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PubMed1998

A double-headed Gly-Pro-Arg-Pro ligand mimics the functions of the E domain of fibrin for promoting the end-to-end crosslinking of gamma chains by factor XIIIa.

The E domain of fibrinogen represents the central region of the protein that, after the removal of fibrinopeptides from the N-termini of its alpha chains by thrombin, orders the noncovalent assembly of fibrin units into a half-staggered array. This structural organization is accomplished purely through noncovalent binding between the E domain of one molecule and the distal D domains of two others. The process of assembly has a physiologically important up-regulatory effect on the next enzymatic phase of blood coagulation, which is the factor XIIIa-catalyzed end-to-end ligation of the gamma chains at the D domains of the protein. Fibrin assembly, as well as the acceleration of the factor XIIIa reaction, could be prevented by Gly-Pro-Arg-Pro, a homologue of the natural sequence of amino acids at the N termini of alpha chains in the E domain. We have now succeeded with a simple double-headed ligand, bis(Gly-Pro-Arg-Pro-amido)polyethylene glycol, in fully replacing the regulatory functions of the large E domains of the native protein.

Cross-Linking Reagents↗
PubMed1996

Inefficient assembly limits transport and cell surface expression of HLA-Cw4 molecules in C1R.

HLA-C antigens are expressed to the cell surface at roughly 10% the level of HLA-B or -A, and their serological definition remains persistently difficult. To characterize the factors limiting surface expression, the processes of assembly and intracellular transport of HLA-Cw4 molecules were investigated in the C1R cell line. When appropriate peptides were added to cultured cells or in cell lysates significant amounts of conformed HLA-C molecules that associate with beta 2-microglobulin (beta 2 m) are detected, but are indeed not sufficient to restore expression to the level observed for HLA-A or -B molecules. Furthermore, a precursor/product relationship exists between the free class I heavy chain and the mature conformation of HLA-Cw4 molecules. Thus, HLA-C assembly promotes the conversion of HC-10-reactive molecules (weakly-beta 2m-associated non-ligand associated free HC form) into the beta 2m-associated class I molecules recognized by W6/32. To further investigate the factors that regulate cell surface expression, intracellular transport of HLA-Cw4 was studied in pulse chase analysis. In contrast to some HLA-A and B, maturation of HLA-Cw4 heavy chains and their export to the medial and trans-Golgi compartments are quite inefficient. After 4 h of chase period, roughly half of the pulse-labeled HLA-Cw4 molecules have transited to the medial-Golgi and acquired complex oligosaccharides characteristic of mature form. In addition, treatment with gamma-interferon does not appear to improve maturation of HLA-Cw4 heavy chains, suggesting that increased supply of peptides does not influence intracellular transport. Moreover, only a small fraction in the pool of HLA-Cw4 molecules was subsequently transported through the trans-Golgi network, as indicated by their acquisition of sialic acids. Taken together these studies show that HLA-Cw4 molecules are inefficiently transported through the Golgi apparatus and presumably retained in the endoplasmic reticulum or cis-Golgi compartment.

Biological Transport↗
PubMed1992

Insertion and assembly of the precursor of subunit II into the photosystem I complex may precede its processing.

The biogenesis and assembly of subunit II of photosystem I (PSI) (psaD gene product) were studied and characterized. The precursor and the mature form were produced in vitro and incubated with intact plastids or isolated thylakoids. Following import of the precursor into isolated plastids, mostly the mature form of subunit II was found in the thylakoids. However, when the processing activity was inhibited only the precursor form was present in the membranes. The precursor was processed by a stromal peptidase and processing could occur before or after insertion of the precursor into the thylakoids. Following insertion into isolated thylakoids, both the precursor and the mature form of subunit II were confined to the PSI complex. Insertion of the mature form of subunit II was much less efficient than that of the precursor. Kinetic studies showed that the precursor was inserted into the membrane. Only at a later stage, the mature form began to accumulate. These results suggest that in vivo the precursor of subunit II is inserted and embedded in the thylakoids, as part of the PSI complex. Only later, it is processed to the mature form through the action of a stromal peptidase.

Base Sequence↗
PubMed2006

Physical map-assisted whole-genome shotgun sequence assemblies.

