Synthesis and Some Biological Properties of Carbon-Bridged Cyclonucleosides and Their Phosphates

Abstract

Methods for the synthesis of carbon-bridged 6,5′-, 6,6′-, 6,3′-, and 6,2′-cyclopyrimidine and 8,2′-cycloadenine nucleosides and some of their phosphates are described. The 6,5′- and 6,6′-cyclouridine 2′,3′-cyclic phosphates were the substrates and their 3′-phosphates were strong inhibitors of pancreatic ribonuclease.     

Crystal complexes of a predicted S-adenosylmethionine-dependent methyltransferase reveal a typical AdoMet binding domain and a substrate recognition domain

S-adenosyl-L-methionine-dependent methyltransferases (MTs) are abundant, and highly conserved across phylogeny. These enzymes use the cofactor AdoMet to methylate a wide variety of molecular targets, thereby modulating important cellular and metabolic activities. Thermotoga maritima protein 0872 (TM0872) belongs to a large sequence family of predicted MTs, ranging phylogenetically from relatively simple bacteria to humans. The genes for many of the bacterial homologs are located within operons involved in cell wall synthesis and cell division. Despite preliminary biochemical studies in E. coli and B. subtilis, the substrate specificity of this group of more than 150 proteins is unknown. As part of the Midwest Center for Structural Genomics initiative (www.mcsg.anl.gov), we have determined the structure of TM0872 in complexes with AdoMet and with S-adenosyl-L-homocysteine (AdoHcy). As predicted, TM0872 has a typical MT domain, and binds endogenous AdoMet, or co-crystallized AdoHcy, in a manner consistent with other known MT structures. In addition, TM0872 has a second domain that is novel among MTs in both its location in the sequence and its structure. The second domain likely acts in substrate recognition and binding, and there is a potential substrate-binding cleft spanning the two domains. This long and narrow cleft is lined with positively charged residues which are located opposite the S(+)-CH(3) bond, suggesting that a negatively charged molecule might be targeted for catalysis. However, AdoMet and AdoHcy are both buried, and access to the methyl group would presumably require structural rearrangement. These TM0872 crystal structures offer the first structural glimpses at this phylogenetically conserved sequence family.     

Overview: Protein palmitoylation in the nervous system: Current views and unsolved problems

Palmitoylation refers to a dynamic post-translational modification of proteins involving the covalent attachment of long-chain fatty acids to the side chains of cysteine, threonine or serine residues. In recent years, palmitoylation has been identified as a widespread modification of both viral and cellular proteins. Because of its dynamic nature, protein palmitoylation, like phosphorylation, appears to have a crucial role in the functioning of the nervous system. Several important questions regarding the post-translational acylation of cysteine residues in proteins are briefly discussed: (a) What are the molecular mechanisms involved in dynamic acylation? (b) What are the determinants of the fatty acid specificity and the structural requirements of the acceptor proteins? (c) What are the physiological signals regulating this type of protein modification, and (d) What is the biological role(s) of this reaction with respect to the functioning of specific nervous system proteins? We also present the current experimental obstacles that have to be overcome to fully understand the biology of this dynamic modification.     

Protein arginine methyltransferase 7 has a novel homodimer-like structure formed by tandem repeats

Protein arginine methyltransferase 7 (PRMT7) is a member of a family of enzymes that catalyze the transfer of methyl groups from S-adenosyl-l-methionine to nitrogen atoms on arginine residues. Here, we describe the crystal structure of Caenorhabditis elegans PRMT7 in complex with its reaction product S-adenosyl-l-homocysteine. The structural data indicated that PRMT7 harbors two tandem repeated PRMT core domains that form a novel homodimer-like structure. S-adenosyl-l-homocysteine bound to the N-terminal catalytic site only; the C-terminal catalytic site is occupied by a loop that inhibits cofactor binding. Mutagenesis demonstrated that only the N-terminal catalytic site of PRMT7 is responsible for cofactor binding.     

