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PubMed2005

Formation of Bombyx mori nucleopolyhedrovirus IE2 nuclear foci is regulated by the functional domains for oligomerization and ubiquitin ligase activity.

Baculovirus IE2 functions as a transregulator and is also involved in viral DNA replication. However, the mechanism for these functions remains unknown. It has previously been reported that Bombyx mori nucleopolyhedrovirus (BmNPV) IE2 has a ubiquitin ligase activity that is dependent on the RING finger domain and that IE2 can oligomerize through its C-terminal coiled-coil region. Here, confocal microscopy analysis demonstrated that IE2 formed nuclear foci only during the early phase of infection (2-6 h post-infection). Therefore, it was determined whether the IE2 functional regions described above could affect this characteristic distribution. Transient expression of ie2 also showed focus formation, suggesting that IE2 does not require any other viral factors. IE2 mutants lacking the C-terminal coiled-coil region did not form foci, while a mutant of the RING finger domain showed nuclear foci that appeared larger and brighter than those formed by wild-type IE2. In addition, IE2 exhibited enlarged foci in infected cells following treatment with a proteasome inhibitor, suggesting that foci enlargement resulted from accumulation of IE2 due to inhibition of the ubiquitin-proteasome pathway. These results suggest that BmNPV IE2 oligomerization and ubiquitin ligase activity functional domains regulate nuclear foci formation.

Animals↗
PubMed2006

Structural analysis of the human respiratory syncytial virus phosphoprotein: characterization of an alpha-helical domain involved in oligomerization.

Human respiratory syncytial virus (HRSV) phosphoprotein (P), an essential cofactor of the viral polymerase, is much shorter (241 aa) than and has no sequence similarity to P of other paramyxoviruses. Nevertheless, bioinformatic analysis of HRSV P sequence revealed a modular organization, reminiscent of other paramyxovirus Ps, with a central structured domain (aa 100-200), flanked by two intrinsically disordered regions (1-99 and 201-241). To test the predicted structure experimentally, HRSV P was purified from cell extracts infected with recombinant vaccinia virus or HRSV. The estimated molecular mass of P by gel filtration (approximately 500 kDa) greatly exceeded the theoretical mass of a homotetramer, proposed as the oligomeric form of native P. Nevertheless, the profile of cross-linked products obtained with purified P resembled that reported by others with P purified from bacteria or mammalian cells. Thus, the shape of HRSV P probably influences its elution from the gel filtration column, as reported for other paramyxovirus Ps. Digestion of purified HRSV P with different proteases identified a trypsin-resistant fragment (X) that reacted with a previously characterized monoclonal antibody (021/2P). N-terminal sequencing and mass spectrometry analysis placed the X fragment boundaries (Glu-104 and Arg-163) within the predicted structured domain of P. Cross-linking and circular dichroism analyses indicated that fragment X was oligomeric, with a high alpha-helical content, properties resembling those of the multimerization domain of Sendai and rinderpest virus P. These results denote structural features shared by HRSV and other paramyxovirus Ps and should assist in elucidation of the HRSV P structure.

Cell Line↗
PubMed2006

Transmembrane domain-dependent functional oligomerization of syndecans.

Cell surface adhesion receptors of the syndecan family initiate intracellular events through clustering of receptors. This crucial clustering occurs through receptor dimerization or oligomerization, which is mediated by receptor transmembrane domains. However, the exact role of the transmembrane domain during receptor activation is not fully understood. Researchers have not yet determined whether the transmembrane domain functions solely in the physical aspects of receptor clustering, or whether the domain has additional functional roles. Here we review recent advances in understanding the functionality of transmembrane domain-dependent oligomerization of syndecan cell adhesion receptor.

Animals↗
PubMed1997

Nuclear entry, oligomerization, and DNA binding of the Drosophila heat shock transcription factor are regulated by a unique nuclear localization sequence.

