Efficacy of Alkaline Phosphatase in Critically Ill Patients with COVID-19: A Multicentre Investigator-Initiated Double-Blind Randomised Placebo-Controlled Trial.

BACKGROUND: Efforts to identify therapies to treat hospitalised patients with COVID-19 are being continued. Alkaline phosphatase (AP) dephosphorylates pro-inflammatory adenosine triphosphate (ATP) into anti-inflammatory adenosine. METHODS: In a randomised controlled trial, we investigated the safety and efficacy of AP in patients with SARS-CoV-2 infection admitted to the ICU. AP or a placebo was administered for four days following admission to the ICU. The primary outcome was the duration of mechanical ventilation. Mortality in 28 days, acute kidney injury, need for reintubation, safety, and inflammatory markers relevant to the described high cytokine release associated with SARS-CoV-2 infection were the secondary outcomes. RESULTS: Between December 2020 and March 2022, 97 patients (of the intended 132) were included, of which 51 were randomised to AP. The trial was terminated prematurely based on meeting the threshold for futility. Compared to the placebo, AP did not affect the duration of mechanical ventilation (9.0 days vs. 9.3 days, p = 1.0). No safety issues were observed. After 28 days, mortality was 9 (18%) in the AP group versus 6 (13%) in the placebo group (p = 0.531). Additionally, no statistically significant differences between the AP and the placebo were observed for the other secondary outcomes. CONCLUSIONS: Alkaline phosphatase (AP) therapy in COVID-19 ICU patients showed no significant benefits in this trial.

Tibial Nerve Block as Treatment of Chronic Foot Pain.

Background: Minimally invasive therapies can alleviate pain and improve walking in individuals with persistent foot and ankle pain. Objectives: The current study aimed to define the protracted consequences of tibial nerve blocks with steroids for individuals with persistent foot pain and to investigate the link between the thermography of the plantar foot and the beneficial effect of a tibial nerve block with steroids. Methods: All patients with chronic foot pain (n = 45) in this cohort underwent a block of the tibial nerve in the Department of Pain Therapy of Pain Clinic De Bilt, Utrecht, Netherlands, within November 2019 to April 2020. The thermographic images of patients were taken before and after injection. Results were retrospectively evaluated after 18 months. Results: In this study, 53% of the patients had pain relief at 7 weeks of follow-up with a unilateral or bilateral block of the tibial nerve. An improvement in walking distance was reported by 22% of the patients. Side effects of the tibial nerve block reported at 7 weeks of follow-up increased pain (5%) and the occurrence of leg cramps (5%) among the treated patients. At 18 months, 45% of the successfully treated feet still had benefits. A difference between the big toe's temperature and the foot's average temperature of less than -0.9°C on thermography before and after the tibial nerve block can predict a beneficial result of therapy. Conclusions: Tibial nerve block provides a safe, minimally invasive treatment option for almost half of the patients with painful feet in this cohort, and when successful, it can last a long term. Thermographic imaging of the plantar foot can predict only to a small extent the beneficial effect of the tibial nerve block with steroids on foot pain. Tibial nerve block should be considered when custom foot orthoses have been inadequate for pain relief or restricted walking distance.

A multimodal pain protocol for treatment of vaso-occlusive crisis in patients with sickle cell disease: Implementation and evaluation.

OBJECTIVES: Pain management during a vaso-occlusive crisis (VOC) for patients with sickle cell disease (SCD) remains a major challenge and strongly depends on opioids. We developed a multimodality pain protocol for rapid, opioid-sparing pain treatment of VOC and evaluated its feasibility. METHODS: Patients were included for evaluation if they were ≥18 years, diagnosed with SCD and visited the emergency department (ED) because of VOC between July 2018 and December 2020. Primary evaluation outcome was the feasibility of multimodal pain analgesia (i.e., the use of at least two analgesics with different underlying mechanisms of action). RESULTS: A total of 131 SCD patients visited the ED because of VOC with a total of 550 ED presentations, of which 377 were eventually hospitalised. A total of 508 (92.4%) ED presentations and 374 (99.2%) hospital admissions received multimodal pain treatment. Time to first administration of an opioid was median [IQR] 34.0 [21.0-62.0] minutes. CONCLUSION: The implementation of a pain protocol using multimodal analgesia for VOC in patients with SCD appeared to be feasible and facilitated rapid administration of opioids. Controlled trials are needed to investigate the effectiveness of multimodal analgesia on pain and should focus on patient reported outcome measures.

