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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.
2
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.
3
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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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.
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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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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.
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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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Stoneman, Michael R., Naomi Raicu, Gabriel Biener et Valerică Raicu. « Fluorescence-based Methods for the Study of Protein-Protein Interactions Modulated by Ligand Binding ». Current Pharmaceutical Design 26, no 44 (22 décembre 2020) : 5668–83. http://dx.doi.org/10.2174/1381612826666201116120934.
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Background: The growing evidence that G protein-coupled receptors (GPCRs) not only form oligomers but that the oligomers also may modulate the receptor function provides a promising avenue in the area of drug design. Highly selective drugs targeting distinct oligomeric sub-states offer the potential to increase efficacy while reducing side effects. In this regard, determining the various oligomeric configurations and geometric sub-states of a membrane receptor is of utmost importance. Methods: In this report, we have reviewed two techniques that have proven to be valuable in monitoring the quaternary structure of proteins in vivo: Fӧrster resonance energy transfer (FRET) spectrometry and fluorescence intensity fluctuation (FIF) spectrometry. In FRET spectrometry, distributions of pixel-level FRET efficiency are analyzed using theoretical models of various quaternary structures to determine the geometry and stoichiometry of protein oligomers. In FIF spectrometry, spatial fluctuations of fluorescent molecule intensities are analyzed to reveal quantitative information on the size and stability of protein oligomers. Results: We demonstrate the application of these techniques to a number of different fluorescence-based studies of cells expressing fluorescently labeled membrane receptors, both in the presence and absence of various ligands. The results show the effectiveness of using FRET spectrometry to determine detailed information regarding the quaternary structure receptors form, as well as FIF and FRET for determining the relative abundance of different-sized oligomers when an equilibrium forms between such structures. Conclusion: FRET and FIF spectrometry are valuable techniques for characterizing membrane receptor oligomers, which are of great benefit to structure‐based drug design.
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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.
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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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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.
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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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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.
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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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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.
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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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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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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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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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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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Milligan, G. « Oligomerisation of G-protein-coupled receptors ». Journal of Cell Science 114, no 7 (1 avril 2001) : 1265–71. http://dx.doi.org/10.1242/jcs.114.7.1265.
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A range of approaches have recently provided evidence that G-protein-coupled receptors can exist as oligomeric complexes. Both homo-oligomers, comprising multiple copies of the same gene product, and hetero-oligomers containing more than one receptor have been detected. In several, but not all, examples, the extent of oligomerisation is regulated by the presence of agonist ligands, and emerging evidence indicates that receptor hetero-oligomers can display distinct pharmacological characteristics. A chaperonin-like role for receptor oligomerisation in effective delivery of newly synthesised receptors to the cell surface is a developing concept, and recent studies have employed a series of energy-transfer techniques to explore the presence and regulation of receptor oligomerisation in living cells. However, the majority of studies have relied largely on co-immunoprecipitation techniques, and there is still little direct information on the fraction of receptors existing as oligomers in intact cells.
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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.
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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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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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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.
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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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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.
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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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Di Domizio, Jeremy, Ran Zhang, Ming Zhuo, Loren Stagg, John Ladbury, Michel Gilliet et Wei Cao. « A novel class of host-derived etiological agent for autoimmunity : oligomers of endogenous proteins bind to self-nucleic acids and trigger type I IFN production by plasmacytoid dendritic cells (44.2) ». Journal of Immunology 186, no 1_Supplement (1 avril 2011) : 44.2. http://dx.doi.org/10.4049/jimmunol.186.supp.44.2.
