Synthesis of S100 Protein on Free and Membrane-Bound Polysomes of the Rabbit Brain

Abstract: Free and membrane-bound polysomes were isolated from the cerebral hemispheres and cerebellum of the young adult rabbit. The two polysomal populations were translated in an mRNA-dependent cell-free system derived from rabbit reticulocytes. Analysis of the [³⁵S]methionine-labeled translation products on two-dimensional polyacrylamide gels indicated an efficient separation of the two classes of brain polysomes. The relative synthesis of S100 protein by free and membrane- bound polysomes was determined by direct immuno-precipitation of the cell-free translation products in the presence of detergents to reduce nonspecific trapping. Synthesis of S100 protein was found to be twofold greater on membrane-bound polysomes compared with free polysomes isolated from either the cerebral hemispheres or the cerebellum. In addition, the proportion of poly- (A+)mRNA coding for SlOO protein was also twofold greater in membrane-bound polysomes compared with free polysomes isolated from the cerebral hemispheres. These results indicate that the cytoplasmic S100 protein is synthesized predominantly on membrane-bound polysomes in the rabbit brain. We suggest that the nascent S100 polypeptide chain translation complex is attached to the rough endoplasmic reticulum by an ionic interaction involving a sequence of 13 basic amino acids in S100 protein.     

Fate of mRNA following disaggregation of brain polysomes after administration of (+)-lysergic acid diethylamide in vivo

Intravenous injection of (+)-lysergic acid diethylamide into young rabbits induced a transient brain-specific disaggregation of polysomes to monosomes. Investigation of the fate of mRNA revealed that brain poly(A+)mRNA was conserved. In particular, mRNA coding for brain-specific S100 protein was not degraded, nor was it released into free ribonucleoprotein particles. Following the (+)-lysergic acid diethylamide-induced disaggregation of polysomes, mRNA shifted from polysomes and accumulated on monosomes. Formation of a blocked monosome complex, which contained intact mRNA and 40-S plus 60-S ribosomal subunits but lacked nascent peptide chains, suggested that (+)-lysergic acid diethylamide inhibited brain protein synthesis at a specific stage of late initiation or early elongation.     

Identification of rat brain polysomes synthesizing the brain specific enolase (14.3.2 protein), S100 protein and alpha and beta tubulin subunits

Polysomes prepared from frozen rat brain powder were fractionated by centrifugation in a sucrose gradient. Individual fractions were used to program a reticulocyte lysate in a run-off reaction. The products of cell-free synthesis were assayed for the brain-specific enolase (14.3.2 protein) and S100 protein by immunoprecipitation with specific antisera and for tubulin by two-dimensional electrophoresis in polyacrylamide slab gels. The relative synthesis of these proteins by unfractionated free brain polysomes were 0.1 per cent, 0.05 per cent and 0.7 per cent respectively. After centrifugation in a sucrose gradient polysomes synthesizing S100 protein were separated from those synthesizing the other two markers. There was a threefold enrichment in the specific messenger RNA activity for each of the three proteins studied in their respective peak fractions of polysomes.     

In Vitro Synthesis of Polypeptides of Moderately Large Size by Poly(A)-Containing Messenger RNA from Postmortem Human Brain and Mouse Brain

Studies were undertaken to optimize the conditions for isolation and in vitro translation of poly(A)-containing mRNA from human postmortem brain. The comparison of several methods for preparation of mRNA from frozen mouse brain indicated that although the yield of mRNA was increased using polysomes prepared in the presence of ribonucleoside vanadyl complexes and subsequently extracted with guanidinium thiocyanate, the translation products were indistinguishable from those synthesized by total cellular RNA directly extracted from tissue with guanidinium thiocyanate. The oligo d(T)-cellulose-purified poly(A)-containing mRNA preparations were translated in vitro in a rabbit reticulocyte lysate in the presence of L-[35S]methionine. Messenger RNA from frozen mouse brain stimulated protein synthesis from 9- to 20-fold over endogenous mRNA. Over 450 polypeptides were reproducibly synthesized and separated by two-dimensional polyacrylamide gel electrophoresis (PAGE); size classes up to 130,000 daltons were present. Direct extraction of RNA from frozen human cerebral cortex and cerebellum with guanidinium thiocyanate followed by oligo d(T)-cellulose chromatography yielded 1.8 micrograms/g and 2.0 micrograms/g, respectively, of poly(A)-containing mRNA; this represents a two- to fourfold increase over our earlier results. In the rabbit reticulocyte translation system human brain mRNA stimulated protein synthesis nearly threefold over endogenous mRNA. Compared with earlier studies, the number of newly synthesized polypeptides was increased by 30%. Over 300 species were separated by two-dimensional PAGE, and size classes up to 130,000 daltons were present, as compared to 70,000 in an earlier report. The polypeptides synthesized by human cerebral cortex and cerebellum were indistinguishable. However, several appeared to be uniquely human when compared with the products synthesized by mouse brain mRNA. The method described for the preparation of postmortem human brain mRNA eliminates the need to prepare polysomes, which are recovered in variable and low yield from the postmortem human brain. The procedure appears applicable to studies on the synthesis of moderately large human brain polypeptides and for investigations of brain protein polymorphism when relatively large numbers of products are required for analysis.     

