Cellular Membrane Proteins Research Articles

Abstract 483: Unraveling the impact of cancer cell derived membrane and cytosolic proteins using synthetic small extracellular vesicles

Abstract Background: Extracellular vesicles (EVs) are couriers in cell-cell communication. Evidence suggests that EV biology, from cargo packing, biodistribution, to cell uptake is a non-random and well-orchestrated process. Substantial efforts have been devoted to studying the pathological roles and therapeutic potential of EVs. It is speculated that surface properties, such as membrane proteins, mediate the interaction between EVs and recipient cells, while the enclosed “messages” exert downstream effects. This concept is exemplified in cancer metastasis, where cancer cell-derived EVs were shown to establish pre-metastatic niches and guide cancer organotropism. We propose selectively loading cancer cell membrane and cytosolic proteins into synthetic vesicles (SVs) like liposomes. This approach allows for the isolated study of individual factors, in contrast to the complexity of naturally occurring EVs. Methods: Cellular membrane and cytosolic proteins were extracted separately from a cancer cell line (MP41) (Mem-PERTM Plus kit). Empty-SV, Mem-SV (with membrane proteins), Cyto-SV (with cytosolic proteins), and Complete-SV (with both) were synthesized in a 3D-printed microfluidic chip and subsequently dialyzed (1000 KDa) overnight. The volume of protein buffer was controlled in all SV syntheses. EVs were isolated from the conditioned medium by ultrafiltration (Amicon) followed by ultracentrifugation. The size (Nanosight), zeta potential (ZetaView), and morphology (Transmission Electron Microscopy) of SVs and EVs were analyzed. Proteins incorporated into SVs were validated by stain-free gel, western blot, and CytoFLEX. SVs and EVs were stained by sp-DiIC18 fluorescent dye. The number of fluorescent particles was quantified (CytoFLEX) and controlled in cell uptake analysis on hepatocytes (IHH) and fibroblasts (BJ) (Incucyte). Results: All SVs were successfully synthesized with a mean size of ~100 nm. The zeta potential of both Mem-SV (-27.03 mV) and Complete-SV (-24.78 mV) is close to MP41 EVs (-26.01 mV) (P > 0.05) and are more negative than Empty-SV (-11.30 mV) and Cargo-SV (-13.38 mV) (P < 0.05). Total protein incorporation efficiency was estimated to be ~30%. The relative level of specific proteins (e.g. integrin av, TSG101) in Mem-SV and Cyto-SV matched the level in the raw membrane and cytosolic proteins by western blot. Integrin avb5 was detected on the surface of Mem-SV. Membrane and cytosolic protein incorporation increased the uptake efficiency of SVs compared to Empty-SVs, and the efficiency varied with cell type and protein content. Conclusion: In this study, we demonstrate that membrane and cytosolic proteins differentially impact the physical (zeta potential) and functional (cellular uptake) properties of SVs. Investigating these distinct roles could further our understanding of cancer EV biology, as well as advance drug delivery systems. Citation Format: Yunxi Chen, Rubén R. López, Thupten Tsering, Chaymaa Zouggari Ben El Khyat, Vahé Nerguizian, Julia V. Burnier. Unraveling the impact of cancer cell derived membrane and cytosolic proteins using synthetic small extracellular vesicles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 483.

Preferential electrostatic interactions of phosphatidic acid with arginines.

Phosphatidic acid (PA) is an anionic lipid that preferentially interacts with proteins in a diverse set of cellular processes such as transport, apoptosis, and neurotransmission. One such interaction is that of the PA lipids with the proteins of voltage-sensitive ion channels. In comparison to several other similarly charged anionic lipids, PA lipids exhibit much stronger interactions. Intrigued and motivated by this finding, we sought out to gain deeper understanding into the electrostatic interactions of anionic lipids with charged proteins. Using the voltage sensor domain (VSD) of the KvAP channel as a model system, we performed long-timescale atomistic simulations to analyze the interactions of POPA, POPG, and POPI lipids with arginines (ARGs). Our simulations reveal two mechanisms. First, POPA is able to interact not only with surface ARGs but is able to snorkel and interact with a buried arginine. POPG and POPI lipids on the other hand show weak interactions even with both the surface and buried ARGs. Second, deprotonated POPA with -2 charge is able to break the salt-bridge connection between VSD protein segments and establish its own electrostatic bond with the ARG. Based on these findings, we propose a headgroup size hypothesis for preferential solvation of proteins by charged lipids. These findings may be valuable in understanding how PA lipids could be modulating kinematics of transmembrane proteins in cellular membranes.

