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PubMed2001

Oligomeric structure of virion-associated and soluble forms of the simian immunodeficiency virus envelope protein in the prefusion activated conformation.

The envelope proteins (env) of simian immunodeficiency virus (SIV) and HIV type 1 assemble to form noncovalently associated oligomers in the endoplasmic reticulum. After cleavage in a Golgi compartment, oligomeric env complexes are transported to the surface of infected cells, where incorporation into budding virions can occur. Difficulties in obtaining adequate quantities of virions retaining env, as well as the unstable nature and hydrophobicity of the oligomer, may account for the absence of previous biophysical studies to determine the oligomeric valency of membrane-associated env. The aim of this study was to evaluate the oligomeric state of SIV env before membrane-fusion activation. Virion-associated env, obtained by crosslinking and detergent extraction, and non-crosslinked secreted env ectodomain (recombinant gp140) were purified by lentil-lectin chromatography and gel filtration as single predominant species. Sedimentation equilibrium-derived mass values for both forms of SIV env were close to those predicted for trimeric assemblies. Determination of the mass of individual molecules by scanning transmission electron microscopy confirmed that SIV virion-associated env and gp140 formed largely homogeneous populations of trimers. Furthermore, a triangular or tri-lobed morphology was clearly visualized in a subset of the trimers.

Biopolymers↗
PubMed1999

Toward a cancer therapy with boron-rich oligomeric phosphate diesters that target the cell nucleus.

The viability of boron neutron capture therapy depends on the development of tumor-targeting agents that contain large numbers of boron-10 (10B) atoms and are readily taken up by cells. Here we report on the selective uptake of homogeneous fluorescein-labeled nido-carboranyl oligomeric phosphate diesters (nido-OPDs) by the cell nucleus and their long-term retention after their delivery into the cytoplasm of TC7 cells by microinjection. All nido-OPDs accumulated in the cell nucleus within 2 h after microinjection. However, nido-OPDs in which the carborane cage was located on a side chain attached to the oligomeric backbone were redistributed between both the cytoplasm and nucleus after 24 h of incubation, whereas nido-OPDs in which the carborane cage was located along the oligomeric backbone remained primarily in the nucleus. Furthermore, cell-free incubation of digitonin-permeabilized TC7 cells with the nido-OPDs resulted in nuclear accumulation of the compounds, thus corroborating the microinjection studies. Our observation of fluorescence primarily located in the cell nucleus indicates that nuclear-specific uptake of sufficient amounts of 10B for effective boron neutron capture therapy ( approximately 10(8)-10(9) 10B atoms/tumor cell) via nido-OPDs is achievable.

Biological Transport↗
PubMed1999

Mutually compensatory mutations during evolution of the tetramerization domain of tumor suppressor p53 lead to impaired hetero-oligomerization.

We have measured the stability and stoichiometry of variants of the human p53 tetramerization domain to assess the effects of mutation on homo- and hetero-oligomerization. The residues chosen for mutation were those in the hydrophobic core that we had previously found to be critical for its stability but are not conserved in human p73 or p51 or in p53-related proteins from invertebrates or vertebrates. The mutations introduced were either single natural mutations or combinations of mutations present in p53-like proteins from different species. Most of the mutations were substantially destabilizing when introduced singly. The introduction of multiple mutations led to two opposite effects: some combinations of mutations that have occurred during the evolution of the hydrophobic core of the domain in p53-like proteins had additive destabilizing effects, whereas other naturally occurring combinations of mutations had little or no net effect on the stability, there being mutually compensating effects of up to 9.5 kcal/mol of tetramer. The triple mutant L332V/F341L/L344I, whose hydrophobic core represents that of the chicken p53 domain, was nearly as stable as the human domain but had impaired hetero-oligomerization with it. Thus, engineering of a functional p53 variant with a reduced capacity to hetero-oligomerize with wild-type human p53 can be achieved without any impairment in the stability and subunit affinity of the engineered homo-oligomer.

Animals↗
PubMed1997

Oligomerization of hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase, an enzyme with multiple transmembrane spans.

Hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT), which initiates N-linked glycosylation by catalyzing the synthesis of GlcNAc-P-P-dolichol, has multiple transmembrane spans and a catalytic site that probably exists on the cytosolic face of the endoplasmic reticulum membrane (Dan, N., Middleton, R. M., and Lehrman, M. A. (1996) J. Biol. Chem. 271, 30717-30725). In this report, we demonstrate that GPT forms functional oligomers, probably dimers. Oligomers were detected by chemical cross-linking of GPT and by a dominant-negative effect caused by co-expression of enzymatically inactive (but properly folded) GPT mutants. The GPT mutants had no effect on two other dolichol-P-dependent endoplasmic reticulum enzymes. Mixing experiments indicated that mature GPT was competent for oligomerization. Oligomerization appeared to be favored in detergent extracts compared with intact microsomes. Detergent treatments were found to prevent, rather than promote, nonspecific aggregation of GPT. These results demonstrate that GPT subunits can physically interact and influence each other. The implications of oligomerization for enzyme function are discussed. From these results, we conclude that GPT is one of a very small number of multitransmembrane span enzymes that can form multimers.

Animals↗
PubMed1997

Membrane insertion, glycosylation, and oligomerization of inositol trisphosphate receptors in a cell-free translation system.

In order to study the membrane topology, processing, and oligomerization of inositol trisphosphate receptor (IP3R) isoforms, we have utilized RNA templates encoding putative transmembrane domains to program a cell-free translation system of rabbit reticulocyte lysates supplemented with canine pancreas microsomes. In the absence of microsomes, translation of the RNA templates encoding all the putative transmembrane domains present in the C-terminal segment of the type I (1TM) and type III (3TM) IP3R isoforms resulted in a 62- and 59-kDa polypeptide, respectively. In both cases, an additional band approximately 3 kDa larger was observed upon the addition of microsomes. Both bands in the translation doublet were integrated into microsomal membranes and were full-length translation products, as shown by sedimentation through a sucrose cushion and immunoprecipitation with C-terminal isoform-specific antibodies. With both isoforms, N-glycopeptidase F digestion indicates that the upper band in the doublet corresponds to a glycosylated translation product. A 17-kDa protected fragment was observed after proteinase-K digestion of 1TM translated in the presence of microsomes. The pattern and size of protected fragments was consistent with the current six-transmembrane domain model of IP3R topology. Cotranslation of both 1TM and 3TM RNA templates in the presence of microsomes followed by immunoprecipitation with isoform specific antibodies revealed coimmunoprecipitation of translation products. This was not observed when the isoforms were translated separately and then mixed, suggesting that heteroligomerization occurs cotranslationally. A construct encoding only the first putative transmembrane domain of the type I isoform was found to be sufficient for integration into membranes but was unable to oligomerize with either 1TM or 3TM. Cotranslation experiments using additional constructs indicate that the major structural determinant for homoligomerization lies between putative transmembrane domain 5 and the C terminus. A second oligomerization domain involved in stabilization of heteroligomers is present within the first four transmembrane domains.

Animals↗
PubMed1997

TOUSLED is a nuclear serine/threonine protein kinase that requires a coiled-coil region for oligomerization and catalytic activity.

The TOUSLED (TSL) gene is essential for the proper morphogenesis of leaves and flowers in Arabidopsis thaliana. Protein sequence analysis predicts TSL is composed of a carboxyl-terminal protein kinase catalytic domain and a large amino-terminal regulatory domain. TSL fusion proteins, expressed in and purified from yeast, were used to demonstrate TSL protein kinase activity in vitro. TSL trans-autophosphorylates on serine and threonine residues, and phosphorylates exogenous substrates. Using the yeast two-hybrid system, TSL was found to oligomerize via its NH2-terminal domain. A deletion series indicates that a region containing two alpha-helical segments predicted to participate in a coiled-coil structure is essential for oligomerization. TSL localizes to the nucleus in plant cells through an essential NH2-terminal nuclear localization signal; however, this signal is not necessary for protein kinase activity. Finally, deletion mutants demonstrate a strict correlation between catalytic activity and the ability to oligomerize, arguing that activation of the protein kinase requires interaction between TSL molecules.

Amino Acid Sequence↗
PubMed1998

An oligonucleotide inhibits oligomerization of a rolling circle initiator protein at the pT181 origin of replication.

