The Hybrid Type Polyketide Synthase SteelyA Is Required for cAMP Signalling in Early Dictyostelium Development

Background

In our previous study we found that the expression of stlA showed peaks both in the early and last stages of development and that a product of SteelyA, 4-methyl-5-pentylbenzene-1,3-diol (MPBD), controlled Dictyostelium spore maturation during the latter. In this study we focused on the role of SteelyA in early stage development.

Principal Findings

Our stlA null mutant showed aggregation delay and abnormally small aggregation territories. Chemotaxis analysis revealed defective cAMP chemotaxis in the stlA null mutant. cAMP chemotaxis was restored by MPBD addition during early stage development. Assay for cAMP relay response revealed that the stlA null mutant had lower cAMP accumulation during aggregation, suggesting lower ACA activity than the wild type strain. Exogenous cAMP pulses rescued the aggregation defect of the stlA null strain in the absence of MPBD. Expression analysis of cAMP signalling genes revealed lower expression levels in the stlA null mutant during aggregation.

Conclusion

Our data indicate a regulatory function by SteelyA on cAMP signalling during aggregation and show that SteelyA is indispensable for full activation of ACA.     

Dissecting the functional role of polyketide synthases in Dictyostelium discoideum: biosynthesis of the differentiation regulating factor 4-methyl-5-pentylbenzene-1,3-diol

Dictyostelium discoideum exhibits the largest repository of polyketide synthase (PKS) proteins of all known genomes. However, the functional relevance of these proteins in the biology of this organism remains largely obscure. On the basis of computational, biochemical, and gene expression studies, we propose that the multifunctional Dictyostelium PKS (DiPKS) protein DiPKS1 could be involved in the biosynthesis of the differentiation regulating factor 4-methyl-5-pentylbenzene-1,3-diol (MPBD). Our cell-free reconstitution studies of a novel acyl carrier protein Type III PKS didomain from DiPKS1 revealed a crucial role of protein-protein interactions in determining the final biosynthetic product. Whereas the Type III PKS domain by itself primarily produces acyl pyrones, the presence of the interacting acyl carrier protein domain modulates the catalytic activity to produce the alkyl resorcinol scaffold of MPBD. Furthermore, we have characterized an O-methyltransferase (OMT12) from Dictyostelium with the capability to modify this resorcinol ring to synthesize a variant of MPBD. We propose that such a modification in vivo could in fact provide subtle variations in biological function and specificity. In addition, we have performed systematic computational analysis of 45 multidomain PKSs, which revealed several unique features in DiPKS proteins. Our studies provide a new perspective in understanding mechanisms by which metabolic diversity could be generated by combining existing functional scaffolds.     

The polyketide MPBD initiates the SDF-1 signaling cascade that coordinates terminal differentiation in Dictyostelium

Dictyostelium uses a wide array of chemical signals to coordinate differentiation as it switches from a unicellular to a multicellular organism. MPBD, the product of the polyketide synthase encoded by stlA, regulates stalk and spore differentiation by rapidly stimulating the release of the phosphopeptide SDF-1. By analyzing specific mutants affected in MPBD or SDF-1 production, we delineated a signal transduction cascade through the membrane receptor CrlA coupled to Gα1, leading to the inhibition of GskA so that the precursor of SDF-1 is released. It is then processed by the extracellular protease of TagB on prestalk cells. SDF-1 apparently acts through the adenylyl cyclase ACG to activate the cyclic AMP (cAMP)-dependent protein kinase A (PKA) and trigger the production of more SDF-1. This signaling cascade shows similarities to the SDF-2 signaling pathway, which acts later to induce rapid spore encapsulation.     

Maintenance of the engrailed expression pattern by Polycomb group genes in Drosophila.

