Calcineurin-fusion facilitates cryo-EM structure determination of a Family A GPCR.

Advances in singe-particle cryo-electron microscopy (cryo-EM) have made it possible to solve the structures of numerous Family A and Family B G protein-coupled receptors (GPCRs) in complex with G proteins and arrestins, as well as several Family C GPCRs. Determination of these structures has been facilitated by the presence of large extramembrane components (such as G protein, arrestin, or Venus flytrap domains) in these complexes that aid in particle alignment during the processing of the cryo-EM data. In contrast, determination of the inactive state structure of Family A GPCRs is more challenging due to the relatively small size of the seven transmembrane domain (7TM) and to the surrounding detergent micelle that, in the absence of other features, make particle alignment impossible.

Signaling Modulation Mediated by Ligand Water Interactions with the Sodium Site at μOR.

The mu opioid receptor (μOR) is a target for clinically used analgesics. However, adverse effects, such as respiratory depression and physical dependence, necessitate the development of alternative treatments. Recently we reported a novel strategy to design functionally selective opioids by targeting the sodium binding allosteric site in μOR with a supraspinally active analgesic named .

Neuroanatomical and clinical factors predicting future cognitive impairment.

Identifying cognitively normal (CN) older adults who will convert to cognitive impairment (CI) due to Alzheimer's disease is crucial for early intervention. Clinical and neuroimaging measures were acquired from 301 CN adults who converted to CI within 15 years of baseline, and 294 who did not. Regional volumes and brain age measures were extracted from T-weighted magnetic resonance images.

A µ-opioid receptor modulator that works cooperatively with naloxone.

The µ-opioid receptor (µOR) is a well-established target for analgesia, yet conventional opioid receptor agonists cause serious adverse effects, notably addiction and respiratory depression. These factors have contributed to the current opioid overdose epidemic driven by fentanyl, a highly potent synthetic opioid. µOR negative allosteric modulators (NAMs) may serve as useful tools in preventing opioid overdose deaths, but promising chemical scaffolds remain elusive.

Microscale geometrical modulation of PIEZO1 mediated mechanosensing through cytoskeletal redistribution.

The microgeometry of the cellular microenvironment profoundly impacts cellular behaviors, yet the link between it and the ubiquitously expressed mechanosensitive ion channel PIEZO1 remains unclear. Herein, we describe a fluorescent micropipette aspiration assay that allows for simultaneous visualization of intracellular calcium dynamics and cytoskeletal architecture in real-time, under varied micropipette geometries. By integrating elastic shell finite element analysis with fluorescent lifetime imaging microscopy and employing PIEZO1-specific transgenic red blood cells and HEK cell lines, we demonstrate a direct correlation between the microscale geometry of aspiration and PIEZO1-mediated calcium signaling.

Stepwise activation of a metabotropic glutamate receptor.

Metabotropic glutamate receptors belong to a family of G protein-coupled receptors that are obligate dimers and possess a large extracellular ligand-binding domain that is linked via a cysteine-rich domain to their 7-transmembrane domain. Upon activation, these receptors undergo a large conformational change to transmit the ligand binding signal from the extracellular ligand-binding domain to the G protein-coupling 7-transmembrane domain. In this manuscript, we propose a model for a sequential, multistep activation mechanism of metabotropic glutamate receptor subtype 5.

Protein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis.

The essence of difference between hemostasis and thrombosis is that the clotting reaction is a highly fine-tuned process. Vascular protein disulfide isomerase (PDI) represents a critical mechanism regulating the functions of hemostatic proteins. Herein we show that histidine-rich glycoprotein (HRG) is a substrate of PDI.

Structure and dynamics determine G protein coupling specificity at a class A GPCR.

G protein coupled receptors (GPCRs) exhibit varying degrees of selectivity for different G protein isoforms. Despite the abundant structures of GPCR-G protein complexes, little is known about the mechanism of G protein coupling specificity. The β2-adrenergic receptor is an example of GPCR with high selectivity for Gαs, the stimulatory G protein for adenylyl cyclase, and much weaker for the Gαi family of G proteins inhibiting adenylyl cyclase.

Fluorescence-coupled Micropipette Aspiration Assay to Investigate Red Blood Cell Mechanosensing.

