The signal peptide peptidase SppA is involved in sterol regulatory element-binding protein cleavage and hypoxia adaptation in Aspergillus nidulans.

Using forward genetics, we revealed that the signal peptide peptidase (SPP) SppA, an aspartyl protease involved in regulated intramembrane proteolysis (RIP), is essential for hypoxia adaptation in Aspergillus nidulans, as well as hypoxia-sensitive mutant alleles of a sterol regulatory element-binding protein (SREBP) srbA and the Dsc ubiquitin E3 ligase complex dscA-E. Both null and dead activity [D337A] mutants of sppA failed to grow in hypoxia, and the growth defect of ΔsppA was complemented by nuclear SrbA-N381 expression. Additionally, SppA interacted with SrbA in the endoplasmic reticulum, where SppA localized in normoxia and hypoxia. Expression of the truncated SrbA-N414 covering the SrbA sequence prior to the second transmembrane region rescued the growth of ΔdscA but not of ΔsppA in hypoxia. Unlike ΔdscA and ΔdscA;ΔsppA double mutants, in which SrbA cleavage was blocked, the molecular weight of cleaved SrbA increased in ΔsppA compared to the control strain in immunoblot analyses. Overall, our data demonstrate the sequential cleavage of SrbA by Dsc-linked proteolysis followed by SppA, proposing a new model of RIP for SREBP cleavage in fungal hypoxia adaptation. Furthermore, the function of SppA in hypoxia adaptation was consistent in Aspergillus fumigatus, suggesting the potential roles of SppA in fungal pathogenesis.

Genome-Wide Analysis of Hypoxia-Responsive Genes in the Rice Blast Fungus, Magnaporthe oryzae.

Rice blast fungus, Magnaporthe oryzae, is the most destructive pathogen in the rice-growing area. This fungus has a biotrophic phase early in infection and later switches to a necrotrophic lifestyle. During the biotrophic phase, the fungus competes with its host for nutrients and oxygen. Continuous uptake of oxygen is essential for successful establishment of blast disease of this pathogen. Here, we report transcriptional responses of the fungus to oxygen limitation. Transcriptome analysis using RNA-Seq identified that 1,047 genes were up-regulated in response to hypoxia. Those genes are involved in mycelial development, sterol biosynthesis, and metal ion transport based on hierarchical GO terms, and are well-conserved among three fungal species. In addition, null mutants of two hypoxia-responsive genes were generated and their roles in fungal development and pathogenicity tested. The mutant for the sterol regulatory element-binding protein gene, MoSRE1, exhibited increased sensitivity to a hypoxia-mimicking agent, increased conidiation, and delayed invasive growth within host cells, which is suggestive of important roles in fungal development. However, such defects did not cause any significant decrease in disease severity. The other null mutant, for the alcohol dehydrogenase gene MoADH1, showed no defect in the hypoxia-mimicking condition (using cobalt chloride) and fungal development. Taken together, this comprehensive transcriptional profiling in response to a hypoxic condition with experimental validations would provide new insights into fungal development and pathogenicity in plant pathogenic fungi.

Autoregulation of ZEB2 expression for zearalenone production in Fusarium graminearum.

Several Fusarium species produce the polyketide mycotoxin zearalenone (ZEA), a causative agent of hyperestrogenic syndrome in animals that is often found in F. graminearum-infected cereals in temperate regions. The ZEA biosynthetic cluster genes PKS4, PKS13, ZEB1 and ZEB2 encode a reducing polyketide synthase, a non-reducing polyketide synthase, an isoamyl alcohol oxidase and a transcription factor respectively. In this study, the production of two isoforms (ZEB2L and ZEB2S) from the ZEB2 gene in F. graminearum via an alternative promoter was characterized. ZEB2L contains a basic leucine zipper (bZIP) DNA-binding domain at the N-terminus, whereas ZEB2S is an N-terminally truncated form of ZEB2L that lacks the bZIP domain. Interestingly, ZEA triggers the induction of both ZEB2L and ZEB2S transcription. ZEB2L and ZEB2S interact with each other to form a heterodimer that regulates ZEA production by reducing the binding affinity of ZEB2L for the ZEB2L gene promoter. Our study provides insight into the autoregulation of ZEB2 expression by alternative promoter usage and a feedback loop during ZEA production; this regulatory mechanism is similar to that observed in higher eukaryotes.

