The organ-specific differential roles of rice DXS and DXR, the first two enzymes of the MEP pathway, in carotenoid metabolism in Oryza sativa leaves and seeds.

BACKGROUND: Deoxyxylulose 5-phosphate synthase (DXS) and deoxyxylulose 5-phosphate reductoisomerase (DXR) are the enzymes that catalyze the first two enzyme steps of the methylerythritol 4-phosphate (MEP) pathway to supply the isoprene building-blocks of carotenoids. Plant DXR and DXS enzymes have been reported to function differently depending on the plant species. In this study, the differential roles of rice DXS and DXR genes in carotenoid metabolism were investigated. RESULTS: The accumulation of carotenoids in rice seeds co-expressing OsDXS2 and stPAC was largely enhanced by 3.4-fold relative to the stPAC seeds and 315.3-fold relative to non-transgenic (NT) seeds, while the overexpression of each OsDXS2 or OsDXR caused no positive effect on the accumulation of either carotenoids or chlorophylls in leaves and seeds, suggesting that OsDXS2 functions as a rate-limiting enzyme supplying IPP/DMAPPs to seed carotenoid metabolism, but OsDXR doesn't in either leaves or seeds. The expressions of OsDXS1, OsPSY1, OsPSY2, and OsBCH2 genes were upregulated regardless of the reductions of chlorophylls and carotenoids in leaves; however, there was no significant change in the expression of most carotenogenic genes, even though there was a 315.3-fold increase in the amount of carotenoid in rice seeds. These non-proportional expression patterns in leaves and seeds suggest that those metabolic changes of carotenoids were associated with overexpression of the OsDXS2, OsDXR and stPAC transgenes, and the capacities of the intermediate biosynthetic enzymes might be much more important for those metabolic alterations than the transcript levels of intermediate biosynthetic genes are. Taken together, we propose a 'Three Faucets and Cisterns Model' about the relationship among the rate-limiting enzymes OsDXSs, OsPSYs, and OsBCHs as a "Faucet", the biosynthetic capacity of intermediate metabolites as a "Cistern", and the carotenoid accumulations as the content of "Cistern". CONCLUSION: Our study suggests that OsDXS2 plays an important role as a rate-limiting enzyme supplying IPP/DMAPPs to the seed-carotenoid accumulation, and rice seed carotenoid metabolism could be largely enhanced without any significant transcriptional alteration of carotenogenic genes. Finally, the "Three Faucets and Cisterns model" presents the extenuating circumstance to elucidate rice seed carotenoid metabolism.

PhiA, a Peptidoglycan Hydrolase Inhibitor of Brucella Involved in the Virulence Process.

The peptidoglycan in Gram-negative bacteria is a dynamic structure in constant remodeling. This dynamism, achieved through synthesis and degradation, is essential because the peptidoglycan is necessary to maintain the structure of the cell but has to have enough plasticity to allow the transport and assembly of macromolecular complexes in the periplasm and outer membrane. In addition, this remodeling has to be coordinated with the division process. Among the multiple mechanisms bacteria have to degrade the peptidoglycan are the lytic transglycosidases, enzymes of the lysozyme family that cleave the glycan chains generating gaps in the mesh structure increasing its permeability. Because these enzymes can act as autolysins, their activity has to be tightly regulated, and one of the mechanisms bacteria have evolved is the synthesis of membrane bound or periplasmic inhibitors. In the present study, we identify a periplasmic lytic transglycosidase inhibitor (PhiA) in Brucella abortus and demonstrate that it inhibits the activity of SagA, a lytic transglycosidase we have previously shown is involved in the assembly of the type IV secretion system. A phiA deletion mutant results in a strain with the incapacity to synthesize a complete lipopolysaccharide but with a higher replication rate than the wild-type parental strain, suggesting a link between peptidoglycan remodeling and speed of multiplication.

Crosstalk between the lipopolysaccharide and phospholipid pathways during outer membrane biogenesis in Escherichia coli.