We describe a targeted approach to improve the contiguity of whole-genome shotgun sequence (WGS) assemblies at run-time, using information from Bacterial Artificial Chromosome (BAC)-based physical maps. Clone sizes and overlaps derived from clone fingerprints are used for the calculation of length constraints between any two BAC neighbors sharing 40% of their size. These constraints are used to promote the linkage and guide the arrangement of sequence contigs within a sequence scaffold at the layout phase of WGS assemblies. This process is facilitated by FASSI, a stand-alone application that calculates BAC end and BAC overlap length constraints from clone fingerprint map contigs created by the FPC package. FASSI is designed to work with the assembly tool PCAP, but its output can be formatted to work with other WGS assembly algorithms able to use length constraints for individual clones. The FASSI method is simple to implement, potentially cost-effective, and has resulted in the increase of scaffold contiguity for both the Drosophila melanogaster and Cryptococcus gattii genomes when compared to a control assembly without map-derived constraints. A 6.5-fold coverage draft DNA sequence of the Pan troglodytes (chimpanzee) genome was assembled using map-derived constraints and resulted in a 26.1% increase in scaffold contiguity.

Animals↗
PubMed1984

Asymmetric acetylcholinesterase is assembled in the Golgi apparatus.

The synthesis, assembly, and processing of the multiple molecular forms of acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, EC 3.1.1.7) in quail muscle cultures was studied by using lectins to distinguish enzyme molecules residing in different subcellular compartments. Special emphasis was given to the assembly of asymmetric AcChoEase molecules because these appear to be the predominant, if not unique, forms of AcChoEase at the vertebrate neuromuscular junction. All cell surface and secreted AcChoEase forms bind to immobilized wheat germ agglutinin, ricin, and concanavalin A, indicating that they have complex oligosaccharides. After treatment of muscle cells with a membrane-permeable irreversible AcChoEase inhibitor, there is a rapid reappearance of the globular monomeric, dimeric, and tetrameric AcChoEase forms. However, the collagen-tailed asymmetric form does not appear until about 90 min after treatment. Analysis of the AcChoEase oligosaccharides with lectins indicates maturation to complex forms over a 90-min period. A large fraction of the intracellular globular AcChoEase molecules bind only to concanavalin A, indicating that they are assembled in the rough endoplasmic reticulum. In contrast, all intracellular asymmetric AcChoEase binds to wheat germ agglutinin, and a significant fraction binds to ricin, indicating that this unique AcChoEase form is assembled from subunits that have previously acquired complex sugars. I conclude that assembly of asymmetric AcChoEase, hence acquisition of information specifying basal lamina localization, occurs in the Golgi apparatus.

Acetylcholinesterase↗
PubMed1993

Signal sequence processing is required for the assembly of LamB trimers in the outer membrane of Escherichia coli.

Proteins destined for either the periplasm or the outer membrane of Escherichia coli are translocated from the cytoplasm by a common mechanism. It is generally assumed that outer membrane proteins, such as LamB (maltoporin or lambda receptor), which are rich in beta-structure, contain additional targeting information that directs proper membrane insertion. During transit to the outer membrane, these proteins may pass, in soluble form, through the periplasm or remain membrane associated and reach their final destination via sites of inner membrane-outer membrane contact (zones of adhesion). We report lamB mutations that slow signal sequence cleavage, delay release of the protein from the inner membrane, and interfere with maltoporin biogenesis. This result is most easily explained by proposing a soluble, periplasmic LamB assembly intermediate. Additionally, we found that such lamB mutations confer several novel phenotypes consistent with an abortive attempt by the cell to target these tethered LamB molecules. These phenotypes may allow isolation of mutants in which the process of outer membrane protein targeting is altered.

Bacterial Outer Membrane Proteins↗
PubMed1993

Deletion analysis of K+ channel assembly.

An understanding of K+ channel structure is a critical step in developing an appreciation of the function and regulation of these proteins. We have begun a biochemical analysis of the early steps in K+ channel formation following translation into endoplasmic reticulum membranes. In our experiments, a series of K+ channel subunit protein deletions were constructed and then tested for posttranslational processing and assembly. We find that all deletions containing the S1 domain are inserted into the membrane. The loop between S1 and S2 is glycosylated; thus, this segment is topologically extracellular. Translated subunit proteins mix in the membrane, then assemble into tetramers. This subunit assembly is critically driven by a conserved, self-tetramerizing sequence in the N-terminal cytoplasmic region, which we have named the tetramerization 1 domain.

Animals↗
PubMed1990

Mutational analysis of muscle nicotinic acetylcholine receptor subunit assembly.