FgFlbD regulates hyphal differentiation required for sexual and asexual reproduction in the ascomycete fungus Fusarium graminearum.

Fusarium graminearum is a filamentous fungal plant pathogen that infects major cereal crops. The fungus produces both sexual and asexual spores in order to endure unfavorable environmental conditions and increase their numbers and distribution across plants. In a model filamentous fungus, Aspergillus nidulans, early induction of conidiogenesis is orchestrated by the fluffy genes. The objectives of this study were to characterize fluffy gene homologs involved in conidiogenesis and their mechanism of action in F. graminearum. We characterized five fluffy gene homologs in F. graminearum and found that FlbD is the only conserved regulator for conidiogenesis in A. nidulans and F. graminearum. Deletion of fgflbD prevented hyphal differentiation and the formation of perithecia. Successful interspecies complementation using A. nidulans flbD demonstrated that the molecular mechanisms responsible for FlbD functions are conserved in F. graminearum. Moreover, abaA-wetA pathway is positively regulated by FgFlbD during conidiogenesis in F. graminearum. Deleting fgflbD abolished morphological effects of abaA overexpression, which suggests that additional factors for FgFlbD or an AbaA-independent pathway for conidiogenesis are required for F. graminearum conidiation. Importantly, this study led to the construction of a genetic pathway of F. graminearum conidiogenesis and provides new insights into the genetics of conidiogenesis in fungi.

WetA is required for conidiogenesis and conidium maturation in the ascomycete fungus Fusarium graminearum.

Fusarium graminearum, a prominent fungal pathogen that infects major cereal crops, primarily utilizes asexual spores to spread disease. To understand the molecular mechanisms underlying conidiogenesis in F. graminearum, we functionally characterized the F. graminearum ortholog of Aspergillus nidulans wetA, which has been shown to be involved in conidiogenesis and conidium maturation. Deletion of F. graminearum wetA did not alter mycelial growth, sexual development, or virulence, but the wetA deletion mutants produced longer conidia with fewer septa, and the conidia were sensitive to acute stresses, such as oxidative stress and heat stress. Furthermore, the survival rate of aged conidia from the F. graminearum wetA deletion mutants was reduced. The wetA deletion resulted in vigorous generation of single-celled conidia through autophagy-dependent microcycle conidiation, indicating that WetA functions to maintain conidial dormancy by suppressing microcycle conidiation in F. graminearum. Transcriptome analyses demonstrated that most of the putative conidiation-related genes are expressed constitutively and that only a few genes are specifically involved in F. graminearum conidiogenesis. The conserved and distinct roles identified for WetA in F. graminearum provide new insights into the genetics of conidiation in filamentous fungi.

Caspase-mediated cleavage and DNase activity of the translation initiation factor 3, subunit G (eIF3g).

Eukaryotic translation initiation factor 3 is composed of 13 subunits (eIF3a through eIF3m) and plays an essential role in translation. During apoptosis, several caspases rapidly down-regulate protein synthesis by cleaving eIF4G, -4B, -3j, and -2α. In this study, we found that the activation of caspases by cisplatin in T24 cells induces the cleavage of subunit G of the eIF3 complex (eIF3g). The cleavage site (SLRD(220)G) was identified, and we found that the cleaved N-terminus was translocated to the nucleus, activating caspase-3, and that it also showed a strong DNase activity. These data demonstrate the important roles of eIF3g in the translation initiation machinery and in DNA degradation during apoptosis.

AbaA regulates conidiogenesis in the ascomycete fungus Fusarium graminearum.

