Control of the phosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in the immature rat hippocampus by glutamate and calcium ions: possible key factor in astrocytic plasticity

The present review describes recent research on the regulation by glutamate and Ca2+ of the phosphorylation state of the intermediate filament protein of the astrocytic cytoskeleton, glial fibrillary acidic protein (GFAP), in immature hippocampal slices. The results of this research are discussed against a background of modern knowledge of the functional importance of astrocytes in the brain and of the structure and dynamic properties of intermediate filament proteins. Astrocytes are now recognized as partners with neurons in many aspects of brain function with important roles in neural plasticity. Site-specific phosphorylation of intermediate filament proteins, including GFAP, has been shown to regulate the dynamic equilibrium between the polymerized and depolymerized state of the filaments and to play a fundamental role in mitosis. Glutamate was found to increase the phosphorylation state of GFAP in hippocampal slices from rats in the post-natal age range of 12-16 days in a reaction that was dependent on external Ca2+. The lack of external Ca2+ in the absence of glutamate also increased GFAP phosphorylation to the same extent. These effects of glutamate and Ca2+ were absent in adult hippocampal slices, where the phosphorylation of GFAP was completely Ca2+-dependent. Studies using specific agonists of glutamate receptors showed that the glutamate response was mediated by a G protein-linked group II metabotropic glutamate receptor (mGluR). Since group II mGluRs do not act by liberating Ca2+ from internal stores, it is proposed that activation of the receptor by glutamate inhibits Ca2+ entry into the astrocytes and consequently down-regulates a Ca2+-dependent dephosphorylation cascade regulating the phosphorylation state of GFAP. The functional significance of these results may be related to the narrow developmental window when the glutamate response is present. In the rat brain this window corresponds to the period of massive synaptogenesis during which astrocytes are known to proliferate. Possibly, glutamate liberated from developing synapses during this period may signal an increase in the phosphorylation

The quality control of glycoprotein folding in the endoplasmic reticulum, a trip from trypanosomes to mammals

The present review deals with the stages of synthesis and processing of asparagine-linked oligosaccharides occurring in the lumen of the endoplasmic reticulum and their relationship to the acquisition by glycoproteins of their proper tertiary structures. Special emphasis is placed on reactions taking place in trypanosomatid protozoa since their study has allowed the detection of the transient glucosylation of glycoproteins catalyzed by UDP-Glc:glycoprotein glucosyltransferase and glucosidase II. The former enzyme has the unique property of covalently tagging improperly folded conformations by catalyzing the formation of protein-linked Glc1Man7GlcNAc2, Glc1Man8GlcNac2 and Glc1Man9GlcNAc2 from the unglucosylated proteins. Glucosyltransferase is a soluble protein of the endoplasmic reticulum that recognizes protein domains exposed in denatured but not in native conformations (probably hydrophobic amino acids) and the innermost N-acetylglucosamine unit that is hidden from macromolecular probes in most native glycoproteins. In vivo, the glucose units are removed by glucosidase II. The influence of oligosaccharides in glycoprotein folding is reviewed as well as the participation of endoplasmic reticulum chaperones (calnexin and calreticulin) that recognize monoglucosylated species in the same process. A model for the quality control of glycoprotein folding in the endoplasmic reticulum, i.e., the mechanism by which cells recognize the tertiary structure of glycoproteins and only allow transit to the Golgi apparatus of properly folded species, is discussed. The main elements of this control are calnexin and calreticulin as retaining components, the UDP-Glc:glycoprotein glucosyltransferase as a sensor of tertiary structures and glucosidase II as the releasing agent.

Molecular biology of the kallikrein-kinin system: from structure to function

The participation of the kallikrein-kinin system, comprising the serine proteases kallikreins, the protein substrates kininogens and the effective peptides kinins, in some pathological processes like hypertension and cardiovascular diseases is still a matter of controversy. The use of different experimental set-ups in concert with the development of potent and specific inhibitors and antagonists for the system has highlighted its importance but the results still lack conclusivity. Over the last few years, transgenic and gene-targeting technologies associated with molecular biology tools have provided specific information about the elusive role of the kallikrein-kinin system in the control of blood pressure and electrolyte homeostasis. cDNA and genomic sequences for kinin receptors B2 and B1 from different species were isolated and shown to encode G-protein-coupled receptors and the structure and pharmacology of the receptors were characterized. Transgenic animals expressing an overactive kallikrein-kinin system were established to study the cardiovascular effects of these alterations and the results of these investigations further corroborate the importance of this system in the maintenance of normal blood pressure. Knockout animals for B2 and B1 receptors are available and their analysis also points to the role of these receptors in cardiovascular regulation and inflammatory processes. In this paper the most recent and relevant genetic animal models developed for the study of the kallikrein-kinin system are reviewed, and the advances they brought to the understanding of the biological role of this system are discussed.

