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PubMed1996

Effect of subunit composition on GABAA receptor complex characteristics in a baculovirus expression system.

A baculovirus expression system was used to produce functional human recombinant GABAA receptors in Sf-9 insect cells in order to study the biochemistry, pharmacology and functional characteristics of this receptor complex. We have identified and characterized various factors which influence the level of receptor expression in multiple virus infections. We have shown that the level of expression of the GABAA receptor complex varies with the levels of expression of the individual subunits. We have also shown that the assembly process has a defined timecourse, and it is dependent upon the ratio of the number of infectious virus particles (MOI ratio) of each subunit in multi-virus infections. In multiple infections, the capacity for expression of the infected cell is shared proportionally by entering virus particles and, there is a direct correlation between the amounts of subunit mRNA and levels of subunit protein expression, and the amount of ligand binding to expressed protein. Finally, reinfection of previously infected cells does not result in subsequent protein expression. Knowledge of these various factors allows us to construct recombinant GABAA receptor complexes with reproducibility and flexibility with regard to subunit composition. By co-expression of alpha, beta, and gamma subunits, both the recognition site for GABA and the allosteric (benzodiazepine) modulatory site are formed and appear to reproduce the pharmacology of endogenously expressed receptors as measured in mammalian CNS. Only a single receptor is produced irrespective of the expression levels of the subunits, showing that GABAA receptor assembly is highly regulated.

Animals↗
PubMed1999

Thick filament assembly occurs after the formation of a cytoskeletal scaffold.

The development of myofibrils involves the formation of contractile filaments and their assembly into the strikingly regular structure of the sarcomere. We analysed this assembly process in cultured human skeletal muscle cells and in rat neonatal cardiomyocytes by immunofluorescence microscopy using antibodies directed against cytoskeletal and contractile proteins. In particular, the question in which temporal order the respective proteins are integrated into developing sarcomeres was addressed. Although sarcomeric myosin heavy chain is expressed as one of the first myofibrillar proteins, its characteristic A band arrangement is reached at a very late stage. In contrast, titin, then myomesin and finally C-protein (MyBP-C) gradually form a regularly arranged scaffold on stress fiber-like structures (SFLS), on non-striated myofibrils (NSMF) and on nascent striated myofibrils (naSMF). Immediately subsequent to the completion of sarcomere cytoskeleton formation, the labeling pattern of myosin changes from the continuous staining of SFLS to the periodic staining characteristic for mature myofibrils. This series of events can be seen most clearly in the skeletal muscle cell cultures and--probably due to a faster developmental progression less well in cardiomyocytes. We therefore conclude that the correct assembly of a cytoskeletal scaffold is a prerequisite for correct thick filament assembly and for the integration of the contractile apparatus into the myofibril.

Actin Cytoskeleton↗
PubMed1998

The role of charged amino acids in the alpha1-beta4 loop of the iron-sulfur protein of the cytochrome bc1 complex of yeast mitochondria.

Previous experiments using deletion mutants of the iron-sulfur protein had indicated that amino acid residues 138-153 might be involved in the assembly of this protein into the cytochrome bc1 complex. To determine which specific residues might be involved in the assembly process, charged amino acids located in the alpha1-beta4 loop of the iron-sulfur protein were mutated to uncharged residues and tryptophan 152 to phenylalanine. The mutant genes were used to transform yeast cells (JPJ1) lacking the iron-sulfur protein gene. Mutants R146I and W152F had almost undetectable growth in medium containing glycerol/ethanol, whereas mutants D143A, K148I, and D149A grew more slowly than the wild type. Activity of the cytochrome bc1 complex was decreased 50, 90, 67, 89, and 90% in mutants D143A, R146I, K148I, D149A, and W152F, respectively, but unchanged in mutants D139A, Q141I, D145L, and V147S. In all of these mutants except W152F, the cytochrome c1 content, determined by immunoblotting, was comparable with that of wild-type cells. However, immunoblotting revealed that the content of the iron-sulfur protein was decreased proportionately in the five mutants with lowered enzymatic activity and growth suggesting that these amino acids are critical for maintaining the stability of the iron-sulfur protein. The efficiency of assembly in vitro compared with the wild type determined by selective immunoprecipitation was unchanged in the mutants with the exception of R146I, D149A, and W152F where decreases of 80, 60, and 60%, respectively, were observed suggesting that these amino acids are critical for the proper assembly of the iron-sulfur protein into the bc1 complex.

