Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Assembly process”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 433 records · Page 24Linked to original sources

A biochemical logic gate using an enzyme and its inhibitor. 1. The inhibitor as switching element.

Molecular-scale logic systems will allow for further miniaturization of information processing assemblies and contribute to a better understanding of brain function. Of much interest are the pertinent biological systems, some of the basic components of which are biomolecular switching elements and enzyme-based logic gates. In this series of accounts, results of investigations are presented on the implementation of an enzyme/inhibitor logic gate operating under the rules of Boolean algebra. In this report (part 1 of the series), consideration is given to the experimental conditions-particularly the irradiation mode-that affect the performance of proflavine as inhibitor of alpha-chymotrypsin. Also, assessments are made on the reversibility of the process involved and the long-term stability of the system. Moreover, using a theoretical conformational analysis of proflavine and its reduction products, detailed features were established regarding their three-dimensional structure, partial charge distribution, and hydrophobicity. Accordingly, an understanding was reached as to the factors affecting the interaction between these compounds and the enzyme. In part 2 of this series, the actual implementation of an AND logic gate will be presented. This gate involves proflavine and a chemically derivatized alpha-chymotrypsin, and its operation relies on the conclusions reached in this report regarding the optimal mode for controlling the inhibitory activity of proflavine.

Animals↗

Defective assembly of the mitochondrial ribosomes in yeast cells grown in the presence of mitochondrial protein synthesis inhibitors.

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 the presence of mitochondrial protein synthesis inhibitors erythromycin and chloramphenicol. Yeast cells grown in the presence of erythromycin (2 mg/ml) do not appear to contain any detectable amounts of the mitochondrial small (37 S) ribosomal subunit. Instead, a ribonucleoparticle with a sedimentation coefficient of 30 S was observed; this particle could be shown to be related to the mitochondrial small ribosomal subunit by two-dimensional gel electrophoretic analysis of its protein components. Since the var1 protein is the only mitochondrial translation product known to be associated with the mitochondrial ribosome, our results suggest that this protein is essential for the assembly of the mature small subunit, and that the var1 protein enters the pathway for the assembly of the small subunit at a late step. In at least one strain of yeast the accumulation of the 30-S particle appears to be very sensitive to catabolite repression. When yeast cells are grown in the presence of chloramphenicol instead of erythromycin, assembly of the small subunit appears to be only partially inhibited, and the presence of the 30-S particle could not be clearly demonstrated. This observation is consistent with the fact that in yeast, chloramphenicol inhibits mitochondrial protein synthesis by about 95% only and that the synthesis of the var1 protein appears to be the least sensitive to this inhibition.

Cell Fractionation↗

The assembly pathway of outer membrane protein PhoE of Escherichia coli.

The assembly of the wild-type and several mutant forms of the trimeric outer membrane porin PhoE of Escherichia coli was investigated in vitro and in vivo. In in vivo pulse-chase experiments, approximately half of the wild-type PhoE molecules assembled within the 30-s pulse in the native conformation in the cell envelope. The other half of the molecules followed slower kinetics, and three intermediates in this multistep assembly process were detected: a soluble trypsin-sensitive monomer, a trypsin-sensitive monomeric form that was loosely associated with the cell envelope and a metastable trimer, which was integrated into the membranes and converted to the stable trimeric configuration within minutes. The metastable trimers disassembled during sample preparation for standard SDS/PAGE into folded monomers. In vitro, the isolated PhoE protein could efficiently be folded in the presence of N,N-dimethyldodecylamine-N-oxide (LDAO). A mutant PhoE protein, DeltaF330, which lacks the C-terminal phenylalanine residue, mainly followed the slower kinetic pathway observed in vivo, resulting in increased amounts of the various assembly intermediates. It appears that the DeltaF330 mutant protein is intrinsically able to fold, because it was able to fold in vitro with LDAO with similar efficiencies as the wild-type protein. Therefore, we propose that the conserved C-terminal Phe is (part of) a sorting signal, directing the protein efficiently to the outer membrane. Furthermore, we analysed a mutant protein with a hydrophilic residue introduced at the hydrophobic side of one of the membrane-spanning amphipathic beta strands. The assembly of this mutant protein was not affected in vivo or in vitro in the presence of LDAO. However, it was not able to form folded monomers in a previously established in vitro folding system, which requires the presence of lipopolysaccharides and Triton. Hence, a folded monomer might not be a true assembly intermediate of PhoE in vivo.