Fusarium graminearum (teleomorph Gibberella zeae) is a prominent pathogen that infects major cereal crops such as wheat, barley, and maize. Both sexual (ascospores) and asexual (conidia) spores are produced in F. graminearum. Since conidia are responsible for secondary infection in disease development, our objective of the present study was to reveal the molecular mechanisms underlying conidiogenesis in F. graminearum based on the framework previously described in Aspergillus nidulans. In this study, we firstly identified and functionally characterized the ortholog of AbaA, which is involved in differentiation from vegetative hyphae to conidia and known to be absent in F. graminearum. Deletion of abaA did not affect vegetative growth, sexual development, or virulence, but conidium production was completely abolished and thin hyphae grew from abnormally shaped phialides in abaA deletion mutants. Overexpression of abaA resulted in pleiotropic defects such as impaired sexual and asexual development, retarded conidium germination, and reduced trichothecene production. AbaA localized to the nuclei of phialides and terminal cells of mature conidia. Successful interspecies complementation using A. nidulans AbaA and the conserved AbaA-WetA pathway demonstrated that the molecular mechanisms responsible for AbaA activity are conserved in F. graminearum as they are in A. nidulans. Results from RNA-sequencing analysis suggest that AbaA plays a pivotal role in conidiation by regulating cell cycle pathways and other conidiation-related genes. Thus, the conserved roles of the AbaA ortholog in both A. nidulans and F. graminearum give new insight into the genetics of conidiation in filamentous fungi.

NDRG2 and PRA1 interact and synergistically inhibit T-cell factor/ß-catenin signaling.

NDRG2 is a member of the N-myc downstream regulated gene (NDRG) family, implicated in cell growth and differentiation. Investigation of NDRG2 molecular interactions by yeast two-hybrid screening identified prenylated Rab acceptor-1 (PRA1), involved in vesicle trafficking and protein transport, as binding partner. Binding of NDRG2 (and NDRG1-4) with PRA1 in vitro was confirmed by GST pull-down assay and immunoprecipitation, and colocalization was verified by confocal microscopy in HCT116 cells. Intracellular coexpression showed that NDRG2 and PRA1 synergistically downregulate T-cell factor (TCF) promoter activity and GSK3ß phosphorylation. Results suggest that NDRG2 and PRA1 might act synergistically to prevent signaling of TCF/ß-catenin.

Polyamine biosynthesis regulated by StARD expression plays an important role in potato wound periderm formation.

An acireductone dioxygenase (ARD) gene of potatoes was isolated from the expressed sequence tags (ESTs) of potato post-suberization cDNA libraries. The highest expression levels of the StARD gene and the protein appeared 36 h after suberization. An approximate 9-fold increase in ARD activity was detected at 36 h after wounding. Real-time reverse transcription-PCR (RT-PCR) analysis and immunolocalization studies revealed that StARD transcripts increase at the wound surface of potato tubers. The polyamine (PA) contents increased significantly after wounding at the wound surface. The increased PA content and ARD activity may play an important role in wound periderm formation.

uvsF RFC1, the large subunit of replication factor C in Aspergillus nidulans, is essential for DNA replication, functions in UV repair and is upregulated in response to MMS-induced DNA damage.

uvsF201 was the first highly UV-sensitive repair-defective mutation isolated in Aspergillus nidulans. It showed epistasis only with postreplication repair mutations, but caused lethal interactions with many other repair-defective strains. Unexpectedly, closest homology of uvsF was found to the large subunit of human DNA replication factor RFC that is essential for DNA replication. Sequencing of the uvsF201 region identified changes at two close base pairs and the corresponding amino acids in the 5'-region of uvsF(RFC1). This viable mutant represents a novel and possibly important type. Additional sequencing of the uvsF region confirmed a mitochondrial ribosomal protein gene, mrpA(L16), closely adjacent, head-to-head with a 0.2kb joint promoter region. MMS-induced transcription of both the genes, but especially uvsF(RFC1), providing evidence for a function in DNA damage response.

Phospholipase C-epsilon augments epidermal growth factor-dependent cell growth by inhibiting epidermal growth factor receptor down-regulation.

The down-regulation of the epidermal growth factor (EGF) receptor is critical for the termination of EGF-dependent signaling, and the dysregulation of this process can lead to oncogenesis. In the present study, we suggest a novel mechanism for the regulation of EGF receptor down-regulation by phospholipase C-epsilon. The overexpression of PLC-epsilon led to an increase in receptor recycling and decreased the down-regulation of the EGF receptor in COS-7 cells. Adaptor protein complex 2 (AP2) was identified as a novel binding protein that associates with the PLC-epsilon RA2 domain independently of Ras. The interaction of PLC-epsilon with AP2 was responsible for the suppression of EGF receptor down-regulation, since a perturbation in this interaction abolished this effect. Enhanced EGF receptor stability by PLC-epsilon led to the potentiation of EGF-dependent growth in COS-7 cells. Finally, the knockdown of PLC-epsilon in mouse embryo fibroblast cells elicited a severe defect in EGF-dependent growth. Our results indicated that PLC-epsilon could promote EGF-dependent cell growth by suppressing receptor down-regulation.

