Journal articles on the topic 'Homodimers'
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1
Krause, Jean-Michel, Peter Berger, Jordi Roig, Vinod Singh, and Wolfgang E. Merz. "Rapid Maturation of Glycoprotein Hormone Free α-Subunit (GPHα) and GPHαα Homodimers." Molecular Endocrinology 21, no. 10 (October 1, 2007): 2551–64. http://dx.doi.org/10.1210/me.2007-0051.
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Abstract The dynamics of glycoprotein hormone α-subunit (GPHα) maturation and GPHαα homodimer formation were studied in presence (JEG-3 choriocarcinoma cells) and absence (HeLa cells) of hCGβ. In both cases, the major initially occurring GPHα variant in [35S]Met/Cys-labeled cells carried two N-glycans (Mr app = 22 kDa). Moreover, a mono-N-glycosylated in vivo association-incompetent GPHα variant (Mr app = 18 kDa) was observed. In JEG-3 cells the early 22-kDa GPHα either associated with hCGβ, or showed self-association to yield GPHαα homodimers, or was later converted into heavily glycosylated large free GPHα (Mr app = 24 kDa). Micro-preparative isolation of intracellular GPHαα homodimers of JEG-3 cells and their conversion by reduction revealed that they consisted of 22-kDa GPHα monomers and not of large free GPHα. In HeLa cells, the large free GPHα variant was not observed, whereas GPHαα homodimers were present. Intracellularly, early GPHαα homodimers (35 kDa) and late variants (JEG-3: 44 kDa, HeLa: 39 kDa) were found. Both cell types secreted 45 kDa GPHαα homodimers. Large free GPHα and GPHαα homodimers were more rapidly sialylated than hCG αβ-heterodimers indicating a sequestration mechanism in the secretory pathway. In GPHαα homo- as well as hCG αβ-heterodimers the subunit interaction site, located on loop 2 of GPHα (amino acids 33–42), became immunologically inaccessible indicating similar spatial orientation of GPHα in both types of dimers. The studies demonstrate the formation, in vivo dynamics of GPHαα homodimers, and the pathways of the cellular metabolism of variants of GPHα, monoglycosylated GPHα and large free GPHα.
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Hadarovich, A. Y., A. A. Kalinouski, and A. V. Tuzikov. "Protein homodimers structure prediction based on deep neural network." Informatics 17, no. 2 (June 26, 2020): 44–53. http://dx.doi.org/10.37661/1816-0301-2020-17-2-44-53.
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Structural prediction of protein-protein complexes has important application in such domains as modeling of biological processes and drug design. Homodimers (complexes which consist of two identical proteins) are the most common type of protein complexes in nature but there is still no universal algorithm to predict their 3D structures. Experimental techniques to identify the structure of protein complex require enormous amount of time and resources, and each method has its own limitations. Recently Deep Neural Networks allowed to predict structures of individual proteins greatly prevailing in accuracy over other algorithmic approaches. Building on the idea of this approach, we developed an algorithm to model the 3D structure of homodimer based on deep learning. It consists of two major steps: at the first step a protein complex contact map is predicted with the deep convolutional neural network, and the second stage is used to predict 3D structure of homodimer based on obtained contact map and optimization procedure. The use of the neural network in combination with optimization procedure based on gradient descent method allowed to predict structures for protein homodimers. The suggested approach was tested and validated on a dataset of protein homodimers from Protein Data Bank (PDB). The developed procedure could be also used for evaluating protein homodimer models as one of the stages in drug compounds developing.
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Geng, Jie, and Malini Raghavan. "CD8αα homodimers function as a coreceptor for KIR3DL1." Proceedings of the National Academy of Sciences 116, no. 36 (August 16, 2019): 17951–56. http://dx.doi.org/10.1073/pnas.1905943116.
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Cluster of differentiation 8 (CD8) is a cell surface glycoprotein, which is expressed as 2 forms, αα homodimer or αβ heterodimer. Peptide-loaded major histocompatibility complex class I (pMHC-I) molecules are major ligands for both forms of CD8. CD8αβ is a coreceptor for the T cell receptor (TCR) and binds to the same cognate pMHC-I as the TCR, thus enabling or augmenting T cell responses. The function of CD8αα homodimers is largely unknown. While CD8αβ heterodimer is expressed exclusively on CD8+ T cells, the CD8αα homodimer is present in subsets of T cells and human natural killer (NK) cells. Here, we report that the CD8αα homodimer functions as a coreceptor for KIR3DL1, an inhibitory receptor of NK cells that is specific for certain MHC-I allotypes. CD8αα enhances binding of pMHC-I to KIR3DL1, increases KIR3DL1 clustering at the immunological synapse, and augments KIR3DL1-mediated inhibition of NK cell activation. Additionally, interactions between pMHC-I and CD8αα homodimers regulate KIR3DL1+ NK cell education. Together, these findings reveal another dimension to the modulation of NK cell activity.
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Tajer, Benjamin, James A. Dutko, Shawn C. Little, and Mary C. Mullins. "BMP heterodimers signal via distinct type I receptor class functions." Proceedings of the National Academy of Sciences 118, no. 15 (April 7, 2021): e2017952118. http://dx.doi.org/10.1073/pnas.2017952118.
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Heterodimeric TGF-β ligands outperform homodimers in a variety of developmental, cell culture, and therapeutic contexts; however, the mechanisms underlying this increased potency remain uncharacterized. Here, we use dorsal–ventral axial patterning of the zebrafish embryo to interrogate the BMP2/7 heterodimer signaling mechanism. We demonstrate that differential interactions with BMP antagonists do not account for the reduced signaling ability of homodimers. Instead, we find that while overexpressed BMP2 homodimers can signal, they require two nonredundant type I receptors, one from the Acvr1 subfamily and one from the Bmpr1 subfamily. This implies that all BMP signaling within the zebrafish gastrula, even BMP2 homodimer signaling, requires Acvr1. This is particularly surprising as BMP2 homodimers do not bind Acvr1 in vitro. Furthermore, we find that the roles of the two type I receptors are subfunctionalized within the heterodimer signaling complex, with the kinase activity of Acvr1 being essential, while that of Bmpr1 is not. These results suggest that the potency of the Bmp2/7 heterodimer arises from the ability to recruit both Acvr1 and Bmpr1 into the same signaling complex.
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Peng, K.-C., D. Puett, and J. M. Brewer. "Homodimer formation by the individual subunits of bovine lutropin as determined by sedimentation equilibrium." Journal of Molecular Endocrinology 18, no. 3 (June 1997): 259–65. http://dx.doi.org/10.1677/jme.0.0180259.
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ABSTRACT Although differing in their amino acid sequences, the folding patterns of the α and β subunits of human choriogonadotropin are similar in the crystal structure of the HF-treated glycoprotein hormone. Each subunit forms a cystine-knot motif like that found in several growth factors that form homodimers and heterodimers. In order to ascertain if the α and β subunits can self-associate, e.g. to form homodimers, sedimentation equilibrium at various glycoprotein concentrations and temperatures was used to study the subunits of bovine lutropin, which are expected to exhibit conformations like those of the choriogonadotropin subunits. Each subunit was found to form homodimers with Kd values of 0·3 and 0·1 mm for α and β respectively at 37 °C. Self-association was weakly exothermic for α and endothermic for β; entropic factors made a major contribution for each. It is unlikely that homodimer formation of either subunit would be physiologically important, although homodimers may form to some extent intracellularly because of the relatively high concentrations during biosynthesis.
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Garton, Michael, Stephen S. MacKinnon, Anatoly Malevanets, and Shoshana J. Wodak. "Interplay of self-association and conformational flexibility in regulating protein function." Philosophical Transactions of the Royal Society B: Biological Sciences 373, no. 1749 (May 7, 2018): 20170190. http://dx.doi.org/10.1098/rstb.2017.0190.
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Many functional roles have been attributed to homodimers, the most common mode of protein self-association, notably in the regulation of enzymes, ion channels, transporters and transcription factors. Here we review findings that offer new insights into the different roles conformational flexibility plays in regulating homodimer function. Intertwined homodimers of two-domain proteins and their related family members display significant conformational flexibility, which translates into concerted motion between structural domains. This flexibility enables the corresponding proteins to regulate function across family members by modulating the spatial positions of key recognition surfaces of individual domains, to either maintain subunit interfaces, alter them or break them altogether, leading to a variety of functional consequences. Many proteins may exist as monomers but carry out their biological function as homodimers or higher-order oligomers. We present early evidence that in such systems homodimer formation primes the protein for its functional role. It does so by inducing elevated mobility in protein regions corresponding to the binding epitopes of functionally important ligands. In some systems this process acts as an allosteric response elicited by the self-association reaction itself. Our analysis furthermore suggests that the induced extra mobility likely facilitates ligand binding through the mechanism of conformational selection. This article is part of a discussion meeting issue ‘Allostery and molecular machines’.
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Campbell, Brandon, Marharyta Petukh, Emil Alexov, and Chuan Li. "On the electrostatic properties of homodimeric proteins." Journal of Theoretical and Computational Chemistry 13, no. 03 (May 2014): 1440007. http://dx.doi.org/10.1142/s0219633614400070.
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A large fraction of proteins function as homodimers, but it is not always clear why the dimerization is important for functionality since frequently each monomer possesses a distinctive active site. Recent work (PLoS Computational Biology9(2):e1002924) indicates that homodimerization may be important for forming an electrostatic funnel in the spermine synthase homodimer which guides changed substrates toward the active centers. This prompted us to investigate the electrostatic properties of a large set of homodimeric proteins and resulted in an observation that in a vast majority of the cases the dimerization indeed results in specific electrostatic features, although not necessarily in an electrostatic funnel. It is demonstrated that the electrostatic dipole moment of the dimer is predominantly perpendicular to the axis connecting the centers of the mass of the monomers. In addition, the surface points with highest potential are located in the proximity of the interfacial plane of the homodimeric complexes. These findings indicate that frequent homodimerization provides specific electrostatic features needed for the function of proteins.
