bZIP Proteins Research Articles

현사시나무에서 Basic Leucine Zipper 유전자의 분리와 특성 구명

Basic leucine zipper (bZIP) protein is a regulatory transcription factor that plays crucial roles in growth, development and stress response of plant. In this study, we isolated a PagbZIP1 gene that belonged to Group SE3 of bZIP from Populus alba × P. glandulosa, and investigated its expressional characteristics. The PagbZIP1 is 844 base pairs long and encodes a putative 144-amino-acid protein with an expected molecular mass of 16.6 kDa. The PagbZIP1 has two conserved domains including the basic and leucine zipper portions. Southern blot analysis revealed that two copies of the gene are presented in the poplar genome. PagbZIP1 was specifically expressed in the root and suspension cells. Moreover, the expression of PagbZIP1 was induced by drought, salt, cold and ABA. Therefore, our results indicated that PagbZIP1 might be expressed in response to abiotic stress through the ABA-mediated signaling pathway in poplar.

Genome-wide analysis of the bZIP transcription factors in cucumber.

bZIP proteins are one of the largest transcriptional regulators playing crucial roles in plant development, physiological processes, and biotic/abiotic stress responses. Despite the availability of recently published draft genome sequence of Cucumis sativus, no comprehensive investigation of these family members has been presented for cucumber. We have identified 64 bZIP transcription factor-encoding genes in the cucumber genome. Based on structural features of their encoded proteins, CsbZIP genes could be classified into 6 groups. Cucumber bZIP genes were expanded mainly by segmental duplication rather than tandem duplication. Although segmental duplication rate of the CsbZIP genes was lower than that of Arabidopsis, rice and sorghum, it was observed as a common expansion mechanism. Some orthologous relationships and chromosomal rearrangements were observed according to comparative mapping analysis with other species. Genome-wide expression analysis of bZIP genes indicated that 64 CsbZIP genes were differentially expressed in at least one of the ten sampled tissues. A total of 4 CsbZIP genes displayed higher expression values in leaf, flowers and root tissues. The in silico micro-RNA (miRNA) and target transcript analyses identified that a total of 21 CsbZIP genes were targeted by 38 plant miRNAs. CsbZIP20 and CsbZIP22 are the most targeted by miR165 and miR166 family members, respectively. We also analyzed the expression of ten CsbZIP genes in the root and leaf tissues of drought-stressed cucumber using quantitative RT-PCR. All of the selected CsbZIP genes were measured as increased in root tissue at 24th h upon PEG treatment. Contrarily, the down-regulation was observed in leaf tissues of all analyzed CsbZIP genes. CsbZIP12 and CsbZIP44 genes showed gradual induction of expression in root tissues during time points. This genome-wide identification and expression profiling provides new opportunities for cloning and functional analyses, which may be used in further studies for improving stress tolerance in plants.

Genome-wide analysis and expression profile of the bZIP transcription factor gene family in grapevine (Vitis vinifera).

BackgroundBasic leucine zipper (bZIP) transcription factor gene family is one of the largest and most diverse families in plants. Current studies have shown that the bZIP proteins regulate numerous growth and developmental processes and biotic and abiotic stress responses. Nonetheless, knowledge concerning the specific expression patterns and evolutionary history of plant bZIP family members remains very limited.ResultsWe identified 55 bZIP transcription factor-encoding genes in the grapevine (Vitis vinifera) genome, and divided them into 10 groups according to the phylogenetic relationship with those in Arabidopsis. The chromosome distribution and the collinearity analyses suggest that expansion of the grapevine bZIP (VvbZIP) transcription factor family was greatly contributed by the segment/chromosomal duplications, which may be associated with the grapevine genome fusion events. Nine intron/exon structural patterns within the bZIP domain and the additional conserved motifs were identified among all VvbZIP proteins, and showed a high group-specificity. The predicted specificities on DNA-binding domains indicated that some highly conserved amino acid residues exist across each major group in the tree of land plant life. The expression patterns of VvbZIP genes across the grapevine gene expression atlas, based on microarray technology, suggest that VvbZIP genes are involved in grapevine organ development, especially seed development. Expression analysis based on qRT-PCR indicated that VvbZIP genes are extensively involved in drought- and heat-responses, with possibly different mechanisms.ConclusionsThe genome-wide identification, chromosome organization, gene structures, evolutionary and expression analyses of grapevine bZIP genes provide an overall insight of this gene family and their potential involvement in growth, development and stress responses. This will facilitate further research on the bZIP gene family regarding their evolutionary history and biological functions.

