N-terminal Extracellular Domain Research Articles

Inflammatory risk contributes to post-COVID endothelial dysfunction through anti-ACKR1 autoantibody.

Subclinical vascular impairment can be exacerbated in individuals who experience sustained inflammation after COVID-19 infection. Our study explores the prevalence and impact of autoantibodies on vascular dysfunction in healthy COVID-19 survivors, an area that remains inadequately investigated. Focusing on autoantibodies against the atypical chemokine receptor 1 (ACKR1), COVID-19 survivors demonstrated significantly elevated anti-ACKR1 autoantibodies, correlating with systemic cytokines, circulating damaged endothelial cells, and endothelial dysfunction. An independent cohort linked these autoantibodies to increased vascular disease outcomes during a median 6.7-yr follow-up. We analyzed a single-cell transcriptome atlas of endothelial cells from diverse mouse tissues, identifying enriched Ackr1 expressions in venous regions of the brain and soleus muscle vasculatures, which holds intriguing implications for tissue-specific venous thromboembolism manifestations reported in COVID-19. Functionally, purified immunoglobulin G (IgG) extracted from patient plasma did not trigger cell apoptosis or increase barrier permeability in human vein endothelial cells. Instead, plasma IgG enhanced antibody-dependent cellular cytotoxicity mediated by patient PBMCs, a phenomenon alleviated by blocking peptide or liposome ACKR1 recombinant protein. The blocking peptide uncovered that purified IgG from COVID-19 survivors possessed potential epitopes in the N-terminal extracellular domain of ACKR1, which effectively averted antibody-dependent cellular cytotoxicity. Our findings offer insights into therapeutic development to mitigate autoantibody reactivity in blood vessels in chronic inflammation.

Recombinant expression and tryptophan-assisted analysis of human sweet taste receptor T1R3’s extracellular domain in sweetener interaction studies

The human palate can discern multiple tastes; however, it predominantly perceives five fundamental flavors: sweetness, saltiness, sourness, bitterness, and umami. Sweetness is primarily mediated through the sweet taste receptor, a membrane-bound heterodimeric structure comprising T1R2-T1R3. However, unraveling the structural and mechanistic intricacies of the sweet taste receptor has proven challenging. This study aimed to address this knowledge gap by expressing an extracellular N-terminal domain encompassing the cysteine-rich domain of human hT1R3 (hT1R3-TMD) in Escherichia coli. The expressed protein was obtained as inclusion bodies, purified by metal affinity chromatography, and refolded using the dilution-refolding method. Through rigorous analysis, we confirmed the successful refolding of hT1R3-TMD and elucidated its structural characteristics using circular dichroism spectroscopy. Notably, the refolded protein was found to exist as either a monomer or a dimer, depending on its concentration. A tryptophan fluorescence quenching assay revealed that the dissociation constants for sucrose, sucralose, and brazzein were >9500 μM, 2380 μM and 14.3 μM, respectively. Our findings highlight the utility of this E. coli expression system for producing functional hT1R3-TMD for investigations and demonstrate the efficacy of the tryptophan fluorescence quenching assay in revealing complex interactions between sweet taste receptors and various sweeteners.

Abstract 5888: Novel immune-therapeutic target for lung cancer uncovered by SpliceIO™, an innovative platform that identifies disease-specific alternative splicing

