Studies on the Biosynthesis of Intermediate Filament Proteins in the Rat CNS

Abstract: The biosynthesis of brain intermediate filament proteins [neurofilament proteins and glial fibrillary acidic protein (GFA)] was studied with cell-free systems containing either rat spinal cord polysomes (free polysomes or rough microsomes) and rabbit reticulocyte factors or wheat germ homogenate containing spinal cord messenger RNA. The products of translation were isoated by immunoaffinity chromatography and then analyzed by two-dimensional gel electrophoresis (2DGE) followed by fluorography. The free polysome population was found to synthesize two neurofilament proteins (MW 145K, p15.4, and MW 70K, pl 5.3) and three isomers of GFA (α, β, and γ) that differ in isoelectric point. Wheat germ homogenate containing messenger RNA extracted from free cord polysomes synthesized two proteins that comigrated with neurofilament protein standards at 145K 5.4 and 70K 5.3; these proteins were partially purified by neurofilament affinity chromatography. The wheat germ system also synthesized the α, β, and γ isomers of GFA as characterized by immunoaffinity chromatographic purification and comigration with standards in 2DGE analysis. Our data are consistent with the conclusion that synthesis of neurofilament proteins requires multiple messenger RNAs. Also, synthesis of intermediate filament proteins occurs in the free polysome population; detectable amounts of these proteins were not synthcsized by the rough microsomes.     

Characterization of Postmortem Human Brain Proteins by Two-Dimensional Gel Electrophoresis

Abstract: The proteins of membrane and cytosol fractions from frozen human postmortem brain were analyzed by two-dimensional gel electrophoresis (isoelectric range: 5.1–6.0) and both Coomassie-blue and ammoniacal silver staining. Cytosol preparations were analyzed from six different postmortem brains from patients with various neurologic diagnoses and immediate causes of death. Intervals between death and brain freezing (−70^oC) ranged from 2 to 20 h. The vast majority of proteins detected in these cytosol fractions had identical molecular weights and isoelectric points in each of six human brains examined. However, in some tissue samples tubulin was either quantitatively decreased or undetectable. The possibility that this partial or complete depletion of tubulin was related to postmortem interval and/or brain freezing was studied using rat forebrain tissue. Rat brain incubated at room temperature for up to 24 h did not reproduce the changes seen in the region of human cytosol tubulin. However, other changes seen in the two-dimensional electrophoretic pattern of rat cytosol proteins did relate to postmortem interval, brain freezing, or both. Rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum were prepared from three human brains, with highly reproducible two-dimensional patterns. Protein analysis of these membrane fractions revealed that human RER contained significant amounts of tubulin, in contrast to rat RER which contained no detectable tubulin. This discrepancy was elucidated by allowing rat brains to remain at room temperature for 24 h before freezing; gels of rat RER prepared from this tissue showed that tubulin subunits were present.     

The Characterization of Tubulin in CNS Membrane Fractions

Abstract— Rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SER), and a plasma membrane (PM) fraction enriched in synaptic membranes were isolated from rat forebrain. The proteins in these membrane fractions were analyzed by two-dimensional gel electrophoresis (2DGE) in the isoelectric range of 5.1 to 6.0 by a modification of the O'Farrell procedure. Proteins were detected by Coomassie Brilliant Blue staining of the electrophoretograms. The results of these analyses were compared with 2DGE analysis of cytosol proteins, with particular attention given to tubulin subunits and actin. The RER contained one major protein (53K 5.4) in the β-tubulin region with a molecular weight of 53,000 and an isoelectric point of 5.4. The SER contained at least two major proteins in the β-tubulin region; one with a migration identical to 53K 5.4 and other proteins with slightly higher apparent molecular weights and more acidic isoelectric points (54K, 5.4 to 5.3), identical to cytoplasmic β-tubulin. The PM fraction also contained multiple overlapping proteins (54K, 5.4 to 5.3) in the β-tubulin area and a trace amount of the 53K 5.4 protein. The proteins in the β-tubulin region were removed from the 2DGE electrophoretogram and digested by Staphylococcus aureus V8 protease, and the peptides separated on one-dimensional polyacrylamide gels. The peptide patterns of 53K 5.4 protein from RER and SER were almost identical and differed significantly from the cytoplasmic β-tubulin pattern; however, the peptide maps of the PM and SER β-tubulin region were identical to the cytoplasmic β-tubulin. The 2DGE analysis of RER did not contain proteins in the region of cytoplasmic α-tubulin. SER and PM contained proteins in the α-tubulin region with a similar, but not identical, peptide analysis to cytoplasmic α-tubulin. Significant amounts of actin were detected in 2DGE analysis of SER and PM, and the peptide analysis of the actin was identical to the cytoplasmic actin analysis. The RER fraction contained only trace amounts of actin. The cytosol and all membrane fractions contained a protein (68K 5.6) found among microtubule-associated proteins, as judged by molecular weight and isoelectric point. Several proteins present in all membrane fractions (61K 5.1 and 58K 5.1) bound to concanavalin A agarose.     