Protein kinase and protein phosphatase expression in the central nervous system of G93A mSOD over-expressing mice

The expressions of 78 protein kinases, 24 protein phosphatases and 31 phosphoproteins were investigated by Kinetworks trade mark analysis in brain and spinal cord tissue of transgenic mice over-expressing G93A mutant superoxide dismutase (mSOD), a murine model of amyotrophic lateral sclerosis (ALS). In the brains of affected mSOD mice, we observed increased expression of cAMP-dependent protein kinase (PKA, 111% increase compared with control), and protein phosphatase 2B Aalpha-catalytic subunit (calcineurin, 109% increase), and reductions in the levels of PAK3 (76% decrease) and protein phosphatase 2C Cbeta-subunit (32% decrease). Increased Ser259 phosphorylation of Raf1 (126% increase) in mSOD mice correlated with higher expression of p73 Raf1 (147% increase). There was also increased p73 Raf1 (69% increase) and Ser259 phosphorylation (45% increase) in the spinal cords of mSOD mice. While adducin underwent enhanced phosphorylation (alphaS724, 90% increase; gammaS662, 290% increase) in mSOD brain, its phosphorylation was lower in the mSOD spinal cord (alphaS724, 53% decrease; gammaS662, 46% decrease). In spinal cords of affected mSOD mice, we also observed elevated expression of casein kinase 1delta (CK1delta, 157% increase), JAK2 (84% increase), PKA (183% increase), protein kinase C (PKC) delta (123% increase), p124 PKC micro (142% increase), and RhoA kinase (221% increase), and enhanced phosphorylation of extracellular regulated kinases 1 (ERK1, T202/Y204, 90% increase), and 2 (ERK2, T185/Y187, 73% increase), p38 MAP kinase (T180/Y182, 1570% increase), and PKBalpha (T308, 154% increase; S473, 61% increase). There was also reduced phosphorylation of RB (S780, 45% decrease; S807/S811, 65% decrease), Src (Y418, 63% decrease) and p40 SAPK/JNKbeta (T183/Y185, 43% decrease). Variability in the expression of kinases, phosphatases and phosphorylation of their substrates was observed even in mutant animals having a similar phenotype. The expression and phosphorylation differences between mSOD and control mice were dissimilar to those between ALS patients and controls. This finding indicates that the activation of protein kinases and phosphoproteins is different with neuron loss in the mSOD mouse compared with that seen in patients with the sporadic form of ALS.     

Formation and function of apolipoprotein E-containing lipoproteins in the nervous system

The strongest known genetic risk factor for the development of late-onset Alzheimer disease is inheritance of the apolipoprotein (apo) E4 (ε4 allele) although the mechanisms underlying this connection are still not entirely clear. In this review, we shall discuss the role of apo E in the brain, particularly in relation to Alzheimer disease. Cholesterol transport and homeostasis in the central nervous system (CNS) are separated from that in the peripheral circulation by the blood–brain barrier. However, the brain operates its own lipoprotein transport system that is mediated by high density lipoprotein-sized, apo E-containing lipoproteins that are synthesized and secreted by glial cells (primarily astrocytes). Several ATP-binding cassette (ABC) transporters are expressed in the brain, including ABCA1 and ABCG1 which play important roles in the transfer of phospholipids and cholesterol to apo E. The astrocyte-derived apo E-containing lipoproteins can bind to, and be internalized by, receptors of the low density lipoprotein receptor superfamily that are located on the surface of neurons. In addition to these receptors serving as endocytosis receptors for lipoproteins, several of these receptors also act as signaling receptors in neurons and activate pathways involved in axonal growth, as well as neuronal survival. These beneficial pathways appear to be enhanced to a greater extent by apo E3 than by apo E4. Apo E has also been implicated in the deposition of amyloid plaques since apo E3, more readily than apo E4, forms a complex with Aß peptides, and mediates the degradation of amyloid deposits.     

Glutamate Receptor-interacting Protein 1 Protein Binds to the Microtubule-associated Protein

To identify the interaction proteins for the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit glutamate receptor-interacting protein 1 (GRIP1), GRIP1 interactions with microtubule-associated protein (MAP)-1B light chain (LC) were investigated. GRIP1 interacts with MAP-1A and MAP-1B in the yeast two-hybrid assay, as is indicated also by glutathione S-transferase (GST) pull-down and coimmunoprecipitation with MAP-1B LC antibody in brain fractions. These results suggest a novel mechanism for localizing AMPA receptors to synaptic sites.     