Differential brain expression of the Alzheimer''s amyloid precursor protein.

The expression of the Alzheimer amyloid protein precursor (AAPP) was examined in human, monkey, dog and rat brains. Two proteins, one identified as AAPP695 and the other as AAPP751, were immunoprecipitated from the in vitro translation of human, dog and rat brain polysomes. The AAPP751 to AAPP695 ratio was highest in human, intermediate in dog and lowest in rat brain polysomes. Human cerebral cortex contained higher levels of the AAPP751 mRNA than either dog or rat cortex. AAPP695 was detected in both cerebral cortex and cerebellum of all species examined. In contrast, AAPP751 was detected predominantly in the cortex of human, monkey and to a lesser extent dog brains while it was not detected in rat brain. These findings indicate that the amyloid precursors are differentially expressed in different mammalian brains and suggest that AAPP751 is mainly expressed in the brain regions involved in plaque formation.     

CHARACTERIZATION OF BRAIN RIBONUCLEOPROTEIN PARTICLES

Abstract— Brain RNP particles were characterized to determine whether they play a role in the regulation of brain protein synthesis. RNP particles were isolated from the postribosomal supernatant of cerebral hemispheres of young rabbits, employing conditions which minimize adventitious protein-RNA interactions. Brain RNP particles consist of a different set of proteins compared to proteins associated with either 40 and 60s ribosomal subunits or polysomal mRNA. Poly(A+)mRNA from brain RNP particles stimulates the incorporation of [³⁵S]methionine in a wheat embryo cell-free system and codes for a different set of proteins compared to poly(A+)mRNA isolated from polysomes (with some overlap; i.e. mRNA coding for brain-specific S100 protein is present in both RNP particles and polysomes).
Addition of total brain RNP particles to a cell-free wheat embryo system inhibits the endogenous incorporation of [³⁵S]methionine. Total RNP particles were fractionated by sucrose density gradient centrifugation into a'light'and a'heavy'fraction. The light RNP fraction inhibited while the heavy RNP fraction stimulated protein synthesis in the wheat embryo cell-free system. Analysis of the protein composition of fractionated RNP particles revealed that the light and heavy RNP particles contained different sets of proteins. Together these results suggested that one class of brain RNP particles may contain a translational inhibitor and may be involved in the regulation of protein synthesis in the brain.     

Developmental changes in S100B content in brain tissue,cerebrospinal fluid,and astrocyte cultures of rats

1. We investigated the content of S100B protein by ELISA in three brain regions (hippocampus, cerebral cortex, and cerebellum) and in cerebrospinal fluid of rats during postnatal development as well as the content and secretion of S100B in pre- and postconfluent primary astrocyte cultures.2. An accumulation of S100B occurred in all brain regions with similar ontogenetic pattern between second and fourth postnatal weeks. However, we observed a decrease in the cerebrospinal fluid S100B after the critical period for synaptogenesis in rodents.3. A similar profile of cell accumulation and decrease in basal secretion was also observed during aging of astrocyte cultures.4. These data contribute to the proposal that S100B is an important glial-derived protein during brain development and that changes in extracellular levels of S100B may be related to glial proliferation and synaptogenesis.     

ON THE MECHANISM OF ELECTROSHOCK-INDUCED INHIBITION OF PROTEIN SYNTHESIS IN RABBIT CEREBRAL CORTEX

Abstract— The mechanism of electroshock (ES)-induced inhibition of protein synthesis in rabbit cerebral cortex has been investigated by using a cell-free system. The protein biosynthetic activity of the post-mitochondrial supernatant (PMS) obtained from the cerebral cortex of ES-treated animals was found to be markedly lower than in controls (C). This inhibition was accompanied by a decrease of polysomes and an increase of monomers. In addition, a relative increase in light polysomes was evident at short intervals after ES treatment. No difference was found in the total soluble activity and in the activity of the elongation factors and ribonuclease present in the cell sap of C and ES animals. The biosynthetic activity of ES-total. free and membrane-bound ribosomes was approx 45% lower than that of the corresponding C fractions: polysome/monomer ratios were similarly reduced. The total content of cortical ribosomes was not affected by ES. Following ES treatment there was no change in the ribo-somal ability to elongate, terminate and release polypeptide chains, nor a decrease in the polysomal content of poly(A)-containing mRNA. These data strongly suggest that the ES-induced inhibition of protein synthesis results from a defect in the initiation process. The possible mechanisms mediating this defect have been discussed.     

