Receptor Assembly Research Articles

Gatekeepers of the brain: Identifying hidden mechanisms of type A GABA receptor signaling and assembly.

Identifying hidden mechanisms of type A GABA receptor signaling and assembly.

PH-Dependent Assembly and Stability of Toll-Like Receptor 3/dsRNA Signaling Complex: Insights from Constant pH Molecular Dynamics and Metadynamics Simulations.

The pH-dependent assembly of Toll-like receptors (TLRs), which triggers a threshold-like response, is a key principle in immune signaling. While crystallography has revealed the intricate structure of these assembly complexes, the mechanisms underlying their pH dependency remain unclear. Herein, constant pH simulations and metadynamics are employed to investigate the pH-dependent assembly and stability of the TLR3/dsRNA signaling complex. The findings demonstrate that system pH regulates complex assembly and stability by modulating the protonation and charge states of histidines. Histidines in TLR3 act as pH-dependent, positively charged binding sites that capture negatively charged dsRNA. Additionally, these histidines form a [H682⁺]-[E626⁻] dipole, facilitating the assembly of two TLR3 molecules into an antisymmetric dimer through dipole-dipole interactions. Surprisingly, TLR3 can shift the pKa values of key histidines from their model pKa of 6.5, increasing protonation likelihood and enhancing ligand binding. Notably, the aromatic residue Phe84, located within the dsRNA binding site [His39⁺-His60⁺-Phe84-His108⁺], alters the pKa of His60 through cation-π interactions with its protonated state. This study offers new insights into the molecular mechanisms underlying pH-dependent immune signaling via higher-order assemblies and suggests potential applications for histidine in self-assembling biomaterials.

Quantitative Single-Molecule Analysis of Ryanodine Receptor 2 Subunit Assembly in Cardiac and Neuronal Tissues.

We developed a method for ex vivo receptor encapsulation and single-molecule imaging techniques from neuronal and cardiac tissues, illustrating the method's broad applicability for measuring membrane receptor assembly. Ryanodine receptor 2 (RyR2) is a tetrameric Ca2+ channel governing intracellular Ca2+ dynamics, which is critical for muscle contraction. Employing GFP-RyR2 knock-in mice, we isolated individual receptor proteins in tissue-specific nanovesicles and performed subunit counting analyses to yield quantitative assessment of stoichiometric distributions across different organs. With this method, we explored the potential heterogeneity of brain-derived RyR2, which has been reported to form heteromeric assemblies with other ryanodine receptor isoforms.

TMX1, a disulfide oxidoreductase, is necessary for T cell function through regulation of CD3ζ.

T cell-targeted therapies are commonly used to manage T cell hyperactivity in autoimmune disorders, graft-versus-host diseases (GVHD), and transplant rejections. However, many patients experience significant side effects or inadequate responses to current treatments, highlighting the urgent need for alternative strategies. In this study, we searched for regulators of T cells through proximity labeling with APEX2 to detect proteins interacting with CD8α, a coreceptor of the T-cell receptor (TCR). This screen revealed TMX1, an ER resident transmembrane disulfide oxidoreductase, is essential for T cell cytotoxicity and NFAT, NFκB, and AP1 signaling but not cell proliferation. TMX1 deletion decreases surface TCR expression and destabilizes CD3ζ, a subunit of TCR complex; however, overexpression of CD3ζ rescues the phenotype, suggesting that TMX1 is not required for CD3ζ function. Mechanistically, TMX1 was found to directly engage the CxxC motif of CD3δ, which has been reported to be essential for proper TCR assembly and function. We hypothesize that the loss of TMX1 interaction with CD3δ leads to impaired TCR assembly and subsequent CD3ζ destabilization. These findings identify TMX1 as a novel regulator of T-cell receptor assembly and a potential target for immunosuppressive therapy.

A versatile approach to quality control of protein-based receptor layers by reversible, nonspecific staining for multiplex SPRi immunosensing