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Ibrahim, Mahmoud A. A., Rehab R. A. Saeed, Mohammed N. I. Shehata, Muhammad Naeem Ahmed, Ahmed M. Shawky, Manal M. Khowdiary, Eslam B. Elkaeed, Mahmoud E. S. Soliman, and Nayra A. M. Moussa. "Type I–IV Halogen⋯Halogen Interactions: A Comparative Theoretical Study in Halobenzene⋯Halobenzene Homodimers." International Journal of Molecular Sciences 23, no. 6 (March 14, 2022): 3114. http://dx.doi.org/10.3390/ijms23063114.
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In the current study, unexplored type IV halogen⋯halogen interaction was thoroughly elucidated, for the first time, and compared to the well-established types I–III interactions by means of the second-order Møller–Plesset (MP2) method. For this aim, the halobenzene⋯halobenzene homodimers (where halogen = Cl, Br, and I) were designed into four different types, parodying the considered interactions. From the energetic perspective, the preference of scouted homodimers was ascribed to type II interactions (i.e., highest binding energy), whereas the lowest binding energies were discerned in type III interactions. Generally, binding energies of the studied interactions were observed to decline with the decrease in the σ-hole size in the order, C6H5I⋯IC6H5 > C6H5Br⋯BrC6H5 > C6H5Cl⋯ClC6H5 homodimers and the reverse was noticed in the case of type IV interactions. Such peculiar observations were relevant to the ample contributions of negative-belt⋯negative-belt interactions within the C6H5Cl⋯ClC6H5 homodimer. Further, type IV torsional trans → cis interconversion of C6H5X⋯XC6H5 homodimers was investigated to quantify the π⋯π contributions into the total binding energies. Evidently, the energetic features illustrated the amelioration of the considered homodimers (i.e., more negative binding energy) along the prolonged scope of torsional trans → cis interconversion. In turn, these findings outlined the efficiency of the cis configuration over the trans analog. Generally, symmetry-adapted perturbation theory-based energy decomposition analysis (SAPT-EDA) demonstrated the predominance of all the scouted homodimers by the dispersion forces. The obtained results would be beneficial for the omnipresent studies relevant to the applications of halogen bonds in the fields of materials science and crystal engineering.
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Bonsor, Daniel A., Sebastian Günther, Robert Beadenkopf, Dorothy Beckett, and Eric J. Sundberg. "Diverse oligomeric states of CEACAM IgV domains." Proceedings of the National Academy of Sciences 112, no. 44 (October 19, 2015): 13561–66. http://dx.doi.org/10.1073/pnas.1509511112.
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Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) comprise a large family of cell surface adhesion molecules that bind to themselves and other family members to carry out numerous cellular functions, including proliferation, signaling, differentiation, tumor suppression, and survival. They also play diverse and significant roles in immunity and infection. The formation of CEACAM oligomers is caused predominantly by interactions between their N-terminal IgV domains. Although X-ray crystal structures of CEACAM IgV domain homodimers have been described, how CEACAMs form heterodimers or remain monomers is poorly understood. To address this key aspect of CEACAM function, we determined the crystal structures of IgV domains that form a homodimeric CEACAM6 complex, monomeric CEACAM8, and a heterodimeric CEACAM6–CEACAM8 complex. To confirm and quantify these interactions in solution, we used analytical ultracentrifugation to measure the dimerization constants of CEACAM homodimers and isothermal titration calorimetry to determine the thermodynamic parameters and binding affinities of CEACAM heterodimers. We found the CEACAM6–CEACAM8 heterodimeric state to be substantially favored energetically relative to the CEACAM6 homodimer. Our data provide a molecular basis for the adoption of the diverse oligomeric states known to exist for CEACAMs and suggest ways in which CEACAM6 and CEACAM8 regulate the biological functions of one another, as well as of additional CEACAMs with which they interact, both in cis and in trans.
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Mou, Yun, Po-Ssu Huang, Fang-Ciao Hsu, Shing-Jong Huang, and Stephen L. Mayo. "Computational design and experimental verification of a symmetric protein homodimer." Proceedings of the National Academy of Sciences 112, no. 34 (August 12, 2015): 10714–19. http://dx.doi.org/10.1073/pnas.1505072112.
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Homodimers are the most common type of protein assembly in nature and have distinct features compared with heterodimers and higher order oligomers. Understanding homodimer interactions at the atomic level is critical both for elucidating their biological mechanisms of action and for accurate modeling of complexes of unknown structure. Computation-based design of novel protein–protein interfaces can serve as a bottom-up method to further our understanding of protein interactions. Previous studies have demonstrated that the de novo design of homodimers can be achieved to atomic-level accuracy by β-strand assembly or through metal-mediated interactions. Here, we report the design and experimental characterization of a α-helix–mediated homodimer with C2 symmetry based on a monomeric Drosophila engrailed homeodomain scaffold. A solution NMR structure shows that the homodimer exhibits parallel helical packing similar to the design model. Because the mutations leading to dimer formation resulted in poor thermostability of the system, design success was facilitated by the introduction of independent thermostabilizing mutations into the scaffold. This two-step design approach, function and stabilization, is likely to be generally applicable, especially if the desired scaffold is of low thermostability.
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Hwang, Eunha, Hae-Kap Cheong, Ameeq Ul Mushtaq, Hye-Yeon Kim, Kwon Joo Yeo, Eunhee Kim, Woo Cheol Lee, Kwang Yeon Hwang, Chaejoon Cheong, and Young Ho Jeon. "Structural basis of the heterodimerization of the MST and RASSF SARAH domains in the Hippo signalling pathway." Acta Crystallographica Section D Biological Crystallography 70, no. 7 (June 29, 2014): 1944–53. http://dx.doi.org/10.1107/s139900471400947x.
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Despite recent progress in research on the Hippo signalling pathway, the structural information available in this area is extremely limited. Intriguingly, the homodimeric and heterodimeric interactions of mammalian sterile 20-like (MST) kinases through the so-called `SARAH' (SAV/RASSF/HPO) domains play a critical role in cellular homeostasis, dictating the fate of the cell regarding cell proliferation or apoptosis. To understand the mechanism of the heterodimerization of SARAH domains, the three-dimensional structures of an MST1–RASSF5 SARAH heterodimer and an MST2 SARAH homodimer were determined by X-ray crystallography and were analysed together with that previously determined for the MST1 SARAH homodimer. While the structure of the MST2 homodimer resembled that of the MST1 homodimer, the MST1–RASSF5 heterodimer showed distinct structural features. Firstly, the six N-terminal residues (Asp432–Lys437), which correspond to the short N-terminal 310-helix h1 kinked from the h2 helix in the MST1 homodimer, were disordered. Furthermore, the MST1 SARAH domain in the MST1–RASSF5 complex showed a longer helical structure (Ser438–Lys480) than that in the MST1 homodimer (Val441–Lys480). Moreover, extensive polar and nonpolar contacts in the MST1–RASSF5 SARAH domain were identified which strengthen the interactions in the heterodimer in comparison to the interactions in the homodimer. Denaturation experiments performed using urea also indicated that the MST–RASSF heterodimers are substantially more stable than the MST homodimers. These findings provide structural insights into the role of the MST1–RASSF5 SARAH domain in apoptosis signalling.
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Neugebauer, Judith M., Sunjong Kwon, Hyung-Seok Kim, Nathan Donley, Anup Tilak, Shailaja Sopory, and Jan L. Christian. "The prodomain of BMP4 is necessary and sufficient to generate stable BMP4/7 heterodimers with enhanced bioactivity in vivo." Proceedings of the National Academy of Sciences 112, no. 18 (April 20, 2015): E2307—E2316. http://dx.doi.org/10.1073/pnas.1501449112.
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Bone morphogenetic proteins 4 and 7 (BMP4 and BMP7) are morphogens that signal as either homodimers or heterodimers to regulate embryonic development and adult homeostasis. BMP4/7 heterodimers exhibit markedly higher signaling activity than either homodimer, but the mechanism underlying the enhanced activity is unknown. BMPs are synthesized as inactive precursors that dimerize and are then cleaved to generate both the bioactive ligand and prodomain fragments, which lack signaling activity. Our study reveals a previously unknown requirement for the BMP4 prodomain in promoting heterodimer activity. We show that BMP4 and BMP7 precursor proteins preferentially or exclusively form heterodimers when coexpressed in vivo. In addition, we show that the BMP4 prodomain is both necessary and sufficient for generation of stable heterodimeric ligands with enhanced activity and can enable homodimers to signal in a context in which they normally lack activity. Our results suggest that intrinsic properties of the BMP4 prodomain contribute to the relative bioactivities of homodimers versus heterodimers in vivo. These findings have clinical implications for the use of BMPs as regenerative agents for the treatment of bone injury and disease.
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Thompson, PD, LS Remus, JC Hsieh, PW Jurutka, GK Whitfield, MA Galligan, C. Encinas Dominguez, CA Haussler, and MR Haussler. "Distinct retinoid X receptor activation function-2 residues mediate transactivation in homodimeric and vitamin D receptor heterodimeric contexts." Journal of Molecular Endocrinology 27, no. 2 (October 1, 2001): 211–27. http://dx.doi.org/10.1677/jme.0.0270211.
