Retinoid signalling and gene expression in neuroblastoma cells: RXR agonist and antagonist effects on CRABP-II and RARbeta expression

9-cis Retinoic acid (RA) induces gene expression in neuroblastoma cells more effectively and with different kinetics than other RA isomers, and could be acting in part through Retinoid X Receptors (RXRs). The aim of this study was to characterise the effects of an RXR agonist and RXR homodimer antagonist on the induction of cellular RA binding protein II (CRABP-II) and RA receptor-beta (RARbeta) in neuroblastoma cells in response to different retinoids. The RXR agonist, LDG1069, was as effective as all-trans RA in inducing gene expression, but less effective than 9-cis RA. The RXR-homodimer antagonist, LG100754, inhibited the induction of CRABP-II mRNA in SH-SY5Y neuroblastoma cells by 9-cis RA or the RXR-specific agonist LGD1069, but had no effect when used with all-trans RA. Conversely, LG100754 did not inhibit induction of RARbeta mRNA by 9-cis or all-trans RA, or by LGD1069. RAR- and RXR-specific ligands used together induced CRABP-II and RARbeta as effectively as 9-cis RA. These results demonstrate the value of combining RXR- and RAR-specific ligands to regulate RA-inducible gene expression. The possibility that RXR-homodimers mediate, in part, the induction of CRABP-II by 9-cis RA and RXR-specific ligands is discussed.     

c-Abl蛋白酪氨酸激酶形成同源二聚体

c-Abl是非受体酪氨酸激酶,它在细胞内被一些基因毒性的、氧化的及其它形式的压力所激活。目前研究证明：应用标记的c-Abl发现其在细胞内可以相互形成同源二聚体,并且一分子c-Abl的N末端区域与相应的另一分子的C末端相互作用形成二聚体。实验进一步表明： cAbl SH3 结构域结合到另一c-Abl 分子富含脯氨酸的C-末端约958-982氨基酸区域。如果去除c-Abl 富含脯氨酸的结构域,就会阻止二聚体的形成。这些结果首先证实了c-Abl在细胞内可以相互形成同源二聚体,并暗示着二聚体的形成可能影响着c-Abl活性的调节。     

Discriminating physiological from non-physiological interfaces in structures of protein complexes: A community-wide study

Reliably scoring and ranking candidate models of protein complexes and assigning their oligomeric state from the structure of the crystal lattice represent outstanding challenges. A community-wide effort was launched to tackle these challenges. The latest resources on protein complexes and interfaces were exploited to derive a benchmark dataset consisting of 1677 homodimer protein crystal structures, including a balanced mix of physiological and non-physiological complexes. The non-physiological complexes in the benchmark were selected to bury a similar or larger interface area than their physiological counterparts, making it more difficult for scoring functions to differentiate between them. Next, 252 functions for scoring protein-protein interfaces previously developed by 13 groups were collected and evaluated for their ability to discriminate between physiological and non-physiological complexes. A simple consensus score generated using the best performing score of each of the 13 groups, and a cross-validated Random Forest (RF) classifier were created. Both approaches showed excellent performance, with an area under the Receiver Operating Characteristic (ROC) curve of 0.93 and 0.94, respectively, outperforming individual scores developed by different groups. Additionally, AlphaFold2 engines recalled the physiological dimers with significantly higher accuracy than the non-physiological set, lending support to the reliability of our benchmark dataset annotations. Optimizing the combined power of interface scoring functions and evaluating it on challenging benchmark datasets appears to be a promising strategy.     

Analysis of the quaternary structure of the MutL C-terminal domain

The dimeric DNA mismatch repair protein MutL has a key function in communicating mismatch recognition by MutS to downstream repair processes. Dimerization of MutL is mediated by the C-terminal domain, while activity of the protein is modulated by the ATP-dependent dimerization of the highly conserved N-terminal domain. Recently, a crystal structure analysis of the Escherichia coli MutL C-terminal dimerization domain has been reported and a model for the biological dimer was proposed. In this model, dimerization is mediated by the internal (In) subdomain comprising residues 475-569. Here, we report a computational analysis of all protein interfaces observed in the crystal structure and suggest that the biological dimer interface is formed by a hydrophobic surface patch of the external (Ex) subdomain (residues 432-474 and 570-615). Moreover, sequence analysis revealed that this surface patch is conserved among the MutL proteins. To test this hypothesis, single and double-cysteine variants of MutL were generated and tested for their ability to be cross-linked with chemical cross-linkers of various size. Finally, deletion of the C-terminal residues 605-615 abolished homodimerization. The biochemical data are fully compatible with a revised model for the biological dimer, which has important implications for understanding the heterodimerization of eukaryotic MutL homologues, modeling the MutL holoenzyme and predicting protein-protein interaction sites.     