Isolation of PGP 9.5, a New Human Neurone-Specific Protein Detected by High-Resolution Two-Dimensional Electrophoresis

Protein gene product (PGP) 9.5 is a new brain-specific protein originally detected by high-resolution two-dimensional electrophoresis of the soluble proteins of human brain and other organs. We have purified this protein from human brain and raised a rabbit antihuman PGP 9.5 antiserum. The protein has a monomer molecular weight of approximately 27,000 and is present in brain at concentrations at least 50 times greater than in other organs. Immunoperoxidase labelling has localised PGP 9.5 to neurones in the human cerebral cortex with no evidence of staining of glial elements. PGP 9.5 is estimated to be present in brain at concentrations of 200-500 micrograms/g wet weight and represents a major protein component of neuronal cytoplasm. This new neurone-specific cytoplasmic marker may prove useful in studies of neuronal development and in the detection of neuronal damage in disease of the nervous system.     

Comparison of cell-free protein synthesis by different regions of chicken brain

The cell-free protein synthesis by the postmitochondrial supernatant from chicken cerebrum was twofold greater than protein synthesis by the cerebellum or optic lobes. Ribosomal aggregation of mRNA and ribonuclease activity of the postmitochondrial supernatant from the three brain regions was not statistically different. The higher protein synthetic activity of the cerebral postmitochondrial supernatant was associated with both the postribosomal supernatant (cell sap) and microsomal fractions. Cerebral monomeric ribosomes were more active in polyuridylic acid directed polyphenylalanine synthesis than monomeric ribosomes from either the cerebellum or optic lobes. The ability of cerebral cell sap to support polyuridylic acid directed polyphenylalanine synthesis was 1.6 to 2 times greater than cell sap from the other two regions. Cell sap factors other than tRNA^phe or phenylalanyl-tRNA synthetases appear to be responsible for the higher protein synthetic activity of the cbr cell sap.     

The termination of the synthesis of proteins in vitro with extracts from the undeveloped cyst of Artemia salina

Soluble fractions (S-100) from both undeveloped cysts and developing embryos of Artemia salina promoted elongation of polypeptides initiated in vivo on polysomes of developing embryos or nauplius larvae. The ability of the extract from the undeveloped cyst to terminate correctly the synthesis of polypeptides has been determined indirectly from the distribution of polysomes before and after in vitro translation and, more directly, from the nature of the protein product released from rabbit reticulocyte polysomes. The extract from the undeveloped cyst and also, as expected, that from the developing embryo catalyzed a reduction in the amount of the polysomes of larger size and an increase in the amount of 80 S ribosomes. The soluble extract from the undeveloped cyst can terminate the synthesis of rabbit globin on reticulocyte polysomes. The major polypeptide product released from the polysomes had an electrophoretic mobility identical with that of the subunit of isolated rabbit globin. This indicated that the cyst contained the components necessary to complete and terminate the synthesis of polypeptides correctly and that the released protein product was not predominantly as a result of premature chain termination. The size distribution of Artemia salina proteins released from polysomes from developing embryos was similar when the synthesis was directed by the S-100 at each stage of development.     

Evidence for the presence of an inhibitor on ribosomes in mouse L cells infected with mengovirus.