The stable maintenance of expression patterns of homeotic genes depends on the function of a number of negative trans-regulators, termed the Polycomb (Pc) group of genes. We have examined the pattern of expression of the Drosophila segment polarity gene, engrailed (en), in embryos mutant for several different members of the Pc group. Here we report that embryos mutant for two or more Pc group genes show strong ectopic en expression, while only weak derepression of en occurs in embryos mutant for a single Pc group gene. This derepression is independent of two known activators of en expression: en itself and wingless. Additionally, in contrast to the strong ectopic expression of homeotic genes observed in extra sex combs- (esc-) mutant embryos, the en expression pattern is nearly normal in esc- embryos. This suggests that the esc gene product functions in a pathway independent of the other genes in the group. The data indicate that the same group of genes is required for stable restriction of en expression to a striped pattern and for the restriction of expression of homeotic genes along the anterior-posterior axis, and support a global role for the Pc group genes in stable repression of activity of developmental selector genes.     

Truncated products of the vestigial proliferation gene induce apoptosis.

The vestigial (vg) gene in D. melanogaster, whose mutant phenotype is characterized by wing atrophy, encodes a novel nuclear protein involved in cell proliferation. The original vg mutant (vgBG) displays massive apoptosis in the wing imaginal disc. Here we tested the hypothesis that the vg mutant phenotype could be due: (i) to lack of cell proliferation in null mutants due to the absence of the Vg product and, (ii) to apoptosis in vgBG and other mutants due to the presence of a major Vg truncated product. In agreement with our hypothesis no cell death was observed in null vg mutants, and the anticell death baculovirus P35 product is unable to rescue the mutant phenotype caused by absence of the Vg product. In addition, expression of the antiproliferative gene dacapo, the homolog of p21, induces a mutant wing phenotype without inducing cell death. In contrast the wing phenotype of the original vg mutant could be reproduced by the ectopic expression of the reaper cell death gene when expressed by vg regulatory sequences. In agreement with the hypothesis, the classic vg mutant spontaneously displays an increase in reaper expression in the wing disc and its phenotype can be partially rescued by the P35 product. Finally, we showed that ectopic expression of a truncated Vg product is able on its own to induce ectopic cell death and reaper expression. Our results shed new light on the function of the vg gene, in particular, they suggest that the normal and truncated products affect vg target genes in different ways.     

Analysis of JNK, Mdm2 and p14(ARF) contribution to the regulation of mutant p53 stability

Identification of Mdm2 and JNK as proteins that target degradation of wt p53 prompted us to examine their effect on mutant p53, which exhibits a prolonged half-life. Of five mutant p53 forms studied for association with the targeting molecules, two no longer bound to Mdm2 and JNK. Three mutant forms, which exhibit high expression levels, showed lower affinity for association with Mdm2 and JNK in concordance with greater affinity to p14(ARF), which is among the stabilizing p53 molecules. Monitoring mutant p53 stability in vitro confirmed that, while certain forms of mutant p53 are no longer affected by either JNK or Mdm2, others are targeted for degradation by JNK/Mdm2, albeit at lower efficiency when compared with wt p53. Expression of wt p53 in tumor cells revealed a short half-life, suggesting that the targeting molecules are functional. Forced expression of mutant p53 in p53 null cells confirmed pattern of association with JNK/Mdm2 and prolonged half-life, as found in the tumor cells. Over-expression of Mdm2 in either tumor (which do express endogenous functional Mdm2) or in p53 null cells decreased the stability of mutant p53 suggesting that, despite its expression, Mdm2/JNK are insufficient (amount/affinity) for targeting mutant p53 degradation. Based on both in vitro and in vivo analyses, we conclude that the prolonged half-life of mutant p53 depends on the nature of the mutation, which either alters association with targeting molecules, ratio between p53 and targeting/stabilizing molecules or targeting efficiency.     

