Glutamate synthase: a complex iron-sulfur flavoprotein

Glutamate synthase is a complex iron-sulfur flavoprotein that forms l-glutamate from l-glutamine and 2-oxoglutarate. It participates with glutamine synthetase in ammonia assimilation processes. The known structural and biochemical properties of glutamate synthase from Azospirillum brasilense, a nitrogen-fixing bacterium, will be discussed in comparison to those of the ferredoxin-dependent enzyme from photosynthetic tissues and of the eukaryotic reduced pyridine nucleotide-dependent form of glutamate synthase in order to gain insight into the mechanism of the glutamate synthase reaction. Sequence analyses also revealed that the small subunit of bacterial glutamate synthase may be the prototype of a novel class of flavin adenine dinucleotide- and iron-sulfur-containing oxidoreductase widely used as an enzyme subunit or domain to transfer reducing equivalents from NAD(P)H to an acceptor protein or protein domain. Received 10 November 1998, received after revision 10 December 1998; accepted 10 December 1998     

Chitin in the epidermal cuticle of a vertebrate (Paralipophrys trigloides,Blenniidae, Teleostei)

Lectin binding, endo-chitinase binding and enzymatic degradation studies show that the epidermal cuticle of the bony fishParalipophrys trigloides (Blenniidae) is chitinous. This is the first evidence that a vertebrate species possesses a chitinous tissue. Recently aXenopus gene has been identified which has significant sequence similarity to the catalytic domain of yeast chitin synthase III, a chitin producing enzyme^1,2. Taken together these two findings imply that chitin synthesis capability may be a basic vertebrate feature.     

Mechanism of polyamine tolerance in yeast: novel regulators and insights

Polyamines are small charged molecules essential for various cellular functions, but at high levels they are cytotoxic. Two yeast kinases, SKY1 and PTK2, have been demonstrated to regulate polyamine tolerance. Here we report the identification and characterization of additional genes involved in regulating polyamine tolerance: YGL007W, FES1 and AGP2. Deletion of YGL007W, an open reading frame located within the promoter of the membrane proton pump PMA1, decreased Pma1p expression. Deletion of FES1 or AGP2 resulted in reduced polyamine uptake. While high-affinity spermine uptake was practically absent in agp2Δ cells, fes1Δ cells displayed only reduced affinity towards spermine. Despite the reduced uptake, the resistant strains accumulated significant levels of polyamines and displayed increased ornithine decarboxylase activity, suggesting reduced polyamine sensing. Interestingly, fes1Δ cells were highly sensitive to salt ions, suggesting different underlying mechanisms. These results indicate that mechanisms leading to polyamine tolerance are complex, and involve components other than uptake. Received 31 July 2005; received after revision 7 October 2005; accepted 19 October 2005     

Modulation of E-cadherin function and dysfunction by N-glycosylation

Several mechanisms have been proposed to explain the E-cadherin dysfunction in cancer, including genetic and epigenetic alterations. Nevertheless, a significant number of human carcinomas have been seen that show E-cadherin dysfunction that cannot be explained at the genetic/epigenetic level. A substantial body of evidence has appeared recently that supports the view that other mechanisms operating at the post-translational level may also affect E-cadherin function. The present review addresses molecular aspects related to E-cadherin N-glycosylation and evidence is presented showing that the modification of N-linked glycans on E-cadherin can affect the adhesive function of this adhesion molecule. The role of glycosyltransferases involved in the remodeling of N-glycans on E-cadherin, including N-acetylglucosaminyltransferase III (GnT-III), N-acetylglucosaminyltransferase V (GnT-V), and the α1,6 fucosyltransferase (FUT8) enzyme, is also discussed. Finally, this review discusses an alternative functional regulatory mechanism for E-cadherin operating at the post-translational level, N-glycosylation, that may underlie the E-cadherin dysfunction in some carcinomas.     

Chromatin domain boundaries in the Bithorax complex

Eukaryotic chromosomes are thought to be organized into a series of discrete higher-order chromatin domains. This organization is believed to be important not only in the compaction of the chromatin fibre, but also in the utilization of genetic information. Critical to this model are the domain boundaries that delimit and segregate the chromosomes into units of independent gene activity. In Drosophila, such domain boundaries have been identified through two different approaches. On the one hand, elements like scs/scs′ and the reiterated binding site for the SU(HW) protein have been characterized through their activity of impeding enhancer-promoter interactions when intercalated between them. Their role of chromatin insulators can protect transgenes from genomic position effects, thereby establishing in dependent functional domains within the chromosome. On the other hand, domain boundaries of the Bithorax complex (BX-C) like Fab-7 and Mcp have been identified through mutational analysis. Mcp and Fab-7, however, may represent a specific class of boundary elements; instead of separating adjacent domains that contain separate structural genes, Mcp and Fab-7 delimit adjacent cis-regulatory domains, each of which interacts independently with their target promoters. In this article, we review the genetic and molecular characteristics of the domain boundaries of the BX-C. We describe how Fab-7 functions to confine activating as well as repressive signals to the flanking regulatory domains. Although the mechanisms by which Fab-7 works as a domain boundary remain an open issue, we provide preliminary evidence that Fab-7 is not a mere insulator like scs or the reiterated binding site for the SU(HW) protein.     