A receptor for infectious and cellular prion protein

Prions are an unconventional form of infectious agents composed only of protein and involved in transmissible spongiform encephalopathies in humans and animals. The infectious particle is composed by PrPsc which is an isoform of a normal cellular glycosyl-phosphatidylinositol (GPI) anchored protein, PrPc, of unknown function. The two proteins differ only in conformation, PrPc is composed of 40% a helix while PrPsc has 60% ß-sheet and 20% a helix structure. The infection mechanism is trigged by interaction of PrPsc with cellular prion protein causing conversion of the latter's conformation. Therefore, the infection spreads because new PrPsc molecules are generated exponentially from the normal PrPc. The accumulation of insoluble PrPsc is probably one of the events that lead to neuronal death. Conflicting data in the literature showed that PrPc internalization is mediated either by clathrin-coated pits or by caveolae-like membranous domains. However, both pathways seem to require a third protein (a receptor or a prion-binding protein) either to make the connection between the GPI-anchored molecule to clathrin or to convert PrPc into PrPsc. We have recently characterized a 66-kDa membrane receptor which binds PrPc in vitro and in vivo and mediates the neurotoxicity of a human prion peptide. Therefore, the receptor should have a role in the pathogenesis of prion-related diseases and in the normal cellular process. Further work is necessary to clarify the events triggered by the association of PrPc/PrPsc with the receptor.

P-glycoprotein-mediated resistance to chemotherapy in cancer cells: using recombinant cytosolic domains to establish structure-function relationships

Resistance to chemotherapy in cancer cells is mainly mediated by overexpression of P-glycoprotein (Pgp), a plasma membrane ATP-binding cassette (ABC) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. Pgp consists of two homologous halves each containing a transmembrane domain and a cytosolic nucleotide-binding domain (NBD) which contains two consensus Walker motifs, A and B, involved in ATP binding and hydrolysis. The protein also contains an S signature characteristic of ABC transporters. The molecular mechanism of Pgp-mediated drug transport is not known. Since the transporter has an extraordinarily broad substrate specificity, its cellular function has been described as a "hydrophobic vacuum cleaner". The limited knowledge about the mechanism of Pgp, partly due to the lack of a high-resolution structure, is well reflected in the failure to efficiently inhibit its activity in cancer cells and thus to reverse multidrug resistance (MDR). In contrast to the difficulties encountered when studying the full-length Pgp, the recombinant NBDs can be obtained in large amounts as soluble proteins. The biochemical and biophysical characterization of recombinant NBDs is shown here to provide a suitable alternative route to establish structure-function relationships. NBDs were shown to bind ATP and analogues as well as potent modulators of MDR, such as hydrophobic steroids, at a region close to the ATP site. Interestingly, flavonoids also bind to NBDs with high affinity. Their binding site partly overlaps both the ATP-binding site and the steroid-interacting region. Therefore flavonoids constitute a new promising class of bifunctional modulators of Pgp.

Structure and function of the cystic fibrosis transmembrane conductance regulator

Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR). Mutations in the CFTR gene may result in a defective processing of its protein and alter the function and regulation of this channel. Mutations are associated with different symptoms, including pancreatic insufficiency, bile duct obstruction, infertility in males, high sweat Cl-, intestinal obstruction, nasal polyp formation, chronic sinusitis, mucus dehydration, and chronic Pseudomonas aeruginosa and Staphylococcus aureus lung infection, responsible for 90% of the mortality of CF patients. The gene responsible for the cellular defect in CF was cloned in 1989 and its protein product CFTR is activated by an increase of intracellular cAMP. The CFTR contains two membrane domains, each with six transmembrane domain segments, two nucleotide-binding domains (NBDs), and a cytoplasmic domain. In this review we discuss the studies that have correlated the role of each CFTR domain in the protein function as a chloride channel and as a regulator of the outwardly rectifying Cl- channels (ORCCs).

Gap junctions in cells of the immune system: structure, regulation and possible functional roles

Gap junction channels are sites of cytoplasmic communication between contacting cells. In vertebrates, they consist of protein subunits denoted connexins (Cxs) which are encoded by a gene family. According to their Cx composition, gap junction channels show different gating and permeability properties that define which ions and small molecules permeate them. Differences in Cx primary sequences suggest that channels composed of different Cxs are regulated differentially by intracellular pathways under specific physiological conditions. Functional roles of gap junction channels could be defined by the relative importance of permeant substances, resulting in coordination of electrical and/or metabolic cellular responses. Cells of the native and specific immune systems establish transient homo- and heterocellular contacts at various steps of the immune response. Morphological and functional studies reported during the last three decades have revealed that many intercellular contacts between cells in the immune response present gap junctions or "gap junction-like" structures. Partial characterization of the molecular composition of some of these plasma membrane structures and regulatory mechanisms that control them have been published recently. Studies designed to elucidate their physiological roles suggest that they might permit coordination of cellular events which favor the effective and timely response of the immune system.