Loss- and gain-of-function mutations in the F1-HAMP region of the Escherichia coli aerotaxis transducer Aer

The Escherichia coli Aer protein contains an N-terminal PAS domain that binds flavin adenine dinucleotide (FAD), senses aerotactic stimuli, and communicates with the output signaling domain. To explore the roles of the intervening F1 and HAMP segments in Aer signaling, we isolated plasmid-borne aerotaxis-defective mutations in a host strain lacking all chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family. Under these conditions, Aer alone established the cell's run/tumble swimming pattern and modulated that behavior in response to oxygen gradients. We found two classes of Aer mutants: null and clockwise (CW) biased. Most mutant proteins exhibited the null phenotype: failure to elicit CW flagellar rotation, no aerosensing behavior in MCP-containing hosts, and no apparent FAD-binding ability. However, null mutants had low Aer expression levels caused by rapid degradation of apparently nonnative subunits. Their functional defects probably reflect the absence of a protein product. In contrast, CW-biased mutant proteins exhibited normal expression levels, wild-type FAD binding, and robust aerosensing behavior in MCP-containing hosts. The CW lesions evidently shift unstimulated Aer output to the CW signaling state but do not block the Aer input-output pathway. The distribution and properties of null and CW-biased mutations suggest that the Aer PAS domain may engage in two different interactions with HAMP and the HAMP-proximal signaling domain: one needed for Aer maturation and another for promoting CW output from the Aer signaling domain. Most aerotaxis-defective null mutations in these regions seemed to affect maturation only, indicating that these two interactions involve structurally distinct determinants.     

Thyroid hormone inhibits slow skeletal TnI expression in cardiac TnI-null myocardial cells

A cardiac troponin I (cTnI) gene knockout mouse model has been created and the phenotype of the cTnI null mice is an acute heart failure resulting from the deficiency of TnI and a diastolic dysfunction. Two isoforms of TnI (the fetal form ssTnI and the adult form cTnI) are mainly expressed in the heart under a developmentally regulated program. In our previous studies, we demonstrated that thyroid hormone could alter the time course of ssTnI gene expression in the heart. In the present study, we have successfully cultured neonatal cardiac myocytes from wild type and cTnI null mouse hearts. The ssTnI gene expression pattern has been investigated in these cells. By using Western blotting assays, a TnI isoform switching has been observed in the wild type cardiac myocytes. The pattern of TnI isoform switching is very similar to that of in vivo study we reported previously. In cTnI null cardiac myocytes cultured from day 1 to day 7, there is a continuous decline in ssTnI concentration in the cells. The time course of ssTnI decline in cTnI null cells is similar to that of wild type cardiac myocytes, suggesting that there is no significant compensation of ssTnI gene expression for the absence of the cTnI. This observation is different from what we found previously at a whole heart level. In addition, when thyroid hormone T3 (20 ng/ml) is added to cultured cTnI null cardiac myocytes, the decline of ssTnI concentration occurs earlier. This is inconsistent with our observations from previous in vivo studies. The data demonstrate that thyroid hormone can alter the time course of ssTnI gene expression in cultured cardiac myocytes and TnI gene regulation is also controlled by some unknown programmed events inside of cardiac myocytes.     

Equivalent genetic roles for bmp7/snailhouse and bmp2b/swirl in dorsoventral pattern formation

A bone morphogenetic protein (BMP) signaling pathway acts in the establishment of the dorsoventral axis of the vertebrate embryo. Here we demonstrate the genetic requirement for two different Bmp ligand subclass genes for dorsoventral pattern formation of the zebrafish embryo. From the relative efficiencies observed in Bmp ligand rescue experiments, conserved chromosomal synteny, and isolation of the zebrafish bmp7 gene, we determined that the strongly dorsalized snailhouse mutant phenotype is caused by a mutation in the bmp7 gene. We show that the original snailhouse allele is a hypomorphic mutation and we identify a snailhouse/bmp7 null mutant. We demonstrate that the snailhouse/bmp7 null mutant phenotype is identical to the presumptive null mutant phenotype of the strongest dorsalized zebrafish mutant swirl/bmp2b, revealing equivalent genetic roles for these two Bmp ligands. Double mutant snailhouse/bmp7; swirl/bmp2b embryos do not exhibit additional or stronger dorsalized phenotypes, indicating that these Bmp ligands do not function redundantly in early embryonic development. Furthermore, overexpression experiments reveal that Bmp2b and Bmp7 synergize in the ventralization of wild-type embryos through a cell-autonomous mechanism, suggesting that Bmp2b/Bmp7 heterodimers may act in vivo to specify ventral cell fates in the zebrafish embryo.     