Adaptive Gating Enhances Intelligent Membranes for Cellular Functions and Precise Separations

AbstractIntelligent membranes with adaptive gating channels play a crucial role in cellular functions and separation processes. This study introduces a novel approach to controlling channel expansion using biomimetic adaptive aggregates, fundamentally overcoming the drawbacks of existing methods relying on intertwined polyelectrolyte chains for channel blockage. Specifically, a pH‐responsive 2D material membrane is constructed through the self‐assembling of protein‐mimetic zwitterionic polydopamine (ZPDA) soft molecular aggregates within graphene oxide (GO) interlayers. Similar to the conformational switch of inserted proteins in cellular membranes, the embedded ZPDA in GO nanofluidic channels undergoes an adaptive conformational transformation in response to pH changes. This dynamic adjustment of ZPDA physicochemical properties allows for reversible regulation of GO nanofluidic channel size and charge by external pH modulation. The resulting GO/ZPDA membrane exhibits programmable separation performance for dye molecules (i.e., water permeance of 18.8–35.4 L m−2 h−1 bar−1 and molecular selectivity of 73.7–32.9) and salt ions (i.e., ionic permeance of 0.69–1.25 mol m−2 h−1 and ionic selectivity of 20.8–21.9). This work sheds new light on the engineering of intelligent stimuli‐responsive 2D nanofluidic channels with adaptive gating properties, holding promise across a wide range of applications including separation, drug delivery, sensing, and catalysis.

Solid-state NMR structure determination of a membrane protein in E. coli cellular inner membrane.

Structure determination of membrane proteins in native cellular membranes is critical to precisely reveal their structures in physiological conditions. However, it remains challenging for solid-state nuclear magnetic resonance (ssNMR) due to the low sensitivity and high complexity of ssNMR spectra of cellular membranes. Here, we present the structure determination of aquaporin Z (AqpZ) by ssNMR in Escherichia coli inner membranes. To enhance the signal sensitivity of AqpZ, we optimized protein overexpression and removed outer membrane components. To suppress the interference of background proteins, we used a "dual-media" expression approach and antibiotic treatment. Using 1017 distance restraints obtained from two-dimensional 13C-13C spectra based on the complete chemical shift assignments, the 1.7-Å ssNMR structure of AqpZ is determined in E. coli inner membranes. This cellular ssNMR structure determination paves the way for analyzing the atomic structural details for membrane proteins in native cellular membranes.

Improved polarizable force field for modeling Mg2+ binding to proteins and nucleotides.

Magnesium (Mg2+) is an indispensable regulatory cation for numerous cellular enzymes and membrane proteins that participate in a variety of biological processes, including cell signaling, glycolysis, cell differentiation, and genome stability. Molecular mechanics (MM) simulations have the potential to provide detailed insights into the mechanisms of these Mg-dependent proteins. However, MM force fields continue to suffer from large inaccuracies when dealing with divalent cations. The inclusion of explicit polarization in MM models, such as in CHARMM Drude and AMOEBA models, has been promising, but despite substantial improvements over non-polarizable models, errors in interactions of proteins with divalent cations remain in excess of 10 kcal/mol. Such errors are present even with ‘nonbonded-fix (NB-fix)’ corrections. Modeling becomes even more problematic when dealing with enzymes like kinases and phosphatases, where Mg2+ ions interact directly with both proteins and nucleotides. Toward addressing this long-standing challenge, we recently presented a recalibration of the AMOEBA model (Delgado et al. J Chem. Info. Model. 2022). Notably, polarization terms of cation-coordinating groups were improved to respond better to the high electric fields present near cations. This systematically reduced errors in interactions of monovalent cations with proteins. Here we show that this revised model, along with our many body NB-fix (MB-NB-fix) corrections, reduces errors in Mg2+-protein interaction energies to 5 kcal/mol. MB-NB-fix corrections also improve predictions of Mg2+-ATP interactions substantially. Errors are computed with respect to estimates from vdW-inclusive density functional theory that we benchmark against "gold standard" CCSD(T) calculations and experiments. We will also present the ramifications of these improvements in local interactions on Mg2+-ATP binding free energies and binding modes in bulk water, and on complexation of Mg2+ and ATP with proteins.