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Abstract Under certain conditions, some soluble endogenous proteins can form ordered oligomeric structures leading to the assembly of stable insoluble amyloids, a process that increases with age. The presence of such structures is associated with cellular toxicity and diseases development, e.g. Alzheimer disease, type II diabetes. Recent studies clearly show that the primary toxic species in such pathologies is the soluble oligomers of proteins, precursors of amyloids. Yet, it is unknown if such aberrant forms of proteins would elicit any immune reaction. Here we obtained oligomers derived from several human endogenous proteins and characterized their biochemical and immunological functions. The oligomeric proteins preferentially bind to both DNA and RNA and can be effectively internalized by cells. Surprisingly, the soluble protein oligomers enable prominent IFNα production by PBMCs to self-DNA, self-RNA and necrotic cell debris in a pDC-dependent manner. Consistently, peptides from amyloid β and prion protein, two known etiological agents associated with amyloid diseases, display similar innate immune functions by complexing with nucleic acids and inducing IFN production by pDCs. Therefore, oligomers of endogenous proteins formed during the aging process may strongly promote the host’s type I IFN response to self-nucleic acids, a reaction likely favoring the development of autoimmunity, such as SLE where strong type I IFN presence correlates with disease pathology.
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Martellucci, Stefano, Letizia Clementi, Samantha Sabetta, Paola Muzi, Vincenzo Mattei, Mauro Bologna et Adriano Angelucci. « Tau oligomers accumulation sensitizes prostate cancer cells to docetaxel treatment ». Journal of Cancer Research and Clinical Oncology 147, no 7 (2 avril 2021) : 1957–71. http://dx.doi.org/10.1007/s00432-021-03598-3.
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Abstract Purpose Human tau is a highly dynamic, multifunctional protein expressed in different isoforms and conformers, known to modulate microtubule turnover. Tau oligomers are considered pathologic forms of the protein able to initiate specific protein accumulation diseases, called tauopathies. In our study, we investigated the potential association between autophagy and tau oligomers accumulation and its role in the response of prostate cancer cells to docetaxel. Methods We evaluated in vitro the expression of tau oligomers in prostate cancer cell lines, PC3 and DU145, in presence of autophagy inhibitors and investigated the role of tau oligomers accumulation in resistance to docetaxel treatment. Results Tau protein was basally expressed in prostate cancer lines as several monomeric and oligomeric forms. The pharmacologic inhibition of autophagy induced in cancer cells the accumulation of tau protein, with a prevalent expression of oligomeric forms. Immunofluorescence analysis of untreated cells revealed that tau was visible mainly in dividing cells where it was localized on the mitotic spindle. Inhibition of autophagy determined an evident upregulation of tau signal in dividing cells and the presence of aberrant monoastral mitotic spindles. The accumulation of tau oligomers was associated with DNA DSB and increased cytotoxic effect by docetaxel. Conclusions Our data indicate that autophagy could exert a promoting role in cancer growth and during chemotherapy facilitating degradation of tau protein and thus blocking the antimitotic effect of accumulated tau oligomers. Thus, therapeutic strategies aimed at stimulating tau oligomers formation, such as autophagy inhibition, could be an effective adjuvant in cancer therapy.
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Iljina, Marija, Gonzalo A. Garcia, Mathew H. Horrocks, Laura Tosatto, Minee L. Choi, Kristina A. Ganzinger, Andrey Y. Abramov et al. « Kinetic model of the aggregation of alpha-synuclein provides insights into prion-like spreading ». Proceedings of the National Academy of Sciences 113, no 9 (16 février 2016) : E1206—E1215. http://dx.doi.org/10.1073/pnas.1524128113.
Texte intégralRésumé :
The protein alpha-synuclein (αS) self-assembles into small oligomeric species and subsequently into amyloid fibrils that accumulate and proliferate during the development of Parkinson’s disease. However, the quantitative characterization of the aggregation and spreading of αS remains challenging to achieve. Previously, we identified a conformational conversion step leading from the initially formed oligomers to more compact oligomers preceding fibril formation. Here, by a combination of single-molecule fluorescence measurements and kinetic analysis, we find that the reaction in solution involves two unimolecular structural conversion steps, from the disordered to more compact oligomers and then to fibrils, which can elongate by further monomer addition. We have obtained individual rate constants for these key microscopic steps by applying a global kinetic analysis to both the decrease in the concentration of monomeric protein molecules and the increase in oligomer concentrations over a 0.5–140-µM range of αS. The resulting explicit kinetic model of αS aggregation has been used to quantitatively explore seeding the reaction by either the compact oligomers or fibrils. Our predictions reveal that, although fibrils are more effective at seeding than oligomers, very high numbers of seeds of either type, of the order of 104, are required to achieve efficient seeding and bypass the slow generation of aggregates through primary nucleation. Complementary cellular experiments demonstrated that two orders of magnitude lower numbers of oligomers were sufficient to generate high levels of reactive oxygen species, suggesting that effective templated seeding is likely to require both the presence of template aggregates and conditions of cellular stress.
