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Oligomerization
Kinds of Oligomerization Terms modified by Oligomerization Selected Abstracts(4-Acyl-5-pyrazolonato)titanium Derivatives: Oligomerization, Hydrolysis, Voltammetry, and DFT StudyEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 17 2003Francesco Caruso Abstract Twenty 4-acyl-5-pyrazolonato (Q) titanium derivatives of varied nuclearity have been synthesized from Ti(OR)4 or TiCl4 and characterized with spectroscopic methods (IR, NMR, ESI-MS). While Ti,(,-diketonato) cleavage is not seen in isolated solids, Ti,O(alkoxy) (or Ti,Cl) bonds cleave upon hydrolysis, leading to several structural forms, including oligomers. Ionic Q species with no Ti, i.e., obtained after Ti,Q cleavage, are seen for some Ti,Q derivatives by ESI-MS, which also indicates a varied nuclearity for a given species, e.g., the isolated polynuclear [Q2Ti-,-O]n has several "n" values. Mononuclear Ti complexes are obtained under rigorous anhydrous conditions. The cis structures of the mononuclear species (QT)2Ti(OCH3)2, QT = 3-methyl-4-(neopentylcarbonyl)-1-phenylpyrazol-5-onato have been analyzed with DFT methods. A trans influence is a major driving force that accounts for several sets of Ti,O bonds. One of the cis stereoisomers is 56 kcal/mol higher in energy than the other two. In contrast, all (QT)2TiCl2cis isomers show similar energies. Voltammetry of the mononuclear species (QT)2Ti(OnPr)2 and the antitumor tetranuclear compound [(QB)2Ti-,-O]4, (QB = 4-benzoyl-3-methyl-1-phenylpyrazol-5-onato) indicate that the TiIV is less prone to reduction to TiIII in the latter (Epc for the TiIV/TiIII couple is ,1.71 V and ,1.46 V versus Fc+/Fc, respectively). Potential antitumor compounds having a Ti/Q ratio of 1:1 do not disproportionate, unlike the equivalent acetylacetonato derivatives, and are water-soluble. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003) [source] Randomization of Amyloid-,-Peptide(1-42) Conformation by Sulfonated and Sulfated Nanoparticles Reduces Aggregation and CytotoxicityMACROMOLECULAR BIOSCIENCE, Issue 10 2010Ana M. Saraiva Abstract The amyloid-, peptide (A,) plays a central role in the mechanism of Alzheimer's disease, being the main constituent of the plaque deposits found in AD brains. A, amyloid formation and deposition are due to a conformational switching to a ,-enriched secondary structure. Our strategy to inhibit A, aggregation involves the re-conversion of A, conformation by adsorption to nanoparticles. NPs were synthesized by sulfonation and sulfation of polystyrene, leading to microgels and latexes. Both polymeric nanostructures affect the conformation of A, inducing an unordered state. Oligomerization was delayed and cytotoxicity reduced. The proper balance between hydrophilic moieties and hydrophobic chains seems to be an essential feature of effective NPs. [source] Titanium-Catalyzed Norbornene Oligomerization.MACROMOLECULAR RAPID COMMUNICATIONS, Issue 22 20063- exo -Disyndiotactic Structure, Isolation of a Crystalline Heptamer with a Abstract Summary: Norbornene (NB) was oligomerized at 0,°C using AlEt2Cl-TiCl4 at a monomer/titanium molar ratio of about 11. The oligomerization product consists of a fraction soluble in diethyl ether, amorphous by X-ray examination, and of a crystalline fraction, insoluble in diethyl ether. The crystalline fraction was shown by powder X-ray diffraction to consist of a NB heptamer. Single-crystal X-ray analysis indicated that the heptamer had a stereoregular 2,3- exo -disyndiotactic structure. The heptamer adopts a strained, highly irregular, folded conformation in the crystalline state. Structural differences with respect to NB oligomers obtained with zirconocene catalysts are discussed. A view of the molecular structure of the crystalline NB heptamer. [source] The intrinsic ATPase activity of Mycobacterium tuberculosis DnaA promotes rapid oligomerization of DnaA on oriCMOLECULAR MICROBIOLOGY, Issue 6 2006Murty V. V. S. Madiraju Summary Oligomerization of the initiator protein, DnaA, on the origin of replication (oriC) is crucial for initiation of DNA replication. Studies in Escherichia coli (Gram-negative) have revealed that binding of DnaA to ATP, but not hydrolysis of ATP, is sufficient to promote DnaA binding, oligomerization and DNA strand separation. To begin understanding the initial events involved in the initiation of DNA replication in Mycobacterium tuberculosis (Gram-positive), we investigated interactions of M. tuberculosis DnaA (DnaATB) with oriC using surface plasmon resonance in the presence of ATP and ADP. We provide evidence that, in contrast to what is observed in E. coli, ATPase activity of DnaATB promoted rapid oligomerization on oriC. In support, we found that a recombinant mutant DnaATB proficient in binding to ATP, but deficient in ATPase activity, did not oligomerize as rapidly. The corresponding mutation in the dnaA gene of M. tuberculosis resulted in non-viability, presumably due to a defect in oriC,DnaA interactions. Dimethy sulphate (DMS) footprinting experiments revealed that DnaATB bound to DnaA boxes similarly with ATP or ADP. DnaATB binding to individual DnaA boxes revealed that rapid oligomerization on oriC is triggered only after the initial interaction of DnaA with individual DnaA boxes. We propose that ATPase activity enables the DnaA protomers on oriC to rapidly form oligomeric complexes competent for replication initiation. [source] The oligomerization of CynR in Escherichia coliPROTEIN SCIENCE, Issue 11 2009Gwendowlyn S. Knapp Abstract Deletion analysis and alanine-scanning based on a homology-based