The structural elements required for normal maturation and assembly of the nicotinic acetylcholine receptor alpha subunit were investigated by expression of mutated subunits in transfected fibroblasts. Normally, the wild-type alpha subunit acquires high affinity alpha bungarotoxin binding in a time-dependent manner; however, mutation of the 128 and/or 142 cysteines to either serine or alanine, as well as deletion of the entire 14 amino acids in this region abolished all detectable high affinity binding. Nonglycosylated subunits that had a serine to glycine mutation in the consensus sequence also did not efficiently attain high affinity binding to toxin. In contrast, mutation of the proline at position 136 to glycine or alanine, or a double mutation of the cysteines at position 192 and 193 to serines had no effect on the acquisition of high affinity toxin binding. These data suggest that a disulfide bridge between cysteines 128 and 142 and oligosaccharide addition at asparagine 141 are required for the normal maturation of alpha subunit as assayed by high affinity toxin binding. The unassembled wild-type alpha subunit expressed in fibroblasts is normally degraded with a t1/2 of 2 h; upon assembly with the delta subunit, the degradation rate slows significantly (t1/2 greater than 13 h). All mutated alpha subunits retained the capacity to assemble with a delta subunit coexpressed in fibroblasts; however, mutated alpha subunits that were not glycosylated or did not acquire high affinity toxin binding were rapidly degraded (t1/2 = 20 min to 2 h) regardless of whether or not they assembled with the delta subunit. Assembly and rapid degradation of nonglycosylated acetylcholine receptor (AChR) subunits and subunit complexes were also observed in tunicamycin-treated BC3H-1 cells, a mouse musclelike cell line that normally expresses functional AChR. Hence, rapid degradation may be one form of regulation assuring that only correctly processed and assembled subunits accumulate, and ultimately make functional receptors in AChR-expressing cells.

Animals↗
PubMed1998

The role of assembly in insulin's biosynthesis.

Insulin is synthesised as a single-chain precursor, preproinsulin, that contains an N-terminal signal sequence and a connecting peptide linking the A and B chains of the insulin molecule. Nascent proinsulin is directed into the regulated secretory pathway, converted to insulin and stored as microcrystals. These processes exploit assembly to the zinc-containing hexamer. Structural, chemical and genetic studies, and experiments with transgenic animals and transfected cells are providing new details about the molecular events in insulin's biosynthesis.

Animals↗
PubMed1990

Protein transport into mitochondria is conserved between plant and yeast species.

Protein targeting into plant mitochondria was investigated by in vitro translocation experiments. The precursor of the mitochondrial F1-ATPase beta subunit from Nicotiana plumbaginifolia was synthesized in vitro, translocated to, processed, and assembled in purified Vicia faba mitochondria. Transport (but not binding) required a membrane potential and external nucleotides and was conserved among plant species. beta subunit precursors from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe were imported and correctly processed in plant mitochondria. This translocation used protease-sensitive components of the outer membrane. Conversely, the N. plumbaginifolia beta subunit precursor was efficiently translocated and cleaved in yeast mitochondria. However, a precursor for a chloroplast protein was not targeted to plant or yeast mitochondria. We conclude that the machinery for protein import into mitochondria is specific and conserved in plant and yeast organisms. These results are discussed in the context of a poly- or monophyletic origin of mitochondria.

Amino Acid Sequence↗
PubMed2006

Molecular genetic analysis of Escherichia coli type I adhesins.

Protein FimH is an adhesive terminal subunit of Escherichia coli type I pili. The content of FimH on the surface of a bacterial cell does not directly depend on FimH expression, but is limited by expression of other fim cluster genes encoding structural subunits and proteins responsible for assembly of pili. Piline domain, a component of FimH, cannot incorporate into the fimbria independently, without lectin domain. It seems that fimbriae containing only piline domain are not formed because of inability of the piline domain to initiate the process of assembly.

Adhesins, Bacterial↗
PubMedDate not supplied

Overexpression of a cytosolic chaperone to improve solubility and secretion of a recombinant IgG protein in insect cells.