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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.
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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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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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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.
Texte intégralRésumé :
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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Tsukakoshi, Kaori, Rikako Kubo et Kazunori Ikebukuro. « Development of Alkaline Phosphatase-Fused Mouse Prion Protein and Its Application in Toxic Aβ Oligomer Detection ». International Journal of Molecular Sciences 23, no 23 (23 novembre 2022) : 14588. http://dx.doi.org/10.3390/ijms232314588.
Texte intégralRésumé :
Amyloid β (Aβ) oligomers play a key role in the progression of Alzheimer’s disease (AD). Multiple forms of Aβ assemblies have been identified by in vitro and in vivo analyses; however, it is uncertain which oligomer is highly neurotoxic. Thus, understanding the pathogenesis of AD by detecting toxic Aβ oligomers is crucial. In this study, we report a fusion protein of cellular prion protein (PrPc) and alkaline phosphatase (ALP) from Escherichia coli as a sensing element for toxic Aβ oligomers. Since the N-terminus domain of PrPc (residue 23–111) derived from mice is known to bind to toxic Aβ oligomers in vitro, we genetically fused PrPc23–111 to ALP. The developed fusion protein, PrP–ALP, retained both the binding ability of PrPc and enzymatic activity of ALP. We showed that PrP–ALP strongly bound to high molecular weight (HMW) oligomers but showed little or no affinity toward monomers. The observation that PrP–ALP neutralized the toxic effect of Aβ oligomers indicated an interaction between PrP–ALP and toxic HMW oligomers. Based on ALP activity, we succeeded in detecting Aβ oligomers. PrP–ALP may serve as a powerful tool for detecting toxic Aβ oligomers that may be related to AD progression.
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Gaber, Aljaž, et Miha Pavšič. « Modeling and Structure Determination of Homo-Oligomeric Proteins : An Overview of Challenges and Current Approaches ». International Journal of Molecular Sciences 22, no 16 (23 août 2021) : 9081. http://dx.doi.org/10.3390/ijms22169081.
Texte intégralRésumé :
Protein homo-oligomerization is a very common phenomenon, and approximately half of proteins form homo-oligomeric assemblies composed of identical subunits. The vast majority of such assemblies possess internal symmetry which can be either exploited to help or poses challenges during structure determination. Moreover, aspects of symmetry are critical in the modeling of protein homo-oligomers either by docking or by homology-based approaches. Here, we first provide a brief overview of the nature of protein homo-oligomerization. Next, we describe how the symmetry of homo-oligomers is addressed by crystallographic and non-crystallographic symmetry operations, and how biologically relevant intermolecular interactions can be deciphered from the ordered array of molecules within protein crystals. Additionally, we describe the most important aspects of protein homo-oligomerization in structure determination by NMR. Finally, we give an overview of approaches aimed at modeling homo-oligomers using computational methods that specifically address their internal symmetry and allow the incorporation of other experimental data as spatial restraints to achieve higher model reliability.
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Fernández-Silva, Arline, Fernando Lledías, Jonathan Rodríguez-López, Juan E. Olivares, Leidys French-Pacheco, Marcela Treviño, Carlos Amero et Claudia Díaz-Camino. « The Common Bean Small Heat Shock Protein Nodulin 22 from Phaseolus vulgaris L. Assembles into Functional High-Molecular-Weight Oligomers ». Molecules 27, no 24 (8 décembre 2022) : 8681. http://dx.doi.org/10.3390/molecules27248681.