SPRi protein arrays for rapid, label-free immunodetection offer an attractive approach for high throughput biosensing and analysis of molecular interactions. However, their routine use for quantitative analysis requires adequate reproducibility and homogeneity of receptor assemblies manufactured ex-situ using microdispensing techniques. There is also an emerging need to develop versatile and non-destructive methods for the determination of protein surface density for quality control of the immobilization process. Here, we report on a simple, one-step method of surface proteins visualization by their reversible, non-specific staining with anionic dyes: Ponceau S (PS), Amido Black (AB) or Coomassie Brilliant Blue G (CBB-G). Using rabbit IgG antibody and transferrin as model receptors, we demonstrate the possibility of protein surface density determination in a broad range up to ∼ 3 ng∙mm−2 (for a 2D monolayer) and up to at least 12.5 ng∙mm−2 (for a 3D hydrogel-coated substrate). The ranges of dynamic response for the developed methods cover the typical surface densities required for biosensing and studies of the binding kinetics. It was demonstrated that each of the investigated dyes manifests advantages in specified applications. While CBB-G shows the highest sensitivity of SPRi response, AB requires the gentlest labeling conditions, and PS shows the highest association rate and ease of washing out, even from protein layers of high density. The feasibility of the developed method for the quality control of SPRi microarray was confirmed. The advantages of the new, dye-based, non-specific labeling method over immunolabeling in terms of simplicity of implementation and versatility have also been highlighted.

Structural insights into the high-affinity IgE receptor FcεRI complex.

Immunoglobulin E (IgE) plays a pivotal role in allergic responses1,2. The high-affinity IgE receptor, FcεRI, found on mast cells and basophils, is central to the effector functions of IgE. FcεRI is a tetrameric complex, comprising FcεRIα, FcεRIβ and a homodimer of FcRγ (originally known asFcεRIγ), with FcεRIα recognizing the Fc region of IgE (Fcε) and FcεRIβ-FcRγ facilitating signal transduction3. Additionally, FcRγ is a crucial component of other immunoglobulin receptors, including those for IgG (FcγRI and FcγRIIIA) and IgA (FcαRI)4-8. However, the molecular basis of FcεRI assembly and the structure of FcRγ have remained elusive. Here we elucidate the cryogenic electron microscopy structure of the Fcε-FcεRI complex. FcεRIα has an essential rolein the receptor's assembly, interacting with FcεRIβ and both FcRγ subunits. FcεRIβ is structured as a compact four-helix bundle, similar to the B cell antigen CD20. The FcRγ dimer exhibits an asymmetric architecture, and coils with the transmembrane region of FcεRIα to form a three-helix bundle. A cholesterol-like molecule enhances the interaction between FcεRIβ and the FcεRIα-FcRγ complex. Our mutagenesis analyses further indicate similarities between the interaction of FcRγ with FcεRIα and FcγRIIIA, but differences in that with FcαRI. These findings deepen our understanding of the signalling mechanisms of FcεRI and offer insights into the functionality of other immune receptors dependent on FcRγ.

Dynamics and Activation of Membrane-Bound B Cell Receptor Assembly.

B-cell receptor complexes (BCR) are expressed on the surface of a B-cell and are the critical regulators of adaptive immune response. Even though the relevance of antibodies has been known for almost a hundred years, the antigen-dependent activation of antibody-producing B-cells has remained elusive. Several models have been proposed for BCR activation, including cross-linking, conformation-induced oligomerization, and dissociation activation models. Recently, the first cryo-EM structure of the human B-cell antigen receptor of the IgM isotype was published. Given the new asymmetric BCR complex, we have carried out extensive molecular dynamics simulations to probe the conformational changes upon antigen binding and the influence of the membrane. We identified two critical dynamical events that could be associated with antigen-dependent activation of BCR. First, antigen binding caused increased flexibility in regions distal to the antigen binding site. Second, this increased flexibility led to the rearrangement of helices in transmembrane helices, including the relative interaction of Igα/Igβ, which has been responsible for intracellular signaling. Further, these transmembrane rearrangements led to changes in localized lipid composition. Even though the simulations considered only a single BCR complex, our work indirectly supports the dissociation activation model.

Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum.

Protein homeostasis (proteostasis) deficiency is an important contributing factor to neurological and metabolic diseases. However, how the proteostasis network orchestrates the folding and assembly of multi-subunit membrane proteins is poorly understood. Previous proteomics studies identified Hsp47 (Gene: SERPINH1), a heat shock protein in the endoplasmic reticulum lumen, as the most enriched interacting chaperone for gamma-aminobutyric acid type A (GABAA) receptors. Here, we show that Hsp47 enhances the functional surface expression of GABAA receptors in rat neurons and human HEK293T cells. Furthermore, molecular mechanism study demonstrates that Hsp47 acts after BiP (Gene: HSPA5) and preferentially binds the folded conformation of GABAA receptors without inducing the unfolded protein response in HEK293T cells. Therefore, Hsp47 promotes the subunit-subunit interaction, the receptor assembly process, and the anterograde trafficking of GABAA receptors. Overexpressing Hsp47 is sufficient to correct the surface expression and function of epilepsy-associated GABAA receptor variants in HEK293T cells. Hsp47 also promotes the surface trafficking of other Cys-loop receptors, including nicotinic acetylcholine receptors and serotonin type 3 receptors in HEK293T cells. Therefore, in addition to its known function as a collagen chaperone, this work establishes that Hsp47 plays a critical and general role in the maturation of multi-subunit Cys-loop neuroreceptors.