Siah proteins induce the epidermal growth factor-dependent degradation of phospholipase Cepsilon.

Phospholipase Cepsilon (PLCepsilon) is activated by various growth factors or G-protein-coupled receptor ligands via different activation mechanisms. The Ras association (RA) domain of PLCepsilon is known to be important for its ability to bind with Ras-family GTPase upon growth factor stimulation. In the present study, we identified Siah1 and Siah2 as novel binding partners of the PLCepsilon RA domain. Both Siah1 and Siah2 interacted with the RA2 domain of PLCepsilon, and the mutation of Lys-2186 of the PLCepsilon RA2 domain abolished this association. Moreover, Siah induced the ubiquitination and degradation of PLCepsilon upon epidermal growth factor (EGF) stimulation, and Siah proteins were phosphorylated on multiple tyrosine residues via an Src-dependent pathway upon EGF treatment. The Src inhibitor abolished the EGF-dependent ubiquitination of PLCepsilon, and the Siah1 phosphorylation-deficient mutant could not increase the EGF-dependent ubiquitination and degradation of PLCepsilon. The EGF-dependent degradation of PLCepsilon was blocked in mouse embryonic fibroblast (MEF) cells derived from Siah1a/Siah2 double knockout mice, and the extrinsic expression of wild-type Siah1 restored the degradation of PLCepsilon, whereas the phosphorylation-deficient mutant did not. Siah1 expression abolished PLCepsilon-dependent potentiation of EGF-dependent cell growth. In addition, the expression of wild-type Siah1 in Siah1a/Siah2-double knockout MEF cells inhibited EGF-dependent cell growth, and this inhibition was abolished by PLCepsilon knockdown. Our results suggest that the Siah-dependent degradation of PLCepsilon plays a role in the regulation of growth factor-dependent cell growth.

Apoptosis-inducing factor (AIF) inhibits protein synthesis by interacting with the eukaryotic translation initiation factor 3 subunit p44 (eIF3g).

Apoptosis-inducing factor (AIF) is a ubiquitous FAD-binding flavoprotein comprised of 613 amino acids and plays an important role in caspase-independent apoptosis. During apoptotic induction, AIF is translocated from the mitochondrial intermembrane space to the nucleus, where it interacts with DNA and activates a nuclear endonuclease. By performing a yeast two-hybrid screen with mature AIF, we have isolated the eukaryotic translation initiation factor 3 subunit p44 (eIF3g). Our deletion mutant analysis revealed that the eIF3g N-terminus interacts with the C-terminal region of AIF. The direct interaction between AIF and eIF3g was confirmed in a GST pull-down assay and also verified by the results of co-immunoprecipitation and confocal microscopy studies. Using an in vitro TNT coupled transcription-translation system, we found that mature AIF could inhibit newly-translated protein synthesis and this inhibition was significantly blocked by eIF3g competitively. These results were also confirmed in cells. In addition, mature AIF overexpression specifically resulted in the activation of caspase-7, thereby amplifying the inhibition of protein synthesis including eIF3g cleavage. Our data suggest that eIF3g is one of the cytosolic targets that interacts with mature AIF, and provide insight into the AIF's cellular functions of the inhibition of protein synthesis during apoptosis.

Prenylated Rab acceptor 1 (PRA1) inhibits TCF/beta-catenin signaling by binding to beta-catenin.