Abstract Alternative splicing (AS) plays a critical role in generating tumor-specific neoantigens targetable through an arsenal of immunotherapeutic modalities. Previous studies have demonstrated tumor-promoting activity of deregulated AS isoforms and mutated splicing factors, both of which increase the yield of neoantigen-encoding AS isoforms. Therefore, the identification of AS isoforms has the potential to produce novel therapeutic targets, especially in lung tumors where splicing deregulation is frequent. Envisagenics has developed SpliceIO, a proprietary software platform for AS-derived neoantigen discovery. SpliceIO uses RNA-seq data to uncover extracellular neoantigens encoded by transmembrane proteins. This predictive ensemble maximizes the value of RNA-seq data to predict fundamental aspects of neoantigen activity, such as protein localization, epitope accessibility, and cancer pathway relevance. Here, we summarize the discovery and validation of novel AS-derived neoantigens identified using SpliceIO in non-small cell lung cancer (NSCLC). Our novel SpliceIO-identified neoantigens in NSCLC were validated following our well-established experimental pipeline. We have confirmed RNA expression of the neoantigen-expressing transcripts in lung cancer cell lines and patient samples using a combination of RT-PCR as well as RNA-ISH. Further, we have validated the membrane-bound extracellular localization of neoantigen-containing isoforms at the protein level using Western blotting, GFP and c-Myc tagged isoform generation, eukaryotic cells transfection, and microscopy. Our top target showed high tumor specificity, heterogeneous distribution in tumor samples, high prevalence, confirmed novel extracellular N-terminal domain, and strong biological relevance in lung cancer. This demonstrates the potential of utilizing RNA splicing for identifying many more membrane associated proteins for tumor specific immunotherapeutic targeting. The combination of computational and experimental approaches to neoantigen identification using SpliceIO allows for novel target discovery amenable for targeted immunotherapeutic development for lung cancer treatment and other indications driven by deregulated AS. Citation Format: Miguel A. Manzanares, Kendall Anderson, Hasan Zumrut, Sakshi Gera, Nayab Habib, Nicole Williams, Shaleigh Stanton, Taylor Floyd, Sanjana Shah, Devon Trahan, Martin Akerman, Gayatri Arun. Novel immune-therapeutic target for lung cancer uncovered by SpliceIO™, an innovative platform that identifies disease-specific alternative splicing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5888.

Abstract 5894: Novel splicing-derived neoantigen in triple negative breast cancer uncovered by SpliceIO™ a robust platform for the discovery and validation of disease-specific immunotherapies

Abstract Splicing dysregulation is a major hallmark of cancer, affecting tumor progression, metastasis, and therapy resistance. Aberrant alternative splicing (AS) plays a critical role in generating tumor-specific neoantigens targetable through an arsenal of immunotherapeutic modalities. Such neoantigens cannot be discovered using conventional DNA-based tools; they require specialized software for AS analysis using RNA-seq data. SpliceIO is Envisagenics’ AI platform for AS-derived neoantigen discovery. SpliceIO uses a “predictive ensemble” approach to uncover neoantigens that are both stably expressed and located on the extracellular membrane of cancer cells. Using SpliceIO, we have analyzed more than 10,000 RNA-seq samples from 5 patient cohorts covering 5 breast cancer subtypes as well as 2,647 normal RNA-seq samples from GTEx. Our analysis has identified tumor-specific neoantigens present in AS transmembrane proteins specific for Triple Negative Breast Cancer. SpliceIO target candidates were validated experimentally using our well-established pipeline in which isoform specific transcripts are tested in cancer cell lines by PCR and primary tumor tissues by qPCR to confirm RNA expression. Tumor heterogeneity and prevalence is confirmed by RNA in-situ hybridization in Tissue Microarrays. Extracellular localization of the novel epitope is confirmed by clone generation with GFP and c-Myc tagging followed by microscopy, Western blotting, and proteomic analysis. Our top validated target presents high TNBC tumor specificity and heterogeneous distribution, and contains a novel extracellular N-terminal domain. These results strongly support the role of aberrant AS as a major source of tumor-specific neoantigens. Our combined computational and experimental approach promotes novel target discovery, enabling the development of novel immunotherapeutic for TNBC patients who are in critical need of better treatment options. Citation Format: Nicole Williams, Miguel A. Manzanares, Kendall Anderson, Hasan Zunrut, Sakshi Gera, Nayab Habib, Shaleigh Stanton, Taylor Floyd, Sanjana Shah, Devon Trahan, Martin Akerman, Gayatri Arun. Novel splicing-derived neoantigen in triple negative breast cancer uncovered by SpliceIO™ a robust platform for the discovery and validation of disease-specific immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5894.

Glycine Receptor β-Targeting Autoantibodies Contribute to the Pathology of Autoimmune Diseases.

