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PubMed1979

In vitro assembly of intermediate filaments from baby hamster kidney (BHK-21) cells.

Intermediate filaments (IF) from baby hamster kidney (BHK-21) cells can be disassembled at low ionic strength and reassembled upon addition of salt. Turbidimetric analyses show that reassembled IF exhibit the light scattering properties of long rods under physiological conditions (5 mM Na+/K+ phosphate, pH 7.2/170 mM NaCl at 21 degrees C). IF weight concentration, determined by centrifugation, is directly proportional to the optical density at 3000 nm. Thus, turbidity can be used as a quantitative assay for IF assembly. Turbidimetric and centrifugation analyses both indicate that IF assembly exhibits a critical protein concentration of 0.05-0.15 mg/ml. Above the critical concentration, IF weight concentration at steady-state is a linear function of the total protein concentration. Negative stain observations at early stages of the assembly process suggest lateral association of protofilaments to form short IF. This lateral association is accompanied by a rapid turbidity increase which is then followed by IF elongation and a slower turbidity increase to plateau. Further purification of IF by low/high-NaCl-induced cycles of disassembly/reassembly results in retention of 54- and 55-kilodalton (decamin) polypeptides. These results constitute a quantitative description of in vitro reassembly of IF from homogeneous cultures of nonkeratinizing cells and establish conditions for further studies on the regulation of IF assembly.

Animals↗
PubMed1981

Identification of a lymphocyte enzyme that catalyzes pentamer immunoglobulin M assembly.

A protein with immunoglobulin M-polymerizing activity was isolated from the membrane fraction of mouse plasmacytoma cells secreting pentamer IgM. The isolation was achieved by taking advantage of the solubility of the protein in 50% ammonium sulfate, its relatively high net negative charge, and its sedimentation at 4.2 S. Analyses of the purified preparations showed that the polymerizing protein catalyzes the assembly of pentamer IgM in vitro; less than 1 mol of enzyme/10 mol of monomer IgM and 2 ml of J chain were found to promote 50% polymerization. Evidence that the enzyme also plays an essential role in the in vivo assembly process was obtained from the reaction rates of the polymerization catalyzed in vitro, the similarity between the pentamer IgM molecules synthesized in vitro and in vivo, and the finding that polymerizing enzyme is a specific product of B lymphocytes. Analyses of the mechanism of polymerization suggested that polymerizing enzyme is a sulfhydryl oxidase; it was found to be inactivated by chelating agents and to resemble Cu2+ in catalyzing the formation of IgM intersubunit disulfide bonds. These results raise the possibility that the assembly of pentamer IgM does not involve disulfide interchange is previously thought, but proceeds by the direct oxidation of monomer IgM and J chain sulfhydryls.

Animals↗
PubMed2002

Structural requirements for the assembly of Norwalk virus-like particles.

Norwalk virus (NV) is the prototype strain of a group of human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. While these viruses do not grow in tissue culture cells or animal models, expression of the capsid protein in insect cells results in the self-assembly of recombinant NV virus-like particles (rNV VLPs) that are morphologically and antigenically similar to native NV. The X-ray structure of the rNV VLPs has revealed that the capsid protein folds into two principal domains: a shell (S) domain and a protruding (P) domain (B. V. V. Prasad, M. E. Hardy, T. Dokland, J. Bella, M. G. Rossmann, and M. K. Estes, Science 286:287-290, 1999). To investigate the structural requirements for the assembly of rNV VLPs, we performed mutational analyses of the capsid protein. We examined the ability of 10 deletion mutants of the capsid protein to assemble into VLPs in insect cell cultures. Deletion of the N-terminal 20 residues, suggested by the X-ray structure to be involved in a switching mechanism during assembly, did not affect the ability of the mutant capsid protein to self-assemble into 38-nm VLPs with a T=3 icosahedral symmetry. Further deletions in the N-terminal region affected particle assembly. Deletions in the C-terminal regions of the P domain, involved in the interactions between the P and S domains, did not block the assembly process, but they affected the size and stability of the particles. Mutants carrying three internal deletion mutations in the P domain, involved in maintaining dimeric interactions, produced significantly larger 45-nm particles, albeit in low yields. The complete removal of the protruding domain resulted in the formation of smooth particles with a diameter that is slightly smaller than the 30-nm diameter expected from the rNV structure. These studies indicate that the shell domain of the NV capsid protein contains everything required to initiate the assembly of the capsid, whereas the entire protruding domain contributes to the increased stability of the capsid by adding intermolecular contacts between the dimeric subunits and may control the size of the capsid.

