Degradation of the cleaved leader peptide of thiolase by a peroxisomal proteinase.

A peroxisomal location for insulin-degrading enzyme (IDE) has been defined by confocal immunofluorescence microscopy of stably transfected CHO cells overexpressing IDE and digitonin-permeabilization studies in normal nontransfected fibroblasts. The functional significance of IDE in degrading cleaved leader peptides of peroxisomal proteins targeted by the type II motif was evaluated with a synthetic peptide corresponding to the type II leader peptide of prethiolase. The peptide effectively competed for degradation and cross-linking of the high-affinity substrate 125I-labeled insulin to IDE. Direct proteolysis of the leader peptide of prethiolase was confirmed by HPLC; degradation was inhibited by immunodepletion with an antibody to IDE. Phylogenetic analysis of proteinases related to IDE revealed sequence similarity to mitochondrial processing peptidases.

Repressible cation-phosphate symporters in Neurospora crassa.

The filamentous fungus Neurospora crassa possesses two nonhomologous high-affinity phosphate permeases, PHO-4 and PHO-5. We have isolated separate null mutants of these permeases, allowing us to study the remaining active transporter in vivo in terms of phosphate uptake and sensitivity to inhibitors. The specificity for the cotransported cation differs for PHO-4 and PHO-5, suggesting that these permeases employ different mechanisms for phosphate translocation. Phosphate uptake by PHO-4 is stimulated 85-fold by the addition of Na+, which supports the idea that PHO-4 is a Na(+)-phosphate symporter. PHO-5 is unaffected by Na+ concentration but is much more sensitive to elevated pH than is PHO-4. Presumably, PHO-5 is a H(+)-phosphate symporter. Na(+)-coupled symport is usually associated with animal cells. The finding of such a system in a filamentous fungus is in harmony with the idea that the fungal and animal kingdoms are more closely related to each other than either is to the plant kingdom.

Expression cloning of dSR-CI, a class C macrophage-specific scavenger receptor from Drosophila melanogaster.

Mammalian class A macrophage-specific scavenger receptors (SR-A) exhibit unusually broad binding specificity for a wide variety of polyanionic ligands. The properties of these receptors suggest that they may be involved in atherosclerosis and host defense. We have previously observed a similar receptor activity in Drosophila melanogaster embryonic macrophages and in the Drosophila macrophage-like Schneider L2 cell line. Expression cloning was used to isolate from L2 cells a cDNA that encodes a third class (class C) of scavenger receptor, Drosophila SR-CI (dSR-CI). dSR-CI expression was restricted to macrophages/hemocytes during embryonic development. When expressed in mammalian cells, dSR-CI exhibited high affinity and saturable binding of 125I-labeled acetylated low density lipoprotein and mediated its chloroquine-dependent, presumably lysosomal, degradation. Although the broad polyanionic ligand-binding specificity of dSR-CI was similar to that of SR-A, their predicted protein sequences are not similar. dSR-CI is a 609-residue type I integral membrane protein containing several well-known sequence motifs, including two complement control protein (CCP) domains and somatomedin B, MAM, and mucin-like domains. Macrophage scavenger receptors apparently mediate important, well-conserved functions and may be pattern-recognition receptors that arose early in the evolution of host-defense mechanisms. Genetic and physiologic analysis of dSR-CI function in Drosophila should provide further insights into the roles played by scavenger receptors in host defense and development.

Bipartite function of a small RNA hairpin in transcription antitermination in bacteriophage lambda.