Identification of domains within megalomicin and erythromycin polyketide synthase modules responsible for differences in polyketide production levels in Escherichia coli

The megalomicin and erythromycin polyketide synthases (PKSs) produce the same aglycon product, 6-deoxyerythronolide B (6-dEB). Both PKSs were examined in an Escherichia coli strain metabolically engineered to support complex polyketide biosynthesis. Production of 6-dEB in shake flask fermentations was undetectable by mass spectrometry in the strain expressing the megalomicin (Meg) PKS genes, whereas 31 mg/L 6-dEB was produced by the strain with the erythromycin (DEBS) PKS. The genes for each of the three subunits comprising the PKSs were expressed in different combinations from three compatible expression vectors (e.g., DEBS1, DEBS2, and MegA3) to identify two Meg PKS subunits, MegA1 and MegA3, which conferred lower 6-dEB titers than their DEBS counterparts. Comparison of protein expression levels and 6-dEB titers by engineered hybrid DEBS/Meg PKS genes further defined regions within modules 2 and 6 of MegA1 and MegA3, respectively, which limit protein expression and 6-dEB production in E. coli. Meg module 2 + TE (M2 + TE) and a hybrid DEBS M2/Meg M2 + TE protein were engineered and purified for in vitro comparisons with DEBS M2 + TE. The specific activity of the hybrid M2 + TE was approximately 16-fold lower than DEBS M2 + TE and only twice as high as the Meg M2 + TE enzyme in diketide elongation assays. Since the hybrid M2 worked comparably to DEBS M2 in vivo, this suggests that boosting subunit concentration could serve as a useful approach to overcome enzyme deficiencies in heterologous polyketide production.     

Molecular Cloning,Modeling, and Site-Directed Mutagenesis of Type III Polyketide Synthase from <Emphasis Type="Italic">Sargassum binderi</Emphasis> (Phaeophyta)

Type III polyketide synthases (PKSs) produce an array of metabolites with diverse functions. In this study, we have cloned the complete reading frame encoding type III PKS (SbPKS) from a brown seaweed, Sargassum binderi, and characterized the activity of its recombinant protein biochemically. The deduced amino acid sequence of SbPKS is 414 residues in length, sharing a higher sequence similarity with bacterial PKSs (38% identity) than with plant PKSs. The Cys-His-Asn catalytic triad of PKS is conserved in SbPKS with differences in some of the residues lining the active and CoA binding sites. The wild-type SbPKS displayed broad starter substrate specificity to aliphatic long-chain acyl-CoAs (C₆–C₁₄) to produce tri- and tetraketide pyrones. Mutations at H³³¹ and N³⁶⁴ caused complete loss of its activity, thus suggesting that these two residues are the catalytic residues for SbPKS as in other type III PKSs. Furthermore, H227G, H227G/L366V substitutions resulted in increased tetraketide-forming activity, while wild-type SbPKS produces triketide α-pyrone as a major product. On the other hand, mutant H227G/L366V/F93A/V95A demonstrated a dramatic decrease of tetraketide pyrone formation. These observations suggest that His²²⁷ and Leu³⁶⁶ play an important role for the polyketide elongation reaction in SbPKS. The conformational changes in protein structure especially the cavity of the active site may have more significant effect to the activity of SbPKS compared with changes in individual residues.     

Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo

Within the vertebrate nervous system, the presence of many different lineages of neurons and glia complicates the molecular characterization of single neuronal populations. In order to elucidate molecular mechanisms underlying the specification and development of corticospinal motor neurons (CSMN), we purified CSMN at distinct stages of development in vivo and compared their gene expression to two other pure populations of cortical projection neurons: callosal projection neurons and corticotectal projection neurons. We found genes that are potentially instructive for CSMN development, as well as genes that are excluded from CSMN and are restricted to other populations of neurons, even within the same cortical layer. Loss-of-function experiments in null mutant mice for Ctip2 (also known as Bcl11b), one of the newly characterized genes, demonstrate that it plays a critical role in the development of CSMN axonal projections to the spinal cord in vivo, confirming that we identified central genetic determinants of the CSMN population.     

