High-Throughput NanoBiT-Based Screening for Inhibitors of HIV-1 Vpu and Host BST-2 Protein Interaction

Bone marrow stromal cell antigen 2 (BST-2), also known as CD317 or tetherin, has been identified as a host restriction factor that suppresses the release of enveloped viruses from host cells by physically tethering viral particles to the cell surface; however, this host defense can be subverted by multiple viruses. For example, human immunodeficiency virus (HIV)-1 encodes a specific accessory protein, viral protein U (Vpu), to counteract BST-2 by binding to it and directing its lysosomal degradation. Thus, blocking the interaction between Vpu and BST-2 will provide a promising strategy for anti-HIV therapy. Here, we report a NanoLuc Binary Technology (NanoBiT)-based high-throughput screening assay to detect inhibitors that disrupt the Vpu-BST-2 interaction. Out of more than 1000 compounds screened, four inhibitors were identified with strong activity at nontoxic concentrations. In subsequent cell-based BST-2 degradation assays, inhibitor Y-39983 HCl restored the cell-surface and total cellular level of BST-2 in the presence of Vpu. Furthermore, the Vpu-mediated enhancement of pesudotyped viral particle production was inhibited by Y-39983 HCl. Our findings indicate that our newly developed assay can be used for the discovery of potential antiviral molecules with novel mechanisms of action.     

Two Motors and One Spring: Hypothetic Roles of Non-Muscle Myosin II and Submembrane Actin-Based Cytoskeleton in Cell Volume Sensing

Changes in plasma membrane curvature and intracellular ionic strength are two key features of cell volume perturbations. In this hypothesis we present a model of the responsible molecular apparatus which is assembled of two molecular motors [non-muscle myosin II (NMMII) and protrusive actin polymerization], a spring [a complex between the plasma membrane (PM) and the submembrane actin-based cytoskeleton (smACSK) which behaves like a viscoelastic solid] and the associated signaling proteins. We hypothesize that this apparatus senses changes in both the plasma membrane curvature and the ionic strength and in turn activates signaling pathways responsible for regulatory volume increase (RVI) and regulatory volume decrease (RVD). During cell volume changes hydrostatic pressure (HP) changes drive alterations in the cell membrane curvature. HP difference has opposite directions in swelling versus shrinkage, thus allowing distinction between them. By analogy with actomyosin contractility that appears to sense stiffness of the extracellular matrix we propose that NMMII and actin polymerization can actively probe the transmembrane gradient in HP. Furthermore, NMMII and protein-protein interactions in the actin cortex are sensitive to ionic strength. Emerging data on direct binding to and regulating activities of transmembrane mechanosensors by NMMII and actin cortex provide routes for signal transduction from transmembrane mechanosensors to cell volume regulatory mechanisms.     

Adenylate Kinase and AMP Signaling Networks: Metabolic Monitoring,Signal Communication and Body Energy Sensing

Adenylate kinase and downstream AMP signaling is an integrated metabolic monitoring system which reads the cellular energy state in order to tune and report signals to metabolic sensors. A network of adenylate kinase isoforms (AK1-AK7) are distributed throughout intracellular compartments, interstitial space and body fluids to regulate energetic and metabolic signaling circuits, securing efficient cell energy economy, signal communication and stress response. The dynamics of adenylate kinase-catalyzed phosphotransfer regulates multiple intracellular and extracellular energy-dependent and nucleotide signaling processes, including excitation-contraction coupling, hormone secretion, cell and ciliary motility, nuclear transport, energetics of cell cycle, DNA synthesis and repair, and developmental programming. Metabolomic analyses indicate that cellular, interstitial and blood AMP levels are potential metabolic signals associated with vital functions including body energy sensing, sleep, hibernation and food intake. Either low or excess AMP signaling has been linked to human disease such as diabetes, obesity and hypertrophic cardiomyopathy. Recent studies indicate that derangements in adenylate kinase-mediated energetic signaling due to mutations in AK1, AK2 or AK7 isoforms are associated with hemolytic anemia, reticular dysgenesis and ciliary dyskinesia. Moreover, hormonal, food and antidiabetic drug actions are frequently coupled to alterations of cellular AMP levels and associated signaling. Thus, by monitoring energy state and generating and distributing AMP metabolic signals adenylate kinase represents a unique hub within the cellular homeostatic network.     

