Cytoplasmic Side Research Articles

Transmembrane helix 6b links proton and metal release pathways and drives conformational change in an Nramp-family transition metal transporter

Transmembrane helix 6b links proton and metal release pathways and drives conformational change in an Nramp-family transition metal transporter

Identification and prediction of molecular targets related to space radiation protection based on associated networks

The effect of cosmic radiation on astronauts is a major barrier for human space exploration. In particular, medical protection against heavy ion radiation-induced injury is one of the key approaches in counteracting cosmic radiation to humans. In recent years, with the rapid accumulation of high-throughput data including gene expression profiles and omics-based data such as protein-protein interactions (PPI), large-scaled network pharmacology analyses can now be conducted. Exploring human genes with potentially high correlation to protection against radiation is becoming an important component and a new approach in heavy ion radiation study owing to advances in rational computational methods based on systems biology techniques. In this study, heavy ion accelerators were employed to simulate cosmic radiation and a pipeline was constructed to identify activated subnetworks of significantly differentially expressed genes and to predict essential genes related to radiation-induced injury protection. Human lymphoblastoid AHH-1 cells were irradiated with 0.5 or 2 Gy of carbon ions (12C6+). Two kinds of existing radioprotective agents 523 and VND3207 were separately used in vitro 0.5 h before irradiation. Gene expression profiles of the cells were obtained by microarray 4 h after irradiation without and with the treatment of radioprotective agents. The networks of gene expression regulation were constructed based on PPI integrated from Human Protein Reference Database (HPRD). Simulated annealing algorithm was then used to extract activated subnetworks and potential candidate molecular targets including PPM1A , RIF1 , CASP3 , TFE3 and RIN1 were proposed according to their degrees of connection. Further gene set enrichment analysis (GSEA) indicated significantly enriched clusters of the identified genes in molecular functions including transcription regulator activity, transcription activator activity, enzyme activator activity, protein kinase activity and zinc ion binding. The most enriched clusters in cell components included nuclear body, transcription factor complex, cell junction, chromosome and cytoplasmic side of membrane. The most enriched biological processes were regulation of RNA metabolism, intracellular signal transduction, regulation of neuron death, regulation of cellular component and phosphorylation. We further investigated the expression of RIF1 before and after irradiation with 0.5 and 2 Gy of 12C6+ in Hela cells by western blotting. Expression of RIF1 increased significantly after irradiation and the relative expression in cells 4h after irradiation was greater than 2-fold that of the control. This study took gene correlation into consideration in addition to their differential expression. It provides promising candidates for further discovery of anti-radiation drugs and will be of importance in promoting cosmic radiation protection techniques in long-term manned space programs and deep space exploration missions. In addition, considerable support and clues may also be revealed from the data generated in this study for future research on the underlying mechanisms in radiation-induced injury and medical protection against cosmic radiation.

Myelin basic protein (MBP) charge variants show different sphingomyelin-mediated interactions with myelin-like lipid monolayers

Multiple sclerosis (MS) is correlated with increased deimination of myelin basic protein (MBP) in the central nervous system. Here, the interaction of MBP C1 (charge: +19) and MBP C8 (charge: +13) with the major lipids of the cytoplasmic side of the oligodendrocyte membrane is analysed using monolayer adsorption experiments and epifluorescence microscopy. Our findings show that the electrostatic attraction between the positively charged proteins and negatively charged lipids in the myelin-like monolayers competes with the incorporation of MBP into regions directly bordering cholesterol-rich domains. The latter is favoured to avoid additional lipid condensation and reduction in fluidity of the phospholipid layer. We find that MBP C1 does not incorporate at the cholesterol-rich domains if sphingomyelin (SM) is absent from the lipid composition. In contrast, MBP C8 is still incorporated near cholesterol-enriched regions without SM. Thus, the highly charged C1 variant needs a specific interaction with SM, whereas for C8 the incorporation at the cholesterol-rich regions is ensured due to its reduced net positive charge. This phenomenon may be relevant for the correlation of higher amounts of MBP C8 in brains of adult MS patients and healthy children, in which the amount of SM is reduced compared to healthy adults.

Analyzing the Redox Status of Intracellular Glutathione and Its Application to an Intestinal Bowel Disease Model

Oxidative stress, including reactive oxygen species (ROS) generation and resulting glutathione oxidation, have been implicated in numerous aspects of cell physiology and human pathology such as cell senescence, cell differentiation, and inflammation. Significant effort has been made to establish methods of analyzing ROS levels and glutathione oxidation within a living cell. The recent development of redox-sensitive green fluorescent protein (GFP) variants enables a robust and accurate estimation of ROS level and glutathione oxidation at subcellular resolution. We created membrane-targeted versions of glutathione and hydrogen peroxide sensors by attaching palmitoylation signals to existing sensors (Grx1-roGFP2 and roGFP2-Orp1, respectively), and demonstrated the nonuniform distribution of these oxidative elements within cytosol. In living cells, cytosolic glutathione is highly reduced, and hydrogen peroxide is barely detected. Nevertheless, near the cytoplasmic side of intracellular vesicular membranes, significant glutathione oxidation and hydrogen peroxide were successfully probed by our sensors, clearly showing the difference between various areas within cytosol. Currently, these sensors are being applied to an intestinal inflammation model which is constituted by co-culturing intestinal epithelial cells and macrophage-like inflammatory cells derived from THP-1. This review covers the current status of studies regarding the association of oxidative stress and intestinal inflammation, with a focus on the redox regulation of intracellular glutathione.

