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PubMed2006

A novel tricyclic pyrone compound ameliorates cell death associated with intracellular amyloid-beta oligomeric complexes.

The neurotoxicity of amyloid-beta protein (Abeta) is widely regarded as one of the fundamental causes of neurodegeneration in Alzheimer's disease (AD). This toxicity is related to Abeta aggregation into oligomers, protofibrils and fibrils. Recent studies suggest that intracellular Abeta, which causes profound toxicity, could be one of the primary therapeutic targets in AD. So far, no compounds targeting intracellular Abeta have been identified. We have investigated the toxicity induced by intracellular Abeta in a neuroblastoma MC65 line and found that it was closely related to intracellular accumulation of oligomeric complexes of Abeta (Abeta-OCs). We further identified a cell-permeable tricyclic pyrone named CP2 that ameliorates this toxicity and significantly reduces the levels of Abeta-OCs. In aqueous solution, CP2 attenuates Abeta oligomerization and prevents the oligomer-induced death of primary cortical neurons. CP2 analogs represent a new class of promising compounds for the amelioration of Abeta toxicities within both intracellular and extracellular sites.

Amyloid beta-Peptides↗
PubMed2007

Oligomerization is a specific requirement for apical sorting of glycosyl-phosphatidylinositol-anchored proteins but not for non-raft-associated apical proteins.

Protein apical sorting in polarized epithelial cells is mediated by two different mechanisms, raft dependent and raft independent. In Madin-Darby canine kidney (MDCK) cells, an essential step for apical sorting of glycosyl-phosphatidylinositol (GPI)-anchored proteins (GPI-APs) is their coalescence into high-molecular-weight (HMW) oligomers. Here we show that this mechanism is also functional in Fischer rat thyroid cells, which possess a different sorting phenotype compared with MDCK cells. We demonstrate that, as in MDCK cells, both apical and basolateral GPI-APs associate with detergent-resistant microdomains, but that only the apical proteins are able to oligomerize into HMW complexes during their passage through the medial Golgi. We also show that oligomerization is a specific requirement for apical sorting of GPI-APs and is not used by transmembrane, non-raft-associated apical proteins.

Animals↗
PubMed2004

Multiple interactions between transmembrane helices generate the oligomeric alpha1b-adrenoceptor.

Combinations of coimmunoprecipitation, single-cell fluorescence resonance energy transfer, and cell-surface time-resolved fluorescence resonance energy transfer demonstrated protein-protein interactions and quaternary structure for the alpha(1b)-adrenoceptor. Self-association of transmembrane domain 1 and its interaction with the full-length receptor indicated a symmetrical interface provided by this domain. Lack of effect of mutation of the glycophorin-A dimerization-like region within this helix demonstrated that this did not provide the molecular mechanism. Multiple interactions were observed between the alpha(1b)-adrenoceptor and fragments derived from its sequence. Fragments comprising transmembrane domains 3 and 4 and transmembrane domains 5 and 6, but not transmembrane domain 7, were also able to interact with the full-length receptor. Transmembrane domain 7 failed to interact significantly with any element of the receptor and was not transported to the cell surface after coexpression with the full-length receptor. Symmetrical interactions were also noted between fragments incorporating transmembrane domain 4, but this segment of the receptor failed to interact with transmembrane domains 1 and 2 or transmembrane domains 5 and 6. Time-resolved fluorescence resonance energy transfer studies were also consistent with contributions of transmembrane domains 1 and/or 2 and transmembrane domains 3 and/or 4 to protein-protein interactions within the quaternary structure of the alpha(1b)-adrenoceptor, and with a contribution of transmembrane domains 5 and/or 6. These data are consistent with a complex oligomeric quaternary structure of the alpha(1b)-adrenoceptor in which major, symmetrical interactions may define intradimeric contacts with other contributions, providing interdimer contacts to generate oligomeric complexes akin to those observed for murine rhodopsin. A model derived from this was developed.

Animals↗
PubMed1985

Oligomerization of intervening sequence RNA molecules in the absence of proteins.

