A new ß-galactosidase from Paenibacillus wynnii with potential for industrial applications.

Commercial ß-galactosidases exhibit undesirable kinetic properties regarding substrate affinity (Michaelis-Menten constant [KM] for lactose) and product inhibition (inhibitor constant [Ki] for galactose). An in silico screening of gene sequences was done and identified a putative ß-galactosidase (Paenibacillus wynnii ß-galactosidase, BgaPw) from the psychrophilic bacterium Paenibacillus wynnii. The cultivation of the wild-type P. wynnii strain resulted in very low ß-galactosidase activities of a maximum of 150 nkat per liter of medium with o-nitrophenyl-ß-d-galactopyranoside (oNPGal) as substrate. The recombinant production of BgaPw in Escherichia coli BL21(DE3) increased the yield ∼9,000-fold. Here, a volumetric activity of 1,350.18 ± 11.82 µkatoNPGal/Lculture was achieved in a bioreactor cultivation. The partly purified BgaPw showed a pH optimum at 7.0, a temperature maximum at 40°C, and an excellent stability at 8°C with a half-life of 77 d. Kinetic studies with BgaPw were done in milk or in milk-imitating synthetic buffer (Novo buffer), respectively. Remarkably, the KM value of BgaPw with lactose was as low as 0.63 ± 0.045 mM in milk. It was found that the resulting products of lactose hydrolysis, namely galactose and glucose, did not inhibit the ß-galactosidase activity of BgaPw, but instead showed a striking activating effect in both cases (up to 144%). In a comparison study in milk, lactose was completely hydrolyzed by BgaPw in 72 h at 8°C, whereas 2 other known ß-galactosidases were less powerful and converted only about 90% of lactose in the same time. Finally, the formation of galactooligosaccharides (GOS) was demonstrated with the new BgaPw, starting with pharma-lactose (400 g/L). A GOS production of about 144 g/L was achieved after 24 h (36.0% yield).

Phosphorylation of Suppressor of Hairless impedes its DNA-binding activity.

Notch signalling activity governs cellular differentiation in higher metazoa, where Notch signals are transduced by the transcription factor CSL, called Suppressor of Hairless [Su(H)] in Drosophila. Su(H) operates as molecular switch on Notch target genes: within activator complexes, including intracellular Notch, or within repressor complexes, including the antagonist Hairless. Mass spectrometry identified phosphorylation on Serine 269 in Su(H), potentially serving as a point of cross-regulation by other signalling pathways. To address the biological significance, we generated phospho-deficient [Su(H)S269A] and phospho-mimetic [Su(H)S269D] variants: the latter displayed reduced transcriptional activity despite unaltered protein interactions with co-activators and -repressors. Based on the Su(H) structure, Ser269 phosphorylation may interfere with DNA-binding, which we confirmed by electro-mobility shift assay and isothermal titration calorimetry. Overexpression of Su(H)S269D during fly development demonstrated reduced transcriptional regulatory activity, similar to the previously reported DNA-binding defective mutant Su(H)R266H. As both are able to bind Hairless and Notch proteins, Su(H)S269D and Su(H)R266H provoked dominant negative effects upon overexpression. Our data imply that Ser269 phosphorylation impacts Notch signalling activity by inhibiting DNA-binding of Su(H), potentially affecting both activation and repression. Ser269 is highly conserved in vertebrate CSL homologues, opening the possibility of a general and novel mechanism of modulating Notch signalling activity.

Hairless-binding deficient Suppressor of Hairless alleles reveal Su(H) protein levels are dependent on complex formation with Hairless.

Cell fate choices during metazoan development are driven by the highly conserved Notch signalling pathway. Notch receptor activation results in release of the Notch intracellular domain (NICD) that acts as transcriptional co-activator of the DNA-binding protein CSL. In the absence of signal, a repressor complex consisting of CSL bound to co-repressors silences Notch target genes. The Drosophila repressor complex contains the fly CSL orthologue Suppressor of Hairless [Su(H)] and Hairless (H). The Su(H)-H crystal structure revealed a large conformational change within Su(H) upon H binding, precluding interactions with NICD. Based on the structure, several sites in Su(H) and H were determined to specifically engage in complex formation. In particular, three mutations in Su(H) were identified that affect interactions with the repressor H but not the activator NICD. To analyse the effects these mutants have on normal fly development, we introduced these mutations into the native Su(H) locus by genome engineering. We show that the three H-binding deficient Su(H) alleles behave similarly. As these mutants lack the ability to form the repressor complex, Notch signalling activity is strongly increased in homozygotes, comparable to a complete loss of H activity. Unexpectedly, we find that the abundance of the three mutant Su(H) protein variants is altered, as is that of wild type Su(H) protein in the absence of H protein. In the presence of NICD, however, Su(H) mutant protein persists. Apparently, Su(H) protein levels depend on the interactions with H as well as with NICD. Based on these results, we propose that in vivo levels of Su(H) protein are stabilised by interactions with transcription-regulator complexes.