A large number of plasmids have been shown to replicate by a rolling circle (RC) mechanism. The initiators encoded by these plasmids have origin-specific, nicking-closing activity that is required for the initiation and termination of RC replication. Since the initiators of many RC plasmids are rate-limiting for replication, these proteins are usually inactivated after supporting one round of replication. In the case of the pT181 plasmid, inactivation of the initiator RepC protein occurs by the attachment of an oligonucleotide to its active tyrosine residue. We have generated the inactivated form of RepC, termed RepC*, in vitro and investigated the effects of attachment of the oligonucleotide on its various biochemical activities. Our results demonstrate that while RepC* is inactive in nicking-closing and replication activities due to the blockage of its active tyrosine residue, it is competent in origin DNA binding and DNA religation activities. We have investigated the oligomeric state of RepC and RepC* and found that RepC exists as a dimer in solution and can oligomerize on the DNA. We have generated heterodimers in vitro between the wild-type and epitope-tagged RepC proteins. In electrophoretic mobility shift experiments, the initiator heterodimers generated a novel DNA-protein complex, demonstrating that it binds to DNA as a dimer. We have shown that a DNA binding mutant of RepC can be targeted to the origin in the presence of the wild-type protein primarily through a protein-protein interaction. Interestingly, RepC* is defective in its ability to oligomerize on the DNA. RepC* inhibited the DNA binding and replication activity of wild-type RepC to only a very limited extent, suggesting that it may play only a minor regulatory role in replication in vivo. Based on these and earlier results, we propose a model for the role of RepC during the initiation and termination of pT181 RC replication.

Bacterial Proteins↗
PubMed1998

Oligomeric nature of the integral membrane protein stomatin.

The 31-kDa integral membrane protein stomatin (protein 7.2b) is not only an important component of the red cell membrane but can also be found in abundance in different tissues and cell lines. The protein is thought to be anchored to the membrane by a hydrophobic domain while both N and C termini are exposed to the cytoplasm. We have previously shown in the human cell line UAC that stomatin concentrates preferentially in plasma membrane folds and protrusions. There is also evidence that stomatin is linked to the cortical actin cytoskeleton, suggesting a role in cortical morphogenesis of the cell. In this study, we demonstrate that the fundamental structure of stomatin is oligomeric. Whereas interaction of stomatin with itself was suggested by cross-linking experiments, we show by density gradient centrifugation analysis that soluble homo-oligomeric complexes of this protein are present in Triton X-100 extracts of UAC cells. We also show the existence of these oligomers by co-immunoprecipitation of the endogenous stomatin and a recombinantly expressed myc-tagged stomatin, using an anti-myc antibody. The data indicate that these complexes comprise between 9 and 12 monomers of stomatin. Two C-terminally truncated forms of stomatin do not incorporate into these oligomers, suggesting an involvement of the C terminus in the homo-oligomeric interaction.

Amino Acid Sequence↗
PubMed1998

LSF and NTF-1 share a conserved DNA recognition motif yet require different oligomerization states to form a stable protein-DNA complex.

The mammalian transcription factor LSF (also known as CP2 and LBP-1c) binds as a homo-oligomer to directly repeated elements in viral and cellular promoters. LSF and the Drosophila transcription factor NTF-1 (also known as Elf-1 and Grainyhead) share a similar DNA binding region, which is unlike any established DNA binding motifs. However, we demonstrate that dimeric NTF-1 can bind an LSF half-site, whereas LSF cannot. To characterize further the DNA binding and oligomerization characteristics of LSF, truncation mutants were used to demonstrate that between 234 and 320 amino acids of LSF are required for high affinity DNA binding. Mixing of a truncation mutant with full-length LSF in a DNA binding assay established that the form of LSF that binds DNA is larger than a dimer. Unexpectedly, one C-terminal deletion derivative, partially defective in oligomerization properties, could occupy odd numbers of adjacent, tandem LSF half-sites, unlike full-length LSF. The numbers of DNA-protein complexes formed on multiple half-sites with this mutant indicated that LSF binds DNA as a tetramer, although cross-linking experiments confirmed a previous report concluding that LSF is primarily dimeric in solution. The DNA binding and oligomerization properties of LSF support models depicting novel mechanisms to prevent continual, adjacent binding by a protein that recognizes directly repeated DNA sequences.