Animals↗
PubMed2001

Structure determination of micelle-like intermediates in amyloid beta -protein fibril assembly by using small angle neutron scattering.

Increasing evidence supports the hypothesis that amyloid beta-protein (Abeta) assembly is a key pathogenic feature of Alzheimer's disease. Thus, understanding the assembly process offers opportunities for the development of strategies for treating this devastating disease. In prior studies, Abeta was found to form micelle-like aggregates under acidic conditions. These structures exhibited an average observed hydrodynamic radius of 7 nm. They were found to be in rapid equilibrium with Abeta monomers or low molecular weight oligomers, and were centers of fibril nucleation. Here the technique of small angle neutron scattering has been used to determine the structure of these Abeta micelles. The data reveal that the micellar assemblies comprise 30-50 Abeta monomers and have elongated geometries. The best fit of the data to a uniform spherocylinder yields a radius approximately 2.4 nm and cylinder length approximately 11 nm. These structure parameters remain constant over more than a decade in concentration range. The concentration independence of the length of the cylindrical aggregate indicates the presence of an internal nonrepetitive structure that spans the entire length of the Abeta assembly.

Amyloid beta-Peptides↗
PubMed1985

Genetic analysis of subunit assembly of the tail fiber of bacteriophage T3.

Bacteriophage T3 virions have six tail fibers composed of the product of gene 17 (gp17). Each tail fiber is a trimer of gp17 polypeptide. To characterize the assembly process of the tail fiber, temperature-sensitive (ts) mutants of gene 17 (ts17) were analyzed by SDS-polyacrylamide gel electrophoresis and by extract complementation. Newly synthesized gp17 polypeptide chains matured to SDS-resistant native trimers with a half time of about 7.5 min at 30 degrees. Although all ts17 mutants had similar plating efficiencies at restrictive temperature (41.5 degrees or 42 degrees), they showed different phenotypes. tsNG75, whose mutation was located in the carboxyl-terminal region of gene 17, was defective in trimer assembly at 41.5 degrees. The ts tail fibers formed at 30 degrees lost the ability to attach to the tail upon treatment at 41.5 degrees. There was a change in temperature sensitivity of tsNG75 tail fibers upon attachment to the tail, suggesting that the tail fiber may change conformation after attachment to the tail. tsNG215 and tsNG169, whose mutation sites were located in the amino-terminal region of gene 17, were not defective in the trimer assembly and attachment to the tail at the restrictive temperature. tsNG215 tail fibers formed at 41.5 degrees appear to be aberrant because they were not active in extract complementation and their attachment to fiberless particles resulted in production of noninfectious phage. Tail fibers produced by cells infected with tsNG169 at the restrictive temperature were active in extract complementation. Phage particles were formed in tsNG169-infected cells at the restrictive temperature. These particles were infectious at the permissive temperature and the mutant was non-infectious only if infection was continued at the restrictive temperature. These phenotypic differences exhibited by different gene 17 mutants may indicate the regions within the gene 17 polypeptide that play a role(s) in the folding and assembly of gp17 and in the biological activity of the mature tail fiber.

Electrophoresis, Polyacrylamide Gel↗
PubMed2002

A chemical genetic screen for direct v-Src substrates reveals ordered assembly of a retrograde signaling pathway.