After infection of mouse L cells with mengovirus, there is a rapid inhibition of protein synthesis, a concurrent disaggregation of polysomes, and an accumulation of 80S ribosomes. These 80S ribosomes could not be chased back into polysomes under an elongation block. The infected-cell 80S-ribosome fraction contained twice as much initiator methionyl-tRNA and mRNA as the analogous fraction from uninfected cells. Since the proportion of 80S ribosomes that were resistant to pronase digestion also increased after infection, these data suggest that the accumulated 80S ribosomes may be in the form of initiation complexes. The specific protein synthetic activity of polysomal ribosomes also decreased with time of infection. However, the transit times in mock-infected and infected cells remained the same. Cell-free translation systems from infected cells reflected the decreased protein synthetic activity of intact cells. The addition of reticulocyte initiation factors to such systems failed to relieve the inhibition. Fractionation of the infected-cell lysate revealed that the ribosomes were the predominant target affected. Washing the infected-cell ribosomes with 0.5 M KCI restored their translational activity. In turn, the salt wash from infected-cell ribosomes inhibited translation in lysates from mock-infected cells. The inhibitor in the ribosomal salt wash was temperature sensitive and micrococcal nuclease resistant. A model is proposed wherein virus infection activates (or induces the synthesis of) an inhibitor that binds to ribosomes and stops translation after the formation of the 80S-ribosome initiation complex but before elongation. The presence of such an inhibitor on ribosomes could prevent them from being remobilized into polysomes in the presence of an inhibitor of polypeptide elongation.     

The regulation of protein synthesis by translation control RNA

The mechanism by which translational control RNA (tcRNA) inhibits protein synthesis was investigated. In the presence of heme the inhibitory role of muscle tcRNA on hemoglobin synthesis was confirmed. Upon the addition of muscle tcRNA to a rabbit reticulocyte cell-free system the binding of [³²P]-globin mRNA to 40S ribosomal subunits and its subsequent incorporation into polysomes was inhibited. Furthermore, muscle tcRNA inhibits met-tRNA binding to polysomes and yet stimulates the formation of methionine-puromycin. These results suggest that muscle tcRNA blocks the binding of globin mRNA to ribosomes resulting in an abortive initiation complex that is, however, still capable of the methionine-puromycin reaction.     

Translational control and the cytoskeleton in Physarum polycephalum

Translationally active plasmodia of the syncytial slime mold Physarum polycephalum develop into translationally dormant sclerotia during starvation. Although functional mRNA and ribosomes exist in sclerotia, protein synthesis is suppressed at the level of initiation. To test the possibility that alterations in the cytoskeleton may limit protein synthesis, we have examined the distribution of polysomes and actin mRNA in the cytoskeletal (CSK) and soluble (SOL) fractions of Triton X-100-extracted plasmodia and sclerotia. Most of the polysomes and actin mRNA were located in the CSK of plasmodia, while most of the ribosomes and actin mRNA were located in the SOL of sclerotia. The results suggest that ribosomes and mRNA shift from the CSK to the SOL as protein synthesis is suppressed during starvation. Plasmodia and sclerotia can be induced to accumulate excess polysomes by treatment with low levels of the elongation inhibitor cycloheximide. Treatment of plasmodia with cycloheximide caused excess polysomes to accumulate in the SOL, suggesting that the CSK contains a limited capacity for binding translational components and that the association of polysomes with the cytoskeleton is not required for protein synthesis. Treatment of sclerotia with cycloheximide, however, caused polysomes and actin mRNA to accumulate in the CSK, suggesting that the sclerotial cytoskeleton, although depleted in ribosomes and mRNA, is capable of binding translational components. It is concluded that alterations in the sclerotial cytoskeleton are not involved in translational control.     

The distribution of functional bacteriophage T4 mRNA on polysomes.

Functional bacteriophage T4 deoxynucleotide kinase and α-glucosyl transferase mRNAs can be isolated from polysomes extracted from cells 8 min after infection. At least 55% of the 8-min deoxynucleotide kinase mRNA is associated with polysomes and is released from the cell membrane by deoxyribonuclease (DNase) treatment (soluble mRNA). Approximately 20% of the kinase mRNA remains tightly bound to membrane after DNase treatment (membrane mRNA) and 25% of the kinase mRNA is routinely lost during fractionation. The membrane-bound kinase mRNA is about three times as stable in vitro as the soluble kinase mRNA. Soluble kinase mRNA (14.5S) is found associated with as few as one ribosome and as many as 22 ribosomes; however, 14.5S α-glucosyl transferase mRNA is found predominantly in six ribosome polysomes. The size of the α-glucosyl transferase mRNA is heterogenous, ranging between 14.5 and 20S. The larger α-glucosyl transferase mRNAs are never found on small polysomes but appear only in polysomes containing at least nine ribosomes (18S α-glucosyl transferase mRNA). Maximum-size α-glucosyl transferase mRNA (approximately 20S) appears on polysomes containing at least 14 ribosomes. The relationships between decay of T4 mRNA and polysome size and the location of ribosome loading sites on the 20S α-glucosyl transferase message are also discussed.     

