and antagonizes conidiophore development [60]. Our results indicate that Gba1 encoded by fadA is homologous to Gpa2 of S. cerevisiae which is involved in the cAMP-mediated signal transduction mechanism [54, 55]. This implies that the cAMP-PKA pathway in A. nidulans functions in parallel with the filamentation-invasion pathway homolog to regulate conidiation. Also, protein kinase A regulatory subunit has been isolated in A. nidulans [30]. The possible involvement of a cAMP-dependent protein kinase, PkaC, in sporulation has been studied in the related fungus Aspergillus niger [61]. The cAMP signaling pathway in the filamentous fungus Neurospora crassa is known to regulate the conidiation, morphogenesis, mating and stress tolerance [14]. Hence the A. nidulans cAMP signaling pathway may regulate similar cellular responses. Together, these results imply the cAMP-PKA pathway in A. nidulans is involved in regulation of conidiation.

Table 3: S. cerevisiae cAMP-PKA pathway proteins and their A. nidulans homologs
(Click for viewing complete Table 3)


Figure 2: In silico reconstruction of the S. cerevisiae cAMP-PKA pathway [37, 54, 62] in A. nidulans. A. nidulans sequence homologs of S. cerevisiae proteins are listed in square brackets. | | | | Binding. Notations as in Fig. 1.


Since we observed the lack of sequence homolog for GPCR in A. nidulans which is similar to the S. pombe, we decided to identify the A. nidulans homologs for S. pombe cAMP-PKA pathway proteins. The results are shown in Table 4. As we can see from the results, the central part of the signaling mechanism is very well conserved between A. nidulans and S. pombe. This comparison between S. pombe and A. nidulans genome also doesn’t show a sequence homolog for the glucose receptor protein. This indicates that the glucose receptor protein molecule in A. nidulans doesn’t show a sequence homology with glucose receptor proteins in both S. pombe and S. cerevisiae. It has been established that the cAMP-PKA pathways in S. pombe and S. cerevisiae are very similar [14]. The lack of sequence homology of the glucose receptor proteins can mainly be attributed to asparagine-rich third intracellular loop found in S. cerevisiae Gpr1 and its absence in S. pombe Git3 . Hence, we can predict that the cAMP-PKA pathway in A. nidulans is also similar to the cAMP-PKA pathways in S. cerevisiae and S. pombe but has a glucose receptor protein which doesn’t show significant sequence homology. From our results in Table 3, we find that the all the three cAMP-dependent protein kinase subunits show only one sequence homolog in A. nidulans. The same protein from A. nidulans shows homology with the cAMP-dependent protein kinase catalytic subunit of S. pombe. This probably means that A. nidulans has only one cAMP-dependent protein kinase catalytic subunit similar to the S. pombe genome and not three cAMP-dependent protein kinase catalytic subunits as in the case of S. cerevisiae. Due to the probable high similarity of A. nidulans cAMP-PKA pathway to the S. pombe cAMP-PKA pathway we have reconstructed the S. pombe cAMP-PKA pathway in A. nidulans. This is shown in Fig. 3.

Table 4: S. pombe cAMP-PKA pathway proteins and their A. nidulans homologs
(Click for viewing complete Table 4)


Figure 3: In silico reconstruction of S. pombe cAMP-PKA pathway [14, 63] in A. nidulans. A. nidulans sequence homologs of S. pombe proteins are listed in square brackets. Notations as in Fig. 1.



Pheromone response pathway

In S. cerevisiae, the pheromone response pathway induces polarized growth towards the mating partner and cell cycle arrest [35]. In addition, expression of proteins needed for cell adhesion, cell fusion and nuclear fusion are increased [35]. While this is not a nutrient sensing pathway, we include it here because of its similarity to the filamentation-invasion pathway.

The S. cerevisiae pheromone response pathway is also a MAPK cascade, which uses components common to the filamentation-invasion pathway [14]. The Ste2p and Ste3p receptors sense the pheromone and activate the MAPK cascade through the upstream protein kinase Ste20p [35]. Ste20p activates Ste11p (MEKK), which phosphorylates Ste7p (MEK), which in turn phosphorylates Fus3p (MAPK). S. cerevisiae Fus3p regulates cell cycle arrest and the transcription of pheromone response genes [35]. In addition the pathway is controlled by a scaffold protein, Ste5p [35].

