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PubMed2001

In vitro reconstitution of recognition and activation complexes between interleukin-6 and gp130.

Gp130 is a shared signal-transducing receptor for a family of four-helix cytokines, of which interleukin-6 is a prototypic member. IL-6-type cytokines activate gp130 to elicit downstream intracellular JAK/STAT signaling cascades through formation of hetero-oligomeric receptor complexes. Interleukin-6 must first complex with its specific alpha-receptor (Ralpha) in order to bind and activate gp130. We have dissected the extracellular activation pathway of human gp130 by human IL-6 through reconstitution of soluble complexes representing intermediate and final states in the hierarchical assembly of the IL-6/IL-6Ralpha/gp130 signaling complex. To isolate these hetero-complexes, we have applied a protein engineering strategy of covalently linking IL-6 to its Ralpha, which results in a "hyperactive" single-chain complex (hyper-IL-6) which we express in both Escherichia coli and insect cells. We have determined that IL-6/IL-Ralpha and the cytokine-binding homology region (CHR) of gp130 (D2D3) form a stable trimolecular "recognition" complex (trimer) consisting of 1IL-6,1 IL-6Ralpha, and 1 gp130-CHR. Addition of the N-terminal (D1) Ig-like domain (IGD) of gp130 to the CHR results in a transition to a hexameric "activation" complex containing 2 IL-6, 2IL-6Ralpha, and 2 gp130. These results clearly demonstrate that the recognition and activation complexes are disparate hetero-oligomeric molecular species linked by the recruitment of the gp130 IGD by the unique site III epitope present on all gp130-class cytokines. The results of these studies are relevant to other members of the IL-6 family of gp130-cytokines and address a longstanding question concerning the respective roles of the gp130 CHR and IGD in assembly of the active signaling oligomer.

Antigens, CD↗
PubMed2003

C-terminal extension of phaseolin with a short methionine-rich sequence can inhibit trimerisation and result in high instability.

In an attempt to increase the content in essential amino acids methionine and tryptophan of the trimeric storage protein phaseolin, we fused a Met- and Trp-rich sequence to the C-terminus of a phaseolin variant lacking its vacuolar sorting signal, with the aim to target the protein for secretion and accumulation into the apoplast. The fate of the mutant protein, denominated Y3, was studied in transiently transfected tobacco protoplasts. We report that the presence of the additional sequence causes structural defects which inhibit trimerization and lead to partial aggregation of Y3. The protein interacts with the ER chaperone BiP prior to being degraded very rapidly, in a process that does not require vesicular transport from the ER. The rate of degradation of Y3 is higher than that observed for another assembly defective mutant of phaseolin, delta360, which remains monomeric and does not aggregate. This indicates that the plant ER quality control machinery can dispose of defective proteins with different kinetics and perhaps mechanisms, depending on the nature of their defect.

Binding Sites↗
PubMed2004

The PM2 virion has a novel organization with an internal membrane and pentameric receptor binding spikes.

Biological membranes are notoriously resistant to structural analysis. Excellent candidates to tackle this problem in situ are membrane-containing viruses where the membrane is constrained by an icosahedral capsid. Cryo-EM and image reconstruction of bacteriophage PM2 revealed a membrane bilayer following the internal surface of the capsid. The viral genome closely interacts with the inner leaflet. The capsid, at a resolution of 8.4 A, reveals 200 trimeric capsomers with a pseudo T = 21 dextro organization. Pentameric receptor-binding spikes protrude from the surface. It is evident from the structure that the PM2 membrane has at least two important roles in the life cycle. First, it acts as a scaffold to nucleate capsid assembly. Second, after host recognition, it fuses with the host outer membrane to promote genome entry. The structure also sheds light on how the viral supercoiled circular double-stranded DNA genome might be packaged and released.

Bacteriophages↗
PubMed2005

Ku is a novel transcriptional recycling coactivator of the androgen receptor in prostate cancer cells.