Characterization and Biosynthesis of Cyclic-AMP-Binding Proteins in the Rat Central Nervous System

Cyclic-AMP-binding proteins in membrane and soluble fractions from rat forebrain were compared; membrane fractions included smooth and rough microsomes and a plasma membrane fraction enriched in synaptic membranes. Protein fractions were treated with 8-azido-[32P]cyclic AMP and ultraviolet irradiation to covalently tag cyclic-AMP-binding proteins. Labeled proteins were then analyzed by two-dimensional gel electrophoresis (2DGE) and fluorography. The soluble CNS proteins contained two major cyclic-AMP-binding species at 48K (48K 5.5 and 48K 5.45), differing slightly in their isoelectric points. Another protein was seen at 54K (54K 5.3) adjacent to the beta-tubulin subunits in the 2D electrophoretogram. The analysis of the smooth microsome and plasma membrane fractions differed from the soluble fraction in that there were two cyclic-AMP-binding proteins adjacent to the beta-tubulin region (54K 5.3 and 52K 5.3) differing slightly in apparent molecular weight. The membrane fractions also contained a cyclic-AMP-binding protein at 54K 5.8. The 52K 5.3 and 54K 5.8 species were unique to the membrane fractions. The rough microsomes did not contain detectable amounts of cyclic-AMP-binding proteins. Free polysomes were isolated from brain tissue, and translation products were analyzed by cyclic AMP affinity chromatography and immunopurification with antibodies to the brain specific type II regulatory subunit. The translation products that were found to bind cyclic AMP Sepharose are as follows: 48K 5.5, 48K 5.45, 52K 5.3, and 54K 5.8. These species comigrated with proteins that were photoaffinity-labeled in cytosol and membrane fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterizaton and Biosynthesis of the Plasma Membrane Proteolipid Protein in Neural Tissue

In this study we have characterized, in brain, the expression of a plasma membrane proteolipid protein (PM-PLP) complex that can form cation-selective channels in lipid bilayers. We isolated PLP fractions from synaptic plasma membrane and glial microsomes and found a high degree of similarity in both size and amino acid composition to the complex we had previously isolated from kidney. Antibodies specific to the kidney PM-PLP were prepared, and, on the basis of immunoblot and immunoprecipitation studies, the PM-PLP complex isolated from neural membranes was shown to be immunologically related to the kidney PM-PLP. These proteolipid proteins exhibited a molecular weight of approximately 14K and contained a high percentage of hydrophobic amino acids with an apparent absence of cysteine. The biogenesis of PM-PLP in brain was studied by in vitro translation of free and bound polysomes and total RNA in a rabbit reticulocyte lysate followed by immunoprecipitation of the translation products. From these studies it is concluded that the PM-PLP complex is synthesized on the rough endoplasmic reticulum. On the basis of the identical electrophoretic mobility of material isolated from plasma membranes and material immunoprecipitated after translation of bound polysomes and isolated RNA, it appears that the PM-PLP does not undergo detectable posttranslational processing between its site of synthesis and its incorporation into the plasma membrane.     