Texte intégralRésumé :
Small heat shock proteins (sHsps) are present in all domains of life. These proteins are responsible for binding unfolded proteins to prevent their aggregation. sHsps form dynamic oligomers of different sizes and constitute transient reservoirs for folding competent proteins that are subsequently refolded by ATP-dependent chaperone systems. In plants, the sHsp family is rather diverse and has been associated with the ability of plants to survive diverse environmental stresses. Nodulin 22 (PvNod22) is an sHsp of the common bean (Phaseolus vulgaris L.) located in the endoplasmic reticulum. This protein is expressed in response to stress (heat or oxidative) or in plant roots during mycorrhizal and rhizobial symbiosis. In this work, we study its oligomeric state using a combination of in silico and experimental approaches. We found that recombinant PvNod22 was able to protect a target protein from heat unfolding in vitro. We also demonstrated that PvNod22 assembles into high-molecular-weight oligomers with diameters of ~15 nm under stress-free conditions. These oligomers can cluster together to form high-weight polydisperse agglomerates with temperature-dependent interactions; in contrast, the oligomers are stable regarding temperature.
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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.
Texte intégralRésumé :
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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Pokrajac, Lisa, Clara Baik, J. Robin Harris, Naghmeh S. Sarraf et Michael Palmer. « Partial oligomerization of pyolysin induced by a disulfide-tethered mutant ». Biochemistry and Cell Biology 90, no 6 (décembre 2012) : 709–17. http://dx.doi.org/10.1139/o2012-029.
Texte intégralRésumé :
The bacterial toxin pyolysin (PLO) belongs to the family of cholesterol-dependent cytolysins (CDCs), which form large, ring-shaped oligomeric pores in cholesterol-containing membranes. Monomeric CDC molecules have a structure of four domains, with domains 2 and 3 packed against each other. After binding to target membranes containing cholesterol, toxin monomers oligomerize into pre-pore complexes. Trans-membrane pores form when the pre-pores insert into the lipid bilayer. Membrane insertion requires each subunit in the pre-pore to undergo a significant change in conformation, including the separation of domains 2 and 3. We here characterize a pyolysin mutant with an engineered disulfide bond between domains 2 and 3. The disulfide-tethered mutant binds to membranes but does not form oligomers. When mixed with wild type PLO, the two proteins form hybrid oligomers, which are reduced in size and arc-shaped rather than ring-shaped. With equimolar mixtures or the disulfide mutant in slight excess, the hybrid oligomers retain pore-forming activity, while a larger excess of the mutant suppresses pore formation. These results support a “partially cooperative” mode of protein activity, in which a limited number of functional subunits within an oligomer have to cooperate to initiate membrane insertion and pore formation.
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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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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.
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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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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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Carugo, Oliviero. « A structural proteomics filter : prediction of the quaternary structural type of hetero-oligomeric proteins on the basis of their sequences ». Journal of Applied Crystallography 40, no 6 (10 novembre 2007) : 986–89. http://dx.doi.org/10.1107/s0021889807041076.
Texte intégralRésumé :
A protein chain can correspond to a monomeric protein or it can form, together with other chains, oligomeric assemblies, which can be either homo-oligomers or hetero-oligomers. In the latter case, the three-dimensional structure of the single protein chain is unlikely to be determined, since it will probably be difficult to express and crystallize. A computational method is presented here that allows one to predict if a chain participates in hetero-oligomeric assemblies, on the basis of its amino acid composition, with accuracy close to 80%. Such a technique should improve the success rate of structural biology projects.
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Gurevich, Vsevolod V., et Eugenia V. Gurevich. « Solo vs Chorus : Monomers and Oligomers of Arrestin Proteins ». International Journal of Molecular Sciences 23, no 13 (29 juin 2022) : 7253. http://dx.doi.org/10.3390/ijms23137253.
