Inducing apoptosis of cancer cells using small-molecule plant compounds that bind to GRP78.

BACKGROUND: Glucose regulated protein 78 (GRP78) functions as a sensor of endoplasmic reticulum (ER) stress. The aim of this study was to test the hypothesis that molecules that bind to GRP78 induce the unfolded protein response (UPR) and enhance cell death in combination with ER stress inducers. METHODS: Differential scanning calorimetry (DSC), measurement of cell death by flow cytometry and the induction of ER stress markers using western blotting. RESULTS: Epigallocatechin gallate (EGCG), a flavonoid component of Green Tea Camellia sinensis, and honokiol (HNK), a Magnolia grandiflora derivative, bind to unfolded conformations of the GRP78 ATPase domain. Epigallocatechin gallate and HNK induced death in six neuroectodermal tumour cell lines tested. Levels of death to HNK were twice that for EGCG; half-maximal effective doses were similar but EGCG sensitivity varied more widely between cell types. Honokiol induced ER stress and UPR as predicted from its ability to interact with GRP78, but EGCG was less effective. With respect to cell death, HNK had synergistic effects on melanoma and glioblastoma cells with the ER stress inducers fenretinide or bortezomib, but only additive (fenretinide) or inhibitory (bortezomib) effects on neuroblastoma cells. CONCLUSION: Honokiol induces apoptosis due to ER stress from an interaction with GRP78. The data are consistent with DSC results that suggest that HNK binds to GRP78 more effectively than EGCG. Therefore, HNK may warrant development as an antitumour drug.

Crystal structure of the dimer of two essential Salmonella typhimurium proteins, YgjD & YeaZ and calorimetric evidence for the formation of a ternary YgjD-YeaZ-YjeE complex.

YgjD from COG0533 is amongst a small group of highly conserved proteins present in all three domains of life. Various roles and biochemical functions (including sialoprotease and endonuclease activities) have been ascribed to YgjD and orthologs, the most recent, however, is involvement in the post transcriptional modification of certain tRNAs by formation of N6-threonyl-adenosine (t6A) at position 37. In bacteria, YgjD is essential and along with YeaZ, YjeE, and YrdC has been shown to be 'necessary and sufficient' for the tRNA modification. To further define interactions and possible roles for some of this set of proteins we have undertaken structural and biochemical studies. We show that formation of the previously reported heterodimer of YgjD-YeaZ involves ordering of the C-terminal region of YeaZ which extends along the surface of YgjD in the crystal structure. ATPγS or AMP is observed in YgjD while no nucleotide is bound on YeaZ. ITC experiments reveal previously unreported binary and ternary complexes which can be nucleotide dependent. The stoichiometry of the YeaZ-YgjD complex is 1:1 with a K(D) of 0.3 µM. YgjD and YjeE interact only in the presence of ATP, while YjeE binds to YgjD-YeaZ in the presence of ATP or ADP with a K(D) of 6 µM. YgjD doesn't bind the precursors of t6A, threonine, and bicarbonate. These results show a more complex set of interactions than previously thought, which may have a regulatory role. The understanding gained should help in deriving inhibitors of these essential proteins that might have potential as antibacterial drugs.

Structure of the ribosomal interacting GTPase YjeQ from the enterobacterial species Salmonella typhimurium.

The YjeQ class of P-loop GTPases assist in ribosome biogenesis and also bind to the 30S subunit of mature ribosomes. YjeQ ribosomal binding is GTP-dependent and thought to specifically direct protein synthesis, although the nature of the upstream signal causing this event in vivo is as yet unknown. The attenuating effect of YjeQ mutants on bacterial growth in Escherichia coli makes it a potential target for novel antimicrobial agents. In order to further explore the structure and function of YjeQ, the isolation, crystallization and structure determination of YjeQ from the enterobacterial species Salmonella typhimurium (StYjeQ) is reported. Whilst the overall StYjeQ fold is similar to those of the previously reported Thematoga maritima and Bacillus subtilis orthologues, particularly the GTPase domain, there are larger differences in the three OB folds. Although the zinc-finger secondary structure is conserved, significant sequence differences alter the nature of the external surface in each case and may reflect varying signalling pathways. Therefore, it may be easier to develop YjeQ-specific inhibitors that target the N- and C-terminal regions, disrupting the metabolic connectivity rather than the GTPase activity. The availability of coordinates for StYjeQ will provide a significantly improved basis for threading Gram-negative orthologue sequences and in silico compound-screening studies, with the potential for the development of species-selective drugs.