The androgen receptor (AR) dynamically assembles and disassembles multicomponent receptor complexes in order to respond rapidly and reversibly to fluctuations in androgen levels. We are interested in identifying the basal factors that compose the AR aporeceptor and holoreceptor complexes and impact the transcriptional process. Using tandem mass spectroscopy analysis, we identified the trimeric DNA-dependent protein kinase (DNA-PK) complex as the major AR-interacting proteins. AR directly interacts with both Ku70 and Ku80 in vivo and in vitro, as shown by co-immunoprecipitation, glutathione S-transferase pull-down, and Sf9 cell/baculovirus expression. The interaction was localized to the androgen receptor ligand binding domain and is independent of DNA interactions. Ku interacts with AR in the cytoplasm and nucleus regardless of the presence or absence of androgen. Ku acts as a coactivator of AR activity in a luciferase reporter assay employing both Ku-defective cells and Ku small interfering RNA knock-down in a prostate cancer cell line. DNA-PK catalytic subunit (DNA-PKcs) also acts as a coactivator of androgen receptor activity in a luciferase reporter assay employing DNA-PKcs defective cells. AR nuclear translocation is not affected in Ku defective cells, implying Ku functionality may be mainly nuclear. Chromatin immunoprecipitation experiments demonstrated that both Ku70 and Ku80 interact with the prostate-specific antigen promoter in an androgen-dependant manner. Finally, in vitro transcription assays demonstrated Ku involvement in transcriptional recycling with androgen dependent promoters.

Androgens↗
PubMed1995

Molecular dissection of the neurofibrillary lesions of Alzheimer's disease.

Abundant neurofibrillary tangles, neuropil threads and senile plaque neurites constitute the neurofibrillary pathology of Alzheimer's disease. They form in the nerve cells that undergo degeneration in the disease, in which their regional distribution correlates with the degree of dementia. Each lesion contains the paired helical filament (PHF) as its major fibrous component. Recent work has shown that PHFs are composed of the microtubule-associated protein tau in a hyperphosphorylated state. PHF-tau is hyperphosphorylated on six adult brain tau isoforms. As a consequence, tau is unable to bind to microtubules and is believed to self-assemble into the PHF. Current evidence suggests that protein kinases or protein phosphatases with a specificity for serine/threonine-proline residues are instrumental in the hyperphosphorylation of tau. Candidate protein kinases include mitogen-activated protein kinase, glycogen synthase kinase 3 and cyclin-dependent kinase 5, whereas the trimeric form of protein phosphatase 2A is a candidate phosphatase.

Alzheimer Disease↗
PubMed1996

Modeling of a five-stranded coiled coil structure for the assembly domain of the cartilage oligomeric matrix protein.

The three-dimensional structure of the assembly domain of the cartilage oligomeric matrix protein (COMP) has been modeled. The model demonstrates a parallel five-stranded coiled coil and fits well with a large amount of experimental data that describe the oligomerization state, the alpha-helical conformation, the helix directionality and the properties of the (abcdefg)n repeat sequence containing apolar residues at (a) and (d) positions. Comparison of the pentamer model with the known dimer, trimer, and tetramer coiled coils revealed interactions that could mediate the switch to the formation of the pentamer coiled coil. The most distinctive feature of the pentamer model involves ion pair interactions. Charged side chains of the pentamer can form (f-g), (f-b'), and (e-c') interhelical ion pairs, which are neither experimentally observed nor modeled in the di-, tri-, and tetramers. A polar glutamine residue could be adopted at an interior (d) position of the modeled structure due to the formation of a symmetrical network of buried hydrogen bonds between five such glutamines. The pentamer model contains an axial cavity that can accept water molecules. Conformational analysis was carried out in an attempt to determine the three-dimensional structure of the disulfide bonded C-terminal region of the pentamer. Recent data on crystallization of the COMP assembly domain (Efimov et al., Proteins 24:259-262, 1996) indicate that the prediction can be tested experimentally in the near future.

Cartilage↗
PubMed1994

Studies with cross-linking reagents on the oligomeric form of the paramyxovirus fusion protein.

