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1
Schmid, J. A., H. Just et H. H. Sitte. « Impact of oligomerization on the function of the human serotonin transporter ». Biochemical Society Transactions 29, no 6 (1 novembre 2001) : 732–36. http://dx.doi.org/10.1042/bst0290732.
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The formation of oligomeric structures has been proposed for a large number of membrane proteins, including G-protein-coupled receptors and ion channels. Biochemical studies employing gel filtration, cross-linking or co-immunoprecipitation techniques showed that the serotonin [5-hydroxytryptamine (5-HT)] transporter is also capable of forming oligomers. We investigated whether the human serotonin transporter (hSERT) can be visualized as an oligomer in the plasma membrane of intact cells. To test this working hypothesis, we generated fusion proteins of hSERT and spectral variants of green fluorescent protein [cyan and yellow fluorescent proteins (CFP and YFP, respectively)]. When expressed in HeLa or HEK-293 cells, the resulting fusion proteins (CFP-hSERT and YFP-hSERT) were inserted into the plasma membrane and were indistinguishable from wild-type hSERT on functional testing (5-HT uptake assays, inhibition of 5-HT uptake by blockers such as imipramine). Oligomers were visualized by fluorescence resonance energy transfer (FRET) microscopy in living cells using complementary methods. Interestingly, oligomerization was not confined to hSERT; FRET was also observed between CFP-and YFP-labelled rat γ-aminobutyric acid transporter. Gel filtration experiments showed that most of the protein was recovered as higher molecular weight complexes; almost no monomeric form was detected. This indicates that the homo-oligomeric form is the favoured state of hSERT in living cells. The formation of oligomers was not significantly affected by co-incubation with transporter substrates or blockers. Based on our observations, oligomer formation might not be essential for the physiological function of the transporter protein, the re-uptake of substrates. Furthermore, we conclude that constitutive oligomer formation might be a general property of Na+/Cl−-dependent neurotransmitter transporters.
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Kreiser, Ryan P., Aidan K. Wright, Natalie R. Block, Jared E. Hollows, Lam T. Nguyen, Kathleen LeForte, Benedetta Mannini, Michele Vendruscolo et Ryan Limbocker. « Therapeutic Strategies to Reduce the Toxicity of Misfolded Protein Oligomers ». International Journal of Molecular Sciences 21, no 22 (17 novembre 2020) : 8651. http://dx.doi.org/10.3390/ijms21228651.
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The aberrant aggregation of proteins is implicated in the onset and pathogenesis of a wide range of neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. Mounting evidence indicates that misfolded protein oligomers produced as intermediates in the aggregation process are potent neurotoxic agents in these diseases. Because of the transient and heterogeneous nature of these elusive aggregates, however, it has proven challenging to develop therapeutics that can effectively target them. Here, we review approaches aimed at reducing oligomer toxicity, including (1) modulating the oligomer populations (e.g., by altering the kinetics of aggregation by inhibiting, enhancing, or redirecting the process), (2) modulating the oligomer properties (e.g., through the size–hydrophobicity–toxicity relationship), (3) modulating the oligomer interactions (e.g., by protecting cell membranes by displacing oligomers), and (4) reducing oligomer toxicity by potentiating the protein homeostasis system. We analyze examples of these complementary approaches, which may lead to the development of compounds capable of preventing or treating neurodegenerative disorders associated with protein aggregation.
3
Wako, Hiroshi, et Shigeru Endo. « ProMode-Oligomer : Database of Normal Mode Analysis in Dihedral Angle Space for a Full-Atom System of Oligomeric Proteins ». Open Bioinformatics Journal 6, no 1 (21 février 2012) : 9–19. http://dx.doi.org/10.2174/1875036201206010009.
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The database ProMode-Oligomer (http://promode.socs.waseda.ac.jp/promode_oligomer) was constructed by collecting normal-mode-analysis (NMA) results for oligomeric proteins including protein-protein complexes. As in the ProMode database developed earlier for monomers and individual subunits of oligomers (Bioinformatics vol. 20, pp. 2035–2043, 2004), NMA was performed for a full-atom system using dihedral angles as independent variables, and we released the results (fluctuations of atoms, fluctuations of dihedral angles, correlations between atomic fluctuations, etc.). The vibrating oligomer is visualized by animation in an interactive molecular viewer for each of the 20 lowest-frequency normal modes. In addition, displacement vectors of constituent atoms for each normal mode were decomposed into two characteristic motions in individual subunits, i.e., internal and external (deformation and rigid-body movements of the individual subunits, respectively), and then the mutual movements of the subunits and the movement of atoms around the interface regions were investigated. These results released in ProMode-Oligomer are useful for characterizing oligomeric proteins from a dynamic point of view. The analyses are illustrated with immunoglobulin light- and heavy-chain variable domains bound to lysozyme and to a 12-residue peptide.
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Eisenberg, David, Arthur Laganowsky, Cong Liu, Michael Sawaya, Julian Whitelegge, Minglei Zhao, Angela Soriaga et al. « Structural Studies of the Amyloid State of Proteins ». Acta Crystallographica Section A Foundations and Advances 70, a1 (5 août 2014) : C797. http://dx.doi.org/10.1107/s205327331409202x.
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Amyloid diseases, including Alzheimer's, Parkinson's, and the prion conditions, are each associated with a particular protein in fibrillar form. At the morphological level, these fibers appear similar and are termed "amyloid." We found that the adhesive segments of amyloid fibers are short protein sequences which form pairs of interdigitated, in-register beta sheets. These amyloid fibrils were long suspected to be the disease agents, but evidence suggests that in the neurodegenerative diseases, smaller, often transient and polymorphic oligomers are the toxic entities. We have identified a segment of the amyloid-forming protein, alphaB crystallin, which forms an oligomeric complex exhibiting properties of other amyloid oligomers: beta-sheet-rich structure, cytotoxicity, and recognition by an anti-oligomer antibody. The X-ray-derived atomic structure of the oligomer reveals a cylindrical barrel, formed from six anti-parallel, out-of-register protein strands, which we term a cylindrin. The cylindrin structure is compatible with sequence segments from the Abeta protein of Alzheimer's disease and from other amyloid proteins. Cylindrins offer models for the hitherto elusive structures of amyloid oligomers, and are distinct in structure from amyloid fibrils.
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de Klerk, G. J., et D. Engelen. « Assembly of Agrostemma githago (corn-cockle) storage proteins and their precursor proteins into oligomers ». Biochemical Journal 230, no 1 (15 août 1985) : 269–72. http://dx.doi.org/10.1042/bj2300269.
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The major fraction of seed storage proteins of Agrostemma githago (corn-cockle), a non-leguminous dicot, occurs as material with S20,w values of approximately 11S and approximately 2S, and a minor fraction as oligomers with S20,w values of approximately 6.5S. The 11S proteins are of the legumin type and consist of disulphide-linked α- and β-subunits of Mr approximately 39 000 and approximately 23 000 respectively. The oligomeric assembly of the precursor polypeptides of the 11S proteins was examined. The approximately 65 000-Mr precursor polypeptides of two 11S proteins, which consist of 38 000-25 000-Mr subunits and 36 000-22 000-Mr subunits respectively, were assembled into oligomers of approximately 7S and subsequently cleaved. Thereafter the 11S oligomer was formed. The 88 000-Mr precursor of a third 11S protein, which consists of 41 000-23 000-Mr subunits, was assembled into an approximately 8S oligomer and then cleaved, yielding two disulphide-linked intermediates of Mr 59 000 and 24 000. Thereafter, the 11S oligomer was formed. Processing of the 59 000-Mr to the 41 000-Mr polypeptide occurred both in the 8S and in the 11S form.
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Burford, Neil T., Tom Wehrman, Daniel Bassoni, Jonathan O’Connell, Martyn Banks, Litao Zhang et Andrew Alt. « Identification of Selective Agonists and Positive Allosteric Modulators for µ- and δ-Opioid Receptors from a Single High-Throughput Screen ». Journal of Biomolecular Screening 19, no 9 (21 juillet 2014) : 1255–65. http://dx.doi.org/10.1177/1087057114542975.
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Hetero-oligomeric complexes of G protein–coupled receptors (GPCRs) may represent novel therapeutic targets exhibiting different pharmacology and tissue- or cell-specific site of action compared with receptor monomers or homo-oligomers. An ideal tool for validating this concept pharmacologically would be a hetero-oligomer selective ligand. We set out to develop and execute a 1536-well high-throughput screen of over 1 million compounds to detect potential hetero-oligomer selective ligands using a β-arrestin recruitment assay in U2OS cells coexpressing recombinant µ- and δ-opioid receptors. Hetero-oligomer selective ligands may bind to orthosteric or allosteric sites, and we might anticipate that the formation of hetero-oligomers may provide novel allosteric binding pockets for ligand binding. Therefore, our goal was to execute the screen in such a way as to identify positive allosteric modulators (PAMs) as well as agonists for µ, δ, and hetero-oligomeric receptors. While no hetero-oligomer selective ligands were identified (based on our selection criteria), this single screen did identify numerous µ- and δ-selective agonists and PAMs as well as nonselective agonists and PAMs. To our knowledge, these are the first µ- and δ-opioid receptor PAMs described in the literature.
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You, Young Suk, Jae-Hoon Kim, Jong-Soo Lee et Hyeseong Cho. « The mitochodnrial E3 ligase MARCH5 resolves RIG-I and MAVS aggregates in innate immunity ». Journal of Immunology 198, no 1_Supplement (1 mai 2017) : 222.13. http://dx.doi.org/10.4049/jimmunol.198.supp.222.13.