Engineering Mesoscale T Cell Receptor Clustering by Plug-and-Play Nanotools.

Tcell receptor (TCR) clustering and formation of an immune synapse are crucial for TCR signaling. However, limited information is available about these dynamic assemblies and their connection to transmembrane signaling. Here, we controlled TCR clustering via plug-and-play nanotools based on an engineered irreversible conjugation pair and a peptide-loaded major histocompatibility complex (pMHC) molecule to compare receptor assembly in a ligand (pMHC)-induced or ligand-independent manner. A streptavidin-binding peptide displayed in both tools enabled their anchoring in streptavidin-pre-structured matrices. Strikingly, pMHC-induced clustering in the confined regions exhibited higher density and dynamics than the ligand-free approach, indicating that the size and architecture of the pMHC ligand influences TCR assembly. Our approach enables the control of membrane receptor clustering with high specificity and provide the possibility to explore different modalities of receptor activation. This article is protected by copyright. All rights reserved.

Cholesterol inhibits assembly and activation of the EphA2 receptor.

The receptor tyrosine kinase EphA2 drives cancer malignancy by facilitating metastasis. EphA2 can be found in different self-assembly states: as a monomer, dimer, and oligomer. However, our understanding remains limited regarding which EphA2 state is responsible for driving pro-metastatic signaling. To address this limitation, we have developed SiMPull-POP, a single-molecule method for accurate quantification of membrane protein self-assembly. Our experiments revealed that a reduction of plasma membrane cholesterol strongly promoted EphA2 self-assembly. Indeed, low cholesterol caused a similar effect to the EphA2 ligand ephrinA1-Fc. These results indicate that cholesterol inhibits EphA2 assembly. Phosphorylation studies in different cell lines revealed that low cholesterol increased phospho-serine levels, the signature of oncogenic signaling. Investigation of the mechanism that cholesterol uses to inhibit the assembly and activity of EphA2 indicate an in-trans effect, where EphA2 is phosphorylated by protein kinase A downstream of beta-adrenergic receptor activity, which cholesterol also inhibits. Our study not only provides new mechanistic insights on EphA2 oncogenic function, but also suggests that cholesterol acts as a molecular safeguard mechanism that prevents uncontrolled self-assembly and activation of EphA2.

Complexation-driven assembly of imine-linked helical receptors showing adaptive folding and temperature-dependent guest selection

The development of synthetic receptors capable of selectively binding guests with diverse structures and multiple functional groups poses a significant challenge. Here, we present the efficient assembly of foldamer-based receptors for monosaccharides, utilising the principles of complexation-induced equilibrium shifting and adaptive folding. Diimine 4 can be quantitatively assembled from smaller components when d-galactose is added as a guest among monosaccharides we examined. During this assembly, dual complexation-induced equilibrium shifts toward both the formation of diimine 4 and the conversion of d-galactose into α-d-galactofuranose are observed. Diimine 6 is quantitatively assembled in the presence of two different guests, methyl β-d-glucopyranoside and methyl β-d-galactopyranoside, resulting in the formation of two dimeric complexes: (6-MP)2⊃(methyl β-d-glucopyranoside)2 and (6-MM)2⊃(methyl β-d-galactopyranoside∙2H2O)2, respectively. These two complexes exhibit distinct folding structures with domain-swapping cavities depending on the bound guest and temperature. Interestingly, (6-MM)2⊃(methyl β-d-galactopyranoside∙2H2O)2 is exclusively formed at lower temperatures, while (6-MP)2⊃(methyl β-d-glucopyranoside)2 is only formed at higher temperatures.

Dclre1c-Mutation-Induced Immunocompromised Mice Are a Novel Model for Human Xenograft Research.

