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NanoLuc (NLuc) luciferase has found extensive application in designing a range of biological assays including gene expression analysis, protein-protein interaction and protein conformational changes due to its enhanced brightness and small size. However, questions related to its mechanism of interaction with the substrate, furimazine, as well as bioluminescence activity remains elusive. Here, we combined molecular dynamics (MD) simulation and mutational analysis to show that the R162A mutation results in a decreased but stable bioluminescence activity of NLuc in vitro. Specifically, we performed multiple, all-atom, explicit solvent MD simulations of the apo and furimazine-docked (holo) NLuc structures revealing differential dynamics of the protein in the absence and presence of the ligand. Further, analysis of trajectories for hydrogen bonds (H-bonds) formed between NLuc and furimazine revealed substantial H-bond interaction between R162 and Q32 residues. Mutation of the two residues in NLuc revealed a decreased but stable activity of the R162A, but not Q32A, mutant NLuc in in vitro assays performed with furimazine. In addition to highlighting the role of the R162 residue in NLuc activity, we believe that the mutant NLuc will find wide application in designing in vitro assays requiring extended monitoring of NLuc bioluminescence activity.

Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small protein β2-microglobulin (β2m), bind peptides in the endoplasmic reticulum that are generated by the cytosolic turnover of cellular proteins. In virus-infected cells these peptides may include those derived from viral proteins. Peptide-MHC-I complexes then traffic through the secretory pathway and are displayed at the cell surface where those containing viral peptides can be detected by CD8+ T lymphocytes that kill infected cells. Many viruses enhance their in vivo survival by encoding genes that downregulate MHC-I expression to avoid CD8+ T cell recognition. Here we report that two accessory proteins encoded by SARS-CoV-2, the causative agent of the ongoing COVID-19 pandemic, downregulate MHC-I expression using distinct mechanisms. One, ORF3a, a viroporin, reduces global trafficking of proteins, including MHC-I, through the secretory pathway. The second, ORF7a, interacts specifically with the MHC-I heavy chain, acting as a molecular mimic of β2m to inhibit its association. This slows the exit of properly assembled MHC-I molecules from the endoplasmic reticulum. We demonstrate that ORF7a reduces antigen presentation by the human MHC-I allele HLA-A*02:01. Thus, both ORF3a and ORF7a act post-translationally in the secretory pathway to lower surface MHC-I expression, with ORF7a exhibiting a novel and specific mechanism that allows immune evasion by SARS-CoV-2.Significance StatementViruses may down-regulate MHC class I expression on infected cells to avoid elimination by cytotoxic T cells. We report that the accessory proteins ORF7a and ORF3a of SARS-CoV-2 mediate this function and delineate the two distinct mechanisms involved. While ORF3a inhibits global protein trafficking to the cell surface, ORF7a acts specifically on MHC-I by competing with β2m for binding to the MHC-I heavy chain. This is the first account of molecular mimicry of β2m as a viral mechanism of MHC-I down-regulation to facilitate immune evasion.

The cellular cytoskeleton consisting of microtubules, intermediate filaments and the actin filaments is a dynamic structure providing shape and structural stability to cells. Particularly, the actin cytoskeleton formed by a combination of polymerized actin molecules and several other actin binding proteins including myosin is key to sensing and development of mechanical forces in cells. Given this and other features, the actin cytoskeleton has been implicated in a variety of cellular process including cellular motility and migration, cytokinesis, phagocytosis, cytoplasmic streaming, organelle transport, cellular transformation and metastasis, cellular metabolism, cell–matrix adhesion, and cell–cell adhesion. The latter is mediated by E-cadherin in the epithelial tissue and is fundamental to tissue morphogenesis and normal development. Here we discuss the role of the actin cytoskeleton in the assembly and maintenance of E-cadherin-based cell–cell adhesion through the formation of cellular appendages such as filopodia and lamellipodia and thus, impinging on one of the fundamental features of multicellular organisms.

