Computational modeling and in silico analysis of differential regulation of myo-inositol catabolic enzymes in Cryptococcus neoformans
© Mackenzie and Klig; licensee BioMed Central Ltd. 2008
Received: 29 March 2008
Accepted: 14 October 2008
Published: 14 October 2008
Inositol is a key cellular metabolite for many organisms. Cryptococcus neoformans is an opportunistic pathogen which primarily infects the central nervous system, a region of high inositol concentration, of immunocompromised individuals. Through the use of myo-inositol oxygenase C. neoformans can catabolize inositol as a sole carbon source to support growth and viability.
Three myo-inositol oxygenase gene sequences were identified in the C. neoformans genome. Differential regulation was suggested by computational analyses of the three gene sequences. This included examination of the upstream regulatory regions, identifying ORE/TonE and UASINO sequences, conserved introns/exons, and in frame termination sequences. Homology modeling of the proteins encoded by these genes revealed key differences in the myo-inositol active site.
The results suggest there are two functional copies of the myo-inositol oxygenase gene in the C. neoformans genome. The functional genes are differentially expressed in response to environmental inositol concentrations. Both the upstream regulatory regions of the genes and the structure of the specific proteins suggest that MIOX1 would function when inositol concentrations are low, whereas MIOX2 would function when inositol concentrations are high.
Myo-inositol, a key cellular metabolite, is a simple six carbon ring sugar with one hydroxyl group on each carbon. Myo-inositol is the precursor for the synthesis of phosphatidylinositol, an essential membrane lipid, an anchor for proteins, and a core component of signal transduction mechanisms [1, 2]. Inositol and compounds derived from inositol are among the major nonperturbing intracellular osmolytes which accumulate in response to hypertonic stress of the organism or tissue. Stepwise phosphorylation of inositol yields the myo-inositol polyphosphates. Inositol hexakisphosphate, in particular, has been found in soil, bacteria and most animals .
Cryptococcus neoformans is an opportunistic pathogen primarily infecting individuals with compromised immune systems. C. neoformans is found worldwide in soil and pigeon droppings. Under environmentally dry conditions, the quiescent fungal spores in the soil or pigeon guano can become airborne. Once in the air mammals can inhale the dehydrated yeast spores. The immune system of a non-immunocompromised individual typically eliminates C. neoformans with out any symptoms of disease. If the host is immunocompromised the pathogen can cause cryptococcosis. C. neoformans infections often localize to the brain and central nervous system (CNS) . C. neoformans is unusual among the fungi in that the pathogen can use inositol as a sole carbon source to support growth. Catabolism of myo-inositol in C. neoformans is through the action of myo-inositol oxygenase (MIOX), though this has not been confirmed as the only pathway . The inositol concentration in the cerebral spinal fluid (CSF) is high (when compared to plasma levels ). C. neoformans localized in the CNS could utilize myo-inositol as a substrate for myo-inositol oxygenase (MIOX) in order to generate glucuronic acid for energy production.
The conversion of myo-inositol to glucuronic acid by MIOX involves the cleavage of the inositol ring between the 6 C and 1 C, and a four-electron transfer with 1 atom of oxygen incorporated into the glonate, xylulose and xyulose-5-phosphate which can then enter the pentose phosphate pathway resulting in energy production.
This enzyme has been extensively studied in many eukaryotic organisms, recently crystallized, and modeled [6–9]. However, regulation of the MIOX protein in any organism has only recently been examined. Transcription of the MIOX gene in humans has been shown to respond to osmotic response element (ORE) binding proteins and/or the tonicity-responsive enhancer (TonE) binding protein through conserved motifs [7, 10]. Expression of genes regulated by ORE/TonE binding proteins have been shown to increase when an AP-1 protein binding sequence is located downstream. In conditions of high osmolarity, this AP-1 mediated increase in transcription is inhibited by A-Fos or Tam-67 . The promoter of a renal specific oxidoreductase with increased expression in diabetes mellitus, that has been experimentally determined to respond to inositol in media, also contains the conserved ORE motif GGAAA . An additional transcriptional regulating sequence, known as an inositol upstream activation sequence (UASINO), has the conserved core sequence of CANNTG and has been identified upstream of several genes encoding proteins involved in phospholipid metabolism .