膜上tRNA结合蛋白的分离与初步鉴定

用ＴｒｉｔｏｎＸ－１１４分相法分离啤酒酵母的膜总蛋白,经过酵母ｔＲＮＡ分子交联的Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析,用０－０．８ｍｏｌ／Ｌ（ＮＨ４０２ＳＯ４梯度缓冲液洗脱ｔＲＮＡ结合的蛋白质。凝胶阻滞电泳实验室鉴定出两种主要的与ｔＲＮＡ分子特异性结合的蛋白质。     

Interaction of the Two Structural Domains of Calmodulin with Mature and Immature Rat Brain Microtubules

The inhibitory effect of calmodulin on the assembly of mature and immature rat brain microtubules was compared with that of the two major structural domains of this protein, the COOH-terminal fragment (amino acids 78-148) and the NH2-terminal fragment (amino acids 1-77), to determine the calmodulin structural domain responsible for the inhibitory effect on microtubule assembly. Microtubules prepared during the early stages of brain development, i.e., during intensive neurite outgrowth, are more sensitive to inhibition by the Ca2(+)-calmodulin complex than those obtained from adult brain. Significant inhibition of immature microtubule assembly was observed with both fragments in the absence of Ca2+, but the effects were more important when Ca2+ was present. With adult brain microtubules, the two fragments remained without effect on assembly in the absence of Ca2+, whereas some inhibition was seen in its presence but only with the COOH-terminal polypeptide. Under all these conditions, the COOH-terminal fragment was always more active than the NH2-terminal fragment on microtubule polymerization, albeit to a lesser extent than native calmodulin.     

Cellular Origin and Biosynthesis of Rat Optic Nerve Proteins: A Two-Dimensional Gel Analysis

High resolution 2DGE (two-dimensional gel electrophoresis) was used to characterize neuronal and glial proteins of the rat optic nerve, to examine the phases of intraaxonal transport with which the neuronal proteins are associated, and to identify the ribosomal populations on which these proteins are synthesized. Neuronal proteins synthesized in the retinal ganglion cells were identified by injecting the eye with L-[35S]methionine, followed by 2DGE analysis of fast and slow axonally transported proteins in particulate and soluble fractions. Proteins synthesized by the glial cells were labeled by incubating isolated optic nerves in the presence of L-[35S]methionine and then analyzed by 2DGE. A number of differences were seen between filamentous proteins of neurons and glia. Most strikingly, proteins in the alpha- and beta-tubulin region of the 2D gels of glial proteins were distinctly different than was observed for axonal proteins. As expected, neurons but not glia expressed neurofilament proteins, which appeared among the slow axonally transported proteins in the particulate fraction; significant amounts of the glial filamentous protein, GFA, were also labeled under these conditions, which may have been due to transfer of amino acids from the axon to the glial compartment. The fast axonally transported proteins contained relatively large amounts of high-molecular-weight acidic proteins, two of which were shown to comigrate (on 2DGE) with proteins synthesized by rat CNS rough microsomes; this finding suggests that rough endoplasmic reticulum may be a major site of synthesis for fast transported proteins. In contrast, the free polysome population was shown to synthesize the principal components of slow axonal transport, including tubulin subunits, actin, and neurofilament proteins.     

Characterization of Cyclic AMP-Dependent Phosphorylation of Neuronal Membrane Proteins

We examined the patterns of cyclic AMP-dependent protein phosphorylation in membranes prepared from rat cortical synaptosomes following gel electrophoresis and autoradiography. We determined the optimum pH (6.2), time (20 s), Mg2+ concentration (10 mM) and cyclic AMP concentration (5 microM) for the reaction. We also found that the detergents Triton X-100 and gramicidin S enhanced cyclic AMP-dependent protein phosphorylation. Inhibitors of the Na+, K+ ATPase (ouabain, NaF, vanadate) enhanced protein phosphorylation. This effect occurred in the presence but not in the absence of detergent. The addition of purified bovine brain cyclic AMP-dependent protein kinase catalytic subunit enhanced membrane protein phosphorylation. The addition of homogeneous neural (bovine brain) and non-neural (bovine skeletal muscle) cyclic AMP-dependent protein kinase type II regulatory subunit partially inhibited protein phosphorylation. Both neural and non-neural regulatory subunits behaved similarly. In addition to cyclic AMP-dependent phosphorylation, the alpha-subunit of pyruvate dehydrogenase (Mr = 41,000) is phosphorylated in a cyclic AMP-independent fashion. We also examined the phosphorylation pattern of membranes prepared from rat heart and found that the number of acceptor substrates was much less than that from the nervous system.     