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Abstract Ubiquitin proteasome system (UPS) on the mitochondrial is one of quality control systems that works as a first line of defense barrier against aggregated or misfolded proteins. In innate immunity formation of the MAVS(Mitochondrial anti-viral signaling protein) oligomers elicits robust type-I interferon induction upon viral infection and however, persistent RIG-I and MAVS complex rather leads to host immunopathology. We recently reported that mitochondria-resident E3 ligase, MARCH5, recognizes the oligomeric form of RIG-I and MAVS complex. MARCH5+/− mice and MARCH5 deficient immune cells exhibited low viral replication and elevated type-I interferon response to viral infection. MARCH5 bound RIG-I and MAVS complex only during viral infection when MAVS forms oligomer. MARCH5 mediates Lys-48-linked poly-ubiquitination chain addition to RIG-I and MAVS oligomer and induces the RIG-I/MAVS monomer. Next, we studies have suggested that a novel function of MARCH5 in inflammation signaling. Together, these data demonstrates that MARCH5 regulates oligomeric RIG-I and MAVS complex.
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Weisz, OA, AM Swift et CE Machamer. « Oligomerization of a membrane protein correlates with its retention in the Golgi complex ». Journal of Cell Biology 122, no 6 (15 septembre 1993) : 1185–96. http://dx.doi.org/10.1083/jcb.122.6.1185.
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The first membrane-spanning domain (m1) of the M glycoprotein of avian coronavirus (formerly called E1) is sufficient to retain this protein in the cis-Golgi. When the membrane-spanning domain of a protein which is efficiently delivered to the plasma membrane (VSV G protein) is replaced with m1, the resulting chimera (Gm1) is retained in the Golgi (Swift, A. M., and C. E. Machamer. 1991. J. Cell Biol. 115:19-30). When assayed in sucrose gradients, we observed that Gm1 formed a large oligomer, and that much of this oligomer was SDS resistant and stayed near the top of the stacking gel of an SDS-polyacrylamide gel. The unusual stability of the oligomer allowed it to be detected easily. Gm1 mutants with single amino acid substitutions in the m1 domain that were retained in the Golgi complex formed SDS-resistant oligomers, whereas mutants that were rapidly released to the plasma membrane did not. Oligomerization was not detected immediately after synthesis of Gm1, but occurred gradually with a lag of approximately 10 min, suggesting that it is not merely aggregation of misfolded proteins. Furthermore, oligomerization did not occur under several conditions that block ER to Golgi transport. The lumenal domain was not required for oligomerization since another chimera (alpha m1G), where the lumenal domain of Gm1 was replaced by the alpha subunit of human chorionic gonadotropin, also formed an SDS-resistant oligomer, and was able to form hetero-oligomers with Gm1 as revealed by coprecipitation experiments. SDS resistance was conferred by the cytoplasmic tail of VSV G, because proteolytic digestion of the tail in microsomes containing Gm1 oligomers resulted in loss of SDS resistance, although the protease-treated material continued to migrate as a large oligomer on sucrose gradients. Interestingly, treatment of cells with cytochalasin D blocked formation of SDS-resistant (but not SDS-sensitive) oligomers. Our data suggest that SDS-resistant oligomers form as newly synthesized molecules of Gm1 arrive at the Golgi complex and may interact (directly or indirectly) with an actin-based cytoskeletal matrix. The oligomerization of Gm1 and other resident proteins could serve as a mechanism for their retention in the Golgi complex.
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Milošević, Jelica, Radivoje Prodanović et Natalija Polović. « On the Protein Fibrillation Pathway : Oligomer Intermediates Detection Using ATR-FTIR Spectroscopy ». Molecules 26, no 4 (12 février 2021) : 970. http://dx.doi.org/10.3390/molecules26040970.
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Oligomeric intermediates on the pathway of amyloid fibrillation are suspected as the main cytotoxins responsible for amyloid-related pathogenicity. As they appear to be a part of the lag phase of amyloid fibrillation when analyzed using standard methods such as Thioflavin T (ThT) fluorescence, a more sensitive method is needed for their detection. Here we apply Fourier transform infrared spectroscopy (FTIR) in attenuated total reflectance (ATR) mode for fast and cheap analysis of destabilized hen-egg-white lysozyme solution and detection of oligomer intermediates of amyloid fibrillation. Standard methods of protein aggregation analysis— Thioflavin T (ThT) fluorescence, atomic force microscopy (AFM), and 8-anilinonaphthalene-1-sulphonic acid (ANS) fluorescence were applied and compared to FTIR spectroscopy data. Results show the great potential of FTIR for both, qualitative and quantitative monitoring of oligomer formation based on the secondary structure changes. While oligomer intermediates do not induce significant changes in ThT fluorescence, their secondary structure changes were very prominent. Normalization of specific Amide I region peak intensities by using Amide II peak intensity as an internal standard provides an opportunity to use FTIR spectroscopy for both qualitative and quantitative analysis of biological samples and detection of potentially toxic oligomers, as well as for screening of efficiency of fibrillation procedures.
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Eghiaian, Frederic, Thorsten Daubenfeld, Yann Quenet, Marieke van Audenhaege, Anne-Pascale Bouin, Guillaume van der Rest, Jeanne Grosclaude et Human Rezaei. « Diversity in prion protein oligomerization pathways results from domain expansion as revealed by hydrogen/deuterium exchange and disulfide linkage ». Proceedings of the National Academy of Sciences 104, no 18 (18 avril 2007) : 7414–19. http://dx.doi.org/10.1073/pnas.0607745104.
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The prion protein (PrP) propensity to adopt different structures is a clue to its biological role. PrP oligomers have been previously reported to bear prion infectivity or toxicity and were also found along the pathway of in vitro amyloid formation. In the present report, kinetic and structural analysis of ovine PrP (OvPrP) oligomerization showed that three distinct oligomeric species were formed in parallel, independent kinetic pathways. Only the largest oligomer gave rise to fibrillar structures at high concentration. The refolding of OvPrP into these different oligomers was investigated by analysis of hydrogen/deuterium exchange and introduction of disulfide bonds. These experiments revealed that, before oligomerization, separation of contacts in the globular part (residues 127–234) occurred between the S1–H1–S2 domain (residues 132–167) and the H2–H3 bundle (residues 174–230), implying a conformational change of the S2–H2 loop (residues 168–173). The type of oligomer to be formed depended on the site where the expansion of the OvPrP monomer was initiated. Our data bring a detailed insight into the earlier conformational changes during PrP oligomerization and account for the diversity of oligomeric entities. The kinetic and structural mechanisms proposed here might constitute a physicochemical basis of prion strain genesis.
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Chia, Sean, Johnny Habchi, Thomas C. T. Michaels, Samuel I. A. Cohen, Sara Linse, Christopher M. Dobson, Tuomas P. J. Knowles et Michele Vendruscolo. « SAR by kinetics for drug discovery in protein misfolding diseases ». Proceedings of the National Academy of Sciences 115, no 41 (26 septembre 2018) : 10245–50. http://dx.doi.org/10.1073/pnas.1807884115.
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To develop effective therapeutic strategies for protein misfolding diseases, a promising route is to identify compounds that inhibit the formation of protein oligomers. To achieve this goal, we report a structure−activity relationship (SAR) approach based on chemical kinetics to estimate quantitatively how small molecules modify the reactive flux toward oligomers. We use this estimate to derive chemical rules in the case of the amyloid beta peptide (Aβ), which we then exploit to optimize starting compounds to curtail Aβ oligomer formation. We demonstrate this approach by converting an inactive rhodanine compound into an effective inhibitor of Aβ oligomer formation by generating chemical derivatives in a systematic manner. These results provide an initial demonstration of the potential of drug discovery strategies based on targeting directly the production of protein oligomers.
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Roh, Soung-Hun, Corey F. Hryc, Hyun-Hwan Jeong, Xue Fei, Joanita Jakana, George H. Lorimer et Wah Chiu. « Subunit conformational variation within individual GroEL oligomers resolved by Cryo-EM ». Proceedings of the National Academy of Sciences 114, no 31 (14 juillet 2017) : 8259–64. http://dx.doi.org/10.1073/pnas.1704725114.
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Single-particle electron cryo-microscopy (cryo-EM) is an emerging tool for resolving structures of conformationally heterogeneous particles; however, each structure is derived from an average of many particles with presumed identical conformations. We used a 3.5-Å cryo-EM reconstruction with imposed D7 symmetry to further analyze structural heterogeneity among chemically identical subunits in each GroEL oligomer. Focused classification of the 14 subunits in each oligomer revealed three dominant classes of subunit conformations. Each class resembled a distinct GroEL crystal structure in the Protein Data Bank. The conformational differences stem from the orientations of the apical domain. We mapped each conformation class to its subunit locations within each GroEL oligomer in our dataset. The spatial distributions of each conformation class differed among oligomers, and most oligomers contained 10–12 subunits of the three dominant conformation classes. Adjacent subunits were found to more likely assume the same conformation class, suggesting correlation among subunits in the oligomer. This study demonstrates the utility of cryo-EM in revealing structure dynamics within a single protein oligomer.
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Tanner, John J. « Empirical power laws for the radii of gyration of protein oligomers ». Acta Crystallographica Section D Structural Biology 72, no 10 (15 septembre 2016) : 1119–29. http://dx.doi.org/10.1107/s2059798316013218.
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The radius of gyration is a fundamental structural parameter that is particularly useful for describing polymers. It has been known since Flory's seminal work in the mid-20th century that polymers show a power-law dependence, where the radius of gyration is proportional to the number of residues raised to a power. The power-law exponent has been measured experimentally for denatured proteins and derived empirically for folded monomeric proteins using crystal structures. Here, the biological assemblies in the Protein Data Bank are surveyed to derive the power-law parameters for protein oligomers having degrees of oligomerization of 2–6 and 8. The power-law exponents for oligomers span a narrow range of 0.38–0.41, which is close to the value of 0.40 obtained for monomers. This result shows that protein oligomers exhibit essentially the same power-law behavior as monomers. A simple power-law formula is provided for estimating the oligomeric state from an experimental measurement of the radius of gyration. Several proteins in the Protein Data Bank are found to deviate substantially from power-law behavior by having an atypically large radius of gyration. Some of the outliers have highly elongated structures, such as coiled coils. For coiled coils, the radius of gyration does not follow a power law and instead scales linearly with the number of residues in the oligomer. Other outliers are proteins whose oligomeric state or quaternary structure is incorrectly annotated in the Protein Data Bank. The power laws could be used to identify such errors and help prevent them in future depositions.