Local overexpression of Su(H)-MAPK variants affects Notch target gene expression and adult phenotypes in Drosophila.

In Drosophila, Notch and EGFR signalling pathways are closely intertwined. Their relationship is mostly antagonistic, and may in part be based on the phosphorylation of the Notch signal transducer Suppressor of Hairless [Su(H)] by MAPK. Su(H) is a transcription factor that together with several cofactors regulates the expression of Notch target genes. Here we address the consequences of a local induction of three Su(H) variants on Notch target gene expression. To this end, wild-type Su(H), a phospho-deficient Su(H) (MAPK-) (ko) and a phospho-mimetic Su(H) (MAPK-ac) isoform were overexpressed in the central domain of the wing anlagen. The expression of the Notch target genes cut, wingless, E(spl)m8-HLH and vestigial, was monitored. For the latter two, reporter genes were used (E(spl)m8-lacZ, vg (BE) -lacZ). In general, Su(H) (MAPK-) (ko) induced a stronger response than wild-type Su(H), whereas the response to Su(H) (MAPK-ac) was very weak. Notch target genes cut, wingless and vg (BE) -lacZ were ectopically activated, whereas E(spl)m8-lacZ was repressed by overexpression of Su(H) proteins. In addition, in epistasis experiments an activated form of the EGF-receptor (DER (act) ) or the MAPK (rl (SEM) ) and individual Su(H) variants were co-overexpressed locally, to compare the resultant phenotypes in adult flies (thorax, wings and eyes) as well as to assay the response of the Notch target gene cut in cell clones.

Cyclin G Functions as a Positive Regulator of Growth and Metabolism in Drosophila.

In multicellular organisms, growth and proliferation is adjusted to nutritional conditions by a complex signaling network. The Insulin receptor/target of rapamycin (InR/TOR) signaling cascade plays a pivotal role in nutrient dependent growth regulation in Drosophila and mammals alike. Here we identify Cyclin G (CycG) as a regulator of growth and metabolism in Drosophila. CycG mutants have a reduced body size and weight and show signs of starvation accompanied by a disturbed fat metabolism. InR/TOR signaling activity is impaired in cycG mutants, combined with a reduced phosphorylation status of the kinase Akt1 and the downstream factors S6-kinase and eukaryotic translation initiation factor 4E binding protein (4E-BP). Moreover, the expression and accumulation of Drosophila insulin like peptides (dILPs) is disturbed in cycG mutant brains. Using a reporter assay, we show that the activity of one of the first effectors of InR signaling, Phosphoinositide 3-kinase (PI3K92E), is unaffected in cycG mutants. However, the metabolic defects and weight loss in cycG mutants were rescued by overexpression of Akt1 specifically in the fat body and by mutants in widerborst (wdb), the B'-subunit of the phosphatase PP2A, known to downregulate Akt1 by dephosphorylation. Together, our data suggest that CycG acts at the level of Akt1 to regulate growth and metabolism via PP2A in Drosophila.

Loss of putzig Activity Results in Apoptosis during Wing Imaginal Development in Drosophila.

The Drosophila gene putzig (pzg) encodes a nuclear protein that is an integral component of the Trf2/Dref complex involved in the transcription of proliferation-related genes. Moreover, Pzg is found in a complex together with the nucleosome remodeling factor NURF, where it promotes Notch target gene activation. Here we show that downregulation of pzg activity in the developing wing imaginal discs induces an apoptotic response, accompanied by the induction of the pro-apoptotic gene reaper, repression of Drosophila inhibitor of apoptosis protein accumulation and the activation of the caspases Drice, Caspase3 and Dcp1. As a further consequence 'Apoptosis induced Proliferation' (AiP) and 'Apoptosis induced Apoptosis' (AiA) are triggered. As expected, the activity of the stress kinase Jun N-terminal kinase (JNK), proposed to mediate both processes, is ectopically induced in response to pzg loss. In addition, the expression of the mitogen wingless (wg) but not of decapentaplegic (dpp) is observed. We present evidence that downregulation of Notch activates Dcp1 caspase and JNK signaling, however, neither induces ectopic wg nor dpp expression. In contrast, the consequences of Dref-RNAi were largely indistinguishable from pzg-RNAi with regard to apoptosis induction. Moreover, overexpression of Dref ameliorated the downregulation of pzg compatible with the notion that the two are required together to maintain cell and tissue homeostasis in Drosophila.

MAPK-dependent phosphorylation modulates the activity of Suppressor of Hairless in Drosophila.