Structural characterization of Salmonella typhimurium YeaZ, an M22 O-sialoglycoprotein endopeptidase homolog.

The Salmonella typhimurium "yeaZ" gene (StyeaZ) encodes an essential protein of unknown function (StYeaZ), which has previously been annotated as a putative homolog of the Pasteurella haemolytica M22 O-sialoglycoprotein endopeptidase Gcp. YeaZ has also recently been reported as the first example of an RPF from a gram-negative bacterial species. To further characterize the properties of StYeaZ and the widely occurring MK-M22 family, we describe the purification, biochemical analysis, crystallization, and structure determination of StYeaZ. The crystal structure of StYeaZ reveals a classic two-lobed actin-like fold with structural features consistent with nucleotide binding. However, microcalorimetry experiments indicated that StYeaZ neither binds polyphosphates nor a wide range of nucleotides. Additionally, biochemical assays show that YeaZ is not an active O-sialoglycoprotein endopeptidase, consistent with the lack of the critical zinc binding motif. We present a detailed comparison of YeaZ with available structural homologs, the first reported structural analysis of an MK-M22 family member. The analysis indicates that StYeaZ has an unusual orientation of the A and B lobes which may require substantial relative movement or interaction with a partner protein in order to bind ligands. Comparison of the fold of YeaZ with that of a known RPF domain from a gram-positive species shows significant structural differences and therefore potentially distinctive RPF mechanisms for these two bacterial classes.

Ligand-induced conformational changes and a mechanism for domain closure in Aspergillus nidulans dehydroquinate synthase.

In order to investigate systematically substrate and cofactor-induced conformational changes in the enzyme dehydroquinate synthase (DHQS), eight structures representing a series of differently liganded states have been determined in a total of six crystal forms. DHQS in the absence of the substrate analogue carbaphosphonate, either unliganded or in the presence of NAD or ADP, is in an open form where a relative rotation of 11-13 degrees between N and C-terminal domains occurs. Analysis of torsion angle difference plots between sets of structures reveals eight rearrangements that appear relevant to domain closure and a further six related to crystal packing. Overlapping 21 different copies of the individual N and C-terminal DHQS domains further reveals a series of pivot points about which these movements occur and illustrates the way in which widely separated secondary structure elements are mechanically inter-linked to form "composite elements", which propagate structural changes across large distances. This analysis has provided insight into the basis of DHQS ligand-initiated domain closure and gives rise to the proposal of an ordered sequence of events involving substrate binding, and local rearrangements within the active site that are propagated to the hinge regions, leading to closure of the active-site cleft.

The structure of the negative transcriptional regulator NmrA reveals a structural superfamily which includes the short-chain dehydrogenase/reductases.

NmrA is a negative transcriptional regulator involved in the post-translational modulation of the GATA-type transcription factor AreA, forming part of a system controlling nitrogen metabolite repression in various fungi. X-ray structures of two NmrA crystal forms, both to 1.8 A resolution, show NmrA consists of two domains, including a Rossmann fold. NmrA shows an unexpected similarity to the short-chain dehydrogenase/reductase (SDR) family, with the closest relationship to UDP-galactose 4-epimerase. We show that NAD binds to NmrA, a previously unreported nucleotide binding property for this protein. NmrA is unlikely to be an active dehydrogenase, however, as the conserved catalytic tyrosine in SDRs is absent in NmrA, and thus the nucleotide binding to NmrA could have a regulatory function. Our results suggest that other transcription factors possess the SDR fold with functions including RNA binding. The SDR fold appears to have been adapted for other roles including non-enzymatic control functions such as transcriptional regulation and is likely to be more widespread than previously recognized.

Identification of domains responsible for signal recognition and transduction within the QUTR transcription repressor protein.