Micropipette aspiration assays have long been a cornerstone for the investigation of live-cell mechanics, offering insights into cellular responses to mechanical stress. This paper details an innovative adaptation of the fluorescence-coupled micropipette aspiration (fMPA) assay. The fMPA assay introduces the capability to administer precise mechanical forces while concurrently monitoring the live-cell mechanotransduction processes mediated by ion channels.

Endoplasmic Reticulum Protein 72 Regulates Integrin Mac-1 Activity to Influence Neutrophil Recruitment.

Background: Integrins mediate the adhesion, crawling, and migration of neutrophils during vascular inflammation. Thiol exchange is important in the regulation of integrin functions. ERp72 (endoplasmic reticulum-resident protein 72) is a member of the thiol isomerase family responsible for the catalysis of disulfide rearrangement.

Biomembrane force probe (BFP): Design, advancements, and recent applications to live-cell mechanobiology.

Mechanical forces play a vital role in biological processes at molecular and cellular levels, significantly impacting various diseases such as cancer, cardiovascular disease, and COVID-19. Recent advancements in dynamic force spectroscopy (DFS) techniques have enabled the application and measurement of forces and displacements with high resolutions, providing crucial insights into the mechanical pathways underlying these diseases. Among DFS techniques, the biomembrane force probe (BFP) stands out for its ability to measure bond kinetics and cellular mechanosensing with pico-newton and nano-meter resolutions.

Step-wise activation of a Family C GPCR.

Metabotropic glutamate receptors belong to a family of G protein-coupled receptors that are obligate dimers and possess a large extracellular ligand-binding domain (ECD) that is linked via a cysteine-rich domain (CRDs) to their 7-transmembrane (TM) domain. Upon activation, these receptors undergo a large conformational change to transmit the ligand binding signal from the ECD to the G protein-coupling TM. In this manuscript, we propose a model for a sequential, multistep activation mechanism of metabotropic glutamate receptor subtype 5.

Discovery of VH domains that allosterically inhibit ENPP1.

Ectodomain phosphatase/phosphodiesterase-1 (ENPP1) is overexpressed on cancer cells and functions as an innate immune checkpoint by hydrolyzing extracellular cyclic guanosine monophosphate adenosine monophosphate (cGAMP). Biologic inhibitors have not yet been reported and could have substantial therapeutic advantages over current small molecules because they can be recombinantly engineered into multifunctional formats and immunotherapies. Here we used phage and yeast display coupled with in cellulo evolution to generate variable heavy (VH) single-domain antibodies against ENPP1 and discovered a VH domain that allosterically inhibited the hydrolysis of cGAMP and adenosine triphosphate (ATP).

Engineered symbiotic bacteria interfering Nosema redox system inhibit microsporidia parasitism in honeybees.

Nosema ceranae is an intracellular parasite invading the midgut of honeybees, which causes serious nosemosis implicated in honeybee colony losses worldwide. The core gut microbiota is involved in protecting against parasitism, and the genetically engineering of the native gut symbionts provides a novel and efficient way to fight pathogens. Here, using laboratory-generated bees mono-associated with gut members, we find that Snodgrassella alvi inhibit microsporidia proliferation, potentially via the stimulation of host oxidant-mediated immune response.

Structural basis for activation of CB1 by an endocannabinoid analog.

Endocannabinoids (eCBs) are endogenous ligands of the cannabinoid receptor 1 (CB1), a G protein-coupled receptor that regulates a number of therapeutically relevant physiological responses. Hence, understanding the structural and functional consequences of eCB-CB1 interactions has important implications for designing effective drugs targeting this receptor. To characterize the molecular details of eCB interaction with CB1, we utilized AMG315, an analog of the eCB anandamide to determine the structure of the AMG315-bound CB1 signaling complex.

Negative allosteric modulation of the glucagon receptor by RAMP2.

Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6 transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface.

Facility planning and schedule design in the pandemic: Eliminating contacts at construction workplace.