Changes in the cannabinoid receptor binding, G protein coupling, and cyclic AMP cascade in the CNS of rats tolerant to and dependent on the synthetic cannabinoid compound CP55,940

Chronic exposure to CP55,940 produced a significant down-regulation of cannabinoid receptors in the striatum, cortex, hippocampus, and cerebellum of rat brain. At 24 h after SR141716-precipitated withdrawal, we observed a tendency to return to basal levels in the striatum and cortex, whereas the specific binding remained lower in the hippocampus and cerebellum. When we surveyed cannabinoid receptor-activated G proteins, in chronic CP55,940-treated rats the guanosine 5'-O:-(3-[(35)S]thiotriphosphate) ([(35)S]GTPgammaS) binding assay revealed a decrease of activated G proteins in the striatum, cortex, and hippocampus, whereas no significant changes were seen in the cerebellum. At 24 h after the SR141716-precipitated withdrawal, [(35)S]GTPgammaS binding increased compared with that of rats chronically exposed to CP55,940, attaining the control level except for cerebellum, where we observed a trend to overcome the control amounts. Concerning the cyclic AMP (cAMP) cascade, which represents the major intracellular signaling pathway activated by cannabinoid receptors, in the cerebral areas from rats chronically exposed to CP55,940 we found alteration in neither cAMP levels nor protein kinase A activity. In the brain regions taken from CP55, 940-withdrawn rats, we only observed a significant up-regulation in the cerebellum. Our findings suggest that receptor desensitization and down-regulation are strictly involved in the development of cannabinoid tolerance, whereas alterations in the cAMP cascade in the cerebellum could be relevant in the mediation of the motor component of cannabinoid abstinence.     

Quantitative Proteomics Links the LRRC59 Interactome to mRNA Translation on the ER Membrane

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Highlights

• •Identification of subcellular protein interactomes via proximity labeling and quantitative multiplexed proteomics.
• •SEC61β and RPN1 interactomes overlap with translocon-associated protein networks.
• •SEC62 interacts with redox-linked proteins and ER luminal chaperones.
• •LRRC59 directly interacts with mRNA translation factors and SRP machinery on the ER.

     

Making the Most of Fusion Tags Technology in Structural Characterization of Membrane Proteins

Membrane proteins can be investigated at various structural levels, including the topological structure, the high-resolution three-dimensional structure, and the organization and assembly of membrane protein complexes. Gene fusion technology makes it possible to insert a polynucleotide encoding a protein or polypeptide tag into the gene encoding a membrane protein of interest. Resultant recombinant proteins may possess the functions of the original membrane proteins, together with the biochemical properties of the imported fusion tag, greatly enhancing functional and structural studies of membrane proteins. In this article, the latest literature is reviewed in relation to types, applications, strategies, and approaches to fusion tag technology for structural investigations of membrane proteins.     

Evidence for a distinct ligand binding site on tubulin discovered through inhibition by GDP of paclitaxel-induced tubulin assembly in the absence of exogenous GTP

GDP inhibits paclitaxel-induced tubulin assembly without GTP when the tubulin bears GDP in the exchangeable site (E-site). Initially, we thought inhibition was mediated through the E-site, since small amounts of GTP or Mg²⁺, which favors GTP binding to the E-site, reduced inhibition by GDP. We thought trace GTP released from the nonexchangeable site (N-site) by tubulin denaturation was required for polymer nucleation, but microtubule length was unaffected by GDP. Further, enhancing polymer nucleation reduced inhibition by GDP. Other mechanisms involving the E-site were eliminated experimentally. Upon finding that ATP weakly inhibited paclitaxel-induced assembly, we concluded that another ligand binding site was responsible for these inhibitory effects, and we found that GDP was not binding at the taxoid, colchicine, or vinca sites. There may therefore be a lower affinity site on tubulin to which GDP can bind distinct from the E- and N-sites, possibly on α-tubulin, based on molecular modeling studies.     