Structural, functional and immunological studies on a polymeric bacterial protein

The characterization of proteins from Brucella spp, the causative agent of brucellosis, has been the subject of intensive research. We have described an 18-kDa cytoplasmic protein of Brucella abortus and shown the potential usefulness of this protein as an antigen for the serologic diagnosis of brucellosis. The amino acid sequence of the protein showed a low but significant homology with that of lumazine synthases. Lumazine is an intermediate product in bacterial riboflavin biosynthesis. The recombinant form of the 18-kDa protein (expressed in E. coli) folds like the native Brucella protein and has lumazine-synthase enzymatic activity. Three-dimensional analysis by X-ray crystallography of the homolog Bacillus subtilis lumazine synthase has revealed that the enzyme forms an icosahedral capsid. Recombinant lumazine synthase from B. abortus was crystallized, diffracted X rays to 2.7-Å resolution at room temperature, and the structure successfully solved by molecular replacement procedures. The macromolecular assembly of the enzyme differs from that of the enzyme from B. subtilis. The Brucella enzyme remains pentameric (90 kDa) in its crystallographic form. Nonetheless, the active sites of the two enzymes are virtually identical at the structural level, indicating that inhibitors of these enzymes could be viable pharmaceuticals across a broad species range. We describe the structural reasons for the differences in their quaternary arrangement and also discuss the potential use of this protein as a target for the development of acellular vaccines.

Aggrecan structure in amphibian cartilage

The structure of the large proteoglycan present in the bullfrog epiphyseal cartilage was studied by immunochemical and biochemical methods. The isolated monomer showed a polydisperse behavior on Sepharose CL2B, with a peak at Kav = 0.14. Chondroitin sulfate chains were identified by HPLC analysis of the products formed by chondroitinase digestion and mercuric acetate treatment. These chains have approximately 38 disaccharides, a Di45:Di68 ratio of 1.6 and GalNAc4S + GalNAc4,6S are the main non-reducing terminals. Keratan sulfate was identified by the use of two monoclonal antibodies in Western blots after chondroitinase ABC treatment. A keratan sulfate-rich region (~110 kDa) was isolated by sequential treatment with chondroitinase ABC and proteases. We also employed antibodies in Western blotting experiments and showed that the full length deglycosylated core protein is about 300 kDa after SDS-PAGE. Domain-specific antibodies revealed the presence of immunoreactive sites corresponding to G1/G2 and G3 globular domains and the characterization of this large proteoglycan as aggrecan. The results indicate the high conservation of the aggrecan domain structure in this lower vertebrate.

Identification of the divergent calmodulin binding motif in yeast Ssb1/Hsp75 protein and in other HSP70 family members

Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37% of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89% similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed.

In silico analysis identifies a C3HC4-RING finger domain of a putative E3 ubiquitin-protein ligase located at the C-terminus of a polyglutamine-containing protein

Almost identical polyglutamine-containing proteins with unknown structures have been found in human, mouse and rat genomes (GenBank AJ277365, AF525300, AY879229). We infer that an identical new gene (RING) finger domain of real interest is located in each C-terminal segment. A three-dimensional (3-D) model was generated by remote homology modeling and the functional implications are discussed. The model consists of 65 residues from terminal position 707 to 772 of the human protein with a total length of 796 residues. The 3-D model predicts a ubiquitin-protein ligase (E3) as a binding site for ubiquitin-conjugating enzyme (E2). Both enzymes are part of the ubiquitin pathway to label unwanted proteins for subsequent enzymatic degradation. The molecular contact specificities are suggested for both the substrate recognition and the residues at the possible E2-binding surface. The predicted structure, of a ubiquitin-protein ligase (E3, enzyme class number 6.3.2.19, CATH code 3.30.40.10.4) may contribute to explain the process of ubiquitination. The 3-D model supports the idea of a C3HC4-RING finger with a partially new pattern. The putative E2-binding site is formed by a shallow hydrophobic groove on the surface adjacent to the helix and one zinc finger (L722, C739, P740, P741, R744). Solvent-exposed hydrophobic amino acids lie around both zinc fingers (I717, L722, F738, or P765, L766, V767, V733, P734). The 3-D structure was deposited in the protein databank theoretical model repository (2B9G, RCSB Protein Data Bank, NJ).