interaction model were used to identify determinants of oligomerization in the transcriptional regulator CynR, a member of the LysR-type transcriptional regulator (LTTR) family. Deletion analysis confirmed that the putative regulatory domain of CynR was essential for driving the oligomerization of , repressor-CynR fusion proteins. The interaction surface of a different LTTR and OxyR was mapped onto a multiple sequence alignment of the LTTR family. This mapping identified putative contacts in the CynR regulatory domain dimer interface, which were targeted for alanine-scanning mutagenesis. Oligomerization was assayed by the ability of mutant , repressor-CynR fusions to assemble in E. coli revealing interesting similarities and differences between OxyR and CynR. [source] Oligomerization of BenM, a LysR-type transcriptional regulator: structural basis for the aggregation of proteins in this familyACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2007Sandra Haddad LysR-type transcriptional regulators comprise the largest family of homologous regulatory DNA-binding proteins in bacteria. A problematic challenge in the crystallization of LysR-type regulators stems from the insolubility and precipitation difficulties encountered with high concentrations of the full-length versions of these proteins. A general oligomerization scheme is proposed for this protein family based on the structures of the effector-binding domain of BenM in two different space groups, P4322 and C2221. These structures used the same oligomerization scheme of dimer,dimer interactions as another LysR-type regulator, CbnR, the full-length structure of which is available [Muraoka et al. (2003), J. Mol. Biol.328, 555,566]. Evaluation of packing relationships and surface features suggests that BenM can form infinite oligomeric arrays in crystals through these dimer,dimer interactions. By extrapolation to the liquid phase, such dimer,dimer interactions may contribute to the significant difficulty in crystallizing full-length members of this family. The oligomerization of dimeric units to form biologically important tetramers appears to leave unsatisfied oligomerization sites. Under conditions that favor association, such as neutral pH and concentrations appropriate for crystallization, higher order oligomerization could cause solubility problems with purified proteins. A detailed model by which BenM and other LysR-type transcriptional regulators may form these arrays is proposed. [source] ChemInform Abstract: Rhodium-Catalyzed Aryl- and Alkylation,Oligomerization of Alkynoates with Organoboron Reagents Giving Salicylates.CHEMINFORM, Issue 30 2010Yuichi Yasuhara Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] A Novel Catalyst for Isobutene Oligomerization to High Quality GasolineCHINESE JOURNAL OF CHEMISTRY, Issue 6 2003Zhou Peng Abstract Dimerization of isobutene in liquid state to form high-octane value gasoline components was investigated over a homemade novel ion exchange resin, SPPESK, which shows high activity and selectivity in isobutene dimerization under mild conditions. [source] Exendin-4 protects pancreatic beta cells from human islet amyloid polypeptide-induced cell damage: potential involvement of AKT and mitochondria biogenesisDIABETES OBESITY & METABOLISM, Issue 9 2010R. Fan Aim: Glucagon-like peptide-1 (GLP-1) stimulates beta-cell proliferation and enhances beta-cell survival, whereas oligomerization of human islet amyloid polypeptide (hIAPP) may induce beta-cell apoptosis and reduce beta-cell mass. Type 2 diabetes is associated with increased expression of IAPP. As GLP-1-based therapy is currently developed as a novel antidiabetic therapy, we examined the potential protective action of the GLP-1 receptor agonist exendin-4 on hIAPP-induced beta-cell apoptosis. Methods: The study was performed in clonal insulinoma (INS-1E) cells. Both method of transcriptional and translational and sulphorhodamine B (SRB) assays were used to evaluate cell viability and cell mass. Western blot analysis was applied to detect protein expression. Transfection of constitutively active protein kinase B (PKB/AKT) was performed to examine the role of AKT. Mitochondrial biogenesis was quantified by mitogreen staining and RT-PCR. Results: First, we confirmed that hIAPP induced cell apoptosis and growth inhibition in INS-1E cells. These effects were partially protected by exendin-4 in association with partial recovery of the hIAPP-mediated AKT inhibition. Furthermore, AKT constitutive activation attenuated hIAPP-induced apoptosis, whereas PI3K/AKT inhibition abrogated exendin-4-mediated effects. These findings suggest that the antiapoptotic and proliferative effects of exendin-4 in hIAPP-treated INS-1E cells were partially mediated through AKT pathway. Moreover, hIAPP induced FOXO1 but inhibited pdx-1 nucleus translocation. These effects were restored by exendin-4. Finally, mitogreen staining and RT-PCR revealed enhanced mitochondrial biogenesis by exendin-4 treatment. Conclusions: Collectively, these results suggest that GLP-1 receptor agonist protects beta cells from hIAPP-induced cell death partially through the activation of AKT pathway and improved mitochondrial function. [source] Progress in the development of new treatments for combined Alzheimer's and Parkinson's diseasesDRUG DEVELOPMENT RESEARCH, Issue 3 2002Eliezer Masliah Abstract Misfolding of synaptic molecules such as amyloid , peptide and ,-synuclein has been proposed to play a key role in the mechanisms of neurodegeneration in Alzheimer's and Parkinson's disease, respectively. Notably, the majority of patients with