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Dey, Simli, Anirban Das, Arpan Dey et Sudipta Maiti. « Membrane affinity of individual toxic protein oligomers determined at the single-molecule level ». Physical Chemistry Chemical Physics 22, no 26 (2020) : 14613–20. http://dx.doi.org/10.1039/d0cp00450b.
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Holt, J. T., R. L. Redner et A. W. Nienhuis. « An oligomer complementary to c-myc mRNA inhibits proliferation of HL-60 promyelocytic cells and induces differentiation ». Molecular and Cellular Biology 8, no 2 (février 1988) : 963–73. http://dx.doi.org/10.1128/mcb.8.2.963-973.1988.
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To study the role of a nuclear proto-oncogene in the regulation of cell growth and differentiation, we inhibited HL-60 c-myc expression with a complementary antisense oligomer. This oligomer was stable in culture and entered cells, forming an intracellular duplex. Incubation of cells with the anti-myc oligomer decreased the steady-state levels of c-myc protein by 50 to 80%, whereas a control oligomer did not significantly affect the c-myc protein concentration. Direct inhibition of c-myc expression with the anti-myc oligomer was associated with a decreased cell growth rate and an induction of myeloid differentiation. Related antisense oligomers with 2- to 12-base-pair mismatches with c-myc mRNA did not influence HL-60 cells. Thus, the effects of the antisense oligomer exhibited sequence specificity, and furthermore, these effects could be reversed by hybridization competition with another complementary oligomer. Antisense inhibition of a nuclear proto-oncogene apparently bypasses cell surface events in affecting cell proliferation and differentiation.
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Holt, J. T., R. L. Redner et A. W. Nienhuis. « An oligomer complementary to c-myc mRNA inhibits proliferation of HL-60 promyelocytic cells and induces differentiation. » Molecular and Cellular Biology 8, no 2 (février 1988) : 963–73. http://dx.doi.org/10.1128/mcb.8.2.963.
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To study the role of a nuclear proto-oncogene in the regulation of cell growth and differentiation, we inhibited HL-60 c-myc expression with a complementary antisense oligomer. This oligomer was stable in culture and entered cells, forming an intracellular duplex. Incubation of cells with the anti-myc oligomer decreased the steady-state levels of c-myc protein by 50 to 80%, whereas a control oligomer did not significantly affect the c-myc protein concentration. Direct inhibition of c-myc expression with the anti-myc oligomer was associated with a decreased cell growth rate and an induction of myeloid differentiation. Related antisense oligomers with 2- to 12-base-pair mismatches with c-myc mRNA did not influence HL-60 cells. Thus, the effects of the antisense oligomer exhibited sequence specificity, and furthermore, these effects could be reversed by hybridization competition with another complementary oligomer. Antisense inhibition of a nuclear proto-oncogene apparently bypasses cell surface events in affecting cell proliferation and differentiation.
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Burghout, Peter, Ria van Boxtel, Patrick Van Gelder, Philippe Ringler, Shirley A. Müller, Jan Tommassen et Margot Koster. « Structure and Electrophysiological Properties of the YscC Secretin from the Type III Secretion System of Yersinia enterocolitica ». Journal of Bacteriology 186, no 14 (15 juillet 2004) : 4645–54. http://dx.doi.org/10.1128/jb.186.14.4645-4654.2004.
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ABSTRACT YscC is the integral outer membrane component of the type III protein secretion machinery of Yersinia enterocolitica and belongs to the family of secretins. This group of proteins forms stable ring-like oligomers in the outer membrane, which are thought to function as transport channels for macromolecules. The YscC oligomer was purified after solubilization from the membrane with a nonionic detergent. Sodium dodecyl sulfate did not dissociate the oligomer, but it caused a change in electrophoretic mobility and an increase in protease susceptibility, indicating partial denaturation of the subunits within the oligomer. The mass of the homo-oligomer, as determined by scanning transmission electron microscopy, was approximately 1 MDa. Analysis of the angular power spectrum from averaged top views of negatively stained YscC oligomers revealed a 13-fold angular order, suggesting that the oligomer consists of 13 subunits. Reconstituted in planar lipid bilayers, the YscC oligomer displayed a constant voltage-independent conductance of approximately 3 nS, thus forming a stable pore. However, in vivo, the expression of YscC did not lead to an increased permeability of the outer membrane. Electron microscopy revealed that the YscC oligomer is composed of three domains, two stacked rings attached to a conical domain. This structure is consistent with the notion that the secretin forms the upper part of the basal body of the needle structure of the type III secreton.
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Green, Todd J., Silvia Macpherson, Shihong Qiu, Jacob Lebowitz, Gail W. Wertz et Ming Luo. « Study of the Assembly of Vesicular Stomatitis Virus N Protein : Role of the P Protein ». Journal of Virology 74, no 20 (15 octobre 2000) : 9515–24. http://dx.doi.org/10.1128/jvi.74.20.9515-9524.2000.
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ABSTRACT To derive structural information about the vesicular stomatitis virus (VSV) nucleocapsid (N) protein, the N protein and the VSV phosphoprotein (P protein) were expressed together in Escherichia coli. The N and P proteins formed soluble protein complexes of various molar ratios when coexpressed. The major N/P protein complex was composed of 10 molecules of the N protein, 5 molecules of the P protein, and an RNA. A soluble N protein-RNA oligomer free of the P protein was isolated from the N/P protein-RNA complex using conditions of lowered pH. The molecular weight of the N protein-RNA oligomer, 513,879, as determined by analytical ultracentrifugation, showed that it was composed of 10 molecules of the N protein and an RNA of approximately 90 nucleotides. The N protein-RNA oligomer had the appearance of a disk with outer diameter, inner diameter, and thickness of 148 ± 10 Å, 78 ± 9 Å, and 83 ± 8 Å, respectively, as determined by electron microscopy. RNA in the complexes was protected from RNase digestion and was stable at pH 11. This verified that N/P protein complexes expressed in E. coli were competent for encapsidation. In addition to coexpression with the full-length P protein, the N protein was expressed with the C-terminal 72 amino acids of the P protein. This portion of the P protein was sufficient for binding to the N protein, maintaining it in a soluble state, and for assembly of N protein-RNA oligomers. With the results provided in this report, we propose a model for the assembly of an N/P protein-RNA oligomer.
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Cole, Harriet, Massimiliano Porrini, Ryan Morris, Tom Smith, Jason Kalapothakis, Stefan Weidt, C. Logan Mackay, Cait E. MacPhee et Perdita E. Barran. « Early stages of insulin fibrillogenesis examined with ion mobility mass spectrometry and molecular modelling ». Analyst 140, no 20 (2015) : 7000–7011. http://dx.doi.org/10.1039/c5an01253h.
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Mass spectrometry shows insulin oligomers [I]nwhere n ranges from 1-12, and ion mobility analysis reveals ∼60 structurally distinct species across this oligomer distribution. Experimental data trains MD simulations to characterize a persistent prefibrillar protein oligomer that is a dimer enriched in β sheets.
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Sanganna Gari, Raghavendar Reddy, Patrick Seelheim, Brendan Marsh, Volker Kiessling, Carl E. Creutz et Lukas K. Tamm. « Quaternary structure of the small amino acid transporter OprG from Pseudomonas aeruginosa ». Journal of Biological Chemistry 293, no 44 (20 septembre 2018) : 17267–77. http://dx.doi.org/10.1074/jbc.ra118.004461.
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Pseudomonas aeruginosa is an opportunistic human pathogen that causes nosocomial infections. The P. aeruginosa outer membrane contains specific porins that enable substrate uptake, with the outer membrane protein OprG facilitating transport of small, uncharged amino acids. However, the pore size of an eight-stranded β-barrel monomer of OprG is too narrow to accommodate even the smallest transported amino acid, glycine, raising the question of how OprG facilitates amino acid uptake. Pro-92 of OprG is critically important for amino acid transport, with a P92A substitution inhibiting transport and the NMR structure of this variant revealing that this substitution produces structural changes in the barrel rim and restricts loop motions. OprG may assemble into oligomers in the outer membrane (OM) whose subunit interfaces could form a transport channel. Here, we explored the contributions of the oligomeric state and the extracellular loops to OprG's function. Using chemical cross-linking to determine the oligomeric structures of both WT and P92A OprG in native outer membranes and atomic force microscopy, and single-molecule fluorescence of the purified proteins reconstituted into lipid bilayers, we found that both protein variants form oligomers, supporting the notion that subunit interfaces in the oligomer could provide a pathway for amino acid transport. Furthermore, performing transport assays with loop-deleted OprG variants, we found that these variants also can transport small amino acids, indicating that the loops are not solely responsible for substrate transport. We propose that OprG functions as an oligomer and that conformational changes in the barrel–loop region might be crucial for its activity.
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Wang, Yu, Karen S. L. Lam, Ming-hon Yau et Aimin Xu. « Post-translational modifications of adiponectin : mechanisms and functional implications ». Biochemical Journal 409, no 3 (15 janvier 2008) : 623–33. http://dx.doi.org/10.1042/bj20071492.