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The vitamin D receptor (VDR) stimulates transcription as a 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3))-activated heterodimer with retinoid X receptor (RXR). RXR also forms homodimers to mediate 9-cis retinoic acid (9-cis RA)-induced gene expression. Both receptors possess a C-terminal hormone-dependent activation function-2 (AF-2), a highly conserved region that binds coactivators to transduce the transcriptional signal. By replacing single amino acids within the AF-2 of human RXR alpha (hRXR alpha) or mouse RXR beta (mRXR beta), the contribution of these residues to transactivation by the RXR-VDR heterodimer and the RXR-RXR homodimer was evaluated. In 9-cis RA-responsive homodimers, the second and fourth positions of the AF-2 (leucine and glutamate respectively) are essential. However, in the context of an RXR-VDR heterodimer activated by 1,25(OH)(2)D(3), alteration of these two RXR residues has little effect. Instead, AF-2 residues located towards the C-terminus, such as the penultimate position (L455 in hRXR alpha or L441 in mRXR beta), are crucial for RXR-VDR heterodimers. Indeed, L455A mutant RXR exerts a dominant negative effect on RXR-VDR transcriptional responsiveness to 1,25(OH)(2)D(3). Further experiments with a mutant hRXR alpha (F313A) which elicits 9-cis RA-independent transactivation as a homodimer demonstrate that, when heterodimerized with VDR, this RXR mutant is incapable of activating the RXR-VDR heterocomplex in the absence of the VDR ligand. Taken together, these results indicate that RXR is a subordinate, yet essential transcriptional partner in RXR-VDR-mediated activation of gene expression. Furthermore, a functional switch in RXR AF-2 signaling occurs between RXR residues in the homodimeric versus the heterodimeric states, likely reflecting different interactions between subregions of the AF-2 and coactivator(s).
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Liss, Andrew S., and Henry R. Bose,. "Mutational Analysis of the v-Rel Dimerization Interface Reveals a Critical Role for v-Rel Homodimers in Transformation." Journal of Virology 76, no. 10 (May 15, 2002): 4928–39. http://dx.doi.org/10.1128/jvi.76.10.4928-4939.2002.
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ABSTRACT The v-rel oncogene encoded by reticuloendotheliosis virus strain T is the acutely transforming member of the Rel/NF-κB family of transcription factors. In v-Rel-transformed cells, v-Rel exists as homodimers or heterodimers with the endogenous Rel/NF-κB proteins c-Rel, NF-κB1, NF-κB2, and RelA. To examine the contribution of these complexes to v-Rel-mediated transformation, mutations were introduced into the dimerization interface of v-Rel to generate v-Rel mutants with selective dimerization properties. Nine mutants are described in this study that are defective in homodimer and/or heterodimer formation with specific Rel/NF-κB family members. Viruses expressing mutants that failed to homodimerize but were able to form heterodimeric complexes were unable to transform splenic lymphocytes in vitro, indicating that the dimerization of v-Rel with endogenously expressed Rel/NF-κB proteins is not in itself sufficient for transformation. In addition, two partially transforming mutants were identified that exhibited an impaired ability to form homodimers. Sequence analysis of the proviral DNA from cells transformed by these mutants revealed the presence of multiple secondary mutations in sequences responsible for dimerization and DNA binding. Two of these mutations either enhanced or restored the ability of these proteins to bind DNA as a homodimer. Viruses expressing these proteins transformed cells at levels comparable to or slightly less than v-Rel, suggesting that a threshold level of DNA binding by v-Rel homodimers is required for transformation.
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Peckys, Diana B., Ulrike Korf, and Niels de Jonge. "Local variations of HER2 dimerization in breast cancer cells discovered by correlative fluorescence and liquid electron microscopy." Science Advances 1, no. 6 (July 2015): e1500165. http://dx.doi.org/10.1126/sciadv.1500165.
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The formation of HER2 homodimers plays an important role in breast cancer aggressiveness and progression; however, little is known about its localization. We have studied the intra- and intercellular variation of HER2 at the single-molecule level in intact SKBR3 breast cancer cells. Whole cells were visualized in hydrated state with correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM). The locations of individual HER2 receptors were detected using an anti-HER2 affibody in combination with a quantum dot (QD), a fluorescent nanoparticle. Fluorescence microscopy revealed considerable differences of HER2 membrane expression between individual cells and between different membrane regions of the same cell (that is, membrane ruffles and flat areas). Subsequent ESEM of the corresponding cellular regions provided images of individually labeled HER2 receptors. The high spatial resolution of 3 nm and the close proximity between the QD and the receptor allowed quantifying the stoichiometry of HER2 complexes, distinguishing between monomers, dimers, and higher-order clusters. Downstream data analysis based on calculating the pair correlation function from receptor positions showed that cellular regions exhibiting membrane ruffles contained a substantial fraction of HER2 in homodimeric state. Larger-order clusters were also present. Membrane areas with homogeneous membrane topography, on the contrary, displayed HER2 in random distribution. Second, HER2 homodimers appeared to be absent from a small subpopulation of cells exhibiting a flat membrane topography, possibly resting cells. Local differences in homodimer presence may point toward functional differences with possible relevance for studying metastasis and drug response.
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Zhang, X. K., G. Salbert, M. O. Lee, and M. Pfahl. "Mutations that alter ligand-induced switches and dimerization activities in the retinoid X receptor." Molecular and Cellular Biology 14, no. 6 (June 1994): 4311–23. http://dx.doi.org/10.1128/mcb.14.6.4311-4323.1994.
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The retinoid X receptor (RXR) heterodimerizes with a variety of nuclear receptors. In addition, RXR forms homodimers in the presence of its ligand, 9-cis-retinoic acid. From deletion and point mutation analysis we present evidence that a short region (amino acids 413 to 443) in the carboxy terminus of RXR alpha is critical for both homo- and heterodimeric interactions as well as for diverse functional activities. In addition, we present evidence that homo- and heterodimer functions can be separated. The deletion of 19 amino acids from the C-terminal end of RXR dramatically reduced the transcriptional activation function of RXR. The removal of 10 additional amino acids resulted in a receptor (delta RXR3) that had completely lost its ligand-dependent homodimer function but retained its heterodimer activities. Heterodimer function was abolished by the deletion of an additional 20 amino acids. Single amino acid substitutions in the region generated receptors with altered RXR homodimer DNA binding, while simultaneous mutation of three Leu residues (Leu-418, -419 and -422) completely abolished both RXR homodimer and heterodimer DNA binding activities. Mutation of Leu-430 to Phe (L430-F) resulted in a receptor that bound to DNA strongly as homodimers in a ligand-independent manner, while another single amino acid exchange (L422-Q) led to a mutant that behaved in a manner exactly opposite to that of wild-type RXR in that the homodimerization of the mutant occurred in the absence of ligand and was inhibited by 9-cis-retinoic acid. In transfection assays, both L422-Q and L430-F failed to act as homodimers but retained their heterodimer function. Our studies demonstrate the unique properties of the RXR ligand binding domain and point to specific residues that mediate homo- and heterodimer activities and ligand-induced conformational switches.
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Zhang, X. K., G. Salbert, M. O. Lee, and M. Pfahl. "Mutations that alter ligand-induced switches and dimerization activities in the retinoid X receptor." Molecular and Cellular Biology 14, no. 6 (June 1994): 4311–23. http://dx.doi.org/10.1128/mcb.14.6.4311.
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The retinoid X receptor (RXR) heterodimerizes with a variety of nuclear receptors. In addition, RXR forms homodimers in the presence of its ligand, 9-cis-retinoic acid. From deletion and point mutation analysis we present evidence that a short region (amino acids 413 to 443) in the carboxy terminus of RXR alpha is critical for both homo- and heterodimeric interactions as well as for diverse functional activities. In addition, we present evidence that homo- and heterodimer functions can be separated. The deletion of 19 amino acids from the C-terminal end of RXR dramatically reduced the transcriptional activation function of RXR. The removal of 10 additional amino acids resulted in a receptor (delta RXR3) that had completely lost its ligand-dependent homodimer function but retained its heterodimer activities. Heterodimer function was abolished by the deletion of an additional 20 amino acids. Single amino acid substitutions in the region generated receptors with altered RXR homodimer DNA binding, while simultaneous mutation of three Leu residues (Leu-418, -419 and -422) completely abolished both RXR homodimer and heterodimer DNA binding activities. Mutation of Leu-430 to Phe (L430-F) resulted in a receptor that bound to DNA strongly as homodimers in a ligand-independent manner, while another single amino acid exchange (L422-Q) led to a mutant that behaved in a manner exactly opposite to that of wild-type RXR in that the homodimerization of the mutant occurred in the absence of ligand and was inhibited by 9-cis-retinoic acid. In transfection assays, both L422-Q and L430-F failed to act as homodimers but retained their heterodimer function. Our studies demonstrate the unique properties of the RXR ligand binding domain and point to specific residues that mediate homo- and heterodimer activities and ligand-induced conformational switches.
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Krouwels, Anita, Juvita D. Iljas, Angela H. M. Kragten, Wouter J. A. Dhert, F. Cumhur Öner, Marianna A. Tryfonidou, and Laura B. Creemers. "Bone Morphogenetic Proteins for Nucleus Pulposus Regeneration." International Journal of Molecular Sciences 21, no. 8 (April 14, 2020): 2720. http://dx.doi.org/10.3390/ijms21082720.
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Matrix production by nucleus pulposus (NP) cells, the cells residing in the center of the intervertebral disc, can be stimulated by growth factors. Bone morphogenetic proteins (BMPs) hold great promise. Although BMP2 and BMP7 have been used most frequently, other BMPs have also shown potential for NP regeneration. Heterodimers may be more potent than single homodimers, but it is not known whether combinations of homodimers would perform equally well. In this study, we compared BMP2, BMP4, BMP6, and BMP7, their combinations and heterodimers, for regeneration by human NP cells. The BMPs investigated induced variable matrix deposition by NP cells. BMP4 was the most potent, both in the final neotissue glysosaminoglycan content and incorporation efficiency. Heterodimers BMP2/6H and BMP2/7H were more potent than their respective homodimer combinations, but not the BMP4/7H heterodimer. The current results indicate that BMP4 might have a high potential for regeneration of the intervertebral disc. Moreover, the added value of BMP heterodimers over their respective homodimer BMP combinations depends on the BMP combination applied.