AP-2 Adaptor Complex-Dependent Enhancement of HIV-1 Replication by Nef in the Absence of the Nef/AP-2 Targets SERINC5 and CD4.

Human immunodeficiency virus type 1 (HIV-1) Nef hijacks the clathrin adaptor complex 2 (AP-2) to downregulate the viral receptor CD4 and the antiviral multipass transmembrane proteins SERINC3 and SERINC5, which inhibit the infectivity of progeny virions when incorporated. In Jurkat Tag T lymphoid cells lacking SERINC3 and SERINC5, Nef is no longer required for full progeny virus infectivity and for efficient viral replication. However, in MOLT-3 T lymphoid cells, HIV-1 replication remains highly dependent on Nef even in the absence of SERINC3 and SERINC5. Using a knockout (KO) approach, we now show that the Nef-mediated enhancement of HIV-1 replication in MOLT-3 cells does not depend on the Nef-interacting kinases LCK and PAK2. Furthermore, Nef substantially enhanced HIV-1 replication even in triple-KO MOLT-3 cells that simultaneously lacked the three Nef/AP-2 targets, SERINC3, SERINC5, and CD4, and were reconstituted with a Nef-resistant CD4 to permit HIV-1 entry. Nevertheless, the ability of Nef mutants to promote HIV-1 replication in the triple-KO cells correlated strictly with the ability to bind AP-2. In addition, knockdown and reconstitution experiments confirmed the involvement of AP-2. These observations raise the possibility that MOLT-3 cells express a novel antiviral factor that is downregulated by Nef in an AP-2-dependent manner. IMPORTANCE The HIV-1 Nef protein hijacks a component of the cellular endocytic machinery called AP-2 to downregulate the viral receptor CD4 and the antiviral cellular membrane proteins SERINC3 and SERINC5. In the absence of Nef, SERINC3 and SERINC5 are taken up into viral particles, which reduces their infectivity. Surprisingly, in a T cell line called MOLT-3, Nef remains crucial for HIV-1 spreading in the absence of SERINC3 and SERINC5. We now show that this effect of Nef also does not depend on the cellular signaling molecules and Nef interaction partners LCK and PAK2. Nef was required for efficient HIV-1 spreading even in triple-knockout cells that completely lacked Nef/AP-2-sensitive CD4, in addition to the Nef/AP-2 targets SERINC3 and SERINC5. Nevertheless, our results indicate that the enhancement of HIV-1 spreading by Nef in the triple-knockout cells remained AP-2 dependent, which suggests the presence of an unknown antiviral factor that is sensitive to Nef/AP-2-mediated downregulation.

Genetically Encoded 1,2-Aminothiol for Site-Specific Modification of a Cellular Membrane Protein via TAMM Condensation.

Site-specific modification of proteins has wide applications in probing and perturbing biological systems. A popular means to achieve such a modification on a target protein is through a reaction between bioorthogonal functionalities. Indeed, various bioorthogonal reactions have been developed, including a recently reported reaction between 1,2-aminothiol and ((alkylthio)(aryl)methylene)malononitrile (TAMM). Here, we describe the procedure that combines genetic code expansion and TAMM condensation for site-specific modification of cellular membrane proteins. The 1,2-aminothiol functionality is introduced through a genetically incorporated noncanonical amino acid to a model membrane protein on mammalian cells. Treatment of the cells with a fluorophore-TAMM conjugate leads to fluorescent labeling of the target protein. This method can be applied to modify different membrane proteins on live mammalian cells.