Using an ATP analog that is a specific substrate for an analog-specific allele of v-Src, we identified several novel cytoskeletal substrates that control actin assembly processes. A screen for less abundant v-Src substrates revealed the scaffolding protein Dok-1 as a direct substrate of v-Src. Further studies suggest that v-Src phosphorylation sites on Dok-1 are critical for its binding to RasGAP and Csk, negative regulators of Src signaling. This results in the downregulation of growth-promoting signals of the Src family kinases and the Ras pathway. Identification of the direct substrates of v-Src leads to a model for the precise order of assembly of a retrograde signaling pathway in v-Src-transformed cells and has provided new insight into the balance between those signals that promote cell transformation mediated by v-Src catalyzed tyrosine phosphorylation and those that inhibit it.

3T3 Cells↗
PubMed2000

Functional dissection of the major structural protein of bluetongue virus: identification of key residues within VP7 essential for capsid assembly.

A lattice of VP7 trimers forms the surface of the icosahedral bluetongue virus (BTV) core. To investigate the role of VP7 oligomerization in core assembly, a series of residues for substitution were predicted based on crystal structures of BTV type 10 VP7 molecule targeting the monomer-monomer contacts within the trimer. Seven site-specific substitution mutations of VP7 have been created using cDNA clones and were employed to produce seven recombinant baculoviruses. The effects of these mutations on VP7 solubility, ability to trimerize and formation of core-like particles (CLPs) in the presence of the scaffolding VP3 protein, were investigated. Of the seven VP7 mutants examined, three severely affected the stability of CLP, while two other mutants had lesser effect on CLP stability. Only one mutant had no apparent effect on the formation of the stable capsid. One mutant in which the conserved tyrosine at residue 271 (lower domain helix 6) was replaced by arginine formed insoluble aggregates, implying an effect in the folding of the molecule despite the prediction that such a change would be accommodated. All six soluble VP7 mutants were purified, and their ability to trimerize was examined. All mutants, including those that did not form stable CLPs, assembled into stable trimers, implying that single substitution may not be sufficient to perturb the complex monomer-monomer contacts, although subtle changes within the VP7 trimer could destabilize the core. The study highlights some of the key residues that are crucial for BTV core assembly and illustrates how the structure of VP7 in isolation underrepresents the dynamic nature of the assembly process at the biological level.

Amino Acid Sequence↗
PubMed2000

Structure of the coat protein-binding domain of the scaffolding protein from a double-stranded DNA virus.

Scaffolding proteins are required for high fidelity assembly of most high T number dsDNA viruses such as the large bacteriophages, and the herpesvirus family. They function by transiently binding and positioning the coat protein subunits during capsid assembly. In both bacteriophage P22 and the herpesviruses the extreme scaffold C terminus is highly charged, is predicted to be an amphipathic alpha-helix, and is sufficient to bind the coat protein, suggesting a common mode of action. NMR studies show that the coat protein-binding domain of P22 scaffolding protein exhibits a helix-loop-helix motif stabilized by a hydrophobic core. One face of the motif is characterized by a high density of positive charges that could interact with the coat protein through electrostatic interactions. Results from previous studies with a truncation fragment and the observed salt sensitivity of the assembly process are explained by the NMR structure.

Amino Acid Sequence↗
PubMed1990

Structure and assembly of calf hoof keratin filaments.