QUTR (qutR-encoded transcription-repressing protein) is a multi-domain repressor protein active in the signal-transduction pathway that regulates transcription of the quinic acid utilization (qut) gene cluster in Aspergillus nidulans. In the presence of quinate, production of mRNA from the eight genes of the qut pathway is stimulated by the activator protein QUTA (qutA-encoded transcription-activating protein). Mutations in the qutR gene alter QUTR function such that the transcription of the qut gene cluster is permanently on (constitutive phenotype) or is insensitive to the presence of quinate (super-repressed phenotype). These mutant phenotypes imply that the QUTR protein plays a key role in signal recognition and transduction, and we have used deletion analysis to determine which regions of the QUTR protein are involved in these functions. We show that the QUTR protein recognizes and binds to the QUTA protein in vitro and that the N-terminal 88 amino acids of QUTR are sufficient to inactivate QUTA function in vivo. Deletion analysis and domain-swap experiments imply that the two C-terminal domains of QUTR are mainly involved in signal recognition.

Deletion of the N-terminal region of the AREA protein is correlated with a derepressed phenotype with respect to nitrogen metabolite repression.

The entire areA gene and a truncated version lacking the sequence encoding the N-terminal 389 amino acids were expressed from the qutE promoter and terminator in an Aspergillus nidulans strain with the endogenous areA gene deleted. This expression system was used to decouple the effects of transcription regulation and mRNA stability mediated by the native promoter and terminator from any posttranslational modulation of AREA activity. Both the full-length AREA protein and the truncated form were able to function in the deletion strain, conferring the ability to use alternate nitrogen sources. Transformants containing the entire areA gene had a repressible phenotype with respect to nitrogen metabolite repression, whereas those containing the truncated form of the areA gene had a derepressed phenotype. The truncated areA gene was expressed in an A. nidulans strain containing a normally regulated wild-type areA gene, and transformants displayed a quinate-inducible nitrogen metabolite derepressed phenotype. Northern blot analysis of transformed strains showed that areA-specific mRNAs of the expected sizes were being produced. The truncated AREA protein was overproduced in Escherichia coli as a fusion protein and purified to homogeneity by a single-step immobilized metal affinity chromatography, and the purified protein was shown to bind specifically to the niaD promoter. Revised sequences of the 5' region of the areA gene and the entire meaB gene are reported.

Differential expression of ryanodine receptor RyR2 mRNA in the non-pregnant and pregnant human myometrium.

We describe here the expression of the ryanodine receptor isoforms RyR2 and RyR3 in human non-pregnant and pregnant (non-labouring) myometrium, and in isolated cultured myometrial cells. The mRNA encoding the RyR3 isoform was found in both non-pregnant and pregnant myometrial tissue samples; however, the mRNA for RyR2 was found only in pregnant samples. It can be speculated that the appearance of this additional isoform in the pregnant myometrium may increase the ability of this tissue to contract at term. Control of expression of the RyR2 gene may therefore be another example of an up-regulated signalling system in pregnancy. Although the mRNA for RyR3 was expressed in cultured myometrial cells, the mRNA for RyR2 could not be detected. Thus cultured myometrial cells appear to be similar to the non-pregnant myometrium. The cytokine transforming growth factor beta (TGF-beta) has been reported to alter RyR mRNA expression in many cell types. After treatment with TGF-beta, both RyR2 and RyR3 mRNAs could be detected in cultured myometrial cells. These observations support the idea that the expression of the RyR2 isoform is up-regulated both in pregnancy and in TGF-beta-treated cultured myometrial cells. Using measurements of 45Ca2+ release, we have further demonstrated that cultured human myometrial cells show a significant augmentation of both the Ca2+-induced Ca2+ release (CICR) mechanism and ryanodine-induced Ca2+ release after treatment with TGF-beta. Additionally, caffeine was able to induce Ca2+ release and sensitize the CICR mechanism to ryanodine. Thus we suggest that the appearance of RyR2 mRNA leads to the expression of this receptor/channel protein with identifiable pharmacological characteristics. These results are discussed in the context of the potential role of gene activation in the process of maturation of the human myometrium during pregnancy.

Control of metabolic flux through the quinate pathway in Aspergillus nidulans.