Evolutionary and Expression Analyses of Basic Zipper Transcription Factors in the Highly Homozygous Model Grape PN40024 (Vitis vinifera L.)

Basic leucine zipper (bZIP) proteins, which function as transcription factors and play important regulatory roles in all eukaryotic organisms, have been identified and classified in plants based on the sequenced genomes of model species such as Arabidopsis thaliana and rice (Oryza sativa). However, far less is currently known about the evolutionary relationships and expression patterns of bZIP genes in nonmodel plants. In this study, we performed a genome-wide analysis and identified a total of 47 bZIP transcription factors from grape (Vitis vinifera L., cv PN40024). Phylogenetic analysis of grape bZIP transcription factors along with their Arabidopsis and rice counterparts indicated that they can be classified into 13 different groups. Furthermore, evolutionary analysis of the grape bZIP transcription factors demonstrated that segmental duplications have contributed substantially to the expansion of this family in grape. In addition, synteny analysis between grape and Arabidopsis suggested that some of the bZIP members were present in their most recent common ancestor and that the major expansion occurred before the divergence of the two species. Gene expression analysis of the grape bZIP transcription factor-encoding genes revealed tissue-specific, biotic and abiotic stress and hormone-responsive expression profiles. Taken together, the genome-wide identification and characterization of grape bZIP transcription factors provide insights into their evolutionary history and a resource for further functional characterization in the context of crop improvement and stress tolerance.

Nuclear Translocation and Regulation of Intranuclear Distribution of Cytoplasmic Poly(A)-Binding Protein Are Distinct Processes Mediated by Two Epstein Barr Virus Proteins

Many viruses target cytoplasmic polyA binding protein (PABPC) to effect widespread inhibition of host gene expression, a process termed viral host-shutoff (vhs). During lytic replication of Epstein Barr Virus (EBV) we observed that PABPC was efficiently translocated from the cytoplasm to the nucleus. Translocated PABPC was diffusely distributed but was excluded from viral replication compartments. Vhs during EBV infection is regulated by the viral alkaline nuclease, BGLF5. Transfection of BGLF5 alone into BGLF5-KO cells or uninfected 293 cells promoted translocation of PAPBC that was distributed in clumps in the nucleus. ZEBRA, a viral bZIP protein, performs essential functions in the lytic program of EBV, including activation or repression of downstream viral genes. ZEBRA is also an essential replication protein that binds to viral oriLyt and interacts with other viral replication proteins. We report that ZEBRA also functions as a regulator of vhs. ZEBRA translocated PABPC to the nucleus, controlled the intranuclear distribution of PABPC, and caused global shutoff of host gene expression. Transfection of ZEBRA alone into 293 cells caused nuclear translocation of PABPC in the majority of cells in which ZEBRA was expressed. Co-transfection of ZEBRA with BGLF5 into BGLF5-KO cells or uninfected 293 cells rescued the diffuse intranuclear pattern of PABPC seen during lytic replication. ZEBRA mutants defective for DNA-binding were capable of regulating the intranuclear distribution of PABPC, and caused PABPC to co-localize with ZEBRA. One ZEBRA mutant, Z(S186E), was deficient in translocation yet was capable of altering the intranuclear distribution of PABPC. Therefore ZEBRA-mediated nuclear translocation of PABPC and regulation of intranuclear PABPC distribution are distinct events. Using a click chemistry-based assay for new protein synthesis, we show that ZEBRA and BGLF5 each function as viral host shutoff factors.

