Sequence-structure analysis of FAD-containing proteins

We have analyzed structure-sequence relationships in 32 families of flavin adenine dinucleotide (FAD)-binding proteins, to prepare for genomic-scale analyses of this family. Four different FAD-family folds were identified, each containing at least two or more protein families. Three of these families, exemplified by glutathione reductase (GR), ferredoxin reductase (FR), and p-cresol methylhydroxylase (PCMH) were previously defined, and a family represented by pyruvate oxidase (PO) is newly defined. For each of the families, several conserved sequence motifs have been characterized. Several newly recognized sequence motifs are reported here for the PO, GR, and PCMH families. Each FAD fold can be uniquely identified by the presence of distinctive conserved sequence motifs. We also analyzed cofactor properties, some of which are conserved within a family fold while others display variability. Among the conserved properties is cofactor directionality: in some FAD-structural families, the adenine ring of the FAD points toward the FAD-binding domain, whereas in others the isoalloxazine ring points toward this domain. In contrast, the FAD conformation and orientation are conserved in some families while in others it displays some variability. Nevertheless, there are clear correlations among the FAD-family fold, the shape of the pocket, and the FAD conformation. Our general findings are as follows: (a) no single protein 'pharmacophore' exists for binding FAD; (b) in every FAD-binding family, the pyrophosphate moiety binds to the most strongly conserved sequence motif, suggesting that pyrophosphate binding is a significant component of molecular recognition; and (c) sequence motifs can identify proteins that bind phosphate-containing ligands.     

Necrotic core in EMT6/Ro tumour spheroids: Is it caused by an ATP deficit?

Although commonly related to nutrient deprivation, the cause of the formation of the necrotic core in the multicellular tumour spheroids is still a controversial issue. We propose a simple model for the cell ATP production that assumes glucose and lactate as the only fuel substrates, and describes the main reactions occurring in the glycolytic and the oxidative pathways. Under the key assumption that cell death occurs when ATP production falls to a critical level, we formulate a multiscale model that integrates the energy metabolism at the cellular level with the diffusive transport of the metabolites in the spheroid mass. The model has been tested by predicting the measurements of the necrotic radius obtained by Freyer and Sutherland (1986a) in EMT6/Ro spheroids under different concentrations of glucose and oxygen in the culture medium. The results appear to be in agreement with the hypothesis that necrosis is caused by ATP deficit.     

Esrom, an ortholog of PAM (protein associated with c-myc), regulates pteridine synthesis in the zebrafish

Zebrafish esrom mutants have an unusual combination of phenotypes: in addition to a defect in the projection of retinal axons, they have reduced yellow pigmentation. Here, we investigate the pigment phenotype and, from this, provide evidence for an unexpected defect in retinal neurons. Esrom is not required for the differentiation of neural crest precursors into pigment cells, nor is it essential for cell migration, pigment granule biogenesis, or translocation. Instead, loss of yellow color is caused by a deficiency of sepiapterin, a yellow pteridine. The level of several other pteridines is also affected in mutants. Importantly, the cofactor tetrahydrobiopterin (BH4) is drastically reduced in esrom mutants. Mutant retinal neurons also appear deficient in this pteridine. BH4-synthesizing enzymes are active in mutants, indicating a defect in the regulation rather than production of enzymes. Esrom has recently been identified as an ortholog of PAM (protein associated with c-myc), a very large protein involved in synaptogenesis in Drosophila and C. elegans. These data thus introduce a new regulator of pteridine synthesis in a vertebrate and establish a function for the Esrom protein family outside synaptogenesis. They also raise the possibility that neuronal defects are due in part to an abnormality in pteridine synthesis.     

Compensatory and long-range changes in picosecond-nanosecond main-chain dynamics upon complex formation: 15N relaxation analysis of the free and bound states of the ubiquitin-like domain of human plexin-B1 and the small GTPase Rac1