Biochemistry↗

Deletions in epidermal keratins leading to alterations in filament organization in vivo and in intermediate filament assembly in vitro.

To investigate the sequences important for assembly of keratins into 10-nm filaments, we used a combined approach of (a) transfection of mutant keratin cDNAs into epithelial cells in vivo, and (b) in vitro assembly of mutant and wild-type keratins. Keratin K14 mutants missing the nonhelical carboxy- and amino-terminal domains not only integrated without perturbation into endogenous keratin filament networks in vivo, but they also formed 10-nm filaments with K5 in vitro. Surprisingly, keratin mutants missing the highly conserved L L E G E sequence, common to all intermediate filament proteins and found at the carboxy end of the alpha-helical rod domain, also assembled into filaments with only a somewhat reduced efficiency. Even a carboxy K14 mutant missing approximately 10% of the rod assembled into filaments, although in this case filaments aggregated significantly. Despite the ability of these mutants to form filaments in vitro, they often perturbed keratin filament organization in vivo. In contrast, small truncations in the amino-terminal end of the rod domain more severely disrupted the filament assembly process in vitro as well as in vivo, and in particular restricted elongation. For both carboxy and amino rod deletions, the more extensive the deletion, the more severe the phenotype. Surprisingly, while elongation could be almost quantitatively blocked with large mutations, tetramer formation and higher ordered lateral interactions still occurred. Collectively, our in vitro data (a) provide a molecular basis for the dominance of our mutants in vivo, (b) offer new insights as to why different mutants may generate different phenotypes in vivo, and (c) delineate the limit sequences necessary for K14 to both incorporate properly into a preexisting keratin filament network in vivo and assemble efficiently into 10-nm keratin filaments in vitro.

Amino Acid Sequence↗

Hybrid organic/inorganic reverse osmosis (RO) membrane for bactericidal anti-fouling. 1. Preparation and characterization of TiO2 nanoparticle self-assembled aromatic polyamide thin-film-composite (TFC) membrane.

Hybrid organic/inorganic reverse osmosis (RO) membranes composed of aromatic polyamide thin films underneath titanium dioxide (TiO2) nanosized particles have been fabricated by a self-assembly process, aiming at breakthrough of biofouling problems. First, positively charged particles of the colloidal TiO2 were synthesized by a sol-gel process, and the diameter of the resulting particles in acidic aqueous solution was estimated to be approximately 2 nm by analyzing the UV-visible absorption characteristics with a quantum mechanical model developed by Brus. Transmission electron microscopy (TEM) further confirmed the formation of the quantum-sized TiO2 particles (approximately 10 nm or less). The TiO2 particles appeared to exist in the crystallographic form of anatase as observed with the X-ray diffraction (XRD) pattern in comparison with those of commercial 100% rutile and commercial 70:30% anatase-to-rutile mixture. The hybrid thin-film-composite (TFC) aromatic polyamide membranes were prepared by self-assembly of the TiO2 nanoparticles on the polymer chains with COOH groups along the surface. They showed improved RO performance in which the water flux even increased, though slightly. Field-emission scanning electron microscopy (FESEM) exhibited the TiO2 nanoparticles well adsorbed onto the surface. X-ray photoelectron spectroscopy (XPS) demonstrated quantitatively that a considerable amount of the adsorbed particles were tightly self-assembled at the expense of the initial loss of those that were loosely bound, and became stabilized even after exposure to the various washing and harsh RO operating conditions. The antibacterial fouling potential of the TiO2 hybrid membrane was examined and verified by measuring the viable numbers and determining the survival ratios of the Escherichia coli (E. coli) as a model bacterium, both with and without UV light illumination. The photocatalytic bactericidal efficiency was remarkably higher for the TiO2 hybrid membrane under UV illumination, compared to that of the same membrane in darkness, as well as those for the neat membranes under either light condition.

Biofilms↗

Complementation of in vitro-assembled spliceosomes.