Animals↗
PubMed1998

The pleckstrin homology domains of dynamin isoforms require oligomerization for high affinity phosphoinositide binding.

The dynamins are 100-kDa GTPases involved in the scission event required for formation of endocytotic vesicles. The two main described mammalian dynamins (dynamin-1 and dynamin-2) both contain a pleckstrin homology (PH) domain, which has been implicated in dynamin binding to (and activation by) acidic phospholipids, most notably phosphoinositides. We demonstrate that the PH domains of both dynamin isoforms require oligomerization for high affinity phosphoinositide binding. Strong phosphoinositide binding was detected only when the PH domains were dimerized by fusion to glutathione S-transferase, or via a single engineered intermolecular disulfide bond. Phosphoinositide binding specificities agreed reasonably with reported effects of different phospholipids on dynamin GTPase activity. Although they differ in their ability to inhibit rapid endocytosis in adrenal chromaffin cells, the dynamin-1 and dynamin-2 PH domains showed identical phosphoinositide binding specificities. Since oligomerization is required for binding of the dynamin PH domain to phosphoinositides, it follows that PH domain-mediated phosphoinositide binding will favor oligomerization of intact dynamin (which has an inherent tendency to self-associate). We propose that the dynamin PH domain thus mediates the observed cooperative binding of dynamin to membranes containing acidic phospholipids and promotes the self-assembly that is critical for both stimulation of its GTPase activity and its ability to achieve membrane scission.

Amino Acid Sequence↗
PubMed1999

Oligomerization is required for p53 to be efficiently ubiquitinated by MDM2.

Wild-type p53 is degraded in part through the ubiquitin proteolysis pathway. Recent studies indicate that MDM2 can bind p53 and promote its rapid degradation although the molecular basis for this degradation has not been clarified. This report demonstrates that MDM2 can promote the ubiquitination of wild-type p53 and cancer-derived p53 mutants in transiently transfected cells. Deletion mutants that disrupted the oligomerization domain of p53 displayed low binding affinity for MDM2 and were poor substrates for ubiquitination. However, efficient MDM2 binding and ubiquitination were restored when an oligomerization-deficient p53 mutant was fused to the dimerization domain from another protein. These results indicate that oligomerization is required for p53 to efficiently bind and be ubiquitinated by MDM2. p53 ubiquitination was inhibited in cells exposed to UV radiation, and this inhibition coincided with a decrease in MDM2 protein levels and p53.MDM2 complex formation. In contrast, p53 dimerization was unaffected following UV treatment. These results suggest that UV radiation may stabilize p53 by blocking the ubiquitination and degradation of p53 mediated by MDM2.

Humans↗
PubMed1999

Oligomerization of a MutS mismatch repair protein from Thermus aquaticus.

The MutS DNA mismatch protein recognizes heteroduplex DNAs containing mispaired or unpaired bases. We have examined the oligomerization of a MutS protein from Thermus aquaticus that binds to heteroduplex DNAs at elevated temperatures. Analytical gel filtration, cross-linking of MutS protein with disuccinimidyl suberate, light scattering, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry establish that the Taq protein is largely a dimer in free solution. Analytical equilibrium sedimentation showed that the oligomerization of Taq MutS involves a dimer-tetramer equilibrium in which dimer predominates at concentrations below 10 microM. The DeltaG(0)(2-4) for the dimer to tetramer transition is approximately -6.9 +/- 0.1 kcal/mol of tetramer. Analytical gel filtration of native complexes and gel mobility shift assays of an maltose-binding protein-MutS fusion protein bound to a short, 37-base pair heteroduplex DNA reveal that the protein binds to DNA as a dimer with no change in oligomerization upon DNA binding.

Adenosine Triphosphatases↗
PubMed2001

Stabilization of partially folded conformation during alpha-synuclein oligomerization in both purified and cytosolic preparations.