The secretion of heterologous IgG proteins in the baculovirus-insect cell expression system is accompanied by substantial insoluble immunoglobulin in the infected cells. The accumulation of these insoluble forms suggests a limitation in the processing and secretory pathway of the infected cells. As a result, cytosolic hsp70 chaperones, which are known to associate and prevent aggregation of polypeptides in vitro, have been coexpressed in the infected cells. The hsp70 protein coprecipitated with the immunoglobulin to indicate the formation of a specific hsp70-immunoglobulin complex in vivo. Immunoblot and pulse chase studies indicated that coexpression of hsp70 increased intracellular immunoglobulin solubility. Metabolic labeling experiments revealed that hsp70 increased secreted immunoglobulin levels after several days infection as compared to infection with control baculoviruses. Pulse chase studies indicated that hsp70 increases the solubility of immunoglobulin precursors that are then processed and assembled into the complete antibody oligomer. A comparison of the action of cytosolic hsp70 chaperone to the endoplasmic reticulum chaperone BiP suggests sequential action in which hsp70 increases the solubility of preprocessed immunoglobulin, while BiP enhances the solubility of processed immunoglobulin chains.

Animals↗
PubMed2004

The bounty of RAGs: recombination signal complexes and reaction outcomes.

V(D)J recombination is a form of site-specific DNA rearrangement through which antigen receptor genes are assembled. This process involves the breakage and reunion of DNA mediated by two lymphoid cell-specific proteins, recombination activating genes RAG-1 and RAG-2, and ubiquitously expressed architectural DNA-binding proteins and DNA-repair factors. Here I review the progress toward understanding the composition, assembly, organization, and activity of the protein-DNA complexes that support the initiation of V(D)J recombination, as well as the molecular basis for the sequence-specific recognition of recombination signal sequences (RSSs) that are the targets of the RAG proteins. Parallels are drawn between V(D)J recombination and Tn5/Tn10 transposition with respect to the reactions, the proteins, and the protein-DNA complexes involved in these processes. I also consider the relative roles of the different sequence elements within the RSS in recognition, cleavage, and post-cleavage events. Finally, I discuss alternative DNA transactions mediated by the V(D)J recombinase, the protein-DNA complexes that support them, and factors and forces that control them.

Animals↗
PubMed1993

Assembly of in vitro synthesized large subunits into ribulose-bisphosphate carboxylase/oxygenase. Formation and discharge of an L8-like species.

Ribulose-bisphosphate carboxylase/oxygenase (Rubisco) from higher plants consists of eight approximately 53-kDa large subunits and eight approximately 14-kDa small subunits. Cytosolic ribosomes synthesize the small subunits as precursors, which enter the chloroplast, undergo proteolytic processing, and assemble with large subunits. Large subunits, synthesized in the chloroplast, first form a complex with the chloroplast chaperonin 60 (Cpn60(14)). In the presence of ATP, large subunits dissociate from Cpn60(14) and assemble into Rubisco. We now describe partial characterization of a new species, Z, containing radiotracer-labeled, newly synthesized pea Rubisco large subunits. Rubisco assembly occurs in low salt in the presence of small subunits and ATP. As with Rubisco assembly, the formation of Z is ATP-dependent and is inhibited by high chloride. Once formed, Z is stable except in high chloride. Z does not appear to interact directly with small subunits. However, after Z formation, Rubisco assembly occurs in an ATP-independent reaction that requires KCl and small subunits. These results are consistent with the hypothesis that Z is a large subunit containing structure that can contribute large subunits to Rubisco under appropriate conditions. Z shares some physical characteristics with reported cyanobacterial L8 core particles. However, formation of Rubisco from Z in the absence of ATP and the presence of small subunits appears to require conditions that otherwise destabilize Z.

Adenosine Triphosphate↗
PubMed2004

Local and remote functional connectivity of neocortex under the inhibition influence.

The current paper focuses on a relatively new and promising area of the study of EEG transformations during brain information processing based on the reduction of the signal to the discrete quasi-stationary segment sequences which may reflect individual brain microstates or discrete operations. In this framework, the complex brain functions require integration of several operations throughout the whole neocortex. However, the role of inhibitory brain systems in such processes is still unsettled. The effects of a single dose (30 microg/kg) of lorazepam on the operational activity of neuronal populations and on the temporal binding between them were examined in a double-blind randomized crossover placebo-controlled study with eight healthy volunteers. EEG measures at 20 channels were evaluated on two occasions: (1) eyes closed, (2) eyes open. In short, we conducted a two-by-two factorial study where one factor manipulated GABAergic neurotransmission (lorazepam vs. placebo), and the other factor was simply brain state (eyes closed vs. eyes opened). We were primarily interested in the main effect of lorazepam. In the present study, a connection between the mesoscopic level, described by the local functional processes (neuronal assemblies or populations) and the macroscopic level, described as a sequence of metastable brain states (remote functionally synchronized neuronal populations) was established. The role of inhibitory brain systems facilitated by lorazepam in the operational dynamics of neuronal populations and in the process of EEG structural synchrony (SS) (topological peculiarities) was addressed for the first time. It was shown that GABA signaling reorganized the dynamics of local neuronal populations and the remote functional connectivity between them.