Tremendous changes have been witnessed in the post-COVID-19 world. Global efforts were initiated to reach a successful treatment for this emerging disease. These efforts have focused on developing vaccinations and/or finding therapeutic agents that can be used to combat the virus or reduce its accompanying symptoms. Gulf Cooperation Council (GCC) countries have initiated efforts on many clinical trials to address the efficacy and the safety of several therapeutic agents used for COVID-19 treatment. In this article, we provide an overview of the GCC's clinical trials and associated drugs' discovery process in the pursuit of an effective medication for COVID-19.

Tremendous changes have been witnessed in the post-COVID-19 world. Global efforts were initiated to reach a successful treatment for this emerging disease. These efforts have focused on developing vaccinations and/or finding therapeutic agents that can be used to combat the virus or reduce its accompanying symptoms. Gulf Cooperation Council (GCC) countries have initiated efforts on many clinical trials to address the efficacy and the safety of several therapeutic agents used for COVID-19 treatment. In this article, we provide an overview of the GCC’s clinical trials and associated drugs’ discovery process in the pursuit of an effective medication for COVID-19.

Fanconi–Bickel Syndrome (FBS) is a rare disorder of carbohydrate metabolism that is characterized mainly by the accumulation of glycogen in the liver and kidney. It is inherited as an autosomal recessive disorder caused by mutations in the SLC2A2 gene, which encodes for GLUT2. Patients with FBS have dysglycemia but the molecular mechanisms of dysglycemia are still not clearly understood. Therefore, we aimed to understand the underlying molecular mechanisms of dysglycemia in a patient with FBS. Genomic DNA was isolated from a peripheral blood sample and analyzed by whole genome and Sanger sequencing. CRISPR-Cas9 was used to introduce a mutation that mimics the patient’s mutation in a human kidney cell line expressing GLUT2 (HEK293T). Mutant cells were used for molecular analysis to investigate the effects of the mutation on the expression and function of GLUT2, as well as the expression of other genes implicated in dysglycemia. The patient was found to have a homozygous nonsense mutation (c.901C>T, R301X) in the SLC2A2 gene. CRISPR-Cas9 successfully mimicked the patient’s mutation in HEK293T cells. The mutant cells showed overexpression of a dysfunctional GLUT2 protein, resulting in reduced glucose release activity and enhanced intracellular glucose accumulation. In addition, other glucose transporters (SGLT1 and SGLT2 in the kidney) were found to be induced in the mutant cells. These findings suggest the last loops (loops 9-12) of GLUT2 are essential for glucose transport activity and indicate that GLUT2 dysfunction is associated with dysglycemia in FBS.

Cells sense a variety of extracellular growth factors and signaling molecules through numerous distinct receptor tyrosine kinases (RTKs) on the cell surface. In many cases, the same intracellular signaling molecules interact with more than one type of RTK. How signals from different RTKs retain the identity of the triggering receptor and how (or if) different receptors may synergize or compete remain largely unknown. Here we utilize an experimental strategy, combining microscale patterning and single-molecule imaging, to measure the competition between ephrin-A1:EphA2 and epidermal growth factor (EGF):EGF receptor (EGFR) ligand-receptor complexes for the shared downstream signaling molecules, Grb2 and SOS. The results reveal a distinct hierarchy, in which newly formed EGF:EGFR complexes outcompete ephrin-A1:EphA2 for Grb2 and SOS, revealing a type of negative crosstalk interaction fundamentally controlled by chemical mass action and protein copy number limitations.