Autoregulation of GPCR signalling through the third intracellular loop.

The third intracellular loop (ICL3) of the G protein-coupled receptor (GPCR) fold is important for the signal transduction process downstream of receptor activation1-3. Despite this, the lack of a defined structure of ICL3, combined with its high sequence divergence among GPCRs, complicates characterization of its involvement in receptor signalling4. Previous studies focusing on the ß2 adrenergic receptor (ß2AR) suggest that ICL3 is involved in the structural process of receptor activation and signalling5-7. Here we derive mechanistic insights into the role of ICL3 in ß2AR signalling, observing that ICL3 autoregulates receptor activity through a dynamic conformational equilibrium between states that block or expose the receptor's G protein-binding site. We demonstrate the importance of this equilibrium for receptor pharmacology, showing that G protein-mimetic effectors bias the exposed states of ICL3 to allosterically activate the receptor. Our findings additionally reveal that ICL3 tunes signalling specificity by inhibiting receptor coupling to G protein subtypes that weakly couple to the receptor. Despite the sequence diversity of ICL3, we demonstrate that this negative G protein-selection mechanism through ICL3 extends to GPCRs across the superfamily, expanding the range of known mechanisms by which receptors mediate G protein subtype selective signalling. Furthermore, our collective findings suggest ICL3 as an allosteric site for receptor- and signalling pathway-specific ligands.

Multimodal regulation of myosin VI ensemble transport by cargo adaptor protein GIPC.

A range of cargo adaptor proteins are known to recruit cytoskeletal motors to distinct subcellular compartments. However, the structural impact of cargo recruitment on motor function is poorly understood. Here, we dissect the multimodal regulation of myosin VI activity through the cargo adaptor GAIP-interacting protein, C terminus (GIPC), whose overexpression with this motor in cancer enhances cell migration. Using a range of biophysical techniques, including motility assays, FRET-based conformational sensors, optical trapping, and DNA origami-based cargo scaffolds to probe the individual and ensemble properties of GIPC-myosin VI motility, we report that the GIPC myosin-interacting region (MIR) releases an autoinhibitory interaction within myosin VI. We show that the resulting conformational changes in the myosin lever arm, including the proximal tail domain, increase the flexibility of the adaptor-motor linkage, and that increased flexibility correlates with faster actomyosin association and dissociation rates. Taken together, the GIPC MIR-myosin VI interaction stimulates a twofold to threefold increase in ensemble cargo speed. Furthermore, the GIPC MIR-myosin VI ensembles yield similar cargo run lengths as forced processive myosin VI dimers. We conclude that the emergent behavior from these individual aspects of myosin regulation is the fast, processive, and smooth cargo transport on cellular actin networks. Our study delineates the multimodal regulation of myosin VI by the cargo adaptor GIPC, while highlighting linkage flexibility as a novel biophysical mechanism for modulating cellular cargo motility.

ß2-adrenoceptor ligand efficacy is tuned by a two-stage interaction with the Gαs C terminus.