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Vidilaseris, Keni, Alexandros Kiriazis, Ainoleena Turku, Ayman Khattab, Niklas G. Johansson, Teppo O. Leino, Paula S. Kiuru, et al. "Asymmetry in catalysis by Thermotoga maritima membrane-bound pyrophosphatase demonstrated by a nonphosphorus allosteric inhibitor." Science Advances 5, no. 5 (May 2019): eaav7574. http://dx.doi.org/10.1126/sciadv.aav7574.
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Membrane-bound pyrophosphatases are homodimeric integral membrane proteins that hydrolyze pyrophosphate into orthophosphates, coupled to the active transport of protons or sodium ions across membranes. They are important in the life cycle of bacteria, archaea, plants, and parasitic protists, but no homologous proteins exist in vertebrates, making them a promising drug target. Here, we report the first nonphosphorus allosteric inhibitor of the thermophilic bacterium Thermotoga maritima membrane-bound pyrophosphatase and its bound structure together with the substrate analog imidodiphosphate. The unit cell contains two protein homodimers, each binding a single inhibitor dimer near the exit channel, creating a hydrophobic clamp that inhibits the movement of β-strand 1–2 during pumping, and thus prevents the hydrophobic gate from opening. This asymmetry of inhibitor binding with respect to each homodimer provides the first clear structural demonstration of asymmetry in the catalytic cycle of membrane-bound pyrophosphatases.
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Ivanov, Yuri D., Amir Taldaev, Andrey V. Lisitsa, Elena A. Ponomarenko, and Alexander I. Archakov. "Prediction of Monomeric and Dimeric Structures of CYP102A1 Using AlphaFold2 and AlphaFold Multimer and Assessment of Point Mutation Effect on the Efficiency of Intra- and Interprotein Electron Transfer." Molecules 27, no. 4 (February 18, 2022): 1386. http://dx.doi.org/10.3390/molecules27041386.
Full textAbstract:
The three-dimensional structure of monomers and homodimers of CYP102A1/WT (wild-type) proteins and their A83F and A83I mutant forms was predicted using the AlphaFold2 (AF2) and AlphaFold Multimer (AFMultimer) programs, which were compared with the rate constants of hydroxylation reactions of these enzyme forms to determine the efficiency of intra- and interprotein electron transport in the CYP102A1 hydroxylase system. The electron transfer rate constants (ket), which determine the rate of indole hydroxylation by the CYP102A1 system, were calculated based on the distances (R) between donor-acceptor prosthetic groups (PG) FAD→FMN→HEME of these proteins using factor β, which describes an exponential decay from R the speed of electron transport (ET) according to the tunnelling mechanism. It was shown that the structure of monomers in the homodimer, calculated using the AlpfaFold Multimer program, is in good agreement with the experimental structures of globular domains (HEME-, FMN-, and FAD-domains) in CYP102A1/WT obtained by X-ray structural analysis, and the structure of isolated monomers predicted in AF2 does not coincide with the structure of monomers in the homodimer, although a high level of similarity in individual domains remains. The structures of monomers and homodimers of A83F and A83I mutants were also calculated, and their structures were compared with the wild-type protein. Significant differences in the structure of all isolated monomers with respect to the structures of monomers in homodimers were also found for them, and at the same time, insignificant differences were revealed for all homodimers. Comparative analysis for CYP102A1/WT between the calculated intra- and interprotein distances FAD→FMN→HEME and the rate constants of hydroxylation in these proteins showed that the distance between prosthetic groups both in the monomer and in the dimer allows the implementation of electron transfer between PGs, which is consistent with experimental literature data about kcat. For the mutant form of monomer A83I, an increase in the distance between PGs was obtained, which can restrict electron transportation compared to WT; however, for the dimer of this protein, a decrease in the distance between PGs was observed compared to the WT form, which can lead to an increase in the electron transfer rate constant and, accordingly, kcat. For the monomer and homodimer of the A83F mutant, the calculations showed an increase in the distance between the PGs compared to the WT form, which should have led to a decrease in the electron transfer rate, but at the same time, for the homodimer, the approach of the aromatic group F262 with heme can speed up transportation for this form and, accordingly, the rate of hydroxylation.
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Ganchi, P. A., S. C. Sun, W. C. Greene, and D. W. Ballard. "A novel NF-kappa B complex containing p65 homodimers: implications for transcriptional control at the level of subunit dimerization." Molecular and Cellular Biology 13, no. 12 (December 1993): 7826–35. http://dx.doi.org/10.1128/mcb.13.12.7826-7835.1993.
Full textAbstract:
The predominant inducible form of the NF-kappa B transcription factor is a heteromeric complex containing two Rel-related DNA-binding subunits, termed p65 and p50. Prior transfection studies have shown that when these p65 and p50 subunits are expressed independently as stable homodimers, p65 stimulates kappa B-directed transcription, whereas p50 functions as a kappa B-specific repressor. While authentic p50 homodimers (previously termed KBF1) have been detected in nuclear extracts from nontransfected cells, experimental evidence supporting the existence of p65 homodimers in vivo was lacking. We now provide direct biochemical evidence for the presence of an endogenous pool of inducible p65 homodimers in intact human T cells. As with the prototypical NF-kappa B p50-p65 heterodimer, this novel p65 homodimeric form of NF-kappa B is functionally sequestered in the cytoplasm but rapidly appears in the nuclear compartment following cellular stimulation. Site-directed mutagenesis studies indicate that the homodimerization function of p65 is dependent upon the presence of cysteine 216 and a conserved recognition motif for protein kinase A (RRPS; amino acids 273 to 276), both of which reside within a 91-amino-acid segment of the Rel homology domain that mediates self-association. In contrast, mutations at these two sites do not affect heterodimerization of p65 with p50 or its functional interaction with I kappa B alpha. These later findings indicate that neither homo- nor heterodimer formation is an absolute prerequisite for I kappa B alpha recognition of p65. Taken together with prior in vivo transcription studies, these results suggest that the biological activities of p65 and p50 homodimers are independently regulated, thereby providing an integrated and flexible control mechanism for the rapid activation and repression of NF-kappa B/Rel-directed gene expression.
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Ganchi, P. A., S. C. Sun, W. C. Greene, and D. W. Ballard. "A novel NF-kappa B complex containing p65 homodimers: implications for transcriptional control at the level of subunit dimerization." Molecular and Cellular Biology 13, no. 12 (December 1993): 7826–35. http://dx.doi.org/10.1128/mcb.13.12.7826.
Full textAbstract:
The predominant inducible form of the NF-kappa B transcription factor is a heteromeric complex containing two Rel-related DNA-binding subunits, termed p65 and p50. Prior transfection studies have shown that when these p65 and p50 subunits are expressed independently as stable homodimers, p65 stimulates kappa B-directed transcription, whereas p50 functions as a kappa B-specific repressor. While authentic p50 homodimers (previously termed KBF1) have been detected in nuclear extracts from nontransfected cells, experimental evidence supporting the existence of p65 homodimers in vivo was lacking. We now provide direct biochemical evidence for the presence of an endogenous pool of inducible p65 homodimers in intact human T cells. As with the prototypical NF-kappa B p50-p65 heterodimer, this novel p65 homodimeric form of NF-kappa B is functionally sequestered in the cytoplasm but rapidly appears in the nuclear compartment following cellular stimulation. Site-directed mutagenesis studies indicate that the homodimerization function of p65 is dependent upon the presence of cysteine 216 and a conserved recognition motif for protein kinase A (RRPS; amino acids 273 to 276), both of which reside within a 91-amino-acid segment of the Rel homology domain that mediates self-association. In contrast, mutations at these two sites do not affect heterodimerization of p65 with p50 or its functional interaction with I kappa B alpha. These later findings indicate that neither homo- nor heterodimer formation is an absolute prerequisite for I kappa B alpha recognition of p65. Taken together with prior in vivo transcription studies, these results suggest that the biological activities of p65 and p50 homodimers are independently regulated, thereby providing an integrated and flexible control mechanism for the rapid activation and repression of NF-kappa B/Rel-directed gene expression.
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Gresock, Michael G., Kyle A. Kastead, and Kathleen Postle. "From Homodimer to Heterodimer and Back: Elucidating the TonB Energy Transduction Cycle." Journal of Bacteriology 197, no. 21 (August 17, 2015): 3433–45. http://dx.doi.org/10.1128/jb.00484-15.
Full textAbstract:
ABSTRACTThe TonB system actively transports large, scarce, and important nutrients through outer membrane (OM) transporters of Gram-negative bacteria using the proton gradient of the cytoplasmic membrane (CM). InEscherichia coli, the CM proteins ExbB and ExbD harness and transfer proton motive force energy to the CM protein TonB, which spans the periplasmic space and cyclically binds OM transporters. TonB has two activity domains: the amino-terminal transmembrane domain with residue H20 and the periplasmic carboxy terminus, through which it binds to OM transporters. TonB is inactivated by all substitutions at residue H20 except H20N. Here, we show that while TonB trapped as a homodimer through its amino-terminal domain retained full activity, trapping TonB through its carboxy terminus inactivated it by preventing conformational changes needed for interaction with OM transporters. Surprisingly, inactive TonB H20A had little effect on homodimerization through the amino terminus and instead decreased TonB carboxy-terminal homodimer formation prior to reinitiation of an energy transduction cycle. That result suggested that the TonB carboxy terminus ultimately interacts with OM transporters as a monomer. Our findings also suggested the existence of a separate equimolar pool of ExbD homodimers that are not in contact with TonB. A model is proposed where interaction of TonB homodimers with ExbD homodimers initiates the energy transduction cycle, and, ultimately, the ExbD carboxy terminus modulates interactions of a monomeric TonB carboxy terminus with OM transporters. After TonB exchanges its interaction with ExbD for interaction with a transporter, ExbD homodimers undergo a separate cycle needed to re-energize them.IMPORTANCECanonical mechanisms of active transport across cytoplasmic membranes employ ion gradients or hydrolysis of ATP for energy. Gram-negative bacterial outer membranes lack these resources. The TonB system embodies a novel means of active transport across the outer membrane for nutrients that are too large, too scarce, or too important for diffusion-limited transport. A proton gradient across the cytoplasmic membrane is converted by a multiprotein complex into mechanical energy that drives high-affinity active transport across the outer membrane. This system is also of interest since one of its uses in pathogenic bacteria is for competition with the host for the essential element iron. Understanding the mechanism of the TonB system will allow design of antibiotics targeting iron acquisition.