Alzheimer's disease also have ,-synuclein-immunoreactive Lewy bodies, and a substantial proportion of them develop a form of parkinsonism also known as Lewy body disease, that defies conventional therapies. Thus, factors involved in the pathogenesis of Alzheimer's disease might promote the development of particularly recalcitrant forms of Lewy body disease. We have shown that the amyloid , peptide 1-42, of Alzheimer's disease, promotes the toxic conversion of ,-synuclein and accelerates ,-synuclein-dependent deficits in transgenic mice. Understanding the mechanisms promoting the toxic conversion of ,-synuclein is of critical importance for the design of rationale treatments for Lewy body disease and transgenic models hold the promise for the development of such novel therapies. In this context therapies aimed at: (1) reducing amyloid , peptide 1-42 production, (2) blocking toxic ,-synuclein oligomerization (e.g., ,-synuclein, antioxidants), (3) promoting ,-synuclein protofibril degradation, and (4) protecting neurons (e.g., anti-oxidants, neurotrophic agents) against toxic ,-synuclein aggregates might prove to be significantly useful in the treatment of Lewy body disease. We characterized ,-synuclein, the non-amyloidogenic homologue of ,-synuclein, as an inhibitor of aggregation of ,-synuclein. Our results raise the intriguing possibility that ,-synuclein might be a natural negative regulator of ,-synuclein aggregation, and that a similar class of endogenous factors might modulate the toxic conversion of other molecules involved in neurodegeneration. Such an anti-amyloidogenic property of ,-synuclein in combination with other treatments might also provide a novel strategy for the treatment of neurodegenerative disorders. Drug Dev. Res. 56:282,292, 2002. © 2002 Wiley-Liss, Inc. [source] ,-Cyclodextrin as novel chiral probe for enantiomeric separation by electromigration methodsELECTROPHORESIS, Issue 21 2006Dorothee Wistuba Abstract Native ,-CD has been employed as chiral selector in CE and MEKC. To investigate the potential of the enantiodiscriminating properties of ,-CD, negatively charged 5-dimethylamino-1-naphthalene-sulfonyl (dansyl)-, 2,4-dinitrophenyl (DNP)- and FMOC-derivatives of several amino acids, 1,1'-binaphthyl-2,2'-diylhydrogenphosphate, flavanones and three positively charged drugs have been selected as testing samples. Enantioresolution factors up to 4.82 have been observed. The results were compared with those achieved by the conventional running buffer additives ,-, ,- and ,-CDs. For several examples a steady increase of enantioresolution with increasing degree of oligomerization has been detected. [source] Mutation in hotfoot-4J mice results in retention of ,2 glutamate receptors in EREUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2002Shinji Matsuda Abstract The orphan glutamate receptor ,2 is selectively expressed in Purkinje cells and plays a critical role in cerebellar function. Recently, the ataxia of hotfoot-4J (ho-4J) mice was shown to be caused by a 170,amino acid deletion in the N-terminal region of ,2 receptors. To understand ,2 receptor function, we characterized these mutant receptors (,2ho) in Purkinje cells. Immunohistochemical staining showed that ,2ho receptors of the ho-4J homozygotes were abundantly expressed but localized to the Purkinje cell soma; in wild-type mice, ,2 receptors were predominantly present at distal dendrites of Purkinje cells. In addition, ,2ho receptors of the ho-4J mice were sensitive to endoglycosidase H, a finding suggesting that ,2ho receptors were not transported beyond the endoplasmic reticulum (ER) or cis -Golgi apparatus. To gain further insights into the mechanisms of this phenomenon, we characterized ,2ho receptors in transfected HEK293 cells. ,2ho receptors expressed in HEK293 cells were also sensitive to endoglycosidase H. Immunohistochemical staining showed that ,2ho receptors colocalized with proteins retained in the ER. Furthermore, ,2ho receptors were not labelled by membrane-impermeable biotinylation reagents. Coimmunoprecipitation assays showed that the intermolecular interaction of ,2ho receptors was significantly weaker than those of wild-type ,2 receptors, a finding suggesting that the ho-4J region is involved in oligomerization of ,2 receptors. Thus, ,2ho receptors were retained in the ER, probably by the quality control mechanism that detects unstable oligomers. We conclude that the absence of ,2 receptors on the cell surface by failed transport from the ER of Purkinje cells causes ataxia. [source] An Improved Synthesis of Procyanidin Dimers: Regio- and Stereocontrol of the Interflavan BondEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 23 2006Isabelle Tarascou Abstract A direct and general synthesis of procyanidin dimers B1, B2, B3 and B4 (10a,d) is presented. The approach is based on the stoichiometric coupling of two protected monomeric units (the nucleophilic 2a,b and electrophilic 4a,b partners) and deals with the regio- and stereocontrol of the C4,C8 interflavan bond as well as the control of the degree of oligomerization. The synthesis involves a five-step pathway starting from the native catechin (1a) or epicatechin (1b) to the fully deprotected dimers 10a,d. Furthermore, the process appears to be iterative as the coupling intermediates 9a,d themselves can be readily used in further selective syntheses of trimers or higher oligomers. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006) [source] Structure of the HIV-1 Rev response element alone and in complex with regulator of virion (Rev) studied by atomic force microscopyFEBS JOURNAL, Issue 15 2009Jesper Pallesen The interaction of multiple HIV-1 regulator of virion (Rev) proteins with the viral RNA target, the Rev response element (RRE), is critical for nuclear export of incompletely spliced and unspliced viral RNA, and for the onset of the late phase in the viral replication cycle. The heterogeneity of the Rev,RRE complex has made it difficult to study using conventional structural methods. In the present study, atomic force microscopy is applied to directly visualize the tertiary structure of the RRE RNA alone and in complex with Rev proteins. The appearance of the RRE is compatible with the earlier proposed RRE secondary structure in dimensions and overall shape, including a stalk and a head interpreted as stem I, and stem-loops II,V in the secondary structure model, respectively. Atomic force microscopy imaging of the Rev,RRE complex revealed an increased height of the structure both in the stalk and head regions, which is in accordance with a binding model in which Rev binding to a high affinity site in stem IIB triggers oligomerization of Rev proteins through cooperative binding along stem I in RRE. The present study demonstrates that atomic force microscopy comprises a useful technique to study complex biological structures of nucleic acids at high resolution. [source] Phosphorylation of the arginine/serine dipeptide-rich motif of the severe acute respiratory syndrome coronavirus nucleocapsid protein modulates its multimerization, translation inhibitory activity and cellular localizationFEBS JOURNAL, Issue 16 2008Tsui-Yi Peng Coronavirus nucleocapsid protein is abundant in infected cells and participates in viral RNA replication and transcription. The central domain of the nucleocapsid protein contains several arginine/serine (RS) dipeptides, the biological significance of which has not been well investigated. In the present study, we demonstrate that the severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated primarily within the RS-rich region in cells and by SR protein kinase 1 in vitro. The nucleocapsid protein could suppress translation and its RS motif is essential for such an activity. Moreover, phosphorylation of the RS motif could modulate the translation inhibitory activity of the nucleocapsid protein. We further found that RS motif phosphorylation did not significantly affect RNA binding of the nucleocapsid protein but impaired its multimerization ability. We observed that the nucleocapsid protein could translocate to cytoplasmic stress granules in response to cellular stress. Deletion or mutations of the RS motif enhanced stress granule localization of the nucleocapsid protein, whereas overexpression of SR protein kinase 1 inhibited nucleocapsid protein localization to stress granules. The nucleocapsid protein lacking the RS motif formed high-order RNP complexes, which may also account for its enhanced stress granule localization. Taken together, phosphorylation of the severe acute respiratory syndrome-CoV nucleocapsid protein modulates its activity in translation control and also interferes with its oligomerization and aggregation in stress granules. [source] Salt-resistant homodimeric bactenecin, a cathelicidin-derived antimicrobial peptideFEBS JOURNAL, Issue 15 2008Ju Y. Lee The cathelicidin antimicrobial peptide bactenecin is a ,-hairpin molecule with a single disulfide bond and broad antimicrobial activity. The proform of bactenecin exists as a dimer, however, and it has been proposed that bactenecin is released as a dimer in vivo, although there has been little study of the dimeric form of bactenecin. To investigate the effect of bactenecin dimerization on its biological activity, we characterized the dimer's effect on phospholipid membranes, the kinetics of its bactericidal activity, and its salt sensitivity. We initially synthesized two bactenecin dimers (antiparallel and parallel) and two monomers (,-hairpin and linear). Under oxidative folding conditions, reduced linear bactenecin preferentially folded into a dimer forming a ladder-like structure via intermolecular disulfide bonding. As compared to the monomer, the dimer had a greater ability to induce lysis of lipid bilayers and was more rapidly bactericidal. Interestingly, the dimer retained antimicrobial activity at physiological salt concentrations (150 mm NaCl), although the monomer was inactivated. This salt resistance was also seen with bactenecin dimer containing one intermolecular disulfide bond, and the bactenecin dimer appears to undergo multimeric oligomerization at high salt concentrations. Overall, dimeric bactenecin shows potent and rapid antimicrobial activity, and resists salt-induced inactivation under physiological conditions through condensation and oligomerization. These characteristics shed light on the features that a peptide would need to serve as an effective therapeutic agent. [source] Lipid-induced conformational transition of the amyloid core fragment A,(28,35) and its A30G and A30I mutantsFEBS JOURNAL, Issue 10 2008Sureshbabu Nagarajan The interaction of the ,-amyloid peptide (A,) with neuronal membranes could play a key role in the pathogenesis of Alzheimer's disease. Recent studies have focused on the interactions of A, oligomers to explain the neuronal toxicity accompanying Alzheimer's disease. In our study, we have investigated the role of lipid interactions with soluble A,(28,35) (wild-type) and its mutants A30G and A30I in their aggregation and conformational preferences. CD and Trp fluorescence spectroscopic studies indicated that, immediately on dissolution, these peptides adopted a random coil structure. Upon