Stiff-person syndrome (SPS) and progressive encephalomyelitis with rigidity and myoclonus (PERM) are rare neurologic disorders of the CNS. Until now, exclusive GlyRα subunit-binding autoantibodies with subsequent changes in function and surface numbers were reported. GlyR autoantibodies have also been described in patients with focal epilepsy. Autoimmune reactivity against the GlyRβ subunits has not yet been shown. Autoantibodies against GlyRα1 target the large extracellular N-terminal domain. This domain shares a high degree of sequence homology with GlyRβ making it not unlikely that GlyRβ-specific autoantibody (aAb) exist and contribute to the disease pathology. In this study, we investigated serum samples from 58 patients for aAb specifically detecting GlyRβ. Studies in microarray format, cell-based assays, and primary spinal cord neurons and spinal cord tissue immunohistochemistry were performed to determine specific GlyRβ binding and define aAb binding to distinct protein regions. Preadsorption approaches of aAbs using living cells and the purified extracellular receptor domain were further used. Finally, functional consequences for inhibitory neurotransmission upon GlyRβ aAb binding were resolved by whole-cell patch-clamp recordings. Among 58 samples investigated, cell-based assays, tissue analysis, and preadsorption approaches revealed 2 patients with high specificity for GlyRβ aAb. Quantitative protein cluster analysis demonstrated aAb binding to synaptic GlyRβ colocalized with the scaffold protein gephyrin independent of the presence of GlyRα1. At the functional level, binding of GlyRβ aAb from both patients to its target impair glycine efficacy. Our study establishes GlyRβ as novel target of aAb in patients with SPS/PERM. In contrast to exclusively GlyRα1-positive sera, which alter glycine potency, aAbs against GlyRβ impair receptor efficacy for the neurotransmitter glycine. Imaging and functional analyses showed that GlyRβ aAbs antagonize inhibitory neurotransmission by affecting receptor function rather than localization.

TMEM25 is a Par3-binding protein that attenuates claudin assembly during tight junction development

The tight junction (TJ) in epithelial cells is formed by integral membrane proteins and cytoplasmic scaffolding proteins. The former contains the claudin family proteins with four transmembrane segments, while the latter includes Par3, a PDZ domain-containing adaptor that organizes TJ formation. Here we show the single membrane-spanning protein TMEM25 localizes to TJs in epithelial cells and binds to Par3 via a PDZ-mediated interaction with its C-terminal cytoplasmic tail. TJ development during epithelial cell polarization is accelerated by depletion of TMEM25, and delayed by overexpression of TMEM25 but not by that of a C-terminally deleted protein, indicating a regulatory role of TMEM25. TMEM25 associates via its N-terminal extracellular domain with claudin-1 and claudin-2 to suppress their cis- and trans-oligomerizations, both of which participate in TJ strand formation. Furthermore, Par3 attenuates TMEM25–claudin association via binding to TMEM25, implying its ability to affect claudin oligomerization. Thus, the TJ protein TMEM25 appears to negatively regulate claudin assembly in TJ formation, which regulation is modulated by its interaction with Par3.

Regulation of AMPA receptor trafficking by secreted protein factors

AMPA receptors (AMPARs) mediate the majority of fast excitatory transmission in the brain. Regulation of AMPAR levels at synapses controls synaptic strength and underlies information storage and processing. Many proteins interact with the intracellular domain of AMPARs to regulate their trafficking and synaptic clustering. However, a growing number of extracellular factors important for glutamatergic synapse development, maturation and function have emerged that can also regulate synaptic AMPAR levels. This mini-review highlights extracellular protein factors that regulate AMPAR trafficking to control synapse development and plasticity. Some of these factors regulate AMPAR clustering and mobility by interacting with the extracellular N-terminal domain of AMPARs whereas others regulate AMPAR trafficking indirectly via their respective signaling receptors. While several of these factors are secreted from neurons, others are released from non-neuronal cells such as glia and muscle. Although it is apparent that secreted factors can act locally on neurons near their sites of release to coordinate individual synapses, it is less clear if they can diffuse over longer ranges to coordinate related synapses within a circuit or region of the brain. Given that there are hundreds of factors that can be secreted from neuronal and non-neuronal cells, it will not be surprising if more extracellular factors that modulate AMPARs and glutamatergic synapses are discovered. Many open questions remain including where and when the factors are expressed, what regulates their secretion from different cell types, what controls their diffusion, stability, and range of action, and how their cognate receptors influence intracellular signaling to control AMPAR trafficking.