The outer membrane of gram-negative bacteria is composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. LPS is an endotoxin that elicits a strong immune response from humans, and its biosynthesis is in part regulated via degradation of LpxC (EC 3.5.1.108) and WaaA (EC 2.4.99.12/13) enzymes by the protease FtsH (EC 3.4.24.-). Because the synthetic pathways for both molecules are complex, in addition to being produced in strict ratios, we developed a computational model to interrogate the regulatory mechanisms involved. Our model findings indicate that the catalytic activity of LpxK (EC 2.7.1.130) appears to be dependent on the concentration of unsaturated fatty acids. This is biologically important because it assists in maintaining LPS/phospholipids homeostasis. Further crosstalk between the phospholipid and LPS biosynthetic pathways was revealed by experimental observations that LpxC is additionally regulated by an unidentified protease whose activity is independent of lipid A disaccharide concentration (the feedback source for FtsH-mediated LpxC regulation) but could be induced in vitro by palmitic acid. Further experimental analysis provided evidence on the rationale for WaaA regulation. Overexpression of waaA resulted in increased levels of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) sugar in membrane extracts, whereas Kdo and heptose levels were not elevated in LPS. This implies that uncontrolled production of WaaA does not increase the LPS production rate but rather reglycosylates lipid A precursors. Overall, the findings of this work provide previously unidentified insights into the complex biogenesis of the Escherichia coli outer membrane.

cis-Prenyltransferase atCPT6 produces a family of very short-chain polyisoprenoids in planta.

cis-Prenyltransferases (CPTs) comprise numerous enzymes synthesizing isoprenoid hydrocarbon skeleton with isoprenoid units in the cis (Z) configuration. The chain-length specificity of a particular plant CPT is in most cases unknown despite thecomposition of the accumulated isoprenoids in the tissue of interest being well established. In this report AtCPT6, one of the nine Arabidopsis thaliana CPTs, is shown to catalyze the synthesis of a family of very short-chain polyisoprenoid alcohols of six, seven, and eight isoprenoid units, those of seven units dominating The product specificity of AtCPT6 was established in vivo following its expression in the heterologous system of the yeast Saccharomyces cerevisiae and was confirmed by the absence of specific products in AtCPT6 T-DNA insertion mutants and their overaccumulation in AtCPT6-overexpressing plants. These observations are additionally validated in silico using an AtCPT6 model obtained by homology modeling. AtCPT6 only partially complements the function of the yeast homologue of CPT-Rer2 since it restores the growth but not protein glycosylation in rer2delta yeast.This is the first in planta characterization of specific products of a plant CPT producing polyisoprenoids. Their distribution suggests that a joint activity of several CPTs is required to produce the complex mixture of polyisoprenoid alcohols found in Arabidopsis roots.

Zng1 is a GTP-dependent zinc transferase needed for activation of methionine aminopeptidase.

The evolution of zinc (Zn) as a protein cofactor altered the functional landscape of biology, but dependency on Zn also created an Achilles' heel, necessitating adaptive mechanisms to ensure Zn availability to proteins. A debated strategy is whether metallochaperones exist to prioritize essential Zn-dependent proteins. Here, we present evidence for a conserved family of putative metal transferases in human and fungi, which interact with Zn-dependent methionine aminopeptidase type I (MetAP1/Map1p/Fma1). Deletion of the putative metal transferase in Saccharomyces cerevisiae (ZNG1; formerly YNR029c) leads to defective Map1p function and a Zn-deficiency growth defect. In vitro, Zng1p can transfer Zn2+ or Co2+ to apo-Map1p, but unlike characterized copper chaperones, transfer is dependent on GTP hydrolysis. Proteomics reveal mis-regulation of the Zap1p transcription factor regulon because of loss of ZNG1 and Map1p activity, suggesting that Zng1p is required to avoid a compounding effect of Map1p dysfunction on survival during Zn limitation.

Characterization of a Cul9-Parkin double knockout mouse model for Parkinson's disease.