Biological Transport↗
PubMed1993

The dTAFII80 subunit of Drosophila TFIID contains beta-transducin repeats.

A key component of the RNA polymerase II transcriptional apparatus, TFIID, is a multi-protein complex containing the TATA box-binding protein (TBP) and at least seven tightly associated factors (TAFs). Although the functions of most TFIID subunits are unknown, it is clear that TAFs are not necessary for basal activity but that one or more are required for regulated transcription, and so behave as coactivators. The presence of multiple subunits indicates that there is an intricate assembly process and that TAFs may be responsible for other activities. We have described the properties of the subunit dTAFII110, which can interact directly with the transcriptional activator Sp1 (ref. 5). In addition, the largest subunit, dTAFII250, binds directly to TBP and links other TAFs to the complex. Here we describe the cloning, expression and partial characterization of the Drosophila TAF of M(r) 80,000, dTAFII80. Sequence analysis reveals that dTAFII80 contains several copies of the WD40 (beta-transducin) repeat. Moreover, dTAFII80 shares extended sequence similarity with an Arabidopsis gene, COP1, which encodes a putative transcription factor that is though to regulate development. We have expressed recombinant dTAFII80 and begun to characterize its interaction with other members of the TFIID complex. Purified recombinant dTAFII80 is unable to bind TBP directly or to interact strongly with the C-terminal domain of dTAFII250 (delta N250). Instead, dTAFII80 is only able to recognize and interact with a higher-order complex containing TBP, delta N250, 110 and 60. These findings suggest the formation of TFIID may require an ordered assembly of the TAFs, some of which bind directly to TBP and others that are tethered to the complex as a result of specific TAF/TAF interactions.

Amino Acid Sequence↗
PubMed1983

Involvement of the N-terminal polypeptide of vimentin in the formation of intermediate filaments.

The potential to form intermediate filaments of a 54 X 10(3) molecular weight (Mr) polypeptide derived from vimentin by cleavage by the intermediate filament-specific, Ca2+-activated proteinase was investigated. Under physiological conditions of assembly, the breakdown product did not form intermediate filaments. Electron microscopy revealed short, rod-like structures similar to those described by Geisler et al. for a 38 X 10(3) Mr alpha-helical core particle derived from desmin. Since the specific, Ca2+-activated proteinase degrades vimentin preferentially from its N terminus, this result suggests the involvement of the basic, N-terminal polypeptide of vimentin in the assembly of intermediate filaments. This was supported by the observation that arginine inhibits the formation of intermediate filaments from intact vimentin. Whereas lysine had very little effect on the assembly process, guanidinium hydrochloride was effective at the same concentration as arginine. On the basis of these findings, an affinity chromatography method for the identification and isolation of intermediate filament subunit proteins was developed. Beside vimentin, desmin, the 68 X 10(3) Mr neurofilament triplet protein, the glial fibrillary acidic protein and cytokeratins also bound to arginine methylester Sepharose 4B in a salt-stable manner and could be eluted with arginine. The 145 X 10(3) Mr neurofilament triplet protein exhibited reduced binding activity, whereas the 210 X 10(3) Mr subunit did not bind to the affinity matrix. Among the degradation products of vimentin produced by the specific, Ca2+-activated proteinase, only those with molecular weights higher than 40 X 10(3) bound to arginine methylester Sepharose 4B. The same applied to the high molecular weight degradation products of desmin with a protein-resistant 37 X 10(3) Mr polypeptide as the major component. The results suggest that arginine residues of the non-alpha-helical, N-terminal polypeptides of intermediate filament subunit proteins play an important role in filament assembly.

Animals↗
PubMed1993

The yeast PRP19 protein is not tightly associated with small nuclear RNAs, but appears to associate with the spliceosome after binding of U2 to the pre-mRNA and prior to formation of the functional spliceosome.