We describe the development and application of a system of in vitro-assembled splicing complexes that can be used for the identification of protein splicing factors which become associated with the spliceosome at the end of the assembly process ("late" splicing components). A splicing reaction performed in the presence of polyvinyl alcohol is interrupted after 15 to 20 minutes, before the appearance of splicing intermediates and products in significant amounts. Following low-speed centrifugation, a pellet is obtained containing splicing complexes that can be solubilized with 0.6 M-KCl. These complexes can be rapidly complemented for splicing in the presence of ATP and Mg2+ with protein factors that are present in HeLa cell nuclear extracts or in chromatographic extract fractions. Biochemical features of the complementation reactions, and conditions for reversible uncoupling of the two splicing steps, are described and discussed. These conditions are used to generate fully assembled spliceosomes in which splicing of the pre-mRNA can occur in the presence of ATP and Mg2+, but in the absence of nuclear extract ("autonomous splicing").

Adenosine Triphosphate↗

Colloidal assemblies on patterned silane layers.

The site-selective assembly of colloidal polymer particles onto laterally patterned silane layers was studied as a model system for the object assembly process at mesoscale dimensions. The structured silane monolayers on silicon oxide substrates were fabricated by a combination of liquid- and gas-phase deposition of different trialkoxysilanes with a photolithographic patterning technique. By using this method various types of surface functionalizations such as regions with amino functions next to areas of the bare silica surface or positively charged regions of a quaternary ammonium silane surrounded by a hydrophobic octadecylsilane film could be obtained. Furthermore, a triethoxysilane with a photoprotected amino group was synthesized, which allowed direct photopatterning after monolayer preparation, leading to free NH(2) groups at the irradiated regions. The different silane monolayer patterns were used to study the surface assembly behavior of carboxylated methacrylate particles by optical and scanning electron microscopy. In dependence of the assembly conditions (different surface functionalizations, pH, and drying conditions), a selective preference of the particles for a specific surface type versus others was found. Site-specific colloid adsorption could be observed also on the photosensitive silane layers after local deprotection with light. From the photosensitive silane and positively charged ammonium silane, molecularly mixed monolayers were prepared, which allowed particle adsorption and photoactivation within the same monolayer as shown by fluorescence labeling.

Carboxylic Acids↗

pH-Dependent Monomer <--> Oligomer Interconversion of Copper(II) Complexes with N-(2-R-imidazol-4-ylmethylidene)-2-aminoethylpyridine (R = Methyl, Phenyl).

The monomer <--> oligomer interconversion of the reported metal complexes is generated by proton abstraction/supply as a common external information input. The mononuclear copper(II) complexes 1 and 2 with [CuCl(2)(HL(n)())] chemical formula have been prepared (HL(1) = N-(2-methylimidazol-4-ylmethylidene)-2-aminoethylpyridine; HL(2) = N-(2-phenylimidazol-4-ylmethylidene)-2-aminoethylpyridine). The crystal structures were determined. 1.H(2)O, C(12)H(16)N(4)OCl(2)Cu: a = 13.773(2) Å, b = 8.245(2) Å, c = 13.861(2) Å, beta = 110.10(1) degrees, monoclinic, P2(1)/n, and Z = 4. 2, C(17)H(16)N(4)Cl(2)Cu: a = 7.6659(7) Å, b = 16.287(1) Å, c = 14.103(1) Å, beta = 95.058(7) degrees, monoclinic, P2(1)/c, and Z = 4. Complexes 1.H(2)O and 2 assume a pentacoordinated square pyramidal geometry with a N(3)Cl(2) donor set consisting of the nitrogen atoms of the protonated tridentate ligand and two chloride ions in the solid state, while in aqueous solution the Cu(II) ion is tetracoordinated (N(3)Ow donor set). When 1 and 2 are treated with an equimolar amount of sodium hydroxide or triethylamine, the deprotonation of the imidazole moiety promotes a self-assembly process, arising from coordination of the imidazolate nitrogen atom to a Cu(II) ion of an adjacent unit, to yield compounds 1'.4H(2)O as the perchlorate salt, and 2'a.6H(2)O as the perchlorate salt and( )()2'b as the hexafluorophosphate salt, respectively. 1'.4H(2)O, C(12)H(15)N(4)O(5)ClCu: a = b = 13.966(2) Å, c = 33.689(3) Å, tetragonal, I4(1)/a, and Z = 16. 2'a.6H(2)O, C(51)H(51)N(12)O(15)Cl(3)Cu(3): a = 15.177(3) Å, b = 15.747(3) Å, c = 14.128(3) Å, alpha = 100.06(2) degrees, beta = 110.37(2) degrees, gamma = 63.54(1) degrees, triclinic, P&onemacr;, and Z = 2. 2'b, C(17)H(15)N(9)F(6)PCu: a = b = 29.812(5) Å, c = 11.484(3) Å, trigonal, R&thremacr;, and Z = 18. The nuclearity of the self-assembled molecules and their detailed structure were confirmed to be cyclic imidazolate-bridged tetranuclear for 1'.4H(2)O and hexanuclear for 2'a.6H(2)O and 2'b, respectively, through single-crystal X-ray analyses and FAB-MS spectra. Variable-temperature experimental magnetic susceptibility data were well reproduced by using the Heisenberg model based on a cyclic tetranuclear structure for 1' and a cyclic hexanuclear structure for 2'a and 2'b. The reversible interconversion between the protonated monomeric and deprotonated oligomeric species were confirmed by pH-dependent potentiometric and electronic spectral titrations in aqueous solution, whereas the Pd(II) complex did not show a perfect disassembly process.

