Identification and partial characterization of a glycoprotein species with high affinity for methylmercury in peripheral nervous tissues of man and experimental animals

A small amount of a glycoprotein species (21-kDa glycoprotein) with high affinity for methylmercury (MeHg) was detected in the post-nuclear or post-mitochondrial supernatant fraction of the homogenate of rat sciatic nerve on electrophoresis and autoradiography after binding of Me203Hg to the fraction. The 21-kDa glycoprotein was also found in the subcellular fractions of mouse, hamster, guinea pig, rabbit and human peripheral nervous tissues. Experiments with the cellular fractions of the tissues revealed that the 21-kDa glycoprotein is localized mainly in the myelin fraction, whereas it was not found in the cellular fractions of brain, spinal cord and nonneural tissues, such as kidney and liver. The specific binding activity of the 21-kDa glycoprotein with MeHg was 12-15 fold that of the major myelin protein, Po. It was shown that the interaction of the 21-kDa glycoprotein with MeHg was mediated through sulfhydryl groups in experiments with iodoacetamide and dithiothreitol. The amino acid compositions of the rat and human 21-kDa glycoproteins were similar but very different from that of a typical metallothionein. The N-terminal amino acid sequences of the two components of the rat 21-kDa glycoprotein were identical to those of P0 and PMP-22, respectively. The in vitro binding of MeHg was also observed in the myelin fraction obtained from the sciatic nerves of MeHg-dosed rats.     

Oligodendrocyte-specific protein (OSP) is a major component of CNS myelin

An oligodendrocyte-specific protein (OSP) cDNA was recently identified and found to be expressed primarily in oligodendrocytes and has a deduced amino acid sequence similar to that of peripheral myelin protein 22 (PMP-22). We raised antibodies against a synthetic peptide corresponding to OSP amino acid residues 179-194 which reacted with a 22 kd protein in mouse CNS. OSP immunoreactivity localized to spinal cord white matter tracts using immunohistochemistry in a similar distribution to that of MBP. OSP localized to CNS myelin biochemically with more than a 30-fold enrichment measured in purified myelin. We further purified the proteolipid fraction of myelin and determined that OSP contributes approximately 7% of total myelin protein making it the third most abundant protein in CNS myelin. No binding was found to several agglutinins or a HNK1-specific antibody suggesting that OSP is not a glycoprotein.     

Zinc binding to the HIV-1 nucleocapsid protein: a thermodynamic investigation by fluorescence spectroscopy

The HIV-1 nucleocapsid protein, NCp7, is characterized by two CCHC zinc finger motifs which have been shown to stoichiometrically bind zinc in mature virions. Moreover, this binding of zinc proves to be critical in various NCp7 functions, especially in the encapsidation process. To further understand the central role of zinc binding to NCp7, we closely investigated the zinc binding properties of NCp7 and various deleted or substituted derivatives. To this end, the fluorescence of wither the naturally occurring Trp37 or the conservatively substituted Trp16 was used to monitor the binding of zinc to the N- and C-terminal finger motifs, respectively. At pH 7.5, the NCp7 proximal motif was found to bind zinc strongly with 2.8 x 10(14) M-1 binding constant about five times higher than the NCp7 distal motif. Moreover, the binding of zinc to one finger motif decreased the affinity of the second one, and this negative cooperativity was shown to be related to the spatial proximity of the zinc-saturated finger motifs. The binding seemed to be almost equally driven by entropy and enthalpy, and the binding information was essentially encoded by the finger motifs themselves whereas the other parts of the protein only played a marginal stabilization role. As expected, the Cys and His residues of the CCHC motifs were critical and competition between protons and zinc ions to these residues induced a steep pH-dependence of the zinc binding constants to both sites. Taken together, our data provide further evidence for the nonequivalence of the two NCp7 finger motifs.     