The formation of a complex between Rac1 and the cytoplasmic domain of plexin-B1 is one of the first documented cases of a direct interaction between a small guanosine 5′-triphosphatase (GTPase) and a transmembrane receptor. Structural studies have begun to elucidate the role of this interaction for the signal transduction mechanism of plexins. Mapping of the Rac1 GTPase surface that contacts the Rho GTPase binding domain of plexin-B1 by solution NMR spectroscopy confirms the plexin domain as a GTPase effector protein. Regions neighboring the GTPase switch I and II regions are also involved in the interaction and there is considerable interest to examine the changes in protein dynamics that take place upon complex formation. Here we present main-chain nitrogen-15 relaxation measurements for the unbound proteins as well as for the Rho GTPase binding domain and Rac1 proteins in their complexed state. Derived order parameters, S², show that considerable motions are maintained in the bound state of plexin. In fact, some of the changes in S² on binding appear compensatory, exhibiting decreased as well as increased dynamics. Fluctuations in Rac1, already a largely rigid protein on the picosecond-nanosecond timescale, are overall diminished, but isomerization dynamics in the switch I and II regions of the GTPase are retained in the complex and appear to be propagated to the bound plexin domain. Remarkably, fluctuations in the GTPase are attenuated at sites, including helices α6 (the Rho-specific insert helix), α7 and α8, that are spatially distant from the interaction region with plexin. This effect of binding on long-range dynamics appears to be communicated by hinge sites and by subtle conformational changes in the protein. Similar to recent studies on other systems, we suggest that dynamical protein features are affected by allosteric mechanisms. Altered protein fluctuations are likely to prime the Rho GTPase-plexin complex for interactions with additional binding partners.     

Considering the role of pyruvate in tumor cells during hypoxia

Impairment of oxygen supply occurs in many pathological situations. In the case of cancer, both chronic and acute hypoxic areas are found in the tumor. Tumor hypoxia is associated with poor clinical prognoses and is correlated with tumor growth and metastasis development.     

Enhancement of riboflavin production with Bacillus subtilis by expression and site-directed mutagenesis of zwf and gnd gene from Corynebacterium glutamicum

Zwf (code for glucose-6-phosphate dehydrogenase) and gnd (code for 6-phosphogluconate dehydrogenase) genes from Corynebacterium glutamicum were firstly cloned, and then site-directed mutagenesis was successfully introduced to remove allosteric inhibition by intracellular metabolites. Expression of the mutant zwf and gnd in Bacillus subtilis RH33 resulted in significant enhancement of riboflavin productivity, while the specific growth rate decreased slightly and the specific glucose uptake rate was unchanged. Introduction of the mutant zwf and gnd led to approximately 18% and 22% increased riboflavin production, respectively. An improvement by 31% and 39% of the riboflavin production was obtained by co-expression of the mutated dehydrogenases in shaker flask and fed-batch cultivation. Intracellular metabolites analysis indicated that metabolites detected in pentose phosphate pathway or riboflavin synthesis pathway of engineered strains showed higher concentration, while TCA cycle and glycolysis metabolites detected were lower abundance than that of parent strain.     

Studies of regulatory metabolism in Moniezia expansa: Glutamate,and the absence of the γ-aminobutyrate pathway

Moniezia expansa takes up radioactive glutamate from an isotonic medium, and radiocarbon appears primarily in α-ketoglutarate and succinate. Glutamate-oxaloacetate and glutamate-pyruvate transaminase activities were present in subcellular preparations; however, the enzymes of the γ-aminobutyrate pathway were absent. Further attempts to obtain indirect evidence for the operation of this pathway failed, and the metabolism of glutamate appears to take place via a preliminary transamination to α-ketoglutarate, followed by oxidation to succinate.     

Long-term daily access to alcohol alters dopamine-related synthesis and signaling proteins in the rat striatum

Chronic alcohol exposure can adversely affect neuronal morphology, synaptic architecture and associated neuroplasticity. However, the effects of moderate levels of long-term alcohol intake on the brain are a matter of debate. The current study used 2-DE (two-dimensional gel electrophoresis) proteomics to examine proteomic changes in the striatum of male Wistar rats after 8 months of continuous access to a standard off-the-shelf beer in their home cages. Alcohol intake under group-housed conditions during this time was around 3–4 g/kg/day, a level below that known to induce physical dependence in rats. After 8 months of access rats were euthanased and 2-DE proteomic analysis of the striatum was conducted. A total of 28 striatal proteins were significantly altered in the beer drinking rats relative to controls. Strikingly, many of these were dopamine (DA)-related proteins, including tyrosine hydroxylase (an enzyme of DA biosynthesis), pyridoxal phosphate phosphatase (a co-enzyme in DA biosynthesis), DA and cAMP regulating phosphoprotein (a regulator of DA receptors and transporters), protein phosphatase 1 (a signaling protein) and nitric oxide synthase (which modulates DA uptake). Selected protein expression changes were verified using Western blotting. We conclude that long-term moderate alcohol consumption is associated with substantial alterations in the rat striatal proteome, particularly with regard to dopaminergic signaling pathways. This provides potentially important evidence of major neuroadaptations in dopamine systems with daily alcohol consumption at relatively modest levels.     