Severe combined immunodeficient (SCID) mice serve as a critical model for human xenotransplantation studies, yet they often suffer from low engraftment rates and susceptibility to graft-versus-host disease (GVHD). Moreover, certain SCID strains demonstrate 'immune leakage', underscoring the need for novel model development. Here, we introduce an SCID mouse model with a targeted disruption of the dclre1c gene, encoding Artemis, which is essential for V(D)J recombination and DNA repair during T cell receptor (TCR) and B cell receptor (BCR) assembly. Artemis deficiency precipitates a profound immunodeficiency syndrome, marked by radiosensitivity and compromised T and B lymphocyte functionality. Utilizing CRISPR/Cas9-mediated gene editing, we generated dclre1c-deficient mice with an NOD genetic background. These mice exhibited a radiosensitive SCID phenotype, with pronounced DNA damage and defective thymic, splenic and lymph node development, culminating in reduced T and B lymphocyte populations. Notably, both cell lines and patient-derived tumor xenografts were successfully engrafted into these mice. Furthermore, the human immune system was effectively rebuilt following peripheral blood mononuclear cells (PBMCs) transplantation. The dclre1c-knockout NOD mice described herein represent a promising addition to the armamentarium of models for xenotransplantation, offering a valuable platform for advancing human immunobiological research.

Dynamics of B cell receptor assembly in a complex membrane revealed by multiscale simulations

Dynamics of B cell receptor assembly in a complex membrane revealed by multiscale simulations

Structures of complete extracellular receptor assemblies mediated by IL-12 and IL-23.

Cell-surface receptor complexes mediated by pro-inflammatory interleukin (IL)-12 and IL-23, both validated therapeutic targets, are incompletely understood due to the lack of structural insights into their complete extracellular assemblies. Furthermore, there is a paucity of structural details describing the IL-12-receptor interaction interfaces, in contrast to IL-23-receptor complexes. Here we report structures of fully assembled mouse IL-12/human IL-23-receptor complexes comprising the complete extracellular segments of the cognate receptors determined by electron cryo-microscopy. The structures reveal key commonalities but also surprisingly diverse features. Most notably, whereas IL-12 and IL-23 both utilize a conspicuously presented aromatic residue on their α-subunit as a hotspot to interact with the N-terminal Ig domain of their high-affinity receptors, only IL-12 juxtaposes receptor domains proximal to the cell membrane. Collectively, our findings will help to complete our understanding of cytokine-mediated assemblies of tall cytokine receptors and will enable a cytokine-specific interrogation of IL-12/IL-23 signaling in physiology and disease.

Quantitative Super-Resolution Imaging of ER-Phagy Initiation in Cells.

Selective autophagy of the endoplasmic reticulum (ER-phagy) is a mechanism that is necessary for degrading damaged ER components and preventing cells from experiencing ER stress. Various ER-phagy receptors orchestrate this process by building protein assemblies with dedicated functions. In order to understand the molecular building principles of ER-phagy, it is important to reveal the assembly of ER-phagy receptors in a temporal and functional context. However, direct visualization is hampered by the diffraction limit in light microscopy. Super-resolution microscopy (SRM) can bypass this limitation and resolve single proteins and nanoscale protein clusters in cells. In particular, DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful technology that can resolve individual protein clusters in cells and provide information on their molecular composition. Here, we report a step-by-step protocol on how to utilize DNA-PAINT to perform super-resolution imaging of ER-phagy receptors in fixed cells. In addition, we provide a detailed explanation of image generation, cluster analysis, and molecular quantification.

Estimation of plasma soluble interleukin-2 receptor alpha chain level in adults with acute myeloid leukemia

Abstract: BACKGROUND: Acute myeloid leukemia (AML) is a clonal malignant condition of immature hematopoietic cells, characterized by clonal proliferation of abnormal cells (blasts) in the marrow leading to impairment of the normal blood cell production giving rise to failure of the bone marrow. Soluble interleukin-2 (IL-2) receptor alpha chain is a protein that is involved in the assembly of the high-affinity IL-2 receptor, and it has a critical role in controlling immune system homeostasis. The overexpression of sIL-2RA was investigated in many hematopoietic malignancies, and it was correlated with poor outcome. OJECTIVES: The aim of this study was to assess the sIL-2RA level as a prognostic factor and assess its impact on survival and if it can be used as a targeted treatment for a better outcome. MATERIALS AND METHODS: Sixty newly diagnosed adults with AML before starting therapy were included in the study, and they were followed up for 6 months to document survival status. Thirty healthy adults were taken as a control group. Using an enzyme-linked immunosorbent assay, the plasma sIL-2RA level was measured. Statistical analysis was done using Microsoft Excel 2019 and version 26 SPSS statistical software. P <0.05 was considered statistically significant. RESULTS: A considerable difference in the plasma sIL-2RA level between AML patients and controls also was more elevated in patients who died after 6-month follow-up. According to the blast percentage, total white blood cell count, and M0-M2 subgroups, the sIL-2RA level correlated positively. Irrelevant association was found regarding the patients’ age, the count of platelet, and the hemoglobin. CONCLUSIONS: Plasma sIL-2RA level is higher in AML patients than the control group at the time of diagnosis. Patients with a high level of plasma sIL-2RA have an inferior (overall survival) and poor outcome. SIL-2RA level is higher in M0-M2 subgroups than other subtypes. There is a significant association between sIL-2RA level and the absolute count of leukemic blasts.