Glutamate receptors as seen by light: spectroscopic studies of structure-function relationships

Ionotropic glutamate receptors are major excitatory receptors in the central nervous system and also have a far reaching influence in other areas of the body. Their modular nature has allowed for the isolation of the ligand-binding domain and for subsequent structural studies using a variety of spectroscopic techniques. This review will discuss the role of specific ligand:protein interactions in mediating activation in the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype of glutamate receptors as established by various spectroscopic investigations of the GluR2 and GluR4 subunits of this receptor. Specifically, this review will provide an introduction to the insight gained from X-ray crystallography and nuclear magnetic resonance investigations and then go on to focus on studies utilizing vibrational spectroscopy and fluorescence resonance energy transfer to study the behavior of the isolated ligand-binding domain in solution and discuss the importance of specific ligand:protein interactions in the mechanism of receptor activation.

Griscelli syndrome-type 2 in twin siblings: case report and update on RAB27A human mutations and gene structure

Griscelli syndrome (GS) is a rare autosomal recessive disorder caused by mutation in the MYO5A (GS1, Elejalde), RAB27A (GS2) or MLPH (GS3) genes. Typical features of all three subtypes of this disease include pigmentary dilution of the hair and skin and silvery-gray hair. Whereas the GS3 phenotype is restricted to the pigmentation dysfunction, GS1 patients also show primary neurological impairment and GS2 patients have severe immunological deficiencies that lead to recurrent infections and hemophagocytic syndrome. We report here the diagnosis of GS2 in 3-year-old twin siblings, with silvery-gray hair, immunodeficiency, hepatosplenomegaly and secondary severe neurological symptoms that culminated in multiple organ failure and death. Light microscopy examination of the hair showed large, irregular clumps of pigments characteristic of GS. A homozygous nonsense mutation, C-T transition (c.550C>T), in the coding region of the RAB27A gene, which leads to a premature stop codon and prediction of a truncated protein (R184X), was found. In patient mononuclear cells, RAB27A mRNA levels were the same as in cells from the parents, but no protein was detected. In addition to the case report, we also present an updated summary on the exon/intron organization of the human RAB27A gene, a literature review of GS2 cases, and a complete list of the human mutations currently reported in this gene. Finally, we propose a flow chart to guide the early diagnosis of the GS subtypes and Chédiak-Higashi syndrome.

Roles of mitogen-activated protein kinases and angiotensin II in renal development

Experimental and clinical evidence suggests that angiotensin II (AII) participates in renal development. Renal AII content is several-fold higher in newborn rats and mice than in adult animals. AII receptors are also expressed in higher amounts in the kidneys of newborn rats. The kidneys of fetuses whose mother received a type 1 AII receptor (AT1) antagonist during gestation present several morphological alterations. Mutations in genes that encode components of the renin-angiotensin system are associated with autosomal recessive renal tubular dysgenesis. Morphological changes were detected in the kidneys of 3-week-old angiotensin-deficient mice. Mitogen-activated protein kinases (MAPKs) are important mediators that transduce extracellular stimuli to intracellular responses. The MAPK family comprises three major subgroups, namely extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinases (JNK), and p38 MAPK (p38). Important events in renal growth during nephrogenesis such as cellular proliferation and differentiation accompanied by apoptosis on a large scale can be mediated by MAPK pathways. A decrease in glomerulus number was observed in embryos cultured for 48 and 120 h with ERK or p38 inhibitors. Many effects of AII are mediated by MAPK pathways. Treatment with losartan during lactation provoked changes in renal function and structure associated with alterations in AT1 and type 2 AII (AT2) receptors and p-JNK and p-p38 expression in the kidney. Several studies have shown that AII and MAPKs play an important role in renal development. However, the relationship between the effects of AII and MAPK activation on renal development is still unclear.

cDNA and deduced primary structure of basic phospholipase A2 with neurotoxic activity from the venom secretion of the Crotalus durissus collilineatus rattlesnake

To illustrate the construction of precursor complementary DNAs, we isolated mRNAs from whole venom samples. After reverse transcription polymerase chain reaction (RT-PCR), we amplified the cDNA coding for a neurotoxic protein, phospholipase A2 D49 (PLA2 D49), from the venom of Crotalus durissus collilineatus (Cdc PLA2). The cDNA encoding Cdc PLA2 from whole venom was sequenced. The deduced amino acid sequence of this cDNA has high overall sequence identity with the group II PLA2 protein family. Cdc PLA2 has 14 cysteine residues capable of forming seven disulfide bonds that characterize this group of PLA2 enzymes. Cdc PLA2 was isolated using conventional Sephadex G75 column chromatography and reverse-phase high performance liquid chromatography (RP-HPLC). The molecular mass was estimated using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. We tested the neuromuscular blocking activities on chick biventer cervicis neuromuscular tissue. Phylogenetic analysis of Cdc PLA2 showed the existence of two lines of N6-PLA2, denominated F24 and S24. Apparently, the sequences of the New World’s N6-F24-PLA2 are similar to those of the agkistrodotoxin from the Asian genus Gloydius. The sequences of N6-S24-PLA2 are similar to the sequence of trimucrotoxin from the genus Protobothrops, found in the Old World.