The intervening sequence RNA excised from the ribosomal RNA precursor of Tetrahymena forms linear and circular oligomers when exposed to a heating-cooling treatment in vitro. The reactions require no protein or external energy source. Oligomerization is different from other self-catalyzed reactions of the intervening sequence RNA in that it involves intermolecular rather than intramolecular recombination, producing RNA molecules that are substantially larger than the original. The observation that RNA molecules can catalyze their own oligomerization has possible implications for the evolution of chromosomes and for the replicative cycle of plant viroids and virus-associated RNA's.

Animals↗
PubMed1997

Structural basis for ligand-regulated oligomerization of AraC.

The crystal structure of the arabinose-binding and dimerization domain of the Escherchia coli gene regulatory protein AraC was determined in the presence and absence of L-arabinose. The 1.5 angstrom structure of the arabinose-bound molecule shows that the protein adopts an unusual fold, binding sugar within a beta barrel and completely burying the arabinose with the amino-terminal arm of the protein. Dimer contacts in the presence of arabinose are mediated by an antiparallel coiled-coil. In the 2.8 angstrom structure of the uncomplexed protein, the amino-terminal arm is disordered, uncovering the sugar-binding pocket and allowing it to serve as an oligomerization interface. The ligand-gated oligomerization as seen in AraC provides the basis of a plausible mechanism for modulating the protein's DNA-looping properties.

AraC Transcription Factor↗
PubMed1987

Quantitative analysis of the binding and oligomerization of staphylococcal alpha-toxin in target erythrocyte membranes.

The binding of staphylococcal alpha-toxin to rabbit and human erythrocytes was quantitated over a wide range of toxin concentrations (3 x 10(-11) to 3 x 10(-6) M) with the use of an enzyme-linked immunosorbent assay that permitted simultaneous quantitation of monomeric and oligomeric toxin forms. Three basic observations were made. First, in no range of concentrations did the binding of alpha-toxin to rabbit erythrocytes display characteristics of a receptor-ligand interaction. Net binding to rabbit cells was nil at sublytic concentrations (10(-10) M or 3 ng/ml). The onset of binding occurred at around 10 ng/ml and remained fairly constant and ineffective (5 to 8% of toxin offered) over a wide concentration range (up to 10 micrograms/ml). Second, hemolysis of rabbit and human erythrocytes at 37 degrees C was always accompanied by the formation of toxin oligomers in the membrane. Third, overall toxin binding at 0 degree C followed a pattern similar to that at 37 degrees C. However, oligomer formation and cell lysis were retarded (but not totally inhibited) at 0 degree C. When rabbit erythrocytes were incubated with low levels of toxin at 0 degree C (0.5 microgram/ml) for 30 min, the toxin became bound exclusively in monomer form, and no lysis occurred. When cells thus treated were washed and suspended at 37 degrees C, lysis rapidly ensued, and native monomeric toxin was replaced by oligomeric toxin. The collective results directly support the oligomer pore concept of toxin action and also indicate that toxin oligomers form by lateral aggregation of bound monomers in the bilayer. They speak against the existence of specific binding sites for alpha-toxin on rabbit erythrocytes.

Animals↗
PubMed1988

Identification of monomeric and oligomeric forms of a major Leishmania infantum antigen by using monoclonal antibodies.

Ten monoclonal antibodies (MAbs) produced against isolated Leishmania infantum membranes were used as probes of L. infantum membrane antigens. Western blots of L. infantum membranes, sodium dodecyl sulfate solubilized and heated at 100 degrees C before analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed that all 10 MAbs recognized a band at 58 kilodaltons (kDa). However, when solubilized membranes were not heated, 2 of the 10 MAbs recognized, in addition to the 58-kDa band, bands of higher molecular weight. Limited digestion of heated or nonheated membranes showed that both groups of MAbs (i.e., not capable or capable of binding to the high-molecular-weight bands) recognized the same proteolytic digests. Hydrophilic forms of the above proteins, possessing proteolytic activity, were detected and isolated by gel filtration. Protein staining of the isolated monomer analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under reducing and heating conditions, revealed incomplete reduction of the 58-kDa protein. The reduced form of the 58-kDa protein migrated at 63 to 65 kDa and was not recognized by the MAbs. These results suggest the existence of a monomeric and an oligomeric form of the 58-kDa antigen. The observed inhibition of Leishmania promastigote-macrophage binding caused by MAbs representative of the two groups (capable of oligomeric and/or monomeric antigen recognition) suggest that the 58-kDa monomer and oligomer play an important role in promastigote-macrophage interaction. We suggest that the 58-kDa L. infantum antigen is the major surface Leishmania antigen (p63) identified by others.