Texte intégralRésumé :
Adiponectin is an insulin-sensitizing adipokine with anti-diabetic, anti-atherogenic, anti-inflammatory and cardioprotective properties. This adipokine is secreted from adipocytes into the circulation as three oligomeric isoforms, including trimeric, hexameric and the HMW (high-molecular-mass) oligomeric complex consisting of at least 18 protomers. Each oligomeric isoform of adiponectin exerts distinct biological properties in its various target tissues. The HMW oligomer is the major active form mediating the insulin-sensitizing effects of adiponectin, whereas the central actions of this adipokine are attributed primarily to the hexameric and trimeric oligomers. In patients with Type 2 diabetes and coronary heart disease, circulating levels of HMW adiponectin are selectively decreased due to an impaired secretion of this oligomer from adipocytes. The biosynthesis of the adiponectin oligomers is a complex process involving extensive post-translational modifications. Hydroxylation and glycosylation of several conserved lysine residues in the collagenous domain of adiponectin are necessary for the intracellular assembly and stabilization of its high-order oligomeric structures. Secretion of the adiponectin oligomers is tightly controlled by a pair of molecular chaperones in the ER (endoplasmic reticulum), including ERp44 (ER protein of 44 kDa) and Ero1-Lα (ER oxidoreductase 1-Lα). ERp44 inhibits the secretion of adiponectin oligomers through a thiol-mediated retention. In contrast, Ero1-Lα releases HMW adiponectin trapped by ERp44. The PPARγ (peroxisome-proliferator-activated receptor γ) agonists thiazolidinediones selectively enhance the secretion of HMW adiponectin through up-regulation of Ero1-Lα. In the present review, we discuss the recent advances in our understanding of the structural and biological properties of the adiponectin oligomeric isoforms and highlight the role of post-translational modifications in regulating the biosynthesis of HMW adiponectin.
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Avery, Ross A., et William J. Bettger. « The oligomeric state of spectrin in the rat erythrocyte membrane skeleton ». Biochemistry and Cell Biology 68, no 6 (1 juin 1990) : 936–43. http://dx.doi.org/10.1139/o90-138.
Texte intégralRésumé :
The oligomeric state of spectrin in the erythrocyte membrane skeleton of the rat was investigated following extraction in a low ionic strength buffer for 24 and 96 h. All analyses were quantitively compared with preparations from human erythrocyte membranes. After nondenaturing agarose–polyacrylamide gel electrophoresis, the human samples revealed their characteristic spectrin oligomer pattern; there were high molecular weight complexes near the origin of the gel, followed by several high order oligomers, tetramers, and dimers. The pattern in the rat membrane skeleton also included tetramers and a high molecular weight complex band, but had only one oligomer and no dimers. With time the high molecular weight complex diminished and oligomers accumulated in both the rat and human, while dimers accumulated only in the human and tetramers accumulated only in the rat. Tetramers decreased with time in the human. Extraction of spectrin increased with time and was greater from rat than the human red cell membrane at both time points. The percentage of spectrin and actin in the low ionic strength extract was similar between species, as analyzed by SDS–polyacrylamide electrophoresis, staining, and densitometry. Proteins 4.1 and 4.9 were present in greater percentages in the human. The only temporal effect on monomeric protein composition was an increase of protein A in the rat. There was no species difference in protein A percentage at 24 h, but at 96 h the rat was greater than the human. The results suggest that there are significant differences in the structural arrangement of the rat and human erythrocyte membrane skeleton.Key words: spectrin, erythrocyte, membrane, cytoskeleton.
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Haataja, Leena, Tatyana Gurlo, Chang J. Huang et Peter C. Butler. « Islet Amyloid in Type 2 Diabetes, and the Toxic Oligomer Hypothesis ». Endocrine Reviews 29, no 3 (27 février 2008) : 303–16. http://dx.doi.org/10.1210/er.2007-0037.
Texte intégralRésumé :
Abstract Type 2 diabetes (T2DM) is characterized by insulin resistance, defective insulin secretion, loss of β-cell mass with increased β-cell apoptosis and islet amyloid. The islet amyloid is derived from islet amyloid polypeptide (IAPP, amylin), a protein coexpressed and cosecreted with insulin by pancreatic β-cells. In common with other amyloidogenic proteins, IAPP has the propensity to form membrane permeant toxic oligomers. Accumulating evidence suggests that these toxic oligomers, rather than the extracellular amyloid form of these proteins, are responsible for loss of neurons in neurodegenerative diseases. In this review we discuss emerging evidence to suggest that formation of intracellular IAPP oligomers may contribute to β-cell loss in T2DM. The accumulated evidence permits the amyloid hypothesis originally developed for neurodegenerative diseases to be reformulated as the toxic oligomer hypothesis. However, as in neurodegenerative diseases, it remains unclear exactly why amyloidogenic proteins form oligomers in vivo, what their exact structure is, and to what extent these oligomers play a primary or secondary role in the cytotoxicity in what are now often called unfolded protein diseases.
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Bokor, Mónika, et Ágnes Tantos. « Protein–Protein Connections—Oligomer, Amyloid and Protein Complex—By Wide Line 1H NMR ». Biomolecules 11, no 5 (18 mai 2021) : 757. http://dx.doi.org/10.3390/biom11050757.
Texte intégralRésumé :
The amount of bonds between constituting parts of a protein aggregate were determined in wild type (WT) and A53T α-synuclein (αS) oligomers, amyloids and in the complex of thymosin-β4–cytoplasmic domain of stabilin-2 (Tβ4-stabilin CTD). A53T αS aggregates have more extensive βsheet contents reflected by constant regions at low potential barriers in difference (to monomers) melting diagrams (MDs). Energies of the intermolecular interactions and of secondary structures bonds, formed during polymerization, fall into the 5.41 kJ mol−1 ≤ Ea ≤ 5.77 kJ mol−1 range for αS aggregates. Monomers lose more mobile hydration water while forming amyloids than oligomers. Part of the strong mobile hydration water–protein bonds break off and these bonding sites of the protein form intermolecular bonds in the aggregates. The new bonds connect the constituting proteins into aggregates. Amyloid–oligomer difference MD showed an overall more homogeneous solvent accessible surface of A53T αS amyloids. From the comparison of the nominal sum of the MDs of the constituting proteins to the measured MD of the Tβ4-stabilin CTD complex, the number of intermolecular bonds connecting constituent proteins into complex is 20(1) H2O/complex. The energies of these bonds are in the 5.40(3) kJ mol−1 ≤ Ea ≤ 5.70(5) kJ mol−1 range.
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Poon, G. M. K. « Enhancement of oligomeric stability by covalent linkage and its application to the human p53tet domain : thermodynamics and biological implications ». Biochemical Society Transactions 35, no 6 (23 novembre 2007) : 1574–78. http://dx.doi.org/10.1042/bst0351574.
Texte intégralRésumé :
The formation of oligomeric proteins proceeds at a major cost of reducing the translational and rotational entropy for their subunits in order to form the stabilizing interactions found in the oligomeric state. Unlike site-directed mutations, covalent linkage of subunits represents a generically applicable strategy for enhancing oligomeric stability by reducing the entropic driving force for dissociation. Although this can be realized by introducing de novo disulfide cross-links between subunits, issues with irreversible aggregation limit the utility of this approach. In contrast, tandem linkage of subunits in a single polypeptide chain offers a universal method of pre-paying the entropic cost of oligomer formation. In the present paper, thermodynamic, structural and experimental aspects of designing and characterizing tandem-linked oligomers are discussed with reference to engineering a stabilized tetramer of the oligomerization domain of the human p53 tumour-suppressor protein by tandem dimerization.
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Hale, Martha L., Jean-Christophe Marvaud, Michel R. Popoff et Bradley G. Stiles. « Detergent-Resistant Membrane Microdomains Facilitate Ib Oligomer Formation and Biological Activity of Clostridium perfringens Iota-Toxin ». Infection and Immunity 72, no 4 (avril 2004) : 2186–93. http://dx.doi.org/10.1128/iai.72.4.2186-2193.2004.
Texte intégralRésumé :
ABSTRACT Clostridium perfringens iota-toxin consists of two separate proteins identified as a cell binding protein, iota b (Ib), which forms high-molecular-weight complexes on cells generating Na+/K+-permeable pores through which iota a (Ia), an ADP-ribosyltransferase, presumably enters the cytosol. Identity of the cell receptor and membrane domains involved in Ib binding, oligomer formation, and internalization is currently unknown. In this study, Vero (toxin-sensitive) and MRC-5 (toxin-resistant) cells were incubated with Ib, after which detergent-resistant membrane microdomains (DRMs) were extracted with cold Triton X-100. Western blotting revealed that Ib oligomers localized in DRMs extracted from Vero, but not MRC-5, cells while monomeric Ib was detected in the detergent-soluble fractions of both cell types. The Ib protoxin, previously shown to bind Vero cells but not form oligomers or induce cytotoxicity, was detected only in the soluble fractions. Vero cells pretreated with phosphatidylinositol-specific phospholipase C before addition of Ib indicated that glycosylphosphatidyl inositol-anchored proteins were minimally involved in Ib binding or oligomer formation. While pretreatment of Vero cells with filipin (which sequesters cholesterol) had no effect, methyl-β-cyclodextrin (which extracts cholesterol) reduced Ib binding and oligomer formation and delayed iota-toxin cytotoxicity. These studies showed that iota-toxin exploits DRMs for oligomer formation to intoxicate cells.
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Lin, Jiajia, Jie Yu, Jiaying Zhao, Ke Zhang, Jiachen Zheng, Jialing Wang, Chunhui Huang et al. « Fucoxanthin, a Marine Carotenoid, Attenuates β-Amyloid Oligomer-Induced Neurotoxicity Possibly via Regulating the PI3K/Akt and the ERK Pathways in SH-SY5Y Cells ». Oxidative Medicine and Cellular Longevity 2017 (2017) : 1–10. http://dx.doi.org/10.1155/2017/6792543.
Texte intégralRésumé :
Alzheimer’s disease (AD), the most common neurodegenerative disorder, is characterized by neurofibrillary tangles, synaptic impairments, and loss of neurons. Oligomers of β-amyloid (Aβ) are widely accepted as the main neurotoxins to induce oxidative stress and neuronal loss in AD. In this study, we discovered that fucoxanthin, a marine carotenoid with antioxidative stress properties, concentration dependently prevented Aβ oligomer-induced increase of neuronal apoptosis and intracellular reactive oxygen species in SH-SY5Y cells. Aβ oligomers inhibited the prosurvival phosphoinositide 3-kinase (PI3K)/Akt cascade and activated the proapoptotic extracellular signal-regulated kinase (ERK) pathway. Moreover, inhibitors of glycogen synthase kinase 3β (GSK3β) and mitogen-activated protein kinase (MEK) synergistically prevented Aβ oligomer-induced neuronal death, suggesting that the PI3K/Akt and ERK pathways might be involved in Aβ oligomer-induced neurotoxicity. Pretreatment with fucoxanthin significantly prevented Aβ oligomer-induced alteration of the PI3K/Akt and ERK pathways. Furthermore, LY294002 and wortmannin, two PI3K inhibitors, abolished the neuroprotective effects of fucoxanthin against Aβ oligomer-induced neurotoxicity. These results suggested that fucoxanthin might prevent Aβ oligomer-induced neuronal loss and oxidative stress via the activation of the PI3K/Akt cascade as well as inhibition of the ERK pathway, indicating that further studies of fucoxanthin and related compounds might lead to a useful treatment of AD.