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Cheskis, B., and L. P. Freedman. "Ligand modulates the conversion of DNA-bound vitamin D3 receptor (VDR) homodimers into VDR-retinoid X receptor heterodimers." Molecular and Cellular Biology 14, no. 5 (May 1994): 3329–38. http://dx.doi.org/10.1128/mcb.14.5.3329-3338.1994.
Full textAbstract:
Protein dimerization facilitates cooperative, high-affinity interactions with DNA. Nuclear hormone receptors, for example, bind either as homodimers or as heterodimers with retinoid X receptors (RXR) to half-site repeats that are stabilized by protein-protein interactions mediated by residues within both the DNA- and ligand-binding domains. In vivo, ligand binding among the subfamily of steroid receptors unmasks the nuclear localization and DNA-binding domains from a complex with auxiliary factors such as the heat shock proteins. However, the role of ligand is less clear among nuclear receptors, since they are constitutively localized to the nucleus and are presumably associated with DNA in the absence of ligand. In this study, we have begun to explore the role of the ligand in vitamin D3 receptor (VDR) function by examining its effect on receptor homodimer and heterodimer formation. Our results demonstrate that VDR is a monomer in solution; VDR binding to a specific DNA element leads to the formation of a homodimeric complex through a monomeric intermediate. We find that 1,25-dihydroxyvitamin D3, the ligand for VDR, decreases the amount of the DNA-bound VDR homodimer complex. It does so by significantly decreasing the rate of conversion of DNA-bound monomer to homodimer and at the same time enhancing the dissociation of the dimeric complex. This effectively stabilizes the bound monomeric species, which in turn serves to favor the formation of a VDR-RXR heterodimer. The ligand for RXR, 9-cis retinoic acid, has the opposite effect of destabilizing the heterodimeric-DNA complex. These results may explain how a nuclear receptor can bind DNA constitutively but still act to regulate transcription in a fully hormone-dependent manner.
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Cheskis, B., and L. P. Freedman. "Ligand modulates the conversion of DNA-bound vitamin D3 receptor (VDR) homodimers into VDR-retinoid X receptor heterodimers." Molecular and Cellular Biology 14, no. 5 (May 1994): 3329–38. http://dx.doi.org/10.1128/mcb.14.5.3329.
Full textAbstract:
Protein dimerization facilitates cooperative, high-affinity interactions with DNA. Nuclear hormone receptors, for example, bind either as homodimers or as heterodimers with retinoid X receptors (RXR) to half-site repeats that are stabilized by protein-protein interactions mediated by residues within both the DNA- and ligand-binding domains. In vivo, ligand binding among the subfamily of steroid receptors unmasks the nuclear localization and DNA-binding domains from a complex with auxiliary factors such as the heat shock proteins. However, the role of ligand is less clear among nuclear receptors, since they are constitutively localized to the nucleus and are presumably associated with DNA in the absence of ligand. In this study, we have begun to explore the role of the ligand in vitamin D3 receptor (VDR) function by examining its effect on receptor homodimer and heterodimer formation. Our results demonstrate that VDR is a monomer in solution; VDR binding to a specific DNA element leads to the formation of a homodimeric complex through a monomeric intermediate. We find that 1,25-dihydroxyvitamin D3, the ligand for VDR, decreases the amount of the DNA-bound VDR homodimer complex. It does so by significantly decreasing the rate of conversion of DNA-bound monomer to homodimer and at the same time enhancing the dissociation of the dimeric complex. This effectively stabilizes the bound monomeric species, which in turn serves to favor the formation of a VDR-RXR heterodimer. The ligand for RXR, 9-cis retinoic acid, has the opposite effect of destabilizing the heterodimeric-DNA complex. These results may explain how a nuclear receptor can bind DNA constitutively but still act to regulate transcription in a fully hormone-dependent manner.
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Liu, Ying, Yue Zhang, Jing Jiang, Peter E. Lobie, Ramasamy Paulmurugan, John F. Langenheim, Wen Y. Chen, Kurt R. Zinn, and Stuart J. Frank. "GHR/PRLR Heteromultimer Is Composed of GHR Homodimers and PRLR Homodimers." Molecular Endocrinology 30, no. 5 (May 2016): 504–17. http://dx.doi.org/10.1210/me.2015-1319.
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Barkalow, Fern J., Kurt L. Barkalow, and Tanya N. Mayadas. "Dimerization of P-selectin in platelets and endothelial cells." Blood 96, no. 9 (November 1, 2000): 3070–77. http://dx.doi.org/10.1182/blood.v96.9.3070.
Full textAbstract:
Abstract P-selectin is a leukocyte adhesion receptor stored in platelets and endothelial cells and is translocated to the surface upon cell activation. Purified P-selectin is oligomeric and has increased avidity for its ligand relative to the monomeric form, but whether P-selectin self-associates in the membrane of intact cells is not known. A chemical cross-linking approach was used to show that P-selectin is present as noncovalent dimers in resting platelets, human umbilical vein endothelial cells, and heterologous RIN5F cells expressing P-selectin. The results of 2-dimensional isoelectric focusing are consistent in showing P-selectin dimers as homodimers, but they are composed of a more basic subset of P-selectin than the monomers. This suggests that the dimers are a biochemically distinct subset of P-selectin. P-selectin dimers form in the endoplasmic reticulum and Golgi compartments of human umbilical vein endothelial cells only after synthesis of the mature P-selectin subunit, and are not preferentially stored in Weibel-Palade bodies as compared with the monomeric form. Platelet activation with thrombin receptor–activating peptide leads to the presence of P-selectin monomers and homodimers on the cell surface as well as P-selectin heterodimers, which are composed of P-selectin and an unidentified protein of approximately 81 kd molecular weight. In summary, these studies demonstrate that P-selectin is homodimeric in situ and that platelet activation leads to the formation of an additional activation-specific heterodimeric species. In addition, the homodimer has unique biochemical characteristics compared with the monomeric form, and dimerization occurs in the endoplasmic reticulum and Golgi compartments of endothelial cells.
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Barkalow, Fern J., Kurt L. Barkalow, and Tanya N. Mayadas. "Dimerization of P-selectin in platelets and endothelial cells." Blood 96, no. 9 (November 1, 2000): 3070–77. http://dx.doi.org/10.1182/blood.v96.9.3070.h8003070_3070_3077.
Full textAbstract:
P-selectin is a leukocyte adhesion receptor stored in platelets and endothelial cells and is translocated to the surface upon cell activation. Purified P-selectin is oligomeric and has increased avidity for its ligand relative to the monomeric form, but whether P-selectin self-associates in the membrane of intact cells is not known. A chemical cross-linking approach was used to show that P-selectin is present as noncovalent dimers in resting platelets, human umbilical vein endothelial cells, and heterologous RIN5F cells expressing P-selectin. The results of 2-dimensional isoelectric focusing are consistent in showing P-selectin dimers as homodimers, but they are composed of a more basic subset of P-selectin than the monomers. This suggests that the dimers are a biochemically distinct subset of P-selectin. P-selectin dimers form in the endoplasmic reticulum and Golgi compartments of human umbilical vein endothelial cells only after synthesis of the mature P-selectin subunit, and are not preferentially stored in Weibel-Palade bodies as compared with the monomeric form. Platelet activation with thrombin receptor–activating peptide leads to the presence of P-selectin monomers and homodimers on the cell surface as well as P-selectin heterodimers, which are composed of P-selectin and an unidentified protein of approximately 81 kd molecular weight. In summary, these studies demonstrate that P-selectin is homodimeric in situ and that platelet activation leads to the formation of an additional activation-specific heterodimeric species. In addition, the homodimer has unique biochemical characteristics compared with the monomeric form, and dimerization occurs in the endoplasmic reticulum and Golgi compartments of endothelial cells.
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Nakayama, K., H. Shimizu, K. Mitomo, T. Watanabe, S. Okamoto, and K. Yamamoto. "A lymphoid cell-specific nuclear factor containing c-Rel-like proteins preferentially interacts with interleukin-6 kappa B-related motifs whose activities are repressed in lymphoid cells." Molecular and Cellular Biology 12, no. 4 (April 1992): 1736–46. http://dx.doi.org/10.1128/mcb.12.4.1736-1746.1992.
Full textAbstract:
The proto-oncoprotein c-Rel is a member of the nuclear factor kappa B transcription factor family, which includes the p50 and p65 subunits of nuclear factor kappa B. We show here that c-Rel binds to kappa B sites as homodimers as well as heterodimers with p50. These homodimers and heterodimers show distinct DNA-binding specificities and affinities for various kappa B motifs. In particular, the c-Rel homodimer has a high affinity for interleukin-6 (IL-6) and beta interferon kappa B sites. In spite of its association with p50 in vitro, however, we found a lymphoid cell-specific nuclear factor in vivo that contains c-Rel but not p50 epitopes; this factor, termed IL-6 kappa B binding factor II, appears to contain the c-Rel homodimer and preferentially recognizes several IL-6 kappa B-related kappa B motifs. Although it has been previously shown that the IL-6 kappa B motif functions as a potent IL-1/tumor necrosis factor-responsive element in nonlymphoid cells, its activity was found to be repressed in lymphoid cells such as a Jurkat T-cell line. We also present evidence that IL-6 kappa B binding factor II functions as a repressor specific for IL-6 kappa B-related kappa B motifs in lymphoid cells.