Clustering of ligand:receptor complexes on the cell membrane is widely presumed to have functional consequences for subsequent signal transduction. However, it is experimentally challenging to selectively manipulate receptor clustering without altering other biochemical aspects of the cellular system. Here, we develop a microfabrication strategy to produce substrates displaying mobile and immobile ligands that are separated by roughly 1 µm, and thus experience an identical cytoplasmic signaling state, enabling precision comparison of downstream signaling reactions. Applying this approach to characterize the ephrinA1:EphA2 signaling system reveals that EphA2 clustering enhances both receptor phosphorylation and downstream signaling activity. Single-molecule imaging clearly resolves increased molecular binding dwell times at EphA2 clusters for both Grb2:SOS and NCK:N-WASP signaling modules. This type of intracellular comparison enables a substantially higher degree of quantitative analysis than is possible when comparisons must be made between different cells and essentially eliminates the effects of cellular response to ligand manipulation.

A wide range of cell–microenvironmental interactions are mediated by membrane-localized receptors that bind ligands present on another cell or the extracellular matrix. This situation introduces a number of physical effects including spatial organization of receptor–ligand complexes and development of mechanical forces in cells. Unlike traditional experimental approaches, hybrid live cell–supported lipid bilayer (SLB) systems, wherein a live cell interacts with a synthetic substrate supported membrane, allow interrogation of these aspects of receptor signaling. The SLB system directly offers facile control over the identity, density, and mobility of ligands used for engaging cellular receptors. Further, application of various nano- and micropatterning techniques allows for spatial patterning of ligands. In this review, we describe the hybrid live cell–SLB system and its application in uncovering a range of spatial and mechanical aspects of receptor signaling. We highlight the T cell immunological synapse, junctions formed between EphA2- and ephrinA1-expressing cells, and adhesions formed by cadherin and integrin receptors.

Corona Virus Disease of 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) has caused a massive health crisis across the globe, with some genetic variants gaining enhanced infectivity and competitive fitness, and thus significantly aggravating the global health concern. In this regard, the recent SARS-CoV-2 alpha variant, B.1.1.7 lineage, reported from the United Kingdom (UK), is of great significance in that it contains several mutations that increase its infection and transmission rates as evident from clinical reports. Specifically, the N501Y mutation in the SARS-CoV-2 spike S1 receptor binding domain (S1-RBD) has been shown to possess an increased affinity for ACE2, although the basis for this is not entirely clear yet. Here, we dissect the mechanism underlying the increased affinity using molecular dynamics (MD) simulations of the available ACE2-S1-RBD complex structure (6M0J) and show a prolonged and stable interfacial interaction of the N501Y mutant S1-RBD with ACE2 compared to the wild type S1-RBD. Additionally, we find that the N501Y mutant S1-RBD displays altered dynamics that likely aids in its enhanced interaction with ACE2. By elucidating a mechanistic basis for the increased affinity of the N501Y mutant S1-RBD for ACE2, we believe that the results presented here will aid in developing therapeutic strategies against SARS-CoV-2 including designing drugs targeting the ACE2-S1-RBD interaction.SignificanceThe emergence of the new SARS-CoV-2 lineage in the UK in December 2020 has further aggravated the COVID-19 pandemic due to an increased ability of the variant to infect human hosts, likely due to mutations in the viral S1 spike protein including the N501Y S1-RBD mutation that is located at the interface of S1-RBD and ACE2, the host cell receptor for SARS-CoV-2. Given its location at the interface, N501Y S1-RBD mutation can therefore potentially alter the interfacial interaction. Multiple, all-atom, explicit solvent MD simulations of the ACE2-S1-RBD complex carried here indicated a more stable interaction between the N501Y mutant S1-RBD and ACE2 through stabilizing interfacial interactions of residues at one end of the interface that are either sequentially or physically near the mutation site. These mechanistic details will aid in better understanding the mechanism by which the alpha variant has increased infectivity as well as in designing better therapeutics including ACE2-S1 spike protein inhibitors that will, in turn, help thwarting the current and future pandemic.HighlightsN501 in the wild type SARS-CoV-2 S1-RBD forms unsustained hydrogen bonds with residues in the ACE2, namely Y41 and K353Y501 in the N501Y mutant SARS-CoV-2 S1-RBD is not capable of forming substantial hydrogen bonds with ACE2 within the time span of the current simulationEvidence from analyzing the simulation results suggests that Y501 of S1-RBD could form other types of non-covalent interactions with ACE2, such as van der Waals interactionsN501Y S1-RBD mutation stabilizes the position of interfacial residues neighboring to the mutation site, as well as other non-interfacial residues that are distant from the mutation siteThese altered dynamics results in more stable interaction of S1-RBD with ACE2 which could be the main reason underlying the reported enhanced affinity of S1-RBD in the SARS-CoV-2 alpha variant (UK B.1.1.7 lineage) to ACE2

Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation, phase separation, and cell volume. It could be altered by changes in the activity of cellular signaling pathways, such as those that impact the activity of membrane-localized ion channels or by alterations in the microenvironmental osmolarity. Therefore, there is a demand for the development of sensitive tools for real-time monitoring of intracellular ionic strength. Here, we developed a bioluminescence-based intracellular ionic strength sensing strategy using the Nano Luciferase (NanoLuc) protein that has gained tremendous utility due to its high, long-lived bioluminescence output and thermal stability. Biochemical experiments using a recombinantly purified protein showed that NanoLuc bioluminescence is dependent on the ionic strength of the reaction buffer for a wide range of ionic strength conditions. Importantly, the decrease in the NanoLuc activity observed at higher ionic strengths could be reversed by decreasing the ionic strength of the reaction, thus making it suitable for sensing intracellular ionic strength alterations. Finally, we used an mNeonGreen&ndash, NanoLuc fusion protein to successfully monitor ionic strength alterations in a ratiometric manner through independent fluorescence and bioluminescence measurements in cell lysates and live cells. We envisage that the biosensing strategy developed here for detecting alterations in intracellular ionic strength will be applicable in a wide range of experiments, including high throughput cellular signaling, ion channel functional genomics, and drug discovery.

Phosphodiesterase 5 (PDE5) is one of the most well-studied phosphodiesterases (PDEs) that specifically targets cGMP typically generated by nitric oxide (NO)-mediated activation of the soluble guanylyl cyclase. Given the crucial role of cGMP generated through the activation of this cellular signaling pathway in a variety of physiologically processes, pharmacological inhibition of PDE5 has been demonstrated to have several therapeutic applications including erectile dysfunction and pulmonary arterial hypertension. While they are designed to inhibit PDE5, the inhibitors show different affinities and specificities against all PDE subtypes. Additionally, they have been shown to induce allosteric structural changes in the protein. These are mostly attributed to their chemical structure and, therefore, binding interactions with PDE catalytic domains. Therefore, understanding how these inhibitors interact with PDE5 and the structural basis of their selectivity is critically important for the design of novel, highly selective PDE5 inhibitors. Here, we review the structure of PDE5, how its function is regulated, and discuss the clinically available inhibitors that target phosphodiesterase 5, aiming to better understand the structural bases of their affinity and specificity. We also discuss the therapeutic indications of these inhibitors and the potential of repurposing for a wider range of clinical applications.

The pathway of vesicle adsorption onto a solid support depends on the material composition of the underlying support, and there is significant interest in developing material-independent strategies to modulate the spectrum of vesicle-substrate interactions on a particular surface. Herein, using the quartz crystal microbalance-dissipation (QCM-D) technique, we systematically investigated how solution pH and membrane surface charge affect vesicle adsorption onto a silicon dioxide surface. While vesicle adsorption and spontaneous rupture to form complete supported lipid bilayer (SLBs) occurred in acidic conditions, it was discovered that a wide range of adsorption pathways occurred in alkaline conditions, including (i) vesicle adsorption and spontaneous rupture to form complete SLBs, (ii) vesicle adsorption and spontaneous rupture to form incomplete SLBs, (iii) irreversible adsorption of intact vesicles, (iv) reversible adsorption of intact vesicles, and (v) negligible adsorption. In general, SLB formation became more favorable with increasingly positive membrane surface charge although there were certain conditions at which attractive electrostatic forces were insufficient to promote vesicle rupture. To rationalize these findings, we discuss how solution pH and membrane surface charge affect interfacial forces involved in vesicle-substrate interactions. Taken together, our findings present a comprehensive picture of how interfacial forces dictate the pathway of phospholipid vesicle adsorption onto silicon dioxide surfaces and offer a broadly applicable framework to characterize the interactions between phospholipid vesicles and inorganic material surfaces. NRF (Natl Research Foundation, S’pore)