Lipid-protein interactions with native and modified myelin basic protein

The basic protein of central nervous system myelin has been shown to form complexes with acidic lipids in vitro. We measured the interaction of myelin basic protein with several charged and neutral lipids in a biphasic chloroform/methanol/water system and investigated the effect of decreasing the electrical charge of the basic amino groups of the myelin basic protein by acetylation. The modified myelin basic protein, which has an average of eight acetyl residues incorporated, was characterised by gel electrophoresis and circular dichroism. Complexes formed between the acetylated myelin basic protein and acidic lipids exhibited a reduction in the amount of lipids bound, a value that could be correlated with the number of modified amino groups. The significance of these experiments with reference to protein-lipid interaction in the myelin membrane is discussed.     

Solution structure and backbone dynamics of Mason-Pfizer monkey virus (MPMV) nucleocapsid protein

Retroviral nucleocapsid proteins (NCPs) are CCHC-type zinc finger proteins that mediate virion RNA binding activities associated with retrovirus assembly and genomic RNA encapsidation. Mason-Pfizer monkey virus (MPMV), a type D retrovirus, encodes a 96-amino acid nucleocapsid protein, which contains two Cys-X2-Cys-X4-His-X4-Cys (CCHC) zinc fingers connected by an unusually long 15-amino acid linker. Homonuclear, two-dimensional sensitivity-enhanced 15N-1H, three-dimensional 15N-1H, and triple resonance NMR spectroscopy have been used to determine the solution structure and residue-specific backbone dynamics of the structured core domain of MPMV NCP containing residues 21-80. Structure calculations and spectral density mapping of N-H bond vector mobility reveal that MPMV NCP 21-80 is best described as two independently folded, rotationally uncorrelated globular domains connected by a seven-residue flexible linker consisting of residues 42-48. The N-terminal CCHC zinc finger domain (residues 24-37) appears to adopt a fold like that described previously for HIV-1 NCP; however, residues within this domain and the immediately adjacent linker region (residues 38-41) are characterized by extensive conformational averaging on the micros-ms time scale at 25 degrees C. In contrast to other NCPs, residues 49-77, which includes the C-terminal CCHC zinc-finger (residues 53-66), comprise a well-folded globular domain with the Val49-Pro-Gly-Leu52 sequence and C-terminal tail residues 67-77 characterized by amide proton exchange properties and 15N R1, R2, and (1H-15N) NOE values indistinguishable to residues in the core C-terminal finger. Twelve refined structural models of MPMV NCP residues 49-80 (pairwise backbone RMSD of 0.77 A) reveal that the side chains of the conserved Pro50 and Trp62 are in van der Waals contact with one another. Residues 70-73 in the C-terminal tail adopt a reverse turn-like structure. Ile77 is involved in extensive van der Waals contact with the core finger domain, while the side chains of Ser68 and Asn75 appear to form hydrogen bonds that stabilize the overall fold of this domain. These residues outside of the core finger structure are conserved in D-type and related retroviral NCPs, e.g., MMTV NCP, suggesting that the structure of MPMV NCP may be representative of this subclass of retroviral NCPs.     

Fission yeast pak1+ encodes a protein kinase that interacts with Cdc42p and is involved in the control of cell polarity and mating

A STE20/p65pak homolog was isolated from fission yeast by PCR. The pak1+ gene encodes a 72 kDa protein containing a putative p21-binding domain near its amino-terminus and a serine/threonine kinase domain near its carboxyl-terminus. The Pak1 protein autophosphorylates on serine residues and preferentially binds to activated Cdc42p both in vitro and in vivo. This binding is mediated through the p21 binding domain on Pak1p and the effector domain on Cdc42p. Overexpression of an inactive mutant form of pak1 gives rise to cells with markedly abnormal shape with mislocalized actin staining. Pak1 overexpression does not, however, suppress lethality associated with cdc42-null cells or the morphologic defeat caused by overexpression of mutant cdc42 alleles. Gene disruption of pak1+ establishes that, like cdc42+, pak1+ function is required for cell viability. In budding yeast, pak1+ expression restores mating function to STE20-null cells and, in fission yeast, overexpression of an inactive form of Pak inhibits mating. These results indicate that the Pak1 protein is likely to be an effector for Cdc42p or a related GTPase, and suggest that Pak1p is involved in the maintenance of cell polarity and in mating.     