Arabidopsis CAM7 and HY5 physically interact and directly bind to the HY5 promoter to regulate its expression and thereby promote photomorphogenesis.

Arabidopsis thaliana CALMODULIN7 (CAM7), a unique member of the calmodulin gene family, plays a crucial role as a transcriptional regulator in seedling development. The elongated HYPOCOTYL5 (HY5) bZIP protein, an integrator of multiple signaling pathways, also plays an important role in photomorphogenic growth and light-regulated gene expression. CAM7 acts synergistically with HY5 to promote photomorphogenesis at various wavelengths of light. Although the genetic relationships between CAM7 and HY5 in light-mediated seedling development have been demonstrated, the molecular connectivity between CAM7 and HY5 is unknown. Furthermore, whereas HY5-mediated gene regulation has been fairly well investigated, the transcriptional regulation of HY5 is largely unknown. Here, we report that HY5 expression is regulated by HY5 and CAM7 at various wavelengths of light and also at various stages of development. In vitro and in vivo DNA-protein interaction studies suggest that HY5 and CAM7 bind to closely located T/G- and E-box cis-acting elements present in the HY5 promoter, respectively. Furthermore, CAM7 and HY5 physically interact and regulate the expression of HY5 in a concerted manner. Taken together, these results demonstrate that CAM7 and HY5 directly interact with the HY5 promoter to mediate the transcriptional activity of HY5 during Arabidopsis seedling development.

Ribosomal Protein S6, a Target of Rapamycin, Is Involved in the Regulation of rRNA Genes by Possible Epigenetic Changes in Arabidopsis

The target of rapamycin (TOR) kinase pathway regulates various biological processes, including translation, synthesis of ribosomal proteins, and transcription of rRNA. The ribosomal protein S6 (RPS6) is one of the well known downstream components of the TOR pathway. Ribosomal proteins have been known to have diverse functions in regulating cellular metabolism as well as protein synthesis. So far, however, little is known about other possible role(s) of RPS6 in plants, besides being a component of the 40 S ribosomal subunit and acting as a target of TOR. Here, we report that RPS6 may have a novel function via interaction with histone deacetylase 2B (AtHD2B) that belongs to the plant-specific histone deacetylase HD2 family. RPS6 and AtHD2B were localized to the nucleolus. Co-expression of RPS6 and AtHD2B caused a change in the location of both RPS6 and AtHD2B to one or several nucleolar spots. ChIP analysis suggests that RPS6 directly interacts with the rRNA gene promoter. Protoplasts overexpressing both AtHD2B and RPS6 exhibited down-regulation of pre-18 S rRNA synthesis with a concomitant decrease in transcription of some of the ribosomal proteins, suggesting their direct role in ribosome biogenesis and plant development. This is consistent with the mutation in rps6b that results in reduction in 18 S rRNA transcription and decreased root growth. We propose that the interaction between RPS6 and AtHD2B brings about a change in the chromatin structure of rDNA and thus plays an important role in linking TOR signaling to rDNA transcription and ribosome biogenesis in plants.

Distinct functions of COAR and B3 domains of maize VP1 in induction of ectopic gene expression and plant developmental phenotypes in Arabidopsis

Arabidopsis ABI3 and maize VP1 are orthologous transcription factors that regulate seed maturation. ABI3 and VP1 have a C-terminal B3 DNA binding domain and a conserved N-terminal co-activator/co-repressor (COAR) domain consisting of A1, B1, B2 sub-domains. The COAR domain mediates abscisic acid signaling via a physical interaction with ABI5-related bZIP proteins. In order to delineate the COAR and B3 domain dependent functions of VP1, we created site directed mutations in the B3 domain that disrupted DNA binding activity and characterized gene regulation by the mutant proteins in transgenic abi3 mutant Arabidopsis plants. In seeds, COAR domain function of VP1 mutants that lacked B3 DNA binding activity was sufficient for complementation of the desiccation intolerant seed phenotype of abi3. Similarly in seedlings, the B3 domain was dispensable for most VP1 induced gene expression and ectopic developmental phenotypes, except for a small subset of the genes that showed B3 dependent regulation. Unexpectedly, over-expression of the DNA-binding deficient VP1-K519R mutant protein caused quantitative changes in floral organ size including elongation of pistils and shortened stamen filaments that resulted in a self-incompatible longistyly flower morphology, a key component of heterostyly type self-incompatibility.