Classical pharmacological models have incorporated an "intrinsic efficacy" parameter to capture system-independent effects of G protein-coupled receptor (GPCR) ligands. However, the nonlinear serial amplification of downstream signaling limits quantitation of ligand intrinsic efficacy. A recent biophysical study has characterized a ligand "molecular efficacy" that quantifies the influence of ligand-dependent receptor conformation on G protein activation. Nonetheless, the structural translation of ligand molecular efficacy into G protein activation remains unclear and forms the focus of this study. We first establish a robust, accessible, and sensitive assay to probe GPCR interaction with G protein and the Gα C terminus (G-peptide), an established structural determinant of G protein selectivity. We circumvent the need for extensive purification protocols by the single-step incorporation of receptor and G protein elements into giant plasma membrane vesicles (GPMVs). We use previously established SPASM FRET sensors to control the stoichiometry and effective concentration of receptor-G protein interactions. We demonstrate that GPMV-incorporated sensors (v-SPASM sensors) provide enhanced dynamic range, expression-insensitive readout, and a reagent level assay that yields single point measurements of ligand molecular efficacy. Leveraging this technology, we establish the receptor-G-peptide interaction as a sufficient structural determinant of this receptor-level parameter. Combining v-SPASM measurements with molecular dynamics (MD) simulations, we elucidate a two-stage receptor activation mechanism, wherein receptor-G-peptide interactions in an intermediate orientation alter the receptor conformational landscape to facilitate engagement of a fully coupled orientation that tunes G protein activation.

KIF13A motors are regulated by Rab22A to function as weak dimers inside the cell.

Endocytic recycling is a complex itinerary, critical for many cellular processes. Membrane tubulation is a hallmark of recycling endosomes (REs), mediated by KIF13A, a kinesin-3 family motor. Understanding the regulatory mechanism of KIF13A in RE tubulation and cargo recycling is of fundamental importance but is overlooked. Here, we report a unique mechanism of KIF13A dimerization modulated by Rab22A, a small guanosine triphosphatase, during RE tubulation. A conserved proline between neck coil-coiled-coil (NC-CC1) domains of KIF13A creates steric hindrance, rendering the motors as inactive monomers. Rab22A plays an unusual role by binding to NC-CC1 domains of KIF13A, relieving proline-mediated inhibition and facilitating motor dimerization. As a result, KIF13A motors produce balanced motility and force against multiple dyneins in a molecular tug-of-war to regulate RE tubulation and homeostasis. Together, our findings demonstrate that KIF13A motors are tuned at a single-molecule level to function as weak dimers on the cellular cargo.

Kinase inhibitors allosterically disrupt a regulatory interaction to enhance PKCα membrane translocation.

The eukaryotic kinase domain has multiple intrinsically disordered regions whose conformation dictates kinase activity. Small molecule kinase inhibitors (SMKIs) rely on disrupting the active conformations of these disordered regions to inactivate the kinase. While SMKIs are selected for their ability to cause this disruption, the allosteric effects of conformational changes in disordered regions is limited by a lack of dynamic information provided by traditional structural techniques. In this study, we integrated multiscale molecular dynamics simulations with FRET sensors to characterize a novel allosteric mechanism that is selectively triggered by SMKI binding to the protein kinase Cα domain. The indole maleimide inhibitors BimI and sotrastaurin were found to displace the Gly-rich loop (G-loop) that normally shields the ATP-binding site. Displacement of the G-loop interferes with a newly identified, structurally conserved binding pocket for the C1a domain on the N lobe of the kinase domain. This binding pocket, in conjunction with the N-terminal regulatory sequence, masks a diacylglycerol (DAG) binding site on the C1a domain. SMKI-mediated displacement of the G-loop released C1a and exposed the DAG binding site, enhancing protein kinase Cα translocation both to synthetic lipid bilayers and to live cell membranes in the presence of DAG. Inhibitor chemotype determined the extent of the observed allosteric effects on the kinase domain and correlated with the extent of membrane recruitment. Our findings demonstrate the allosteric effects of SMKIs beyond the confines of kinase catalytic conformation and provide an integrated computational-experimental paradigm to investigate parallel mechanisms in other kinases.

ER/K-link-Leveraging a native protein linker to probe dynamic cellular interactions.

ER/K α-helices are a subset of single alpha helical domains, which exhibit unusual stability as isolated protein secondary structures. They adopt an elongated structural conformation, while regulating the frequency of interactions between proteins or polypeptides fused to their ends. Here we review recent advances on the structure, stability and function of ER/K α-helices as linkers (ER/K linkers) in native proteins. We describe methodological considerations in the molecular cloning, protein expression and measurement of interaction strengths, using sensors incorporating ER/K linkers. We highlight biological insights obtained over the last decade by leveraging distinct biophysical features of ER/K-linked sensors. We conclude with the outlook for the use of ER/K linkers in the selective modulation of dynamic cellular interactions.