The mammalian NudC-like genes: a family with functions other than regulating nuclear distribution

Nuclear distribution gene C homolog (NudC) is a highly conserved gene. It has been identified in different species from fungi to mammals. The high degree of conservation, in special in the nudC domain, suggests that they are genes with essential functions. Most of the identified genes in the family have been implicated in cell division through the regulation of cytoplasmic dynein. As for mammalian genes, human NUDC has been implicated in the migration and proliferation of tumor cells and has therefore been considered a possible therapeutic target. There is evidence suggesting that mammalian NudC is also implicated in the regulation of the inflammatory response and in thrombopoiesis. The presence of these other functions not related to the interaction with molecular motors agrees with that these genes and their products are larger in size than their microbial orthologous, indicating that they have evolved to convey additional features.     

Effect of spermine on the kinetics of estradiol hydroxylation by rat liver microsomes

Résumé L'addition de spermine au microsomes du foie de rats femelles modifie leK _m du système enzymatique qui transforme 1'estradiol en 2-hydroxyestradiol et aussi en des produits aqueux. La nouvelle valeur resemble à celle qu'ont trouve avec des rats mâles, mais leV _max de la réaction reste inchangé. L'action de la spermine dans ce système est discutée.     

Signal regulation by family conspiracy

The signal regulating proteins (SIRPs) are a family of ubiquitously expressed transmembrane glycoproteins composed of two subgroups: SIRPα and SIRPβ, containing more than ten members. SIRPα has been shown to inhibit signalling through a variety of receptors including receptor tyrosine kinases and cytokine receptors. This function involves protein tyrosine kinases and is dependent on immunoreceptor tyrosine-based inhibition motifs which recruit key protein tyrosine phosphatases to the membrane. Negative regulation by SIRPα may also involve its ligand, CD47, in a bi-directional signalling mechanism. The SIRPβ subtype has no cytoplasmic domain but instead associates with at least one other transmembrane protein (DAP-12, or KARAP). DAP-12 possesses immunoreceptor tyrosine-based activation motifs within its cytoplasmic domain that are thought to link SIRPβ to activating machinery. SIRPα and SIRPβ thus have complementary roles in signal regulation and may conspire to tune the response to a stimulus. Received 6 July 2000; revised 2 August 2000; accepted 5 August 2000     

Stimulation ofDugesia tigrina auricle regeneration by exogenous putrescine,spermine or spermidine

Summary Specimens of a flatworm,Dugesia tigrina were decapitated and then cultured in a solution of 1×10^–4 M putrescine, spermine or spermidine. Subsequent observation for the reappearance of auricles indicates that the amine treatment stimulates the flatworm regeneration process.     

The FN3 and BRCT motifs in the exomer component Chs5p define a conserved module that is necessary and sufficient for its function

Chs5p is a component of the exomer, a coat complex required to transport the chitin synthase Chs3p from the trans-Golgi network to the plasma membrane. The Chs5p N-terminal region exhibits fibronectin type III (FN3) and BRCT domains. FN3 domains are present in proteins that mediate adhesion processes, whereas BRCT domains are involved in DNA repair. Several fungi—including Schizosaccharomyces pombe, which has no detectable amounts of chitin—have proteins similar to Chs5p. Here we show that the FN3 and BRCT motifs in Chs5p behave as a module that is necessary and sufficient for Chs5p localization and for cargo delivery. The N-terminal regions of S. cerevisiae Chs5p and S. pombe Cfr1p are interchangeable in terms of Golgi localization, but not in terms of exomer assembly, showing that the conserved function of this module is protein retention in this organelle and that the interaction between the exomer components is organism-specific.     