Mini review roles of the bZIP gene family in rice.

The basic leucine zipper (bZIP) genes encode transcription factors involved in the regulation of various biological processes. Similar to WRKY, basic helix-loop-helix, and several other groups of proteins, the bZIP proteins form a superfamily of transcription factors that mediate plant stress responses. In this review, we present the roles of bZIP proteins in multiple biological processes that include pathogen defense; responses to abiotic stresses; seed development and germination; senescence; and responses to salicylic, jasmonic, and abscisic acids in rice. We also examined the characteristics of the bZIP proteins and their genetic composition. To ascertain the evolutionary changes in and functions of this supergene family, we performed an exhaustive comparison among the 89 rice bZIP genes that were previously described and those more recently listed in the MSU Rice Genome Annotation Project Database using a Hidden Markov Model. We excluded 3 genes from the list, resulting in a total of 86 bZIP genes in japonica rice.

Molecular study of HBZ and gp21 Human T cell lymphotropic virus type 1 proteins isolated from different clinical profile infected individuals

The HTLV-1 gp21 glycoprotein is involved in envelope trafficking and membrane targeting while bZIP protein is indispensable for cell growth and proliferation. This study aimed to assess the molecular diversity of gp21 and HBZ proteins, in TSP/HAM and healthy carriers. DNA samples from HTLV-1 infected individuals were submitted to PCR, sequencing, and the molecular analyses were performed using bioinformatics tools. From eight gp21 analyzed sequences one amino acid change (Y477H) was associated to the switch of helix to coil structure at secondary structure prediction. From ten HBZ analyzed sequences, two amino acid changes were identified (S9P and T95I) at the activation domain. One mutation (R112C) located at nuclear localization signal was present in 66.7% and 25% of HC and TSP/HAM groups, respectively. This is the first report of mutations in HBZ region. These polymorphisms might be important for viral fitness.

Phylogenetic and transcriptional analysis of an expanded bZIP transcription factor family in Phytophthora sojae

BackgroundBasic leucine zipper (bZIP) transcription factors are present exclusively in eukaryotes and constitute one of the largest and most diverse transcription factor families. The proteins are responsible for central developmental and physiological processes in plants, animals, and fungi, including the pathogenicity of fungal plant pathogens. However, there is limited understanding of bZIPs in oomycetes, which are fungus-like organisms in the kingdom Stramenopila. Oomycetes include many destructive plant pathogens, including the well-studied species Phytophthora sojae, which causes soybean stem and root rot.ResultsCandidate bZIPs encoded in the genomes of P. sojae and four other oomycetes, two diatoms, and two fungal species were predicted using bioinformatic methods. Comparative analysis revealed expanded numbers of bZIP candidates in oomycetes, especially the Phytophthora species, due to the expansion of several novel bZIP classes whose highly conserved asparagines in basic DNA-binding regions were substituted by other residues such as cysteine. The majority of these novel bZIP classes were mostly restricted to oomycetes. The large number of novel bZIPs appears to be the result of widespread gene duplications during oomycete evolution. The majority of P. sojae bZIP candidates, including both conventional and novel bZIP classes, were predicted to contain canonical protein secondary structures. Detection of gene transcripts using digital gene expression profiling and qRT-PCR suggested that most of the candidates were not pseudogenes. The major transcriptional shifts of bZIPs occurred during the zoosporangia/zoospore/cyst and host infection stages. Several infection-associated bZIP genes were identified that were positively regulated by H2O2 exposure.ConclusionsThe identification of large classes of bZIP proteins in oomycetes with novel bZIP motif variants, that are conserved and developmentally regulated and thus presumably functional, extends our knowledge of this important family of eukaryotic transcription factors. It also lays the foundation for detailed studies of the roles of these proteins in development and infection in P. sojae and other oomycetes.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-14-839) contains supplementary material, which is available to authorized users.