We have previously shown that the yeast PRP19 protein is associated with the spliceosome during the splicing reaction by immunoprecipitation studies with anti-PRP19 antibody. We have extended such studies by using extracts depleted of specific splicing factors to investigate the step of the spliceosome assembly process that PRP19 is involved in. PRP19 was not associated with the splicing complexes formed in U2- or U6-depleted extracts but was associated with the splicing complex formed in heat-inactivated prp2 extracts. This finding indicates that PRP19 becomes associated with the splicing complexes after or concomitant with binding of the U6 small nuclear ribonucleoprotein particle (snRNP) to the precursor RNA and before formation of the functional spliceosome. We further analyzed whether PRP19 is an integral component of snRNPs. We have constructed a strain in which an epitope of nine amino acid residues recognized by a well-characterized monoclonal antibody, 12CA5, is linked to the carboxyl terminus of the wild-type PRP19 protein. Immunoprecipitation of the splicing extracts with anti-PRP19 antibody or precipitation of the extracts prepared from the epitope-tagged strain with the 12CA5 antibody did not precipitate significant amounts of snRNAs. Addition of micrococcal nuclease-treated extracts to the PRP19-depleted extract restored its splicing activity. These results indicate that PRP19 is not tightly associated with any of the snRNAs required for the splicing reaction. No non-snRNP protein factor has been demonstrated to participate in either step of the spliceosome assembly pathway that PRP19 might be involved in. Thus, PRP19 represents a novel splicing factor.

Antibodies, Monoclonal↗
PubMed1984

[Synthesis of 10S particles in cells infected with aphthovirus].

In the present study, evidence is presented for the existence of a morphogenetic intermediary that may be a precursor of the procapsids in the assembling process. BHK21 clone 13S cells were infected with Aphthovirus A24 (Cruzeiro strain), and pulse-chase experiments were carried out using 3H-leucine. Cytoplasmic extracts were then prepared at appropriate times, and analyzed by sucrose-gradient ultracentrifugation. After preliminary assays (Fig. 1), working conditions were standardized so as to obtain maximal recovery of the morphogenetic intermediary, as well as consistency of results. Only in the presence of DOC-Brij58 and Mg++ could a 10S sedimentation coefficient peak be seen (Fig. 1 a). A heterogeneous zone, with 4,5-5S as sedimentation coefficient, was also observed. The degree of labeling in the region 4,5-5S compared with that in the 10S portion, depends on the time within the infectious cycle when cells were pulse-labeled. Maximal levels for the ratio 10S/4,5-5S are reached when pulse-labeling takes place at the time when the amount of RNA viral synthesis reaches 80% of its total value (Fig. 2). Similar experiments, performed with third passage bovine fetal kidney cells, were confirmatory of the presence of a structure sedimenting at 10S, as well as of a heterogeneous zone of 4,5-5S (Fig. 3). It would appear that assembling of Aphthoviruses is accomplished through an intermediary unit which differs from that found for other Picornaviruses, the latter being the result of the union of 12 pentamers. The capsid of Aphthoviruses, also composed of 60 identical sub-units, would instead derive from the joining of 20 trimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

The formation of a defective small subunit of the mitochondrial ribosomes in petite mutants of Saccharomyces cerevisiae.

The involvement of mitochondrial protein synthesis in the assembly of the mitochondrial ribosomes was investigated by studying the extent to which the assembly process can proceed in petite mutants of Saccharomyces cerevisiae which lack mitochondrial protein synthetic activity due to the deletion of some tRNA genes and/or one of the rRNA genes on the mtDNA. Petite strains which retain the 15-S rRNA gene can synthesize this rRNA species, but do not contain any detectable amounts of the small mitochondrial ribosomal subunit. Instead, a ribonucleoparticle with a sedimentation coefficient of 30 S (instead of 37 S) was observed. This ribonucleoparticle contained all the small ribosomal subunit proteins with the exception of the var1 and three to five other proteins, which indicates that the 30-S ribonucleoparticle is related to the small mitochondrial ribosomal subunit (37 S). Reconstitution experiments using the 30-S particle and the large mitochondrial ribosomal subunit from a wild-type yeast strain indicate that the 30-S particle is not active in translating the artificial message poly(U). The large mitochondrial ribosomal subunit was present in petite strains retaining the 21-S rRNA gene. The petite 54-S subunit is biologically active in the translation of poly(U) when reconstituted with the small subunit (37 S) from a wild-type strain. The above results indicate that mitochondrial protein synthetic activity is essential for the assembly of the mature small ribosomal subunit, but not for the large subunit. Since the var1 protein is the only mitochondrial translation product known to date to be associated with the mitochondrial ribosomes, the results suggest that this protein is essential for the assembly of the mature small subunit.