Multiple toll-like receptors (TLRs) display differential bacterial and ligand specificity in the earthworm, Eisenia andrei

Toll-like receptors (TLRs), an ancient and well-conserved group of pattern recognition receptors (PRRs), recognize conserved pathogen-associated molecular patterns. TLRs consist of three domains: the extracellular N-terminal domain, containing one or more leucine-rich repeats (LRRs), responsible for the recognizing and binding of antigens; the type-I transmembrane domain; and the intracellular domain known as the Toll/Interleukin-1 receptor (TIR) domain required for the downstream signaling pathway. We identified six new full-length complementary DNA (cDNA) sequences, Ean-TLR1/2/3/4/5/6. The deduced amino acid sequences indicate that Ean-TLRs consist of one signal peptide, one LRR N-terminal domain (Ean-TLR4/5), varying numbers of LRRs, one (Ean-TLR1/2/3/4/5) or two (Ean-TLR6) LRR C-terminal domains, one type-I transmembrane domain, and a TIR domain. In addition, a TIR domain alignment revealed that three conserved motifs, designated as Box 1, Box 2, and Box 3, contain essential amino acid residues for downstream signaling activity. Phylogenetic analysis of earthworm TLRs generated two separate evolutionary branches representing single (sccTLR) and multiple (mccTLR) cysteine cluster TLRs. Ean-TLR1/2/3/4 (sccTLR type) and Ean-TLR6 (mccTLR type) were clustered with corresponding types of previously reported earthworm TLRs as well as TLRs from Clitellata and Polychaete. As PRRs, earthworm TLRs should be capable of sensing a diverse range of pathogens. Except for Ean-TLR3, which was not responsive to any bacteria, earthworm TLR expression was significantly induced by Gram-positive but not Gram-negative bacteria. Moreover, it is likely that earthworms can differentiate between different species of Gram-positive bacteria via their TLR responses. The ligand specificity of earthworm TLRs suggests that their pathogenic ligand recognition is likely to be as specific and diverse as the mammalian TLR pathogen-sensing system.

Lack of evidence for coevolution between oxytocin receptor N-terminal variants and monogamy in placental mammals

Oxytocin (OXT) is a neurohypophyseal hormone that influences a wide range of affiliative behaviors, such as pair-bonding and infant care, across mammals. The effects of OXT depend significantly on an adequate interaction with its receptor, OXTR. OXTR belongs to the G-protein coupled receptor family. The extracellular N-terminal domain of OXTR interacts with the linear C-terminal tail of OXT and is required for OXT binding. Across mammalian species there is a genetic diversity in OXTR terminal sequence. Previous work on primates has shown an association between OXTR phylogeny and monogamy. However, it is not clear whether this variation coevolved with either mating system (monogamy) or infant care behaviors (such as allomaternal care). Here, we take a phylogenetic comparative and evolutionary modeling approach across a wide range of placental mammals (n = 60) to test whether OXTR N-terminal variants co-evolved with either monogamy or allomaternal care behaviors. Our results indicate that the diversity in OXTR N-terminal region is unlikely to provide the underlying genetic bases for variation in mating system and/or allomaternal behavior as we find no evidence for co-evolution between protein sequence and affiliative behaviors. Hence, the role played by OXT in influencing affiliative behaviors is unlikely to be mediated by the genetic diversity of its receptor.

Emerging Roles of Receptor-like Protein Kinases in Plant Response to Abiotic Stresses.

The productivity of plants is hindered by unfavorable conditions. To perceive stress signals and to transduce these signals to intracellular responses, plants rely on membrane-bound receptor-like kinases (RLKs). These play a pivotal role in signaling events governing growth, reproduction, hormone perception, and defense responses against biotic stresses; however, their involvement in abiotic stress responses is poorly documented. Plant RLKs harbor an N-terminal extracellular domain, a transmembrane domain, and a C-terminal intracellular kinase domain. The ectodomains of these RLKs are quite diverse, aiding their responses to various stimuli. We summarize here the sub-classes of RLKs based on their domain structure and discuss the available information on their specific role in abiotic stress adaptation. Furthermore, the current state of knowledge on RLKs and their significance in abiotic stress responses is highlighted in this review, shedding light on their role in influencing plant-environment interactions and opening up possibilities for novel approaches to engineer stress-tolerant crop varieties.

Structural Space of the Duffy Antigen/Receptor for Chemokines' Intrinsically Disordered Ectodomain 1 Explored by Temperature Replica-Exchange Molecular Dynamics Simulations.

