Mouse glucose-6-phosphate isomerase and neuroleukin have identical 3' sequences

Neuroleukin is a neurotrophic factor of relative molecular mass (Mr) 56,000 (56K) found in skeletal muscle, brain, heart and kidneys which supports the survival of embryonic spinal neurones, skeletal motor neurones and sensory neurones. Neuroleukin is also a lymphokine product of lectin-stimulated T cells and induces immunoglobulin secretion by cultured human peripheral blood mononuclear cells. Mouse neuroleukin has been cloned, the complete nucleotide sequence has been determined and its complementary DNA has been transiently expressed in monkey COS-1 cells. The serum-free supernatant of the transfected, but not of control mock-transfected, cells was shown to mimic the properties of neuroleukin isolated from mouse salivary glands. In our work on the molecular genetics of carbohydrate metabolism we have recently isolated a mouse glucose-6-phosphate isomerase (or phosphoglucose isomerase, PGI) cDNA clone using the yeast PGI gene (PGI 1) as a probe. We report here that there is complete sequence identity between the 759 nucleotides at the 3' end of this clone (coding and non-coding) and the sequence of mouse neuroleukin.     

Protease nexin-II, a potent antichymotrypsin, shows identity to amyloid beta-protein precursor

Protease nexin-II (PN-II) is a protease inhibitor that forms SDS-resistant inhibitory complexes with the epidermal growth factor (EGF)-binding protein, the gamma-subunit of nerve growth factor, and trypsin. The properties of PN-II indicate that it has a role in the regulation of certain proteases in the extracellular environment. Here we describe more of the amino-acid sequence of PN-II and its identity to the deduced sequence of the amyloid beta-protein precursor (APP). Amyloid beta-protein is present in neuritic plaques and cerebrovascular deposits in individuals with Alzheimer's disease and Down's syndrome. A monoclonal antibody against PN-II (designated mAbP2-1) recognized PN-II in immunoblots of serum-free culture medium from human glioblastoma cells and neuroblastoma cells, as well as in homogenates of normal and Alzheimer's disease brains. In addition, mAbP2-1 stained neuritic plaques in Alzheimer's disease brain. PN-II was a potent inhibitor of chymotrypsin with an inhibition constant Ki of 6 x 10(-10)M. Together, these data demonstrate that PN-II and APP are probably the same protein. The regulation of extracellular proteolysis by PN-II and the deposition of at least parts of the molecule in senile plaques is consistent with previous reports that implicate altered proteolysis in the pathogenesis of Alzheimer's disease.     

Localization of dystrophin to postsynaptic regions of central nervous system cortical neurons

Moderate non-progressive cognitive impairment is a consistent feature of Duchenne muscular dystrophy (DMD), although no central nervous system (CNS) abnormality has been identified. Recent studies have elucidated the molecular defect in DMD, including the absence of the protein dystrophin in affected individuals. Normal brain tissue contains dystrophin messenger RNA and dystrophin is present in low abundance in the brain and seems to be regulated in this tissue, at least in part, by a promoter that differs from that in muscle. Until now, antibodies and immunocytochemical methods used to demonstrate dystrophin at the plasma membrane of mouse and human muscle have proven inadequate to localize precisely dystrophin in the mammalian CNS. We have now made an antibody (anti 6-10) which is much more sensitive than those previously available to immunolabel dystrophin in the CNS. Using this antibody, we found that in the mouse, dystrophin is particularly abundant in the neurons of the cerebral and cerebellar cortices, and that it is localized at postsynaptic membrane specializations. Dystrophin may have a different role in neurons than in muscle, and an alteration at the synaptic level may be the basis of the cognitive impairment in DMD.     