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Larson, Megan E., Susan J. Greimel, Fatou Amar, Michael LaCroix, Gabriel Boyle, Mathew A. Sherman, Hallie Schley et al. « Selective lowering of synapsins induced by oligomeric α-synuclein exacerbates memory deficits ». Proceedings of the National Academy of Sciences 114, no 23 (22 mai 2017) : E4648—E4657. http://dx.doi.org/10.1073/pnas.1704698114.
Texte intégralRésumé :
Mounting evidence indicates that soluble oligomeric forms of amyloid proteins linked to neurodegenerative disorders, such as amyloid-β (Aβ), tau, or α-synuclein (αSyn) might be the major deleterious species for neuronal function in these diseases. Here, we found an abnormal accumulation of oligomeric αSyn species in AD brains by custom ELISA, size-exclusion chromatography, and nondenaturing/denaturing immunoblotting techniques. Importantly, the abundance of αSyn oligomers in human brain tissue correlated with cognitive impairment and reductions in synapsin expression. By overexpressing WT human αSyn in an AD mouse model, we artificially enhanced αSyn oligomerization. These bigenic mice displayed exacerbated Aβ-induced cognitive deficits and a selective decrease in synapsins. Following isolation of various soluble αSyn assemblies from transgenic mice, we found that in vitro delivery of exogenous oligomeric αSyn but not monomeric αSyn was causing a lowering in synapsin-I/II protein abundance. For a particular αSyn oligomer, these changes were either dependent or independent on endogenous αSyn expression. Finally, at a molecular level, the expression of synapsin genes SYN1 and SYN2 was down-regulated in vivo and in vitro by αSyn oligomers, which decreased two transcription factors, cAMP response element binding and Nurr1, controlling synapsin gene promoter activity. Overall, our results demonstrate that endogenous αSyn oligomers can impair memory by selectively lowering synapsin expression.
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RAMSAY, Douglas, Elaine KELLETT, Mary McVEY, Stephen REES et Graeme MILLIGAN. « Homo- and hetero-oligomeric interactions between G-protein-coupled receptors in living cells monitored by two variants of bioluminescence resonance energy transfer (BRET) : hetero-oligomers between receptor subtypes form more efficiently than between less closely related sequences ». Biochemical Journal 365, no 2 (15 juillet 2002) : 429–40. http://dx.doi.org/10.1042/bj20020251.
Texte intégralRésumé :
Homo- and hetero-oligomerization of G-protein-coupled receptors (GPCRs) were examined in HEK-293 cells using two variants of bioluminescence resonance energy transfer (BRET). BRET2 (a variant of BRET) offers greatly improved separation of the emission spectra of the donor and acceptor moieties compared with traditional BRET. Previously recorded homo-oligomerization of the human δ-opioid receptor was confirmed using BRET2. Homo-oligomerization of the κ-opioid receptor was observed using both BRET techniques. Both homo- and hetero-oligomers, containing both δ- and κ-opioid receptors, were unaffected by the presence of receptor ligands. BRET detection of opioid receptor homo- and hetero-oligomers required expression of 50000–100000 copies of the receptor energy acceptor construct per cell. The effectiveness of δ—κ-opioid receptor hetero-oligomer formation was as great as for homomeric interactions. The capacity of the two opioid receptors to form oligomeric complexes with the β2-adrenoceptor was also assessed. Although such interactions were detected, at least 250000 copies per cell of the energy acceptor were required. Requirement for high levels of receptor expression was equally pronounced in attempts to measure hetero-oligomer formation between the κ-opioid receptor and the thyrotropin-releasing hormone receptor-1. These studies indicate that constitutively formed homo- and hetero-oligomers of opioid receptor subtypes can be detected in living cells containing less than 100000 copies of the receptors. However, although hetero-oligomeric interactions between certain less closely related GPCRs can be detected, they appear to be of lower affinity than homo- or hetero-oligomers containing closely related sequences. Interactions recorded between certain GPCR family members in heterologous expression systems are likely to be artefacts of extreme levels of overexpression.
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Chase, Anna R., Ethan Laudermilch, Jimin Wang, Hideki Shigematsu, Takeshi Yokoyama et Christian Schlieker. « Dynamic functional assembly of the Torsin AAA+ ATPase and its modulation by LAP1 ». Molecular Biology of the Cell 28, no 21 (15 octobre 2017) : 2765–72. http://dx.doi.org/10.1091/mbc.e17-05-0281.
Texte intégralRésumé :
TorsinA is an essential AAA+ ATPase requiring LAP1 or LULL1 as cofactors. The dynamics of the Torsin/cofactor system remain poorly understood, with previous models invoking Torsin/cofactor assemblies with fixed stoichiometries. Here we demonstrate that TorsinA assembles into homotypic oligomers in the presence of ATP. Torsin variants mutated at the “back” interface disrupt homo-oligomerization but still show robust ATPase activity in the presence of its cofactors. These Torsin mutants are severely compromised in their ability to rescue nuclear envelope defects in Torsin-deficient cells, suggesting that TorsinA homo-oligomers play a key role in vivo. Engagement of the oligomer by LAP1 triggers ATP hydrolysis and rapid complex disassembly. Thus the Torsin complex is a highly dynamic assembly whose oligomeric state is tightly controlled by distinctively localized cellular cofactors. Our discovery that LAP1 serves as a modulator of the oligomeric state of an AAA+ protein establishes a novel means of regulating this important class of oligomeric ATPases.
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NEMOTO, Takayuki, et Nobuko SATO. « Oligomeric forms of the 90-kDa heat shock protein ». Biochemical Journal 330, no 2 (1 mars 1998) : 989–95. http://dx.doi.org/10.1042/bj3300989.
Texte intégralRésumé :
Two isoforms of the 90-kDa heat shock protein, HSP90α and HSP90β, are present in the cytosol of mammalian cells. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions (native PAGE) revealed that HSP90α predominantly exists as a homodimer and that HSP90β is present mainly as a monomer [Minami, Kawasaki, Miyata, Suzuki and Yahara (1991) J. Biol. Chem. 266, 10099-10103]. However, only the dimeric form has been observed under other analytical conditions such as gradient centrifugation. In this study, therefore, we investigated native forms of HSP90 by use of immunochemical techniques with isoform-specific monoclonal antibodies recently developed in our laboratory. Glycerol gradient centrifugation at the physiological salt concentration as well as native PAGE analysis of rat liver cytosol revealed oligomeric forms of HSP90α sedimenting at 8-10S as predominant ones. On the other hand, the glycerol gradient centrifugation revealed multiple forms of HSP90β oligomers sedimenting at 6-12S. All of the HSP90β oligomers, however, migrated at 100-kDa monomer and 190-kDa dimer positions on native PAGE. A novel two-dimensional double native PAGE revealed that the entity was converted from the HSP90β dimer to monomers during the electrophoresis. The same PAGE further revealed that the HSP90α oligomer also dissociated into dimers during the electrophoresis. Full-length form of bacterially-expressed human HSP90α migrated as dimers, but a considerable amount did not penetrate into the gel under native PAGE conditions, indicating the existence of oligomeric forms. Electrophoretic studies of deletion mutants of HSP90 demonstrated that the C-terminal 200 amino acids were capable of forming oligomers. Taken together, we conclude that both of the HSP90 isoforms predominantly exist as oligomeric forms in the cytosol even under unstressed conditions but that they artificially dissociate into smaller forms when subjected to native PAGE.
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Van, Pham Thi, Phan Trong Hoang, Ho Thi Thuong, Nguyen Thu Giang, Pham Bich Ngoc, Vu Huyen Trang, Udo Conrad et Chu Hoang Ha. « Bio-functional enhancement of plant based-haemagglutinin by fusion with IgMFc ». Vietnam Journal of Biotechnology 18, no 2 (3 novembre 2020) : 293–305. http://dx.doi.org/10.15625/1811-4989/18/2/14479.
Texte intégralRésumé :
The creation of recombinant oligomeric haemagglutinin proteins from A/H5N1 virus is a new concern for many scientists. In this study, the gene encoding the haemagglutinin protein (H5TG) derived from virus A/duck/Vietnam/TG24-01/2005 was fused with three different motifs (GCN4pII, GCN4pII-IgMFc and GCN4pII-ELP-IgMFc) in order to form three recombinant proteins (trimeric H5TGpII, oligomeric H5TGpII-IgMFc and ELPylated oligomeric H5TGpII-ELP-IgMFc, respectively) for enhancing protein expression, bio-function and purification of H5TG. These H5TG fragments have been attached to expression cassettes in the pCB301 shuttle vector, transiently expressed in Nicotiana benthamiana by agroinfiltration, then the protein expression was confirmed by SDS-PAGE and Western blot analysis. The results showed that the expression level of the H5TGpII-ELP-IgMFc protein in plant raw extract was stronger than that of two other proteins analyzed in this paper. Evaluation of bio-function showed that the haemagglutination titre (HA titre) of the total solution protein extract containing H5TGpII-IgMFc protein was highest (64 HAU) compared to that containing two remaining proteins (8 HAU). Subsequently, the H5TGpII and H5TGpII-IgMFc proteins were purified by immobilized metal ion affinity chromatography (IMAC), while the H5TGpII-ELP-IgMFc protein was purified using membrane-based Inverse Transition Cycling (mITC). The oligomer state of the purified proteins was then determined by non-reducing SDS-PAGE. The haemagglutination assay analysis of purified proteins showed that the lowest protein amount causing erythrocyte agglutination (1 HAU) of the H5TGpII-IgMFc protein was 0.06 µg and lower four times than that of H5TGpII and H5TGpII-ELP-IgMFc proteins (both of them were 0.24 µg). This indicates that the fusing of the GCN4pII-IgMFc motif into the H5TG protein gives the stronger bio-function than the fusing of two remaining motifs into this protein. This result opens up the applicability of the IgMFc for generation of oligomer proteins to enhance the bio-function of target proteins in the study of recombinant vaccine production.