The oligomeric form of the paramyxovirus simian virus 5 (SV5) fusion (F) glycoprotein has been examined by using chemical cross-linking and sucrose density gradient fractionation. In addition, chemical cross-linking was used to examine the kinetics of assembly of the F oligomer. Analysis by SDS-PAGE on 3.5% gels of the cross-linked F molecules indicated three major species with calculated molecular weights of M(r) approximately 65, M(r) approximately 130, and M(r) approximately 195 kDa, suggesting F monomers, dimers, and trimers, respectively. The cross-linked F species of M(r) approximately 195 kDa migrated on gels faster than influenza virus hemagglutinin trimers and between the dimeric and tetrameric forms of paramyxovirus hemagglutinin-neuraminidase (HN). Furthermore, the F protein oligomer was found to sediment slower than the HN tetramer on sucrose gradient centrifugation. SDS-PAGE analysis of the cross-linked F protein of Newcastle disease virus and human parainfluenza virus 3 showed a pattern very similar to that found for SV5. The data are consistent with those expected for the paramyxovirus F protein being a homotrimer.

Animals↗
PubMed1996

Synthesis of Linear Amphipathic Porphyrin Dimers and Trimers: An Approach to Bilayer Lipid Membrane Spanning Porphyrin Arrays.

A modular building-block approach has been developed for the construction of linear amphipathic porphyrin arrays. The reaction of meso-(trifluoromethyl)dipyrromethane and an aldehyde under the conditions of the two-step room temperature porphyrin synthesis affords the trans-substituted porphyrin (13-56% yields). A similar reaction with two different aldehydes provides access to porphyrins bearing two different functional groups. An ethyne porphyrin and an iodo porphyrin (either free base or zinc) are selectively joined via Pd(0)-catalyzed coupling reactions, affording a linear array with porphyrins in defined metalation states. Coupling of a zinc-porphyrin bearing iodo and ester groups with a free base porphyrin bearing ethyne and ester groups yielded the zinc-free base porphyrin dimer. Coupling of a bis-ethyne porphyrin with a porphyrin bearing iodo and ester groups afforded the porphyrin trimer. Cleavage of the esters yielded the amphipathic porphyrin dimer and trimer arrays. The arrays with adjacent zinc and free base porphyrins undergo efficient electronic energy transfer. Both amphipathic porphyrin arrays have been incorporated into L-alpha-phosphatidylcholine vesicles. This versatile synthetic strategy provides access to a family of porphyrin arrays for studies of photophysical processes in supramolecular assemblies.

Journal Article↗
PubMed2006

Aggregation studies of the water-soluble gadofullerene magnetic resonance imaging contrast agent: [Gd@C82O6(OH)16(NHCH2CH2COOH)8]x.

The aggregation behavior of the newly synthesized gadofullerene magnetic resonance imaging (MRI) contrast agent, i.e., Gd@C(82)O(6)(OH)(16)(NHCH(2)CH(2)COOH)(8) (abbreviated as AAD-EMF), was studied in detail by dynamic light scattering, scanning electron microscopy, T(1)-weighted magnetic resonance, and atomic force microscopy. It was revealed that the AAD-EMF aggregation in aqueous solution is pH-dependent. At pH 2, the AAD-EMF first self-assemble to form ca. 30 nm small clusters, and then dozens of the small clusters further aggregate to form large grapelike particles. At pH 7, the aggregates are also ca. 30 nm small clusters, but they are hard to further aggregate except for forming some cluster dimers or trimers, so AAD-EMF aggregates have a narrow size distribution by this time. At pH 9, the AAD-EMF aggregations cover a large range of continuous hydrodynamic diameters from 30 to 2000 nm. On the basis of the above observations, the aggregating mechanism of AAD-EMF under different pH values was proposed by concurrently considering the hydrogen-bonding effect and the dipolar interactions between AAD-EMF.

Contrast Media↗
PubMed2001

Biochemical characterization of the helper component of Cauliflower mosaic virus.