Aggregation of alpha-synuclein is tightly associated with many neurodegenerative diseases, such as Parkinson's disease, dementia with Lewy body, Lewy body variant of Alzheimer's disease, multiple system atrophy, and Hallervorden-Spatz disease, implicating a crucial role of aggregated forms of alpha-synuclein in the pathogenesis. Here, we examined the effect of elevated temperature on the oligomerization and structural changes of alpha-synuclein in the early stage of aggregation and show that self-assembly is crucial for the stabilization of a partially folded conformation. The efficiency of alpha-synuclein oligomerization increased proportional to the temperature increase, both in purified form and in crude cytosolic preparation. This oligomerization coincided with a small but reproducible change in the circular dichroism spectrum and an increase in the 1-anilinonaphthalene-8-sulfonic acid binding. The hydrodynamic dimensions of the dimer measured by size exclusion chromatography suggest a pre-molten globule-like structure. These data suggest that partially folded alpha-synuclein, which is unstable in the monomeric form, is stabilized by self-assembly and that these oligomers may evolve into the fibril nucleus.

Animals↗
PubMed2000

Changes of matrilin forms during endochondral ossification. Molecular basis of oligomeric assembly.

To understand the molecular properties of matrilin-3, a newly discovered member of the novel extracellular matrix protein family, we cloned a MAT-3 cDNA from developing chicken sterna. Real time quantitative reverse-transcription polymerase chain reaction indicates that MAT-3 mRNA is mainly expressed in the proliferation zone of a growth plate. It is also expressed in the maturation zone, overlapping with that of the mature chondrocyte-abundant matrilin-1 mRNA. This suggests that matrilin-3 may self-assemble in the proliferation zone, in addition to its co-assembly with matrilin-1 during endochondral ossification. Transfection of a MAT-3 cDNA into COS-7 cells shows that MAT-3 predominantly forms a homotetramer but also a trimer and a dimer. Co-transfection of both MAT-3 and MAT-1 cDNAs results in three major matrilins as follows: (MAT-1)(3), (MAT-3)(4), and (MAT-1)(2)(MAT-3)(2). Thus matrilin-3 may assemble into both homotypic and heterotypic oligomers. Our analysis shows that the assembly of MAT-3 does not depend on the number of epidermal growth factor repeats within the molecule, but the presence of Cys(412) and Cys(414) within the coiled-coil domain, which form covalent disulfide linkage responsible for both homo-oligomerization of MAT-3 and hetero-oligomerization of MAT-3 and MAT-1. Our data suggest that the varying synthetic levels of matrilins in different zones of a growth plate may result in a change of matrilin oligomeric forms during endochondral ossification.

Animals↗
PubMed2000

Zipper-mediated oligomerization of the mixed lineage kinase SPRK/MLK-3 is not required for its activation by the GTPase cdc 42 but Is necessary for its activation of the JNK pathway. Monomeric SPRK L410P does not catalyze the activating phosphorylation of Thr258 of murine MITOGEN-ACTIVATED protein kinase kinase 4.

Src homology 3 domain-containing proline-rich kinase (SPRK)/mixed lineage kinase-3 is a serine/threonine kinase that has been identified as an upstream activator of the c-Jun NH(2)-terminal kinase (JNK) pathway. SPRK is capable of activating MKK4 by phosphorylation of serine and threonine residues, and mutant forms of MKK4 that lack the phosphorylation sites Ser(254) and Thr(258) block SPRK-induced JNK activation. A region of 63 amino acids following the kinase domain of SPRK is predicted to form a leucine zipper. The leucine zipper domain of SPRK has been shown to be necessary and sufficient for SPRK oligomerization, but its role in regulating activation of SPRK and downstream signaling remains unclear. In this study, we substituted a proposed stabilizing leucine residue in the zipper domain with a helix-disrupting proline to abrogate zipper-mediated SPRK oligomerization. We demonstrate that constitutively activated Cdc42 fully activates this monomeric SPRK mutant in terms of both autophosphorylation and histone phosphorylation activity and induces the same in vivo phosphorylation pattern as wild type SPRK. However, this catalytically active SPRK zipper mutant is unable to activate JNK. Our data show that the monomeric SPRK mutant fails to phosphorylate one of the two activating phosphorylation sites, Thr(258), of MKK4. These studies suggest that zipper-mediated SPRK oligomerization is not required for SPRK activation by Cdc42 but instead is critical for proper interaction and phosphorylation of a downstream target, MKK4.