In normally growing Drosophila cultured cells the Drosophila heat shock transcription factor (dHSF) is localized in the cytosol and translocates into the nucleus after heat shock. In the cytosol of nonshocked cells, the dHSF is present as a monomer that cannot bind DNA. Upon stress, the dHSF enters the nucleus where it is observed to be a trimer. A novel nuclear localization sequence (NLS) in the dHSF was found to be responsible for stress-dependent nuclear entry. Deletion of the NLS prevents nuclear entry, as expected, yet surprisingly also allows constitutive oligomerization and DNA binding in the cytosol. Further analysis of the NLS by mutagenesis suggests that the two functions of nuclear entry and oligomerization are separable in that distinct residues present in the NLS are responsible for each. Mutations in certain basic residues completely block nuclear entry, as expected for a constitutive NLS. In addition, two residues were found in the NLS that, when altered, allowed constitutive nuclear entry of dHSF independent of stress. These residues may interact with a putative cellular component or possibly other domains of the HSF to prevent nuclear entry in normally growing cells. The NLS can also function autonomously to target a beta-galactosidase fusion protein into the nucleus in a heat shock-dependent fashion.

Amino Acid Sequence↗
PubMed1999

Signaling by proinflammatory cytokines: oligomerization of TRAF2 and TRAF6 is sufficient for JNK and IKK activation and target gene induction via an amino-terminal effector domain.

Interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) stimulate transcription factors AP-1 and NF-kappaB through activation of the MAP kinases JNK and p38 and the IkappaB kinase (IKK), respectively. The TNF-alpha and IL-1 signals are transduced through TRAF2 and TRAF6, respectively. Overexpressed TRAF2 or TRAF6 activate JNK, p38, or IKK in the absence of extracellular stimulation. By replacing the carboxy-terminal TRAF domain of TRAF2 and TRAF6 with repeats of the immunophilin FKBP12, we demonstrate that their effector domains are composed of their amino-terminal Zn and RING fingers. Oligomerization of the TRAF2 effector domain results in specific binding to MEKK1, a protein kinase capable of JNK, p38, and IKK activation, and induction of TNF-alpha and IL-1 responsive genes. TNF-alpha also enhances the binding of native TRAF2 to MEKK1 and stimulates the kinase activity of the latter. Thus, TNF-alpha and IL-1 signaling is based on oligomerization of TRAF2 and TRAF6 leading to activation of effector kinases.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1988

Identification of the DNA binding domain of the phage lambda cII transcriptional activator and the direct correlation of cII protein stability with its oligomeric forms.

The bacteriophage lambda transcriptional activator protein cII is a DNA-binding protein that coordinately regulates transcription from phage promoters important for lysogenic growth. We have genetically and structurally characterized more than 80 different single amino acid substitutions in this 97-amino-acid protein. A subset of 25 of these variant proteins was utilized for detailed biochemical analysis, which allows us to define specific domains critical for sequence-selective DNA recognition, nonspecific DNA binding, and protein oligomerization. The mutation studies also demonstrated the remarkable correlation of oligomeric structure of cII protein to its stability within the bacterial host. An Escherichia coli HtpR- strain has been identified that greatly stabilizes these highly unstable cII mutants.

Amino Acid Sequence↗
PubMed2002

Characterization of SP1, a stress-responsive, boiling-soluble, homo-oligomeric protein from aspen.

sp1 cDNA was isolated from aspen (Populus tremula) plants by immunoscreening an expression library using polyclonal antibodies against BspA protein. BspA, which is a boiling-stable protein, accumulates in aspen plants in response to water stress and abscisic acid application (Pelah et al., 1995). The sp1 cDNA was found to encode a 12.4-kD generally hydrophilic protein with a hydrophobic C terminus, which is different from the BspA protein and was termed SP1 (stable protein 1). Northern-blot analysis revealed that sp1 encodes a small mRNA (about 0.6 kb) that is expressed in aspen plants under non-stress conditions and is accumulated after salt, cold, heat, and desiccation stress, and during the recovery from stress. The SP1 detected in plants remained soluble upon boiling, migrated both as a 12.4-kD band and a much higher mass of 116 kD on a 17% (w/v) Tricine-sodium dodecyl sulfate-polyacrylamide gel. Comparative protease digestion patterns, amino acid analyses, and the N-terminal sequences of the 12.4- and 116-kD proteins revealed that SP1 is homo-oligomeric. Furthermore, gel filtration chromatography analysis indicated that SP1 exists in aspen plants as a complex, composed of 12 subunits of 12.4 kD. A large number of sequences deduced from expressed sequence tags and genomic sequences of other organisms with unknown function show high homology to SP1. Thus, SP1 may represent a new protein family. Here, we present the first report on this putative protein family: the cloning, isolation, and characterization of SP1, a stress-responsive, boiling-soluble, oligomeric protein.