Keratin filament polypeptides were purified from calf hoof stratum corneum with the aim of studying the in vitro assembly process and determining structural parameters of reconstituted filaments. Anion exchange chromatography was used to obtain the most complete fractionation and identification of the acidic and basic components in the purified polypeptide mixture to date. The reassembly products of the fractionated components were investigated by electron microscopy. Fully reconstituted filaments yield homogeneous solutions, and values of 9.8 nm for the filament diameter and 25 kDa/nm for the mass per unit length (M/L) were obtained by X-ray solution scattering. The structures formed in solution at various stages of filament assembly were not sufficiently homogeneous to be studied by this technique. X-ray diffraction patterns from native stratum corneum display strong maxima at 3.6 and 5.4 nm. Contrary to previous reports, these maxima do not appear to be due to lipids since they are also observed with delipidated rehydrated specimens. A series of weak maxima is also detected in the patterns of dry tissue. The absence of these features in the patterns of reconstituted filaments suggests that, in contrast to some electron microscopic observations, there are no prominent regularities in the structure of calf hoof keratin filaments.

Animals↗
PubMedDate not supplied

The protrusive phase and full development of integrin-dependent adhesions in colon epithelial cells require FAK- and ERK-mediated actin spike formation: deregulation in cancer cells.

Integrins play an important role in tumour progression by influencing cellular responses and matrix-dependent adhesion. However, the regulation of matrix-dependent adhesion assembly in epithelial cells is poorly understood. We have investigated the integrin and signalling requirements of cell-matrix adhesion assembly in colon carcinoma cells after plating on fibronectin. Adhesion assembly in these, and in the adenoma cells from which they were derived, was largely dependent on alpha v beta 6 integrin and required phosphorylation of FAK on tyrosine-397. The rate of fibronectin-induced adhesion assembly and the expression of both alpha v beta 6 integrin and FAK were increased during the adenoma-to-carcinoma transition. The matrix-dependent adhesion assembly process, particularly the final stages of complex protrusion that is required for optimal cell spreading, required the activity of extracellular signal-regulated kinase (ERK). Furthermore, phosphorylated ERK was targeted to newly forming cell--matrix adhesions in the carcinoma cells but not the adenoma cells, and inhibition of FAK--tyrosine-397 phosphorylation or MEK suppressed the appearance of phosphorylated ERK at peripheral sites. In addition, inhibition of MEK--ERK activation blocked the formation of peripheral actin microspikes that were necessary for the protrusive phase of cell-matrix adhesion assembly. Thus, MEK--ERK--dependent peripheral actin re-organization is required for the full development of integrin-induced adhesions and this pathway is stimulated in an in vitro model of colon cancer progression.

Actins↗
PubMed1991

Immunological characterization of chromatin assembly factor I, a human cell factor required for chromatin assembly during DNA replication in vitro.

Chromatin assembly factor I (CAF-I) is a multisubunit protein complex purified from the nuclei of human cells and required for chromatin assembly during DNA replication in vitro. Purified CAF-I promotes chromatin assembly in a reaction that is dependent upon, and coupled with, DNA replication and is therefore likely to reflect events that occur during S phase in vivo. In order to investigate the regulation and mechanism of CAF-I and the replication-dependent chromatin assembly process, we have used the purified protein to raise monoclonal antibodies. In this report we describe the characterization of a panel of monoclonal antibodies which recognize different subunits of the CAF-I complex. We use immunoprecipitation analysis to show that CAF-I exists as a multiprotein complex in vivo and that some of the polypeptides are phosphorylated. In addition, immunocytochemistry demonstrates that CAF-I is localized to the nucleus of human cells. Finally, monoclonal antibodies directed against the individual subunits of CAF-I immunodeplete chromatin assembly activity from nuclear extracts, confirming that CAF-I is a multisubunit protein required for chromatin assembly in vitro.

Antibodies, Monoclonal↗
PubMed1991

Tissue-specific control of expression of the tight junction polypeptide ZO-1 in the mouse early embryo.