C1n3缺失对酿酒酵母生长的影响

Cln3是酿酒酵母G1期周期蛋白中的一种,为了研究Cln3在细胞周期与形态发生中的作用,我们构建了酿酒酵母CLN3基因的缺失株,并对其表型进行了分析。结果显示,cln3缺失株对α信息素的敏感性增强,α信息素诱导的细胞周期停滞现象明显大于野生型菌株,这种增强作用不受Sgvl因子的影响。同时,与野生菌相比cln3缺失株的细胞形态也有明显变化,双倍体cln3缺失株细胞的顶端生长能力增强而单倍体细胞的侵入生长能力则受到抑制。结果表明,与酿酒酵母的另外两个G1期周期蛋白Clnl、Cln2不同,Cln3在形态发生中有其独特的功能与作用方式。     

Identification of new differentiation inducing factors from Dictyostelium discoideum

Developing Dictyostelium discoideum amoebae form a stalked fruiting body in which individual cells differentiate into either stalk cells or spores. The major known inducer of stalk cell differentiation is the chlorinated polyketide DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one); however a mutant blocked in the terminal step of DIF-1 biosynthesis still produces one of the prestalk cell subtypes – the pstA cells – as well as some mature stalk cells. We therefore searched for additional stalk cell-inducing factors in the medium supporting development of this mutant. These factors were purified by solvent extraction and HPLC and identified by mass spectroscopy and NMR. The mutant lacked detectable DIF-2 and DIF-3 (the pentanone and deschloro homologues of DIF-1) but four major stalk cell-inducing activities were detected, of which three were identified. Two compounds were predicted intermediates in DIF-1 biosynthesis: the desmethyl, and desmethyl-monochloro analogues of DIF-1 (dM-DIF-1 and Cl-THPH, respectively), supporting the previously proposed pathway of DIF-1 biosynthesis. The third compound was a novel factor and was identified as 4-methyl-5-pentylbenzene-1,3-diol (MPBD) with the structure confirmed by chemical synthesis. To investigate the potential roles of these compounds as signal molecules, their effects on morphological stalk and spore differentiation were examined in cell culture. All three induced morphological stalk cell differentiation. We found that synthetic MPBD also stimulated spore cell differentiation. Now that these factors are known to be produced and released during development, their biological roles can be pursued further.     

Isoform-specific complementation of the yeast sac6 null mutation by human fimbrin.

The actin cytoskeleton is a fundamental component of eukaryotic cells, with both structural and motile roles. Actin and many of the actin-binding proteins found in different cell types are highly conserved, showing considerable similarity in both primary structure and biochemical properties. To make detailed comparisons between homologous proteins, it is necessary to know whether the various proteins are functionally, as well as structurally, conserved. Fimbrin is an example of a cytoskeletal component that, as shown by sequence determinations and biochemical characterizations, is conserved between organisms as diverse as Saccharomyces cerevisiae and humans. In this study, we examined whether the human homolog can substitute for the yeast protein in vivo. We report here that two isoforms of human fimbrin, also referred to as T- and L-plastin, can both substitute in vivo for yeast fimbrin, also known as Sac6p, whereas a third isoform, I-fimbrin (or I-plastin), cannot. We demonstrate that the human T- and L-fimbrins, in addition to complementing the temperature-sensitive growth defect of the sac6 null mutant, restore both normal cytoskeletal organization and cell shape to the mutant cells. In addition, we show that human T- and L-fimbrins can complement a sporulation defect caused by the sac6 null mutation. These findings indicate that there is a high degree of functional conservation in the cytoskeleton, even between organisms as diverse as S. cerevisiae and humans.