The Caenorhabditis elegans lin-12 gene encodes a transmembrane protein with overall similarity to Drosophila Notch

The lin-12 gene seems to control certain binary decisions during Caenorhabditis elegans development, from genetic and anatomical studies of lin-12 mutants that have either elevated or reduced levels of lin-12 activity. We report here the complete DNA sequence of lin-12: 13.5 kilobases (kb) derived from genomic clones and 4.5 kb from complementary DNA clones. It is of interest that the predicted product is a putative transmembrane protein, given that many of the decisions controlled by lin-12 activity require cell-cell interactions for the correct choice of cell fate. In addition, the predicted lin-12 product may be classified into several regions, based on amino acid sequence similarities to other proteins. These include extensive overall sequence similarity to the Drosophila Notch protein, which also is involved in cell-cell interactions that specify cell fate; a repeated motif found in proteins encoded by the yeast cell-cycle control genes cdc10 (Schizosaccharomyces pombe) and SWI6 (Saccharomyces cerevisiae); and a repeated motif exemplified by epidermal growth factor, found in many mammalian proteins.     

Control of gene expression by a natural metabolite-responsive ribozyme

Most biological catalysts are made of protein; however, eight classes of natural ribozymes have been discovered that catalyse fundamental biochemical reactions. The central functions of ribozymes in modern organisms support the hypothesis that life passed through an 'RNA world' before the emergence of proteins and DNA. We have identified a new class of ribozymes that cleaves the messenger RNA of the glmS gene in Gram-positive bacteria. The ribozyme is activated by glucosamine-6-phosphate (GlcN6P), which is the metabolic product of the GlmS enzyme. Additional data indicate that the ribozyme serves as a metabolite-responsive genetic switch that represses the glmS gene in response to rising GlcN6P concentrations. These findings demonstrate that ribozyme switches may have functioned as metabolite sensors in primitive organisms, and further suggest that modern cells retain some of these ancient genetic control systems.     

SGK and 14-3-3 protein are involved in the serine/ threonine phosphorylation mechanism for TPO/MPL signal transduction

Thrombopioetin (TPO), the critical regulator of platelet production, acts by binding to its cell surface receptor, c-Mpl. Yeast two-hybrid screening was performed to isolate the proteins interacting with the cytoplasmic domain of c-Mpl. 48 positive clones were isolated from 5 × 10⁶ independent transformants. The results of sequence analysis demonstrate that they represent 13 different protein encoding sequences. Among them there are a partial coding sequence of serine/threonine protein kinase SGK (serum and glucocorticoid-inducible kinase) and 14-3-3 theta protein partial coding sequence. GST-pull-down assay and co-immunoprecipitation in mammal cells have confirmed the interaction between these two proteins and c-Mpl. By constructing a series of deleted c-Mpl cytoplasmic domain, the interaction region in c-Mpl cytoplasmic tail was localized in amino acids 523–554. At the same time, the directed interaction between SGK and 14-3-3 proteins also has been verified by yeast two-hybrid assay. The present note is the first time to report that two proteins act with c-Mpl at the same time and put forward that SGK and 14-3-3 protein may be involved in the serine/threonine phosphorylation mechanism for signal transduction.     

Existence of distinct sodium channel messenger RNAs in rat brain

The sodium channel is a voltage-gated ionic channel essential for the generation of action potentials. It has been reported that the sodium channels purified from the electric organ of Electrophorus electricus (electric eel) and from chick cardiac muscle consist of a single polypeptide of relative molecular mass (Mr) approximately 260,000 (260K), whereas those purified from rat brain and skeletal muscle contain, in addition to the large polypeptide, two or three smaller polypeptides of Mr 37-45K. Recently, we have elucidated the primary structure of the Electrophorus sodium channel by cloning and sequencing the DNA complementary to its messenger RNA. Despite the apparent homogeneity of the purified sodium channel preparations, several types of tetrodotoxin (or saxitoxin) binding sites or sodium currents have been observed in many excitable membranes. The occurrence of distinguishable populations of sodium channels may be attributable to different states of the same channel protein or to distinct channel proteins. We have now isolated complementary DNA clones derived from two distinct rat brain mRNAs encoding sodium channel large polypeptides and present here the complete amino-acid sequences of the two polypeptides (designated sodium channels I and II), as deduced from the cDNA sequences. A partial DNA sequence complementary to a third homologous mRNA from rat brain has also been cloned.     