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Nakayama, K., H. Shimizu, K. Mitomo, T. Watanabe, S. Okamoto, and K. Yamamoto. "A lymphoid cell-specific nuclear factor containing c-Rel-like proteins preferentially interacts with interleukin-6 kappa B-related motifs whose activities are repressed in lymphoid cells." Molecular and Cellular Biology 12, no. 4 (April 1992): 1736–46. http://dx.doi.org/10.1128/mcb.12.4.1736.
Full textAbstract:
The proto-oncoprotein c-Rel is a member of the nuclear factor kappa B transcription factor family, which includes the p50 and p65 subunits of nuclear factor kappa B. We show here that c-Rel binds to kappa B sites as homodimers as well as heterodimers with p50. These homodimers and heterodimers show distinct DNA-binding specificities and affinities for various kappa B motifs. In particular, the c-Rel homodimer has a high affinity for interleukin-6 (IL-6) and beta interferon kappa B sites. In spite of its association with p50 in vitro, however, we found a lymphoid cell-specific nuclear factor in vivo that contains c-Rel but not p50 epitopes; this factor, termed IL-6 kappa B binding factor II, appears to contain the c-Rel homodimer and preferentially recognizes several IL-6 kappa B-related kappa B motifs. Although it has been previously shown that the IL-6 kappa B motif functions as a potent IL-1/tumor necrosis factor-responsive element in nonlymphoid cells, its activity was found to be repressed in lymphoid cells such as a Jurkat T-cell line. We also present evidence that IL-6 kappa B binding factor II functions as a repressor specific for IL-6 kappa B-related kappa B motifs in lymphoid cells.
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Sun, J., and P. B. Kavathas. "Comparison of the roles of CD8 alpha alpha and CD8 alpha beta in interaction with MHC class I." Journal of Immunology 159, no. 12 (December 15, 1997): 6077–82. http://dx.doi.org/10.4049/jimmunol.159.12.6077.
Full textAbstract:
Abstract The CD8 molecule is expressed either as an alpha/alpha homodimer or an alpha/beta heterodimer on thymocytes and cytotoxic T cells, and functions as a coreceptor in concert with TCR for binding the MHC class I/peptide complex. Although CD8alpha/beta heterodimers have been shown to be more effective coreceptors, the precise role of the beta-chain in TCR-mediated thymic maturation and T cell activation is not understood. To understand the role of CD8beta in mediating CD8/MHC class I interaction, we examined whether cell surface CD8alpha/beta heterodimer promotes better cell-cell adhesion with MHC class I than the CD8alpha/alpha homodimer. The abilities of different forms of CD8 to adhere to MHC class I were measured with a cell-cell binding assay. Using a wild-type CD8beta and -alpha, we found that CD8alphabeta heterodimers did not mediate greater cell-cell adhesion than CD8alphaalpha homodimers. Furthermore, we found that chimeric CD8beta-alpha homodimers afforded no detectable binding. These results do not support the idea that CD8alphabeta binding to MHC class I is greater than that of CD8alphaalpha. Rather, they point to an alternative explanation in which CD8beta may play an role in promoting CD8/TCR interaction and/or in signaling/regulatory pathways.
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SINHA, Dipali, Mariola MARCINKIEWICZ, David GAILANI, and Peter N. WALSH. "Molecular cloning and biochemical characterization of rabbit factor XI." Biochemical Journal 367, no. 1 (October 1, 2002): 49–56. http://dx.doi.org/10.1042/bj20020232.
Full textAbstract:
Human factor XI, a plasma glycoprotein required for normal haemostasis, is a homodimer (160kDa) formed by a single interchain disulphide bond linking the Cys-321 of each Apple 4 domain. Bovine, porcine and murine factor XI are also disulphide-linked homodimers. Rabbit factor XI, however, is an 80kDa polypeptide on non-reducing SDS/PAGE, suggesting that rabbit factor XI exists and functions physiologically either as a monomer, as does prekallikrein, a structural homologue to factor XI, or as a non-covalent homodimer. We have investigated the structure and function of rabbit factor XI to gain insight into the relation between homodimeric structure and factor XI function. Characterization of the cDNA sequence of rabbit factor XI and its amino acid translation revealed that in the rabbit protein a His residue replaces the Cys-321 that forms the interchain disulphide linkage in human factor XI, explaining why rabbit factor XI is a monomer in non-reducing SDS/PAGE. On size-exclusion chromatography, however, purified plasma rabbit factor XI, like the human protein and unlike prekallikrein, eluted as a dimer, demonstrating that rabbit factor XI circulates as a non-covalent dimer. In functional assays rabbit factor XI and human factor XI behaved similarly. Both monomeric and dimeric factor XI were detected in extracts of cells expressing rabbit factor XI. We conclude that the failure of rabbit factor XI to form a covalent homodimer due to the replacement of Cys-321 with His does not impair its functional activity because it exists in plasma as a non-covalent homodimer and homodimerization is an intracellular process.
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Takeshita, Akira, Yasunori Ozawa, and William W. Chin. "Nuclear Receptor Coactivators Facilitate Vitamin D Receptor Homodimer Action on Direct Repeat Hormone Response Elements." Endocrinology 141, no. 3 (March 1, 2000): 1281–84. http://dx.doi.org/10.1210/endo.141.3.7441.
Full textAbstract:
Abstract Vitamin D receptor (VDR) is a ligand-dependent transcription factor that regulates target gene expression. Although VDR forms stable heterodimer complex with retinoid X receptors (RXRs) on vitamin D-response elements (VDREs), it is still not clear whether VDR/RXR heterodimers are the only VDR complexes responsible for vitamin D-mediated gene transcription. In this report, we analyzed the effect of nuclear receptor coactivators (SRC-1 and TRAM-1) on VDR homodimer and VDR/RXR heterodimer formation by electrophoretic mobility shift assay. We found that VDR forms stable homodimers after interaction with the coactivators on a VDRE (DR+3). Of particular note, DR+4 and DR+5 hormone-response elements (HREs) may also support such interactions. Cotransfection experiments revealed further that the coactivators enhance ligand-induced VDR transcription on these elements. Our studies suggest the important role of VDR homodimers, in addition to VDR/RXR heterodimers, in vitamin D-induced transactivation. Thus, specific coactivator-VDR interactions on HREs may determine target gene transactivation.
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Magyar, Csaba, Anikó Mentes, Erzsébet Fichó, Miklós Cserző, and István Simon. "Physical Background of the Disordered Nature of “Mutual Synergetic Folding” Proteins." International Journal of Molecular Sciences 19, no. 11 (October 26, 2018): 3340. http://dx.doi.org/10.3390/ijms19113340.
Full textAbstract:
Intrinsically disordered proteins (IDPs) lack a well-defined 3D structure. Their disordered nature enables them to interact with several other proteins and to fulfil their vital biological roles, in most cases after coupled folding and binding. In this paper, we analyze IDPs involved in a new mechanism, mutual synergistic folding (MSF). These proteins define a new subset of IDPs. Recently we collected information on these complexes and created the Mutual Folding Induced by Binding (MFIB) database. These protein complexes exhibit considerable structural variation, and almost half of them are homodimers, but there is a significant amount of heterodimers and various kinds of oligomers. In order to understand the basic background of the disordered character of the monomers found in MSF complexes, the simplest part of the MFIB database, the homodimers are analyzed here. We conclude that MFIB homodimeric proteins have a larger solvent-accessible main-chain surface area on the contact surface of the subunits, when compared to globular homodimeric proteins. The main driving force of the dimerization is the mutual shielding of the water-accessible backbones and the formation of extra intermolecular interactions.
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Heinzel, F. P., A. M. Hujer, F. N. Ahmed, and R. M. Rerko. "In vivo production and function of IL-12 p40 homodimers." Journal of Immunology 158, no. 9 (May 1, 1997): 4381–88. http://dx.doi.org/10.4049/jimmunol.158.9.4381.
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Abstract The bioactivity of IL-12 is mediated by heterodimers of disulfide-linked p35 and p40 protein subunits. Homodimeric p40 competes with heterodimer for binding to the high affinity IL-12R and inhibits IL-12 bioactivity in vitro. However, the production and significance of p40 homodimer as a cytokine antagonist in vivo have not been determined. In these studies, we observed increased amounts of both IL-12 p40 monomer and homodimer in the serum of C57BL/6 mice following injection of 300 microg of Salmonella enteritidis LPS. Homodimer constituted between 20 and 40% of the total circulating p40 in endotoxemic sera, as confirmed by both Sephacryl S-100 gel filtration and p40-specific immunoprecipitation analyses. Similar relative amounts of homodimer and monomer were observed in endotoxemic BALB/c, C57BL/6, IFN-gamma-deficient C57BL/6 mice and C57BL/6 mice previously infected with bacille Calmette-Guérin. To determine whether IL-12 p40 homodimer was capable of antagonizing IL-12-dependent IFN-gamma responses in vivo, we pretreated C57BL/6 mice with purified rIL-12 p40 homodimer before i.p. challenge with endotoxin. Mice treated with 40 to 80 microg of p40 homodimer generated 80 to 82% less circulating IFN-gamma during acute endotoxemia than saline controls (p < 0.01). We conclude that p40 homodimer is produced in vivo, functions as a cytokine antagonist in the context of the mouse model of acute endotoxemia, and may represent a novel form of self-regulating cytokine response.
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Watowich, S. S., D. J. Hilton, and H. F. Lodish. "Activation and inhibition of erythropoietin receptor function: role of receptor dimerization." Molecular and Cellular Biology 14, no. 6 (June 1994): 3535–49. http://dx.doi.org/10.1128/mcb.14.6.3535-3549.1994.