Ratiometric Zinc Biosensor Based on Bioluminescence Resonance Energy Transfer: Trace Metal Ion Determination with Tunable Response

Determination of metal ions such as zinc in solution remains an important task in analytical and biological chemistry. We describe a novel zinc ion biosensing approach using a carbonic anhydrase–Oplophorus luciferase fusion protein that employs bioluminescence resonance energy transfer (BRET) to transduce the level of free zinc as a ratio of emission intensities in the blue and orange portions of the spectrum. In addition to high sensitivity (below nanomolar levels) and selectivity, this approach allows both quantitative determination of “free” zinc ion (also termed “mobile” or “labile”) using bioluminescence ratios and determination of the presence of the ion above a threshold simply by the change in color of bioluminescence, without an instrument. The carbonic anhydrase metal ion sensing platform offers well-established flexibility in sensitivity, selectivity, and response kinetics. Finally, bioluminescence labeling has proven an effective approach for molecular imaging in vivo since no exciting light is required; the expressible nature of this sensor offers the prospect of imaging zinc fluxes in vivo.     

模拟酸雨中离子强度与pH值影响土壤缓冲力

酸雨是一种强电解质溶液,它与土壤接触发生液-固反应,酸雨的离子强度对土壤中电性吸附的各种离子起着至关重要的作用.本试验通过运用两种离子强度的模拟酸雨溶液,分别进行四种pH值的模拟酸雨淋滤试验,探讨离子强度与氢离子浓度对土壤缓冲力的影响.结果发现,氢离子浓度和离子强度越大,土壤的缓冲能力就越弱.     

Examination of Intracellular GPCR-Mediated Signaling with High Temporal Resolution

The GTP-binding protein-coupled receptors (GPCRs) play important roles in physiology and neuronal signaling. More than a thousand genes, excluding the olfactory receptors, have been identified that encode these integral membrane proteins. Their pharmacological and functional properties make them fascinating targets for drug development, since various disease states can be treated and overcome by pharmacologically addressing these receptors and/or their downstream interacting partners. The activation of the GPCRs typically causes transient changes in the intracellular second messenger concentrations as well as in membrane conductance. In contrast to ion channel-mediated electrical signaling which results in spontaneous cellular responses, the GPCR-mediated metabotropic signals operate at a different time scale. Here we have studied the kinetics of two common GPCR-induced signaling pathways: (a) Ca²⁺ release from intracellular stores and (b) cyclic adenosine monophosphate (cAMP) production. The latter was monitored via the activation of cyclic nucleotide-gated (CNG) ion channels causing Ca²⁺ influx into the cell. Genetically modified and stably transfected cell lines were established and used in stopped-flow experiments to uncover the individual steps of the reaction cascades. Using two homologous biogenic amine receptors, either coupling to G_o/q or G_s proteins, allowed us to determine the time between receptor activation and signal output. With ~350 ms, the release of Ca²⁺ from intracellular stores was much faster than cAMP-mediated Ca²⁺ entry through CNG channels (~6 s). The measurements with caged compounds suggest that this difference is due to turnover numbers of the GPCR downstream effectors rather than the different reaction cascades, per se.     

Chemical probes of signal-transducing proteins

Protein kinases are key participants in signal transduction pathways. A direct assessment of the relationship between the activity of any given protein kinase and the corresponding cellular phenotype has proven challenging. This is due to the large number of protein kinases encoded by the human genome coupled with intracellular temporal and spatial constraints that appear to further regulate the ultimate response of a cell to a stimulus. Our work has focused on the development of chemical probes to address the complexities associated with protein kinase-mediated cell signaling. These include the acquisition of highly selective substrates and inhibitors for specific members of the protein kinase family, the design and synthesis of light-activated signaling proteins and their corresponding inhibitors, and the preparation of fluorescent reporters of intracellular protein kinase action.     