Animals↗
PubMed2001

The critical role of the stem region as a functional domain responsible for the oligomerization and Golgi localization of N-acetylglucosaminyltransferase V. The involvement of a domain homophilic interaction.

We demonstrated that a region in the stem of N-acetylglucosaminyltransferase V (GnT-V), a Golgi resident protein, is not required for enzyme activity but serves as functional domain, responsible for intracellular localization. Deletion of the domain led to complete retention of the kinetic properties but resulted in the cell surface localization of the enzyme as well as its efficient secretion into the medium. The lack of this domain concomitantly abolished the disulfide-mediated oligomerization of GnT-V, which appears to confer the Golgi retention. When the domain was inserted into the stem region of a cell surface-localized type II membrane protein, the resulting chimeric protein was substantially oligomerized and predominantly localized in the intracellular organelle. Furthermore, it was found that the presence of this domain is exclusively responsible for homo-oligomer formation. This homophilic interaction appears to involve a hydrophobic cluster of residues in the alpha-helix of the domain, as indicated by secondary structure predictions. These findings suggest that the domain specifically participates in the Golgi retention of GnT-V, probably via inducing homo-oligomer formation, and would also provide a possible mechanism for the oligomerization, which is critical for localization in the Golgi.

Animals↗
PubMed2001

Excluded volume effects on the refolding and assembly of an oligomeric protein. GroEL, a case study.

We have studied the effect of macromolecular crowding reagents, such as polysaccharides and bovine serum albumin, on the refolding of tetradecameric GroEL from urea-denatured protein monomers. The results show that productive refolding and assembly strongly depends on the presence of nucleotides (ATP or ADP) and background macromolecules. Nucleotides are required to generate an assembly-competent monomeric conformation, suggesting that proper folding of the equatorial domain of the protein subunits into a native-like structure is essential for productive assembly. Crowding modulates GroEL oligomerization in two different ways. First, it increases the tendency of refolded, monomeric GroEL to undergo self-association at equilibrium. Second, crowding can modify the relative rates of the two competing self-association reactions, namely, productive assembly into a native tetradecameric structure and unproductive aggregation. This kinetic effect is most likely exerted by modifications of the diffusion coefficient of the refolded monomers, which in turn determine the conformational properties of the interacting subunits. If they are allowed to become assembly-competent before self-association, productive oligomerization occurs; otherwise, unproductive aggregation takes place. Our data demonstrate that the spontaneous refolding and assembly of homo-oligomeric proteins, such as GroEL, can occur efficiently (70%) under crowding conditions similar to those expected in vivo.

Adenosine Diphosphate↗
PubMed2001

Vacuolar localization of oligomeric alpha-mannosidase requires the cytoplasm to vacuole targeting and autophagy pathway components in Saccharomyces cerevisiae.

One challenge facing eukaryotic cells is the post-translational import of proteins into organelles. This problem is exacerbated when the proteins assemble into large complexes. Aminopeptidase I (API) is a resident hydrolase of the vacuole/lysosome in the yeast Saccharomyces cerevisiae. The precursor form of API assembles into a dodecamer in the cytosol and maintains this oligomeric form during the import process. Vacuolar delivery of the precursor form of API requires a vesicular mechanism termed the cytoplasm to vacuole targeting (Cvt) pathway. Many components of the Cvt pathway are also used in the degradative autophagy pathway. alpha-Mannosidase (Ams1) is another resident hydrolase that enters the vacuole independent of the secretory pathway; however, its mechanism of vacuolar delivery has not been established. We show vacuolar localization of Ams1 is blocked in mutants that are defective in the Cvt and autophagy pathways. We have found that Ams1 forms an oligomer in the cytoplasm. The oligomeric form of Ams1 is also detected in subvacuolar vesicles in strains that are blocked in vesicle breakdown, indicating that it retains its oligomeric form during the import process. These results identify Ams1 as a second biosynthetic cargo protein of the Cvt and autophagy pathways.

Autophagy↗