The construction industry has been severely affected by the COVID-19 pandemic and the associated restrictions on person-to-person contacts issued by the government. A construction site usually has a high number of workers working at the same time; therefore, the question of how to ensure their safety during the pandemic-that is, how to protect them from getting infected-has become an urgent problem. In this study, we propose a bi-objective integer programming model to establish the optimal schedule plan under COVID-19 regulations.

Structural and dynamic insights into supra-physiological activation and allosteric modulation of a muscarinic acetylcholine receptor.

The M2 muscarinic receptor (M2R) is a prototypical G-protein-coupled receptor (GPCR) that serves as a model system for understanding GPCR regulation by both orthosteric and allosteric ligands. Here, we investigate the mechanisms governing M2R signaling versatility using cryo-electron microscopy (cryo-EM) and NMR spectroscopy, focusing on the physiological agonist acetylcholine and a supra-physiological agonist iperoxo, as well as a positive allosteric modulator LY2119620. These studies reveal that acetylcholine stabilizes a more heterogeneous M2R-G-protein complex than iperoxo, where two conformers with distinctive G-protein orientations were determined.

Acoustic Force-Based Cell-Matrix Avidity Measurement in High Throughput.

Cancer cells interacting with the extracellular matrix (ECM) in the tumor microenvironment is pivotal for tumorigenesis, invasion, and metastasis. Cell-ECM adhesion has been intensively studied in cancer biology in the past decades to understand the molecular mechanisms underlying the adhesion events and extracellular mechanosensing, as well as develop therapeutic strategies targeting the cell adhesion molecules. Many methods have been established to measure the cell-ECM adhesion strength and correlate it with the metastatic potential of certain cancer types.

Structure-based design of bitopic ligands for the µ-opioid receptor.

Mu-opioid receptor (µOR) agonists such as fentanyl have long been used for pain management, but are considered a major public health concern owing to their adverse side effects, including lethal overdose. Here, in an effort to design safer therapeutic agents, we report an approach targeting a conserved sodium ion-binding site found in µOR and many other class A G-protein-coupled receptors with bitopic fentanyl derivatives that are functionalized via a linker with a positively charged guanidino group. Cryo-electron microscopy structures of the most potent bitopic ligands in complex with µOR highlight the key interactions between the guanidine of the ligands and the key Asp residue in the Na site.

Insights into distinct signaling profiles of the µOR activated by diverse agonists.

Drugs targeting the μ-opioid receptor (μOR) are the most effective analgesics available but are also associated with fatal respiratory depression through a pathway that remains unclear. Here we investigated the mechanistic basis of action of lofentanil (LFT) and mitragynine pseudoindoxyl (MP), two μOR agonists with different safety profiles. LFT, one of the most lethal opioids, and MP, a kratom plant derivative with reduced respiratory depression in animal studies, exhibited markedly different efficacy profiles for G protein subtype activation and β-arrestin recruitment.

MASR: A novel monitoring method coupled with interpretation platform for near-term management in thermal stratified reservoirs.

Good water quality is critical to public health and aquatic ecological security of global reservoirs. In stratified reservoirs, increasing near-term management demands foster extremely high monitoring and forecasting needs. In this study, a management assistant for stratified reservoirs (MASR) was developed, including a wave-driven monitoring platform and interpretation platform for multiple reservoir water quality variables.

3,4-Bis(hydroxymethyl)hexane-1,6-diol-based Maltosides (HDMs) for Membrane-Protein Study: Importance of Detergent Rigidity-Flexibility Balance in Protein Stability.

Detergents have been major contributors to membrane-protein structural study for decades. However, membrane proteins solubilized in conventional detergents tend to aggregate or denature over time. Stability of large eukaryotic membrane proteins with complex structures tends to be particularly poor, necessitating development of novel detergents with improved properties.

Mechano-covalent protection of coagulation factor VIII by von Willebrand factor.

von Willebrand factor (VWF) is the protective carrier of procoagulant factor VIII (FVIII) in the shear forces of the circulation, prolonging its half-life and delivering it to the developing thrombus. Using force spectroscopy, VWF-FVIII complex formation is characterized by catch-bond behavior in which force first decelerates then accelerates bond dissociation. Patients with mutations in VWF at the FVIII binding site phenocopies hemophilia A and the most common mutations are of cysteine residues involving multiple disulfide bonds.