Dynamic multimerization of Dab2-Myosin VI complexes regulates cargo processivity while minimizing cortical actin reorganization.

Myosin VI ensembles on endocytic cargo facilitate directed transport through a dense cortical actin network. Myosin VI is recruited to clathrin-coated endosomes via the cargo adaptor Dab2. Canonically, it has been assumed that the interactions between a motor and its cargo adaptor are stable. However, it has been demonstrated that the force generated by multiple stably attached motors disrupts local cytoskeletal architecture, potentially compromising transport. In this study, we demonstrate that dynamic multimerization of myosin VI-Dab2 complexes facilitates cargo processivity without significant reorganization of cortical actin networks. Specifically, we find that Dab2 myosin interacting region (MIR) binds myosin VI with a moderate affinity (184 nM) and single-molecule kinetic measurements demonstrate a high rate of turnover (1 s-1) of the Dab2 MIR-myosin VI interaction. Single-molecule motility shows that saturating Dab2-MIR concentration (2 µM) promotes myosin VI homodimerization and processivity with run lengths comparable with constitutive myosin VI dimers. Cargo-mimetic DNA origami scaffolds patterned with Dab2 MIR-myosin VI complexes are weakly processive, displaying sparse motility on single actin filaments and "stop-and-go" motion on a cellular actin network. On a minimal actin cortex assembled on lipid bilayers, unregulated processive movement by either constitutive myosin V or VI dimers results in actin remodeling and foci formation. In contrast, Dab2 MIR-myosin VI interactions preserve the integrity of a minimal cortical actin network. Taken together, our study demonstrates the importance of dynamic motor-cargo association in enabling cargo transportation without disrupting cytoskeletal organization.

Minute-scale persistence of a GPCR conformation state triggered by non-cognate G protein interactions primes signaling.

Despite the crowded nature of the cellular milieu, ligand-GPCR-G protein interactions are traditionally viewed as spatially and temporally isolated events. In contrast, recent reports suggest the spatial and temporal coupling of receptor-effector interactions, with the potential to diversify downstream responses. In this study, we combine protein engineering of GPCR-G protein interactions with affinity sequestration and photo-manipulation of the crucial Gα C terminus, to demonstrate the temporal coupling of cognate and non-cognate G protein interactions through priming of the GPCR conformation. We find that interactions of the Gαs and Gαq C termini with the ß2-adrenergic receptor (ß2-AR), targeted at the G-protein-binding site, enhance Gs activation and cyclic AMP levels. ß2-AR-Gα C termini interactions alter receptor conformation, which persists for ~90 s following Gα C terminus dissociation. Non-cognate G-protein expression levels impact cognate signaling in cells. Our study demonstrates temporal allostery in GPCRs, with implications for the modulation of downstream responses through the canonical G-protein-binding interface.

Engaging myosin VI tunes motility, morphology and identity in endocytosis.

While unconventional myosins interact with different stages of the endocytic pathway, they are ascribed a transport function that is secondary to the protein complexes that control organelle identity. Endosomes are subject to a dynamic, continuous flux of proteins that control their characteristic properties, including their motility within the cell. Efforts to describe the changes in identity of this compartment have largely focused on the adaptors present on the compartment and not on the motile properties of the compartment itself. In this study, we use a combination of optogenetic and chemical-dimerization strategies to target exogenous myosin VI to early endosomes, and probe its influence on organelle motility, morphology and identity. Our analysis across timescales suggests a model wherein the artificial engagement of myosin VI motility on early endosomes restricts microtubule-based motion, followed by morphological changes characterized by the rapid condensation and disintegration of organelles, ultimately leading to the enhanced overlap of markers that demarcate endosomal compartments. Together, our findings show that synthetic engagement of myosin VI motility is sufficient to alter organelle homeostasis in the endocytic pathway.

ER/K linked GPCR-G protein fusions systematically modulate second messenger response in cells.