Sphingolipids and gangliosides of the nervous system in membrane function and dysfunction

Simple sphingolipids such as ceramide and sphingomyelin (SM) as well as more complex glycosphingolipids play very important roles in cell function under physiological conditions and during disease development and progression. Sphingolipids are particularly abundant in the nervous system. Due to their amphiphilic nature they localize to cellular membranes and many of their roles in health and disease result from membrane reorganization and from lipid interaction with proteins within cellular membranes. In this review we discuss some of the functions of sphingolipids in processes that entail cellular membranes and their role in neurodegenerative diseases, with an emphasis on SM, ceramide and gangliosides.     

Protein tyrosine kinases and phosphatases in the nervous system.

Evidence in the past year has provided support for a prominent role of tyrosine phosphorylation in the regulation of neuronal function. The discovery that many novel forms of protein tyrosine kinases and phosphatases are expressed in the brain has revealed that the regulation of tyrosine phosphorylation is highly complex. The recent identification of substrate proteins in the brain for the protein tyrosine kinases and phosphatases has begun to clarify the functional role of tyrosine phosphorylation in the development and modulation of the nervous system.     

Isoprenylation and carboxylmethylation in small GTP-binding proteins of pheochromocytoma (PC-12) cells

1. A group of 21 to 24-kDa proteins of pheochromocytoma (PC-12) cells was found in blot overlay assays to bind specifically [alpha-32P]GTP. Binding was inhibited by GTP analogues but not by ATP. Such small GTP-binding proteins were found in the cytosolic and in the particulate fraction of the cells, but they were unevenly distributed: about 75% of the small GTP-binding proteins were localized within the particulate fraction of the cells. Separation of these proteins by two-dimensional gel electrophoresis revealed the existence of seven distinct [alpha-32P]GTP-binding proteins. 2. Targeting of the small GTP-binding proteins to the particulate fraction of PC-12 cells requires modification by isoprenoids, since depleting the cells of the isoprenoid precursor mevalonic acid (MVA) by the use of lovastatin resulted in a 50% decrease in membrane-bound small GTP-binding proteins, with a proportionate increase in the cytosolic form. This blocking effect of lovastatin was reversed by exogenously added MVA. 3. In addition, metabolic labeling of PC-12 cells with [3H]MVA revealed incorporation of [3H]MVA metabolites into the cluster of 21 to 24-kDa proteins in a form typical of isoprenoids; the label was not removed from the proteins by hydroxylamine, and labeling was enhanced in cells incubated with lovastatin. The latter effect reflects a decrease in the isotopic dilution of the exogenously added [3H]MVA, as the addition of exogenous MVA reversed the effect of lovastatin on [3H]MVA-metabolite incorporation into the 21 to 24-kDa proteins. 4. Additional experiments demonstrated that isoprenylation is required not only for membrane association of small GTP-binding proteins, but also for their further modification by a methylation enzyme. This was evident in experiments in which the cells were metabolically labeled with [methyl-3H]methionine, a methylation precursor. The group of 21 to 24-kDa proteins was labeled with a methyl-3H group in a form typical of C-terminal-cysteinyl carboxylmethyl esters. Their methylation was blocked by the methylation inhibitors methylthioadenosine (MTA), 3-deazadenosine and homocysteine thiolactone as well as by lovastatin. MVA reversed the lovastatin block of methylation. 5. Two-dimensional gel analysis of the [3H]methylated proteins detected seven methylated small GTP-binding proteins that correspond to the isoprenylated proteins. Levels of the small GTP-binding proteins as well as isoprenylation and methylation were reduced by cycloheximide. 6. Distribution of the methylated proteins between particulate and cytosolic fractions was found to be similar to that of the small GTP-binding proteins (i.e., a 4:1 ratio).(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of serotonin receptor function in the nervous system by lipid rafts and adaptor proteins

5-HT is a phylogenetically conserved monoaminergic neurotransmitter which is crucial for a number of physiological processes and is dysregulated in several disease states including depression, anxiety and schizophrenia. 5-HT neurons in the central nervous system are localized in the raphe nuclei and project to a wide range of target areas. 5-HT exerts its functions through 14 subtypes of 5-HT receptors. The tertiary structures of seven transmembrane 5-HT receptors contain several important features, including cholesterol consensus motifs, prominent intracellular loops and free C-termini. Alterations of cholesterol levels affect binding of ligands to 5-HT receptors and cholesterol-enriched microdomains in the cell membrane, termed lipid rafts, regulate 5-HT receptor internalization and signaling. The intracellular loops and the C-termini of 5-HT receptors provide binding sites for interacting adaptor proteins. Adaptor proteins affect internalization, desensitization as well as G-protein dependent and independent signaling via 5-HT receptors. We will here briefly review recent progress on the role of lipid rafts and adaptor proteins in the regulation of localization, trafficking, signaling and ligand bias of 5-HT receptors.     