Full textAbstract:
Members of the cytokine receptor superfamily have structurally similar extracellular ligand-binding domains yet diverse cytoplasmic regions lacking any obvious catalytic domains. Many of these receptors form ligand-induced oligomers which are likely to participate in transmembrane signaling. A constitutively active (factor-independent) mutant of the erythropoietin receptor (EPO-R), R129C in the exoplasmic domain, forms disulfide-linked homodimers, suggesting that the wild-type EPO-R is activated by ligand-induced homodimerization. Here, we have taken two approaches to probe the role EPO-R dimerization plays in signal transduction. First, on the basis of the crystal structure of the ligand-bound, homodimeric growth hormone receptor (GH-R) and sequence alignment between the GH-R and EPO-R, we identified residues of the EPO-R which may be involved in intersubunit contacts in an EPO-R homodimer. Residue 129 of the EPO-R corresponds to a residue localized to the GH-R dimer interface region. Alanine or cysteine substitutions were introduced at four other residues of the EPO-R predicted to be in the dimer interface region. Substitution of residue E-132 or E-133 with cysteine renders the EPO-R constitutively active. Like the arginine-to-cysteine mutation at position 129 in the exoplasmic domain (R129C), E132C and E133C form disulfide-linked homodimers, suggesting that constitutive activity is due to covalent dimerization. In the second approach, we have coexpressed the wild-type EPO-R with inactive mutants of the receptor missing all or part of the cytosolic domain. These truncated receptors have a dominant inhibitory effect on the proliferative action of the wild-type receptor. Taken together, these results strengthen the hypothesis that an initial step in EPO- and EPO-R-mediated signal transduction is ligand-induced receptor dimerization.
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Watowich, S. S., D. J. Hilton, and H. F. Lodish. "Activation and inhibition of erythropoietin receptor function: role of receptor dimerization." Molecular and Cellular Biology 14, no. 6 (June 1994): 3535–49. http://dx.doi.org/10.1128/mcb.14.6.3535.
Full textAbstract:
Members of the cytokine receptor superfamily have structurally similar extracellular ligand-binding domains yet diverse cytoplasmic regions lacking any obvious catalytic domains. Many of these receptors form ligand-induced oligomers which are likely to participate in transmembrane signaling. A constitutively active (factor-independent) mutant of the erythropoietin receptor (EPO-R), R129C in the exoplasmic domain, forms disulfide-linked homodimers, suggesting that the wild-type EPO-R is activated by ligand-induced homodimerization. Here, we have taken two approaches to probe the role EPO-R dimerization plays in signal transduction. First, on the basis of the crystal structure of the ligand-bound, homodimeric growth hormone receptor (GH-R) and sequence alignment between the GH-R and EPO-R, we identified residues of the EPO-R which may be involved in intersubunit contacts in an EPO-R homodimer. Residue 129 of the EPO-R corresponds to a residue localized to the GH-R dimer interface region. Alanine or cysteine substitutions were introduced at four other residues of the EPO-R predicted to be in the dimer interface region. Substitution of residue E-132 or E-133 with cysteine renders the EPO-R constitutively active. Like the arginine-to-cysteine mutation at position 129 in the exoplasmic domain (R129C), E132C and E133C form disulfide-linked homodimers, suggesting that constitutive activity is due to covalent dimerization. In the second approach, we have coexpressed the wild-type EPO-R with inactive mutants of the receptor missing all or part of the cytosolic domain. These truncated receptors have a dominant inhibitory effect on the proliferative action of the wild-type receptor. Taken together, these results strengthen the hypothesis that an initial step in EPO- and EPO-R-mediated signal transduction is ligand-induced receptor dimerization.
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Caillat, Christophe, Alexander Fish, Dafni-Eleftheria Pefani, Stavros Taraviras, Zoi Lygerou, and Anastassis Perrakis. "The structure of the GemC1 coiled coil and its interaction with the Geminin family of coiled-coil proteins." Acta Crystallographica Section D Biological Crystallography 71, no. 11 (October 31, 2015): 2278–86. http://dx.doi.org/10.1107/s1399004715016892.
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GemC1, together with Idas and Geminin, an important regulator of DNA-replication licensing and differentiation decisions, constitute a superfamily sharing a homologous central coiled-coil domain. To better understand this family of proteins, the crystal structure of a GemC1 coiled-coil domain variant engineered for better solubility was determined to 2.2 Å resolution. GemC1 shows a less typical coiled coil compared with the Geminin homodimer and the Geminin–Idas heterodimer structures. It is also shown that both in vitro and in cells GemC1 interacts with Geminin through its coiled-coil domain, forming a heterodimer that is more stable that the GemC1 homodimer. Comparative analysis of the thermal stability of all of the possible superfamily complexes, using circular dichroism to follow the unfolding of the entire helix of the coiled coil, or intrinsic tryptophan fluorescence of a unique conserved N-terminal tryptophan, shows that the unfolding of the coiled coil is likely to take place from the C-terminus towards the N-terminus. It is also shown that homodimers show a single-state unfolding, while heterodimers show a two-state unfolding, suggesting that the dimer first falls apart and the helices then unfold according to the stability of each protein. The findings argue that Geminin-family members form homodimers and heterodimers between them, and this ability is likely to be important for modulating their function in cycling and differentiating cells.
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Kristensen, Kristian K., Katrine Zinck Leth-Espensen, Haydyn D. T. Mertens, Gabriel Birrane, Muthuraman Meiyappan, Gunilla Olivecrona, Thomas J. D. Jørgensen, Stephen G. Young, and Michael Ploug. "Unfolding of monomeric lipoprotein lipase by ANGPTL4: Insight into the regulation of plasma triglyceride metabolism." Proceedings of the National Academy of Sciences 117, no. 8 (February 7, 2020): 4337–46. http://dx.doi.org/10.1073/pnas.1920202117.
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The binding of lipoprotein lipase (LPL) to GPIHBP1 focuses the intravascular hydrolysis of triglyceride-rich lipoproteins on the surface of capillary endothelial cells. This process provides essential lipid nutrients for vital tissues (e.g., heart, skeletal muscle, and adipose tissue). Deficiencies in either LPL or GPIHBP1 impair triglyceride hydrolysis, resulting in severe hypertriglyceridemia. The activity of LPL in tissues is regulated by angiopoietin-like proteins 3, 4, and 8 (ANGPTL). Dogma has held that these ANGPTLs inactivate LPL by converting LPL homodimers into monomers, rendering them highly susceptible to spontaneous unfolding and loss of enzymatic activity. Here, we show that binding of an LPL-specific monoclonal antibody (5D2) to the tryptophan-rich lipid-binding loop in the carboxyl terminus of LPL prevents homodimer formation and forces LPL into a monomeric state. Of note, 5D2-bound LPL monomers are as stable as LPL homodimers (i.e., they are not more prone to unfolding), but they remain highly susceptible to ANGPTL4-catalyzed unfolding and inactivation. Binding of GPIHBP1 to LPL alone or to 5D2-bound LPL counteracts ANGPTL4-mediated unfolding of LPL. In conclusion, ANGPTL4-mediated inactivation of LPL, accomplished by catalyzing the unfolding of LPL, does not require the conversion of LPL homodimers into monomers. Thus, our findings necessitate changes to long-standing dogma on mechanisms for LPL inactivation by ANGPTL proteins. At the same time, our findings align well with insights into LPL function from the recent crystal structure of the LPL•GPIHBP1 complex.
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Marui, Nobuyuki, Russell M. Medford, and Mushtaq Ahmad. "Activation of RelA homodimers by tumour necrosis factor α: a possible transcriptional activator in human vascular endothelial cells." Biochemical Journal 390, no. 1 (August 9, 2005): 317–24. http://dx.doi.org/10.1042/bj20041659.
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In vascular endothelial cells, cytokines induce genes that are expressed in inflammatory lesions partly through the activation of transcription factor NF-κB (nuclear factor-κB). Among the members of the NF-κB/rel protein family, homodimers of the RelA subunit of NF-κB can also function as strong transactivators when expressed in cells. However, the functional role of endogenous RelA homodimers has not been clearly elucidated. We investigated whether RelA homodimers are induced in cytokine-treated vascular endothelial cells. Gel mobility-shift and supershift assays revealed that a cytokine TNFα (tumour necrosis factor α) activated both NF-κB1/RelA heterodimers and RelA homodimers that bound to a canonical κB sequence, IgκB (immunoglobulin κB), in SV40 (simian virus 40) immortalized HMEC-1 (human dermal microvascular endothelial cell line 1). In HMEC-1 and HUVEC (human umbilical-vein endothelial cells), TNFα also induced RelA homodimers that bound to the sequence 65-2κB, which specifically binds to RelA homodimers but not to NF-κB1/RelA heterodimers in vitro. Deoxycholic acid, a detergent that can dissociate the NF-κB–IκB complex (where IκB stands for inhibitory κB), induced the binding of the RelA homodimers to 65-2κB from the cytosolic fraction of resting HMEC-1. Furthermore, TNFα induced the transcriptional activity of a reporter gene that was driven by 65-2κB in HMEC-1. These results suggest that in addition to NF-κB1/RelA heterodimers, TNFα also induces RelA homodimers that are functionally active. Thus RelA homodimers may actively participate in cytokine regulation of gene expression in human vascular endothelial cells.
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Allen, Rachel L., Chris A. O’Callaghan, Andrew J. McMichael, and Paul Bowness. "Cutting Edge: HLA-B27 Can Form a Novel β2-Microglobulin-Free Heavy Chain Homodimer Structure." Journal of Immunology 162, no. 9 (May 1, 1999): 5045–48. http://dx.doi.org/10.4049/jimmunol.162.9.5045.