The bZIP protein from Tamarix hispida, ThbZIP1, is ACGT elements binding factor that enhances abiotic stress signaling in transgenic Arabidopsis

BackgroundTamarix spp. are woody halophyte, which are very tolerant to abiotic stresses such as salinity and drought, but little is known about their specific stress response systems. Basic leucine zipper proteins (bZIPs) play important roles in the ability of plants to withstand adverse environmental conditions. However, their exact roles in abiotic stress tolerance are still not fully known. In the current study, we functionally characterized a bZIP gene (ThbZIP1) from Tamarix hispida in response to abiotic stresses.ResultsWe addressed the regulatory network of ThbZIP1 in three levels, i.e. its upstream regulators, the cis-acting elements recognized by ThbZIP1, and its downstream target genes. Two MYCs were found to bind to E-box, in the promoter of ThbZIP1 to activate its expression. Expression of ThbZIP1 is induced by ABA, salt, drought, methyl viologen and cold. ThbZIP1 can specifically bind to ACGT elements, with the highest binding affinity to the C-box, followed by the G-box and lastly the A-box. Compared with wild-type (Col-0) Arabidopsis, transgenic plants expressing ThbZIP1 had an increased tolerance to drought and salt, but had an increased sensitivity to ABA during seed germination and root growth; meanwhile, ROS level, cell death and water loss rate in transgenic plants were significantly reduced. Microarray analyses showed that many ROS scavenging genes were up-regulated by ThbZIP1 under salt stress conditions.ConclusionsBased on these data, we suggest that ThbZIP1 confers abiotic stress tolerance through activating stress tolerance genes to modulate ROS scavenging ability and other physiological changes involved in stress tolerance, and plays an important role in the ABA-mediated stress response of T. hispida.

Metal-Binding Ability of VIP1: A bZIP Protein in Arabidopsis thaliana

VirE2-interacting protein 1 (VIP1) is an Arabidopsis thaliana bZIP transcription factor which regulates pathogen responses and rehydration responses. VIP1 has transcriptional activation potential, DNA-binding ability, and a nuclear-cytoplasmic shuttling property. These functions are possibly regulated by cofactors and/or post-translational modifications. During an investigation of the functions of VIP1, we discovered that VIP1 can react with an Ni²⁺-activated derivative of horseradish peroxidase, HisProbe-HRP, suggesting that VIP1 can bind Ni²⁺. Using truncated versions and mutated versions of VIP1, the Ni²⁺-binding region was narrowed. Using VIP1 H145Q and H145R mutants, which have H → Q and H → R mutations at the amino acid position 145 of VIP1, a trihistidine site at the amino acid position 144-146 was confirmed to be responsible for the Ni²⁺-binding ability. Immobilized-metal affinity chromatography (IMAC) suggested that VIP1 can bind Zn²⁺ and Co²⁺ as well as Ni²⁺, which is consistent with the known metal-chelating property of polyhistidine. In IMAC, the levels of purified VIP1 were not significantly different between denaturing and non-denaturing conditions, suggesting that the trihistidine is located on the surface of the native form of VIP1. In gel shift assays, VIP1-dependent decreases of electrophoretic mobilities of DNA probes were further decreased by Co²⁺. Among wild-type VIP1 and the H145Q and H145R mutants, H145R was the least sensitive to the effect of Co²⁺ in the gel shift assays. These results suggest that the Co²⁺ and the metal-binding site of VIP1 affect the interaction between VIP1 and DNA.