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Three out of four subtypes of arrestin proteins expressed in mammals self-associate, each forming oligomers of a distinct kind. Monomers and oligomers have different subcellular localization and distinct biological functions. Here we summarize existing evidence regarding arrestin oligomerization and discuss specific functions of monomeric and oligomeric forms, although too few of the latter are known. The data on arrestins highlight biological importance of oligomerization of signaling proteins. Distinct modes of oligomerization might be an important contributing factor to the functional differences among highly homologous members of the arrestin protein family.
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Celej, María Soledad, Rabia Sarroukh, Erik Goormaghtigh, Gerardo D. Fidelio, Jean-Marie Ruysschaert et Vincent Raussens. « Toxic prefibrillar α-synuclein amyloid oligomers adopt a distinctive antiparallel β-sheet structure ». Biochemical Journal 443, no 3 (16 avril 2012) : 719–26. http://dx.doi.org/10.1042/bj20111924.
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Parkinson's disease is an age-related movement disorder characterized by the presence in the mid-brain of amyloid deposits of the 140-amino-acid protein AS (α-synuclein). AS fibrillation follows a nucleation polymerization pathway involving diverse transient prefibrillar species varying in size and morphology. Similar to other neurodegenerative diseases, cytotoxicity is currently attributed to these prefibrillar species rather than to the insoluble aggregates. Nevertheless, the underlying molecular mechanisms responsible for cytotoxicity remain elusive and structural studies may contribute to the understanding of both the amyloid aggregation mechanism and oligomer-induced toxicity. It is already recognized that soluble oligomeric AS species adopt β-sheet structures that differ from those characterizing the fibrillar structure. In the present study we used ATR (attenuated total reflection)–FTIR (Fourier-transform infrared) spectroscopy, a technique especially sensitive to β-sheet structure, to get a deeper insight into the β-sheet organization within oligomers and fibrils. Careful spectral analysis revealed that AS oligomers adopt an antiparallel β-sheet structure, whereas fibrils adopt a parallel arrangement. The results are discussed in terms of regions of the protein involved in the early β-sheet interactions and the implications of such conformational arrangement for the pathogenicity associated with AS oligomers.
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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.
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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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Uguzzoni, Guido, Shalini John Lovis, Francesco Oteri, Alexander Schug, Hendrik Szurmant et Martin Weigt. « Large-scale identification of coevolution signals across homo-oligomeric protein interfaces by direct coupling analysis ». Proceedings of the National Academy of Sciences 114, no 13 (13 mars 2017) : E2662—E2671. http://dx.doi.org/10.1073/pnas.1615068114.
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Proteins have evolved to perform diverse cellular functions, from serving as reaction catalysts to coordinating cellular propagation and development. Frequently, proteins do not exert their full potential as monomers but rather undergo concerted interactions as either homo-oligomers or with other proteins as hetero-oligomers. The experimental study of such protein complexes and interactions has been arduous. Theoretical structure prediction methods are an attractive alternative. Here, we investigate homo-oligomeric interfaces by tracing residue coevolution via the global statistical direct coupling analysis (DCA). DCA can accurately infer spatial adjacencies between residues. These adjacencies can be included as constraints in structure prediction techniques to predict high-resolution models. By taking advantage of the ongoing exponential growth of sequence databases, we go significantly beyond anecdotal cases of a few protein families and apply DCA to a systematic large-scale study of nearly 2,000 Pfam protein families with sufficient sequence information and structurally resolved homo-oligomeric interfaces. We find that large interfaces are commonly identified by DCA. We further demonstrate that DCA can differentiate between subfamilies with different binding modes within one large Pfam family. Sequence-derived contact information for the subfamilies proves sufficient to assemble accurate structural models of the diverse protein-oligomers. Thus, we provide an approach to investigate oligomerization for arbitrary protein families leading to structural models complementary to often-difficult experimental methods. Combined with ever more abundant sequential data, we anticipate that this study will be instrumental to allow the structural description of many heteroprotein complexes in the future.