In this study, three genes on separate chromosomes, encoding the MIOX protein, were identified in the C. neoformans genome. The MIOX promoter region, transcriptional regulatory sequences and myo-inositol binding pocket in C. neoformans were characterized. Examination of the genes revealed differential regulation. This examination includes identification of upstream regulatory sequences such as ORE, UASINO, and TATA boxes, introns, in-frame termination sequences, expressed sequence tags (ESTs) and CpG islands for each sequence. Molecular modeling of the three protein sequences indicated key differences between the isoforms possibly affecting the ability of two isoforms to bind the myo-inositol substrate.
MIOX Gene Identification and Characterization
C. neoformans Upstream Regulatory Sequences.
ORE/TonE Sequence location
Identification of Expressed Sequence Tags That Align with C. neoformans MIOX Gene Sequences.
Expressed sequence Tags (EST)
-184–37, 86–264, 322–498
540–764, 823–947, 1007–1058
The genomic region (TIGR189.m00292 plus +/- 3000 extracted from chr09.b3501.040506 STGC) upstream of the translational start of MIOX2 contains two conserved ORE sequences AGGAAAGCTG and TGGAAAACTG located at -3714 and -3636. Neither sequence was followed by a conserved AP-1 sequence (Table 1). Two ESTs, from the TIGR database were found to align with the coding region of MIOX2. EST d4e08j2.r1 aligns to bases -184–37, 86–264 and 322–498 (98%). EST a9e06cn.r1 aligns with nucleotides 664–846, and 900–1107 (96%) (Table 2). Three introns with GT/AG splice sites were confirmed by comparison of the genomic sequence to ESTs. The AG at the 3'end of the splice site of a fourth computationally predicted intron was confirmed by comparison of the genomic sequence to EST d4e08j2.r1. The GT at the 5' end of this intron has not yet been confirmed by EST data. Four in frame termination signals were identified.
Analysis of the genomic region (TIGR 177.m03138 plus 3000 +/- extracted from STGC chr07.b3501.040506) of MIOX3 upstream from the transcriptional start site for the MIOX3 protein has several computationally identified possible ORE sequences. However, none of the sequences are strictly identical to the experimentally determined ORE sequences and none are followed by a known AP-1 binding site. The MIOX3 sequence was determined to contain only one TATA box starting at nucleotide 27 (Table 1). No corresponding ESTs were identified in the TIGR C. neoformans database with an alignment better than 65% (Table 2). Computational analysis, along with manual alignment of the coding DNA from TIGR database to the genomic sequence, predicted four introns in MIOX3. MIOX3 genomic 3'sequence has four in frame termination signals.
MIOX1 and MIOX2 have a relatively few CpG islands when compared to MIOX3. MIOX3 has five CpG islands in the upstream region that are approximately evenly spaced and several islands located internal to the gene. The five upstream CpG islands are located at -(51..327), -(752..1006), -(1318..1661), -(1746..2007) and -(2817..3080). MIOX2 has 2 CpG islands located from -(457..684), and -(2555..2754). MIOX1 has three CpG islands. One island is close to the transcriptional start located at -(48–373). The other two CpG islands identified in the MIOX1 gene are further from the start site located at -(2325..2545) and -(2609..2881).
As RNA stabilization can effect functional expression, the predicted RNAs were examined. Secondary and tertiary analysis of the computationally predicted RNA showed no significant variance between the corresponding structures of the three genes (data not shown).
MIOX Sequence alignment and Homology Modeling
Relative Positions of Amino Acids Involved in the Predicted Hairpin Loop Stabilizing the Binding of the Myo-Inositol Substrate.
Amino Acids Involved in Salt-Bridges that Stabilize the myo-Inositol Substrate Lid.
Stabilizing Amino Acids Involved in Main-Chain Hydrogen Bonds.
C. neoformans appears to be the only organism in the animal and fungal kingdoms with multiple MIOX genes. Examination of over 60 completed eukaryotic genomes from the animal and fungal kingdoms revealed that if the MIOX gene is present, there is only one highly conserved copy (data not shown). Perhaps the three copies of the MIOX gene in the C. neoformans genome represents a physiological mechanism for survival in various environmental inositol concentrations.