Plasmodium vivax malaria affects 14 million people each year. Its invasion requires interactions between the parasitic Duffy-binding protein (PvDBP) and the N-terminal extracellular domain (ECD1) of the host's Duffy antigen/receptor for chemokines (DARC). ECD1 is highly flexible and intrinsically disordered, therefore it can adopt different conformations. We computationally modeled the challenging ECD1 local structure. With T-REMD simulations, we sampled its dynamic behavior and collected its most representative conformations. Our results suggest that most of the DARC ECD1 domain remains in a disordered state during the simulated time. Globular local conformations are found in the analyzed local free-energy minima. These globular conformations share an α-helix spanning residues Ser18 to Ser29 and in many cases they comprise an antiparallel β-sheet, whose β-strands are formed around residues Leu10 and Ala49. The formation of a parallel β-sheet is almost negligible. So far, progress in understanding the mechanisms forming the basis of the P. vivax malaria infection of reticulocytes has been hampered by experimental difficulties, along with a lack of DARC structural information. Our collection of the most probable ECD1 structural conformations will help to advance modeling of the DARC structure and to explore DARC-ECD1 interactions with a range of physiological and pathological ligands.

LANCL1, a cell surface protein, promotes liver tumor initiation through FAM49B-Rac1 axis to suppress oxidative stress.

HCC is an aggressive cancer with a poor clinical outcome. Understanding the mechanisms that drive tumor initiation is important for improving treatment strategy. This study aimed to identify functional cell membrane proteins that promote HCC tumor initiation. Tailor-made siRNA library screening was performed for all membrane protein-encoding genes that are upregulated in human HCC (n = 134), with sphere formation as a surrogate readout for tumor initiation. Upon confirmation of membranous localization by immunofluorescence and tumor initiation ability by limiting dilution assay in vivo, LanC-like protein-1 (LANCL1) was selected for further characterization. LANCL1 suppressed intracellular reactive oxygen species (ROS) and promoted tumorigenicity both in vitro and in vivo. Mechanistically, with mass spectrometry, FAM49B was identified as a downstream binding partner of LANCL1. LANCL1 stabilized FAM49B by blocking the interaction of FAM49B with the specific E3 ubiquitin ligase TRIM21, thus protecting FAM49B from ubiquitin-proteasome degradation. The LANCL1-FAM49B axis suppressed the Rac1-NADPH oxidase-driven ROS production, but this suppression of ROS was independent of the glutathione transferase function of LANCL1. Clinically, HCCs with high co-expression of LANCL1 and FAM49B were associated with more advanced tumor stage, poorer overall survival, and disease-free survival. In addition, anti-LANCL1 antibodies targeting the extracellular N-terminal domain were able to suppress the self-renewal ability, as demonstrated by the sphere formation ability of HCC cells. Our data showed that LANCL1 is a cell surface protein and a key contributor to HCC initiation. Targeting the LANCL1-FAM49B-Rac1-NADPH oxidase-ROS signaling axis may be a promising therapeutic strategy for HCC.

CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus causing a high fatality rate of up to 30%. To date, the receptor mediating SFTSV entry remained uncharacterized, hindering the understanding of disease pathogenesis. Here, C-C motif chemokine receptor 2 (CCR2) was identified as a host receptor for SFTSV based on a genome-wide CRISPR-Cas9 screen. Knockout of CCR2 substantially reduced viral binding and infection. CCR2 enhanced SFTSV binding through direct binding to SFTSV glycoprotein N (Gn), which is mediated by its N-terminal extracellular domain. Depletion of CCR2 in C57BL/6J mouse model attenuated SFTSV replication and pathogenesis. The peripheral blood primary monocytes from elderly individuals or subjects with underlying diabetes mellitus showed higher CCR2 surface expression and supported stronger binding and replication of SFTSV. Together, these data indicate that CCR2 is a host entry receptor for SFTSV infection and a novel target for developing anti-SFTSV therapeutics.

Characterization of Glutamate-Gated Chloride Channel in Tribolium castaneum.