Animals↗
PubMed1999

Oligomerization of anthrax toxin protective antigen and binding of lethal factor during endocytic uptake into mammalian cells.

The protective antigen (PA) protein of anthrax toxin binds to a cellular receptor and is cleaved by cell surface furin to produce a 63-kDa fragment (PA63). The receptor-bound PA63 oligomerizes to a heptamer and acts to translocate the catalytic moieties of the toxin, lethal factor (LF) and edema factor (EF), from endosomes to the cytosol. In this report, we used nondenaturing gel electrophoresis to show that each PA63 subunit in the heptamer can bind one LF molecule. Studies using PA immobilized on a plastic surface showed that monomeric PA63 is also able to bind LF. The internalization of PA and LF by cells was studied with radiolabeled and biotinylated proteins. Uptake was relatively slow, with a half-time of 30 min. The number of moles of LF internalized was nearly equal to the number of moles of PA subunit internalized. The essential role of PA oligomerization in LF translocation was shown with PA protein cleaved at residues 313-314. The oligomers formed by these proteins during uptake into cells were not as stable when subjected to heat and detergent as were those formed by native PA. The results show that the structure of the toxin proteins and the kinetics of proteolytic activation, LF binding, and internalization are balanced in a way that allows each PA63 subunit to internalize an LF molecule. This set of proteins has evolved to achieve highly efficient internalization and membrane translocation of the catalytic components, LF and EF.

Animals↗
PubMed2006

A Helicobacter pylori vacuolating toxin mutant that fails to oligomerize has a dominant negative phenotype.

Most Helicobacter pylori strains secrete a toxin (VacA) that causes massive vacuolization of target cells and which is a major virulence factor of H. pylori. The VacA amino-terminal region is required for the induction of vacuolization. The aim of the present study was a deeper understanding of the critical role of the N-terminal regions that are protected from proteolysis when VacA interacts with artificial membranes. Using a counterselection system, we constructed an H. pylori strain, SPM 326-Delta49-57, that produces a mutant toxin with a deletion of eight amino acids in one of these protected regions. VacA Delta49-57 was correctly secreted by H. pylori but failed to oligomerize and did not have any detectable vacuolating cytotoxic activity. However, the mutant toxin was internalized normally and stained the perinuclear region of HeLa cells. Moreover, the mutant toxin exhibited a dominant negative effect, completely inhibiting the vacuolating activity of wild-type VacA. This loss of activity was correlated with the disappearance of oligomers in electron microscopy. These findings indicate that the deletion in VacA Delta49-57 disrupts the intermolecular interactions required for the oligomerization of the toxin.

Bacterial Proteins↗
PubMed1983

Formation of oligomeric structures from plasmid DNA carrying cos lambda that is packaged into bacteriophage lambda heads.

Plasmids that carry cos lambda, the region necessary for lambda phage packaging and that are as small as four kilobases in size can be packaged into lambda phage heads in head-to-tail tandem oligomeric structures. Multimeric oligomers as large as undecamers have been detected. Oligomer formation depends upon the products of red and gam of lambda, and the general recombination occurs between different plasmids that share homologous DNA regions. The packaging efficiency of plasmids depends on its copy number in cells and its genome size. Upon injection into a cell, the DNA establishes itself as a plasmid in a tandem structure. When such a plasmid in a high oligomeric structure is used as the source of packaging DNA, the packaging efficiency of the plasmids is elevated. The oligomers are stable in recA cells, whereas they drift toward lower oligomers in recA+ cells.