The prenylated Rab acceptor 1 (PRA1) is a ubiquitously expressed 21 kDa protein containing two transmembrane domains that possibly induce its localization to the Golgi complex. It binds to prenylated Rab GTPases and VAMP2. In this study, we report that PRA1-overexpressing cells exhibited a significantly retarded growth rate as compared to that of the mock-transfected cells, and the transcriptional activity of TCF, as evaluated by TOPflash luciferase reporter assay, was profoundly reduced in the PRA1-overexpressed cells. These intracellular functions of PRA1 were verified by introducing deletion mutant or site-directed mutants, or small interfering RNA of PRA1. In addition, the translocation of beta-catenin from the cytosol to the nucleus was blocked to a significant degree in the PRA1-cells, and the interaction of PRA1 and beta-catenin was identified by confocal microscopy and immunoprecipitation analysis. Finally, we observed that the inhibition of TCF/beta-catenin signaling by PRA1 is associated with ERK1/2 dephosphorylation. Therefore, our data suggest that the in vivo modulation of PRA1 may be involved in TCF/beta-catenin signaling, as well as cellular proliferation and tumorigenesis.

Redox-regulated cochaperone activity of the human DnaJ homolog Hdj2.

The human DnaJ homolog Hdj2 is a cochaperone containing a cysteine-rich zinc finger domain. We identified a specific interaction of Hdj2 with the cellular redox enzyme thioredoxin using a yeast two-hybrid assay and a coimmunoprecipitation assay, thereby investigating how the redox environment of the cell regulates Hdj2 function. In reconstitution experiments with Hsc70, we found that treatment with H2O2 caused the oxidative inactivation of Hdj2 cochaperone activity. Hdj2 inactivation paralleled the oxidation of cysteine thiols and concomitant release of coordinated zinc, suggesting a role of cysteine residues in the zinc finger domain of Hdj2 as a redox sensor of chaperone-mediated protein-folding machinery. H2O2-induced negative regulation of Hdj2 cochaperone activity was also confirmed in mammalian cells using luciferase as a foreign reporter cotransfected with Hsc70 and Hdj2. The in vivo oxidation of cysteine residues in Hdj2 was detected only in thioredoxin-knockdown cells, implying that thioredoxin is involved in the in vivo reduction. The oxidative inactivation of Hdj2 was reversible. Wild-type thioredoxin notably recovered the oxidatively inactivated Hdj2 activity accompanied by the reincorporation of zinc, whereas the catalytically inactive mutant thioredoxin (Cys32Ser/Cys35Ser) did not. Taken together, we propose that oxidation and reduction reversibly regulate Hdj2 function in response to the redox states of the cell.

Upregulation of Daxx mediates apoptosis in response to oxidative stress.

Oxidative stress induces apoptosis in a variety of cell types by as yet unclear signaling mechanisms. The Daxx protein is reportedly involved in apoptosis through its interactions with Fas, transforming growth factor-beta receptor, and promyelocytic leukemia protein (PML). Here, we explored the possible roles of Daxx in oxidative stress-induced apoptosis. We found that both the mRNA and protein levels of Daxx markedly increased when cells underwent apoptosis after H2O2 treatment. Pretreatment with the cell-permeable antioxidant, N-acetyl cysteine, prevented cells from H2O2-induced Daxx upregulation and subsequent apoptosis, indicating that the endogenous oxidant regulated Daxx expression. Furthermore, suppression of endogenous Daxx expression by antisense oligonucleotide technology inhibited oxidative stress-induced apoptosis in HeLa cells. Taken together, these results suggest that Daxx acts as an intermediary messenger of pro-apoptotic signals triggered by oxidative stress.

Systematic identification of hepatocellular proteins interacting with NS5A of the hepatitis C virus.

The hepatitis C virus is associated with the development of liver cirrhosis and hepatocellular carcinomas. Among the 10 polyproteins produced by the virus, no function has been clearly assigned to the non-structural 5A (NS5A) protein. This study was designed to identify the hepatocellular proteins that interact with NS5A of the HCV. Yeast two-hybrid experiments were performed with a human liver cDNA prey-library, using five different NS5A derivatives as baits, the full-length NS5A (NS5A-F, amino acid (aa) 1 approximately 447) and its four different derivatives, denoted as NS5A-A (aa 1 approximately 150), -B (aa 1 approximately 300), -C (aa 300 approximately 447) and D (aa 150 approximately 447). NS5A-F, NS5A-B and NS5A-C gave two, two and 10 candidate clones, respectively, including an AHNAK-related protein, the secreted frizzled-related protein 4 (SFRP4), the N-myc downstream regulated gene 1 (NDRG1), the cellular retinoic acid binding protein 1 (CRABP-1), ferritin heavy chain (FTH1), translokin, tumor-associated calcium signal transducer 2 (TACSTD2), phosphatidylinositol 4-kinase (PI4K) and centaurindelta 2 (CENTdelta2). However, NS5A-A produced no candidates and NS5A-D was not suitable as bait due to transcriptional activity. Based on an in vitro binding assay, CRABP-1, PI4K, CENTdelta2 and two unknown fusion proteins with maltose binding protein (MBP), were confirmed to interact with the glutathione S-transferase (GST)/NS5A fusion protein. Furthermore, the interactions of CRABP-1, PI4K and CENTdelta2 were not related to the PXXP motif (class II), as judged by a domain analysis. While their biological relevance is under investigation, the results contribute to a better understanding of the possible role of NS5A in hepatocellular signaling pathways.