addition of negatively charged 1,2-dipalmitoyl- syn -glycero-3-phospho- rac -(glycerol) sodium salt (PG) lipid, the wild-type and A30I mutant underwent reorganization into a predominant ,-sheet structure. However, no conformational changes were observed in the A30G mutant on interaction with PG. In contrast, the presence of zwitterionic 1,2-dipalmitoyl- syn -glycero-3-phosphatidylcholine (PC) lipid had no effect on the conformation of these three peptides. These observations were also confirmed with atomic force microscopy and the thioflavin-T assay. In the presence of PG vesicles, both the wild-type and A30I mutant formed fibrillar structures within 2 days of incubation in NaCl/Pi, but not in their absence. Again, no oligomerization was observed with PC vesicles. The Trp studies also revealed that both ends of the three peptides are not buried deep in the vesicle membrane. Furthermore, fluorescence spectroscopy using the environment-sensitive probe 1,6-diphenyl-1,3,5-hexatriene showed an increase in the membrane fluidity upon exposure of the vesicles to the peptides. The latter effect may result from the lipid head group interactions with the peptides. Fluorescence resonance energy transfer experiments revealed that these peptides undergo a random coil-to-sheet conversion in solution on aging and that this process is accelerated by negatively charged lipid vesicles. These results indicate that aggregation depends on hydrophobicity and propensity to form ,-sheets of the amyloid peptide, and thus offer new insights into the mechanism of amyloid neurodegenerative disease. [source] The Vps4 C-terminal helix is a critical determinant for assembly and ATPase activity and has elements conserved in other members of the meiotic clade of AAA ATPasesFEBS JOURNAL, Issue 7 2008Parimala R. Vajjhala Sorting of membrane proteins into intralumenal endosomal vesicles, multivesicular body (MVB) sorting, is critical for receptor down regulation, antigen presentation and enveloped virus budding. Vps4 is an AAA ATPase that functions in MVB sorting. Although AAA ATPases are oligomeric, mechanisms that govern Vps4 oligomerization and activity remain elusive. Vps4 has an N-terminal microtubule interacting and trafficking domain required for endosome recruitment, an AAA domain containing the ATPase catalytic site and a , domain, and a C-terminal , helix positioned close to the catalytic site in the 3D structure. Previous attempts to identify the role of the C-terminal helix have been unsuccessful. Here, we show that the C-terminal helix is important for Vps4 assembly and ATPase activity in vitro and function in vivo, but not endosome recruitment or interactions with Vta1 or ESCRT-III. Unlike the , domain, which is also important for Vps4 assembly, the C-terminal helix is not required in vivo for Vps4 homotypic interaction or dominant-negative effects of Vps4,E233Q, carrying a mutation in the ATP hydrolysis site. Vta1 promotes assembly of hybrid complexes comprising Vps4,E233Q and Vps4 lacking an intact C-terminal helix in vitro. Formation of catalytically active hybrid complexes demonstrates an intersubunit catalytic mechanism for Vps4. One end of the C-terminal helix lies in close proximity to the second region of homology (SRH), which is important for assembly and intersubunit catalysis in AAA ATPases. We propose that Vps4 SRH function requires an intact C-terminal helix. Co-evolution of a distinct Vps4 SRH and C-terminal helix in meiotic clade AAA ATPases supports this possibility. [source] Post-translational cleavage of recombinantly expressed nitrilase from Rhodococcus rhodochrous J1 yields a stable, active helical formFEBS JOURNAL, Issue 8 2007R. Ndoria Thuku Nitrilases convert nitriles to the corresponding carboxylic acids and ammonia. The nitrilase from Rhodococcus rhodochrous J1 is known to be inactive as a dimer but to become active on oligomerization. The recombinant enzyme undergoes post-translational cleavage at approximately residue 327, resulting in the formation of active, helical homo-oligomers. Determining the 3D structure of these helices using electron microscopy, followed by fitting the stain envelope with a model based on homology with other members of the nitrilase superfamily, enables the interacting surfaces to be identified. This also suggests that the reason for formation of the helices is related to the removal of steric hindrance arising from the 39 C-terminal amino acids from the wild-type protein. The helical form can be generated by expressing only residues 1,327. [source] NEMO oligomerization in the dynamic assembly of the I,B kinase core complexFEBS JOURNAL, Issue 10 2007Elisabeth Fontan NF-,B essential modulator (NEMO) plays an essential role in the nuclear factor ,B (NF-,B) pathway as a modulator of the two other subunits of the I,B kinase (IKK) complex, i.e. the protein kinases, IKK, and IKK,. Previous reports all envision the IKK complex to be a static entity. Using glycerol-gradient ultracentrifugation, we observed stimulus-dependent dynamic IKK complex assembly. In wild-type fibroblasts, the kinases and a portion of cellular NEMO associate in a 350-kDa high-molecular-mass complex. In response to constitutive NF-,B stimulation by Tax, we observed NEMO recruitment and oligomerization to a shifted high-molecular-mass complex of 440 kDa which displayed increased IKK activity. This stimulus-dependent oligomerization of NEMO was also observed using fluorescence resonance energy transfer after a transient pulse with interleukin-1,. In addition, fully activated, dimeric kinases not bound to NEMO were detected in these Tax-activated