The quinic acid ulitization (qut) pathway in Aspergillus nidulans is a dispensable carbon utilization pathway that catabolizes quinate to protocatechuate via dehydroquinate and dehydroshikimate(DHS). At the usual in vitro growth pH of 6.5, quinate enters the mycelium by means of a specific permease and is converted into PCA by the sequential action of the enzymes quinate dehydrogenase, 3-dehydroquinase and DHS dehydratase. The extent of control on metabolic flux exerted by the permease and the three pathway enzymes was investigated by applying the techniques of Metabolic Control Analysis. The flux control coefficients for each of the three quinate pathway enzymes were determined empirically, and the flux control coefficient of the quinate permease was inferred by use of the summation theorem. There measurements implied that, under the standard growth conditions used, the values for the flux control coefficients of the components of the quinate pathway were: quinate permease, 0.43; quinate dehydrogenase, 0.36; dehydroquinase, 0.18; DHS dehydratase, <0,03. Attempts to partially decouple quinate permease from the control over flux by measuring flux at pH 3.5 (when a significant percentage of the soluble quinate is protonated and able to enter the mycelium without the aid of a permease) led to an increase of approx. 50% in the flux control coefficient for dehydroquinase. Taken together with the fact that A. nidulans has a very efficient pH homeostasis mechanism, these experiments are consistent with the view that quinate permease exerts a high degree of control over pathway flux under the standard laboratory growth conditions at pH 6.5. The enzymes quinate dehydrogenase and 3-dehydroquinase have previously been overproduced in Escherichia coli, and protocols for their purification published. The remaining qut pathway enzyme DHS dehydratase was overproduced in E. coli and a purification protocol established. The purified DHS dehydratase was shown to have a K(m) of 530 microM for its substrate DHS and a requirement for bivalent metal cations that could be fulfilled by Mg(2+), Mn(2+) or Zn(2+). All three qut pathway enzymes were purified in bulk and their elasticity coefficients with respect to the three quinate pathway intermediates were derived over a range of concentrations in a core tricine/NaOH buffer, augmented with necessary cofactors and bivalent cations as appropriate. Using these empirically determined relative values, in conjunction with the connectivity theorem, the relative ratios of the flux control coefficients for the various quinate pathway enzymes, and how this control shifts between them, was determined over a range of possible metabolic concentrations. These calculations, although clearly subject to caveates about the relationswhip between kinetic measurements in vitro and the situation in vivo, were able to successfully predict the hiearchy of control observed under the standard laboratory growth conditions. The calculations imply that the hierarchy of control exerted by the quinate pathway enzymes is stable and relatively insensitive to changing metabolite concentrations in the ranges most likely to correspond to those found in vivo. The effects of substituting the type I 3-dehydroquinases from Salmonella typhi and the A. nidulans AROM protein (a pentadomain protein catalysing the conversion of 3-deoxy-D-arabinoheptulosonic acid 7-phosphate into 5-enolpyruvylshikimate 3 phosphate), and the Mycobacterium tuberculosis type II 3-dehydroquinase, in the quinate pathway were investigated and found to have an effect. In the case of S. typhi and A. nidulans, overproduction of heterologous dehydroquinase led to a diminuation of pathway flux caused by a lowering of in vivo quinate dehydrogenase levels increased above those of the wild type. We speculate that these changes in qu

Comparative analysis of the QUTR transcription repressor protein and the three C-terminal domains of the pentafunctional AROM enzyme.