Polo-box motif targets a centrosome regulator, RanGTPase.

Mammalian polo-like kinase (Plk) acts at various stages in early and late mitosis. Plk1 localizes in the centrosome, the central spindle, the midbody as well as the kinetochore. The non-catalytic region in the C-terminus of Plk1 has conserved sequence motifs, named polo-boxes. These motifs are important for Plk localization. GFP protein fused with the core sequences of polo-box (50 amino acids) localized Plk to target organelles. We screened for Plk interacting proteins by constructing a tandem repeat of the polo-box motif, and used it as bait in the two-hybrid system with HeLa cell cDNA library. RanGTPase was detected as a positive clone. Through in vitro and in vivo protein binding analysis in synchronized cells by thymidine block and by nocodazole treatment, we confirmed the interaction between endogenous Ran and Plk1. We showed that endogenous Ran and Plk1 proteins were co-localized to centrosomes, which is a major target organelle of endogenous Plk1, in early mitotic cells by immunofluorescence. Finally, we demonstrated that Plk1 phosphorylated RanBPM, a Ran-binding protein in microtubule organizing center, through the interaction with Ran. These data suggested that the core motif of polo-box is sufficient for Plk1-targeting, and that Plk1 may play roles in centrosome through recruitment and/or activation of Ran/RanBPM proteins.

Thioredoxin modulates activator protein 1 (AP-1) activity and p27Kip1 degradation through direct interaction with Jab1.

Thioredoxin (Trx) is a cellular redox enzyme that plays multiple roles in regulating cell growth and apoptosis. Jun activation domain-binding protein 1 (Jab1) was originally identified as a coactivator of activator protein 1 (AP-1) transcription and was also shown to promote degradation of the cyclin-dependent kinase inhibitor, p27Kip1. Recently, Jab1 expression was associated with the progression and poor prognosis of pituitary, epithelial ovarian, and breast cancers, suggesting that it plays a role in oncogenesis. Here, we report that Trx specifically interacts with and modulates the function of Jab1. Fluorescence resonance energy transfer and co-immunoprecipitation studies revealed that Trx and Jab1 colocalize and directly interact with each other. Further, Trx negatively regulates two important Jab1-controlled signaling pathways, activation of AP-1 transcription and degradation of p27Kip1, probably through a direct interaction between Trx and C-terminal of Jab1. The negative effect of Trx on AP-1 activity is Jab1-dependent, as it disappears when Jab1 levels are suppressed by an antisense approach. In addition, Trx competes with p27Kip1 for Jab1 binding. Taken together, our results suggest that Trx may regulate cell cycle and growth through a novel modulation of Jab1-mediated proliferation signals, further indicating that Trx may have the ability to control tumor progression.

Association of the Src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP-76) with the p85 subunit of phosphoinositide 3-kinase.

To investigate additional functions of the T cell adaptor, Src homology 2 (SH2) domain-containing leukocyte protein of 76 kD (SLP-76), we performed a yeast two-hybrid assay using the N-terminal region of SLP-76 fused with the kinase domain of Syk. By screening a human leukemia cDNA library, we identified the p85 subunit of phosphoinositide 3-kinase (PI3K) as one of the interacting molecules. Unlike the SH2 domain of Vav or Nck, tyrosine phosphorylation of SLP-76 at position 113 or 128 was sufficient for it to associate with the N-terminal SH2 of p85. Collectively, these data suggest that SLP-76 may play a role in PI3K signaling pathways.