Abscisic Acid↗
PubMed1994

Assembly of the Light-Harvesting Complexes (LHCs) of Photosystem II (Monomeric LHC IIb Complexes Are Intermediates in the Formation of Oligomeric LHC IIb Complexes).

The light-induced assembly of light-harvesting complex (LHC) II has been followed during the biogenesis of the plastid. Seedlings grown in intermittent light (IML) accumulate only small amounts of chlorophyll b. The minor LHC II apoproteins are present; however, the apoprotein levels of the major LHC II complex, LHC IIb, are severely depressed after exposure to IML. The levels of all LHC II apoproteins increase rapidly upon exposure to continuous illumination. The 25-kD, type 3 LHC IIb subunit appears to be more abundant during the early hours of greening in relation to its level in mature thylakoids. The LHC IIb apoproteins are initially associated with pigments to form monomeric pigment-protein complexes. The abundance of monomeric LHC IIb complexes gradually decreases during exposure to continuous light and a concomitant increase occurs in the amount of the trimeric and higher-order oligomeric forms. Pulse-chase experiments verify that labeled LHC IIb monomeric complexes are intermediates in the formation of trimeric and higher-order oligomeric LHC IIb-pigmented complexes. Therefore, the assembly of LHC II occurs via the initial pigmentation of the apoproteins to form monomeric complexes and proceeds in a sequential manner.

Journal Article↗
PubMed1999

Antifreeze proteins in winter rye leaves form oligomeric complexes

Antifreeze proteins (AFPs) similar to three pathogenesis-related proteins, a glucanase-like protein (GLP), a chitinase-like protein (CLP), and a thaumatin-like protein (TLP), accumulate during cold acclimation in winter rye (Secale cereale) leaves, where they are thought to modify the growth of intercellular ice during freezing. The objective of this study was to characterize the rye AFPs in their native forms, and our results show that these proteins form oligomeric complexes in vivo. Nine proteins were separated by native-polyacrylamide gel electrophoresis from apoplastic extracts of cold-acclimated winter rye leaves. Seven of these proteins exhibited multiple polypeptides when denatured and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After isolation of the individual proteins, six were shown by immunoblotting to contain various combinations of GLP, CLP, and TLP in addition to other unidentified proteins. Antisera produced against individual cold-induced winter rye GLP, CLP, and TLP all dramatically inhibited glucanase activity in apoplastic extracts from cold-acclimated winter rye leaves, and each antiserum precipitated all three proteins. These results indicate that each of the polypeptides may be exposed on the surface of the protein complexes. By forming oligomeric complexes, AFPs may form larger surfaces to interact with ice, or they may simply increase the mass of the protein bound to ice. In either case, the complexes of AFPs may inhibit ice growth and recrystallization more effectively than the individual polypeptides.

Journal Article↗
PubMed2005

Oligomerization of the fifth transmembrane domain from the adenosine A2A receptor.

The human adenosine A2A receptor (A(2A)R) belongs to one of the largest family of membrane proteins, the G-protein coupled receptors (GPCRs), characterized by seven transmembrane (TM) helices. Little is known about the determinants of their structures, folding, assembly, activation mechanisms, and oligomeric states. Previous studies in our group showed that peptides corresponding to all seven TM domains form stable helical structures in detergent micelles and lipid vesicles. However, the peptides behave differently; TM5 is the only peptide to have a ratio [theta]222/[theta]208 obtained by circular dichroism (CD) spectroscopy>1. This finding suggested to us that TM5 might self-associate. In the present study, we investigate the unique properties of the TM5 domain. We performed detailed analyses of TM5 peptide behavior in membrane-mimetic environments using CD spectroscopy, fluorescence spectroscopy and Förster resonance energy transfer, and gel electrophoresis. We find that TM5 peptide has the ability to self-associate to form oligomeric structures in various hydrophobic milieus and that these oligomers are highly resistant to temperature and chemical denaturation. We also find that mutation of the full-length A(2A)R at position M193, which is located in the fifth TM domain, noticeably alters A(2A)R monomer: dimer ratio as observed on SDS-PAGE. Our results suggest that parallel association of TM5 dimers may play a role in the known adenosine A2A receptor dimerization. This study represents the first evidence of an individual GPCR transmembrane domain self-association.