Allostery is a mechanism by which the activity of a large number of proteins is regulated. It is manifested as a change in the activity, either ligand binding or catalysis of one site of a protein due to a ligand binding to another distinct site of the protein. The allosteric effect is transduced by a change in the structural properties of the protein. It has been traditionally understood using either the concerted MWC (Monod, Wyman and Changeux) model, or the sequential KNF (Koshland, Nemethy and Filmer) model of structural changes. However, allostery is fundamentally a thermodynamic process and requires an alteration in the enthalpy or entropy associated with the process.

Cells in epithelial tissues utilize homotypic E-cadherin interaction-mediated adhesions to both physically adhere to each other and sense the physical properties of their microenvironment, such as the presence of other cells in close vicinity or an alteration in the mechanical tension of the tissue. These position E-cadherin centrally in organogenesis and other processes, and its function is therefore tightly regulated through a variety of means including endocytosis and gene expression. How does membrane molecular mobility of E-cadherin, and thus membrane physical properties and associated actin cytoskeleton, impinges on the assembly of adhesive clusters and signaling is discussed.

Organized for action It is becoming increasingly clear that biomolecular condensates, which are concentrations of macromolecules not surrounded by a membrane, are a key organizational structure in eukaryotic cells (see the Perspective by Martin and Mittag). Now, two papers show how such condensates function in actin assembly or in a Ras signaling pathway. In both cases, the condensates form at the plasma membrane and increase the activity of signaling proteins by increasing their membrane dwell times. Case et al. show that the dwell time is dependent on cluster stoichiometry, so that stoichiometry of regulatory proteins can control actin assembly. Huang et al. demonstrate that the longer dwell time allows kinetic proofreading in receptor-mediated activation of Ras. Science , this issue p. 1093 , p. 1098 ; see also p. 1036

Mechanotransduction at E-cadherin junctions has been postulated to be mediated in part by a force-dependent conformational activation of α-catenin. Activation of α-catenin allows it to interact with vinculin in addition to F-actin, resulting in a strengthening of junctions. Here, using E-cadherin adhesions reconstituted on synthetic, nanopatterned membranes, we show that activation of α-catenin is dependent on E-cadherin clustering, and is sustained in the absence of mechanical force or association with F-actin or vinculin. Adhesions were formed by filopodia-mediated nucleation and micron-scale assembly of E-cadherin clusters, which could be distinguished as either peripheral or central assemblies depending on their relative location at the cell-bilayer adhesion. Whereas F-actin, vinculin, and phosphorylated myosin light chain associated only with the peripheral assemblies, activated α-catenin was present in both peripheral and central assemblies, and persisted in the central assemblies in the absence of actomyosin tension. Impeding filopodia-mediated nucleation and micron-scale assembly of E-cadherin adhesion complexes by confining the movement of bilayer-bound E-cadherin on nanopatterned substrates reduced the levels of activated α-catenin. Taken together, these results indicate that although the initial activation of α-catenin requires micron-scale clustering that may allow the development of mechanical forces, sustained force is not required for maintaining α-catenin in the active state.