Transcription of downstream genes in the early operons of phage lambda requires a promoter-proximal element known as nut. This site acts in cis in the form of RNA to assemble a transcription antitermination complex which is composed of lambda N protein and at least four host factors. The nut-site RNA contains a small stem-loop structure called boxB. Here, we show that boxB RNA binds to N protein with high affinity and specificity. While N binding is confined to the 5' subdomain of the stem-loop, specific N recognition relies on both an intact stem-loop structure and two critical nucleotides in the pentamer loop. Substitutions of these nucleotides affect both N binding and antitermination. Remarkably, substitutions of other loop nucleotides also diminish antitermination in vivo, yet they have no detectable effect on N binding in vitro. These 3' loop mutants fail to support antitermination in a minimal system with RNA polymerase (RNAP), N, and the host factor NusA. Furthermore, the ability of NusA to stimulate the formation of the RNAP-boxB-N complex is diminished with these mutants. Hence, we suggest that boxB RNA performs two critical functions in antitermination. First, boxB binds to N and secures it near RNAP to enhance their interaction, presumably by increasing the local concentration of N. Second, boxB cooperates with NusA, most likely to bring N and RNAP in close contact and transform RNAP to the termination-resistant state.

DNA-strand exchange promoted by RecA protein in the absence of ATP: implications for the mechanism of energy transduction in protein-promoted nucleic acid transactions.

DNA-strand exchange promoted by Escherichia coli RecA protein normally requires the presence of ATP and is accompanied by ATP hydrolysis, thereby implying a need for ATP hydrolysis. Previously, ATP hydrolysis was shown not to be required; here we demonstrate furthermore that a nucleoside triphosphate cofactor is not required for DNA-strand exchange. A gratuitous allosteric effector consisting of the noncovalent complex of ADP and aluminum fluoride, ADP.AIF4-, can both induce the high-affinity DNA-binding state of RecA protein and support the homologous pairing and exchange of up to 800-900 bp of DNA. These results demonstrate that induction of the functionally active, high-affinity DNA-binding state of RecA protein is needed for RecA protein-promoted DNA-strand exchange and that there is no requirement for a high-energy nucleotide cofactor for the exchange of DNA strands. Consequently, the free energy needed to activate the DNA substrates for DNA-strand exchange is not derived from ATP hydrolysis. Instead, the needed free energy is derived from ligand binding and is transduced to the DNA via the associated ligand-induced structural transitions of the RecA protein-DNA complex; ATP hydrolysis simply destroys the effector ligand. This concept has general applicability to the mechanism of energy transduction by proteins.

Crystal structure of an H-2Kb-ovalbumin peptide complex reveals the interplay of primary and secondary anchor positions in the major histocompatibility complex binding groove.

Sequence analysis of peptides naturally presented by major histocompatibility complex (MHC) class I molecules has revealed allele-specific motifs in which the peptide length and the residues observed at certain positions are restricted. Nevertheless, peptides containing the standard motif often fail to bind with high affinity or form physiologically stable complexes. Here we present the crystal structure of a well-characterized antigenic peptide from ovalbumin [OVA-8, ovalbumin-(257-264), SIINFEKL] in complex with the murine MHC class I H-2Kb molecule at 2.5-A resolution. Hydrophobic peptide residues Ile-P2 and Phe-P5 are packed closely together into binding pockets B and C, suggesting that the interplay of peptide anchor (P5) and secondary anchor (P2) residues can couple the preferred sequences at these positions. Comparison with the crystal structures of H-2Kb in complex with peptides VSV-8 (RGYVYQGL) and SEV-9 (FAPGNYPAL), where a Tyr residue is used as the C pocket anchor, reveals that the conserved water molecule that binds into the B pocket and mediates hydrogen bonding from the buried anchor hydroxyl group could not be likewise positioned if the P2 side chain were of significant size. Based on this structural evidence, H-2Kb has at least two submotifs: one with Tyr at P5 (or P6 for nonamer peptides) and a small residue at P2 (i.e., Ala or Gly) and another with Phe at P5 and a medium-sized hydrophobic residue at P2 (i.e., Ile). Deciphering of these secondary submotifs from both crystallographic and immunological studies of MHC peptide binding should increase the accuracy of T-cell epitope prediction.

Human autoantibody recognition of DNA.