Bacteriophage lambda↗
PubMed1993

Characterization of cytR mutations that influence oligomerization of mutant repressor subunits.

In Escherichia coli, the transport and catabolism of nucleosides require expression of the genes composing the CytR regulon. The role of the CytR repressor in transcriptional regulation has been examined through a study of mutant CytR proteins. Two important and interrelated CytR mutants are encoded by cytR delta M149, a dominant negative allele, and cytRC289R. Studies with CytR delta M149 indicated that the native, repression-competent CytR protein is multimeric while the CytR amino acid substitution C-289-->R has been proposed to affect subunit oligomerization on the basis of its ability to suppress the transdominance of CytR delta M149. The present study identifies other CytR amino acid residues proximal to Cys-289 that may also participate in normal subunit oligomerization. Mutations in these CytR residues, cytRA307P, cytRM308R, and cytRL309P, encoded inactive repressors in a CytR- background and, when combined with cytR delta M149, yielded hybrid repressors that were recessive in a CytR+ genetic background. Because the stability and solubility observed for the new, mutant CytR proteins and the wild-type CytR protein were indistinguishable, these residue replacements, like the C-289-->R substitution, are envisaged as being located at the subunit interface and thus suppress the CytR delta M149 transdominance by blocking efficient and stable assembly of wild-type and hybrid CytR subunits. The assignment of CytR amino acids to a protein region involved in subunit association is also consistent with the observations that these CytR amino acids are roughly colinear with regions of the LacI repressor that influence monomer-dimer association and would be surface located by alignment to the E. coli galactose-binding protein crystal structure.

Amino Acid Sequence↗
PubMed2003

Subunit oligomerization and substrate recognition of the Escherichia coli ClpYQ (HslUV) protease implicated by in vivo protein-protein interactions in the yeast two-hybrid system.

The Escherichia coli ClpYQ (HslUV) is an ATP-dependent protease that consists of an ATPase large subunit with homology to other Clp family ATPases and a peptidase small subunit related to the proteasomal beta-subunits of eukaryotes. Six identical subunits of both ClpY and ClpQ self-assemble into an oligomeric ring, and two rings of each subunit, two ClpQ rings surrounded by single ClpY rings, form a dumbbell shape complex. The ClpYQ protease degrades the cell division inhibitor, SulA, and a positive regulator of capsule transcription, RcsA, as well as RpoH, a heat shock sigma transcription factor. Using the yeast-two hybrid system, we explored the in vivo protein-protein interactions of the individual subunits of the ClpYQ protease involved in self-oligomerization, as well as in recognition of specific substrates. Interactions were detected with ClpQ/ClpQ, ClpQ/ClpY, and ClpY/SulA. No interactions were observed in experiments with ClpY/ClpY, ClpQ/RcsA, and ClpQ/SulA. However, ClpY, lacking domain I (ClpY(Delta I)) was able to interact with itself and with intact ClpY. The C-terminal region of ClpY is important for interaction with other ClpY subunits. The previously defined PDZ-like domains at the C terminus of ClpY, including both D1 and D2, were determined to be indispensable for substrate binding. Various deletion and random point mutants of SulA were also made to verify significant interactions with ClpY. Thus, we demonstrated in vivo hetero- and homointeractions of ClpQ and ClpY molecules, as well as a direct association between ClpY and substrate SulA, thereby supporting previous in vitro biochemical findings.

ATP-Dependent Proteases↗
PubMed1975

In vitro construction of different oligomeric forms of lambdadv DNA and studies of their transforming activities.