Postnatal Development of Cholecystokinin-Like Immunoreactivity and Its mRNA Level in Rat Brain Regions

Developmental changes of preprocholecystokinin mRNA (CCK mRNA) and cholecystokinin-like immunoreactivity (CCK-LI) were examined in rat brain regions (frontal cortex, colliculi, hippocampus, striatum, and cerebellum) using RNA dot blot assays with cholecystokinin (CCK) cDNA and radioimmunoassay, respectively. The CCK-LI levels in all regions examined were very low at birth. Excluding the cerebellum, the levels in these regions increased postnatally and reached adult values at 28 days of age. In contrast to CCK-LI, CCK mRNA levels changed dramatically during development. A considerable amount of CCK mRNA was detected in the frontal cortex and hippocampus at birth. The changes in the level of CCK mRNA in the frontal cortex and colliculi paralleled those of CCK-LI, including a rapid increase from 7 to 14 days of age. The synthesis of CCK mRNA preceded the appearance of CCK-LI. CCK mRNA levels in the hippocampus and striatum exhibited a transient increase, with a peak at 14 days of age. In the adult brain, the CCK mRNA levels were high in the frontal cortex, moderate in the hippocampus and colliculi, and low in the striatum. The cerebellum contained only a negligible amount of CCK mRNA during development. The relatively high level of CCK-LI compared with the low level of CCK mRNA in the striatum supports the idea that most of the striatal CCK-LI is supplied from extrastriatal regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of protein synthesis in mouse myeloma cells by Ricinus communis toxin.

The mechanism of inhibition of protein synthesis in mouse myeloma cells by Ricinus communis toxin was studied. No significant disaggregation of polysomes into monosomes was detected in the toxin-treated cells. The activity of the polysomes isolated from the cells treated with the toxin in protein synthesis was remarkably lower than that of the untreated cells, while the activity of the supernatant enzyme fraction was retained. The ribosomes derived from the polysomes of the toxin-treated cells were inactive in poly(U)-dependent polyphenylalanine synthesis. The activity of ribosomes reconstituted by hybridizing subunits derived from the ribosomes of normal and toxin-treated cells were measured in poly(U)-dependent polyphenylalanine synthesis, and the 60 S subunit was revealed to be inactive. These results indicate that the target of action of the toxin towards intact cells is the 60 S ribosomal subunit.     

Translatability of mRNA from brain of infant rabbits exposed chronically to aluminium

1. Polysomes were isolated from the brain of infant rabbits at 22 days of age. The animals received s.c. injections 3 times weekly of aluminium (Al) maltolate (3 mg Al/kg body wt) or Al lactate (16 mg Al/kg body wt) from 5 days of age. 2. The polysomes were used to direct the incorporation of [14C]leucine into peptides in a brain protein synthesizing system and exhibited a decreased activity when obtained from aluminum exposed infants. 3. The mRNA obtained from the polysomes was used to direct the incorporation of [35S]methionine into peptides in an mRNA dependent rabbit reticulocyte lysate. The translatability of the mRNA derived from aluminum exposed infant brains was significantly lower than that of preparations from control infant rabbits. 4. Al bound to maltolate, a ligand soluble in lipids as well as water, was considerably more detrimental to brain protein synthesis than Al bound to lactate.     

The isolation, from electromotor neurone perikarya, of messenger RNAs coding for synaptic proteins

Poly(A)-containing mRNA was isolated from the electric lobe, cerebellum and forebrain of Torpedo marmorata and from cholinergic electromotor perikarya isolated from the electric lobe. All the mRNA preparations were translated by a cell-free protein-synthesizing system from rabbit reticulocytes; no brain-specific factors were required. The highest stimulation rate was found with the perikaryal mRNA suggesting that this purely neuronal mRNA is a preferred template in the protein-synthesis system; the molecular basis of this phenomenon remains to be elucidated. The translation products of the perikaryal mRNA were analysed by two-dimensional gel electrophoresis and compared with the proteins of synaptosomes derived from the electromotor nerve terminals. The majority of the synaptosomal proteins comigrated with synthesized products. More than 100 synthesized proteins were detected as individual spots in the gel pattern, among them actin, subunits of neurofilamentous proteins and a protein considered to be a specific component of electromotor synaptic vesicles. Identities were confirmed in some cases by immunochemical methods. The results suggest that protein synthesis in the perikaryon of the electromotor neurone is largely directed to the production of proteins needed to maintain synaptic integrity. A comparison of the translation products of mRNA derived from the highly cholinergic electric lobe and a brain region, the cerebellum, which is non-cholinergic, revealed, as expected, some common translation products and others which appeared to be specific for the brain regions concerned. This approach may lead to the identification of protein specific for neurones of different transmitter types.