Development of multimodal membrane adsorbers for antibody purification using atom transfer radical polymerization

This contribution describes a graft polymerization strategy to prepare multimodal membranes, a new class of high-productivity adsorptive materials for the purification of therapeutic proteins. Surface-initiated atom transfer radical polymerization was used to graft poly(glycidyl methacrylate) ‘tentacles’ from the pore surfaces of macroporous regenerated cellulose membranes. Subsequently, 4-mercaptobenzoic acid was coupled to the membranes by an epoxide ring-opening reaction. ATR-FTIR measurements support successful ligand incorporation. Graft polymerization studies from cellulose-coated silicon substrates were done in parallel to measure the thickness evolution of the polymer coating, which plays an important role on protein binding capacities. Protein binding experiments with bovine immunoglobulin G show that the multimodal membranes have high equilibrium capacities, up to 150 mg IgG/mL. The binding capacities are pH-dependent, with maximum binding at pH near the protein isoelectric point. Characteristic of multimodal adsorbers, the membranes retain about 70% of their equilibrium binding capacity at moderate ionic strength (300 mM) and about 40% at high ionic strength (1.6 M).     

AMPK–mTOR Signaling and Cellular Adaptations in Hypoxia

Cellular energy is primarily provided by the oxidative degradation of nutrients coupled with mitochondrial respiration, in which oxygen participates in the mitochondrial electron transport chain to enable electron flow through the chain complex (I–IV), leading to ATP production. Therefore, oxygen supply is an indispensable chapter in intracellular bioenergetics. In mammals, oxygen is delivered by the bloodstream. Accordingly, the decrease in cellular oxygen level (hypoxia) is accompanied by nutrient starvation, thereby integrating hypoxic signaling and nutrient signaling at the cellular level. Importantly, hypoxia profoundly affects cellular metabolism and many relevant physiological reactions induce cellular adaptations of hypoxia-inducible gene expression, metabolism, reactive oxygen species, and autophagy. Here, we introduce the current knowledge of hypoxia signaling with two-well known cellular energy and nutrient sensing pathways, AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin complex 1 (mTORC1). Additionally, the molecular crosstalk between hypoxic signaling and AMPK/mTOR pathways in various hypoxic cellular adaptions is discussed.     

Peptides Derived from Angiogenin Regulate Cellular Copper Uptake

The angiogenin protein (ANG) is one of the most potent endogenous angiogenic factors. In this work we characterized by means of potentiometric, spectroscopic and voltammetric techniques, the copper complex species formed with peptide fragments derived from the N-terminal domain of the protein, encompassing the sequence 1-17 and having free amino, Ang1-17, or acetylated N-terminus group, AcAng1-17, so to explore the role of amino group in metal binding and cellular copper uptake. The obtained data show that amino group is the main copper anchoring site for Ang1-17. The affinity constant values, metal coordination geometry and complexes redox-potentials strongly depend, for both peptides, on the number of copper equivalents added. Confocal laser scanning microscope analysis on neuroblastoma cells showed that in the presence of one equivalent of copper ion, the free amino Ang1-17 increases cellular copper uptake while the acetylated AcAng1-17 strongly decreases the intracellular metal level. The activity of peptides was also compared to that of the protein normally present in the plasma (wtANG) as well as to the recombinant form (rANG) most commonly used in literature experiments. The two protein isoforms bind copper ions but with a different coordination environment. Confocal laser scanning microscope data showed that the wtANG induces a strong increase in intracellular copper compared to control while the rANG decreases the copper signal inside cells. These data demonstrate the relevance of copper complexes’ geometry to modulate peptides’ activity and show that wtANG, normally present in the plasma, can affect cellular copper uptake.     

Effective factors and kinetics study of zinc ion removal from synthetic wastewater by ion flotation

Zn(II) ion removal from synthetic wastewater was investigated using ion flotation in Hallimond tube followed by mechanical cell. The flotation reaction rate curve on the removal of Zn(II) ions in the separation process was divided into two stages, with n ≈ 1 in the first stage and with n ≈ 0 in the second stage. The kinetics of flotation for Zn(II) ion removal was 25 times faster than the kinetics for water removal. The removal kinetics has an inverse relation with the ionic strength and a direct relation with activity coefficients.     