Our results show that 12 of the 23 S. cerevisiae proteins involved in the pheromone response pathway have significant A. nidulans homologs (Table 5, Fig. 4). In addition, eight other S. cerevisiae pheromone response pathway proteins had less conservative homologs. One of these was the scaffold protein Ste5p. This may imply that the A. nidulans pheromone response pathway is more closely related to that in S. pombe, where the MAPK pathway doesn’t have any scaffold protein [15]. The pheromone response pathway in A. nidulans likely regulates the transcription of genes involved in mating.

Table 5: S. cerevisiae pheromone response pathway proteins and their A. nidulans homologs
(Click for viewing complete Table 5)


Figure 4: In silico reconstruction of the S. cerevisiae pheromone response pathway [35] in A. nidulans. A. nidulans sequence homologs of S. cerevisiae proteins are listed in square brackets. Notation as in Fig. 1.


Since A. nidulans didn’t show a significant sequence homolog for the scaffold protein of S. cerevisiae, we wanted to compare the similarity between the pheromone response pathway in A. nidulans and S. pombe. The results obtained by comparing the pheromone response proteins in S. pombe with the A. nidulans genome is shown in Table 6. The results show that the S. pombe proteins which are involved in the MAPK cascade of pheromone response pathway [15] have significant homologs in A. nidulans. This appears to confirm our prediction that the MAPK cascade of the A. nidulans pheromone pathway may be more closely related to S. pombe than to S. cerevisiae. Our results in Table 6 show that almost all the proteins that are involved in S. pombe pheromone response pathway have significant homologs in A. nidulans. In contrast, most of the S. pombe proteins involved in mating and meiosis do not have significant homologs in A. nidulans. The probable A. nidulans pheromone response pathway is shown in Fig. 5.

Table 6: S. pombe pheromone response pathway proteins and their A. nidulans homologs
(Click for viewing complete Table 6)


Figure 5: In silico reconstruction of S. pombe pheromone response pathway [14, 15] in A. nidulans. A. nidulans sequence homologs of S. pombe proteins are listed in square brackets. Notation as in Fig. 1.



Cell integrity pathway

In S. cerevisiae, the cell integrity pathway regulates the expression of cell wall genes [35] and is stimulated by a variety of factors which include cell cycle regulation, heat stress, hypotonic stress and nutrient availability [35]. This pathway is also called as the Protein Kinase C pathway as it is mediated by members of the family of phospholipid-dependent, serine/threonine-specific protein kinases called protein kinase C. The cell integrity pathway in S. cerevisiae is highly conserved and it is very similar to the mammalian cell integrity pathway [35].

A number of membrane proteins, namely, Wsc1p, Wsc2p, Wsc3p, Wsc4p and Mid2p, have been reported to provide input signals to the cell integrity pathway [35, 64]. These signals activate the MAPK cascade which is mediated by the upstream protein kinase protein kinase C [35]. This kinase activates Bck1p (MEKK), which phosphorylates the functionally redundant threonine/tyrosine kinases Mkk1p and Mkk2p (MEK) [64, 65], which in turn phosphorylates Slt2p (MAPK). Slt2p regulates the transcription of cell wall genes [35]. In addition PKC may be activated through the Tor2p-Rom2p-Rho1p route [64].

Our results show that 15 of the 22 S. cerevisiae cell integrity pathway proteins have significant A. nidulans homologs (Table 7, Fig. 6). In addition, five other S. cerevisiae cell integrity pathway proteins show less conservative A. nidulans homologs.

Homolog of Rho1p in A. nidulans, RhoA has been isolated [66]. Pkc1 homolog Kpc1 has been cloned and characterized from A. niger [67] and it has been identified in A. nidulans based on similarity [34]. Our results show that AN4189.2 shows homology with both Mkk1p and Mkk2p, which act as the mitogen activated protein kinase kinases. This could be due to two reasons. First, since Mkk1p and Mkk2p have redundant functions [65] A. nidulans may use only one gene for the same purpose. Another possible explanation is that both Mkk1p and Mkk2p have high sequence homology and result in the same homolog in A. nidulans although there are two proteins present. Cell cycle regulation of cell integrity pathway is mediated by the activity of the cyclin-dependent kinase Cdc28p (CDK in Fig. 6, A. nidulans homolog is CDC2_EMENI) in complex with G1 cyclins. The downstream substrates of Slt2p are Rlm1p and SBF which regulate the transcription of cell wall related genes [35].