The glutamate-gated chloride channels (GluCls) play essential roles in signal transduction by regulating fast inhibitory synaptic transmission in the nervous system of invertebrates. While there is only one GluCl subunit in the insect, the diversity of insect GluCls is broadened by alternative splicing. In the present study, three TcGluCl variant genes were cloned from the red flour beetle Tribolium castaneum. Analysis of the characteristics of TcGluCls including sequence features, genomic structures, and alternative splicing revealed that TcGluCls had the typical structural features of GluCls and showed high homologies with the GluCls from other insect orders. The TcGluCl-encoding gene consists of nine exons and three variants (TcGluCl-3a, TcGluCl-3b, and TcGluCl-3c) were generated by the alternative splicing of exon 3, which was a highly conserved alternative splicing site in insect GluCls. Homology modeling of TcGluCl-3a showed that the exon 3 coding protein located at the N-terminal extracellular domain, and there were no steric clashes encountered between the exon 3 coding region and ivermectin/glutamate binding pocket, which indicated that the alternative splicing of exon 3 might have no impact on the binding of GluCls to glutamate and insecticide. In addition to the head tissue, TcGluCl-3a and TcGluCl-3c also had high expressions in the ovary and testis of T. castaneum, whereas TcGluCl-3b showed high expression in the midgut, suggesting the diverse physiological functions of TcGluCl variants in T. castaneum. The total TcGluCl and three variants showed the highest expression levels in the early stage larvae. The expressions of TcGluCl, TcGluCl-3b, and TcGluCl-3c were significantly increased from the late-stage larvae to the early stage pupae and indicated that the TcGluCl might be involved in the growth and development of T. castaneum. These results are helpful to further understand the molecular characteristics of insect GluCls and provide foundations for studying the specific function of the GluCl variant.

Voltage-gated sodium channels (Na<sub>V</sub>) in GtoPdb v.2023.1

Sodium channels are voltage-gated sodium-selective ion channels present in the membrane of most excitable cells. Sodium channels comprise of one pore-forming α subunit, which may be associated with either one or two β subunits [179]. α-Subunits consist of four homologous domains (I-IV), each containing six transmembrane segments (S1-S6) and a pore-forming loop. The positively charged fourth transmembrane segment (S4) acts as a voltage sensor and is involved in channel gating. The crystal structure of the bacterial NavAb channel has revealed a number of novel structural features compared to earlier potassium channel structures including a short selectivity filter with ion selectivity determined by interactions with glutamate side chains [278]. Interestingly, the pore region is penetrated by fatty acyl chains that extend into the central cavity which may allow the entry of small, hydrophobic pore-blocking drugs [278]. Auxiliary β1, β2, β3 and β4 subunits consist of a large extracellular N-terminal domain, a single transmembrane segment and a shorter cytoplasmic domain.The nomenclature for sodium channels was proposed by Goldin et al., (2000) [146] and approved by the NC-IUPHAR Subcommittee on sodium channels (Catterall et al., 2005, [53]).

Arabidopsis COPT1 copper transporter uses a single histidine to regulate transport activity and protein stability

Copper acquisition and subsequent delivery to target proteins are essential for many biological processes. However, the cellular levels of this trace element must be controlled because of its potential toxicity. The COPT1 protein rich in potential metal-binding amino acids functions in high affinity copper uptake at the plasma membrane of Arabidopsis cells. The functional role of these putative metal-binding residues is largely unknown. Through truncations and site-directed mutagenesis, we identified His43, a single residue within the extracellular N-terminal domain as absolutely critical for copper uptake of COPT1. Substitution of this residue with leucine, methionine or cysteine almost inactivated transport function of COPT1, implying that His43 fails to serves as a copper ligand in the regulation of COPT1 activity. Deletion of all extracellular N-terminal metal-binding residues completely blocked copper-stimulated degradation but did not alter the subcellular distribution and multimerization of COPT1. Although mutation of His43 to alanine and serine retained the transporter activity in yeast cells, the mutant protein was unstable and degraded in the proteasome in Arabidopsis cells. Our results demonstrate a pivotal role for the extracellular residue His43 in high affinity copper transport activity, and suggest common molecular mechanisms for regulating both metal transport and protein stability of COPT1.

Conformational dynamics of glucagon receptor domains revealed by single-molecule fluorescence microscopy.

G protein-coupled receptors (GPCRs) are integral membrane proteins involved in many cellular signaling processes. Representing the largest family of proteins with approximately 800 members in humans, GPCRs are the target for more than 40% of current pharmaceutical drugs. The structural and dynamic information is vital to understanding the activation mechanism of these receptors. Despite the availability of high-resolution structures of GPCRs at different conformational states, the dynamics of those conformational states at the molecular level are missing. We used total internal reflection fluorescence (TIRF) imaging to investigate the conformational dynamics of the glucagon receptor (GCGR), a class B family GPCR involved in glucose homeostasis. We expressed and purified GCGR from mammalian cells and analyzed them at a single molecule level. We used single-molecule Förster resonance energy transfer (smFRET) to observe the conformational dynamics of the donor and acceptor fluorophore-labeled GCGR molecules. We observed ligand-dependent slow and reversible conformational fluctuation of N-terminal extra cellular domain (ECD) without ligand binding. The addition of a saturated concentration of peptide ligand (Glucagon) altered the dynamics towards the lower FRET states. These results provide new insight into the structural dynamics of glucagon receptor and receptor activation.