fibroblasts. By glycerol gradient ultracentrifugation, we also showed that: (a) in fibroblasts deficient in IKK, and IKK,, NEMO predominantly exists as a monomer; (b) in NEMO-deficient fibroblasts, IKK, dimers are present that are less stable than IKK, dimers. Intriguingly, in resting Rat-1 fibroblasts, 160-kDa IKK,,NEMO and IKK,,NEMO heterocomplexes were observed as well as a significant proportion of NEMO monomer. These results suggest that most NEMO molecules do not form a tripartite IKK complex with an IKK,,IKK, heterodimer as previously reported in the literature but, instead, NEMO is able to form a complex with the monomeric forms of IKK, and IKK,. [source] Docosahexaenoic acid stabilizes soluble amyloid-, protofibrils and sustains amyloid-,-induced neurotoxicity in vitroFEBS JOURNAL, Issue 4 2007Ann-Sofi Johansson Enrichment of diet and culture media with the polyunsaturated fatty acid docosahexaenoic acid has been found to reduce the amyloid burden in mice and lower amyloid-, (A,) levels in both mice and cultured cells. However, the direct interaction of polyunsaturated fatty acids, such as docosahexaenoic acid, with A,, and their effect on A, aggregation has not been explored in detail. Therefore, we have investigated the effect of docosahexaenoic acid, arachidonic acid and the saturated fatty acid arachidic acid on monomer oligomerization into protofibrils and protofibril fibrillization into fibrils in vitro, using size exclusion chromatography. The polyunsaturated fatty acids docosahexaenoic acid and arachidonic acid at micellar concentrations stabilized soluble A,42 wild-type protofibrils, thereby hindering their conversion to insoluble fibrils. As a consequence, docosahexaenoic acid sustained amyloid-,-induced toxicity in PC12 cells over time, whereas A, without docosahexaenoic acid stabilization resulted in reduced toxicity, as A, formed fibrils. Arachidic acid had no effect on A, aggregation, and neither of the fatty acids had any protofibril-stabilizing effect on A,42 harboring the Arctic mutation (A,E22G). Consequently, A,Arctic-induced toxicity could not be sustained using docosahexaenoic acid. These results provide new insights into the toxicity of different A, aggregates and how endogenous lipids can affect A, aggregation. [source] Structural model for an AxxxG-mediated dimer of surfactant-associated protein CFEBS JOURNAL, Issue 11 2004Visvaldas Kairys The pulmonary surfactant prevents alveolar collapse and is required for normal pulmonary function. One of the important components of the surfactant besides phospholipids is surfactant-associated protein C (SP-C). SP-C shows complex oligomerization behavior and a transition to ,-amyloid-like fibril structures, which are not yet fully understood. Besides this nonspecific oligomerization, MS and chemical cross-linking data combined with CD spectra provide evidence of a specific, mainly ,-helical, dimer at low to neutral pH. Furthermore, resistance to CNBr cleavage and dual NMR resonances of porcine and human recombinant SP-C with Met32 replaced by isoleucine point to a dimerization site located at the C-terminus of the hydrophobic ,-helix of SP-C, where a strictly conserved heptapeptide sequence is found. Computational docking of two SP-C helices, described here, reveals a dimer with a helix,helix interface that strikingly resembles that of glycophorin A and is mediated by an AxxxG motif similar to the experimentally determined GxxxG pattern of glycophorin A. It is highly likely that mature SP-C adopts such a dimeric structure in the lamellar bilayer systems found in the surfactant. Dimerization has been shown in previous studies to have a role in sorting and trafficking of SP-C and may also be important to the surfactant function of this protein. [source] Elements of the C-terminal t peptide of acetylcholinesterase that determine amphiphilicity, homomeric and heteromeric associations, secretion and degradationFEBS JOURNAL, Issue 8 2004Stéphanie Belbeoc'h The C-terminal t peptide (40 residues) of vertebrate acetylcholinesterase (AChE) T subunits possesses a series of seven conserved aromatic residues and forms an amphiphilic ,-helix; it allows the formation of homo-oligomers (monomers, dimers and tetramers) and heteromeric associations with the anchoring proteins, ColQ and PRiMA, which contain a proline-rich motif (PRAD). We analyzed the influence of mutations in the t peptide of Torpedo AChET on oligomerization and secretion. Charged residues influenced the distribution of homo-oligomers but had little effect on the heteromeric association with QN, a PRAD-containing N-terminal fragment of ColQ. The formation of homo-tetramers and QN -linked tetramers required a central core of four aromatic residues and a peptide segment extending to residue 31; the last nine residues (32,40) were not necessary, although the formation of disulfide bonds by cysteine C37 stabilized T4 and T4,QN tetramers. The last two residues of the t peptide (EL) induced a partial intracellular retention; replacement of the C-terminal CAEL tetrapeptide by KDEL did not prevent tetramerization and heteromeric association with QN, indicating that these associations take place in the endoplasmic reticulum. Mutations that disorganize the ,-helical structure of the t peptide were found to enhance degradation. Co-expression with QN generally increased secretion, mostly as T4,QN complexes, but reduced it for some mutants. Thus, mutations in this small, autonomous interaction domain bring information on the features that determine oligomeric associations of AChET subunits and the choice between secretion and degradation. [source] Constitutive