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Busija, Anna R., Hemal H. Patel et Paul A. Insel. « Caveolins and cavins in the trafficking, maturation, and degradation of caveolae : implications for cell physiology ». American Journal of Physiology-Cell Physiology 312, no 4 (1 avril 2017) : C459—C477. http://dx.doi.org/10.1152/ajpcell.00355.2016.
Texte intégralRésumé :
Caveolins (Cavs) are ~20 kDa scaffolding proteins that assemble as oligomeric complexes in lipid raft domains to form caveolae, flask-shaped plasma membrane (PM) invaginations. Caveolae (“little caves”) require lipid-lipid, protein-lipid, and protein-protein interactions that can modulate the localization, conformational stability, ligand affinity, effector specificity, and other functions of proteins that are partners of Cavs. Cavs are assembled into small oligomers in the endoplasmic reticulum (ER), transported to the Golgi for assembly with cholesterol and other oligomers, and then exported to the PM as an intact coat complex. At the PM, cavins, ~50 kDa adapter proteins, oligomerize into an outer coat complex that remodels the membrane into caveolae. The structure of caveolae protects their contents (i.e., lipids and proteins) from degradation. Cellular changes, including signal transduction effects, can destabilize caveolae and produce cavin dissociation, restructuring of Cav oligomers, ubiquitination, internalization, and degradation. In this review, we provide a perspective of the life cycle (biogenesis, degradation), composition, and physiologic roles of Cavs and caveolae and identify unanswered questions regarding the roles of Cavs and cavins in caveolae and in regulating cell physiology.1
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Gwyther, Rebecca E. A., D. Dafydd Jones et Harley L. Worthy. « Better together : building protein oligomers naturally and by design ». Biochemical Society Transactions 47, no 6 (5 décembre 2019) : 1773–80. http://dx.doi.org/10.1042/bst20190283.
Texte intégralRésumé :
Protein oligomers are more common in nature than monomers, with dimers being the most prevalent final structural state observed in known structures. From a biological perspective, this makes sense as it conserves vital molecular resources that may be wasted simply by generating larger single polypeptide units, and allows new features such as cooperativity to emerge. Taking inspiration from nature, protein designers and engineers are now building artificial oligomeric complexes using a variety of approaches to generate new and useful supramolecular protein structures. Oligomerisation is thus offering a new approach to sample structure and function space not accessible through simply tinkering with monomeric proteins.
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Dabney-Smith, Carole, et Kenneth Cline. « Clustering of C-Terminal Stromal Domains of Tha4 Homo-oligomers during Translocation by the Tat Protein Transport System ». Molecular Biology of the Cell 20, no 7 (avril 2009) : 2060–69. http://dx.doi.org/10.1091/mbc.e08-12-1189.
Texte intégralRésumé :
The chloroplast Twin arginine translocation (Tat) pathway uses three membrane proteins and the proton gradient to transport folded proteins across sealed membranes. Precursor proteins bind to the cpTatC-Hcf106 receptor complex, triggering Tha4 assembly and protein translocation. Tha4 is required only for the translocation step and is thought to be the protein-conducting component. The organization of Tha4 oligomers was examined by substituting pairs of cysteine residues into Tha4 and inducing disulfide cross-links under varying stages of protein translocation. Tha4 formed tetramers via its transmembrane domain in unstimulated membranes and octamers in membranes stimulated by precursor and the proton gradient. Tha4 formed larger oligomers of at least 16 protomers via its carboxy tail, but such C-tail clustering only occurred in stimulated membranes. Mutational studies showed that transmembrane domain directed octamers as well as C-tail clusters require Tha4's transmembrane glutamate residue and its amphipathic helix, both of which are necessary for Tha4 function. A novel double cross-linking strategy demonstrated that both transmembrane domain directed- and C-tail directed oligomerization occur in the translocase. These results support a model in which Tha4 oligomers dock with a precursor–receptor complex and undergo a conformational switch that results in activation for protein transport. This possibly involves accretion of additional Tha4 into a larger transport-active homo-oligomer.