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Downie, Kelsey, Gbolagade Adetola et Eric B. Carstens. « Characterization of protein–protein interaction domains within the baculovirus Autographa californica multiple nucleopolyhedrovirus late expression factor LEF-3 ». Journal of General Virology 94, no 11 (1 novembre 2013) : 2530–35. http://dx.doi.org/10.1099/vir.0.056267-0.
Texte intégralRésumé :
Autographa californica nucleopolyhedrovirus late expression factor 3 (LEF-3) is required for late viral gene expression probably through its numerous functions related to DNA replication, including nuclear localization of the virus helicase P143 and binding to ssDNA. LEF-3 appears to interact with itself as a homo-oligomer, although the details of this oligomeric structure are not yet known. To examine LEF-3–LEF-3 interactions, a bimolecular fluorescent protein complementation assay was used. Pairs of recombinant plasmids expressing full-length LEF-3 fused to one of two complementary fragments (V1 or V2) of a variant of yellow fluorescent protein named ‘Venus’ were constructed. Plasmids expressing fusions with complementary fragments of Venus were co-transfected into Sf21 cells and analysed by fluorescence microscopy. Co-transfected plasmids expressing full-length V1–LEF-3 and V2–LEF-3 showed positive fluorescence, confirming the formation of homo-oligomers. A series of truncated V1/V2–LEF-3 fusions was constructed and used to investigate interactions with one another as well as with full-length LEF-3.
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Krishnan, Gautam, et Utpal Roy. « Prediction of recombinant Mycobacterium tuberculosis α-crystallin oligomer chaperone activity using polynomial graphs ». F1000Research 7 (15 novembre 2018) : 1801. http://dx.doi.org/10.12688/f1000research.16328.1.
Texte intégralRésumé :
Background: Mycobacterial α-crystallin (Acr) is a chaperone that prevents misfolding of proteins when Mycobacterium tuberculosis is found in a latent form in the host tissue. Methods: Using insulin as a model substrate and utilizing polynomial graphs, we attempted to predict molecular-level interactions that are a function of the oligomeric state of the recombinant protein. The chaperone activity of the recombinant oligomeric Acr was measured at 60°C with Acr samples obtained before gel filtration chromatography and compared with a gel-filtered sample. Results: The polynomial graphs constructed showed improved molecular coverage of the insulin B chain by the oligomer. The 2nd order coefficient is the one that changes with the oligomeric ratio of Acr and improves chaperone activity. Polynomial analysis suggested that it could be a useful parameter to predict chaperone activity for potential in vitro batches of M. tuberculosis Acr based on the dynamic nature of the association and disassociation of oligomers. Conclusions: The results showed that coverage of insulin B chain improved with higher ratio of 9-mer as compared to lower ratios. This was shown by both simulation plots and actual assay data. The polynomial graphs showed increase in the 2nd order coefficient, thus suggesting the important role of oligomerisation in improved molecular coverage of insulin B chain.
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Krishnan, Gautam, et Utpal Roy. « Prediction of recombinant Mycobacterium tuberculosis α-crystallin oligomer chaperone activity using polynomial graphs ». F1000Research 7 (15 juillet 2020) : 1801. http://dx.doi.org/10.12688/f1000research.16328.2.
Texte intégralRésumé :
Background: Mycobacterial α-crystallin (Acr) is a chaperone that prevents misfolding of proteins when Mycobacterium tuberculosis is found in a latent form in the host tissue. Methods: Using insulin as a model substrate and utilizing polynomial graphs, we attempted to predict molecular-level interactions that are a function of the oligomeric state of the recombinant protein. The chaperone activity of the recombinant oligomeric Acr was measured at 60°C with Acr samples obtained before gel filtration chromatography and compared with a gel-filtered sample. Results: The polynomial graphs constructed showed improved molecular coverage of the insulin B chain by the oligomer. The 2nd order coefficient is the one that changes with the oligomeric ratio of Acr and improves chaperone activity. Polynomial analysis suggested that it could be a useful parameter to predict chaperone activity for potential in vitro batches of M. tuberculosis Acr based on the dynamic nature of the association and disassociation of oligomers. Conclusions: The results showed that coverage of insulin B chain improved with higher ratio of 9-mer as compared to lower ratios. This was shown by both simulation plots and actual assay data. The polynomial graphs showed increase in the 2nd order coefficient, thus suggesting the important role of oligomerisation in improved molecular coverage of insulin B chain.
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Brindley, Melinda A., et Richard K. Plemper. « Blue Native PAGE and Biomolecular Complementation Reveal a Tetrameric or Higher-Order Oligomer Organization of the Physiological Measles Virus Attachment Protein H ». Journal of Virology 84, no 23 (22 septembre 2010) : 12174–84. http://dx.doi.org/10.1128/jvi.01222-10.
Texte intégralRésumé :
ABSTRACT Members of the Paramyxovirinae subfamily rely on the concerted action of two envelope glycoprotein complexes, attachment protein H and the fusion (F) protein oligomer, to achieve membrane fusion for viral entry. Despite advances in X-ray information, the organization of the physiological attachment (H) oligomer in functional fusion complexes and the molecular mechanism linking H receptor binding with F triggering remain unknown. Here, we have applied an integrated approach based on biochemical and functional assays to the problem. Blue native PAGE analysis indicates that native H complexes extract predominantly in the form of loosely assembled tetramers from purified measles virus (MeV) particles and cells transiently expressing the viral envelope glycoproteins. To gain functional insight, we have established a bimolecular complementation (BiC) assay for MeV H, on the basis of the hypothesis that physical interaction of H with F complexes, F triggering, and receptor binding constitute distinct events. Having experimentally confirmed three distinct H complementation groups, implementation of H BiC (H-BiC) reveals that a high-affinity receptor-to-paramyxovirus H monomer stoichiometry below parity is sufficient for fusion initiation, that F binding and fusion initiation are separable in H oligomers, and that a higher relative amount of F binding-competent than F fusion initiation- or receptor binding-competent H monomers per oligomer is required for optimal fusion. By capitalizing on these findings, H-BiC activity profiles confirm the organization of H into tetramers or higher-order multimers in functional fusion complexes. Results are interpreted in light of a model in which receptor binding may affect the oligomeric organization of the attachment protein complex.
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BÖDE, Csaba, Ferenc G. TÖLGYESI, László SMELLER, Karel HEREMANS, Sergiy V. AVILOV et Judit FIDY. « Chaperone-like activity of alpha-crystallin is enhanced by high-pressure treatment ». Biochemical Journal 370, no 3 (15 mars 2003) : 859–66. http://dx.doi.org/10.1042/bj20021097.
Texte intégralRésumé :
α-Crystallin, an oligomeric protein in vertebrate eye lens, is a member of the small heat-shock protein family. Several papers pointed out that its chaperone-like activity could be enhanced by increasing the temperature. We demonstrate in the present study that structural perturbations by high hydrostatic pressures up to 300MPa also enhance this activity. In contrast with temperature-induced changes, the pressure-induced enhancement is reversible. After pressure release, the extra activity is lost with a relaxation time of 2.0±0.5h. Structural alterations contributing to the higher activity were studied with IR and fluorescence spectroscopy, and light-scattering measurements. The results suggest that while the secondary structure barely changes under pressure, the interactions between the subunits weaken, the oligomers dissociate, the area of accessible hydrophobic surfaces significantly increases and the environment of tryptophan residues becomes slightly more polar. It seems that structural flexibility and the total surface area of the oligomers are the key factors in the chaperone capacity, and that the increase in the chaperone activity does not require the increase in the oligomer size as was assumed previously [Burgio, Kim, Dow and Koretz (2000) Biochem. Biophys. Res. Commun. 268, 426—432]. After pressure release, the structure of subunits are reorganized relatively quickly, whereas the oligomer size reaches its original value slowly with a relaxation time of 33±4 h. In our interpretation, both the fast and slow structural rearrangements have an impact on the functional relaxation.
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Vaikath, Nishant, Indulekha Sudhakaran, Ilham Abdi, Vijay Gupta, Nour Majbour, Simona Ghanem, Houari Abdesselem, Kostas Vekrellis et Omar El-Agnaf. « Structural and Biophysical Characterization of Stable Alpha-Synuclein Oligomers ». International Journal of Molecular Sciences 23, no 23 (23 novembre 2022) : 14630. http://dx.doi.org/10.3390/ijms232314630.
Texte intégralRésumé :
The aggregation of α-synuclein (α-syn) into neurotoxic oligomers and fibrils is an important pathogenic feature of synucleinopatheis, including Parkinson’s disease (PD). A further characteristic of PD is the oxidative stress that results in the formation of aldehydes by lipid peroxidation. It has been reported that the brains of deceased patients with PD contain high levels of protein oligomers that are cross-linked to these aldehydes. Increasing evidence also suggests that prefibrillar oligomeric species are more toxic than the mature amyloid fibrils. However, due to the heterogenous and metastable nature, characterization of the α-syn oligomeric species has been challenging. Here, we generated and characterized distinct α-syn oligomers in vitro in the presence of DA and lipid peroxidation products 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). HNE and ONE oligomer were stable towards the treatment with SDS, urea, and temperature. The secondary structure analysis revealed that only HNE and ONE oligomers contain β-sheet content. In the seeding assay, both DA and ONE oligomers significantly accelerated the aggregation. Furthermore, all oligomeric preparations were found to seed the aggregation of α-syn monomers in vitro and found to be cytotoxic when added to SH-SY5Y cells. Finally, both HNE and ONE α-syn oligomers can be used as a calibrator in an α-syn oligomers-specific ELISA.