FRET and BRET approaches are well established for detecting ligand induced GPCR-G protein interactions in cells. Currently, FRET/BRET assays rely on co-expression of GPCR and G protein, and hence depend on the stoichiometry and expression levels of the donor and acceptor probes. On the other hand, GPCR-G protein fusions have been used extensively to understand the selectivity of GPCR signaling pathways. However, the signaling properties of fusion proteins are not consistent across GPCRs. In this study, we describe and characterize novel sensors based on the Systematic Protein Affinity Strength Modulation (SPASM) technique. Sensors consist of a GPCR and G protein tethered by an ER/K linker flanked by FRET probes. SPASM sensors are tested for the ß2-, α1-, and α2- adrenergic receptors, and adenosine type 1 receptor (A1R), tethered to Gαs-XL, Gαi2, or Gαq subunits. Agonist stimulation of ß2-AR and α2-AR increases FRET signal comparable to co-expressed FRET/BRET sensors. SPASM sensors also retain signaling through the endogenous G protein milieu. Importantly, ER/K linker length systematically tunes the GPCR-G protein interaction, with consequent modulation of second messenger signaling for cognate interactions. SPASM GPCR sensors serve the dual purpose of detecting agonist-induced changes in GPCR-G protein interactions, and linking these changes to downstream signaling.

Priming GPCR signaling through the synergistic effect of two G proteins.

Although individual G-protein-coupled receptors (GPCRs) are known to activate one or more G proteins, the GPCR-G-protein interaction is viewed as a bimolecular event involving the formation of a ternary ligand-GPCR-G-protein complex. Here, we present evidence that individual GPCR-G-protein interactions can reinforce each other to enhance signaling through canonical downstream second messengers, a phenomenon we term "GPCR priming." Specifically, we find that the presence of noncognate Gq protein enhances cAMP stimulated by two Gs-coupled receptors, ß2-adrenergic receptor (ß2-AR) and D1 dopamine receptor (D1-R). Reciprocally, Gs enhances IP1 through vasopressin receptor (V1A-R) but not α1 adrenergic receptor (α1-AR), suggesting that GPCR priming is a receptor-specific phenomenon. The C terminus of either the Gαs or Gαq subunit is sufficient to enhance Gα subunit activation and cAMP levels. Interaction of Gαs or Gαq C termini with the GPCR increases signaling potency, suggesting an altered GPCR conformation as the underlying basis for GPCR priming. We propose three parallel mechanisms involving (i) sequential G-protein interactions at the cognate site, (ii) G-protein interactions at distinct allosteric and cognate sites on the GPCR, and (iii) asymmetric GPCR dimers. GPCR priming suggests another layer of regulation in the classic GPCR ternary-complex model, with broad implications for the multiplicity inherent in signaling networks.

The Role of Regulatory Domains in Maintaining Autoinhibition in the Multidomain Kinase PKCα.

Resolving the conformational dynamics of large multidomain proteins has proven to be a significant challenge. Here we use a variety of techniques to dissect the roles of individual protein kinase Cα (PKCα) regulatory domains in maintaining catalytic autoinhibition. We find that whereas the pseudosubstrate domain is necessary for autoinhibition it is not sufficient. Instead, each regulatory domain (C1a, C1b, and C2) appears to strengthen the pseudosubstrate-catalytic domain interaction in a nucleotide-dependent manner. The pseudosubstrate and C1a domains, however, are minimally essential for maintaining the inactivated state. Furthermore, disrupting known interactions between the C1a and other regulatory domains releases the autoinhibited interaction and increases basal activity. Modulating this interaction between the catalytic and regulatory domains reveals a direct correlation between autoinhibition and membrane translocation following PKC activation.

Calcium Stimulates Self-Assembly of Protein Kinase C α In Vitro.