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Abstract HLA-B27 has a striking association with inflammatory arthritis. We show that free HLA-B27 heavy chains can form a disulfide-bonded homodimer, dependent on residue Cys67 in their extracellular α1 domain. Despite the absence of β2-microglobulin, HLA-B27 heavy chain homodimers (termed HC-B27) were stabilized by a known peptide epitope. HC-B27 complexes were recognized by the conformation-specific Ab W6/32, but not the ME1 Ab. Surface labeling and immunoprecipitation demonstrated the presence of similar W6/32-reactive free heavy chains at the surface of HLA-B27-transfected T2 cells. HC-B27 homodimer formation might explain the ability of HLA-B27 to induce spondyloarthropathy in β2-microglobulin-deficient mice.
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Muthuswamy, Senthil K., Michael Gilman, and Joan S. Brugge. "Controlled Dimerization of ErbB Receptors Provides Evidence for Differential Signaling by Homo- and Heterodimers." Molecular and Cellular Biology 19, no. 10 (October 1, 1999): 6845–57. http://dx.doi.org/10.1128/mcb.19.10.6845.
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ABSTRACT The four members of the ErbB family of receptor tyrosine kinases are involved in a complex array of combinatorial interactions involving homo- and heterodimers. Since most cell types express more than one member of the ErbB family, it is difficult to distinguish the biological activities of different homo- and heterodimers. Here we describe a method for inducing homo- or heterodimerization of ErbB receptors by using synthetic ligands without interference from the endogenous receptors. ErbB receptor chimeras containing synthetic ligand binding domains (FK506-binding protein [FKBP] or FKBP-rapamycin-binding domain [FRB]) were homodimerized with the bivalent FKBP ligand AP1510 and heterodimerized with the bifunctional FKBP-FRB ligand rapamycin. AP1510 treatment induced tyrosine phosphorylation of ErbB1 and ErbB2 homodimers and recruitment of Src homology 2 domain-containing proteins (Shc and Grb2). In addition, ErbB1 and ErbB2 homodimers activated downstream signaling pathways leading to Erk2 and Akt phosphorylation. However, only ErbB1 homodimers were internalized upon AP1510 stimulation, and only ErbB1 homodimers were able to associate with and induce phosphorylation of c-Cbl. Cells expressing AP1510-induced ErbB1 homodimers were able to associate with and induce phosphorylation of c-Cbl. Cells expressing AP1510-induced ErbB1 homodimers were able to form foci; however, cells expressing ErbB2 homodimers displayed a five- to sevenfold higher focus-forming ability. Using rapamycin-inducible heterodimerization we show that c-Cbl is unable to associate with ErbB1 in a ErbB1-ErbB2 heterodimer most likely because ErbB2 is unable to phosphorylate the c-Cbl binding site on ErbB1. Thus, we demonstrate that ErbB1 and ErbB2 homodimers differ in their abilities to transform fibroblasts and provide evidence for differential signaling by ErbB homodimers and heterodimers. These observations also validate the use of synthetic ligands to study the signaling and biological specificity of selected ErbB dimers in any cell type.
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Inukai, T., T. Inaba, T. Yoshihara, and A. T. Look. "Cell transformation mediated by homodimeric E2A-HLF transcription factors." Molecular and Cellular Biology 17, no. 3 (March 1997): 1417–24. http://dx.doi.org/10.1128/mcb.17.3.1417.
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The E2A-HLF fusion gene, created by the t(17;19)(q22;p13) chromosomal translocation in pro-B lymphocytes, encodes an oncogenic protein in which the E2A trans-activation domain is linked to the DNA-binding and protein dimerization domain of hepatic leukemia factor (HLF), a member of the proline- and acidic amino acid-rich (PAR) subfamily of bZIP transcription factors. This fusion product binds to its DNA recognition site not only as a homodimer but also as a heterodimer with HLF and two other members of the PAR bZIP subfamily, thyrotroph embryonic factor (TEF) and albumin promoter D-box binding protein (DBP). Thus, E2A-HLF could transform cells by direct regulation of downstream target genes, acting through homodimeric or heterodimeric complexes, or by sequestering normal PAR proteins into nonfunctional heterocomplexes (dominant-negative interference). To distinguish among these models, we constructed mutant E2A-HLF proteins in which the leucine zipper domain of HLF was extended by one helical turn or altered in critical charged amino acids, enabling the chimera to bind to DNA as a homodimer but not as a heterodimer with HLF or other PAR proteins. When introduced into NIH 3T3 cells in a zinc-inducible vector, each of these mutants induced anchorage-independent growth as efficiently as unaltered E2A-HLF, indicating that the chimeric oncoprotein can transform cells in its homodimeric form. Transformation also depended on an intact E2A activator region, providing further support for a gain-of-function contribution to oncogenesis rather than one based on a dominant-interfering or dominant-negative mechanism. Thus, the tumorigenic effects of E2A-HLF and its mutant forms in NIH 3T3 cells favor a straightforward model in which E2A-HLF homodimers bind directly to promoter/enhancer elements of downstream target genes and alter their patterns of expression in early B-cell progenitors.
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Ghetie, Maria-Ana, Helen Bright, and Ellen S. Vitetta. "Homodimers but not monomers of Rituxan (chimeric anti-CD20) induce apoptosis in human B-lymphoma cells and synergize with a chemotherapeutic agent and an immunotoxin." Blood 97, no. 5 (March 1, 2001): 1392–98. http://dx.doi.org/10.1182/blood.v97.5.1392.
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In 1997, a chimeric anti-CD20 monoclonal antibody (mAb) (Rituxan) was approved for the treatment of low-grade/follicular B-cell lymphoma. Rituxan has a long half-life and low immunogenicity, and it mediates effector function. Rituxan induces apoptosis in some tumor cell lines in vitro. Previous studies with mAbs that react with neoplastic B cells have demonstrated that homodimers of immunoglobulin G ([IgG]2) often inhibit cell growth more effectively than their monomeric (IgG)1counterparts. In this study, the ability of IgG or F(ab′)2 homodimers vs monomers of Rituxan were compared for their ability to inhibit the growth of several different B-lymphoma cell lines in vitro. It was found that homodimers of Rituxan had superior antigrowth activity in vitro and that F(ab′)2 homodimers were the most active. Homodimers, but not monomers, of Rituxan induced both apoptosis and necrosis of several B-cell lymphoma lines in vitro; the inhibition of cell growth was not dependent upon the presence of Fc receptors or upon 10-fold or greater differences in the density of CD20 on the target cells. Rituxan homodimers, compared with monomers, also rendered drug-resistant CD20+ B-lymphoma cells more sensitive to chemotherapeutic agents and synergized with an anti-CD22 immunotoxin in vitro.
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Staudt, Ryan P., and Thomas E. Smithgall. "Nef homodimers down-regulate SERINC5 by AP-2–mediated endocytosis to promote HIV-1 infectivity." Journal of Biological Chemistry 295, no. 46 (September 1, 2020): 15540–52. http://dx.doi.org/10.1074/jbc.ra120.014668.
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SERINC5 is a multipass intrinsic membrane protein that suppresses HIV-1 infectivity when incorporated into budding virions. The HIV-1 Nef virulence factor prevents viral incorporation of SERINC5 by triggering its down-regulation from the producer cell membrane through an AP-2–dependent endolysosomal pathway. However, the mechanistic basis for SERINC5 down-regulation by Nef remains elusive. Here we demonstrate that Nef homodimers are important for SERINC5 down-regulation, trafficking to late endosomes, and exclusion from newly synthesized viral particles. Based on previous X-ray crystal structures, we mutated three conserved residues in the Nef dimer interface (Leu112, Tyr115, and Phe121) and demonstrated attenuated homodimer formation in a cell-based fluorescence complementation assay. Point mutations at each position reduced the infectivity of HIV-1 produced from transfected 293T cells, the Jurkat TAg T-cell line, and donor mononuclear cells in a SERINC5-dependent manner. In SERINC5-transfected 293T cells, virion incorporation of SERINC5 was increased by dimerization-defective Nef mutants, whereas down-regulation of SERINC5 from the membrane of transfected Jurkat cells by these mutants was significantly reduced. Nef dimer interface mutants also failed to trigger internalization of SERINC5 and localization to Rab7+ late endosomes in T cells. Importantly, fluorescence complementation assays demonstrated that dimerization-defective Nef mutants retained interaction with both SERINC5 and AP-2. These results show that down-regulation of SERINC5 and subsequent enhancement of viral infectivity require Nef homodimers and support a mechanism by which the Nef dimer bridges SERINC5 to AP-2 for endocytosis. Pharmacological disruption of Nef homodimers may control HIV-1 infectivity and viral spread by enhancing virion incorporation of SERINC5.
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Wang, Jun, Marek W. Radomski, Carlos Medina, and John F. Gilmer. "MMP inhibition by barbiturate homodimers." Bioorganic & Medicinal Chemistry Letters 23, no. 2 (January 2013): 444–47. http://dx.doi.org/10.1016/j.bmcl.2012.11.063.
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Kubo, Ken-ichiro, Katsuhiko Mikoshiba, and Kazunori Nakajima. "Secreted Reelin molecules form homodimers." Neuroscience Research 43, no. 4 (August 2002): 381–88. http://dx.doi.org/10.1016/s0168-0102(02)00068-8.
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Bardsley, Ben, Younghoon R. Cho, Martin S. Westwell, and Dudley H. Williams. "Induction of asymmetry into homodimers." Chirality 10, no. 12 (1998): 14–23. http://dx.doi.org/10.1002/(sici)1520-636x(1998)10:1/2<14::aid-chir4>3.3.co;2-v.
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Bardsley, Ben, Younghoon R. Cho, Martin S. Westwell, and Dudley H. Williams. "Induction of asymmetry into homodimers." Chirality 10, no. 1-2 (1998): 14–23. http://dx.doi.org/10.1002/chir.4.
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Kundrotas, Petras J., Ilya A. Vakser, and Joël Janin. "Structural templates for modeling homodimers." Protein Science 22, no. 11 (September 20, 2013): 1655–63. http://dx.doi.org/10.1002/pro.2361.
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