CDC50A might be a novel biomarker of epithelial ovarian cancer-initiating cells

BackgroundThe aim of this work was to screen and validate biomarkers of ovarian cancer-initiating cells to detect the mechanisms of recurrence of epithelial ovarian cancer (EOC).MethodsStably labelled the amino acid in side population (SP) cells of epithelial ovarian cancer which were rich in cancer-initiating cells and non-SP cells with isotope in culture and differentially expressed cellular membrane proteins in SP cells were identified through proteomics technology. The new candidate biomarker was screened and validated through RT-PCR and western blot. Both in cell lines and primary EOC, cancer-initiating biofunctions of CDC50A positive cells were validated. Moreover, the characteristics of mesenchymal transition (EMT) was also detected and the correlation between the biomarker and clinical prognosis was observed.ResultsThrough proteomics technology, candidate protein CDC50A was screened, and its significantly differential expression in SP cells was validated. CDC50A-positive cells from cell lines and primary ovarian cancer tissues were validated to show characteristics of cancer-initiating cells both in vitro and in vivo, including sphere-forming, self-renewal, differentiation, tumor metastasis and tumorigenicity in mice. The relationship between CDC50A-positive cells from primary tissues and tumour metastasis was confirmed based on their mesenchymal transition characteristics. Among 16 high-grade ovarian serous cancer patients, a high ratio of CDC50A-positive cells in primary tumours was correlated with a shorter platinum-free interval (p = 0.031, HR 0.260, 95% CI 0.77 ~ 0.885).ConclusionCDC50A could be used to screen ovarian cancer-initiating cells and might be a new target to resolve tumour development in EOC patients.

NMDA receptor inhibition prevents intracellular sodium elevations in human olfactory neuroepithelial precursors derived from bipolar patients

Dysregulation of ion flux across membranes and glutamate-induced excitotoxicity appear to be important pathophysiologic abnormalities in bipolar illness. Understanding ion control and responses to ionic stress is important to decipher the pathogenesis of this disorder. Monensin alone significantly increased [Na]i in ONPs from bipolar individuals (5.08 ± 0.71 vs baseline 3.13 ± 0.93, P = 0.03) and AP5 had no effect (2.0 ± 1.2 vs baseline 3.13 ± 0.93, P = 0.27). However, the combination of AP5 and monensin resulted in normalization of [Na]i (3.25 ± 1.28 vs baseline 3.13 ± 0.93, P = 0.89). This effect was not observed in cells from non-bipolar individuals (monensin alone, 1.72 ± 1.10 vs baseline 2.42 ± 1.80, P = 0.25; AP5 alone, 1.37 ± 0.74 vs baseline 2.42 ± 1.80; AP5 combined with monensin, 1.53 ± 0.98 vs baseline 2.42 ± 1.80, P = 0.31). Sodium regulation is central to neuronal function and may be disturbed in patients with bipolar disorder. Monensin is an ionophore, meaning that it incorporates itself into the membrane and allows sodium to enter independent of cellular membrane proteins. While the mechanism remains obscure, the observation that the NMDA receptor antagonist, AP5, normalizes [Na]i only in olfactory neuroepithelial precursors obtained from bipolar illness may provide novel insights into ion regulation in tissues from subjects with bipolar illness.

Oxygen sensor of the heart.

How oxygen is sensed by the heart and what mechanisms mediate its sensing remain poorly understood. As recent reports show that low PO2 levels are detected by the cardiomyocytes in a few seconds, the rapid and short applications of low levels of oxygen (acute hypoxia), which avoid multiple effects of chronic hypoxia, may be used to probe the oxygen-sensing pathway of the heart. Here, we explored the oxygen-sensing pathway, focusing primarily on cellular surface membrane proteins that were first exposed to low PO2. Such studies suggest that acute hypoxia primarily targets the cardiac calcium channels, where either the channel itself or moieties closely associated with it, for instance heme-oxygenase-2 (HO-2) interacting through kinase phosphorylation, signal the α-subunit of the channel to the altered levels of PO2. Amino acids 1572-1651, the CaMKII phosphorylation sites (S1487 and S1545), CaM-binding sites (I1624 and Q1625), and Ser1928 of the carboxyl tail of the α-subunit appear to be critical residues that sense oxygen. Future studies on HO-2 knockout mice or CRISPR/Cas9 gene-edited human-induced pluripotent stem-cell-derived cardiomyocytes that reduce CaM-binding affinity are likely to provide deeper insights into the O2-sensing mechanisms.