The relative importance of platelet integrins in hemostasis, thrombosis and beyond.

Integrins are heterodimeric transmembrane receptors composed of α and β chains, with an N-terminal extracellular domain forming a globular head corresponding to the ligand binding site. Integrins regulate various cellular functions including adhesion, migration, proliferation, spreading and apoptosis. On platelets, integrins play a central role in adhesion and aggregation on subendothelial matrix proteins of the vascular wall, thereby ensuring hemostasis. Platelet integrins belong either to the β1 family (α2β1, α5β1 and α6β1) or to the β3 family (αIIbβ3 and αvβ3). On resting platelets, integrins can engage their ligands when the latter are immobilized but not in their soluble form. The effects of various agonists promote an inside-out signal in platelets, increasing the affinity of integrins for their ligands and conveying a modest signal reinforcing platelet activation, called outside-in signaling. This outside-in signal ensures platelet adhesion, shape change, granule secretion and aggregation. In this review, we examine the role of each platelet integrin in hemostatic plug formation, hemostasis and arterial thrombosis and also beyond these classical functions, notably in tumor metastasis and sepsis.

Properties of recombinant extracellular N-terminal domain of human high-affinity copper transporter 1 (hNdCTR1) and its interactions with Cu(II) and Ag(I) ions.

High-affinity copper transporter 1 (CTR1) is a key link in the transfer of copper (Cu) from the extracellular environment to the cell. Violation in the control system of its expression, or mutations in this gene, cause a global copper imbalance. However, the mechanism of copper transfer via CTR1 remains unclear. It has been shown that transformed bacteria synthesizing the fused GB1-NdCTR become resistant to toxic silver ions. According to UV-Vis spectrophotometry and isothermal titration calorimetry, electrophoretically pure GB1-NdCTR specifically and reversibly binds copper and silver ions, and binding is associated with aggregation. Purified NdCTR1 forms SDS-resistant oligomers. The link between nontrivial properties of NdCTR1 and copper import mechanism from extracellular space, as well as potential chelating properties of NdCTR1, are discussed.

A new intracellular targeting motif in the cytoplasmic tail of the spike protein may act as a target to inhibit SARS-CoV-2 assembly

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a threat to global public health, underscoring the urgent need for the development of preventive and therapeutic measures. The spike (S) protein of SARS-CoV-2, which mediates receptor binding and subsequent membrane fusion to promote viral entry, is a major target for current drug development and vaccine design. The S protein comprises a large N-terminal extracellular domain, a transmembrane domain, and a short cytoplasmic tail (CT) at the C-terminus. CT truncation of the S protein has been previously reported to promote the infectivity of SARS-CoV and SARS-CoV-2 pseudoviruses. However, the underlying molecular mechanism has not been precisely elucidated. In addition, the CT of various viral membrane glycoproteins play an essential role in the assembly of virions, yet the role of the S protein CT in SARS-CoV-2 infection remains unclear. In this study, through constructing a series of mutations of the CT of the S protein and analyzing their impact on the packaging of the SARS-CoV-2 pseudovirus and live SARS-CoV-2 virus, we identified V1264L1265 as a new intracellular targeting motif in the CT of the S protein, that regulates the transport and subcellular localization of the spike protein through the interactions with cytoskeleton and vesicular transport-related proteins, ARPC3, SCAMP3, and TUBB8, thereby modulating SARS-CoV-2 pseudovirus and live SARS-CoV-2 virion assembly. Either disrupting the V1264L1265 motif or reducing the expression of ARPC3, SCAMP3, and TUBB8 significantly repressed the assembly of the live SARS-CoV-2 virion, raising the possibility that the V1264L1265 motif and the host responsive pathways involved could be new drug targets for the treatment of SARS-CoV-2 infection. Our results extend the understanding of the role played by the S protein CT in the assembly of pseudoviruses and live SARS-CoV-2 virions, which will facilitate the application of pseudoviruses to the study of SARS-CoV-2 and provide potential strategies for the treatment of SARS-CoV-2 infection.