Anisakis simplex: CO(2)-fixing enzymes and development throughout the in vitro cultivation from third larval stage to adult

We studied the effect of CO(2) on the in vitro cultivation of Anisakis simplex, an aquatic parasitic nematode of cetaceans (final hosts) and fish, squid, crustaceans and other invertebrates (intermediate/paratenic hosts), and, occasionally, of man (accidental host). The results showed that a high pCO(2), at a suitable temperature, is vital for the optimum development of these nematodes, at least from the third larval stage (L3) to adult. After 30 days cultivation in air, molting to L4 (fourth larval stage) was reduced to 1/3, while survival was about 1/3 of that when cultivated in air + 5% CO(2). The activity of the CO(2)-fixing enzymes, PEPCK and PEPC, was also studied. Throughout the development of the worms studied, PEPCK activity was much higher than that of PEPC (e.g., 305 vs. 6.8 nmol/min.mg protein, respectively, in L3 collected from the host fish). The activity of these enzymes in the worms cultivated in air + 5% CO(2) was highest during M3, and was also generally higher than that of those cultivated in air only, especially during molting from L3 to L4 (e.g., in recently molted L4, PEPCK activity was 3.7 times greater than that of PEPC 2.9 times greater than when cultivated in air).     

Molecular analysis of Plasmodium falciparum co-chaperone Aha1 supports its interaction with and regulation of Hsp90 in the malaria parasite

The recent recognition of Plasmodium falciparum Hsp90 (PfHsp90) as a promising anti-malaria drug target has sparked interest in identifying factors that regulate its function and drug-interaction. Co-chaperones are well-known regulators of Hsp90's chaperone function, and certain members have been implicated in conferring protection against lethal cellular effects of Hsp90-specific inhibitors. In this context, studies on PfHsp90's co-chaperones are imperative to gain insight into the regulation of the chaperone in the malaria parasite. In this study, a putative co-chaperone P. falciparum Aha1 (PfAha1) was identified and investigated for its interaction and regulation of PfHsp90. A previous genome-wide yeast two-hybrid study failed to identify PfAha1's association with PfHsp90, which prompted us to use a directed assay to investigate their interaction. PfAha1 was shown to interact with PfHsp90 via the in vivo split-ubiquitin assay and the association was confirmed in vitro by GST pull-down experiments. The GST pull-down assay further revealed PfAha1's interaction with PfHsp90 to be dependent on MgCl₂ and ATP, and was competed by co-chaperone Pfp23 that binds PfHsp90 under the same condition. In addition, the PfHsp90-PfAha1 complex was found to be sensitive to disruption by high salt, indicating a polar interaction between them. Using bio-computational modelling coupled with site-directed mutagenesis, the polar residue N108 in PfAha1 was found to be strategically located and essential for PfHsp90 interaction. The functional significance of PfAha1's interaction was clearly that of exerting a stimulatory effect on the ATPase activity of PfHsp90, likely to be essential for promoting the activation of PfHsp90's client proteins.     

Regulation of cytosolic and mitochondrial ATP levels in mouse eggs and zygotes

Fertilization activates development by stimulating a plethora of ATP consuming processes that must be provided for by an up-regulation of energy production in the zygote. Sperm-triggered Ca²⁺ oscillations are known to be responsible for the stimulation of both ATP consumption and ATP supply but the mechanism of up regulation of energy production at fertilization is still unclear. By measuring [Ca²⁺] and [ATP] in the mitochondria of fertilized mouse eggs we demonstrate that sperm entry triggers Ca²⁺ oscillations in the cytosol that are transduced into mitochondrial Ca²⁺ oscillations pacing mitochondrial ATP production. This results, during fertilization, in an increase in both [ATP]_mito and [ATP]_cyto. We also observe the stimulation of ATP consumption accompanying fertilization by monitoring [Ca²⁺]_cyto and [ATP]_cyto during fertilization of starved eggs. Our observations reveal that lactate, in contrast to pyruvate, does not fuel mitochondrial ATP production in the zygote. Therefore lactate-derived pyruvate is somehow diverted from mitochondrial oxidation and may be channeled to other metabolic routes. Together with our earlier findings, this study confirms the essential role for exogenous pyruvate in the up-regulation of ATP production at the onset of development, and suggests that lactate, which does not fuel energetic metabolism may instead regulate the intracellular redox potential.     