Molecular Study of HBZ and gp21 Human T Cell Leukemia Virus Type 1 Proteins Isolated from Different Clinical Profile Infected Individuals

Human T cell leukemia virus type 1 (HTLV-1) is associated with a neurological syndrome named tropical spastic paraparesis/HTLV-associated myelopathy (TSP/HAM) and the disease progression involves viral factors. The gp21 glycoprotein is involved in envelope trafficking and membrane targeting while the bZIP protein is indispensable for cell growth and proliferation. This study aimed to assess the molecular diversity of gp21 and HBZ proteins in TSP/HAM and healthy carriers. DNA samples from HTLV-1-infected individuals were submitted to PCR and sequencing, and the molecular analyses were performed using bioinformatics tools. From eight gp21-analyzed sequences one amino acid change (Y477H) was associated with the switch of a helix to coil structure at secondary structure prediction. From 10 HBZ analyzed sequences, two amino acid changes were identified (S9P and T95I) at the activation domain. One mutation (R112C) located at the nuclear localization signal was present in 66.7% and 25% of healthy carriers (HC) and TSP/HAM groups, respectively. This is the first report of mutations in the HBZ region. These polymorphisms might be important for viral fitness.

English

  Plant basic leucine zipper (bZIP) proteins play an essential role in the genes expression and regulation  in higher plants. They have been shown to regulate diverse plant specific phenomena, including  germination, floral induction and development, seed maturation, photomorphogenesis, biotic and  abiotic stresses. Resistance response mediated by the rice blast resistance gene Pi36 is a typical signal  transduction, in which 12 OsbZIP genes were differentially expressed by microarray analyses. To  understand the potential function of OsbZIP genes during the defense responses against rice blast, the  expression analysis of these OsbZIP genes, in response to the blast fungus inoculation and the related  defense signal molecules induction, were further conducted using real-time fluorescent quantitative  polymerase chain reaction (PCR) technique. Our data indicates that among the 12 OsbZIP genes, the  expression level eight tested genes were differentially regulated and maintained to 96 h points post  inoculation in rice resistant and susceptible cultivars during Magnaporthe oryzae infection, and all of  them were also significantly up-regulated by one or several kinds of exogenous plant hormones  stresses. Although, these genes were induced only at early time points (1 to 24 h); it is evident that the  OsbZIP genes may be involved in different signaling pathway, and play potential important functions in  the defense response to rice blast.    Key words: OsbZIP transcription factors, rice blast, resistance response, plant hormones stresses.

A bZIP protein, VIP1, interacts with Arabidopsis heterotrimeric G protein β subunit, AGB1

Heterotrimeric G proteins (Gα, Gβ, Gγ) are signaling molecules conserved among eukaryotic species. The G proteins transmit signals via protein-protein interactions. By a yeast two-hybrid screen, we identified a bZIP protein, VIP1, as an Arabidopsis thaliana Gβ (AGB1)-interacting partner. The interaction between AGB1 and VIP1 was confirmed by an in vitro GST pull-down assay and a bimolecular fluorescence complementation (BiFC) assay. VIP1 was previously reported to be a regulator of osmosensory signaling. Interestingly, the BiFC pattern between AGB1 and VIP1 was speckled when cells were incubated in a hypotonic solution, but not when cells were incubated in a mannitol-containing hypertonic solution, suggesting that the subcellular localization of the AGB1-VIP1 complex is regulated by extracellular osmolarity and/or turgor pressure.