Amino Acid Sequence↗
PubMed2006

Stepwise disassembly and apparent nonstepwise reassembly for the oligomeric RbsD protein.

Many cellular proteins exist as homo-oligomers. The mechanism of the assembly process of such proteins is still poorly understood. We have previously observed that Hsp16.3, a protein exhibiting chaperone-like activity, undergoes stepwise disassembly and nonstepwise reassembly. Here, the disassembly and reassembly of a nonchaperone protein RbsD, from Escherichia coli, was studied in vitro. The protein was found to mainly exist as decamers with a small portion of apparently larger oligomeric forms, both of which are able to refold/reassemble effectively in a spontaneous way after being completely unfolded. Disassembly RbsD intermediates including pentamers, tetramers, trimers, dimers, and monomers were detected by using urea-containing pore gradient polyacrylamide gel electrophoresis, while only pentamers were detected for its reassembly. The observation of stepwise disassembly and apparent nonstepwise reassembly for both a chaperone protein (Hsp16.3) and a nonchaperone protein (RbsD) strongly suggests that such a feature is most likely general for homo-oligomeric proteins.

Bacterial Proteins↗
PubMed2006

Oligomerization behavior of the archaeal Sm2-type protein from Archaeoglobus fulgidus.

As part of a functional analysis of archaeal Sm-related proteins, we have studied the oligomerization behavior of the Sm-2 type protein from the euryarchaeon Archaeoglobus fulgidus using gel filtration chromatography and noncovalent mass spectrometry. Our experiments show that the oligomeric state of the protein depends on the pH and presence of RNA. The protein forms a hexamer at acidic pH in the absence of RNA. The addition of RNA (oligo U10) induces the formation of a heptamer over the whole pH range studied. The stability of both the hexamer and the RNA-bound heptamer increases at lower pH.

Archaeal Proteins↗
PubMed2002

Oligomerization of acidic fibroblast growth factor is not a prerequisite for its cell proliferation activity.

Oligomerization of fibroblast growth factors (FGFs) induced on binding to heparin or heparan sulfate proteoglycan is considered to be crucial for receptor activation and initiation of biological responses. To gain insight into the mechanism of activation of the receptor by FGFs, in the present study we investigate the effect(s) of interaction of a heparin analog, sucrose octasulfate (SOS), on the structure, stability, and biological activities of a recombinant acidic FGF from Notophthalmus viridescens (nFGF-1). SOS is found to bind to nFGF-1 and significantly increase the thermodynamic stability of the protein. Using a variety of techniques such as size-exclusion chromatography, sedimentation velocity, and multidimensional nuclear magnetic resonance (NMR) spectroscopy, it is shown that binding of SOS to nFGF-1 retains the protein in its monomeric state. In its monomeric state (complexed to SOS), n-FGF-1 shows significant cell proliferation activity. (15)N and (1)H chemical shift perturbation and the intermolecular nuclear Overhauser effects (NOEs) between SOS and nFGF-1 reveal that the ligand binds to the dense, positively charged cluster located in the groove enclosed by beta-strands 10 and 11. In addition, molecular modeling based on the NOEs observed for the SOS-nFGF-1 complex, indicates that SOS and heparin share a common binding site on the protein. In conclusion, the results of the present study clearly show that heparin-induced oligomerization of nFGF-1 is not mandatory for its cell proliferation activity.

Animals↗
PubMed1992

Effect of lac repressor oligomerization on regulatory outcome.