The processes governing differential protein expression in preimplantation lineages were investigated using a monoclonal antibody recognising the tight junction polypeptide, ZO-1. ZO-1 localises to the maturing tight junction membrane domain in the polarised trophectoderm lineage from compaction (8-cell stage) onwards, ultimately forming a zonular belt around each trophectoderm cell of the blastocyst (32- to 64-cell stage). The protein is usually undetectable within the inner cell mass (ICM) although, in a minority of embryos, punctate ZO-1 sites are present on the surface of one or more ICM cells. Since ICM cells derive from the differentiative division of polarised 8- and 16-cell blastomeres, the distribution of ZO-1 following differentiative division in isolated, synchronised cell clusters of varying size, was examined. In contrast to the apical cytocortical pole, ZO-1 was found to be inherited by nonpolar (prospective ICM) as well as polar (prospective trophectoderm) daughter cells. Following division, polar cells adhere to and gradually envelop nonpolar cells. Prior to envelopment, ZO-1 localises to the boundary between the contact area and free membrane of daughter cells, irrespective of their phenotype. After envelopment, polar cells retain these ZO-1 contact sites whilst nonpolar cells lose them, in which case ZO-1 transiently appears as randomly-distributed punctate sites on the membrane before disappearing. Thus, symmetrical cell contact appears to initiate ZO-1 down-regulation in the ICM lineage. The biosynthetic level at which ZO-1 down-regulation occurs was investigated in immunosurgically isolated ICMs undergoing trophectoderm regeneration. By 6 h in culture, isolated ICMs generated a zonular network of ZO-1 at the contact area between outer cells, thereby demonstrating the reversibility of down-regulation. This assembly process was unaffected by alpha-amanitin treatment but was inhibited by cycloheximide. These results indicate that the ICM inherits and stabilises ZO-1 transcripts which can be utilised for rapid synthesis and assembly of the protein, a capacity that may have significance both in maintaining lineage integrity within the blastocyst and in the subsequent development of the ICM.

Animals↗
PubMed1993

Fragmentation and dispersal of Golgi proteins and redistribution of glycoproteins and glycolipids processed through the Golgi apparatus after infection with herpes simplex virus 1.

In Vero monkey cells and HEp-2 human epidermoid carcinoma cells infected with herpes simplex virus 1 the proteins beta-COP, galactosyltransferase, and alpha-mannosidase II associated with the Golgi apparatus appear to be associated with numerous smaller structures dispersed throughout the cytoplasm. Concomitantly, the intracytoplasmic ligands of lectins normally associated wholly (Helix pomatia or Ricinus communis agglutinin) or in part (wheat germ agglutinin) with the Golgi apparatus increased in amount and became dispersed. This phenomenon was seen in some of the baby hamster kidney cells analyzed but not in others and not in the human 143TK- cells. The fragmentation and dispersal of the Golgi apparatus was a late event in the reproductive cycle coinciding with virion assembly, processing of viral glycoproteins, and exocytosis from infected cells. The fragmentation of the Golgi apparatus is morphologically different from that seen with brefeldin A and may reflect disequilibration between the anterograde and retrograde Golgi transport caused by the huge influx of viral glycoproteins contained in virions and membranes flowing through the exocytic pathway.

Animals↗
PubMed1997

Assembly of protein structures on liposomes by non-specific and specific interactions.

We investigate different schemes for fabrication of nanometer sized assemblies that consist of a liposome core over which a shell of ferritin is attached. Three distinct interactions were used for this assembly: (i) Electrostatic attraction. The liposomes are charged by the presence of cationic surfactant (HTAB) and at an appropriate pH collect the ferritin molecules into a 2D-ordered ferritin shell. The protein shells can be fixed by glutaraldehyde. Next, the liposomes can be removed by solubilisation, leaving behind ordered ferritin clusters. (ii) Specific avidin-biotin or streptavidin-biotin binding. The ferritin molecules are conjugated to avidin or streptavidin and the liposomes incorporate biotinylated lipid. We found that the specific binding can be completely blocked by unfavourable electrostatic repulsion. To adjust the appropriate liposome charge we include cationic surfactant in the lipid layer. Thus, to accomplish the assembly process, we need to design and modify both the specific and non-specific colloid interactions in the system. The result is liposomes heavily coated with a strongly and specifically attached ferritin layer. (iii) Specific polysaccharide/lectin binding. The liposomes are first coated with a cholesterol-anchored mannan layer. The ferritin molecules are conjugated with Con A that binds to the polysaccharide. A smooth and dense coating with ferritin is obtained (Fig. 3b). The acquired data can find application in the future fabrication of microstructured, multicomponent, or functionalised protein and liposome/ protein assemblies.