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Sangwan, Smriti, Anni Zhao, Katrina L. Adams, Christina K. Jayson, Michael R. Sawaya, Elizabeth L. Guenther, Albert C. Pan et al. « Atomic structure of a toxic, oligomeric segment of SOD1 linked to amyotrophic lateral sclerosis (ALS) ». Proceedings of the National Academy of Sciences 114, no 33 (31 juillet 2017) : 8770–75. http://dx.doi.org/10.1073/pnas.1705091114.
Texte intégralRésumé :
Fibrils and oligomers are the aggregated protein agents of neuronal dysfunction in ALS diseases. Whereas we now know much about fibril architecture, atomic structures of disease-related oligomers have eluded determination. Here, we determine the corkscrew-like structure of a cytotoxic segment of superoxide dismutase 1 (SOD1) in its oligomeric state. Mutations that prevent formation of this structure eliminate cytotoxicity of the segment in isolation as well as cytotoxicity of the ALS-linked mutants of SOD1 in primary motor neurons and in a Danio rerio (zebrafish) model of ALS. Cytotoxicity assays suggest that toxicity is a property of soluble oligomers, and not large insoluble aggregates. Our work adds to evidence that the toxic oligomeric entities in protein aggregation diseases contain antiparallel, out-of-register β-sheet structures and identifies a target for structure-based therapeutics in ALS.
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Zhu, Lixin, Yuechueng Liu et John G. Forte. « Ezrin oligomers are the membrane-bound dormant form in gastric parietal cells ». American Journal of Physiology-Cell Physiology 288, no 6 (juin 2005) : C1242—C1254. http://dx.doi.org/10.1152/ajpcell.00521.2004.
Texte intégralRésumé :
Ezrin is a member of ezrin, radixin, moesin (ERM) protein family that links F-actin to membranes. The NH2- and COOH-terminal association domains of ERM proteins, known respectively as N-ERMAD and C-ERMAD, participate in interactions with membrane proteins and F-actin, and intramolecular and intermolecular interactions within and among ERM proteins. In gastric parietal cells, ezrin is heavily represented on the apical membrane and is associated with cell activation. Ezrin-ezrin interactions are presumably involved in functional regulation of ezrin and thus became a subject of our study. Fluorescence resonance energy transfer (FRET) was examined with cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-tagged ezrin incorporated into HeLa cells and primary cultures of parietal cells. Constructs included YFP at the NH2 terminus of ezrin (YFP-Ez), CFP at the COOH terminus of ezrin (Ez-CFP), and double-labeled ezrin (N-YFP-ezrin-CFP-C). FRET was probed using fluorescence microscopy and spectrofluorometry. Evidence of ezrin oligomer formation was found using FRET in cells coexpressing Ez-CFP and YFP-Ez and by performing coimmunoprecipitation of endogenous ezrin with fluorescent protein-tagged ezrin. Thus intermolecular NH2- and COOH-terminal association domain (N-C) binding in vivo is consistent with the findings of earlier in vitro studies. After the ezrin oligomers were separated from monomers, FRET was observed in both forms, indicating intramolecular and intermolecular N-C binding. When the distribution of native ezrin as oligomers vs. monomers was examined in resting and maximally stimulated parietal cells, a shift of ezrin oligomers to the monomeric form was correlated with stimulation, suggesting that ezrin oligomers are the membrane-bound dormant form in gastric parietal cells.
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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.
Texte intégralRésumé :
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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Dulle, Jennifer E., Rachel E. Bouttenot, Lisa A. Underwood et Heather L. True. « Soluble oligomers are sufficient for transmission of a yeast prion but do not confer phenotype ». Journal of Cell Biology 203, no 2 (21 octobre 2013) : 197–204. http://dx.doi.org/10.1083/jcb.201307040.