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Irumagawa, Shin, Keiko Hiemori, Sayoko Saito, Hiroaki Tateno et Ryoichi Arai. « Self-Assembling Lectin Nano-Block Oligomers Enhance Binding Avidity to Glycans ». International Journal of Molecular Sciences 23, no 2 (8 janvier 2022) : 676. http://dx.doi.org/10.3390/ijms23020676.
Texte intégralRésumé :
Lectins, carbohydrate-binding proteins, are attractive biomolecules for medical and biotechnological applications. Many lectins have multiple carbohydrate recognition domains (CRDs) and strongly bind to specific glycans through multivalent binding effect. In our previous study, protein nano-building blocks (PN-blocks) were developed to construct self-assembling supramolecular nanostructures by linking two oligomeric proteins. A PN-block, WA20-foldon, constructed by fusing a dimeric four-helix bundle de novo protein WA20 to a trimeric foldon domain of T4 phage fibritin, self-assembled into several types of polyhedral nanoarchitectures in multiples of 6-mer. Another PN-block, the extender PN-block (ePN-block), constructed by tandemly joining two copies of WA20, self-assembled into cyclized and extended chain-type nanostructures. This study developed novel functional protein nano-building blocks (lectin nano-blocks) by fusing WA20 to a dimeric lectin, Agrocybe cylindracea galectin (ACG). The lectin nano-blocks self-assembled into various oligomers in multiples of 2-mer (dimer, tetramer, hexamer, octamer, etc.). The mass fractions of each oligomer were changed by the length of the linkers between WA20 and ACG. The binding avidity of the lectin nano-block oligomers to glycans was significantly increased through multivalent effects compared with that of the original ACG dimer. Lectin nano-blocks with high avidity will be useful for various applications, such as specific cell labeling.
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Wei, Chih-Jen, Ling Xu, Wing-Pui Kong, Wei Shi, Kevin Canis, James Stevens, Zhi-Yong Yang et al. « Comparative Efficacy of Neutralizing Antibodies Elicited by Recombinant Hemagglutinin Proteins from Avian H5N1 Influenza Virus ». Journal of Virology 82, no 13 (16 avril 2008) : 6200–6208. http://dx.doi.org/10.1128/jvi.00187-08.
Texte intégralRésumé :
ABSTRACT Although the human transmission of avian H5N1 virus remains low, the prevalence of this highly pathogenic infection in avian species underscores the need for a preventive vaccine that can be made without eggs. Here, we systematically analyze various forms of recombinant hemagglutinin (HA) protein for their potential efficacy as vaccines. Monomeric, trimeric, and oligomeric H5N1 HA proteins were expressed and purified from either insect or mammalian cells. The immunogenicity of different recombinant HA proteins was evaluated by measuring the neutralizing antibody response. Neutralizing antibodies to H5N1 HA were readily generated in mice immunized with the recombinant HA proteins, but they varied in potency depending on their multimeric nature and cell source. Among the HA proteins, a high-molecular-weight oligomer elicited the strongest antibody response, followed by the trimer; the monomer showed minimal efficacy. The coexpression of another viral surface protein, neuraminidase, did not affect the immunogenicity of the HA oligomer, as expected from the immunogenicity of trimers produced from insect cells. As anticipated, HA expressed in mammalian cells without NA retained the terminal sialic acid residues and failed to bind α2,3-linked sialic acid receptors. Taken together, these results suggest that recombinant HA proteins as individual or oligomeric trimers can elicit potent neutralizing antibody responses to avian H5N1 influenza viruses.
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Gomez Barroso, Juan Arturo, Mariana Reneé Miranda, Claudio Alejandro Pereira, Richard Charles Garratt et Carlos Fernando Aguilar. « X-ray diffraction and in vivo studies reveal the quinary structure of Trypanosoma cruzi nucleoside diphosphate kinase 1 : a novel helical oligomer structure ». Acta Crystallographica Section D Structural Biology 78, no 1 (1 janvier 2022) : 30–42. http://dx.doi.org/10.1107/s2059798321011219.
Texte intégralRésumé :
Trypanosoma cruzi is a flagellated protozoan parasite that causes Chagas disease, which represents a serious health problem in the Americas. Nucleoside diphosphate kinases (NDPKs) are key enzymes that are implicated in cellular energy management. TcNDPK1 is the canonical isoform in the T. cruzi parasite. TcNDPK1 has a cytosolic, perinuclear and nuclear distribution. It is also found in non-membrane-bound filaments adjacent to the nucleus. In the present work, X-ray diffraction and in vivo studies of TcNDPK1 are described. The structure reveals a novel, multi-hexameric, left-handed helical oligomer structure. The results of directed mutagenesis studies led to the conclusion that the microscopic TcNDPK1 granules observed in vivo in T. cruzi parasites are made up by the association of TcNDPK1 oligomers. In the absence of experimental data, analysis of the interactions in the X-ray structure of the TcNDPK1 oligomer suggests the probable assembly and disassembly steps: dimerization, assembly of the hexamer as a trimer of dimers, hexamer association to generate the left-handed helical oligomer structure and finally oligomer association in a parallel manner to form the microscopic TcNDPK1 filaments that are observed in vivo in T. cruzi parasites. Oligomer disassembly takes place on the binding of substrate in the active site of TcNDPK1, leading to dissociation of the hexamers. This study constitutes the first report of such a protein arrangement, which has never previously been seen for any protein or NDPK. Further studies are needed to determine its physiological role. However, it may suggest a paradigm for protein storage reflecting the complex mechanism of action of TcNDPK1.
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Trevelyan, Sarah J., Jodi L. Brewster, Abigail E. Burgess, Jennifer M. Crowther, Antonia L. Cadell, Benjamin L. Parker, David R. Croucher, Renwick C. J. Dobson, James M. Murphy et Peter D. Mace. « Structure-based mechanism of preferential complex formation by apoptosis signal–regulating kinases ». Science Signaling 13, no 622 (10 mars 2020) : eaay6318. http://dx.doi.org/10.1126/scisignal.aay6318.
Texte intégralRésumé :
Apoptosis signal–regulating kinases (ASK1, ASK2, and ASK3) are activators of the p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways. ASK1–3 form oligomeric complexes known as ASK signalosomes that initiate signaling cascades in response to diverse stress stimuli. Here, we demonstrated that oligomerization of ASK proteins is driven by previously uncharacterized sterile-alpha motif (SAM) domains that reside at the carboxy-terminus of each ASK protein. SAM domains from ASK1–3 exhibited distinct behaviors, with the SAM domain of ASK1 forming unstable oligomers, that of ASK2 remaining predominantly monomeric, and that of ASK3 forming a stable oligomer even at a low concentration. In contrast to their behavior in isolation, the ASK1 and ASK2 SAM domains preferentially formed a stable heterocomplex. The crystal structure of the ASK3 SAM domain, small-angle x-ray scattering, and mutagenesis suggested that ASK3 oligomers and ASK1-ASK2 complexes formed discrete, quasi-helical rings through interactions between the mid-loop of one molecule and the end helix of another molecule. Preferential ASK1-ASK2 binding was consistent with mass spectrometry showing that full-length ASK1 formed hetero-oligomeric complexes incorporating large amounts of ASK2. Accordingly, disrupting the association between SAM domains impaired ASK activity in the context of electrophilic stress induced by 4-hydroxy-2-nonenal (HNE). These findings provide a structural template for how ASK proteins assemble foci that drive inflammatory signaling and reinforce the notion that strategies to target ASK proteins should consider the concerted actions of multiple ASK family members.
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Backes, Anna T., Kathrin Reinmuth-Selzle, Anna Lena Leifke, Kira Ziegler, Carola S. Krevert, Georg Tscheuschner, Kurt Lucas et al. « Oligomerization and Nitration of the Grass Pollen Allergen Phl p 5 by Ozone, Nitrogen Dioxide, and Peroxynitrite : Reaction Products, Kinetics, and Health Effects ». International Journal of Molecular Sciences 22, no 14 (16 juillet 2021) : 7616. http://dx.doi.org/10.3390/ijms22147616.
Texte intégralRésumé :
The allergenic and inflammatory potential of proteins can be enhanced by chemical modification upon exposure to atmospheric or physiological oxidants. The molecular mechanisms and kinetics of such modifications, however, have not yet been fully resolved. We investigated the oligomerization and nitration of the grass pollen allergen Phl p 5 by ozone (O3), nitrogen dioxide (NO2), and peroxynitrite (ONOO–). Within several hours of exposure to atmospherically relevant concentration levels of O3 and NO2, up to 50% of Phl p 5 were converted into protein oligomers, likely by formation of dityrosine cross-links. Assuming that tyrosine residues are the preferential site of nitration, up to 10% of the 12 tyrosine residues per protein monomer were nitrated. For the reaction with peroxynitrite, the largest oligomer mass fractions (up to 50%) were found for equimolar concentrations of peroxynitrite over tyrosine residues. With excess peroxynitrite, the nitration degrees increased up to 40% whereas the oligomer mass fractions decreased to 20%. Our results suggest that protein oligomerization and nitration are competing processes, which is consistent with a two-step mechanism involving a reactive oxygen intermediate (ROI), as observed for other proteins. The modified proteins can promote pro-inflammatory cellular signaling that may contribute to chronic inflammation and allergies in response to air pollution.
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Birchenall-Roberts, M. C., C. Ferrer, D. Ferris, L. A. Falk, J. Kasper, G. White et F. W. Ruscetti. « Inhibition of murine monocyte proliferation by a colony-stimulating factor-1 antisense oligodeoxynucleotide. Evidence for autocrine regulation. » Journal of Immunology 145, no 10 (15 novembre 1990) : 3290–96. http://dx.doi.org/10.4049/jimmunol.145.10.3290.