N-acetylmuramic acid 6-phosphate lyases (MurNAc etherases): role in cell wall metabolism, distribution, structure, and mechanism

MurNAc etherases cleave the uniqued-lactyl ether bond of the bacterial cell wall sugar N-acetylmuramic acid (MurNAc). Members of this newly discovered family of enzymes are widely distributed among bacteria and are required to utilize peptidoglycan fragments obtained either from the environment or from the endogenous cell wall (i.e., recycling). MurNAc etherases are strictly dependent on the substrate MurNAc possessing a free reducing end and a phosphoryl group at C6. They carry a single conserved sugar phosphate isomerase/sugar phosphate- binding (SIS) domain to which MurNAc 6-phosphate is bound. Two subunits form an enzymatically active homodimer that structurally resembles the isomerase module of the double-SIS domain protein GlmS, the glucosamine 6-phosphate synthase. Structural comparison provides insights into the two-step lyase-type reaction mechanism of MurNAc etherases: β-elimination of the D-lactic acid substituent proceeds through a 2,3-unsaturated sugar intermediate to which water is subsequently added. Received 31 August 2007; received after revision 12 October 2007; accepted 1 November 2007     

Spermine protects protein kinase C from phospholipid-induced inactivation

Phosphatidylserine (PS), an activator of protein kinase C (PKC) in the assay of protein phosphorylation, inhibited this enzyme in a time-dependent manner following preincubation in the absence of Ca²⁺. The phospholipid-induced inactivation of kinase activity was dependent on the PS content and on the charge density of liposomes. This inactivation of PKC could be reduced, but not completely eliminated, by addition of Ca²⁺. In the present work the effect of a naturally occurring polyamine (spermine) on the PS-induced inactivation of PKC was investigated. The presence of spermine during preincubation without Ca²⁺ was effective in suppressing the PS-induced inactivation of PKC over the period (20 min) required for PS to inhibit the enzyme by 95%. PKC exists in two membrane-bound states: a reversible one which can be dissociated by Ca²⁺ chelators (membrane-associated form) and an irreversible one which is chelator-stable (membrane-inserted form). Gel filtration experiments on the PKC-PS complex formed in the presence of Ca²⁺ indicated that less insertion of enzyme into liposomes occurred in the presence of spermine and that the kinase activity of the reversibly membrane-associated PKC was protected from PS inactivation.     

<Emphasis Type="Italic">O</Emphasis>-fucose modifications of epidermal growth factor-like repeats and thrombospondin type 1 repeats: unusual modifications in unusual places

Recent discoveries revealing that carbohydrate modifications play critical roles in a wide variety of biological processes have brought wide recognition to the field of glycobiology. Growing attention has focused on the function of unusual O-linked carbohydrate modifications such as O-fucose. O-fucose modifications have been described in several different protein contexts, including epidermal growth factor-like repeats and thrombospondin type 1 repeats. The O-fucose modifications on thrombospondin type 1 repeats have only recently been described, but the site of modification occurs in a region proposed to play a role in cell adhesion. O-fucose modifications on epidermal growth factor-like repeats have been described as important players in several signal transduction systems. For instance, Notch, a cell-surface signaling receptor required for many developmental events, bears multiple O-fucose saccharides on the epidermal growth factor-like repeat of its extracellular domain. The O-fucose moieties serve as a substrate for the β1,3 N-acetylglucosaminyltransferase activity of Fringe, a known modifier of Notch function. The alteration of O-fucose structures by Fringe influences the ability of Notch ligands to activate the receptor and provides a means to regulate Notch signaling. Thus, O-fucose and Fringe provide a clear example of how carbohydrate modifications can have direct functional consequences on the proteins they modify. RID="*" ID="*"Corresponding author.     

The BAG proteins: a ubiquitous family of chaperone regulators

The BAG (Bcl-2 associated athanogene) family is a multifunctional group of proteins that perform diverse functions ranging from apoptosis to tumorigenesis. An evolutionarily conserved group, these proteins are distinguished by a common conserved region known as the BAG domain. BAG genes have been found in yeasts, plants, and animals, and are believed to function as adapter proteins forming complexes with signaling molecules and molecular chaperones. In humans, a role for BAG proteins has been suggested in carcinogenesis, HIV infection, and Parkinson’s disease. These proteins are therefore potential therapeutic targets, and their expression in cells may serve as a predictive tool for such diseases. In plants, the Arabidopsis thaliana genome contains seven homologs of the BAG family, including four with domain organization similar to animal BAGs. Three members contain a calmodulin-binding domain possibly reflecting differences between plant and animal programmed cell death. This review summarizes current understanding of BAG proteins in both animals and plants. Received 21 November 2007; received after revision 17 December 2007; accepted 2 January 2008     

Is ATP synthesized by a vacuolar-ATPase in the extremely halophilic bacteria?