oligomerization of human D2 dopamine receptors expressed in Spodoptera frugiperda 9 (Sf9) and in HEK293 cellsFEBS JOURNAL, Issue 19 2003Analysis using co-immunoprecipitation, time-resolved fluorescence resonance energy transfer Human D2Long (D2L) and D2Short (D2S) dopamine receptor isoforms were modified at their N-terminus by the addition of a human immunodeficiency virus (HIV) or a FLAG epitope tag. The receptors were then expressed in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus system, and their oligomerization was investigated by means of co-immunoprecipitation and time-resolved fluorescence resonance energy transfer (FRET). [3H]Spiperone labelled D2 receptors in membranes prepared from Sf9 cells expressing epitope-tagged D2L or D2S receptors, with a pKd value of , 10. Co-immunoprecipitation using antibodies specific for the tags showed constitutive homo-oligomerization of D2L and D2S receptors in Sf9 cells. When the FLAG-tagged D2S and HIV-tagged D2L receptors were co-expressed, co-immunoprecipitation showed that the two isoforms can also form hetero-oligomers in Sf9 cells. Time-resolved FRET with europium and XL665-labelled antibodies was applied to whole Sf9 cells and to membranes from Sf9 cells expressing epitope-tagged D2 receptors. In both cases, constitutive homo-oligomers were revealed for D2L and D2S isoforms. Time-resolved FRET also revealed constitutive homo-oligomers in HEK293 cells expressing FLAG-tagged D2S receptors. The D2 receptor ligands dopamine, R -(,)propylnorapomorphine, and raclopride did not affect oligomerization of D2L and D2S in Sf9 and HEK293 cells. Human D2 dopamine receptors can therefore form constitutive oligomers in Sf9 cells and in HEK293 cells that can be detected by different approaches, and D2 oligomerization in these cells is not regulated by ligands. [source] Probing the role of oligomerization in the high thermal stability of Pyrococcus furiosus ornithine carbamoyltransferase by site-specific mutantsFEBS JOURNAL, Issue 14 2001Bernard Clantin The Pyrococcus furiosus ornithine carbamoyltransferase (OTCase) is extremely heat stable and maintains 50% of its catalytic activity after 60 min at 100 °C. The enzyme has an unusual quaternary structure when compared to anabolic OTCases from mesophilic organisms. It is built up of four trimers arranged in a tetrahedral manner, while other anabolic enzymes are single trimers. Residues Trp21, Glu25, Met29 and Trp33 are located in the main interfaces that occur between the catalytic trimers within the dodecamer. They participate in either hydrophobic clusters or ionic interactions. In order to elucidate the role played by the oligomerization in the enzyme stability at very high temperatures, we performed mutagenesis studies of these residues. All the variants show similar catalytic activities and kinetic properties when compared to the wild-type enzyme, allowing the interpretation of the mutations solely on heat stability and quaternary structure. The W21A variant has only a slight decrease in its stability, and is a dodecamer. The variants E25Q, M29A, W33A, W21A/W33A and E25Q/W33A show that altering more drastically the interfaces results in a proportional decrease in heat stability, correlated with a gradual dissociation of dodecamers into trimers. Finally, the E25Q/M29A/W33A variant shows a very large decrease in heat stability and is a trimer. These results suggest that extreme thermal stabilization of this OTCase is achieved in part through oligomerization. [source] Mechanism of activation of the double-stranded-RNA-dependent protein kinase, PKRFEBS JOURNAL, Issue 13 2001Role of dimerization, cellular localization in the stimulation of PKR phosphorylation of eukaryotic initiation factor-2 (eIF2) An important defense against viral infection involves inhibition of translation by PKR phosphorylation of the , subunit of eIF2. Binding of viral dsRNAs to two dsRNA-binding domains (dsRBDs) in PKR leads to relief of an inhibitory region and activation of eIF2 kinase activity. Interestingly, while deletion of the regulatory region of PKR significantly induces activity in vitro, the truncated kinase does not inhibit translation in vivo, suggesting that these sequences carry out additional functions required for PKR control. To delineate these functions and determine the order of events leading to activation of PKR, we fused truncated PKR to domains of known function and assayed the chimeras for in vivo activity. We found that fusion of a heterologous dimerization domain with the PKR catalytic domain enhanced autophosphorylation and eIF2 kinase function in vivo. The dsRBDs also mediate ribosome association and we proposed that such targeting increases the localized concentration of PKR, enhancing interaction between PKR molecules. We addressed this premise by linking the truncated PKR to RAS sequences mediating farnesylation and membrane localization and found that the fusion protein was functional in vivo. These results indicate that cellular localization along with oligomerization enhances interaction between PKR molecules. Alanine substitution for the phosphorylation site, threonine 446, impeded in vivo and in vitro activity of the PKR fusion proteins, while aspartate or glutamate substitutions partially restored the function of the truncated kinase. These results indicate that both dimerization and cellular localization play a role in transient protein,protein interactions and that trans -autophosphorylation is the final step in the mechanism of activation of PKR. [source] Oligo(triacetylene) Derivatives with Pendant Long Alkyl ChainsHELVETICA