This computational study suggests there are at least two sequences regulating transcription of the C. neoformans MIOX genes, one involves ORE sequences, the other involves UASINO sequences. ORE sequences were originally identified in vertebrates and UASINO sequences were demonstrated in the yeast Saccharomyces cerevisiae, but these sequences are present in the C. neoformans genome. As a basidiomycete the C. neoformans genome has been shown to contain features similar to other yeasts yet its gene organization is more complex resembling higher eukaryotes . Both MIOX1 and MIOX2 have two ORE/TonE sequences upstream of the genes. MIOX protein expression in humans has been demonstrated to be regulated by ORE/TonE binding proteins [7, 10]. In vertebrates, the experimentally demonstrated binding sites for each transcription factor are slightly different. The core sequence TGGAAA is recognized by ORE binding protein, whereas the core sequence GGAAAA is recognized by TonE binding protein (also known as NFAT5). Interestingly, comparison of the C. neoformans promoter regions of MIOX1 and MIOX2 reveals nucleotide differences in the identified ORE/TonE sequences suggesting differential regulation of the two genes. MIOX1 has two ORE like sequences with the conserved TGGAAA sequence coupled with an AP-1 binding sequence. This would render MIOX1 subject to A-Fos and Tam-67 inhibition. Two TonE like sequences are located in MIOX2 however, no AP-1 sequence was identified. The presence of the TonE and the absence of the AP-1 binding sequences suggests MIOX2 is up-regulated in the presence of inositol but is not subject to inhibition by A-Fos or Tam-67 . Unlike MIOX1 and MIOX2, the MIOX3 gene lacked a conserved ORE/TonE sequence within 3000 base pairs upstream of the transcriptional start site suggesting MIOX3 is not regulated by the ORE or TonE transcription factors.
In addition to the ORE and AP-1 sequences, MIOX1 also contains a UASINO sequence. Several C. neoformans genes involved in phospholipid and phospholipid precursor biosynthesis (including MIOX) have been experimentally found to be regulated by the availability of inositol and choline in the medium [4, 12] Studies of the transcriptional control coordinating expression of these genes in S. cerevisiae led to the identification of an upstream activating sequence (UAS) with a core conserved sequence of CANNTG [1, 15]. The UASINO has been found to specifically regulate genes via the INO2p/INO4p bHLH transcription factor complex in response to inositol and choline . The UASINO sequence experimentally demonstrated to serve for transcription activation is CAT(G/A)TGAA(G/A/T)(T/A) . The presence of a UASINO in the upstream region of the C. neoformans MIOX1 but not MIOX2 or MIOX3 genes further suggests that the genes are differentially regulated. Moreover, MIOX1 may be active when inositol concentration levels are low.
Differential regulation is further supported by the identification of expressed sequence tags (ESTs) for MIOX1 and MIOX2 but not MIOX3. ESTs identified for both MIOX1 and MIOX2 in this study confirmed the computationally predicted start sites. Furthermore the splice site junctions for MIOX1 and MIOX2 are supported by alignment of ESTs to genomic DNA. ESTs for MIOX3 were not found however, the splice sites computationally predicted for MIOX3 are identical to the splice sites predicted by ESTs alignment for MIOX2. Duplicated nonfunctional genes are less likely to have ESTs . Unexpressed genes, genes expressed at very low levels or only under specific conditions are also less likely to have associated ESTs/cDNAs. Therefore the absence of ESTs for MIOX3 suggests either the gene is only expressed under specific conditions or is not expressed at all.
The identity of the MIOX2 and MIOX3 coding sequences (80%) is significantly less than that of their amino acids (91%). This can be attributed to changes in the wobble position. Manual analysis of the aligned sequences revealed the majority (78%) of the base changes to be in the wobble position. The synonymous changes allowed the identity of the DNA sequences to decrease but did not change most of the amino acids encoded, thereby retaining the protein sequence identity. The conservation of the amino acid sequence despite changes in the DNA suggests that the function of the protein is conserved through selective pressure.
The MIOX substrate, myo-inositol, is held in the active site by several key interactions. Three salt bridges created between Asp-85/Lys127, Asp-88/Lys257 and Asp92/Arg-39 form a lid that holds the substrate in the active site, along with main-chain H-bonds with Thr-32, Arg-39 and Gln-136 that stabilize the active site. All residues, essential in myo-inositol binding were found to be conserved in C. neoformans MIOX1 except Thr-32 and Asp-92. Optimal alignment of the Mus musculus and C. neoformans MIOX1 protein sequences indicates a conservative substitution of Thr-32 to Glu and Asp-92 to Ser. Thr and Glu are both neutral residues and capable of participating in hydrogen bonds, indicating that the main chain hydrogen bonds found in Mus musculus are all conserved in MIOX1. Asp-92 interacts with Arg-39 to form a salt bridge (dist 2.98Å) in the Mus musculus MIOX protein. Substitution of Ser for Asp-92 in C. neoformans MIOX1 potentially disrupts the salt-bridge formation however; the homology model generated in this study of MIOX1 indicates the distance between the amino acids (3.44Å) supports the formation of a salt bridge at that location despite the substitution.