Table 7: S. cerevisiae cell integrity pathway proteins and their A. nidulans homologs
(Click for viewing complete Table 7)


Figure 6: In silico reconstruction of the S. cerevisiae cell integrity pathway [35] in A. nidulans. A. nidulans sequence homologs of S. cerevisiae proteins are listed in square brackets. CDK – cyclin dependent kinase. Notation as in Fig. 1.



Target of rapamycin pathway

The target of rapamycin (TOR) pathway is one of the central nutrient-sensing signal transduction pathways in eukaryotic cells [68] and is conserved from yeasts to humans [69]. In S. cerevisiae, TOR responds to nutrient limitation and mediates temporal control of cell growth by positively regulating anabolic processes such as translation, transcription and ribosome biosynthesis, and by negative regulation of catabolic processes such as protein and RNA degradation [70]. S. cerevisiae TOR also mediates spatial control of cell growth by regulating polarization of the actin cytoskeleton [71-73]. Furthermore TOR regulates the expression of proteins required for adaptation to starvation of carbon or nitrogen and utilization of poor nitrogen sources [70, 74]. Recent studies have shown that TOR signaling in S. cerevisiae is required for proper pseudohyphal development [69, 74]. However, it is not clear how TOR signaling affects the filamentation [74]. In S. cerevisiae, TOR also plays a key role in regulating autophagy [75-77] based on the nutrient availability.

In S. cerevisiae, TOR signaling consists of two TOR protein kinases namely Tor1p and Tor2p [68]. S. cerevisiae TOR kinases are activated by nutrient availability and are inactivated by either starvation or the presence of rapamycin [74]. Rapamycin inhibits TOR function by forming a complex with the prolyl isomerase FKBP12 and interacting with the TOR kinases at a conserved FRB (FKBP12-rapamycin binding) domain distinct from the kinase domain [74]. Other members of the TOR signaling cascade include type 2A protein phosphatases (PP2A), a type 2A related phosphatase Sit4p and Tap42p, a direct target of the S. cerevisiae TOR proteins [78]. The complex formed between Tap42p and the phosphatases play a central role in TOR signaling cascade [78]. TOR regulates the formation of these complexes by reversible phosphorylation and dephosphorylation of Tap42p [78].

We identified A. nidulans homologs for all the S. cerevisiae TOR signaling pathway proteins (Table 8, Fig. 7). We found only one homolog for both Tor1p and Tor2p of S. cerevisiae in A. nidulans genome. This is probably because A. nidulans is higher on the evolution ladder and it has been observed that in general higher organisms have a single TOR kinase [79-81].

Table 8: S. cerevisiae TOR pathway proteins and their A. nidulans homologs
(Click for viewing complete Table 8)


Figure 7: In silico reconstruction of the S. cerevisiae TOR signaling pathway [78] in A. nidulans. A. nidulans sequence homologs of S. cerevisiae proteins are listed in square brackets. Reversible arrows represent reversible phosphorylation/dephosphorylation. Notation as in Fig. 1.



Autophagy

Here we concentrate on one of the most important cellular responses to TOR signaling, namely the regulation of autophagy. Autophagy is a cellular response to nutrient starvation and is essential for cell survival under nutrient depleted conditions [82]. During autophagy, the bulk of the cytoplasmic components are non-selectively enclosed within a double-membrane structure called autophagosome, and this is transported in to the vacuole/lysosome to be degraded [83-85]. Autophagy is also characterized as type II programmed cell death and plays a critical role during development [77]. Autophagic dysfunction is also associated with various diseases [77]. For example, investigators have found a correlation between defects in autophagy and carcinogenesis [86]. In addition, defective autophagy is also correlated with numerous neurodegenerative diseases such as Huntington’s and Parkinson’s diseases [75].

The genes essential for autophagy, apg and aut genes, have been isolated [87, 88]. It has been shown that the apg mutant cells lose viability under nutrient starvation [87]. There are two types of autophagy: microautophagy and macroautophagy [77]. Microautophagy involves uptake of cytoplasm or whole organelles directly at the vacuole membrane [77]. While macroautophagy involves a sequestration event that is initiated separate from the vacuole membrane [77]. Macroautophagy can be broken down into a few basic steps: signaling, sequestration of cytoplasm, completion of vesicle formation, targeting of the completed vesicle to the lysosome/vacuole followed by docking and fusion, and breakdown (Fig. 8). The proteins involved in each of these steps have been identified in S. cerevisiae (Fig. 8). We note that homologs of the S. cerevisiae autophagy genes have been discovered in a wide range of cells including human, Drosophila, Dictyostelium, Arabidopsis, and Caenorhabditis elegans [89].