CHIMICA ACTA, Issue 6 2004Jean-François Nierengarten Substituted (E)-2-(ethynyl)but-2-ene and (E)-hex-3-ene-1,5-diyne derivatives 6 and 10, respectively, were prepared by dicyclohexylcarbodiimide(DCC)-mediated esterification of tris(dodecyloxy)benzoic acid (4) with (E)-2-[(triisopropylsilyl)ethynyl]but-2-ene-1,4-diol (3) and (E)-2,3-bis[(trimethylsilyl)ethynyl]but-2-ene-1,4-diol (8), respectively, followed by deprotection with Bu4NF in wet THF (Schemes,1 and 2). Oligomerization reactions of diyne derivative 10 were attempted by treatment with the Hay catalyst in the presence of mono-alkyne 6 as an end-capping reagent. Under these conditions, only compound 7 resulting from the homocoupling of 6 (Scheme,1), and polymers of 10 were obtained due to the difference in reactivity of the alkyne groups in 6 and 10. In contrast, when phenylacetylene was used as the stopper, the oligomerization of 10 afforded a mixture of end-capped oligomers, from which 11,13 were isolated by column chromatography (Scheme,3). The poly(triacetylenes) (PTA) 16,18 were prepared in a similar manner starting from diol 8 and stearic acid (Schemes,4 and 5). Whereas the end-capped monomers and dimers 11, 12, 16, and 17 with pendant long alkyl chains do not exhibit any liquid-crystalline behavior, the trimeric derivatives 13 and 18 show mesomorphic properties, thus demonstrating that the poly(triacetylene) backbone can behave as a mesogenic unit. [source] Bid-dependent generation of oxygen radicals promotes death receptor activation,induced apoptosis in murine hepatocytesHEPATOLOGY, Issue 2 2004Wen-Xing Ding Activation of tumor necrosis factor receptor 1 or Fas leads to the generation of reactive oxygen species, which are important to the cytotoxic effects of tumor necrosis factor , (TNF-,) or Fas ligand. However, how these radicals are generated following receptor ligation is not clear. Using primary hepatocytes, we found that TNF-, or anti,Fas antibody,induced burst of oxygen radicals was mainly derived from the mitochondria. We discovered that Bid,a pro-death Bcl-2 family protein activated by ligated death receptors,was the main intracellular molecule signaling the generation of the radicals by targeting to the mitochondria and that the majority of oxygen radical production was dependent on Bid. Reactive oxygen species contributed to cell death and caspase activation by promoting FLICE-inhibitory protein degradation and mitochondrial release of cytochrome c. For the latter part, the oxygen radicals did not affect Bak oligomerization but instead promoted mitochondrial cristae reorganization and membrane lipid peroxidation. Antioxidants could reverse these changes and therefore protect against TNF-, or anti,Fas-induced apoptosis. In conclusion, our studies established the signaling pathway from death receptor engagement to oxygen radical generation and determined the mechanism by which reactive oxygen species contributed to hepatocyte apoptosis following death receptor activation. (HEPATOLOGY 2004;40:403,413.) [source] Characterization of HCF-1, a determinant of Autographa californica multiple nucleopolyhedrovirus host specificityINSECT MOLECULAR BIOLOGY, Issue 6 2003K. L. Hefferon Abstract Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infects a wide variety of insect species. A number of AcMNPV late expression factors that are involved in replication have been identified as playing a role in determining host specificity. Host cell factor-1, or HCF-1, was previously demonstrated to be essential for viral replication in Tn -368 cells. Here we demonstrate that HCF-1 is an early protein and localizes to the cell nucleus. Coprecipitation experiments revealed that HCF-1 interacts with itself but none of the other late expression factors required for replication in Tn -368 cells. HCF-1 mutants were constructed and utilized to search for the domains involved in HCF-1 biological function and oligomerization. Possible roles of HCF-1 in determining host specificity are discussed. [source] Involvement of HAb18G/CD147 in T cell activation and immunological synapse formationJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 8 2010Jinsong Hu Abstract HAb18G/CD147, a glycoprotein of the immunoglobulin super-family (IgSF), is a T cell activation-associated molecule. In this report, we demonstrated that HAb18G/CD147 expression on both activated CD4+ and CD8+ T cells was up-regulated. In vitro cross-linking of T cells with an anti-HAb18G/CD147 monoclonal antibody (mAb) 5A12 inhibited T cells proliferation upon T cell receptor stimulation. Such co-stimulation inhibited T cell proliferation by down-regulating the expression of CD25 and interleukin-2 (IL-2), decreased production of IL-4 but not interferon-,. Laser confocal imaging analysis indicated that HAb18G/CD147 was recruited to the immunological synapse (IS) during T cell activation; triggering HAb18G/CD147 on activated T cells by anti-HAb18G/CD147 mAb 5A12 strongly dispersed the formation of the IS. Further functional studies showed that the ligation of HAb18G/CD147 with mAb 5A12 decreased the tyrosine phosphorylation and intracellular calcium mobilization levels of T cells. Through docking antibody,antigen interactions, we demonstrated that the function of mAb 5A12 is tightly dependent on its specificity of binding to N-terminal domain I, which plays pivotal role in the oligomerization of HAb18G/CD147. Taken together, we provide evidence that HAb18G/CD147 could act as a co-stimulatory receptor to negatively regulate T cell activation and is functionally linked to the formation of the IS. [source] | |||||||||||||||||||||||||||||||