DNA, Mitochondrial↗
PubMed1993

Relating structure to function in the hepatitis delta virus antigen.

Hepatitis delta virus expresses two forms of a single protein, the small (delta Ag-S) and large (delta Ag-L) antigens, which are identical except for an additional 19 residues present at the C terminus of delta Ag-L. While delta Ag-S is required to promote genome replication, delta Ag-L potently inhibits this process and also facilitates packaging of the viral genome by envelope proteins of the helper virus (hepatitis B virus). Regions within the antigens responsible for nuclear localization, RNA binding, and dimerization have been identified, yet it is not clear how these particular activities contribute to the ultimate replication and packaging phenotypes. Here we report the following findings. (i) Although the removal of the nuclear localization signal from either antigen resulted in significant cytoplasmic accumulation, both proteins still had access to the nucleus. As a consequence, no functional defect was observed with either mutant. (ii) The RNA-binding domain, although necessary for delta Ag-S function, could be deleted from delta Ag-L without compromising its ability to either inhibit replication or promote packaging. (iii) In contrast, the coiled-coil dimerization domain was required for both the activation of replication by delta Ag-S and the inhibition of replication by delta Ag-L. This region, with an additional 20 amino acids C-terminal to it, was necessary and sufficient to potently inhibit replication by interacting with the small antigen. (iv) The packaging property of delta Ag-L required a C-terminal Pro/Gly-rich region which is hypothesized to interact with the hepatitis B virus envelope proteins during the assembly process.

Antigens, Viral↗
PubMed1998

Particle size determinants in the human immunodeficiency virus type 1 Gag protein.

The retroviral Gag protein plays the central role in the assembly process and can form membrane-enclosed, virus-like particles in the absence of any other viral products. These particles are similar to authentic virions in density and size. Three small domains of the human immunodeficiency virus type 1 (HIV-1) Gag protein have been previously identified as being important for budding. Regions that lie outside these domains can be deleted without any effect on particle release or density. However, the regions of Gag that control the size of HIV-1 particles are less well understood. In the case of Rous sarcoma virus (RSV), the size determinant maps to the CA (capsid) and adjacent spacer sequences within Gag, but systematic mapping of the HIV Gag protein has not been reported. To locate the size determinants of HIV-1, we analyzed a large collection of Gag mutants. To our surprise, all mutants with defects in the MA (matrix), CA, and the N-terminal part of NC (nucleocapsid) sequences produced dense particles of normal size, suggesting that oncoviruses (RSV) and lentiviruses (HIV-1) have different size-controlling elements. The most important region found to be critical for determining HIV-1 particle size is the p6 sequence. Particles lacking all or small parts of p6 were uniform in size distribution but very large as measured by rate zonal gradients. Further evidence for this novel function of p6 was obtained by placing this sequence at the C terminus of RSV CA mutants that produce heterogeneously sized particles. We found that the RSV-p6 chimeras produced normally sized particles. Thus, we present evidence that the entire p6 sequence plays a role in determining the size of a retroviral particle.

HIV Core Protein p24↗
PubMed1999

Second site mutations in the N-terminus of the major capsid protein (VP5) overcome a block at the maturation cleavage site of the capsid scaffold proteins of herpes simplex virus type 1.