The proton-dependent synthesis of ATP was demonstrated in representative members of the generaHalobacterium, Haloarcula, andHaloferax. In all cases, synthesis was not inhibited by nitrate or N-ethylmaleimide, inhibitors of the vacuolar-like ATPase found in Archaea, but was affected by azide, an inhibitor of F₀F₁-ATP syntheses. These observations extend the earlier observations withHalobacterium saccharovorum and suggest that ATP synthesis in these organisms is brought about by an F₀F₁-APT synthase.     

SAM domain-dependent activity of PfTKL3, an essential tyrosine kinase-like kinase of the human malaria parasite Plasmodium falciparum

Over the last decade, several protein kinases inhibitors have reached the market for cancer chemotherapy. The kinomes of pathogens represent potentially attractive targets in infectious diseases. The functions of the majority of protein kinases of Plasmodium falciparum, the parasitic protist responsible for the most virulent form of human malaria, remain unknown. Here we present a thorough characterisation of PfTKL3 (PF13_0258), an enzyme that belongs to the tyrosine kinase-like kinase (TKL) group. We demonstrate by reverse genetics that PfTKL3 is essential for asexual parasite proliferation in human erythrocytes. PfTKL3 is expressed in both asexual and gametocytes stages, and in the latter the protein co-localises with cytoskeleton microtubules. Recombinant PfTKL3 displays in vitro autophosphorylation activity and is able to phosphorylate exogenous substrates, and both activities are dramatically dependent on the presence of an N-terminal “sterile α-motif” domain. This study identifies PfTKL3 as a validated drug target amenable to high-throughput screening.     

A human homolog of the yeast gene encoding tRNA 2′-phosphotransferase: cloning,characterization and complementation analysis

The Saccharomyces cerevisiae TPT1 gene plays a role in removing the 2-phosphate from ligated tRNA during the maturation of pre-tRNA. Here we reported the cloning and characterization of the human TRPT1 gene as a homolog of yeast TPT1. The TRPT1 gene is located at human chromosome 11q13 and encodes a polypeptide of 253 amino acids. BLAST searches with its amino acid sequence revealed the ubiquitous occurrence of TRPT1 homologs and their functional relationships with the presence of the DUF60/KptA domain. Northern analysis demonstrated that the gene is primarily expressed in heart and skeletal muscle, with lower or undetectable levels in other tissues studied. A plasmid-shuffling experiment showed that the human TRPT1 gene could complement the tpt1 mutation in S. cerevisiaeReceived 19 March 2003; received after revision 25 April 2003; accepted 22 May 2003     

Identification of tyrosine-phosphorylated proteins of the mitochondrial oxidative phosphorylation machinery

The role of some serine/threonine kinases in the regulation of mitochondrial physiology is now well established, but little is known about mitochondrial tyrosine kinases. We showed that tyrosine phosphorylation of rat brain mitochondrial proteins was increased by in vitro addition of ATP and H₂O₂, and also during in situ ATP production at state 3, and maximal reactive oxygen species production. The Src kinase inhibitor PP2 decreased tyrosine phosphorylation and respiratory rates at state 3. We found that the 39-kDa subunit of complex I was tyrosine phosphorylated, and we identified putative tyrosine-phosphorylated subunits for the other complexes. We also have strong evidence that the FoF1-ATP synthase α chain is probably tyrosine-phosphorylated, but demonstrated that the β chain is not. The tyrosine phosphatase PTP 1B was found in brain but not in muscle, heart or liver mitochondria. Our results suggest that tyrosine kinases and phosphatases are involved in the regulation of oxidative phosphorylation.Received 7 January 2005; received after revision 19 April 2005; accepted 22 April 2005     

Mitochondrial DNA replication and disease: insights from DNA polymerase γ mutations

DNA polymerase γ (pol γ), encoded by POLG, is responsible for replicating human mitochondrial DNA. About 150 mutations in the human POLG have been identified in patients with mitochondrial diseases such as Alpers syndrome, progressive external ophthalmoplegia, and ataxia-neuropathy syndromes. Because many of the mutations are described in single citations with no genotypic family history, it is important to ascertain which mutations cause or contribute to mitochondrial disease. The vast majority of data about POLG mutations has been generated from biochemical characterizations of recombinant pol γ. However, recently, the study of mitochondrial dysfunction in Saccharomyces cerevisiae and mouse models provides important in vivo evidence for the role of POLG mutations in disease. Also, the published 3D-structure of the human pol γ assists in explaining some of the biochemical and genetic properties of the mutants. This review summarizes the current evidence that identifies and explains disease-causing POLG mutations.     

On the antimutagenic effect of spermine

Résumé La spermine n'offre aucune protection contre la réversion des mutants derII chezEscherichia coliphage T4, quoiqu'on ait constaté qu'elle antimutagénique chezE. coli.