Regulation of GSK3β-FBXW7-JUNB Axis

Regulation of GSK3β-FBXW7-JUNB Axis

Predicting protein interactions via parsimonious network history inference

Motivation: Reconstruction of the network-level evolutionary history of protein–protein interactions provides a principled way to relate interactions in several present-day networks. Here, we present a general framework for inferring such histories and demonstrate how it can be used to determine what interactions existed in the ancestral networks, which present-day interactions we might expect to exist based on evolutionary evidence and what information extant networks contain about the order of ancestral protein duplications.Results: Our framework characterizes the space of likely parsimonious network histories. It results in a structure that can be used to find probabilities for a number of events associated with the histories. The framework is based on a directed hypergraph formulation of dynamic programming that we extend to enumerate many optimal and near-optimal solutions. The algorithm is applied to reconstructing ancestral interactions among bZIP transcription factors, imputing missing present-day interactions among the bZIPs and among proteins from five herpes viruses, and determining relative protein duplication order in the bZIP family. Our approach more accurately reconstructs ancestral interactions than existing approaches. In cross-validation tests, we find that our approach ranks the majority of the left-out present-day interactions among the top 2 and 17% of possible edges for the bZIP and herpes networks, respectively, making it a competitive approach for edge imputation. It also estimates relative bZIP protein duplication orders, using only interaction data and phylogenetic tree topology, which are significantly correlated with sequence-based estimates.Availability: The algorithm is implemented in C++, is open source and is available at http://www.cs.cmu.edu/ckingsf/software/parana2.Contact: robp@cs.cmu.edu or carlk@cs.cmu.eduSupplementary information: Supplementary data are available at Bioinformatics online.

Sequence‐Selective DNA Recognition with Peptide–Bisbenzamidine Conjugates

Transcription factors (TFs) are specialized proteins that play a key role in the regulation of genetic expression. Their mechanism of action involves the interaction with specific DNA sequences, which usually takes place through specialized domains of the protein. However, achieving an efficient binding usually requires the presence of the full protein. This is the case for bZIP and zinc finger TF families, which cannot interact with their target sites when the DNA binding fragments are presented as isolated monomers. Herein it is demonstrated that the DNA binding of these monomeric peptides can be restored when conjugated to aza-bisbenzamidines, which are readily accessible molecules that interact with A/T-rich sites by insertion into their minor groove. Importantly, the fluorogenic properties of the aza-benzamidine unit provide details of the DNA interaction that are eluded in electrophoresis mobility shift assays (EMSA). The hybrids based on the GCN4 bZIP protein preferentially bind to composite sequences containing tandem bisbenzamidine-GCN4 binding sites (TCAT⋅AAATT). Fluorescence reverse titrations show an interesting multiphasic profile consistent with the formation of competitive nonspecific complexes at low DNA/peptide ratios. On the other hand, the conjugate with the DNA binding domain of the zinc finger protein GAGA binds with high affinity (KD≈12 nM) and specificity to a composite AATTT⋅GAGA sequence containing both the bisbenzamidine and the TF consensus binding sites.

RsmA Regulates Aspergillus fumigatus Gliotoxin Cluster Metabolites Including Cyclo(L-Phe-L-Ser), a Potential New Diagnostic Marker for Invasive Aspergillosis

Dimeric basic leucine zipper (bZIP) proteins are conserved transcriptional enhancers found in all eukaryotes. A recently reported and novel function for bZIPs is association of these proteins with secondary metabolite production in filamentous fungi. In particular a Yap-like bZIP termed RsmA (restorer of secondary metabolism A) was identified in Aspergillus nidulans that positively regulates the carcinogen sterigmatocystin. To assess for conserved function for RsmA, we examined a role of this protein in secondary metabolism in the pathogen A. fumigatus. RsmA was found to positively regulate gliotoxin where overexpression (OE) of rsmA led to 2–100 fold increases of twelve gli cluster metabolites in culture medium including the newly identified gli metabolite cyclo(L-Phe-L-Ser). Lungs from both wild type and OErsmA infected mice contained gliotoxin (2.3 fold higher in OErsmA treatment) as well as the gliotoxin precursor cyclo(L-Phe-L-Ser) (3.2 fold higher in OErsmA treatment). The data here presents a conserved role for RsmA in secondary metabolite cluster activation and suggests cyclo(L-Phe-L-Ser) may serve as an alternative marker for diagnosis of invasive aspergillosis.