Cell differentiation strictly depends on the epidermal growth factor receptor (EGFR)- and Notch-signalling pathways, which are closely intertwined. Here we address the molecular cross talk at the level of Suppressor of Hairless [Su(H)]. The Drosophila transcription factor Su(H) mediates Notch signalling at the DNA level: in the presence of signalling input Su(H) assembles an activator complex on Notch target genes and a repressor complex in its absence. Su(H) contains a highly conserved mitogen activated protein kinase (MAPK) target sequence. Here we provide evidence that Su(H) is phosphorylated in response to MAPK activity. Mutation of the Su(H) MAPK-site modulated the Notch signalling output: whereas a phospho-deficient Su(H)(MAPK-ko) isoform provoked a stronger Notch signalling activity, a phospho-mimetic Su(H)(MAPK-ac) mutant resulted in its attenuation. In vivo assays in Drosophila cell culture as well as in flies support the idea that Su(H) phosphorylation affects the dynamics of repressor or activator complex formation or the transition from the one into the other complex. In summary, the phosphorylation of Su(H) attenuates Notch signalling in vivo in several developmental settings. Consequently, a decrease of EGFR signal causes an increase of Notch signalling intensity. Hence, the antagonistic relationship between EGFR- and Notch-signalling pathways may involve a direct modification of Su(H) by MAPK in several developmental contexts of fly development. The high sequence conservation of the MAPK target site in the mammalian Su(H) homologues supports the idea that EGFR signalling impacts on Notch activity in a similar way in humans as well.

Gain of function notch phenotypes associated with ectopic expression of the Su(H) C-terminal domain illustrate separability of Notch and hairless-mediated activities.

The Notch signaling pathway is instrumental for cell fate decisions. Signals from the Notch receptor are transduced by CSL-type DNA-binding proteins. In Drosophila, this protein is named Suppressor of Hairless [Su(H)]. Together with the intracellular domain of the activated Notch receptor ICN, Su(H) assembles a transcriptional activator complex on Notch target genes. Hairless acts as the major antagonist of the Notch signaling pathway in Drosophila by means of the formation of a repressor complex together with Su(H) and several co-repressors. Su(H) is characterized by three domains, the N-terminal domain NTD, the beta-trefoil domain BTD and the C-terminal domain CTD. NTD and BTD bind to the DNA, whereas BTD and CTD bind to ICN. Hairless binds to the CTD, however, to sites different from ICN. In this work, we have addressed the question of competition and availability of Su(H) for ICN and Hairless binding in vivo. To this end, we overexpressed the CTD during fly development. We observed a strong activation of Notch signaling processes in various tissues, which may be explained by an interference of CTD with Hairless corepressor activity. Accordingly, a combined overexpression of CTD together with Hairless ameliorated the effects, unlike Su(H) which strongly enhances repression when overexpressed concomitantly with Hairless. Interestingly, in the combined overexpression CTD accumulated in the nucleus together with Hairless, whereas it is predominantly cytoplasmic on its own.

Structural and functional analysis of the repressor complex in the Notch signaling pathway of Drosophila melanogaster.

In metazoans, the highly conserved Notch pathway drives cellular specification. On receptor activation, the intracellular domain of Notch assembles a transcriptional activator complex that includes the DNA-binding protein CSL, a composite of human C-promoter binding factor 1, Suppressor of Hairless of Drosophila melanogaster [Su(H)], and lin-12 and Glp-1 phenotype of Caenorhabditis elegans. In the absence of ligand, CSL represses Notch target genes. However, despite the structural similarity of CSL orthologues, repression appears largely diverse between organisms. Here we analyze the Notch repressor complex in Drosophila, consisting of the fly CSL protein, Su(H), and the corepressor Hairless, which recruits general repressor proteins. We show that the C-terminal domain of Su(H) is necessary and sufficient for forming a high-affinity complex with Hairless. Mutations in Su(H) that affect interactions with Notch and Mastermind have no effect on Hairless binding. Nonetheless, we demonstrate that Notch and Hairless compete for CSL in vitro and in cell culture. In addition, we identify a site in Hairless that is crucial for binding Su(H) and subsequently show that this Hairless mutant is strongly impaired, failing to properly assemble the repressor complex in vivo. Finally, we demonstrate Hairless-mediated inhibition of Notch signaling in a cell culture assay, which hints at a potentially similar repression mechanism in mammals that might be exploited for therapeutic purposes.

Radiation-induced apoptosis in retinal progenitor cells is p53-dependent with caspase-independent DNA fragmentation.

Caspases are important executioners of the endogenous cell death program. However, their function is not restricted to the induction of cell death. Caspases may process cytokines and contribute to cell differentiation or lymphocyte proliferation. In addition to their pleiotropic functions we show evidence that, under certain conditions, caspases are activated during apoptosis without executing the cell death program. Following whole body irradiation, p53 and caspases were activated in both the cerebellum and eye of postnatal day 5 mice. Although p53 activation and cell death kinetics were similar in both the cerebellum and eye, the processing of caspases was protracted and reduced in the eye. In particular, retinal caspase activation appeared not to be the executioner of cell death; incubation of retinal and cerebellar explants in the presence of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone prevented DNA fragmentation, a hallmark of apoptosis, only in cerebellar granule cells. In contrast, in retinal cells no impairment of DNA fragmentation was observed in the presence of N-benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone, indicating p53-dependent but caspase-independent cell death pathways despite caspase activation.