Mitochondrial quality control is essential for the long-term survival of postmitotic neurons. The E3 ubiquitin ligase Parkin promotes the degradation of damaged mitochondria via mitophagy and mutations in Parkin are a major cause of early-onset Parkinson's disease (PD). Surprisingly however, mice deleted for Parkin alone are rather asymptomatic for PD-related pathology, suggesting that other complementary or redundant mitochondrial quality control pathways may exist in neurons. Mitochondrial damage is often accompanied by the release of toxic proteins such as cytochrome c. We have reported that once in the cytosol, cytochrome c is targeted for degradation by the E3 ligase CUL9 in neurons. Here we examined whether CUL9 and Parkin cooperate to promote optimal neuronal survival in vivo. We generated mice deficient for both Cul9 and Parkin and examined them for PD-related phenotypes. Specifically, we conducted assays to examine behavioural deficits (locomotor, sensory, memory and learning) and loss of dopaminergic neurons in both males and females. Our results show that the loss of Cul9 and Parkin together did not enhance the effect of Parkin deficiency alone. These results indicate that while both Parkin and CUL9 participate in mitochondrial quality control, neurons likely have multiple redundant mechanisms to ensure their long-term survival.

MCR-1: a promising target for structure-based design of inhibitors to tackle polymyxin resistance.

The spread of a novel mobile colistin resistance gene (mcr1) has jeopardised the use of polymyxins, last-resort antibiotics that are used increasingly to treat infections caused by multidrug-resistant (MDR) Gram-negative pathogens. In early 2017, the WHO reported the global spread of mcr1 within a few years after its initial discovery in China. The protein encoded by mcr1 is a putative 60-kDa phosphoethanolamine (pEtN) transferase, MCR-1, and has been studied extensively since its discovery. Herein, we present a comprehensive review of MCR-1 covering its structure, function, and mechanism, to call for the rational drug design of molecular inhibitors of MCR-1 to use in colistin-based combination therapies.

Transsulfuration in mammals: fetal and early development of methionine-activating enzyme and its relation to hormonal influences.

The development of activity of methionine-activating enzyme was studied in four organs of the rat. Three different patterns were observed: (a) in the liver, specific activity began to increase in late fetal life and reached a maximum 2 days after birth; (b) in the small intestine, specific activity began to rise in the 2nd wk after birth and reached a maximum at age 18 days; and (c) in the brain and kidney, specific activity did not change markedly from the earliest stage of fetal development studied to adult life. Hydrocortisone increased hepatic methionine-activating enzyme activity as much as 55% in the young rat. However, adrenalectomy in the newborn rat did not prevent the postnatal rise in hepatic methionine-activating enzyme activity, nor did adrenalectomy at age 10 days prevent the developmental rise of intestinal activity at age 18 days. Conjugated estrogens partially inhibited both the neonatal rise in hepatic methionine-activating enzyme activity and the rise in activity after adrenal steroid injection. Injection of L-methionine did not increase hepatic methionine-activating enzyme activity in the developing or adult rat.

Cloning and characterization of the dxs gene, encoding 1-deoxy-d-xylulose 5-phosphate synthase from Agrobacterium tumefaciens, and its overexpression in Agrobacterium tumefaciens.

A newly isolated gene dxs11 from Agrobacterium tumefaciens (KCCM 10413), an organism with potential for the industrial production of ubiquinone-10 (UbiQ(10)), encoding a 1-deoxy-d-xylulose 5-phosphate synthase (Dxs), was cloned in Escherichia coli and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1920bp, capable of encoding a polypeptide of 640 amino acids residues with a calculated isoelectric point of pH 5.63 and a molecular mass of 68,054Da. The homodimeric enzyme was overexpressed in E. coli and purified as an active soluble form. The enzyme required thiamine diphosphate and a divalent metal ion, either Mg(2+) or Mn(2+), for enzymatic activity. The enzyme had an optimal pH and temperature of 8.0 and 37 degrees C, respectively, with a k(cat) of 26.8s(-1) and a k(cat)/K(m) of 0.67 and 1.17s(-1)M(-1) for pyruvate and d-glyceraldehyde 3-phosphate, respectively. A. tumefaciens Dxs showed a comparable catalytic efficiency to other Dxs proteins. The dxs11 gene was transformed into A. tumefaciens KCCM 10413, and the resulting recombinant, A. tumefaciens pGX11, showed higher UbiQ(10) production (502.4mg/l) and content (8.3mg/gDCW) than A. tumefaciens KCCM 10413, by 21.9 and 23.9%, respectively. This work describes Dxs from A. tumefaciens, an organism with the potential for industrial UbiQ(10) production, and the first metabolic engineering study with the non-mevalonate pathway enzyme in A. tumefaciens.