Two cytosolic protein families implicated in lipid-binding: main structural and functional features

1. According to the important biological role of fatty acids and phospholipids in cell membranes, two cytosolic proteins implicated in their binding and transport in brain were considered, namely: Fatty Acid-Binding Protein and basic 21 kDa protein. 2. They were reviewed as well as their related protein families. 3. Although the two protein groups do not present significant sequence homologies, they share several similar properties and might thus be implicated in common physiological functions.     

Purification and characterization of bovine heart glycogen synthase kinase-3

Glycogen Synthase Kinase-3 (GSK-3) was isolated from bovine heart tissue extracts by a procedure involving ammonium sulfate fractionation, followed by chromatography on phosphocellulose, Cibacron blue 3GA-agarose, DEAE-Sephacel, CM-Sepharose, heparin-agarose, myelin basic protein-Sepharose, and LiChrospher 1000 C00-. GSK-3 was identified by its activation of protein phosphatase-1i (PP-1i). The purified enzyme had a specific activity of 25,500 units of protein phosphatase-1i activated/mg protein. The enzyme is an asymmetric monomeric protein of 53 kDa. The molecular size and retention of activity after autophosphorylation indicated that the isolated enzyme was the GSK-3 alpha-isoform.     

The 24-kDa subunit of the bovine mitochondrial NADH:ubiquinone oxidoreductase is a G protein

Based on the results obtained from GTP overlay assay, immunoprecipitation, two dimensional electrophoresis and radiolabeled GTP binding, we provide evidence that the bona fide subunit of Complex I, the long known 24 kDa protein is a G protein. Bacterially expressed 24 kDa protein with additional N-terminal methionine and alanine residues or naturally expressed truncated isoform fail to bind GTP suggesting that secondary modification/ processed N-terminal end is necessary for GTP binding. Competitive inhibition of binding of radiolabeled GTP to electroblotted 24 kDa protein with unlabelled nucleotides showed that the protein binds GTP and GDP with high affinity in presence of Mg2+, and has decreased to very low affinity for ITP, CTP, GMP and UTP. A comparative binding of [gamma-35S]-GTP to Complex I and 24 kDa protein (electroblotted) suggests that the GTP binding in the native Complex is solely due to 24 kDa protein. Further, four fold difference in the binding affinities between native Complex I and 24 kDa protein (electroblotted) as seen by Scatchard analysis of the binding data indicates that protein undergoes structural rearrangement in Complex I bound form, that presumably triggers divalent cation dependent GTPase activity in native complex. We were unable to detect the effect of GTP/ GDP on the ubiquinone/ferricyanide reductase activity. Since the subunit is found missing in tissues affected by mitochondrial respiratory chain diseases, we presume that the subunit has regulatory role in the Complex I function in the electron transport chain.     