The MA Helix Is Important for Receptor Assembly and Function in the α4β2 nACh Receptor.

Pentameric ligand-gated ion channels (pLGICs) are expressed throughout the central and peripheral nervous systems of vertebrates and modulate many aspects of human health and disease. Recent structural and computational data indicate that cation-selective pLGICs contain a long helical extension (MA) of one of the transmembrane helices. The MA helix has been shown to affect both the membrane expression of, and ion conductance levels through, these pLGICs. Here we probe the functional effects of 68 mutations in the MA region of the α4β2 nicotinic acetylcholine receptor (nAChR), using a voltage-sensitive membrane dye and radioligand binding to measure receptor function and expression/assembly. We found seven alanine mutations in a stretch of the MA helix that prevent correct receptor folding and/or assembly, as evidenced by the lack of both function and ligand binding. A further two alanine mutations resulted in receptors that were capable of binding ligand but showed no functional response, and we propose that, in these mutants, ligand binding is insufficient to trigger channel opening. The data clarify the effect of the MA helix, and as the effects of some of our mutations in the α4β2 nAChR differ from the effects of equivalent mutations in other cation-selective pLGICs, we suggest that residues in the MA helix may play subtly different roles in different receptors.

Retraction Note: Progesterone receptor assembly of a transcriptional complex along with activator protein 1, signal transducer and activator of transcription 3 and ErbB-2 governs breast cancer growth and predicts response to endocrine therapy

Retraction Note: Progesterone receptor assembly of a transcriptional complex along with activator protein 1, signal transducer and activator of transcription 3 and ErbB-2 governs breast cancer growth and predicts response to endocrine therapy

Cryo-EM structures reveal native GABAA receptor assemblies and pharmacology.

Type A γ-aminobutyric acid receptors (GABAARs) are the principal inhibitory receptors in the brain and the target of a wide range of clinical agents, including anaesthetics, sedatives, hypnotics and antidepressants1-3. However, our understanding of GABAAR pharmacology has been hindered by the vast number of pentameric assemblies that can be derived from 19 different subunits4 and the lack of structural knowledge of clinically relevant receptors. Here, we isolate native murine GABAAR assemblies containing the widely expressed α1 subunit and elucidate their structures in complex with drugs used to treat insomnia (zolpidem(ZOL) and flurazepam) and postpartum depression (the neurosteroid allopregnanolone(APG)). Using cryo-electron microscopy (cryo-EM)analysis and single-molecule photobleaching experiments, we uncover three major structural populations in the brain: the canonical α1β2γ2 receptor containing two α1 subunits, and two assemblies containing one α1 and either an α2 or α3 subunit, in which the single α1-containing receptors feature a more compact arrangement between the transmembrane and extracellular domains. Interestingly, APG is bound at the transmembrane α/β subunit interface, even when not added to the sample, revealing an important role for endogenous neurosteroids in modulating native GABAARs. Together with structurally engaged lipids, neurosteroids produce global conformational changes throughout the receptor that modify the ion channel pore and the binding sites for GABA and insomnia medications. Our data reveal the major α1-containing GABAAR assemblies, bound with endogenous neurosteroid, thus defining a structural landscape from which subtype-specific drugs can be developed.

Lymphocyte pathway analysis using naturally lymphocyte-deficient fish.

Comparative phylogenetic analyses are of potential value to establish the essential components of genetic networks underlying physiological traits. For species that naturally lack particular lymphocyte lineages, we show here that this strategy readily distinguishes trait-specific actors from pleiotropic components of the genetic network governing lymphocyte differentiation. Previously, three of the four members of the DNA polymerase X family have been implicated in the junctional diversification process during the somatic assembly of antigen receptors. Our phylogenetic analysis indicates that the presence of terminal deoxynucleotidyl transferase is strictly associated with the facility of V(D)J recombination, whereas PolL and PolM genes are retained even in species lacking Rag-mediated somatic diversification of antigen receptor genes.