The AROM protein is a pentadomain protein catalysing steps two to six in the prechorismate section of the shikimate pathway in microbial eukaryotes. On the basis of amino acid sequence alignments and the properties of mutants unable to utilize quinic acid as a carbon source, the AROM protein has been proposed to be homologous throughout its length with the proteins regulating transcription of the genes necessary for quinate catabolism. The QUTR transcription repressor protein has been proposed to be homologous with the three C-terminal domains of the AROM protein and one-fifth of the penultimate N-terminal domain. We report here the results of experiments designed to overproduce the QUTR and AROM proteins and their constituent domains in Escherichia coli, the purpose being to facilitate domain purification and (in the case of AROM), complementation of E. coli aro- mutations in order to probe the degree to which individual domains are stable and functional. The 3-dehydroquinate dehydratase domain of the AROM protein and the 3-dehydroquinate dehydratase-like domain of the QUTR spectroscopy and fluorescence emission spectroscopy. The CD spectra were found to be virtually superimposable. The fluorescence emission spectra of both domains had the signal from the tryptophan residues almost completely quenched, giving a tyrosine-dominated spectrum for both the AROM- and QUTR-derived domains. This unexpected observation was demonstrated to be due to a highly unusual environment provided by the tertiary structure, as addition of the denaturant guanidine hydrochloride gave a typical tryptophan-dominated spectrum for both domains. The spectroscopy experiments had the potential to refute the biologically-based proposal for a common origin for the AROM and QUTR proteins; however, the combined biophysical data are consistent with the hypothesis. We have previously reported that the AROM dehydroquinate synthase and 3-dehydroquinate dehydratase are stable and functional as individual domains, but that the 5-enol-pyruvylshikimate-3-phosphate synthase is only active as part of the complete AROM protein or as a bi-domain fragment with dehydroquinate synthase. Here we report that the aromA gene (encoding the AROM protein) of Aspergillus nidulans contains a 53 nt intron in the extreme C-terminus of the shikimate dehydrogenase domain. This finding accounts for the previously reported observation that the AROM protein was unable to complement aroE- (lacking shikimate dehydrogenase) mutations in E. coli. When the intron is removed the correctly translated AROM protein is able to complement the E. coli aroE- mutation. An AROM-derived shikimate dehydrogenase domain is, however, non-functional, but function is restored in a bi-domain protein with e-dehydroquinate dehydratase. This interaction is not entirely specific, as substitution of the 3-dehydroquinate dehydratase domain with the glutathione S-transferase protein partially restores enzyme activity. Similarly an AROM-derived shikimate kinase domain is non-functional, but is functional as part of the complete AROM protein, or as a bi-domain protein with 3-dehydroquinate dehydratase.

Isolation and partial cloning of ryanodine-sensitive Ca2+ release channel protein isoforms from human myometrial smooth muscle.

Partial cDNAs of the ryanodine receptor were cloned using PCR analysis from reverse transcribed total and mRNA, extracted from freshly isolated pregnant, non-pregnant, and cultured human myometrial smooth muscle. The identity of these clones was confirmed by nucleotide sequencing of the fragments and indicate the expression of both the skeletal and brain ryanodine receptor isoforms in these preparations. In freshly isolated non-pregnant myometrial tissue, membrane fractions displaying specific [3H]ryanodine binding activities were isolated using density gradient centrifugation. SDS-PAGE of the sucrose gradient fractions indicated the specific comigration of a polypeptide with a molecular mass of approximately 544 kDa with the ryanodine binding activity.

The molecular biology of multidomain proteins. Selected examples.

The aim of this review is to give an overview of the contribution molecular biology can make to an understanding of the functions and interactions within multidomain proteins. The contemporary advantages ascribed to multidomain proteins include (a) the potential for metabolite channelling and the protection of unstable intermediates; (b) the potential for interactions between domains catalysing sequential steps in a metabolic pathway, thereby giving the potential for allosteric interactions; and (c) the facility to produce enzymic activities in a fixed stoichiometric ratio. The alleged advantages in (a) and (b) however apply equally well to multi-enzyme complexes; therefore, specific examples of these phenomena are examined in multidomain proteins to determine whether the proposed advantages are apparent. Some transcription-regulating proteins active in the control of metabolic pathways are composed of multiple domains and their control is exerted and modulated at the molecular level by protein-DNA, protein-protein and protein-metabolite interactions. These complex recognition events place strong constraints upon the proteins involved, requiring the recognition of and interaction with different classes of cellular metabolites and macromolecules. Specific examples of transcription-regulating proteins are examined to probe how their multidomain nature facilitates a general solution to the problem of multiple recognition events. A general unifying theme that emerges from these case studies is that a basic unitary design of modules provided by enzymes is exploited to produce multidomain proteins by a complex series of gene duplication and fusion events. Successful modules provided by enzymes are co-opted to new function by selection apparently acting upon duplicated copies of the genes encoding the enzymes. In multidomain transcription-regulating proteins, former enzyme modules can be recruited as molecular sensors that facilitate presumed allosteric interactions necessary for the molecular control of transcription.

Evolution of transcription-regulating proteins by enzyme recruitment: molecular models for nitrogen metabolite repression and ethanol utilisation in eukaryotes.