Structural requirements for PAK activation by Rac GTPases

The Rho family GTPases, Rac1 and Rac2, regulate a variety of cellular functions including cytoskeletal reorganization, the generation of reactive oxygen species, G1 cell cycle progression and, in concert with Ras, oncogenic transformation. Among the many putative protein targets identified for Rac (and/or Cdc42), the Ser/Thr kinase p21-activated kinase (PAK) is a prime candidate for mediating some of Rac's cellular effects. This report shows that Rac1 binds to and stimulates the kinase activity of PAK1 approximately 2- and 4-5-fold, respectively, better than Rac2. Mutational analysis was employed to determine the structural elements on Rac and PAK that are important for optimal binding and activation. The most notable difference between the highly homologous Rac isomers is the composition of their C-terminal polybasic domains. Mutation of these six basic residues in Rac1 to neutral amino acids dramatically decreased the ability of Rac1 to bind PAK1 and almost completely abolished its ability to stimulate PAK activity. Moreover, replacing the highly charged polybasic domain of Rac1 with the less charged domain of Rac2 (and vice versa) completely reversed the PAK binding/activation properties of the two Rac isomers. Thus, polybasic domain differences account for the disparate abilities of Rac1 and Rac2 to activate PAK. PAK proteins also contain a basic region, consisting of three contiguous lysine residues (Lys66-Lys67-Lys68), which lies outside of the previously identified Cdc42/Rac-binding domain. Mutation of these Lys residues to neutral residues decreased PAK binding to activated Rac1 and Rac2 (but not Cdc42) and greatly reduced PAK1 activation by Rac1, Rac2, and Cdc42 proteins in vivo. In contrast, mutation of lysines 66-68 to basic Arg residues did not decrease (and in some cases enhanced) the ability of Rac1, Rac2, and Cdc42 to bind and activate PAK1. Our studies suggest that the polybasic domain of Rac is a novel effector domain that may allow the two Rac isomers to activate different effector proteins. In addition, our results indicate that a basic region in PAK is required for PAK activation and that binding of Rac/Cdc42 to PAK is not sufficient for kinase activation.     

Seric immune complexes in multiple sclerosis do not contain MBP epitopes

Immune complexes from sera of MS patients, other neurological diseases, and healthy donors were precipitated using polyethyleneglycol and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Silver staining evidenced additional protein bands whose molecular weights were 14-16, 38, and 43 kDa. These IC proteins were present in most MS patients studied. To identify their nature, immunoblotting was performed with antihuman immunoglobulins A, M, G antibodies. No immunoreactivity was found below a molecular weight of 66 kDa on a nitrocellulose sheet having the transferred protein pattern of MS IC. Using purified human myelin, MS IC transferred to an immobilon sheet and antihuman myelin basic protein antibodies, an immunoreactivity was seen only on purified human MBP. The small proteins of 14-16 kDa and the others of 38, 43 kDa were not immunoreactive. Identification of the nature of these additional proteins in MS IC is in progress.     

Biosynthesis of chondroitin sulfate. Purification of glucuronosyl transferase II and use of photoaffinity labeling for characterization of the enzyme as an 80-kDa protein

A photoaffinity analogue, [beta-32P]5-azido-UDP-GlcA, was used to photolabel the enzymes that utilize UDP-GlcA in cartilage microsomes and rat liver microsomes. SDS-polyacrylamide gel electrophoresis analysis of photolabeled cartilage microsomes, which are specialized in chondroitin sulfate synthesis, showed a major radiolabeled band at 80 kDa and other minor radiolabeled bands near 40 and 60 kDa. Rat liver microsomes, which are enriched for enzymes of detoxification by glucuronidation, had a different pattern with multiple major labeled bands near 50-60 and 35 kDa. To determine that the photolabeled 80-kDa protein is the GlcA transferase II, we have purified the enzyme from cartilage microsomes. This membrane-bound enzyme, involved in the transfer of GlcA residues to non-reducing terminal GalNAc residues of the chondroitin polymer, has now been solubilized, stabilized, and then purified greater than 1350-fold by sequential chromatography on Q-Sepharose, heparin-Sepharose, and WGA-agarose. The purified enzyme exhibited a conspicuous silver-stained protein band on SDS-polyacrylamide gel electrophoresis that coincided with the major radiolabeled band of 80 kDa. SDS-polyacrylamide gel analysis of photoaffinity-labeled active fractions from the Q-Sepharose, heparin-Sepharose, and WGA-agarose also indicated only the single radiolabeled band at 80 kDa. Intensity of photolabeling in each of the fractions examined coincided with enzyme activity. The photolabeling of this 80-kDa protein was saturable with the photoprobe and could be inhibited by the addition of UDP-GlcA prior to the addition of the photoprobe. Thus, the photolabeling with [beta-32P]5-azido-UDP-GlcA has identified the GlcA transferase II as an 80-kDa protein. The purified enzyme was capable of transferring good amounts of GlcA residues to chondroitin-derived pentasaccharide with negligible transfer to pentasaccharides derived from hyaluronan or heparan.     