Tissue Content and Pattern of Expression of Claudin-3 and Occludin in Normal and Neoplastic Tissues in Patients with Colorectal Cancer

BackgroundMetastasis is the worst prognostic variable of patients with colorectal cancer (CRC). For the development of metastases, it is necessary that cancer cells detach from the primary tumor, migrate into the angiolymphatic system, and invade the tissue where they will develop. The breakdown of the tight junctions (TJs) plays an important role in colorectal metastatic tumors. Claudin-3 and occludin are the main component proteins of TJs. AimTo analyze the expression and tissue content of claudin-3 and occludin in normal and neoplastic tissues of patients with metastatic CRC. MethodsFifty-seven consecutive patients with stage III and IV CRC were included. Fragments of neoplastic tissue were collected from the tumor margins, and samples of the normal tissue were collected from the same patient in a standardized distance of 10 cm from the cranial margin of the tumor. Immunohistochemistry technique was used to identify the tissue staining of claudin-3 and occludin. To measure the content of both proteins in cellular membranes of normal and cancer cells, a validated immunoscore was used. ResultsClaudin-3 and occludin in normal tissues are in the apical and lateral membranes of cells, while in the neoplastic, in cytoplasm. The mean of the tissue content of claudin-3 in the normal tissue was 2.57 ± 0.16, while in the neoplastic tissue was 1.03 ± 0.13. The contents of occludin were 2.77±0.1 in normal tissue, while in the neoplastic were 1.08 ± 0.14. ConclusionThere is a reduction in the content of the claudin-3 and occludin in the cell membranes of the neoplastic tissue in patients with CRC.

Biomimetic Nanoparticle Drug Delivery Systems to Overcome Biological Barriers for Therapeutic Applications.

Targeted drug delivery using nanoparticles has been applied for the treatment of diverse diseases, including cancer and inflammatory diseases. Nanoparticle-mediated delivery of therapeutic agents via the enhanced permeability and retention effect generally augments their therapeutic efficiency; however, limitations with passive entry of nanoparticles into diseased sites, due to the presence of biological barriers represented by the endothelial layer, remain to be addressed. To this end, development of nanoparticles with intrinsic characteristics similar to circulatory cells (e.g., leukocytes, platelets) for use as biomimetic drug delivery systems (DDS) has been focused as a means to overcome the issues of conventional DDS. In particular, synthetic biomimetic nanoparticles coated with cellular membranes were recently prepared and shown to actively overcome the inflamed vessels and tumor microenvironment as a result of the functionality of membrane proteins, which allowed secure drug delivery into diseased sites. We recently developed liposomes modified with leukocyte membrane proteins via intermembrane protein transfer, a simple method to reconstitute cellular membrane proteins onto lipid bilayers. The resultant liposomes demonstrated the ability to cross the inflamed endothelial layer and permeate into tumor tissue by mimicking the properties of leukocytes. Thus, biomimetic DDS offer promise as new therapeutic approaches for various diseases by overcoming biological barriers that typically inhibit drug delivery. Herein, we review recent approaches to develop biomimetic DDS using the cell membrane coating method, and highlight our recent findings on leukocyte-mimetic liposomes prepared via intermembrane protein transfer.

Direct functionalization of cell‐adhesion promoters to hydrogel microparticles synthesized by stop‐flow lithography

AbstractPolyethylene glycol (PEG) hydrogel microparticles generated via stop‐flow lithography can be utilized for efficient microparticle‐based cell culture processes because of their high biocompatibility, the molecular diffusion capability in the gel structure, and the tunability of their shape and size. However, the typical functionalization process of PEG microparticles with cell‐adhesion promoters has inevitable limitations, requiring additional linker molecules and the preconjugation of linkers to cell‐adhesion promoters and microparticles. In this study, a simple and direct cell‐adhesion promoter functionalization process of the PEG microparticles is presented by use of aza‐Michael reaction between remnant unreacted acrylate groups in particles and amine groups in cell‐adhesion promoters. On the basis of proposed process, particles are directly conjugated with poly‐l‐lysine (PLL), a typical cell‐adhesion promoter that can electrostatically interact with cellular membranes, in a controllable manner. We demonstrate enhanced cell‐adhesion capabilities of the particles along with the increased amount of conjugated PLL in the particles. Furthermore, to validate extended applicability, the particles are directly conjugated with Gly‐Arg‐Gly‐Asp‐Ser (GRGDS) peptides, in which RGD sequence is involved in the cell‐adhesion behavior of extracellular matrix proteins, including fibronectin. The introduced GRGDS peptides increase the cell‐adhesion capacity of the microparticles binding to integrin proteins in cellular membranes.