Electrochemical sensing of the ion-channel formation of OmpF

The ion-channel formation of outer membrane protein F (OmpF) can be achieved in phosphatidylcholine bilayer membrane modified on a pyrolytic graphite electrode, which makes it possible to sense the porin functionality conveniently with an electrochemical technique through the change in the redox peaks currents of an electroactive marker. The effect of ionic strength, pH on the ion permeability of OmpF and the effect of the protein concentration on the ion-channel formation have been thus examined. This study may provide a simple and rapid way to probe other similar biological processes within natural cellular membranes.     

Activation of Protease and Luciferase Using Engineered Nostoc punctiforme PCC73102 DnaE Intein with Altered Split Position

Inteins, self-catalytic enzymes, have been widely used in the field of protein engineering and chemical biology. Here, Nostoc punctiforme PCC73102 (Npu) DnaE intein was engineered to have an altered split position. An 11-residue N-intein of DnaE in which Gly and Asp were substituted for Tyr4 and Glu5, respectively, was designed, and the active C-intein variants were acquired by a GFP fluorescence-based screening. The designed N-intein and the obtained active C-intein variants were used to construct a turn-on system for enzyme activities such as human immunodeficiency 1 protease and NanoLuc luciferase. Based on the NanoLuc-intein fusion, we developed two intein pairs, each of which is capable of reacting preferentially, by interchanging the charged amino acids on N- and C-inteins. The specific splicing reactions were easily monitored and discriminated by bioluminescence resonance energy transfer (BRET).     

Ca2+-regulated photoproteins: structural insight into the bioluminescence mechanism

The bioluminescent jellyfish has contributed two famous proteins to modern science: green fluorescent protein or GFP, which finds wide use as a probe in cell biology studies, and aequorin, which has been used for intracellular calcium measurement for more than 30 years. More recently, obelin, a protein from the bioluminescent hydroid and also in the family of what are called "Ca2+-regulated photoproteins", has been shown to have very attractive properties both in general applications and for basic structural biology investigations. This review will survey the new information into their molecular mechanism of bioluminescence action.     

Computational Analysis of Short Linear Motifs in the Spike Protein of SARS-CoV-2 Variants Provides Possible Clues into the Immune Hijack and Evasion Mechanisms of Omicron Variant

Short linear motifs (SLiMs) are short linear sequences that can mediate protein–protein interaction. Mimicking eukaryotic SLiMs to compete with extra- or intracellular binding partners, or to sequester host proteins is the crucial strategy of viruses to pervert the host system. Evolved proteins in viruses facilitate minimal protein–protein interactions that significantly affect intracellular signaling networks. Unfortunately, very little information about SARS-CoV-2 SLiMs is known, especially across SARS-CoV-2 variants. Through the ELM database-based sequence analysis of spike proteins from all the major SARS-CoV-2 variants, we identified four overriding SLiMs in the SARS-CoV-2 Omicron variant, namely, LIG_TRFH_1, LIG_REV1ctd_RIR_1, LIG_CaM_NSCaTE_8, and MOD_LATS_1. These SLiMs are highly likely to interfere with various immune functions, interact with host intracellular proteins, regulate cellular pathways, and lubricate viral infection and transmission. These cellular interactions possibly serve as potential therapeutic targets for these variants, and this approach can be further exploited to combat emerging SARS-CoV-2 variants.     