Combinatorial IgG Fab phage display libraries prepared from a systemic lupus erythematosus (SLE) donor and a healthy donor were affinity selected against human placental DNA. Human monoclonal antibody Fab fragments specific for DNA were isolated from both libraries, although Fabs of the highest affinity were isolated only from the lupus library. Generally, apparent affinities of the Fabs for human placental DNA, purified double-stranded DNA, and denatured DNA were approximately equivalent. Surface plasmon resonance indicated Fab binding constants for a double-stranded oligodeoxynucleotide of 0.2-1.3 x 10(8) M-1. The higher-affinity Fabs, as ranked by binding to human placental DNA or to the oligonucleotide probe, tested positive in the Crithidia luciliae assay commonly used in the diagnosis of SLE, and interestingly the genes encoding the heavy-chain variable regions of these antibodies displayed evidence of only minimal somatic hypermutation. The heavy chains of the SLE Fabs were characterized by a predominance of basic residues toward the N terminus of complementarity-determining region 3 (CDR3). The crucial role of heavy-chain CDR3 (HCDR3) in high-affinity DNA recognition was suggested by the creation of DNA binding in an unrelated antibody by HCDR3 transplantation from SLE antibodies. We propose that high-affinity DNA-binding antibodies can arise in SLE without extensive somatic hypermutation in the variable-region genes because of the expression of inappropriate HCDR3s.

The Menkes/Wilson disease gene homologue in yeast provides copper to a ceruloplasmin-like oxidase required for iron uptake.

The CCC2 gene of the yeast Saccharomyces cerevisiae is homologous to the human genes defective in Wilson disease and Menkes disease. A biochemical hallmark of these diseases is a deficiency of copper in ceruloplasmin and other copper proteins found in extracytosolic compartments. Here we demonstrate that disruption of the yeast CCC2 gene results in defects in respiration and iron uptake. These defects could be reversed by supplementing cells with copper, suggesting that CCC2 mutant cells were copper deficient. However, cytosolic copper levels and copper uptake were normal. Instead, CCC2 mutant cells lacked a copper-dependent oxidase activity associated with the extracytosolic domain of the FET3-encoded protein, a ceruloplasmin homologue previously shown to be necessary for high-affinity iron uptake in yeast. Copper restored oxidase activity both in vitro and in vivo, paralleling the ability of copper to restore respiration and iron uptake. These results suggest that the CCC2-encoded protein is required for the export of copper from the cytosol into an extracytosolic compartment, supporting the proposal that intracellular copper transport is impaired in Wilson disease and Menkes disease.

Interações moleculares no mecanismo de ação da galectina-4 humana

A galectina-4 humana (HGal-4), pertencente à família das galectinas, possui dois domínios de reconhecimento de carboidratos (CRDs) com alta afinidade para β-galactosídeos e se encontra amplamente distribuída em células normais e neoplásicas de diferentes organismos. Suas funções snglobam uma grande variedade de eventos celulares, tais como processos inflamatórios, neoplásicos, progressão tumoral e metástase. Entretanto, muitas perguntas sobre suas interações com diferentes carboidratos, a especificidade destas interações e o papel específico das galectinas permanecem ainda sem resposta. No presente trabalho, propomos a investigação das interações galectina-glicano da galectina-4 humana e de seus domínios CRDs independentes (CRD-I e CRD-II) através de um conjunto de métodos biofísicos. Através do método de dicroísmo circular (CD), usando várias regiões espectrais, e fluorescência fomos capazes de entender mudanças ocorrentes na estrutura secundária e terciária das protéinas quando da interação com lactose/sacarose. Estes dados, juntamente com testes de hemaglutinação, mostraram que a glectina-4 e os CRDs respondem de forma distinta à ligação com açúcar. Por diferentes técnicas (fluorescência, ITC e MST) determinamos as constantes de dissociação para os domínios CRDs (Kd ~0,5 mM) e para HGal-4 e, de forma qualitativa, os valores obtidos indicaram possíveis estados oligoméricos dessas proteínas. A investigação da interação proteína-membrana da HGal-4 foi feita, primeiramente, com miméticos de membranas e monitorada pela técnica de RPE em crescente complexidade de composição de tais miméticos, indo desde composições mais simples, passando por lipid rafts na presença de diferentes glicolipídeos (GM1, LPS) e chegando-se à interação com células tumorais (U87MG, T98G e HT-29). Tais experimentos mostraram que galectina-4 reconhece e se liga naqueles modelos onde existem glicanos complexos na superfície. Investigamos também a participação de HGal-4 endógena e exógena no tratamento quimioterápico de células tumorais e verificamos um papel importante de HGal-4 para células HT-29. Finalizando esta tese, apresentamos o trabalho realizado em um ano de estágio na University of Oxford, durante o qual, investigamos a estrutura da região C-terminal de um receptor da família GPCR, qual seja o receptor de neurotensina NTS1. Aqui, mais uma vez, foi empregada a técnica de RPE que aliada à produção/marcação de mutantes do receptor, permitiu determinar que a hélice H8 se estabiliza quando em proteolipossomos.