Regulatory outcome in a bacterial operon depends on the interactions of all the components which influence mRNA production. Levels of mRNA can be altered profoundly by both negative and positive regulatory elements which modulate initiation of transcription. The occupancy of regulatory sites on the DNA by repressors and activators is determined not only by the affinity of these proteins for their cognate site(s) but also by the oligomeric state of the regulatory protein. The lac operon in Escherichia coli provides an excellent prototypic example of the influence of protein assembly on the transcriptional status of the associated structural genes. DNA loop formation is essential for maximal repression of the lac operon and is contingent upon the presence of multiple operator sites in the DNA and the ability of the repressor to self-associate to form a bidentate tetramer. The stability of this looped complex is enhanced significantly by DNA supercoiling. Tetramer assembly from dimers apparently occurs via interactions of a 'leucine zipper' motif in the C-terminal domain of the protein, and the tetramer is essential to formation of looped complexes. Furthermore, analysis of the DNA-binding characteristics of dimeric mutants has established that the monomer-dimer association and dimer-DNA binding (monomer does not bind to DNA) are coupled equilibria. Thus, dimer assembly is essential for generating a DNA-binding unit, and tetramer assembly is required for formation of the stable looped DNA structure that maximally represses mRNA synthesis. Protein-protein interactions therefore play a pivotal role in the regulatory activities of the lac repressor and must be considered when analysing the activities of any oligomeric DNA-binding protein.

DNA, Bacterial↗
PubMed2004

The Arabidopsis FtsH metalloprotease gene family: interchangeability of subunits in chloroplast oligomeric complexes.

The Arabidopsis At filamentation temperature sensitive (FtsH) metalloprotease gene family comprises 12 members (AtFtsH1-AtFtsH12), including three pairs of closely related genes that are targeted to chloroplasts (AtFtsH2 and AtFtsH8; AtFtsH1 and AtFtsH5; and AtFtsH7 and AtFtsH9). Mutations in AtFtsH5 (var1) and AtFtsH2 (var2) give rise to variegated plants with green- and white-sectored leaves. Cells in the green sectors contain morphologically normal chloroplasts, whereas cells in the white sectors are blocked in chloroplast biogenesis. A major question is how chloroplasts arise in cells that have a mutant genotype. We have found by two-dimensional (2-D) green gel and gel filtration analyses that AtFtsH2/VAR2 forms oligomeric complexes. Two bands in the 2-D green gels that correspond to AtFtsH5/VAR1 + AtFtsH1 and AtFtsH2/VAR2 + AtFtsH8 have been identified, and these bands are coordinately reduced in amount in var1 and var2 thylakoids that lack AtFtsH5/VAR1 and AtFtsH2/VAR2, respectively. These reductions are not because of alterations in transcript abundance. Overexpression of AtFtsH8 in var2-4 (a putative null allele) normalizes the variegation phenotype of the mutant and restores the two bands to their wild-type levels. These results suggest that AtFtsH8 is interchangeable with AtFtsH2/VAR2 in AtFtsH-containing oligomers, and that the two proteins have redundant functions. Consistent with this hypothesis, AtFtsH2 and AtFtsH8 have similar expression patterns, as monitored by promoter-beta-glucuronidase (GUS) fusion and RT-PCR experiments. Based on our findings, we propose that AtFtsH1, AtFtsH2/VAR2, AtFtsH5/VAR1, and AtFtsH8 interact to form oligomeric structures, and that subunit stoichiometry is controlled post-transcriptionally in var1 and var2, perhaps by turnover. A threshold model is presented to explain the pattern of variegation in var2 in which AtFtsH8 provides a compensating activity in the green sectors of the mutant.

Arabidopsis↗
PubMed2004

HLA class I serotypes and cytotoxic T-lymphocyte responses among human immunodeficiency virus-1-uninfected Thai volunteers immunized with ALVAC-HIV in combination with monomeric gp120 or oligomeric gp160 protein boosting.