Plasmid lambdadv1, which is in a dimeric form, was converted to a linear monomer duplex by the action of EcoRI restriction endonuclease that incises at a unique site in this plasmid genome. The resulting products were then joined by Escherichia coli DNA ligase to produce molecules with various oligomeric forms, and from these monomeric, dimeric, or trimeric circular molecules were purified. By transformation of cells with these DNAs, clones were obtained that carried lambdadv1 in a monomeric or dimeric form. The former type of clones have not been generated in vivo, except for one in a different host strain, and carriers of timeric or tetrameric lambdadv1's have not been obtained so far. It was observed that a considerable fraction of these oligomeric circular DNAs were converted to lower oligomers (e.g., from trimer to dimer) during transformation. The characteristics of the monomeric lambdadv1 carriers obtained were compared with those of dimeric lambdadv1 carriers. The stabilities of the plasmids of the two forms were the same. However, the monomeric plasmid carriers were less tolerant to lambdavir phage infection and perpetuated about 30% less plasmid genomes in monomer units. Furthermore, dimeric plasmid carriers appeared spontaneously and accumulated in cultures of the monomeric lambdadv1 carriers.

Coliphages↗
PubMed1992

Oligomerization of hepatitis delta antigen is required for both the trans-activating and trans-dominant inhibitory activities of the delta antigen.

Two forms of hepatitis delta antigen (HDAg) have different roles in the replication cycle of hepatitis delta virus (HDV); the small forms trans activates HDV RNA replication, whereas the large form suppresses it but is needed for virion assembly. To understand the mechanism of these regulatory activities, we studied the possible HDAg oligomerization and its role in HDV replication. In this report, we provide direct biochemical evidence for the in vitro and in vivo formation of homodimers and heterodimers between these two HDAg species. By deletion mutagenesis, we showed that this protein interaction is mediated by the leucine zipper-like sequence residing in the N-terminal one-third of HDAg. Furthermore, site-specific mutants with various substitutions on two of the leucine residues in this stretch of sequence had reduced or no ability to form HDAg dimers. Correspondingly, the small HDAg with mutations in the leucine zipper-like sequence had reduced abilities to trans activate HDV RNA replication. Similar mutations on the leucine zipper-like sequence of the large HDAg also resulted in loss of the ability of large HDAg to inhibit HDV RNA replication. The in vivo biological activities of both forms of HDAg (trans activation and trans-dominant inhibition of HDV RNA replication, respectively) correlated with the extent of HDAg oligomerization in vitro. Thus, we conclude that the small HDAg participates in HDV RNA replication as an oligomer form and that the large HDAg inhibits HDV RNA replication as a result of its complex formation with small HDAg. A "black sheep" model for the mechanism of trans-dominant inhibition by the large HDAg is presented.

Amino Acid Sequence↗
PubMed1993

Generation of a water-soluble oligomeric ectodomain of the Rous sarcoma virus envelope glycoprotein.

Sequences encoding the transmembrane domain of the Rous sarcoma virus envelope (Env) glycoprotein were deleted and replaced with sequences that signal addition of a glycosyl phosphatidylinositol (GPI) membrane anchor. Stable NIH 3T3 cell lines expressing either the wild-type transmembrane-anchored Env or the Env chimera with a GPI tail were established. The GPI-anchored envelope glycoprotein is expressed, oligomerized, and transported to the cell surface in a manner identical to that of its wild-type transmembrane-anchored counterpart. The GPI-linked protein is quantitatively removed from the cell surface by treatment with phosphatidylinositol phospholipase C. The phosphatidylinositol phospholipase C-released, water-soluble Env glycoprotein ectodomain retains the wild-type oligomeric structure and provides a useful tool for studying the subgroup-specific binding and fusion activities of a prototypic retroviral Env glycoprotein.

3T3 Cells↗
PubMed1994

High rates of frameshift mutations within homo-oligomeric runs during a single cycle of retroviral replication.