Receptor-ligand complexes spanning a cell-cell interface inevitably establish a preferred intermembrane spacing based on the molecular dimensions and orientation of the complexes. This couples molecular binding events to membrane mechanics and large-scale spatial organization of receptors on the cell surface. Here, we describe a straightforward, epi-fluorescence-based method to precisely determine intermembrane receptor-ligand dimension at adhesions established by receptor-ligand binding between apposed membranes in vitro. Adhesions were reconstituted between planar and silica microbead supported membranes via specific interaction between cognate receptor/ligand pairs (EphA2/EphrinA1 and E-cadherin/anti-E-cadherin antibody). Epi-fluorescence imaging of the ligand enrichment zone in the supported membrane beneath the adhering microbead, combined with a simple geometrical interpretation, proves sufficient to estimate intermembrane receptor-ligand dimension with better than 1 nm precision. An advantage of this assay is that no specialized equipment or imaging methods are required.

Monoamine oxidase A and B (MAO-A and B) are mitochondrial outer membrane enzymes that are implicated in a number of human diseases, and the pharmacological inhibition of these enzymes is a promising therapeutic strategy to alleviate disease symptoms. It has been suggested that optimal levels of enzymatic activity occur in the membrane-associated state, although details of the membrane association process remain to be understood. Herein, we have developed a supported lipid bilayer platform to study MAO-A and B binding and evaluate the effects of known pharmacological inhibitors on the membrane association process. By utilizing the quartz crystal microbalance-dissipation (QCM-D) technique, it was determined that both MAOs exhibit tight binding to negatively and positively charged bilayers with distinct concentration-dependent binding profiles while only transiently binding to neutral bilayers. Importantly, in the presence of known inhibitors, the MAOs showed increased binding to negatively charged bilayers, although there was no effect of inhibitor treatment on binding to positively charged bilayers. Taken together, our findings establish that the membrane association of MAOs is highly dependent on membrane surface charge, and we outline an experimental platform to support the in vitro reconstitution of monoamine oxidases on synthetic membranes, including the evaluation of pharmacological drug candidates. NRF (Natl Research Foundation, S’pore)

Recent studies have revealed pronounced effects of the spatial distribution of EphA2 receptors on cellular response to receptor activation. However, little is known about molecular mechanisms underlying this spatial sensitivity, in part due to lack of experimental systems. Here, we introduce a hybrid live-cell patterned supported lipid bilayer experimental platform in which the sites of EphA2 activation and integrin adhesion are spatially controlled. Using a series of live-cell imaging and single-molecule tracking experiments, we map the transmission of signals from ephrinA1:EphA2 complexes. Results show that ligand-dependent EphA2 activation induces localized myosin-dependent contractions while simultaneously increasing focal adhesion dynamics throughout the cell. Mechanistically, Src kinase is activated at sites of ephrinA1:EphA2 clustering and subsequently diffuses on the membrane to focal adhesions, where it up-regulates FAK and paxillin tyrosine phosphorylation. EphrinA1:EphA2 signaling triggers multiple cellular responses with differing spatial dependencies to enable a directed migratory response to spatially resolved contact with ephrinA1 ligands.

Intercellular cognate receptor-ligand pairs are major players in cellular signal transduction. The fact that both the receptor and the ligand are present on the membrane in these juxtacrine signaling interactions presents distinct experimental difficulties in their study. One experimental platform that has proven particularly useful is the hybrid live cell-supported lipid bilayer system, wherein a live cell is allowed to interact with a synthetic supported membrane displaying ligands of interest. A synthetic membrane enables control over identity, density, mobility, and spatial patterning of the displayed ligands. This chapter provides insights gained from the reconstitution of the immunological synapse formed by T-cells, junction formed by ephrinA1-EphA2 receptor tyrosine kinase in breast cancer cells, and adhesion formed by E-cadherin in epithelial cells on synthetic supported lipid bilayers.