Journal Article↗

Specific packaging of nodaviral RNA2 requires the N-terminus of the capsid protein.

Flock house virus (FHV), a member of the family Nodaviridae, is a nonenveloped, icosahedral insect virus whose capsids are assembled from 180 copies of a single type of coat protein. The viral genome is split between two segments of single-stranded positive-sense RNA, RNA1 and RNA2, which are packaged into a single virion. We previously demonstrated that synthesis of FHV coat protein in the baculovirus expression system results in assembly of virus-like particles whose capsids are indistinguishable from those of native virions, although the encapsidated RNA represents primarily cellular RNA. In contrast, expression of a deletion mutant lacking N-terminal residues 2-31 results in formation of multiple types of particles which differ in size, shape, and RNA contents. We postulated that the polymorphism was imposed by the type of RNA that the coat protein selected for packaging. In the current study we tested this hypothesis by analyzing the assembly of the mutant coat protein in Drosophila cells in the presence of replicating FHV RNAs. As anticipated, the resulting particles had the same shape and dimensions as wt virions. Surprisingly, however, they contained little RNA2 while packaging of RNA1 was not affected. Small amounts of defective interfering RNAs, which emerged rapidly in the presence of the mutant coat protein, were also detected. Taken together, these observations confirm our earlier hypothesis that selection of nonviral RNAs for packaging can significantly alter the assembly process. In addition, they demonstrate that the N-terminus of the FHV coat protein contains important determinants for recognition and packaging of RNA2. Our results provide the first evidence that encapsidation of the two genomic RNAs occurs independently and that the coat protein uses different regions for the recognition of RNA1 and RNA2.

Amino Acid Sequence↗

Self-assembly mechanism of a bimetallic europium triple-stranded helicate.

We report the self-assembly process of a supramolecular edifice based on the coordination of europium(III) by a ditopic strand L bearing tridentate bis(benzimidazolyl)pyridine subunits. Varying the metal/ligand ratio and using a fruitful combination of electrospray mass spectrometry and absorption spectrophotometry, we characterized three major complexes (EuL(2), Eu(2)L(2), and Eu(2)L(3)) in acetonitrile. Kinetic investigations showed an alternative "braiding" and "keystone" mechanism leading to Eu(2)L(3). The formation mechanism of the dinuclear triple-stranded helicate, which is mainly governed by electrostatic interactions, goes via the "side-by-side" Eu(2)L(2) intermediate. Our thermodynamic and kinetic data allow the prediction of the apparent "magic" self-assembly of Eu(2)L(3) which is fast and efficient only under a strict set of conditions.

Journal Article↗

Arresting and releasing Staphylococcal alpha-hemolysin at intermediate stages of pore formation by engineered disulfide bonds.