A 21-kDa chloroplast heat shock protein assembles into high molecular weight complexes in vivo and in Organelle

The conservation of the carboxyl-terminal "heat shock" domain among small (sm) cytoplasmic and chloroplast heat shock proteins (HSPs) suggests that these smHSPs perform similar functions. Previous studies have established that cytoplasmic smHSPs are found in higher order structures in vivo (approximately 500 kDa). To determine if the chloroplast smHSP is found in similar complexes, we examined the size of the 21-kDa chloroplast smHSP from Pisum sativum, PsHSP21, under non-denaturing conditions. Following sedimentation of chloroplast stromal extracts on sucrose gradients PsHSP21 is detected in fractions corresponding to 10-11 S. Upon non-denaturing gel electrophoresis, PsHSP21 was detected in two high molecular mass complexes of approximately 230 and 200 kDa, in good agreement with the sucrose gradient data. These PsHSP21-containing particles were stable under different salt and Mg2+ conditions, and their integrity was not affected by 1.0% Triton X-100 or 10 mM ATP. To study assembly of the high molecular weight complexes containing PsHSP21, in vitro translated PsHSP21 was imported into chloroplasts and its size was examined. Following import into chloroplasts isolated from heat-stressed plants, greater than 50% of PsHSP21 was recovered in the higher molecular weight forms. In contrast, following import into chloroplasts isolated from control plants the protein was recovered exclusively in a 5 S (approximately 42-kDa) form. These data suggest that preexisting PsHSP21 or other heat-induced factors may be required for assembly of the higher molecular weight particles. We propose that the 10-11 S particles are the functional form of PsHSP21.     

Activation of mitogen-activated protein kinases in oligodendrocytes

The proliferation and differentiation of oligodendrocyte progenitors are stringently controlled by an interacting network of growth and differentiation factors. Not much is known, however, about the intracellular signaling pathways activated in oligodendrocytes. In this study, we have examined the activation of mitogen-activated protein (MAP) kinase [also called extracellular signal-regulated protein kinases (ERKs)] in primary cultures of developing oligodendrocytes and in a primary oligodendrocyte cell line, CG4, in response to platelet-derived growth factor (PDGF) and basic fibroblast growth factor. MAP kinase activation was determined by an ingel protein kinase renaturation assay using myelin basic protein (MBP) as the substrate. The specificity of MAP kinase activation was further confirmed by an immune complex kinase assay using anti-MAP kinase antibodies. Stimulation of oligodendrocyte progenitors with the growth factors PDGF and basic fibroblast growth factor and a protein kinase C-activating tumor promoter, phorbol 12-myristate 13-acetate, resulted in a rapid activation of p42mapk (ERK2) and, to a lesser extent, p44mapk (ERK1). Immunoblot analysis with anti-phosphotyrosine antibodies revealed an increased Tyr phosphorylation of a 42-kDa phosphoprotein band cross-reacting with anti-MAP kinase antibodies. The phosphorylation of p42mapk in PDGF-treated oligodendrocyte progenitors was preceded by a robust autophosphorylation of the growth factor receptor. Immunoblot analysis with anti-pan-ERK antibodies indicated the presence of ERK-immunoreactive species other than p42mapk and p44mapk in oligodendrocytes. The presence of some of the same pan-ERK-immunoreactive species and certain renaturable MBP kinase activities was also demonstrable in myelin preparations from rat brain, suggesting that MAP kinases (and other MBP kinases) may function not only during oligodendrogenesis but also in myelinogenesis.