Zalpha-domains: At the intersection between RNA editing and innate immunity

The involvement of A to I RNA editing in antiviral responses was first indicated by the observation of genomic hyper-mutation for several RNA viruses in the course of persistent infections. However, in only a few cases an antiviral role was ever demonstrated and surprisingly, it turns out that ADARs - the RNA editing enzymes - may have a prominent pro-viral role through the modulation/down-regulation of the interferon response. A key role in this regulatory function of RNA editing is played by ADAR1, an interferon inducible RNA editing enzyme. A distinguishing feature of ADAR1, when compared with other ADARs, is the presence of a Z-DNA binding domain, Zalpha. Since the initial discovery of the specific and high affinity binding of Zalpha to CpG repeats in a left-handed helical conformation, other proteins, all related to the interferon response pathway, were shown to have similar domains throughout the vertebrate lineage. What is the biological function of this domain family remains unclear but a significant body of work provides pieces of a puzzle that points to an important role of Zalpha domains in the recognition of foreign nucleic acids in the cytoplasm by the innate immune system. Here we will provide an overview of our knowledge on ADAR1 function in interferon response with emphasis on Zalpha domains.

LeProT1, a Transporter for Proline, Glycine Betaine, and γ-Amino Butyric Acid in Tomato Pollen

During maturation, pollen undergoes a period of dehydration accompanied by the accumulation of compatible solutes.Solute import across the pollen plasma membrane, which occurs via proteinaceous transporters, is required to support pollen development and also for subsequent germination and pollen tube growth. Analysis of the free amino acid composition of various tissues in tomato revealed that the proline content in flowers was 60 times higher than in any other organ analyzed. Within the floral organs, proline was confined predominantly to pollen, where it represented >70 of total free amino acids. Uptake experiments demonstrated that mature as well as germinated pollen rapidly take up proline. To identify proline transporters in tomato pollen, we isolated genes homologous to Arabidopsis proline transporters. LeProT1 was specifically expressed both in mature and germinating pollen, as demonstrated by RNA in situ hybridization. Expression in a yeast mutant demonstrated that LeProT1 transports proline and γ-amino butyric acid with low affinity and glycine betaine with high affinity. Direct uptake and competition studies demonstrate that LeProT1 constitutes a general transporter for compatible solutes.

Using quantity/intensity relationships to assess the potential for ammonium leaching in a Vertosol

High concentrations of ammonium (up to 0.1 cmol/kg) have been observed below 1 m depth in a Vertosol soil near Warra in south-eastern Queensland. This study examined whether ammonium leaching could be responsible for the ammonium accumulation observed in the Warra soil. This was done by using quantity/intensity (Q/I) relationships to compare the ammonium retention capacity of the Warra soil with other similar soils throughout the region that did not contain elevated subsoil ammonium concentrations. Analysis of Q/I curves revealed that in the concentration range studied, the amount of ammonium retained on high affinity adsorption sites in all 3 soils was low, and the Warra soil was not significantly different from the other 2 soils. The ability of the soils to retain ammonium in the soil solution against leaching [i.e. their potential buffer capacity (PBC)] did differ between soils and was greatest at Warra. This indicates that at any one time the Warra soil holds more ammonium on the exchange complex and less in solution than the other soils examined. It was concluded that ammonium is no more likely to leach through the surface horizons of the Warra soil than the other soils examined. Indeed, the data indicated that the Warra soil probably has greater capacity to retain ammonium against leaching due to its greater PBC. Consequently, it is considered unlikely that leaching of ammonium has been a major contributor to the subsoil ammonium concentrations at Warra.