Abnormal ECA-Binding Membrane Glycans and Galactosylated CAT and P4HB in Lesion Tissues as Potential Biomarkers for Hepatocellular Carcinoma Diagnosis

Hepatocellular carcinoma (HCC) is one of the most common types of cancer. Despite decades of research efforts, the search for novel biomarkers is still urgently needed for the diagnosis of HCC and the improvement of clinical outcomes. Previous studies of HCC clinical biomarkers have usually focused on serum and urine samples (e.g., serum Alpha-fetoprotein (AFP). However, cellular membrane proteins in lesion tissues are less used in HCC diagnosis. The abnormal expression of membrane glycoproteins in tumor lesions are considered as potential targets for tumor diagnosis and tumor therapies. Here, a lectin array has been employed to screen and identify abnormal glycopatterns and cellular membrane glycans in HCC lesion tissues compared with adjacent non-tumor tissues. We found that there was significantly less expression of Erythrina cristagalli (ECA) lectin binding (Galβ1-3/β1-4) glycans on the cellular membrane of HCC lesion tissues compared with those of adjacent non-tumor tissues. Immunohistochemistry analysis further showed that ECA-binding ability on the membrane proteins of HCC tissues progressively decreased in different tumor-node-metastasis (TNM) stages (stage I to stage III) as the malignancy of liver cancer increased. Receiver operating curve (ROC) analysis showed ECA-binding ability yielding a sensitivity of 85% and specificity of 75%, and a combination of ECA and AFP has better clinical diagnostic efficiency, yielding a sensitivity of 90% and specificity of 85%, than ECA or AFP assay alone. ECA pull-down followed by mass spectrometry further showed that there was significantly less expression of ECA binding membrane catalase (CAT) and prolyl 4-hydroxylase beta polypeptide (P4HB) in HCC tissues compared with the adjacent non-tumor tissues. The abnormally increased expression of total CAT and P4HB and decreased expression of galactosylated membrane CAT and P4HB in HCC cell lines were correlated with an HCC metastasis status. Our findings suggest that abnormal declined ECA-binding galatosylated membrane glycans and two galactosylated-CAT and P4HB glycoproteins in lesion tissues are potential biomarkers in the diagnosis and/or metastasis prediction for HCC.

Transfusion-associated graft-versus-host disease, transfusion-associated hyperkalemia, and potassium filtration: advancing safety and sufficiency of the blood supply

Irradiation of cellular blood components is well established as a countermeasure against transfusion-associated graft-versus-host disease (TA-GVHD). Unintended consequences of ionizing radiation are also well established. The red cell “storage lesion” – a progression of metabolic, functional, and morphological changes – may be exacerbated by irradiation rates and doses typically used for TA-GVHD prophylaxis. With or without irradiation, a storage lesion change of clinical concern is the accelerated egress of intracellular potassium. ATP depletion during storage limits the activity of the red cell membrane’s sodium-potassium pump (Na,K-ATPase), which normally maintains intracellular potassium (K+) at levels 30–40 times higher than the extracellular milieu. The natural diffusion of potassium down this concentration gradient proceeds faster if the cell membrane is damaged, and oxidative damage to cellular membranes and membrane proteins – including Na,K-ATPase – is an effect of ionizing radiation.Preventing transfusion-related hyperkalemia is a reason for limiting the shelf life of irradiated red cells. In the absence of specific measurements to assess storage lesion in a particular unit of blood, and in the absence of specific interventions at the time of transfusion to mitigate effects of storage lesion, it is consistent with the precautionary principle to put conservative limits on a blood component’s shelf life. On the other hand, both the safety and sufficiency of a nation’s blood supply might be improved by interventions that benefit specific recipients when they are transfused, and benefit future patients by extending the allowable shelf life of blood components. Potassium filtration of irradiated red blood cell components is one such intervention.