Molecular and structural analysis of C4-specific PEPC isoform from Pennisetum glaucum plays a role in stress adaptation

Phosphoenolpyruvate carboxylase is an ubiquitous cytosolic enzyme that catalyzes the ß-carboxylation of phosphoenolpyruvate (PEP) and is encoded by multigene family in plants. It plays an important role in carbon economy of plants by assimilating CO₂ into organic acids for subsequent C₄ or CAM photosynthesis or to perform several anaplerotic roles in non-photosynthetic tissues. In this study, a cDNA clone encoding for PEPC polypeptide possessing signature motifs characteristic to ZmC₄PEPC was isolated from Pennisetum glaucum (PgPEPC). Deduced amino acid sequence revealed its predicted secondary structure consisting of forty alpha helices and eight beta strands is well conserved among other PEPC homologs irrespective of variation in their primary amino acid sequences. Predicted PgPEPC quartenary structure is a tetramer consisting of a dimer of dimers, which is globally akin to maize PEPC crystal structure with respect to major chain folding wherein catalytically important amino acid residues of active site geometry are conserved. Recombinant PgPEPC protein expressed in E. coli and purified to homogeneity, possessed in vitro ß-carboxylation activity that is determined using a coupled reaction converting PEP into malate. Tetramer is the most active form, however, it exists in various oligomeric forms depending upon the protein concentration, pH, ionic strength of the media and presence of its substrate or effecters. Recombinant PgPEPC protein confers enhanced growth advantage to E. coli under harsh growth conditions in comparison to their respective controls; suggesting that PgPEPC plays a significant role in stress adaptation.     

Systems biology of the metabolic network regulated by the Akt pathway

Cancer has been proposed as an example of systems biology disease or network disease. Accordingly, tumor cells differ from their normal counterparts more in terms of intracellular network dynamics than single markers. Here we shall focus on a recently recognized hallmark of cancer, the deregulation of cellular energetics. The constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway has been confirmed as an essential step toward cell transformation. We will consider how the effects of Akt activation are connected with cell metabolism; more precisely, we will review existing metabolic models and discuss the current knowledge available to construct a kinetic model of the most relevant metabolic processes regulated by the PI3K/Akt pathway. The model will enable a systems biology approach to predict the metabolic targets that may inhibit cell growth under hyper activation of Akt.     

The complete chloroplast genome sequence of Taxus chinensis var. mairei (Taxaceae): loss of an inverted repeat region and comparative analysis with related species

Taxus chinensis var. mairei (Taxaceae) is a domestic variety of yew species in local China. This plant is one of the sources for paclitaxel, which is a promising antineoplastic chemotherapy drugs during the last decade. We have sequenced the complete nucleotide sequence of the chloroplast (cp) genome of T. chinensis var. mairei. The T. chinensis var. mairei cp genome is 129,513 bp in length, with 113 single copy genes and two duplicated genes (trnI-CAU, trnQ-UUG). Among the 113 single copy genes, 9 are intron-containing. Compared to other land plant cp genomes, the T. chinensis var. mairei cp genome has lost one of the large inverted repeats (IRs) found in angiosperms, fern, liverwort, and gymnosperm such as Cycas revoluta and Ginkgo biloba L. Compared to related species, the gene order of T. chinensis var. mairei has a large inversion of ~ 110 kb including 91 genes (from rps18 to accD) with gene contents unarranged. Repeat analysis identified 48 direct and 2 inverted repeats 30 bp long or longer with a sequence identity greater than 90%. Repeated short segments were found in genes rps18, rps19 and clpP. Analysis also revealed 22 simple sequence repeat (SSR) loci and almost all are composed of A or T.     

How Mitochondrial Metabolism Contributes to Macrophage Phenotype and Functions

Metabolic reprogramming of cells from the innate immune system is one of the most noteworthy topics in immunological research nowadays. Upon infection or tissue damage, innate immune cells, such as macrophages, mobilize various immune and metabolic signals to mount a response best suited to eradicate the threat. Current data indicate that both the immune and metabolic responses are closely interconnected. On account of its peculiar position in regulating both of these processes, the mitochondrion has emerged as a critical organelle that orchestrates the coordinated metabolic and immune adaptations in macrophages. Significant effort is now underway to understand how metabolic features of differentiated macrophages regulate their immune specificities with the eventual goal to manipulate cellular metabolism to control immunity. In this review, we highlight some of the recent work that place cellular and mitochondrial metabolism in a central position in the macrophage differentiation program.