Adult↗
PubMed1991

Pairwise, cooperative and inhibitory interactions describe the assembly and probable structure of the T-cell antigen receptor.

The T-cell antigen receptor (TCR) is a multi-subunit complex consisting of clonotypic heterodimers (TCR-alpha beta or TCR-gamma delta) that are non-covalently linked to at least four invariant chains (CD3-delta, -epsilon, -gamma; and zeta or eta). The ordered process of assembly and the final number of individual chains that comprise the TCR is unclear. In this study, we examined the molecular basis of subunit interactions and the component requirements leading to the formation of a complete TCR. Analysis of transient cotransfections in monkey kidney fibroblasts (COS cells) showed assembly between selective chain pairs. Multiple chain cotransfections demonstrated the formation of stable higher order partial complexes. Assembly of such subcomplexes was facilitated by cooperative interactions between clonotypic and invariant CD3 chains. When zeta was cotransfected with any TCR component, no pairwise interaction was detected. Only when there was coexpression of all of the other TCR chains (TCR-alpha, -beta, CD3- epsilon, -gamma, -delta) did zeta assemble with the TCR complex. Not all chain pairs formed stable heterodimers. For one such pair, lack of assembly is due to the inhibitory effects of negatively charged residues within their transmembrane domains. The combined effects of these interactions probably determine the assembly and the quaternary structure of the TCR complex.

Animals↗
PubMed1990

Increased gap junction assembly between cultured cells upon cholesterol supplementation.

Novikoff hepatoma cells provide an excellent model system for the study of gap junction assembly, a process that could be influenced by lipids and other factors at numerous points. Since it is possible to alter the cellular levels of cholesterol in these cells, it was added to the cells in serum-supplemented medium and changes in gap junction assembly were evaluated. Cells were dissociated and reaggregated following exposure to a range of cholesterol concentrations for 24 h. A five- to sixfold increase in the number of aggregated gap junction particles and a 50% increase in cellular cholesterol content were observed with 20 microM added cholesterol. A 1-h exposure to added cholesterol, during cell reaggregation, resulted in a fourfold increase in the number of aggregated gap junction particles, demonstrating that the effect was rapid. The number of aggregated gap junction particles and formation plaque areas were used as measures of junction assembly and assayed by quantitative freeze-fracture and electron microscopy. Junctional permeabilities were evaluated by means of dye transfer times following the intracellular microinjection of Lucifer Yellow. Increased dye transfer was observed between cholesterol-treated cells, which suggested that the increase in assembly was accompanied by an increase in junction permeability. Cells were treated with cycloheximide (100 micrograms ml-1) and actinomycin D (10 micrograms ml-1) to determine whether protein and RNA syntheses were involved in the enhanced gap junction assembly. Cycloheximide but not actinomycin D blocked the increased junction assembly observed with added cholesterol. These results suggested that protein synthesis, but not RNA synthesis, is necessary for the increased gap junction formation observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Human RISC couples microRNA biogenesis and posttranscriptional gene silencing.

RNA interference is implemented through the action of the RNA-induced silencing complex (RISC). Although Argonaute2 has been identified as the catalytic center of RISC, the RISC polypeptide composition and assembly using short interfering RNA (siRNA) duplexes has remained elusive. Here we show that RISC is composed of Dicer, the double-stranded RNA binding protein TRBP, and Argonaute2. We demonstrate that this complex can cleave target RNA using precursor microRNA (pre-miRNA) hairpin as the source of siRNA. Although RISC can also utilize duplex siRNA, it displays a nearly 10-fold greater activity using the pre-miRNA Dicer substrate. RISC distinguishes the guide strand of the siRNA from the passenger strand and specifically incorporates the guide strand. Importantly, ATP is not required for miRNA processing, RISC assembly, or multiple rounds of target-RNA cleavage. These results define the composition of RISC and demonstrate that miRNA processing and target-RNA cleavage are coupled.

Adenosine Triphosphate↗