Our results show that 35 out of the 51 S. cerevisiae autophagy proteins have significant A. nidulans homologs (Table 9, Fig. 8). In addition, we observe that 9 S. cerevisiae autophagy proteins have less conservative homologs in A. nidulans. These results imply that A. nidulans autophagy is similar to S. cerevisiae autophagy. We also observe that with the exception of Apg7, none of the proteins involved in Apg12-Apg5 conjugation had a significant A. nidulans homolog. Whereas all the proteins involved in Aut7 lipid conjugation had a significant A. nidulans homolog.

Table 9: S. cerevisiae autophagy proteins and their A. nidulans homologs
(Click for viewing complete Table 9)


Figure 8: Schematic of the four major steps in the macroautophagy pathway in S. cerevisiae, adapted from [82]. Listed proteins are from S. cerevisiae [82], and correspond to each step in the pathway. Using these S. cerevisiae proteins, we have performed a BLAST search and identified homologous proteins in our model fungal strain (A. nidulans A4). Notation as in Fig. 1.



TOR-mediated regulation of autophagy

In S. cerevisiae, the rapamycin treatment mimics nutrient starvation [90] and induces autophagy showing that the TOR signaling cascade is involved in the regulation of autophagy. The mechanism by which the TOR pathway regulates autophagy is well-understood in S. cerevisiae [77] and appears to act by at least by two mechanisms [77].

A putative nutrient sensor on the cell surface appears to transduce the TOR pathway (Fig. 9). Under starvation conditions, Tor2 protein is inhibited, resulting in the dephosphorylation of Tap42 and its subsequent displacement from the protein phosphatase 2A catalytic subunits (i.e. Sit4) and their resulting activation [91]. These activated phosphatases control the activity of transcriptional activators (Fig. 9). Another mechanism of TOR regulation involves protein kinase Apg1 and phosphoprotein Apg13 [92], which are both involved in vesicle biogenesis. Under nutrient-rich conditions, Apg13 is hyper-phosphorylated and interacts weakly with Apg1. Inhibition of TOR under nutrient starvation leads to dephosphorylation of Apg13, thereby increasing its affinity to Apg1. Interaction with Apg13 is thought to regulate Apg1 kinase activity (Fig. 9).

Significant homologs for Sit4 (E-value 10-108) and TOR (E-value 0.00) are seen in A. nidulans genome (Table 8). Our results show the presence of a significant homolog for Apg1 kinase, but the homolog for Apg13 shows less homology (Table 9). This is not surprising because homologs of proteins that interact with Apg1, including Apg13, are absent in higher eukaryotes [77]. Hence, we assume this is the reason for the lack of a significant A. nidulans homolog for S. cerevisiae Apg13.


Figure 9: Schematic of Tor mediated regulation of autophagy, adapted from [76]. The sequence homologs of the S. cerevisiae protein in A. nidulans genome are listed in the square brackets. Notation as in Fig. 1.



Conclusions

Our results show that most of the signal transduction proteins in S. cerevisiae and S. pombe have significant homologs in A. nidulans indicating that the signal transduction pathways are well conserved. Our work has helped in identifying the probable signal transduction proteins in A. nidulans and in reconstructing the possible pathways using in silico techniques. These identifications can be used for further analysis of the signal transduction pathways. Based on the results obtained from our studies, further studies on deletion mutants can be performed to confirm function and physiological responses in A. nidulans under activation of the different signal transduction pathways. As we have seen from earlier studies [17, 18], the results obtained from computational techniques are highly reliable and have been experimentally validated. But our results had a few discrepancies. The most important was that some of the A. nidulans proteins showed significant homology with more than one yeast protein. For example, the mitogen-activated protein kinase in S. cerevisiae filamentation-invasion pathway, pheromone response pathway and cell integrity pathway have the same A. nidulans homolog. This is likely because the MAPKs have high sequence homology amongst themselves. This implies that some other A. nidulans proteins may act as the functional homolog of the S. cerevisiae MAPKs. On the whole, this study has provided insight on the signal transduction pathways involved in A. nidulans, a model filamentous fungus and will aid in understanding similar pathways in other industrially and medically significant Aspergilli.


Acknowledgements

We would like to thank Broad Institute of MIT and Harvard for allowing us to use the A. nidulans genome for performing in silico analysis of the genome sequence.



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