Studies on the quinic acid utilisation gene (qut) cluster in Aspergillus nidulans showed that the genes encoding transcriptional activator and repressor proteins evolved by co-opting duplicated copies of genes encoding metabolic enzymes. In order to test the hypothesis that this was a general route for the genesis of regulatory proteins, the origins of the major control protein mediating nitrogen metabolite repression (an example of inter-pathway regulation) and ethanol utilisation (an example of intra-pathway regulation) in filamentous fungi were sought. The regulatory proteins mediating nitrogen metabolite repression were deduced to have originated in a duplication of genes encoding the anthranilate synthase complex which is active in the shikimate pathway. The major protein regulating ethanol utilisation was deduced to have its origin in the fusion of duplicated genes encoding the aldehyde and alcohol dehydrogenases (ALDA and ALCA). These data strongly support the view that transcriptional regulatory proteins evolve by the recruitment of functional domains provided by metabolic enzymes.

Genesis of eukaryotic transcriptional activator and repressor proteins by splitting a multidomain anabolic enzyme.

The genes necessary for the correctly regulated catabolism of quinate in Aspergillus nidulans and Neurospora crassa are controlled at the level of transcription by a DNA-binding activator protein and a repressor protein that directly interact with one another. The repressor protein is homologous throughout its length with the three C-terminal domains of a pentafunctional enzyme catalysing five consecutive steps in the related anabolic shikimate pathway. We now report that the activator protein is homologous to the two N-terminal domains of the same pentafunctional enzyme and that this proposed structural similarity suggests a molecular mechanism by which the repressor recognises the activator protein. We believe that this is the first report of the genesis of a pair of interacting eukaryotic regulatory proteins by the splitting of a multidomain anabolic enzyme. The recruitment of preformed enzymatically active domains to a regulatory role may represent a general mechanism for the evolution of pathway-specific regulator proteins in dispensable pathways.

Inducible overproduction of the Aspergillus nidulans pentafunctional AROM protein and the type-I and -II 3-dehydroquinases from Salmonella typhi and Mycobacterium tuberculosis.

The aroQ gene of Mycobacterium tuberculosis, encoding a type-II 3-dehydroquinase, and the aroD gene of Salmonella typhi, encoding a type-I 3-dehydroquinase, have been highly overexpressed in Escherichia coli using the powerful trc promoter contained within the expression vector pKK233-2. The M. tuberculosis type-II 3-dehydroquinase has been purified in bulk from overproducing strains of E. coli to greater than 95% homogeneity. The protein is extremely heat-stable, is active as a homododecamer and has the lowest reported Km value of any type-II 3-dehydroquinase. The pentafunctional aromA gene of Aspergillus nidulans has been overexpressed more than 120-fold in an A. nidulans aromA- qutB- double mutant from a truncated quinate-inducible qutE promoter, such that the AROM protein is visible as a significant fraction (approx. 6%) in cell-free crude extracts. The M. tuberculosis aroQ gene has been fused to the same truncated qutE promoter and shown to encode quinate-inducible 3-dehydroquinase activity that allows a qutE- mutant strain of A. nidulans to utilize quinate as sole carbon source.

Differential flux through the quinate and shikimate pathways. Implications for the channelling hypothesis.

The qutC gene encoding dehydroshikimate dehydratase has been constitutively overexpressed in Aspergillus nidulans from a range of 1-30-fold over the normal wild-type level. This overexpression leads to impaired growth in minimal medium which can be alleviated by the addition of aromatic amino acids to the medium. Overexpression of the qutC gene in mutant strains lacking protocatechuic acid (PCA) oxygenase leads to the build up of PCA in the medium, which can be measured by a simple assay. Measuring the rate of production of PCA in strains overproducing dehydroshikimate dehydratase and correlating this with the level of overproduction and impaired ability to grow in minimal medium lacking aromatic amino acids leads to the conclusion that (a) the metabolites 3-dehydroquinate and dehydroshikimate leak from the AROM protein at a rate comparable with the extent of flux catalysed by the AROM protein, (b) the AROM protein has a low-level channelling function probably as a result of the close juxtaposition of five active sites and (c) this channelling function is only physiologically significant under non-optimal conditions of nutrient supply and oxygenation, when the organism is in situ in its natural environment.