VP5, the major capsid protein of herpes simplex virus type 1 (HSV-1), interacts with the C-terminal residues of the scaffold molecules encoded by the overlapping UL26 and UL26.5 open reading frames. Scaffold molecules are cleaved by a UL26 encoded protease (VP24) as part of the normal capsid assembly process. In this study, residues of VP5 have been identified that alter its interaction with the C-terminal residues of the scaffold proteins. A previously isolated virus (KUL26-610/611) was used that encoded a lethal mutation in the UL26 and UL26.5 open reading frames and required a transformed cell line that expresses these proteins for virus growth. The scaffold maturation cleavage site between amino acids 610 and 611 was blocked by changing Ala-Ser to Glu-Phe, which generated a new EcoRI restriction site. Revertant viruses, that formed small plaques on nontransformed cells, were detected at a frequency of 1:3800. Nine revertants were isolated, and all of them retained the EcoRI site and therefore were due to mutations at a second site. The second site mutations were extragenic. Using marker-transfer techniques, the mutation in one of the revertants was mapped to the 5' region of the gene encoding VP5. DNA sequence analysis was performed for the N-terminal 571 codons encoding VP5 for all of the revertant viruses. Six of the nine revertants showed a single base pair change that caused an amino acid substitution between residues 30 and 78 of VP5. Three of these were identical and changed Ala to Val at residue 78. The data provide a partial map of residues of VP5 that alter its interaction with scaffold proteins blocked at their normal cleavage site. The yeast two-hybrid system was used as a measure of the interaction between mutant VP5 and scaffold molecules and varied from 11% to nearly 100%, relative to wild-type VP5. One revertant gave no detectable interaction by this assay. The amount of UL26 encoded protease (VP24) in B capsids for KUL26-610/611 and for revertants was 7% and 25%, respectively, relative to the amount in capsids for wild-type virus. The lack of retention of the viral protease in the mutant virus and a fourfold increase for the revertants suggest an additional essential function for VP24 in capsid maturation, and a role in DNA packaging is indicated.

Capsid↗
PubMed1998

Protein-like molecular architecture: biomaterial applications for inducing cellular receptor binding and signal transduction.

The development of biomaterials with desirable biocompatibility has presented a difficult challenge for tissue engineering researchers. First and foremost, materials themselves tend to be hydrophobic and/or thrombogenic in nature, and face compatibility problems upon implantation. To mediate this problem, researchers have attempted to graft protein fragments onto biomaterial surfaces to promote endothelial cell attachment and minimize thrombosis. We envisioned a novel approach, based on the capability of biomolecules to self-assemble into well-defined and intricate structures, for creating biomimetic biomaterials that promote cell adhesion and proliferation. One of the most intriguing self-assembly processes is the folding of peptide chains into native protein structures. We have developed a method for building protein-like structural motifs that incorporate sequences of biological interest. A lipophilic moiety is attached onto a N alpha-amino group of peptide chain, resulting in a "peptide-amphiphile." The alignment of amphiphilic compounds at the lipid-solvent interface is used to facilitate peptide alignment and structure initiation and propagation, while the lipophilic region absorbs to hydrophobic surfaces. Peptide-amphiphiles containing potentially triple-helical or alpha-helical structural motifs have been synthesized. The resultant head group structures have been characterized by CD spectroscopy and found to be thermally stable over physiological temperature ranges. Triple-helical peptide-amphiphiles have been applied to studies of surface modification and cell receptor binding. Cell adhesion and spreading was promoted by triple-helical peptide-amphiphiles. Cellular interaction with the type IV collagen sequence alpha 1(IV) 1263-1277 increased signal transduction, with both the time and level of induction dependent upon triple-helical conformation. Collectively, these results suggest that peptide-amphiphiles may be used to form stable molecular structure on biomaterial surfaces that promote cellular activities and improve biocompatibility.

Amino Acid Sequence↗
PubMed1997

Bifunctional fusion between nerve growth factor and a transferrin receptor antibody.

The cDNAs encoding the variable regions of the heavy and light chains of a murine antibody specific for the human TfR were cloned and a human chimera (gamma1, kappa) was produced. A gene fusion was created by joining the 3' end of the coding region of the human nerve growth factor (NGF) precursor to the 5' end of the heavy chain variable region of the chimeric antibody. When expressed with the unmodified light chain in mammalian cells, the protein fusion is properly processed, assembled, and secreted. Subsequent purification and characterization established the uncompromised bifunctional activities of the protein, relative to the unmodified components, as demonstrated by its ability to both bind to the human TfR and induce neurite outgrowth in primary sympathetic or spinal ganglia and in trkA-transfected pheochromocytoma cells. The ability to generate biologically active NGF fused to a TfR targeting antibody, which was previously shown to cross the blood-brain barrier, may offer a novel way to deliver NGF and other neurotrophic factors to the central nervous system.