Protein sequence alignment and the homology models generated for the MIOX2 and MIOX3 proteins of C. neoformans also indicate three key differences from the mouse MIOX protein. Unlike the changes in the MIOX1 protein where the active site is preserved, mutations in the MIOX2 and MIOX3 residues suggest disruption of the active site for myo-inositol possibly leading to decreased protein activity. Optimal alignment of the mouse MIOX and C. neoformans MIOX2 and MIOX3 protein sequences indicates substitutions of Thr-32 to Val and Asp-92 to Ser. The change from Thr to Val disrupts one of the three main-chain hydrogen bonds that helps stabilize the myo-inositol lid. The loss of the H-bond between Val and the main chain could be the cause of the increased distance between the Ser and Arg salt bridge (12.15Å) making the formation of a salt bridge at that location beyond the acceptable range. The destabilization of the lid may not allow MIOX2 and MIOX3 proteins to function efficiently.
Multiple copies of the MIOX gene is unique to C. neoformans among the animal and fungal kingdoms. This study suggests that the C. neoformans genome has multiple copies of the MIOX gene which appear to be differentially expressed under various physiological inositol conditions. MIOX1 protein is predicted to be efficient in binding the myo-inositol substrate . MIOX1 gene expression is probably active in conditions of low inositol via a UASINO, up-regulated in the presence of inositol, yet possible inhibited by high levels of inositol by A-Fos or Tam-67 at the AP-1 site. This inhibition may be compensated for by the up-regulation of MIOX2. Although the MIOX2 enzyme is not predicted to be as efficient at holding the myo-inositol substrate in the active site, due to loss of disulfide bridges between the lid and main-chain and loss of main-chain hydrogen bonds, expression of MIOX2 should not be inhibited in elevated inositol levels due to the absence of an AP-1 binding sequence. This differentiated regulation of inositol catabolism could facilitate the growth and viability of C. neoformans in various environments.
MIOX Gene Identification and Characterization
To locate the MIOX gene within the Cryptococcus neoformans genome the N-terminus region of MIOX1 protein (previously isolated in this laboratory) was submitted as a query search to the TBLASTN program via BLAST at the Stanford Genome Technology Center (STGC). A sequence with 100% identity to the MIOX protein N-terminus region plus 3000 bases +/- was then used to search for cDNA's and ESTs in the TIGR C. neoformans gene indices database using BLAST (blastn), and the were aligned with GAP from the Wisconsin sequencing package on the W-H2 server (GCG). Possible open reading frames (ORF) were located using Map (GCG) Translate (GCG), GENSCAN , and ORF Finder . Putative promoter regions were delineated by Promoter Prediction , WWW Promoter Scan  HCtata  Genie  and NetStart 1.0  and manual examination. Exon boundaries were predicted using GAP (GCG), NetGene2 , Splice Site Prediction , Genie  and GENSCAN. ORE sequences and UASino sequences were determined by manual analysis of the upstream regions of each MIOX gene sequence. CpG islands were identified using the Vista Genome  and European Molecular Biology Laboratory servers .
MIOX Secondary Structure Prediction
Predictions of protein secondary structures for MIOX1, MIOX2, MIOX3 were computed using J-pred , PredictProtein , PSIPRED , Discrimination of protein Secondary structure Class (DSC) , Hydrophobic cluster analysis (HCA) , PSSFinder , and SAM_T02  methods. The results from each method were compared and a consensus structure for each MIOX protein was generated based on regions of similarity. Consensus secondary structure sequences for each MIOX protein were generated by comparing results obtained by all servers, and taking into account only the sequence regions that were predicted at least 50% reliability or higher by at least five servers. All alignments were generated with CLUSTALW or GAP then edited manually using Bioedit .
Template Identification and Protein Modeling
The SWISS-MODEL Comparative Protein Modeling Server , along with Modeler within the Accelerys Insight II program suite, were utilized to generate 3D-models of the putative MIOX proteins. The MIOX protein from Mus musculus (PDB code: 2HUO) was used as a template. Each C. neoformans MIOX protein was manually aligned to the template then submitted to the server. The N-terminal regions of the proteins have a high degree of variation therefore the N-terminal region for each protein was not modeled due to lack of a template. One model was generated in a fully automated way for each MIOX sequence based on user defined alignment then verified using PROCHECK .
The authors are indebted to Beth Eldon and Ilva Cabrera for their comments on the manuscript. These studies were supported by grants from the National Institutes of Health (AI43025) and the Keck foundation.
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