Homo-oligomeric runs were inserted into a spleen necrosis virus-based retrovirus vector to determine the nature and rate of mutations within runs of 10 to 12 identical nucleotides during a single replication cycle. Clones of helper cells containing integrated copies of retroviral vectors were used to produce virus for infection of target (nonhelper) cells. Proviral sequences from target cell clones were compared with proviral sequences from helper cell clones to study mutations that occurred during a single cycle of replication. In addition to the internal region spanning the homo-oligomeric inserts, a naturally occurring run of 10 T's in the long terminal repeat (LTR) also was sequenced. Rates of mutation ranged from < 0.01 to 0.38 frameshift mutations per run per cycle for different nucleotide runs. Frameshift mutations ranged from deletions of 2 bases to additions of 5 bases; the most common mutations were +1 and -1. Frameshift mutation rates did not increase as the run length increased from 10 to 12 bases. Rates of frameshift mutation for runs of T's and A's were significantly higher than rates for runs of C's and G's, and rates for runs of pyrimidines were significantly higher than those for runs of purines. Interestingly, the vast majority of frameshift mutations in the internal region (95%) were positive, suggesting that the primer strand tends to slip backward on the template in this region. LTR runs had a significantly lower number of positive frameshift mutations than the internal runs. By analyzing the types of frameshift mutations within runs and by comparing the patterns of frameshift mutations in the 5' and 3' LTRs of individual proviruses, we conclude that the majority of mutations observed in our system occurred during minus-strand DNA synthesis of reverse transcription.

Animals↗
PubMed1998

Structure-based mutational analysis of the C-terminal DNA-binding domain of human immunodeficiency virus type 1 integrase: critical residues for protein oligomerization and DNA binding.

The C-terminal domain of human immunodeficiency virus type 1 (HIV-1) integrase (IN) is a dimer that binds to DNA in a nonspecific manner. The structure of the minimal region required for DNA binding (IN220-270) has been solved by nuclear magnetic resonance spectroscopy. The overall fold of the C-terminal domain of HIV-1 IN is similar to those of Src homology region 3 domains. Based on the structure of IN220-270, we studied the role of 15 amino acid residues potentially involved in DNA binding and oligomerization by mutational analysis. We found that two amino acid residues, arginine 262 and leucine 234, contribute to DNA binding in the context of IN220-270, as indicated by protein-DNA UV cross-link analysis. We also analyzed mutant proteins representing portions of the full-length IN protein. Amino acid substitution of residues located in the hydrophobic dimer interface, such as L241A and L242A, results in the loss of oligomerization of IN; consequently, the levels of 3' processing, DNA strand transfer, and intramolecular disintegration are strongly reduced. These results suggest that dimerization of the C-terminal domain of IN is important for correct multimerization of IN.

Amino Acid Sequence↗
PubMed1999

The first immunoglobulin-like domain of HveC is sufficient to bind herpes simplex virus gD with full affinity, while the third domain is involved in oligomerization of HveC.

The human herpesvirus entry mediator C (HveC/PRR1) is a member of the immunoglobulin family used as a cellular receptor by the alphaherpesviruses herpes simplex virus (HSV), pseudorabies virus, and bovine herpesvirus type 1. We previously demonstrated direct binding of the purified HveC ectodomain to purified HSV type 1 (HSV-1) and HSV-2 glycoprotein D (gD). Here, using a baculovirus expression system, we constructed and purified truncated forms of the receptor containing one [HveC(143t)], two [HveC(245t)], or all three immunoglobulin-like domains [HveC(346t)] of the extracellular region. All three constructs were equally able to compete with HveC(346t) for gD binding. The variable domain bound to virions and blocked HSV infection as well as HveC(346t). Thus, all of the binding to the receptor occurs within the first immunoglobulin-like domain, or V-domain, of HveC. These data confirm and extend those of Cocchi et al. (F. Cocchi, M. Lopez, L. Menotti, M. Aoubala, P. Dubreuil, and G. Campadelli-Fiume, Proc. Natl. Acad. Sci. USA 95:15700, 1998). Using biosensor analysis, we measured the affinity of binding of gD from HSV strains KOS and rid1 to two forms of HveC. Soluble gDs from the KOS strain of HSV-1 had the same affinity for HveC(346t) and HveC(143t). The mutant gD(rid1t) had an increased affinity for HveC(346t) and HveC(143t) due to a faster rate of complex formation. Interestingly, we found that HveC(346t) was a tetramer in solution, whereas HveC(143t) and HveC(245t) formed dimers, suggesting a role for the third immunoglobulin-like domain of HveC in oligomerization. In addition, the stoichiometry between gD and HveC appeared to be influenced by the level of HveC oligomerization.

Animals↗