Avidin↗
PubMed2000

Measles virus structural components are enriched into lipid raft microdomains: a potential cellular location for virus assembly.

The process of measles virus (MV) assembly and subsequent budding is thought to occur in localized regions of the plasma membrane, to favor specific incorporation of viral components, and to facilitate the exclusion of host proteins. We demonstrate that during the course of virus replication, a significant proportion of MV structural proteins were selectively enriched in the detergent-resistant glycosphingolipids and cholesterol-rich membranes (rafts). Isolated rafts could infect the cell through a membrane fusion step and thus contained all of the components required to create a functional virion. However, they could be distinguished from the mature virions with regards to density and Triton X-100 resistance behavior. We further show that raft localization of the viral internal nucleoprotein and matrix protein was independent of the envelope glycoproteins, indicating that raft membranes could provide a platform for MV assembly. Finally, at least part of the raft MV components were included in the viral particle during the budding process. Taken together, these results strongly suggest a role for raft membranes in the processes of MV assembly and budding.

Cell Line↗
PubMed1998

The use of 3-dimensional models in auricular reconstruction.

Reconstruction of the microtic auricle is a difficult process requiring considerable experience and dedication to detail. It is a multistage proposition requiring the talents of both the reconstructive surgeon and the otologic surgeon. Reconstruction of the external ear usually precedes the reconstruction of the middle ear. Often, a template is used by the reconstructive surgeon to aid in this complicated process. Traditionally, templates used by the reconstructive surgeon have been 2 dimensional (usually x-ray paper) and made from the opposite normal ear or another normal ear in cases of bilateral microtia. Use of a 2-dimensional model only provides a rough estimate of the cartilage framework needed. Considerable experience is therefore needed to get this cartilage framework "just right." We have developed a number of 3-dimensional synthetic templates to aid in the creation of an accurate cartilage framework implant. These templates serve as a more accurate guide in the complex cartilage carving and assembly process. The use of 3-dimensional templates has improved our technical reconstructive results in a small number of patients. We present these results and propose future application of these ideas.

Cartilage↗
PubMed1994

Fibrinogen assembly: insights from chicken hepatocytes.

In all vertebrate species studied, the complex, disulfide-linked structure of fibrinogen is essentially the same: a hexamer assembled from three different subunits (A alpha, B beta, gamma)2. This study utilized species differences in fibrinogen subunit monomer pools to address the question of how these surplus subunit pools may affect the assembly process. We used a chicken model system in which B beta and gamma-subunits are present in excess, in contrast to the A alpha and gamma-subunit surplus found in human model systems. Analysis was based on pulse-chase experiments with electrophoretic separation of intracellular forms and secreted fibrinogen on reducing and nonreducing gels. The chicken liver-derived cells employed for this purpose, primary hepatocytes and a hepatoma cell line with a fortuitous defect in fibrinogen synthesis, together offer advantages over human systems for resolving the complexes formed in the early stages of assembly. The results demonstrate that in chicken hepatocytes there is an initial binding of gamma to A alpha subunits rather than to B beta subunits, as occurs in human hepatoma cells. Nevertheless, the presence of similar intracellular fibrinogen-related forms in both chicken- and human-derived cells, in the context of their differing subunit monomer pools, suggests an assembly pathway common to both species, with the versatility to be regulated by limitation of A alpha or B beta subunit production.

Animals↗
PubMedDate not supplied

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↗