Hyperuniformity and phase enrichment in vortex and rotor assemblies

Ensembles of particles rotating in a two-dimensional fluid can exhibit chaotic dynamics yet develop signatures of hidden order. Such rotors are found in the natural world spanning vastly disparate length scales — from the rotor proteins in cellular membranes to models of atmospheric dynamics. Here we show that an initially random distribution of either driven rotors in a viscous membrane, or ideal vortices with minute perturbations, spontaneously self assemble into a distinct arrangement. Despite arising from drastically different physics, these systems share a Hamiltonian structure that sets geometrical conservation laws resulting in prominent structural states. We find that the rotationally invariant interactions isotropically suppress long-wavelength fluctuations — a hallmark of a disordered hyperuniform material. With increasing area fraction, the system orders into a hexagonal lattice. In mixtures of two co-rotating populations, the stronger population will gain order from the other and both will become phase enriched. Finally, we show that classical 2D point vortex systems arise as exact limits of the experimentally accessible microscopic membrane rotors, yielding a new system through which to study topological defects.

Dengue and Zika Virus Capsid Proteins Contain a Common PEX19-Binding Motif.

Flaviviruses such as dengue virus (DENV) and Zika virus (ZIKV) have evolved sophisticated mechanisms to suppress the host immune system. For instance, flavivirus infections were found to sabotage peroxisomes, organelles with an important role in innate immunity. The current model suggests that the capsid (C) proteins of DENV and ZIKV downregulate peroxisomes, ultimately resulting in reduced production of interferons by interacting with the host protein PEX19, a crucial chaperone in peroxisomal biogenesis. Here, we aimed to explore the importance of peroxisomes and the role of C interaction with PEX19 in the flavivirus life cycle. By infecting cells lacking peroxisomes we show that this organelle is required for optimal DENV replication. Moreover, we demonstrate that DENV and ZIKV C bind PEX19 through a conserved PEX19-binding motif, which is also commonly found in cellular peroxisomal membrane proteins (PMPs). However, in contrast to PMPs, this interaction does not result in the targeting of C to peroxisomes. Furthermore, we show that the presence of C results in peroxisome loss due to impaired peroxisomal biogenesis, which appears to occur by a PEX19-independent mechanism. Hence, these findings challenge the current model of how flavivirus C might downregulate peroxisomal abundance and suggest a yet unknown role of peroxisomes in flavivirus biology.

Light‐Fueled Organic Photoelectrochemical Transistor for Probing Membrane Protein in an H‐Cell

AbstractThough great advances are achieved in the area of organic electrochemical transistor (OECT) biosensors, general issues of shared electrolyte by the channel and gate electrode remain unsolved. On the other hand, to date binding‐free OECT detection of cellular membrane proteins is not reported. On the basis of the advanced technique of organic photoelectrochemical transistor (OPECT), herein the H‐cell‐supported OPECT device that can easily circumvent the issues of abovementioned shared electrolyte and its application toward binding‐free detection of membrane alkaline phosphatase (ALP) of HeLa cells is demonstrated. In such a configuration, usage of a Nafion perfluorinated membrane equipped H‐cell can well retain the ionic circuit of the device but prevent the diffusion of various molecules and minimize the possible interference. In the detection, ALP catalytic chemistry enables the sensitization of the TiO2 nanotubes electrode, triggering enhanced de‐doping of the poly(3,4‐ethylenedioxythiophene):poly(styrene sulfonate) and thus resulting in altered channel currents proportionate to the membrane ALP. This work not only provides a new perspective for binding‐free detection of membrane protein but also represents a general protocol for H‐cell‐supported OECT/OPECT biosensors with enhanced accuracy.

MARCH8 inhibits influenza A virus infection by targeting viral M2 protein for ubiquitination-dependent degradation in lysosomes

The membrane-associated RING-CH (MARCH) proteins are E3 ligases that regulate the stability of various cellular membrane proteins. MARCH8 has been reported to inhibit the infection of HIV-1 and a few other viruses, thus plays an important role in host antiviral defense. However, the antiviral spectrum and the underlying mechanisms of MARCH8 are incompletely defined. Here, we demonstrate that MARCH8 profoundly inhibits influenza A virus (IAV) replication both in vitro and in mice. Mechanistically, MARCH8 suppresses IAV release through redirecting viral M2 protein from the plasma membrane to lysosomes for degradation. Specifically, MARCH8 catalyzes the K63-linked polyubiquitination of M2 at lysine residue 78 (K78). A recombinant A/Puerto Rico/8/34 virus carrying the K78R M2 protein shows greater replication and more severe pathogenicity in cells and mice. More importantly, we found that the M2 protein of the H1N1 IAV has evolved to acquire non-lysine amino acids at positions 78/79 to resist MARCH8-mediated ubiquitination and degradation. Together, our data support the important role of MARCH8 in host anti-IAV intrinsic immune defense by targeting M2, and suggest the inhibitory pressure of MARCH8 on H1N1 IAV transmission in the human population.