Cells in our body utilize a variety of adaptor proteins for transmitting context specific signals that arise from the cellular microenvironment. Adaptor proteins lack enzymatic activity and typically perform their function by acting as scaffolds that bind other signaling proteins. While most adaptor proteins are functionally modulated by biochemical alterations such as phosphorylation, a subset of adaptor proteins are functionally modulated by a mechanical alteration in their structure that makes cryptic sites available for binding to downstream signaling proteins. α-catenin is one such adaptor protein that localizes to cadherin-based cell adhesions by binding the membrane-localized cadherin-β-catenin complex at one side and the cytosolic F-actin on the other side. An increase in actomyosin tension is directly relayed to α-catenin resulting in a change in its conformation making cryptic binding sites accessible to its interacting partners. Here, I describe an updated view of the mechanical regulation of α-catenin in the context of cellular adhesion, including the role of cadherin clustering in its activation. Accepted version

Epithelial (E)-cadherin-mediated cell-cell junctions play important roles in the development and maintenance of tissue structure in multicellular organisms. E-cadherin adhesion is thus a key element of the cellular microenvironment that provides both mechanical and biochemical signaling inputs. Here, we report in vitro reconstitution of junction-like structures between native E-cadherin in living cells and the extracellular domain of E-cadherin (E-cad-ECD) in a supported membrane. Junction formation in this hybrid live cell-supported membrane configuration requires both active processes within the living cell and a supported membrane with low E-cad-ECD mobility. The hybrid junctions recruit α-catenin and exhibit remodeled cortical actin. Observations suggest that the initial stages of junction formation in this hybrid system depend on the trans but not the cis interactions between E-cadherin molecules, and proceed via a nucleation process in which protrusion and retraction of filopodia play a key role.

E-cadherin is a calcium dependent cell adhesion molecule that is key to the organization of cells in the epithelial tissue. It is a multidomain, trans-membrane protein in which the extracellular domain forms the homotypic, adhesive interaction while the intracellular domain interacts with the actin cytoskeleton through the catenin family of adaptor proteins. A number of recent studies have provided novel insights into the mechanism of adhesion formation by this class of adhesion proteins. Here, we describe an updated view of the process of E-cadherin adhesion formation with an emphasis on the role of molecular mobility, clustering, and active cellular processes.

BACKGROUND Familial diarrhea disorders are, in most cases, severe and caused by recessive mutations. We describe the cause of a novel dominant disease in 32 members of a Norwegian family. The affected members have chronic diarrhea that is of early onset, is relatively mild, and is associated with increased susceptibility to inflammatory bowel disease, small-bowel obstruction, and esophagitis. METHODS We used linkage analysis, based on arrays with single-nucleotide polymorphisms, to identify a candidate region on chromosome 12 and then sequenced GUCY2C, encoding guanylate cyclase C (GC-C), an intestinal receptor for bacterial heat-stable enterotoxins. We performed exome sequencing of the entire candidate region from three affected family members, to exclude the possibility that mutations in genes other than GUCY2C could cause or contribute to susceptibility to the disease. We carried out functional studies of mutant GC-C using HEK293T cells. RESULTS We identified a heterozygous missense mutation (c.2519G -> T) in GUCY2C in all affected family members and observed no other rare variants in the exons of genes in the candidate region. Exposure of the mutant receptor to its ligands resulted in markedly increased production of cyclic guanosine monophosphate (cGMP). This may cause hyperactivation of the cystic fibrosis transmembrane regulator (CFTR), leading to increased chloride and water secretion from the enterocytes, and may thus explain the chronic diarrhea in the affected family members. CONCLUSIONS Increased GC-C signaling disturbs normal bowel function and appears to have a proinflammatory effect, either through increased chloride secretion or additional effects of elevated cellular cGMP. Further investigation of the relevance of genetic variants affecting the GC-C-CFTR pathway to conditions such as Crohn's disease is warranted. (Funded by Helse Vest Western Norway Regional Health Authority] and the Department of Science and Technology, Government of India.)