The helper component of Cauliflower mosaic virus is encoded by viral gene II. This protein (P2) is dispensable for virus replication but required for aphid transmission. The purification of P2 has never been reported, and hence its biochemical properties are largely unknown. We produced the P2 protein via a recombinant baculovirus with a His tag fused at the N terminus. The fusion protein was purified by affinity chromatography in a soluble and biologically active form. Matrix-assisted laser desorption time-of-flight mass spectrometry demonstrated that P2 is not posttranslationally modified. UV circular dichroism revealed the secondary structure of P2 to be 23% alpha-helical. Most alpha-helices are suggested to be located in the C-terminal domain. Using size exclusion chromatography and aphid transmission testing, we established that the active form of P2 assembles as a huge soluble oligomer containing 200 to 300 subunits. We further showed that P2 can also polymerize as long paracrystalline filaments. We mapped P2 domains involved in P2 self-interaction, presumably through coiled-coil structures, one of which is proposed to form a parallel trimer. These regions have previously been reported to also interact with viral P3, another protein involved in aphid transmission. Possible interference between the two types of interaction is discussed with regard to the biological activity of P2.

Amino Acid Sequence↗
PubMed1984

[Synthesis of 10S particles in cells infected with aphthovirus].

In the present study, evidence is presented for the existence of a morphogenetic intermediary that may be a precursor of the procapsids in the assembling process. BHK21 clone 13S cells were infected with Aphthovirus A24 (Cruzeiro strain), and pulse-chase experiments were carried out using 3H-leucine. Cytoplasmic extracts were then prepared at appropriate times, and analyzed by sucrose-gradient ultracentrifugation. After preliminary assays (Fig. 1), working conditions were standardized so as to obtain maximal recovery of the morphogenetic intermediary, as well as consistency of results. Only in the presence of DOC-Brij58 and Mg++ could a 10S sedimentation coefficient peak be seen (Fig. 1 a). A heterogeneous zone, with 4,5-5S as sedimentation coefficient, was also observed. The degree of labeling in the region 4,5-5S compared with that in the 10S portion, depends on the time within the infectious cycle when cells were pulse-labeled. Maximal levels for the ratio 10S/4,5-5S are reached when pulse-labeling takes place at the time when the amount of RNA viral synthesis reaches 80% of its total value (Fig. 2). Similar experiments, performed with third passage bovine fetal kidney cells, were confirmatory of the presence of a structure sedimenting at 10S, as well as of a heterogeneous zone of 4,5-5S (Fig. 3). It would appear that assembling of Aphthoviruses is accomplished through an intermediary unit which differs from that found for other Picornaviruses, the latter being the result of the union of 12 pentamers. The capsid of Aphthoviruses, also composed of 60 identical sub-units, would instead derive from the joining of 20 trimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Interactions between PE2, E1, and 6K required for assembly of alphaviruses studied with chimeric viruses.

During the assembly of alphaviruses, a preassembled nucleocapsid buds through the cell plasma membrane to acquire an envelope containing two virally encoded glycoproteins, E2 and E1. Using two chimeric viruses, we have studied interactions between E1, E2, and a viral peptide called 6K, which are required for budding. A chimeric Sindbis virus (SIN) in which the 6K gene had been replaced with that from Ross River virus (RR) produced wild-type levels of nucleocapsids and abundant PE2/E1 heterodimers that were processed and transported to the cell surface. However, only about 10% as much chimeric virus as wild-type virus was assembled, demonstrating that there is a sequence-specific interaction between 6K and the glycoproteins required for efficient virus assembly. In addition, the conformation of E1 in the E2/E1 heterodimer on the cell surface was different for the chimeric virus from that for the wild type, suggesting that one function of 6K is to promote proper folding of E1 in the heterodimer. A second chimeric SIN, in which both the 6K and E1 genes, as well as the 3' nontranslated region, were replaced with the corresponding regions of RR also resulted in the production of large numbers of intracellular nucleocapsids and of PE2/E1 heterodimers that were cleaved and transported to the cell surface. Budding of this chimera was severely impaired, however, and the yield of the chimera was only approximately 10(-7) of the SIN yield in a parallel infection. The conformation of the SIN E2/RR E1 heterodimer on the cell surface was different from that of the SIN E2/SIN E1 heterodimer, and no interaction between viral glycoproteins and nucleocapsids at the cell plasma membrane could be detected in the electron microscope. We suggest that proper folding of the E2/E1 heterodimer must occur before the E2 tail is positioned properly in the cytoplasm for budding and before heterodimer trimerization can occur to drive virus budding.