alpha-Hemolysin (alphaHL) is secreted by Staphylococcus aureus as a water-soluble monomer that assembles into a heptamer to form a transmembrane pore on a target membrane. The crystal structures of the LukF water-soluble monomer and the membrane-bound alpha-hemolysin heptamer show that large conformational changes occur during assembly. However, the mechanism of assembly and pore formation is still unclear, primarily because of the difficulty in obtaining structural information on assembly intermediates. Our goal is to use disulfide bonds to selectively arrest and release alphaHL from intermediate stages of the assembly process and to use these mutants to test mechanistic hypotheses. To accomplish this, we created four double cysteine mutants, D108C/K154C (alphaHL-A), M113C/K147C (alphaHL-B), H48C/ N121C (alphaHL-C), I5C/G130C (alphaHL-D), in which disulfide bonds may form between the pre-stem domain and the beta-sandwich domain to prevent pre-stem rearrangement and membrane insertion. Among the four mutants, alphaHL-A is remarkably stable, is produced at a level at least 10-fold greater than that of the wild-type protein, is monomeric in aqueous solution, and has hemolytic activity that can be regulated by the presence or absence of reducing agents. Cross-linking analysis showed that alphaHL-A assembles on a membrane into an oligomer, which is likely to be a heptamer, in the absence of a reducing agent, suggesting that oxidized alphaHL-A is halted at a heptameric prepore state. Therefore, conformational rearrangements at positions 108 and 154 are critical for the completion of alphaHL assembly but are not essential for membrane binding or for formation of an oligomeric prepore intermediate.

Amino Acid Substitution↗

Self-assembly and supramolecular organization of EMILIN.

The primary structure of human Elastin microfibril interface-located protein (EMILIN), an elastic fiber-associated glycoprotein, consists of a globular C1q domain (gC1q) at the C terminus, a short collagenous stalk, a long region with a high potential for forming coiled-coil alpha helices, and a cysteine-rich N-terminal sequence. It is not known whether the EMILIN gC1q domain is involved in the assembly process and in the supramolecular organization as shown for the similar domain of collagen X. By employing the yeast two-hybrid system the EMILIN gC1q domains interacted with themselves, proving for the first time that this interaction occurs in vivo. The gC1q domain formed oligomers running as trimers in native gels that were less stable than the comparable trimers of the collagen X gC1q domain since they did not withstand heating. The collagenous domain was trypsin-resistant and migrated at a size corresponding to a triple helix under native conditions. In reducing agarose gels, EMILIN also migrated as a trimer, whereas under non-reducing conditions it formed polymers of many millions of daltons. A truncated fragment lacking gC1q and collagenous domains assembled to a much lesser extent, thus deducing that the C-terminal domain(s) are essential for the formation of trimers that finally assemble into large EMILIN multimers.

Cell Line↗

Assembly of the herpes simplex virus capsid: preformed triplexes bind to the nascent capsid.

The herpes simplex virus type 1 (HSV-1) capsid is a T=16 icosahedral shell that forms in the nuclei of infected cells. Capsid assembly also occurs in vitro in reaction mixtures created from insect cell extracts containing recombinant baculovirus-expressed HSV-1 capsid proteins. During capsid formation, the major capsid protein, VP5, and the scaffolding protein, pre-VP22a, condense to form structures that are extended into procapsids by addition of the triplex proteins, VP19C and VP23. We investigated whether triplex proteins bind to the major capsid-scaffold protein complexes as separate polypeptides or as preformed triplexes. Assembly products from reactions lacking one triplex protein were immunoprecipitated and examined for the presence of the other. The results showed that neither triplex protein bound unless both were present, suggesting that interaction between VP19C and VP23 is required before either protein can participate in the assembly process. Sucrose density gradient analysis was employed to determine the sedimentation coefficients of VP19C, VP23, and VP19C-VP23 complexes. The results showed that the two proteins formed a complex with a sedimentation coefficient of 7.2S, a value that is consistent with formation of a VP19C-VP23(2) heterotrimer. Furthermore, VP23 was observed to have a sedimentation coefficient of 4.9S, suggesting that this protein exists as a dimer in solution. Deletion analysis of VP19C revealed two domains that may be required for attachment of the triplex to major capsid-scaffold protein complexes; none of the deletions disrupted interaction of VP19C with VP23. We propose that preformed triplexes (VP19C-VP23(2) heterotrimers) interact with major capsid-scaffold protein complexes during assembly of the HSV-1 capsid.

Animals↗

Mechanistic studies of the process of amyloid fibrils formation by the use of peptide fragments and analogues: implications for the design of fibrillization inhibitors.