Texte intégralRésumé :
Amyloidogenic proteins aggregate through a self-templating mechanism that likely involves oligomeric or prefibrillar intermediates. For disease-associated amyloidogenic proteins, such intermediates have been suggested to be the primary cause of cellular toxicity. However, isolation and characterization of these oligomeric intermediates has proven difficult, sparking controversy over their biological relevance in disease pathology. Here, we describe an oligomeric species of a yeast prion protein in cells that is sufficient for prion transmission and infectivity. These oligomers differ from the classic prion aggregates in that they are soluble and less resistant to SDS. We found that large, SDS-resistant aggregates were required for the prion phenotype but that soluble, more SDS-sensitive oligomers contained all the information necessary to transmit the prion conformation. Thus, we identified distinct functional requirements of two types of prion species for this endogenous epigenetic element. Furthermore, the nontoxic, self-replicating amyloid conformers of yeast prion proteins have again provided valuable insight into the mechanisms of amyloid formation and propagation in cells.
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Walsh, D. M., I. Klyubin, G. M. Shankar, M. Townsend, J. V. Fadeeva, V. Betts, M. B. Podlisny et al. « The role of cell-derived oligomers of Aβ in Alzheimer's disease and avenues for therapeutic intervention ». Biochemical Society Transactions 33, no 5 (26 octobre 2005) : 1087–90. http://dx.doi.org/10.1042/bst0331087.
Texte intégralRésumé :
Burgeoning evidence suggests that soluble oligomers of Aβ (amyloid β-protein) are the earliest effectors of synaptic compromise in Alzheimer's disease. Whereas most other investigators have employed synthetic Aβ peptides, we have taken advantage of a β-amyloid precursor protein-overexpressing cell line (referred to as 7PA2) that secretes sub-nanomolar levels of low-n oligomers of Aβ. These are composed of heterogeneous Aβ peptides that migrate on SDS/PAGE as dimers, trimers and tetramers. When injected into the lateral ventricle of rats in vivo, these soluble oligomers inhibit hippocampal long-term potentiation and alter the memory of a complex learned behaviour. Biochemical manipulation of 7PA2 medium including immunodepletion with Aβ-specific antibodies and fractionation by size-exclusion chromatography allowed us to unambiguously attribute these effects to low-n oligomers. Using this paradigm we have tested compounds directed at three prominent amyloid-based therapeutic targets: inhibition of the secretases responsible for Aβ production, inhibition of Aβ aggregation and immunization against Aβ. In each case, compounds capable of reducing oligomer production or antibodies that avidly bind Aβ oligomers also ameliorate the synaptotoxic effects of these natural, cell-derived oligomers.
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Byrne, Patrick O., Kalina Hristova et Daniel J. Leahy. « EGFR forms ligand-independent oligomers that are distinct from the active state ». Journal of Biological Chemistry 295, no 38 (29 juillet 2020) : 13353–62. http://dx.doi.org/10.1074/jbc.ra120.012852.
Texte intégralRésumé :
The human epidermal growth factor receptor (EGFR/ERBB1) is a receptor tyrosine kinase (RTK) that forms activated oligomers in response to ligand. Much evidence indicates that EGFR/ERBB1 also forms oligomers in the absence of ligand, but the structure and physiological role of these ligand-independent oligomers remain unclear. To examine these features, we use fluorescence microscopy to measure the oligomer stability and FRET efficiency for homo- and hetero-oligomers of fluorescent protein-labeled forms of EGFR and its paralog, human epidermal growth factor receptor 2 (HER2/ERBB2) in vesicles derived from mammalian cell membranes. We observe that both receptors form ligand-independent oligomers at physiological plasma membrane concentrations. Mutations introduced in the kinase region at the active state asymmetric kinase dimer interface do not affect the stability of ligand-independent EGFR oligomers. These results indicate that ligand-independent EGFR oligomers form using interactions that are distinct from the EGFR active state.
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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.
Texte intégralRésumé :
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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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.