Retinoid X receptors and retinoid response in neuroblastoma cells

Retinoic acid (RA) modulates differentiation and apoptosis of neural cells via RA receptors (RARs) and retinoid X receptors (RXRs). Neuroblastoma cells are potentially useful models for elucidating the molecular mechanisms of RA in neural cells, and responses to different isomers of RA have been interpreted in terms of differential homo- and heterodimerization of RXRs. The aim of this study was to identify the RXR types expressed in neuroblast and substrate-adherent neuroblastoma cells, and to study the participation of these RXRs in RAR heterodimers. RXRbeta was the predominant RXR type in N-type SH SY 5Y cells and S-type SH EP cells. Gel shift and supershift assays demonstrated that RARbeta and RARgamma predominantly heterodimerize with RXRbeta. In SH SY 5Y cells, RARgamma/RXRbeta was the predominant heterodimer binding to the DR5 RARE in the absence of 9-cis RA (9C), whereas the balance shifted in favor of RARbeta/RXRbeta in the presence of ligand. There was a marked difference between the N- and S-type neuroblastoma cells in retinoid receptor-DNA interactions, and this may underlie the differential effects of retinoids in these neuroblastoma cell types. There was no evidence to indicate that 9C functions via RXR homodimers in either SH SY 5Y or SH EP neuroblastoma cells. The results of this study suggest that interactions between retinoid receptors and other nuclear proteins may be critical determinants of retinoid responses in neural cells.     

Neutron Reflectometry and Molecular Simulations Demonstrate HIV-1 Nef Homodimer Formation on Model Lipid Bilayers

The HIV-1 Nef protein plays a critical role in viral infectivity, high-titer replication in vivo, and immune escape of HIV-infected cells. Nef lacks intrinsic biochemical activity, functioning instead through interactions with diverse host cell signaling proteins and intracellular trafficking pathways. Previous studies have established an essential role for Nef homodimer formation at the plasma membrane for most if not all its functions. Here we combined neutron reflectometry of full-length myristoylated Nef bound to model lipid bilayers with molecular simulations based on previous X-ray crystal structures of Nef homodimers. This integrated approach provides direct evidence that Nef associates with the membrane as a homodimer with its structured core region displaced from the membrane for partner protein engagement. Parallel studies of a dimerization-defective mutant, Nef-L112D, demonstrate that the helical dimerization interface present in previous crystal structures stabilizes the membrane-bound dimer. X-ray crystallography of the Nef-L112D mutant in complex with the SH3 domain of the Nef-associated host cell kinase Hck revealed a monomeric 1:1 complex instead of the 2:2 dimer complex formed with wild-type Nef. Importantly, the crystal structure of the Nef-L112D core and SH3 interface are virtually identical to the wild-type complex, indicating that this mutation does not affect the overall Nef fold. These findings support the intrinsic capacity of Nef to homodimerize at lipid bilayers using structural features present in X-ray crystal structures of dimeric complexes.     

Allophycocyanin: trimers,monomers, subunits,and homodimers

Allophycocyanin is a photosynthetic light-harvesting pigment-protein complex located in the phycobilisomes of cyanobacteria and red algae. Using dynamic light scattering and circular dichroism, solutions of purified allophycocyanin were shown to consist of homogeneous trimers (alpha3beta3) with a nonspherical shape over a very wide range of protein concentrations at pH 6.0 and 20 degrees C. Deconvolutions of the visible circular dichroism spectrum of the trimer were carried out for the first determination of the individual spectra of all six-component chromophores. The chromophores were shown to be in different microenvironments that helped determine the spectrum of the trimer. Monomers (alpha beta) that were formed in either the presence of 0.50M NaSCN or at 45 degrees C were shown to be completely reversible to trimers. However, subunits (alpha and beta) that were formed in either the presence of 8M urea or at 60 degrees C, using spectroscopy and gel-filtration column chromatography, were observed to only partially reconstitute trimers. Homodimers (alpha2 and/or beta2) formed during the regeneration of trimers. The homodimer, which was detected for the first time when both subunits were present, was shown to be in equilibrium with its subunits. Unlike the trimer situation, subunits were found to fully reconstitute monomers in the presence of 0.50M NaSCN. These results suggest a route to trimer assembly from subunits with monomers serving as intermediaries and the homodimers forming in a nonproductive step that did not interfere with the overall assembly scheme.     

HNF4α Combinatorial Isoform Heterodimers Activate Distinct Gene Targets that Differ from Their Corresponding Homodimers

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Evolutionary and structural analyses of heterodimeric proteins composed of subunits with same fold

Heterodimeric proteins with homologous subunits of same fold are involved in various biological processes. The objective of this study is to understand the evolution of structural and functional features of such heterodimers. Using a non‐redundant dataset of 70 such heterodimers of known 3D structure and an independent dataset of 173 heterodimers from yeast, we note that the mean sequence identity between interacting homologous subunits is only 23–24% suggesting that, generally, highly diverged paralogues assemble to form such a heterodimer. We also note that the functional roles of interacting subunits/domains are generally quite different. This suggests that, though the interacting subunits/domains are homologous, the high evolutionary divergence characterize their high functional divergence which contributes to a gross function for the heterodimer considered as a whole. The inverse relationship between sequence identity and RMSD of interacting homologues in heterodimers is not followed. We also addressed the question of formation of homodimers of the subunits of heterodimers by generating models of fictitious homodimers on the basis of the 3D structures of the heterodimers. Interaction energies associated with these homodimers suggests that, in overwhelming majority of the cases, such homodimers are unlikely to be stable. Majority of the homologues of heterodimers of known structures form heterodimers (51.8%) and a small proportion (14.6%) form homodimers. Comparison of 3D structures of heterodimers with homologous homodimers suggests that interfacial nature of residues is not well conserved. In over 90% of the cases we note that the interacting subunits of heterodimers are co‐localized in the cell. Proteins 2015; 83:1766–1786. © 2015 Wiley Periodicals, Inc.