Protein kinase C α (PKCα) is a nodal regulator in several intracellular signaling networks. PKCα is composed of modular domains that interact with each other to dynamically regulate spatial-temporal function. We find that PKCα specifically, rapidly and reversibly self-assembles in the presence of calcium in vitro. This phenomenon is dependent on, and can be modulated by an intramolecular interaction between the C1a and C2 protein domains of PKCα. Next, we monitor self-assembly of PKC-mCitrine fusion proteins using time-resolved and steady-state homoFRET. HomoFRET between full-length PKCα molecules is observed when in solution with both calcium and liposomes containing either diacylglycerol (DAG) or phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Surprisingly, the C2 domain is sufficient to cluster on liposomes containing PI(4,5)P2, indicating the C1a domain is not required for self-assembly in this context. We conclude that three distinct clustered states of PKCα can be formed depending on what combination of cofactors are bound, but Ca2+ is minimally required and sufficient for clustering.

Correlation between Activity and Domain Complementation in Adenylyl Cyclase Demonstrated with a Novel Fluorescence Resonance Energy Transfer Sensor.

Adenylyl cyclase (AC) activity relies on multiple effectors acting through distinct binding sites. Crystal structures have revealed the location of these sites, and biochemical studies have explored the kinetics of ACs, but the interplay between conformation and activity remains incompletely understood. Here, we describe a novel fluorescence resonance energy transfer (FRET) sensor that functions both as a soluble cyclase and a reporter of complementation within the catalytic domain. We report a strong linear correlation between catalytic domain complementation and cyclase activity upon stimulation with forskolin and Gαs. Exploiting this, we dissect the mechanism of action of a series of forskolin analogs and a P-site inhibitor, 2'-d3'-AMP. Finally, we demonstrate that this sensor is functional in live cells, wherein it reports forskolin-stimulated activity of AC.

Optofluidic lasers with a single molecular layer of gain.

We achieve optofluidic lasers with a single molecular layer of gain, in which green fluorescent protein, dye-labeled bovine serum albumin, and dye-labeled DNA, are used as the gain medium and attached to the surface of a ring resonator via surface immobilization biochemical methods. It is estimated that the surface density of the gain molecules is on the order of 10(12) cm(-2), sufficient for lasing under pulsed optical excitation. It is further shown that the optofluidic laser can be tuned by energy transfer mechanisms through biomolecular interactions. This work not only opens a door to novel photonic devices that can be controlled at the level of a single molecular layer but also provides a promising sensing platform to analyze biochemical processes at the solid-liquid interface.

Natural killer cells eradicate galectin-1-deficient glioma in the absence of adaptive immunity.

Natural killer (NK) cells safeguard against early tumor formation by destroying transformed target cells in a process referred to as NK immune surveillance. However, the immune escape mechanisms used by malignant brain tumors to subvert this innate type of immune surveillance remain unclear. Here we show that malignant glioma cells suppress NK immune surveillance by overexpressing the ß-galactoside-binding lectin galectin-1. Conversely, galectin-1-deficient glioma cells could be eradicated by host NK cells before the initiation of an antitumor T-cell response. In vitro experiments demonstrated that galectin-1-deficient GL26-Cit glioma cells are ∼3-fold more sensitive to NK-mediated tumor lysis than galectin-1-expressing cells. Our findings suggest that galectin-1 suppression in human glioma could improve patient survival by restoring NK immune surveillance that can eradicate glioma cells. Cancer Res; 74(18); 5079-90. ©2014 AACR.

Conserved modular domains team up to latch-open active protein kinase Cα.

Signaling proteins comprised of modular domains have evolved along with multicellularity as a method to facilitate increasing intracellular bandwidth. The effects of intramolecular interactions between modular domains within the context of native proteins have been largely unexplored. Here we examine intra- and intermolecular interactions in the multidomain signaling protein, protein kinase Cα (PKCα). We identify three interactions between two activated PKC molecules that synergistically stabilize a nanomolar affinity homodimer. Disruption of the homodimer results in a loss of PKC-mediated ERK1/2 phosphorylation, whereas disruption of the auto-inhibited state promotes the homodimer and prolongs PKC membrane localization. These observations support a novel regulatory mechanism wherein homodimerization dictates the equilibrium between the auto-inhibited and active states of PKC by sequestering auto-inhibitory interactions. Our findings underscore the physiological importance of context-dependent modular domain interactions in cell signaling.