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Abstract Using a combination of v-myc and v-ras oncogenes, we have established a growth factor-independent monocyte cell line from murine fetal liver (FL-ras/myc). Biologic and molecular characterization demonstrated that the gene for the macrophage growth factor CSF-1 and the c-fms proto-oncogene (CSF-1 receptor) are expressed in this cell line, thus suggesting autocrine regulation as a possible mechanism for the unregulated growth of these cells. To study this possibility, we used 1) mAb, to neutralize the CSF-1 protein produced by the cell line, and 2) antisense oligomers, to inhibit CSF-1 gene products by specific base-pairing of complementary nucleic acids. We report here that both approaches inhibited in vitro cell growth by 60 to 70%, whereas the combination of oligomer and mAb inhibited proliferation by 95%. However, control antisense oligomers (50% bp mismatch with CSF-1 mRNA) did not inhibit FL-ras/myc cell growth. Furthermore, the inhibitory effects of mAb and oligomers were reversible when they were removed from the media. Detection of cell-associated CSF-1 protein by immunofluorescence showed that cells treated with the antisense oligomer expressed significantly less CSF-1 protein. These results indicate that the FL-ras/myc cell line requires CSF-1 for autonomous growth and that oligomers can efficiently block production of autocrine growth factors.
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PEZZA, Roberto J., Andrea M. SMANIA, José L. BARRA et Carlos E. ARGARAÑA. « Nucleotides and heteroduplex DNA preserve the active conformation of Pseudomonas aeruginosa MutS by preventing protein oligomerization ». Biochemical Journal 361, no 1 (17 décembre 2001) : 87–95. http://dx.doi.org/10.1042/bj3610087.
Texte intégralRésumé :
MutS, a component of the mismatch repair system begins the DNA reparation process by recognizing base/base mismatches or small insertion/deletion loops. We have cloned the mutS gene from the human opportunistic pathogen Pseudomonas aeruginosa and analysed the biochemical properties of the encoded protein. Complementation of the hypermutator phenotype of a P. aeruginosa mutS mutant strain indicated that the isolated gene was functional. When purified MutS was incubated at 37°C in the absence of ligands, a rapid inactivation of the oligonucleotide binding capability and ATPase activity occurred. However, the presence of ATP, ADP or heteroduplex oligonucleotides, but not homoduplex oligonucleotides, prevented the protein from being inactivated. The analysis of the protein by native PAGE indicated that the active conformation state correlates with the presence of MutS dimer. Analysis by gel-filtration chromatography showed that the inactive protein formed by incubation at 37°C in the absence of ligands corresponds to the formation of a high molecular mass oligomer. The kinetic analysis of the oligomer formation showed that the extent of the reaction was markedly dependent on the temperature and the presence of MutS ligands. However, the protein inactivation apparently occurred before the maximum extent of MutS oligomerization. Further analysis of the MutS oligomers by electron microscopy showed the presence of regular structures consisting of four subunits, with each subunit probably representing a MutS homodimer. It is concluded that MutS possesses an intrinsic propensity to form oligomeric structures and that the presence of physiological ligands, such as nucleotides or heteroduplex DNA, but not homoduplex DNA, plays an important role in keeping the protein in an active conformation by preventing protein oligomerization.
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Zheng, Weihua, Min-Yeh Tsai, Mingchen Chen et Peter G. Wolynes. « Exploring the aggregation free energy landscape of the amyloid-β protein (1–40) ». Proceedings of the National Academy of Sciences 113, no 42 (3 octobre 2016) : 11835–40. http://dx.doi.org/10.1073/pnas.1612362113.
Texte intégralRésumé :
A predictive coarse-grained protein force field [associative memory, water-mediated, structure, and energy model for molecular dynamics (AWSEM)-MD] is used to study the energy landscapes and relative stabilities of amyloid-β protein (1–40) in the monomer and all of its oligomeric forms up to an octamer. We find that an isolated monomer is mainly disordered with a short α-helix formed at the central hydrophobic core region (L17-D23). A less stable hairpin structure, however, becomes increasingly more stable in oligomers, where hydrogen bonds can form between neighboring monomers. We explore the structure and stability of both prefibrillar oligomers that consist of mainly antiparallel β-sheets and fibrillar oligomers with only parallel β-sheets. Prefibrillar oligomers are polymorphic but typically take on a cylindrin-like shape composed of mostly antiparallel β-strands. At the concentration of the simulation, the aggregation free energy landscape is nearly downhill. We use umbrella sampling along a structural progress coordinate for interconversion between prefibrillar and fibrillar forms to identify a conversion pathway between these forms. The fibrillar oligomer only becomes favored over its prefibrillar counterpart in the pentamer where an interconversion bottleneck appears. The structural characterization of the pathway along with statistical mechanical perturbation theory allow us to evaluate the effects of concentration on the free energy landscape of aggregation as well as the effects of the Dutch and Arctic mutations associated with early onset of Alzheimer’s disease.
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Okreglak, Voytek, et David G. Drubin. « Loss of Aip1 reveals a role in maintaining the actin monomer pool and an in vivo oligomer assembly pathway ». Journal of Cell Biology 188, no 6 (15 mars 2010) : 769–77. http://dx.doi.org/10.1083/jcb.200909176.
Texte intégralRésumé :
Although actin filaments can form by oligomer annealing in vitro, they are assumed to assemble exclusively from actin monomers in vivo. In this study, we show that a pool of actin resistant to the monomer-sequestering drug latrunculin A (lat A) contributes to filament assembly in vivo. Furthermore, we show that the cofilin accessory protein Aip1 is important for establishment of normal actin monomer concentration in cells and efficiently converts cofilin-generated actin filament disassembly products into monomers and short oligomers in vitro. Additionally, in aip1Δ mutant cells, lat A–insensitive actin assembly is significantly enhanced. We conclude that actin oligomer annealing is a physiologically relevant actin filament assembly pathway in vivo and identify Aip1 as a crucial factor for shifting the distribution of short actin oligomers toward monomers during disassembly.
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Marchenkova, Margarita A., Petr V. Konarev, Yuliya V. Kordonskaya, Kseniia B. Ilina, Yury V. Pisarevsky, Alexander V. Soldatov, Vladimir I. Timofeev et Mikhail V. Kovalchuk. « The Role of Cations and Anions in the Formation of Crystallization Oligomers in Protein Solutions as Revealed by Combination of Small-Angle X-ray Scattering and Molecular Dynamics ». Crystals 12, no 6 (24 mai 2022) : 751. http://dx.doi.org/10.3390/cryst12060751.
Texte intégralRésumé :
As is known from molecular dynamics simulation, lysozyme oligomers in crystallization solutions are most stable when taking into account as many precipitant ions as possible embedded in the corresponding crystal structure. Therefore, the number of precipitant ions associated with crystallographic oligomer models can play a role during the modeling of small-angle X-ray scattering (SAXS) data. This hypothesis has been tested in the present work. As a result, it turned out that the best fit quality to the experimental SAXS data is reached when using oligomers without precipitant ions at all or with embedded chlorine ions. Molecular dynamics (MD) simulation shows that the stability of crystallization oligomers depends on the consideration of anions and cations in oligomer structure. Thus, it is chlorine ions which stabilize dimer and octamers in lysozyme crystallization solution. As SAXS is more sensitive to the role of cations and MD shows the role of anions which are “light” for X-rays, it has been shown that precipitant cations most likely do not bind to monomers, but to already-formed oligomers.
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Matos, Anna Lívia Linard, Sergej Kudruk, Johanna Moratz, Milena Heflik, David Grill, Bart Jan Ravoo et Volker Gerke. « Membrane Binding Promotes Annexin A2 Oligomerization ». Cells 9, no 5 (8 mai 2020) : 1169. http://dx.doi.org/10.3390/cells9051169.
Texte intégralRésumé :
Annexin A2 (AnxA2) is a cytosolic Ca2+ regulated membrane binding protein that can induce lipid domain formation and plays a role in exocytosis and endocytosis. To better understand the mode of annexin-membrane interaction, we analyzed membrane-bound AnxA2 assemblies by employing a novel 3-armed chemical crosslinker and specific AnxA2 mutant proteins. Our data show that AnxA2 forms crosslinkable oligomers upon binding to membranes containing negatively charged phospholipids. AnxA2 mutants with amino acid substitutions in residues predicted to be involved in lateral protein–protein interaction show compromised oligomer formation, albeit still being capable of binding to negatively charged membranes in the presence of Ca2+. These results suggest that lateral protein–protein interactions are involved in the formation of AnxA2 clusters on a biological membrane.
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Verrey, F., et K. Drickamer. « Determinants of oligomeric structure in the chicken liver glycoprotein receptor ». Biochemical Journal 292, no 1 (15 mai 1993) : 149–55. http://dx.doi.org/10.1042/bj2920149.
Texte intégralRésumé :
The oligomeric state of the chicken liver receptor (chicken hepatic lectin), which mediates endocytosis of glycoproteins terminating with N-acetylglucosamine, has been investigated using physical methods as well as chemical cross-linking. Receptor isolated from liver and from transfected rat fibroblasts expressing the full-length polypeptide is a homotrimer immediately following solubilization in non-ionic detergent, but forms the previously observed hexamer during purification. These results are most consistent with the presence of a trimer of receptor polypeptides in liver membranes and in transfected cells. Analysis of truncated receptors reveals that the C-terminal extracellular portion of this type-II transmembrane protein does not form stable oligomers when isolated from the membrane anchor and cytoplasmic tail. The behaviour of chimeric receptors, in which the cytoplasmic tail of the glycoprotein receptor is replaced with the corresponding segments of rat liver asialoglycoprotein receptor or the beta-subunit of Na+,K(+)-ATPase, or with unrelated sequences from globin, indicates that the cytoplasmic tail influences oligomer stability. Replacement of N-terminal portions of the receptor with corresponding segments of influenza virus neuraminidase results in formation of tetramers, suggesting that the membrane anchor and flanking sequences are important determinants of oligomer formation.