The process of amyloid fibrils formation is a common mechanism of a large number of unrelated infectious, genetic and spontaneous diseases. A partial list includes the bovine spongiform encephalopathy (BSE), Alzheimer's diseases, Type II diabetes, Creutzfeldt-Jakob disease, and various unrelated amyloidosis diseases. In spite of its significant clinical importance, the mechanism of fibrillization is not fully understood. This review discusses the recent advancements in the mechanistic studies of amyloid formation by the use peptide fragments and analogues of amyloid-forming proteins and polypeptides. The use of short peptide shed much light of the mechanism of amyloid fibrillization. Recent studies clearly prove that very short peptide fragments (as short as pentapeptides) can form well-ordered amyloidal structures. Therefore, the molecular recognition and self-assembly process that lead to the formation of order structures is being mediated by small structural elements. Analysis of short amyloid-related fragment by the use of an alanine-scan and sequence analysis of a variety of unrelated peptide and protein fragments suggest that aromatic interaction may play a central role in the process of amyloid formation. Inhibitors that are based on the short aromatic elements already demonstrated clear potency in arresting the process of amyloid fibrils formation. Taken together, the recent advancement in the mechanistic understanding of the process of amyloid fibrils formation has a major importance in the development of inhibitors of fibrillization that may serve as future therapeutic means to treat amyloid diseases.

Amyloid beta-Peptides↗

Assembly of the herpes simplex virus capsid: characterization of intermediates observed during cell-free capsid formation.

The herpes simplex virus-1 (HSV-1) capsid is an icosahedral shell approximately 15 nm thick and 125 nm in diameter. Three of its primary structural components are a major capsid protein (VP5; coded by the UL19 gene) and two minor proteins, VP19C (UL38 gene) and VP23 (UL18 gene). Assembly of the capsid involves the participation of two additional proteins, the scaffolding protein (UL26.5 gene) and the maturational protease (UL26 gene). With the goal of identifying morphological intermediates in the assembly process, we have examined capsid formation in a cell-free system containing the five HSV-1 proteins mentioned above. Capsids and capsid-related structures formed during progressively longer periods of incubation were examined by electron microscopy of thin-sectioned specimens. After one minute, 90 minutes and eight hours of incubation the structures observed, respectively, were partial capsids, closed spherical capsids and polyhedral capsids. Partial capsids were two-layered structures consisting of a segment of external shell partially surrounding a region of scaffold. They appeared as wedges or angular segments of closed spherical capsids, the angle ranging from less than 30 degrees to greater than 270 degrees. Partial capsids are suggested to be precursors of closed spherical capsids because, whereas partial capsids were the predominant assembly product observed after one minute of incubation, they were rare in reactions incubated for 45 minutes or longer. Closed spherical capsids were highly uniform in morphology, consisting of a closed external shell surrounding a thick scaffold similar in morphology to the same layers seen in partial capsids. In negatively stained specimens, closed spherical capsids appeared round in profile, suggesting that they are spherical rather than polyhedral in shape. A three-dimensional reconstruction computed from cryoelectron micrographs confirmed that closed spherical capsids are spherical with T = 16 icosahedral symmetry. The reconstruction showed further that, compared to mature HSV-1 capsids, closed spherical capsids are more open structures in which the capsid floor layer is less pronounced. In contrast to closed spherical capsids, polyhedral capsids exhibited distinct facets and vertices, indicating that they are icosahedral like the capsids in mature virions. Upon incubation in vitro, purified closed spherical capsids matured into polyhedral capsids, indicating that the latter arise by angularization of the former. Partial capsids, closed spherical capsids and polyhedral capsids were all found to contain VP5, VP19C, VP23, VP21 and the scaffolding protein; the scaffolding protein being predominantly in the immature, uncleaved form in all cases. Polyhedral capsids and closed spherical capsids were found to differ in their sensitivity to disruption at 2 degrees C. Closed spherical capsids were disassembled while polyhedral capsids were unaffected. Our results suggest that HSV-1 capsid assembly begins with the partial capsid and proceeds through a closed, spherical, unstable capsid intermediate to a closed, icosahedral form similar to that found in the mature virion. Structures resembling HSV-1 partial capsids have been described as capsid assembly intermediates in Salmonella typhimurium bacteriophage P22. HSV-1 capsid maturation from a fragile, spherical state to a robust polyhedral form resembles the prohead maturation events undergone by dsDNA bacteriophages including lambda, T4 and P22. Because of this similarity, we propose the name procapsid for the closed spherical capsid intermediate in HSV-1 capsid assembly.

Capsid↗

The gene 59 protein of bacteriophage T4. Characterization of protein-protein interactions with gene 32 protein, the T4 single-stranded DNA binding protein.