Dephosphorylation of Microtubule Proteins by Brain Protein Phosphatases 1 and 2A, and Its Effect on Microtubule Assembly

Protein phosphatase C was purified 140-fold from bovine brain with 8% yield using histone H1 phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase (cyclic AMP-kinase). Brain protein phosphatase C was considered to consist of 10 and 90%, respectively, of the catalytic subunits of protein phosphatases 1 and 2A on the basis of the effects of ATP and inhibitor-2. Protein phosphatase C dephosphorylated microtubule-associated protein 2 (MAP2), tau factor, and tubulin phosphorylated by a multifunctional Ca2+/calmodulin-dependent protein kinase (calmodulin-kinase) and the catalytic subunit of cyclic AMP-kinase. The properties of dephosphorylation of MAP2, tau factor, and tubulin were compared. The Km values were in the ranges of 1.6-2.7 microM for MAP2 and tau factor. The Km value for tubulin decreased from 25 to 10-12.5 microM in the presence of 1.0 mM Mn2+. No difference in kinetic properties of dephosphorylation was observed between the substrates phosphorylated by the two kinases. Protein phosphatase C did not dephosphorylate the native tubulin, but universally dephosphorylated tubulin phosphorylated by the two kinases. The holoenzyme of protein phosphatase 2A from porcine brain could also dephosphorylate MAP2, tau factor, and tubulin phosphorylated by the two kinases. The phosphorylation of MAP2 and tau factor by calmodulin-kinase separately induced the inhibition of microtubule assembly, and the dephosphorylation by protein phosphatase C removed its inhibitory effect. These data suggest that brain protein phosphatases 1 and 2A are involved in the switch-off mechanism of both Ca2+/calmodulin-dependent and cyclic AMP-dependent regulation of microtubule formation.     

Polyamine Regulation of the Microtubule-Associated Protein Kinase

Microtubule protein prepared by cycles of assembly-disassembly contains a cyclic AMP-dependent protein kinase that phosphorylates the high-molecular-weight microtubule-associated protein MAP-2. The polyamine spermine at 2mM affected the phosphorylation of MAP-2 in a manner that depended on the cyclic AMP concentration. At cyclic AMP concentrations below 10(-6) M, spermine increased the rate of phosphorylation, while at cyclic AMP concentrations above 10(-6) M, spermine decreased the rate of phosphorylation. Spermine also decreased the final extent of cyclic AMP-dependent phosphorylation but did not affect the protein substrate specificity of the microtubule-associated protein kinase. MAP-2 was the principal substrate both in the presence and in the absence of spermine. Because of these results, we propose that microtubule protein phosphorylation may be regulated in vivo by spermine as well as by cyclic AMP levels.     

Expression of Microtubule-Associated Proteins During the Early Stages of Neurite Extension by Brain Neurons Cultured in a Defined Medium

Immunoblotting analysis was used to identify the microtubule-associated proteins (MAPs) present in cultures of mouse brain neurons. Polyclonal antibodies were raised against the two main adult brain MAPs, i.e., MAP2 (300 kDa) and tau (60-70 kDa). Whatever the stage of the culture, which was performed in a defined medium (3 or 6 days), the anti-MAP2 serum detected several high-molecular-weight components (including MAP2) and an entity with 62-65 kDa. Anti-tau revealed essentially a major peak of 48 kDa (young tau) but also slightly cross-reacted with the 62-65 kDa entity. During the culture period (0-6 days) the cells developed progressively a dense neuritic network; the concentration of the different MAPs increased in parallel but at different rates depending on the different species. The increase in concentration of the high-molecular-weight components occurred before that of 48-kDa tau. This suggests that high-molecular-weight MAPs and 48-kDa tau might be involved respectively in the initiation and elongation of neurites. In contrast, and since the main developmental changes in tau composition seen in vivo did not occur during the time course of the culture, this transition might be related to later events of neuronal differentiation.     