Plasmid DNA-Based Bioluminescence-Activated System for Photodynamic Therapy in Cancer Treatment

The low depth of tissue penetration by therapeutic light sources severely restricts photodynamic therapy (PDT) in treating deep-seated tumors. Using a luciferase/d -luciferin bioluminescence system to artificially create internal light sources in cells instead of external light sources is an effective means of solving the above problems. However, high-efficiency bioluminescence requires a higher concentration of luciferase in the cell, which poses a considerable challenge to the existing system of enzyme loading, delivery, activity and retention of drugs, and dramatically increases the cost of treatment. We loaded the substrate D-luciferin, and the photosensitizer hypericin into a polyethyleneimine (PEI)-modified nano-calcium phosphate (CaP) to solve this problem. Subsequently, the plasmid DNA containing the luciferase gene was loaded onto it using the high-density positive charge characteristic of PEI from the nanodrug (denoted DHDC). After the DHDC enters the tumor cell, it collapses and releases the plasmid DNA, which uses the intracellular protein synthesis system to continuously and massively express luciferase. Using endogenous ATP, Mg²⁺, and O₂ in cells, luciferase oxidizes d -luciferin and produces luminescence. The luminescence triggers hypericin excitation to generate ROS and kill cancer cells. This study provides a new strategy for the application of bioluminescence in PDT treatment.     

Spatio-Temporal Photoactivation of Cytotoxic Proteins

Protein therapeutics offer exquisite selectivity in targeting cellular processes and behaviors, but are rarely used against non-cell surface targets due to their poor cellular uptake. While cell-penetrating peptides can be used to deliver recombinant proteins to the cytosol, it is generally difficult to selectively deliver active proteins to target cells. Here, we report a recombinantly produced, intracellular protein delivery and targeting platform that uses a photocaged intein to regulate the spatio-temporal activation of protein activity in selected cells upon irradiation with light. The platform was successfully demonstrated for two cytotoxic proteins to selectively kill cancer cells after photoactivation of intein splicing. This platform can generically be applied to any protein whose activity can be disrupted by a fused intein, allowing it to underpin a wide variety of future protein therapeutics.     

Analysis of statistical thermodynamic model for binary protein adsorption equilibria on cation exchange adsorbent

A study of nonlinear competitive adsorption equilibria of proteins is of fundamental importance in understanding the behavior of preparative chromatographic separation. This work describes the nonlinear binary protein adsorption equilibria on ion exchangers by the statistical thermodynamic (ST) model. The single-component and binary protein adsorption isotherms of bovine hemoglobin (Hb) and bovine serum albumin (BSA) on SP Sepharose FF were determined by batch adsorption experiments in 0.05 mol/L sodium acetate buffer at three pH values (4.5, 5.0 and 5.5) and three NaCl concentrations (0.05, 0.10 and 0.15 mol/L) at pH 5.0. The ST model was found to depict the effects of pH and ionic strength on the single-component equilibria well, with model parameters depending on the pH and ionic strength. Moreover, the ST model gave acceptable fitting to the binary adsorption data with the fitted single-component model parameters, leading to the estimation of the binary ST model parameter. The effects of pH and ionic strength on the model parameters are reasonably interpreted by the electrostatic and thermodynamic theories. Results demonstrate the availability of the ST model for describing nonlinear competitive protein adsorption equilibria in the presence of two proteins.     

Comparative Investigation of the Chemiluminescent Properties of a Dibrominated Coelenterazine Analog

Chemi- and bioluminescence are remarkable light-emitting phenomena, in which thermal energy is converted into excitation energy due to a (bio)chemical reaction. Among a wide variety of chemi-/bioluminescent systems, one of the most well-known and studied systems is that of marine imidazopyrazinones, such as Coelenterazine and Cypridina luciferin. Due to the increasing usefulness of their chemi-/bioluminescent reactions in terms of imaging and sensing applications, among others, significant effort has been made over the years by researchers to develop new derivatives with enhanced properties. Herein, we report the synthesis and chemiluminescent characterization of a novel dibrominated Coelenterazine analog. This novel compound consistently showed superior luminescence, in terms of total light output and emission lifetime, to natural imidazopyrazinones and commercially available analogs in aprotic media, while being capable of yellow light emission. Finally, this new compound showed enhanced chemiluminescence in an aqueous solution when triggered by superoxide anion, showing potential to be used as a basis for optimized probes for reactive oxygen species. In conclusion, bromination of the imidazopyrazinone scaffold appears to be a suitable strategy for obtaining Coelenterazines with enhanced properties.