The gene 59 protein (gp59) of bacteriophage T4 stimulates the activities of gene 41 protein (gp41), the T4 replicative DNA helicase, by promoting the assembly of gp41 onto single-stranded (ss)-DNA molecules that are covered with cooperatively bound gene 32 protein (gp32). This helicase-ssDNA assembly process, which is important for the reconstitution of the primosome component of the T4 DNA replication fork, appears to require both gp59-gp41 and gp59-gp32 protein-protein interactions. In this study we characterize the physical and functional interactions of gp59 with gp32, the T4 ssDNA-binding protein. Experimental results presented herein indicate: 1) that gp59 binds specifically to both free and ssDNA-bound gp32 molecules; and 2) that in both cases binding involves contacts between gp59 and the acidic C-terminal domain of gp32 (the so-called "A-domain"). We further show that single-stranded DNA molecules coated with (gp32-A), a truncated form of gp32 lacking the A-domain, are refractory to gp59-dependent helicase assembly. The data indicate that specific contacts between gp59 molecules and the A-domains of gp32 molecules are essential for gp59-dependent assembly of gp41 onto gp32-ssDNA complexes. Our results are consistent with a model in which gp59 binds to gp32 molecules within the gp32-ssDNA complex and therein forms a target site for helicase-ssDNA assembly.

Adenosine Triphosphatases↗

alpha-tocopherol inhibits the respiratory burst in human monocytes. Attenuation of p47(phox) membrane translocation and phosphorylation.

Vitamin E (alpha-tocopherol), one of the most important natural antioxidants, is assumed to be beneficial in the prevention of cardiovascular diseases. alpha-Tocopherol exhibits acyl-peroxyl-radical scavenger properties and exerts cell-mediated actions in the hemovascular compartment, such as inhibition of superoxide anion (O-2) production by leukocytes. The aim of this study was to examine the mechanism underlying the inhibitory effect of alpha-tocopherol on O-2 production by human monocytes. In activated monocytes O-2 is produced by the NADPH-oxidase enzyme complex. The oxidase activation elicited by phorbol myristate acetate (PMA) requires membrane translocation of several cytosolic factors. We found that in human PMA-stimulated adherent monocytes, alpha-tocopherol (but not beta-tocopherol) inhibited O-2 production in intact cells but had no effect on a membrane preparation containing activated NADPH-oxidase, suggesting that alpha-tocopherol impairs the assembly process of the enzyme complex. We showed that translocation and phosphorylation of the cytosolic factor p47(phox) were reduced in monocytes preincubated with alpha-tocopherol. We verified that the tryptic phosphopeptide map of monocyte p47(phox) was similar to that of neutrophil p47(phox), indicating that several serine residues were phosphorylated. Peptides whose phosphorylation is dependent on protein kinase C (PKC) were phosphorylated to a lesser degree when p47(phox) was immunoprecipitated from alpha-tocopherol-treated monocytes. In vitro, the activity of PKC from monocytes was inhibited by alpha-tocopherol in a specific manner compared with that of beta-tocopherol or Trolox(R). Membrane translocation of PKC was not affected. These results show that alpha-tocopherol inhibits O-2 production by human adherent monocytes by impairing the assembly of the NADPH-oxidase and suggest that the inhibition of phosphorylation and translocation of the cytosolic factor p47(phox) results from a decrease in PKC activity.

Biological Transport↗

The cell biology and physiologic relevance of ApoE recycling.

Apolipoprotein E (apoE) is unique among the different apoproteins because of its many functions in the assembly, processing, and removal of plasma lipoproteins. Some of these functions require the presence of apoE inside the cell or in close association with its outer membrane. For example, the presence of apoE increases triglyceride content in newly formed very low-density lipoprotein (VLDL), whereas the fast removal of chylomicrons requires the presence of a cellular pool of apoE ready to surface and bind to membrane proteoglycans and possibly to lipoprotein receptors such as the LDL receptor-related protein (LRP). Our recent discovery that VLDL-apoE internalized by hepatocytes is partially protected from lysosomal degradation and recycles through the Golgi apparatus suggests that the biologic cycle of apoE may not be concluded when the lipoprotein is taken up by the cell, and that recycling apoE may have a physiologic role in lipoprotein assembly, remnant removal, and cholesterol efflux.

Animals↗