SEC65 gene product is a subunit of the yeast signal recognition particle required for its integrity.

Protein targeting to the endoplasmic reticulum (ER) in mammalian cells is catalysed by the signal recognition particle (SRP), which consists of six protein subunits and an RNA subunit. Saccharomyces cerevisiae SRP is a 16S particle, of which only two subunits have been identified: a protein subunit, SRP54p, which is homologous to the mammalian SRP54 subunit, and an RNA subunit, scR1 (ref. 3). The sec65-1 mutant yeast cells are temperature-sensitive for growth and defective in the translocation of several secreted and membrane-bound proteins. The DNA sequence of the SEC65 gene suggests that its product is related to mammalian SRP19 subunit and may have a similar function. Here we show that SEC65p is a subunit of the S. cerevisiae SRP and that it is required for the stable association of another subunit, SRP54p, with SRP. Overexpression of SRP54p suppresses both growth and protein translocation defects in sec65-1 mutant cells.     

Active-site remodelling in the bifunctional fructose-1,6-bisphosphate aldolase/phosphatase

Fructose-1,6-bisphosphate (FBP) aldolase/phosphatase is a bifunctional, thermostable enzyme that catalyses two subsequent steps in gluconeogenesis in most archaea and in deeply branching bacterial lineages. It mediates the aldol condensation of heat-labile dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP) to FBP, as well as the subsequent, irreversible hydrolysis of the product to yield the stable fructose-6-phosphate (F6P) and inorganic phosphate; no reaction intermediates are released. Here we present a series of structural snapshots of the reaction that reveal a substantial remodelling of the active site through the movement of loop regions that create different catalytic functionalities at the same location. We have solved the three-dimensional structures of FBP aldolase/phosphatase from thermophilic Thermoproteus neutrophilus in a ligand-free state as well as in complex with the substrates DHAP and FBP and the product F6P to resolutions up to 1.3??. In conjunction with mutagenesis data, this pinpoints the residues required for the two reaction steps and shows that the sequential binding of additional Mg(2+) cations reversibly facilitates the reaction. FBP aldolase/phosphatase is an ancestral gluconeogenic enzyme optimized for high ambient temperatures, and our work resolves how consecutive structural rearrangements reorganize the catalytic centre of the protein to carry out two canonical reactions in a very non-canonical type of bifunctionality.     

Protease inhibitor domain encoded by an amyloid protein precursor mRNA associated with Alzheimer's disease

Amyloid B-protein/amyloid A4 is a peptide present in the neuritic plaques, neurofibrillary tangles and cerebrovascular deposits in patients with Alzheimer's disease and Down's syndrome (trisomy 21) and may be involved in the pathogenesis of Alzheimer's disease. Recent molecular genetic studies have indicated that amyloid protein is encoded as part of a larger protein by a gene on human chromosome 21 (refs 6-9). The amyloid protein precursor (APP) gene is expressed in brain and in several peripheral tissues, but the specific biochemical events leading to deposition of amyloid are not known. We have now screened complementary DNA libraries constructed from peripheral tissues to determine whether the messenger RNA encoding APP in these tissues is identical to that expressed in brain, and we identify a second APP mRNA that encodes an additional internal domain with a sequence characteristic of a Kunitz-type serine protease inhibitor. The alternative APP mRNA is present in both brain and peripheral tissues of normal individuals and those with Alzheimer's disease, but its pattern of expression differs from that of the previously reported APP mRNA.     