Nf1-deficient mouse Schwann cells are angiogenic and invasive and can be induced to hyperproliferate: reversion of some phenotypes by an inhibitor of farnesyl protein transferase.

We have developed a potential model of Schwann cell tumor formation in neurofibromatosis type 1 (NF1). We show that mouse Schwann cells heterozygous or null at Nf1 display angiogenic and invasive properties, mimicking the behavior of Schwann cells from human neurofibromas. Mutations at Nf1 are insufficient to promote Schwann cell hyperplasia. Here we show that Schwann cell hyperplasia can be induced by protein kinase A activation in mutant cells. Removal of serum from the culture medium also stimulates hyperplasia, but only in some mutant cells. After serum removal, clones of hyperproliferating Schwann cells lose contact with axons in vitro, develop growth factor-independent proliferation, and exhibit decreased expression of the cell differentiation marker P0 protein; hyperproliferating cells develop after a 1-week lag in Schwann cells heterozygous at Nf1. The experiments suggest that events subsequent to Nf1 mutations are required for development of Schwann cell hyperplasia. Finally, an anti-Ras farnesyl protein transferase inhibitor greatly diminished both clone formation and hyperproliferation of null mutant cells, but not invasion; farnesyl transferase inhibitors could be useful in treating benign manifestations of NF1.

The yeast RER2 gene, identified by endoplasmic reticulum protein localization mutations, encodes cis-prenyltransferase, a key enzyme in dolichol synthesis.

As an approach to understand the molecular mechanisms of endoplasmic reticulum (ER) protein sorting, we have isolated yeast rer mutants that mislocalize a Sec12-Mfalpha1p fusion protein from the ER to later compartments of the secretory pathway (S. Nishikawa and A. Nakano, Proc. Natl. Acad. Sci. USA 90:8179-8183, 1993). The temperature-sensitive rer2 mutant mislocalizes different types of ER membrane proteins, suggesting that RER2 is involved in correct localization of ER proteins in general. The rer2 mutant shows several other characteristic phenotypes: slow growth, defects in N and O glycosylation, sensitivity to hygromycin B, and abnormal accumulation of membranes, including the ER and the Golgi membranes. RER2 and SRT1, a gene whose overexpression suppresses rer2, encode novel proteins similar to each other, and their double disruption is lethal. RER2 homologues are found not only in eukaryotes but also in many prokaryote species and thus constitute a large gene family which has been well conserved during evolution. Taking a hint from the phenotype of newly established mutants of the Rer2p homologue of Escherichia coli, we discovered that the rer2 mutant is deficient in the activity of cis-prenyltransferase, a key enzyme of dolichol synthesis. This and other lines of evidence let us conclude that members of the RER2 family of genes encode cis-prenyltransferase itself. The difference in phenotypes between the rer2 mutant and previously obtained glycosylation mutants suggests a novel, as-yet-unknown role of dolichol.

The analysis of cell division and cell wall synthesis genes reveals mutationally inactivated ftsQ and mraY in a protoplast-type L-form of Escherichia coli.