Protein function classification via support vector machine approach

Support vector machine (SVM) is introduced as a method for the classification of proteins into functionally distinguished classes. Studies are conducted on a number of protein classes including RNA-binding proteins; protein homodimers, proteins responsible for drug absorption, proteins involved in drug distribution and excretion, and drug metabolizing enzymes. Testing accuracy for the classification of these protein classes is found to be in the range of 84-96%. This suggests the usefulness of SVM in the classification of protein functional classes and its potential application in protein function prediction.     

Mitotic Protein CSPP1 Interacts with CENP-H Protein to Coordinate Accurate Chromosome Oscillation in Mitosis

Mitotic chromosome segregation is orchestrated by the dynamic interaction of spindle microtubules with the kinetochores. During chromosome alignment, kinetochore-bound microtubules undergo dynamic cycles between growth and shrinkage, leading to an oscillatory movement of chromosomes along the spindle axis. Although kinetochore protein CENP-H serves as a molecular control of kinetochore-microtubule dynamics, the mechanistic link between CENP-H and kinetochore microtubules (kMT) has remained less characterized. Here, we show that CSPP1 is a kinetochore protein essential for accurate chromosome movements in mitosis. CSPP1 binds to CENP-H in vitro and in vivo. Suppression of CSPP1 perturbs proper mitotic progression and compromises the satisfaction of spindle assembly checkpoint. In addition, chromosome oscillation is greatly attenuated in CSPP1-depleted cells, similar to what was observed in the CENP-H-depleted cells. Importantly, CSPP1 depletion enhances velocity of kinetochore movement, and overexpression of CSPP1 decreases the speed, suggesting that CSPP1 promotes kMT stability during cell division. Specific perturbation of CENP-H/CSPP1 interaction using a membrane-permeable competing peptide resulted in a transient mitotic arrest and chromosome segregation defect. Based on these findings, we propose that CSPP1 cooperates with CENP-H on kinetochores to serve as a novel regulator of kMT dynamics for accurate chromosome segregation.     

Central nervous system function in systemic lupus erythematosus

Autoantibodies to three eukaryotic 60S ribosomal phosphoproteins P0, P1 and P2 have been found in the sera of 10–20% of patients with systemic lupus erythematosus (SLE). These antibodies inhibit protein synthesis in vitro and when microinjected into cultured human fibroblasts. The three proteins share a common epitope contained within the carboxyl terminal 22 amino acids of each protein. Because a significant number of SLE patients have central nervous system disturbances with major behavioral disorders, the antiribosomal protein autoantibodies were measured in this subset of SLE individuals to determine whether or not there was an association. These antibodies are present in 90% of SLE patients who were diagnosed as having psychosis, secondary to the disease.Special issue dedicated to Dr. Sidney underfriend     

蛋白磷酸酯酶2A调节微管相关蛋白1b的磷酸化

微管相关蛋白MAP1b的生物学活性受其磷酸化修饰的调节,后者则受相应的蛋白激酶和蛋白磷酸酯酶(PP)调控.为研究蛋白磷酸酯酶在脑内对MAP1b磷酸化的调控作用,采用有代谢活性的大鼠脑片作为模型,分别应用冈田酸(okadaic acid)和cyclosporin A选择性地抑制PP2A 和PP2B活性,来研究其对脑内蛋白磷酸酯酶MAP1b磷酸化的调控.采用特异性的MAP1bⅠ型磷酸化依赖性抗体522和免疫印迹技术检测MAP1bⅠ型磷酸化.结果表明,当PP2A被okadaic acid选择性抑制后,MAP1bⅠ型磷酸化明显增加.而PP2B被选择性地抑制后,MAP1b磷酸化的变化不大.免疫组化染色显示,MAP1b广泛分布于鼠大脑神经元和突起中,与对照组相比,在PP2A抑制的脑片中抗体522的免疫活性在神经元中明显升高.上述结果表明,PP2A是脑中调控MAP1bⅠ型磷酸化的主要蛋白磷酸酯酶.