大熊猫肌红蛋白的纯化及一级结构的研究

本文从大熊猫骨骼肌分离纯化了肌红蛋白,鉴定了纯度,测定了分子量及氨基酸组成。确定了该蛋白的N端及C端氨基酸。采用CNBr裂解的方法,用HPLC分离得到4个肽段,分别测定了大熊猫肌红蛋白1—52位,56—77位及132—153位氨基酸的排列顺序。大熊猫肌红蛋白共含153个氨基酸,本文已完成了96个氨基酸的序列测定。用此结果与食肉目有关动物已知肌红蛋白的氨基酸顺序进行比较,获得了有意义的结论。     

Catalysis of guanine nucleotide exchange on Ran by the mitotic regulator RCC1

The product of the gene RCC1 (regulator of chromosome condensation) in a BHK cell line is involved in the control of mitotic events. Homologous genes have been found in Xenopus, Drosophila and yeast. A human genomic DNA fragment and complementary DNA that complement a temperature-sensitive mutation of RCC1 in BHK21 cells encode a protein of relative molecular mass 45,000 (Mr 45K) which is located in the nucleus and binds to chromatin. We have recently isolated a protein from HeLa cells that strongly binds an anti-RCC1 antibody and has the same molecular mass, DNA-binding properties, and amino-acid sequence as the 205 residues already identified. HeLa cell RCC1 is complexed to a protein of Mr 25K. We have shown that this 25K protein has a sequence homologous to the translated reading frame of TC4, a cDNA found by screening a human teratocarcinoma cDNA library with oligonucleotides coding for a ras consensus sequence, and that the protein binds GDP and GTP. We have referred to this protein as the Ran protein (ras-related nuclear protein). In addition to the fraction of Ran protein complexed to RCC1, a 25-fold molar excess of the protein over RCC1 was found in the nucleoplasm of HeLa cells. Here we show that RCC1 specifically catalyses the exchange of guanine nucleotides on the Ran protein but not on the protein c-Ha-ras p21 (p21ras).     

Human preprovasoactive intestinal polypeptide contains a novel PHI-27-like peptide, PHM-27

Vasoactive intestinal polypeptide (VIP), a 28-amino acid peptide originally isolated from porcine duodenum, is present not only in gastrointestinal tissues but also in neural tissues, possibly as a neurotransmitter, and exhibits a wide range of biological actions (for example, relaxation of smooth muscle, stimulation of intestinal water and electrolyte secretion and release of insulin, glucagon and several anterior pituitary hormones). As the structure of porcine and bovine VIP shows several similarities to those of mammalian glucagon, secretin and gastric inhibitory peptide (GIP), VIP is considered to be a member of the glucagon-secretin family. Recently, we have found that VIP is synthesized from a precursor, pro-VIP (molecular weight (Mr) 17,500), in human neuroblastoma cells and that the primary translation product of the mRNA encoding VIP is prepro-VIP (Mr 20,000). In an attempt to elucidate the primary structure of the precursor, we have now cloned the DNA sequence complementary to the mRNA coding for human VIP and analysed the nucleotide sequence. The entire amino acid sequence of the precursor, deduced from the nucleotide sequence, indicates that the precursor protein contains not only VIP but also a novel peptide of 27 amino acids. The peptide, designated PHM-27, differs by only 2 amino acids from PHI-27, a peptide recently isolated from porcine intestine, and is also closely related in sequence to VIP.     

Identification of scrapie prion protein-specific mRNA in scrapie-infected and uninfected brain

To date no nucleic acid has been found in the purified infectious agent which causes the spongiform encephalopathy known as scrapie. In an attempt to identify a unique scrapie virus-associated messenger RNA in tissues of infected animals, we have synthesized an oligonucleotide probe complementary to the mRNA sequence corresponding to the amino-acid sequence of the prion protein, PrP27-30 (ref. 1). We report here that, with this probe, a complementary DNA clone representing PrP27-30 was obtained from scrapie-infected mouse brain; the DNA sequence of this clone could be translated into a protein that matches exactly the published sequence of PrP27-30. The cDNA clone hybridized to a single 2.4-2.5-kilobase (kb) mRNA from both normal and scrapie-infected brain. Thus, the PrP27-30 mRNA is not uniquely associated with scrapie infectivity, suggesting that PrP27-30 may be a normal component of mouse and hamster brain.