Animals↗
PubMed1996

Oligomeric assembly of thrombospondin in the endoplasmic reticulum of thyroid epithelial cells.

The thyroid endoplasmic reticulum (ER) provides an environment in which conformational maturation of thyroglobulin monomers occurs with progressive dissociation from BiP (a molecular chaperone), prior to thyroglobulin dimerization. This pattern of folding is thought to represent a pathway common to many exportable polypeptides. Thyrocytes also synthesize and secrete thrombospondin, an extracellular matrix glycoprotein that forms disulfide-linked trimers. Using a monoclonal antibody recognizing the N-terminal heparin-binding domain of thrombospondin, pulse-chase/immunoprecipitation experiments indicate that this epitope forms essentially cotranslationally. Dependent upon structural information contained within the N-terminal region, thrombospondin trimers also form and are rapidly stabilized by interchain disulfide bonds in the peritranslational period. Within 30 to 60 sec, a new epitope in the mid-molecule is detected. Additional approaches (including thrombospondin dissociation from BiP-an indirect measure of conformational maturation; t1/2 approximately 20 min) independently suggest that significant folding of monomers occurs within the trimer, i.e., well after oligomerization. These later events appear rate limiting for thrombospondin export from the ER (t1/2 approximately 30 min). The results highlight plasticity in the relationship between oligomerization and specific folding events for different proteins exported from the thyroid ER.

Animals↗
PubMed2001

Self-assembled organometallic [12]metallacrown-3 complexes.

The reaction of [(arene)RuCl2]2 (arene = C6H6, cymene, C6H3Et3, or C6Me6) or [Cp*RhCl2]2 with 3-hydroxy-2-pyridone in the presence of Cs2CO3 gives trinuclear metallamacrocyclic complexes. The self-assembly process was shown to be completely diastereoselective, and a racemic mixture of complexes with M(R)M(R)M(R) or MsMsMs (M=Ru, Rh) configuration was obtained. Plausible mononuclear intermediates of the formula [(arene)RuCl(C5H4NO2)] (arene = cymene, C6Me6) have been isolated and characterized. A structurally related trimer was synthesized by using [(cymene)RuCl2]2 and 3-acetamido-2-pyridone instead of 3-hydroxy-2-pyridone. The macrocycles were shown to be highly potent ionophores for Na+ and/or Li+ with negligible affinities for the larger cation K+. The selectivities of the receptors depend on the pi-ligand present: whereas the (C6H6)Ru- and (cymene)Ru complexes bind both Li+ and Na+, the (C6Me6)Ru-, (C6H3Et3)Ru-, and Cp*Rh complexes bind exclusively Li+. For all receptors, the presence of alkali metal ions can be detected electrochemically: the peak potential is shifted by > 300 mV toward anionic potential upon binding. This behavior was utilized to detect Li+ and Na+ colorimetrically. Single crystal X-ray analyses have been carried out on eight complexes, four of which are bound to an alkali metal halide ion pair. Structural parameters, which affect the affinity and selectivity are discussed. A computational study on [[MX][12]crown-3] complexes (M =Li, Na; X=Cl, Br, I) was performed in order to compare relevant bond lengths and angles of the energy-minimized structures with those of the organometallic receptors.

Journal Article↗
PubMedDate not supplied

Molecular dissection of the paired helical filament.

Abundant neurofibrillary tangles, neuropil threads and plaque neurites constitute the neurofibrillary pathology of Alzheimer's disease. They form in the nerve cells that undergo degeneration in the disease where their regional distribution correlates with the degree of dementia. Each lesion contains the paired helical filament (PHF) as its major fibrous component. Recent work has shown that PHFs are composed of the microtubule-associated protein tau in a hyperphosphorylated state. PHF-tau is hyperphosphorylated on six adult brain tau isoforms. As a consequence, tau is unable to bind to microtubules and is believed to self-assemble into the PHF. Current evidence suggests that protein kinases or protein phosphatases with a specificity for serine/threonine-proline residues play an important role in the hyperphosphorylation of tau. Candidate protein kinases include mitogen-activated protein kinase, glycogen synthase kinase-3 and cyclin-dependent kinase 5, whereas the trimeric form of protein phosphatase 2A is a candidate phosphatase.