FC 045URINARY LEVELS OF SARS-COV-2 NUCLEOCAPSID PROTEIN PREDICT AKI AND FATAL OUTCOME IN COVID-19

Background and AimsAcute kidney injury (AKI) is very common in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) disease 2019 (COVID-19), particularlarly among patients requiring intensive care unit (ICU) supportive care and is considered as an independent risk factor for premature death. SARS-CoV-2 renal tropism and detection of SARS nucleocapsid protein (SARS-CoV-2 N) in renal tubules has been described in COVID-19 patients, suggesting that direct viral injury of the kidneys may contribute to AKI. SARS-CoV-2 renal tropism has been in part attributed to the intrarenal presence of cellular membrane proteins essential for viral entry including angiotensin converting enzyme 2 (ACE2) and transmembrane protease serine subtype 2 (TMPRSS2). Based on the assumption that renal injury is caused by direct viral infection of the kidneys, it remains unclear if markers for viral infection (SARS-CoV-2 N) and shedded cellular membrane proteins (ACE2, TMPRSS2) in urinary samples are useful for early identification of COVID-19 patients at risk for AKI.MethodA single-center prospective observational study was carried out in adult cases with confirmed SARS-CoV-2 infection requiring ICU supportive care. Investigators were blinded to clinical data collection and urinary ELISA measurements. According to the manufacturer’s protocols, urinary levels of SARS-CoV-2 N (KIT40588, Sino Biological), human ACE2 (NBP2-78734, Novus Biologicals) and human TMPRSS2 (NBP2-89170, Novus Biologicals) were analyzed. Measurements were done in triplicates for each urinary sample and compared to the standard curve. Negative test results were declared as not detectable. Urinary levels of SARS-CoV-2 N, ACE2 and TMPRSS2 were analyzed at ICU admission and at day 3 and 8 during the further clinical course of severe COVID-19 for association with AKI and outcome of COVID-19.ResultsROC analysis revealed that a cut-off urinary SARS-CoV-2 N level higher than 512.2 pg/mL at ICU admission effectively identified patients with AKI (AUC 0.81, p=0.0211). Survival analysis for cumulative incidence of AKI revealed that urinary SARS-CoV-2 N levels at ICU admission were not only associated with AKI at ICU admission, but also predicted future development of AKI (HR 5.9, 95% CI 1.4-26, p=0.0095). Because plasma albumin levels at time of ICU admission have previously been established as marker to predict AKI and fatal outcome in COVID-19, we next compared clinical and routine laboratory parameters assessed at ICU admission with urinary SARS-CoV-2 N measurements to predict AKI with higher accuracy. In line with aforementioned findings, hypoalbuminemia at time of ICU admission also predicted AKI among all parameters assessed in our cohort. Confirmed by ROC analysis, combining urinary SARS-CoV-2 N and plasma albumin measurements for risk prediction (2-variable model, AUC 0.94, p=0.0009) outperformed urinary SARS-CoV-2 N alone (AUC 0.81, p=0.0211, comparison of models: p=0.0016) or plasma albumin alone (AUC 0.78, p=0.0343, comparison of models: p=0.0061). Thus, combined urinary SARS-CoV-2 N and plasma albumin levels assessed at ICU admission effectively predicted incidence and early onset of AKI during the further clinical course (2-variable model, HR 11.4, 95% CI 2.7-48, p=0.0016). In addition, combining urinary SARS-CoV-2 N and plasma albumin levels at ICU admission effectively predicted fatal outcome in COVID-19 (2-variable model, HR 7.6, 95% CI 1.3-44, p=0.0240).ConclusionUrinary SARS-CoV-2 N levels associate with risk for AKI and correlated with COVID-19 severity. Therefore, we propose that urinary SARS-CoV-2 N could be used as early and easily assessable marker to identify patients at risk for AKI and premature death in COVID-19.