Antigen-induced cellular immunogenicity may vary between populations due to differences in human leukocyte antigen (HLA) diversity and, hence, may play a critical role in the protection afforded by vaccines. In the setting of two, phase I/II human immunodeficiency virus-1 vaccine trials of a recombinant canarypox prime, and boosting with either recombinant monomeric gp120 or oligomeric gp160, we assessed the association between specific human leukocyte antigen (HLA) class I serotypes and the presence of cytotoxic T-lymphocyte response measured by 51Cr-release assay. HLA class I serotypes A11, A24, A33, B46, and B75 were the most common, present in 10% or more of 245 individuals studied. Forty of 187 (21.4%) Thai adults who received either ALVAC-HIV with gp120 or oligomeric gp160 or ALVAC alone had a precursor cytolytic CD8 T-cell response (pCTL). HLA-B44 was positively and significantly associated with a pCTL response (odds ratio 7.6, 95% CI: 2.7-21.2), whereas B46 was negatively associated but not robust when adjusted for multiple comparisons. Responses to Env proteins accounted for the majority (nine of 11) of pCTL activity among those persons with B44. This HLA class I serotype occurred in 9.4% of participants overall (including the placebo group), less commonly than what is reported from populations of European ancestry. These results strengthen the importance of assessing HLA class I distributions in conjunction with studies of vaccines designed to elicit cellular immunity in different populations.

Adult↗
PubMed1994

Chemical synthesis and characterization of peptides and oligomeric proteins designed to form transmembrane ion channels.

A strategy for the synthesis of peptides and oligomeric proteins designed to form transmembrane ion channels is described. A folding motif that exhibits a functional ionic pore encompasses amphipathic alpha-helices organized as a four-helix bundle around a central hydrophilic pore. The channel-forming activity of monomeric amphipathic peptides may be examined after reconstitution in lipid bilayers in which peptides self-assemble into conductive oligomers. The covalent attachment of channel-forming peptides to the lysine epsilon-amino groups of a template molecule (KKKPGKEKG) specifies oligomeric number and facilitates the study of ionic permeation and channel blockade. Here we describe detailed protocols for the total synthesis of peptides and template-assembled four-helix bundle proteins, exemplified with the sequence of M2 delta (EKM-STAISVLLAQAVFLLLTSQR), considered involved in lining the pore of the nicotinic acetylcholine receptor channel. For comparison, the synthesis of a second four-helix bundle, T4CaIVS3 with the sequence of predicted transmembrane segment S3 (DPWNVFDFLIVIGSIIDVILSE) of the fourth repeat of the L-type voltage-gated calcium channel, is included. Peptides and proteins are synthesized step-wise by solid-phase methods, purified by reversed-phase HPLC, and homogeneity ascertained by analytical HPLC, capillary zone electrophoresis, SDS/PAGE, amino acid analysis and sequencing. Optimization of synthetic procedures for hydrophobic molecules include reducing resin substitution to avoid steric hindrance and aggregation of the final product. Protocols for the preparation of the samples prior to HPLC purification as well as the conditions and columns required for successful purification are presented. The methods developed are generally applicable for the chemical synthesis, purification and characterization of amphipathic peptides and template directed helical bundle proteins.

Amino Acid Sequence↗
PubMed2005

The cholesterol-dependent cytolysin listeriolysin O aggregates rafts via oligomerization.

The pore-forming toxin listeriolysin O (LLO) is the main virulence factor of Listeria monocytogenes. LLO is known to act as a pseudo cytokine/chemokine, which induces a broad spectrum of host responses that ultimately influences the outcome of listeriosis. In the present study we demonstrate that LLO is a potent aggregator of lipid rafts. LLO was found to aggregate the raft associated molecules GM1, the GPI-anchored proteins CD14 and CD16 as well as the tyrosine kinase Lyn. Abrogation of the cytolytic activity of LLO by cholesterol pretreatment was found not to interfere with LLO's ability to aggregate rafts or trigger tyrosine phosphorylation in cells. However, a monoclonal antibody that blocks the oligomerization of LLO was found to inhibit rafts' aggregation as well as the induction of tyrosine phosphorylation. This implies that rafts aggregation by LLO which is independent of cytolytic activity, is due to the oligomerization of its membrane bound toxin monomers. Thus, LLO most likely induces signalling through the coaggregation of rafts' associated receptors, kinases and adaptors.

Animals↗