Aged↗
PubMed2002

Effect of various mild surfactants on the reassembly of an oligomeric integral membrane protein OmpF porin.

Reassembly of OmpF porin from its denatured monomer into the sodium dodecyl sulfate-resistant species was investigated by using 27 kinds of mild surfactants. Polyethyleneoxide-type surfactants with a hydrophilic-lipophilic balance value of 10.8-14.6 induced the trimerization of denatured OmpF porin. Dimerization and trimerization were induced by non-polyethyleneoxide-type mild surfactants that are generally used for membrane protein solubilization. The dependence of surfactant concentrations on reassembly was estimated to obtain a minimal concentration required for the reassembly of the protein. Extensive reassembly (to approximately 85% yield) into dimer (a putative assembly intermediate) was observed at a protein concentration of 0.05 mg/ml in 7 mg/ml n-octyl-beta-D-glucopyranoside and 1 mg/ml sodium dodecyl sulfate. This condition will be useful for the studies of the dimer and dimerization of OmpF porin. The role of mixed micelle system on the protein renaturation was discussed.

Chromatography, Gel↗
PubMed1990

Efficient expression of chicken alpha 1(VI) collagen chain in transiently transfected mammalian cells.

Type VI collagen is a component of the extracellular matrix made of three subunits, alpha 1(VI) and alpha 2(VI) (Mr = 140,000), and alpha 3(VI) (Mr = greater than 300,000). Triple helical monomers assemble intracellularly into disulfide-linked dimers and tetramers, with the tetramers being the "building blocks" that give rise to higher order extracellular structures by head-to-head association, the microfilaments. To study the pattern of assembly and the structure-function relationships of type VI collagen, we transfected mammalian cells with a full-length cDNA coding for chicken alpha 1(VI) under the control of SV40 early and late promoters and assayed the expression, secretion, and assembly of the protein by immunoperoxidase and immunoprecipitation of metabolically labeled cells. First, conditions were determined that allowed efficient transfection both in African monkey kidney COS-1 and CV-1 cells and in mouse fibroblasts. In our hands the late promoter was most efficient in CV-1 cells; whereas the early promoter was efficient in L cells at three days post-transfection. Chicken alpha 1(VI) could be isolated from cell extracts as well as from cell medium. Both the intracellular and the secreted forms of alpha 1(VI) are present as a monomer polypeptide and as disulfide-linked dimers and trimers that migrate in SDS gels with apparent Mr of about 130,000, 240,000 and 360,000, respectively. In L cells, endogenous mouse type VI collagen also was isolated by immunoprecipitation with specific antibodies. However, heterologous molecules made of the chicken alpha 1(VI) chain and the mouse alpha 2(VI) and alpha 3(VI) chains were not detected in the present experiments. Digestion with pepsin of the non-reduced chicken alpha 1(VI) polypeptides immunoprecipitated from the cell medium resulted in the disappearance of the bands, suggesting improper or non-stable assembly of alpha 1(VI) homotrimers. These data support predictions from sequence analysis that type VI collagen heterotrimeric molecules are more stable than other assembly alternatives.

Animals↗
PubMed2005

Amplification of acetylcholine-binding catenanes from dynamic combinatorial libraries.

Directed chemical synthesis can produce a vast range of molecular structures, but the intended product must be known at the outset. In contrast, evolution in nature can lead to efficient receptors and catalysts whose structures defy prediction. To access such unpredictable structures, we prepared dynamic combinatorial libraries in which reversibly binding building blocks assemble around a receptor target. We selected for an acetylcholine receptor by adding the neurotransmitter to solutions of dipeptide hydrazones [proline-phenylalanine or proline-(cyclohexyl)alanine], which reversibly combine through hydrazone linkages. At thermodynamic equilibrium, the dominant receptor structure was an elaborate [2]-catenane consisting of two interlocked macrocyclic trimers. This complex receptor with a 100 nM affinity for acetylcholine could be isolated on a preparative scale in 67% yield.

Acetylcholine↗