Cell division and cell wall synthesis are tightly linked cellular processes for bacterial growth. A protoplast-type L-form Escherichia coli, strain LW1655F+, indicated that bacteria can divide without assembling a cell wall. However, the molecular basis of its phenotype remained unknown. To establish a first phenotype-genotype correlation, we analyzed its dcw locus, and other genes involved in division of E. coli. The analysis revealed defective ftsQ and mraY genes, truncated by a nonsense and a frame-shift mutation, respectively. Missense mutations were determined in the ftsA and ftsW products yielding amino-acid replacements at conserved positions. FtsQ and MraY, obviously nonfunctional in the L-form, are essential for cell division and cell wall synthesis, respectively, in all bacteria with a peptidoglycan-based cell wall. LW1655F+ is able to survive their loss-of-functions. This points to compensatory mechanisms for cell division in the absence of murein sacculus formation. Hence, this L-form represents an interesting model to investigate the plasticity of cell division in E. coli, and to demonstrate how concepts fundamental for bacterial life can be bypassed.

Modification of eukaryotic signaling proteins by C-terminal methylation reactions.

Eukaryotic polypeptides that are initially synthesized with the C-terminal sequence -Cys-Xaa-Xaa-Xaa, including a variety of signal-transducing proteins, such as small G-proteins, large G-proteins and cGMP phosphodiesterases, can be targeted for a series of sequential post-translational modifications. This processing pathway includes the isoprenylation of the cysteine residue with a farnesyl or geranylgeranyl moiety, followed by proteolysis of the three terminal residues and alpha-carboxyl methyl esterification of the cysteine residue. The potential reversibility of the last step suggests that it may be involved in modulating the function of these proteins. Firstly, methylation may play a role in the activation of cellular peptides or proteins. Secondly, this modification may aid in the membrane attachment of cytosolic precursor proteins. Thirdly, methylation may protect the polypeptide from C-terminal proteolytic degradation once the three terminal amino acid residues are removed. Finally, reversible methylation may directly regulate the function of its target proteins. Therapeutically, inhibitors of C-terminal isoprenylcysteine methylation or demethylation reactions may prove to be useful pharmacological tools as anti-cancer and anti-inflammatory agents.

A Synechococcus leopoliensis SAUG 1402-1 operon harboring the 1-deoxyxylulose 5-phosphate synthase gene and two additional open reading frames is functionally involved in the dimethylallyl diphosphate synthesis.

Experiments have been performed to prove the existence and the functionality of the novel mevalonate independent 1-deoxyxylulose 5-phosphate isoprenoid biosynthesis pathway in cyanobacteria. For this purpose, a segment of the 1-deoxyxylulose 5-phosphate synthase gene (dxs) was amplified from Synechococcus leopoliensis SAUG 1402-1 DNA via PCR using oligonucleotides for conserved regions of dxs. Subsequent hybridization screening of a genomic cosmid library of S. leopoliensis with this segment has led to the identification of an 18.7 kbp segment of the S. leopoliensis genome on which a dxs homologous gene and two adjacent open reading frames organized in one operon could be localized by DNA sequencing. The three genes of the operon were separately expressed in Escherichia coli, proving that the identified cyanobacterial dxs is functionally involved in the formation of dimethylallyl diphosphate, one basic intermediate of isoprenoid biosynthesis.

Biochemistry of zinc.

The biochemistry of zinc has come under intensive investigation at the molecular level during the past 20 years. More than 70 zinc metalloenzymes are now known, and they span a broad range of biologic activities. Substitution of zinc by cobalt, for example, serves to locate a paramagnetic probe at the active site of the enzyme which can then provide information regarding the coordination properties of the metal and the active site environment.

Chemoprotection by organosulfur inducers of phase 2 enzymes: dithiolethiones and dithiins.