Fatty acids as haptens: exploring the limits of antigenicity

Fatty acids (FAs) are relatively small, hydrophobic and highly mobile molecular structures with vital biological functions and a ubiquitous distribution. Surprisingly, however, they can be rendered immunogenic. We have synthesised a novel immunogen in which dicarboxylic linoleic acid was conjugated to a carrier protein. Dicarboxylic fatty acids (DCA) differ from their normal counterparts only by their possession of a carboxyl group at each end of the molecule. When conjugated to proteins as haptens, they are, therefore, presented to the immune system with a free carboxyl group at the distal end, instead of a methyl group. Polyclonal IgG antibodies raised in response to this unique immunogen could bind not only conjugated hapten with high affinity, but also the equivalent free FA in mono and dicarboxylic form. Similar conjugates constructed from normal FAs produced much weaker antibody responses and could scarcely be considered antigenic at all. The cross-reactivities of the anti-DCA antibodies with FA variants differing in the number, position and configuration of their double bonds showed that the antibody paratope (binding site) was structured to accommodate the hapten in a way that depended on the precise shape of the acyl chain. We suggest that FAs become much more effective as B-cell epitopes when presented with their hydrophilic carboxyl group exposed on the surface of immunogenic conjugates. This type of epitope is determined by the particular double bond pattern of the unsaturated acyl chain, as well as the polar head group. (C) 2003 Elsevier Ltd. All rights reserved.

Repelling class discrimination: ephrin-A5 binds to and activates EphB2 receptor signaling

The interactions between Eph receptor tyrosine kinases and their ephrin ligands regulate cell migration and axon pathfinding. The EphA receptors are generally thought to become activated by ephrin-A ligands, whereas the EphB receptors interact with ephrin-B ligands. Here we show that two of the most widely studied of these molecules, EphB2 and ephrin-A5, which have never been described to interact with each other, do in fact bind one another with high affinity. Exposure of EphB2-expressing cells to ephrin-A5 leads to receptor clustering, autophosphorylation and initiation of downstream signaling. Ephrin-A5 induces EphB2-mediated growth cone collapse and neurite retraction in a model system. We further show, using X-ray crystallography, that the ephrin-A5-EphB2 complex is a heterodimer and is architecturally distinct from the tetrameric EphB2-ephrin-B2 structure. The structural data reveal the molecular basis for EphB2-ephrin-A5 signaling and provide a framework for understanding the complexities of functional interactions and crosstalk between A- and B-subclass Eph receptors and ephrins.

Retinal glutamate transporter activity persists under simulated ischemic conditions

Elevated extracellular concentrations of the neurotransmitter glutamate are neurotoxic and directly contribute to CNS damage as a result of ischemic pathologies. However, the main contributors to this uncontrolled rise in glutamate are still unconfirmed. It has been reported that the reversal of high-affinity glutamate transporters is a significant contributing factor. Conversely, it has also Peen observed that these transporters continue to take up glutamate, albeit at a reduced saturation concentration, under ischemic conditions. We sought to determine whether glutamate transporters continue to remove glutamate from the extracellular space under ischemic conditions by pharmacologically modulating the activity of high-affinity retinal glutamate transporters during simulated ischemia in vitro. Retinal glutamate transporter activity was significantly reduced under these ischemic conditions. The suppression of retinal glutamate transporter activity, with the protein kinase C inhibitor chelerythrine, significantly reduced ischemic glutamate uptake and enhanced retinal neurodegeneration. These findings imply a limited but protective role for retinal glutamate transporters under certain ischemic conditions, suggesting that pharmacological enhancement of high-affinity glutamate transporter activity may reduce tissue damage and loss of function resulting from toxic extracellular glutamate concentrations. (C) 2004 Wiley-Liss, Inc.