Deoxyribonucleic acid (DNA) has been used as a material for a variety of applications, including surface functionalization for cell biological or in vitro reconstitution studies. Use of DNA-based surface functionalization eliminates limitations of multiplexing posed by traditionally used methods in applications requiring spatially segregated surface functionalization. Recently, we have reported a stochastic, membrane fusion-based strategy to fabricate multicomponent membrane array substrates displaying spatially segregated protein ligands using biotin-streptavidin and Ni-NTA-polyhistidine interactions. Here, we report the delivery of DNA oligonucleotide-conjugated lipid molecules to membrane corrals, allowing spatially segregated membrane corral functionalization in a membrane microarray. Incubation of microbeads coated with the supported membrane resulted in an exchange of lipid contents with planar membrane corrals present on a micropatterned substrate. Increases in the system temperature and membrane corral size resulted in alterations in the rate constant of lipid exchange, which are in agreement with our previously developed analytical model and further confirm that lipid exchange is a diffusion-based process that takes place after the formation of a long "fusion-stalk" between the two membranes. We take advantage of the physical dimensions of the fusion-stalk with a large aspect ratio to deliver DNA oligonucleotide-conjugated lipid molecules to membrane corrals. We believe that the ability to functionalize membrane corrals with DNA oligonucleotides significantly increases the utility of the stochastic fusion-mediated lipid delivery strategy in the functionalization of biomolecules such as DNA or DNA-conjugated protein ligands. NRF (Natl Research Foundation, S’pore)

Cells sense biochemical as well as mechanical signals from their microenvironment by engaging multiple receptors. In many cases, multiple receptors operate in concert, and it can be misleading to attempt to isolate a single ligand–receptor interaction. Supported lipid membranes are employed to reconstitute a number of cell receptor systems. Efforts are also made to fabricate membrane microarrays presenting multiple ligands in a spatially segregated manner. However, such membrane multiplexing methods are generally limited by complex instrumentation and scalability. Here, a straightforward method is presented to produce centimeter‐scale membrane microarrays displaying multiple, spatially segregated membrane‐anchored protein ligands suitable for live single‐cell studies. The method is based on stochastic membrane fusion and subsequent diffusion‐mediated mixing of their lipid content. The result is the delivery of membrane contents into spatially segregated membrane corrals. Utilizing the technology developed here, this work probes the recruitment of an adaptor protein, Shc1, to epidermal growth factor receptor (EGFR) and EphA2 receptors and shows that activation of EGFR results in a decrease in the recruitment of protein to activate EphA2 same cell. National Research Foundation (NRF)

Receptor guanylyl cyclases are multidomain proteins, and ligand binding to the extracellular domain increases the levels of intracellular cGMP. The intracellular domain of these receptors is composed of a kinase homology domain (KHD), a linker of ∼70 amino acids, followed by the C-terminal guanylyl cyclase domain. Mechanisms by which these receptors are allosterically regulated by ligand binding to the extracellular domain and ATP binding to the KHD are not completely understood. Here we examine the role of the linker region in receptor guanylyl cyclases by a series of point mutations in receptor guanylyl cyclase C. The linker region is predicted to adopt a coiled coil structure and aid in dimerization, but we find that the effects of mutations neither follow a pattern predicted for a coiled coil peptide nor abrogate dimerization. Importantly, this region is critical for repressing the guanylyl cyclase activity of the receptor in the absence of ligand and permitting ligand-mediated activation of the cyclase domain. Mutant receptors with high basal guanylyl cyclase activity show no further activation in the presence of non-ionic detergents, suggesting that hydrophobic interactions in the basal and inactive conformation of the guanylyl cyclase domain are disrupted by mutation. Equivalent mutations in the linker region of guanylyl cyclase A also elevated the basal activity and abolished ligand- and detergent-mediated activation. We, therefore, have defined a key regulatory role for the linker region of receptor guanylyl cyclases which serves as a transducer of information from the extracellular domain via the KHD to the catalytic domain.