One of the major mechanisms of protection against carcinogenesis, mutagenesis, and other forms of toxicity mediated by carcinogens is the induction of enzymes involved in their metabolism, particularly phase 2 enzymes such as glutathione S-transferases, UDP-glucuronosyl transferases, and quinone reductases. Animal studies indicate that induction of phase 2 enzymes is a sufficient condition for obtaining chemoprevention and can be achieved by administering any of a diverse array of naturally-occurring and synthetic chemopreventive agents. Alliaceous and cruciferous plants are rich in organosulfur compounds with inducer activity. Indeed, monitoring of enzyme induction has led to the recognition or isolation of novel, potent chemopreventive agents such as 1,2-dithiole-3-thiones, dithiins and the isothiocyanate sulforaphane. For example, oltipraz, a substituted 1,2-dithiole-3-thione originally developed as an antischistosomal agent, possesses chemopreventive activity against different classes of carcinogens targeting multiple organs. Mechanistic studies in rodent models for chemoprevention of aflatoxin B1 (AFB1)-induced hepatocarcinogenesis by oltipraz indicates that increased expression of phase 2 genes is of central importance, although inhibition of phase 1 activation of aflatoxin B1 can also contribute to protection. Exposure of rodents to 1,2-dithiole-3-thiones triggers nuclear accumulation of the transcription factor Nrf2 and its enhanced binding to the Antioxidant Response Element, leading to transcriptional activation of a score of genes involved in carcinogen detoxification and attenuation of oxidative stress. Nrf2-deficient mice fail to induce many of these genes in response to oltipraz and the impact of this genotype on the chemopreventive efficacy of dithiolethiones is currently under investigation. To test the hypothesis that enzyme induction is a useful strategy for chemoprevention in humans, three key elements are necessary: a candidate agent, an at-risk population and modulatable intermediate endpoints. Towards this end, a placebo-controlled, double blind clinical trial of oltipraz was conducted in residents of Qidong, P.R. China who are exposed to dietary aflatoxins and who are at high risk for the development of liver cancer. Oltipraz significantly enhanced excretion of a phase 2 product, aflatoxin-mercapturic acid, a derivative of the aflatoxin-glutathione conjugate, in the urine of study participants administered 125 mg oltipraz by mouth daily. Administration of 500 mg oltipraz once a week led to a significant reduction in the excretion of the primary oxidative metabolite of AFB1, aflatoxin M1, when measured shortly after drug administration. While this study highlighted the general feasibility of inducing phase 2 enzymes in humans, a longer term intervention is addressing whether protective alterations in aflatoxin metabolism can be sustained for extended periods of time in this high-risk population. Food-based approaches to chemoprotection, targeted both to the general population and high-risk individuals, offer many practical advantages compared to the use of pharmaceutical agents. Thus, identification and utilization of naturally-occurring organosulfur chemoprotectors including dithiins should be a high priority.

[Ideal gene therapy: approaches and prospects]. / Ideal'naia genoterapiia: podkhody i perspektivy.

Ideal gene therapy implies correction of a genetic mutation at the site of its localization on the chromosome. This can be done only with homologous recombination. In prokaryotes, two mechanisms of homologous recombination are provided by two types of recombinases, an ATP-dependent RecA protein and an ATP-independent renaturase. In eukaryotes both the ATP-dependent and ATP-independent DNA transferases have been revealed, among which functionally RecA-like enzymes were also found. The unique properties of RecA-like proteins suggest that these enzymes promote recombination both in pro- and eukaryotes. According to recent observations, the mitotic eukaryotic cells do possess a potential for homologous recombination. However to realize it the donor DNA should be reasonably long and be maximally homologous to the recipient DNA. Other possibilities of optimizing the gene therapy process are discussed.

Branched signal wiring of an essential bacterial cell-cycle phosphotransfer protein.

Vital to bacterial survival is the faithful propagation of cellular signals, and in Caulobacter crescentus, ChpT is an essential mediator within the cell-cycle circuit. ChpT functions as a histidine-containing phosphotransfer protein (HPt) that shuttles a phosphoryl group from the receiver domain of CckA, the upstream hybrid histidine kinase (HK), to one of two downstream response regulators (CtrA or CpdR) that controls cell-cycle progression. To understand how ChpT interacts with multiple signaling partners, we solved the crystal structure of ChpT at 2.3 Å resolution. ChpT adopts a pseudo-HK architecture but does not bind ATP. We identified two point mutation classes affecting phosphotransfer and cell morphology: one that globally impairs ChpT phosphotransfer, and a second that mediates partner selection. Importantly, a small set of conserved ChpT residues promotes signaling crosstalk and contributes to the branched signaling that activates the master regulator CtrA while inactivating the CtrA degradation signal, CpdR.