Animals↗
PubMed2000

Immobilization of alliinase on porous aluminum oxide.

Membrane filters prepared from porous aluminum oxide (Anopore) were investigated for their potential use as a durable support for enzymes. Alliinase (EC 4.4.1.4) was chosen as a model enzyme for immobilization experiments. To allow for smooth fixation, the enzyme was immobilized indirectly by sugar-lectin binding. Monomolecular layers of the lectin concanavalin A and alliinase were applied by self-assembling processes. As an anchor for these layers, the sugar, mannan, was covalently coupled to the membrane surface. This procedure exhibits several advantages: (i) enzyme immobilization can be carried out under smooth conditions; (ii) immobilization needs little time; and (iii) protein layers may be renewed.

Absorption↗
PubMed2000

Self-complementary

Three [3]catenanes with cavities large enough to accommodate aromatic guests have been designed and synthesized (yields = 5-20 %) by means of kinetically controlled self-assembly processes. The X-ray structural analysis of one of three [3]catenanes confirmed the presence of a rectangular cavity (dimensions = 7 x 11 A) lined by pi-electron-rich recognition sites and hydrogen-bond acceptor groups. In spite of their apparently ideal recognition features, none of these [3]catenanes bind guests incorporating a pi-electron-deficient bipyridinium unit. However, the template-directed syntheses of the [3]catenanes also produce, in yields of 2-23%, [2]catenanes incorporating a 1,5-dioxynaphtho[38]crown-10 interlocked with a bipyridinium-based tetracationic cyclophane. The X-ray structural analyses of two of these [2]catenanes revealed that a combination of [pi...pi] and [C-H...pi] interactions is responsible for the formation of supramolecular homodimers in the solid state. 1H NMR spectroscopic investigations of the four [2]catenanes demonstrated that supramolecular homodimers are also formed (Ka= 17-31M(-1), T= 185 K) in (CD3)2CO solutions. Dynamic 1H NMR spectroscopy revealed that the 1,5-dioxynaphtho[38]crown-10 and tetracationic cyclophane components in the four [2]catenanes and in the three [3]catenanes circumrotate (deltaGc(not equal to) = 9-14 kcal mol(-1)) through each other's cavity in (CD3)2CO. Similarly, the 1,5-dioxynaphthalene and the bipyridinium ring systems rotate (deltaGc(not equal to) =10-14 kcal mol(-1)) about their [O...O] and [N...N] axes, respectively, in solution.

Journal Article↗
PubMed2000

How do they know? Influencing the relative stereochemistry of the complex units of dinuclear triple-stranded helicate-type complexes

Dinuclear triple-stranded helicates or meso-helicates are formed by self-assembly of linear ligand strands with appropriate metal ions. Very often this self-assembly proceeds with high diastereoselectivity leading either to the helicate or the meso-helicate. The stereoselectivity of the self-assembly process can be influenced by different factors such as the rigidity of the ligand (-->helicate), the number of methylene units in the alkyl spacers (even number of CH2 units --> helicate; odd number --> meso-helicate), template effects, or chiral substituents.

Journal Article↗
PubMed2000

Interactions at the outside faces of calix

Attractive pi-pi interactions between two of the four outside cavity faces of 1,3-bis-pyridylmethylcalix[4]arene (1) and both faces of 1,4-diiodotetrafluorobenzene (2a) form infinite one-dimensional non-covalent ribbons where the two modules alternate. These ribbons are cross-linked by electron donor-acceptor interactions between picolyl nitrogen atoms of calixarene 1 in one chain and iodine atoms of perfluoroarene 2a in another chain and the two-dimensional supramolecular network 3a is formed. A similar behaviour is also shown by 1,4-dibromotetrafluorobenzene (2b). The halogen bonding and the attractive pi-pi interactions occur in directions which are nearly orthogonal each other. Diiodotetrafluorobenzene, being involved in both these interactions, appears to be a particularly interesting tecton. The ability of electron-poor arenes to elicit the exo-receptor potential of calixarene module by connecting their outside faces through pi-pi interactions may be developed as a new and general binding protocol in calixarene self-assembly processes.

Journal Article↗