Direct Demonstration of the Activation of UDP-N-Acetylgalactosamine: [GM3]N-Acetylgalactosaminyltransferase by Cyclic AMP

Treatment of NG108-15 cells in culture with the opiate peptide [D-Ala2,D-Leu5]enkephalin produces maximal inhibition of cyclic AMP synthesis in less than 15 min. The activity of [GM3]:N-acetylgalactosaminyltransferase is similarly inhibited, but maximal inhibition is not observed for at least 30 min following the addition of [D-Ala2,D-Leu5]enkephalin. Conversely, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine rapidly potentiates the intracellular accumulation of cyclic AMP and, in a more gradual fashion, increases [GM3]:N-acetylgalactosaminyltransferase activity. The reductions in the activity of [GM3]:N-acetylgalactosaminyltransferase that occur following treatment of NG108-15 cells with indomethacin argues for a direct role of cyclic AMP in the observed changed in [GM3]:N-acetylgalactosaminyltransferase activity. By adding low concentrations of cyclic AMP (but not cyclic GMP) to microsomes derived from neonatal rat brain, we were able to demonstrate a dose-dependent phosphorylation of membrane protein and subsequent doubling of [GM3]:N-acetylgalactosaminyltransferase activity.     

B群脑膜炎奈瑟氏球菌铁调蛋白的诱导表达及其性质研究

对２株Ｂ群脑膜炎奈瑟氏球菌的铁调蛋白的表达及其性质作了研究。在流脑半综合培养基中加人铁螯合剂,诱导了铁调蛋白的表达,并比较了三种铁螯合剂的效果。结果表明,ＥＤＤＨＡ是最令人满意的螯合剂。ＥＬＩＳＡ实验证实含有铁调蛋白的外膜蛋白可以增强对小白鼠的免疫原性。Ｗｅｓｔｅｒｎ－ｂｌｏｔ实验证明有两种铁调蛋白是有效的抗原成分,分子量分别在７５ｋＤａ和８５ｋＤａ附近。由分子量来推算,分别应为ＦｅＲＰ－７０及ＴＢＰ２。     

Protein and Lipid Composition of Radial Component-Enriched CNS Myelin

Abstract: The radial component is a junctional complex that is believed to stabilize the apposition of myelin membranes in the internode of CNS myelin. Based on our previous finding that the radial component of compact myelin retains its structure in tissue treated with the detergent Triton X-100, we have attempted to isolate the junctional complex from spinal cord myelin treated with this detergent. Using 0.5% Triton X-100, our procedures yielded a fraction of isolated myelin that was enriched in well-preserved radial component. This fraction that contained morphologically well-defined radial component was examined by sodium dodecyl sulfate-polyacrylamide gel electropho-resis and immunoblotting, and TLC, and was found to be significantly and consistently enriched in the 21.5-kDa and 17-kDa isoforms of myelin basic protein, and in cerebro-sides, hydroxy sulfatide, and sphingomyelin. In addition, the myelin-associated enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase, tubulin, and actin tended to be resistant to Triton extraction. The fraction of isolated myelin that contained radial component was deficient in proteolipid protein and DM-20, the 18.5-and 14-kDa isoforms of myelin basic proteins, and in the major phospholipids, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine. Our data indicate that the radial component can be isolated and that certain myelin and cytoskeletal proteins and lipids are closely associated with it.     

Stimulus-Specific Hierarchy of Enhancer Elements Within the Rat Prodynorphin Promoter

Abstract: Induction of the prodynorphin gene occurs in a tissue-specific manner following different physiological stimuli. Using electrophoretic mobility shift assays, we studied the relative activity of the five major regulatory sites in the rat prodynorphin promoter. Prodynorphin cyclic AMP-responsive element 2 (DynCRE2), DynCRE3, and the noncanonical prodynorphin AP-1 (ncDynAP-1) regulatory sites control, in a coordinated manner, prodynorphin induction in the spinal cord after noxious stimulation, whereas prodynorphin up-regulation in supraoptic neurons is regulated predominantly by the ncDynAP-1. Conversely, prodynorphin transactivation in the ovaries upon gonadotrophin stimulation is controlled by DynCRE1 and DynCRE3. Our results support the idea that stimulus-specific changes in nuclear proteins establish a functional hierarchy among regulatory sites in the prodynorphin promoter and provide further insight in the molecular mechanisms that govern prodynorphin gene regulation.