BIK1 and ERECTA Play Opposing Roles in Both Leaf and Inflorescence Development in Arabidopsis.

Plants employ cell-surface receptor-like kinases to detect extrinsic and intrinsic signals, thus make a trade-off between growth and immunity. The receptor-like cytoplasmic kinases on the cytoplasmic side act as downstream components involved in the activation, transmission, and integration of intracellular signals. In Arabidopsis thaliana, the RLCK BOTRYTIS-INDUCED KINASE1 (BIK1) associates with multiple RLKs to regulate pathogen defense responses and brassinosteroid (BR) signaling. However, little is known about the biological functions of BIK1 in developmental processes in Arabidopsis. In this study, we established that mutation of ERECTA (ER), an important RLK, counteracts the developmental effects of loss of BIK1 function. BIK1 and ER play opposing roles in leaf morphogenesis and inflorescence architecture. Moreover, we confirmed that BIK1 is required to maintain appropriate auxin response during leaf margin morphogenesis. Finally, we found that BIK1 interacts with ER-family proteins and directly phosphorylates ER. Our findings might provide novel insight into the function of BIK1 in leaf and inflorescence development.

DHHC20 Palmitoyl-Transferase Reshapes the Membrane to Foster Catalysis

Cysteine palmitoylation, a form of S-acylation, is a key posttranslational modification in cellular signaling. This type of reversible lipidation occurs in both plasma and organellar membranes, and is catalyzed by a family of integral membrane proteins known as DHHC acyltransferases. The first step in the S-acylation process is the recognition of free acyl coenzyme A (acyl-CoA) from the lipid bilayer. The DHHC enzyme then becomes autoacylated at a site defined by a conserved Asp-His-His-Cys motif. This reaction entails ionization of the catalytic Cys. Intriguingly, in known DHHC structures, this catalytic Cys appears to be exposed to the hydrophobic interior of the lipid membrane, which would be highly unfavorable for a negatively charged nucleophile, thus hindering autoacylation. Here, we use biochemical and computational methods to reconcile these seemingly contradictory facts. First, we experimentally demonstrate that human DHHC20 is active when reconstituted in POPC nanodiscs. Microsecond-long all-atom molecular dynamics simulations are then calculated for human DHHC20 and for different acyl-CoA forms, also in a POPC membrane. Strikingly, we observe that human DHHC20 induces a drastic deformation in the membrane, particularly on the cytoplasmic side, where autoacylation occurs. As a result, the catalytic Cys becomes hydrated and optimally positioned to encounter the cleavage site in acyl-CoA. In summary, we hypothesize that DHHC enzymes locally reshape the membrane to foster a morphology that is specifically adapted for acyl-CoA recognition and autoacylation.

A Minimal Membrane Metal Transport System: Dynamics and Energetics of mer Proteins.

The mer operon in bacteria encodes a set of proteins and enzymes that impart resistance to environmental mercury toxicity by importing Hg2+ and reducing it to volatile Hg(0). Because the reduction occurs in the cytoplasm, mercuric ions must first be transported across the cytoplasmic membrane by one of a few known transporters. MerF is the smallest of these, containing only two transmembrane helices and two pairs of vicinal cysteines that coordinate mercuric ions. In this work, we use molecular dynamics simulations to characterize the dynamics of MerF in its apo and Hg2+ -bound states. We find that the apo state positions one of the cysteine pairs closer to the periplasmic side of the membrane, while in the bound state the same pair approaches the cytoplasmic side. This finding is consistent with the functional requirement of accepting Hg2+ from the periplasmic space, sequestering it on acceptance, and transferring it to the cytoplasm. Conformational changes in the TM helices facilitate the functional interaction of the two cysteine pairs. Free-energy calculations provide a barrier of 16 kcal/mol for the association of the periplasmic Hg2+ -bound protein MerP with MerF and 7 kcal/mol for the subsequent association of MerF's two cysteine pairs. Despite the significant conformational changes required to move the binding site across the membrane, coarse-grained simulations of multiple copies of MerF support the expectation that it functions as a monomer. Our results demonstrate how conformational changes and binding thermodynamics could lead to such a small membrane protein acting as an ion transporter. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.

Autoantibodies to synapsin I sequestrate synapsin I and alter synaptic function

Synapsin I is a phosphoprotein that coats the cytoplasmic side of synaptic vesicles and regulates their trafficking within nerve terminals. Autoantibodies against Syn I have been described in sera and cerebrospinal fluids of patients with numerous neurological diseases, including limbic encephalitis and clinically isolated syndrome; however, the effects and fate of autoantibodies in neurons are still unexplored. We found that in vitro exposure of primary hippocampal neurons to patient’s autoantibodies to SynI decreased the density of excitatory and inhibitory synapses and impaired both glutamatergic and GABAergic synaptic transmission. These effects were reproduced with a purified SynI antibody and completely absent in SynI knockout neurons. Autoantibodies to SynI are internalized by FcγII/III-mediated endocytosis, interact with endogenous SynI, and promote its sequestration and intracellular aggregation. Neurons exposed to human autoantibodies to SynI display a reduced density of SVs, mimicking the SynI loss-of-function phenotype. Our data indicate that autoantibodies to intracellular antigens such as SynI can reach and inactivate their targets and suggest that an antibody-mediated synaptic dysfunction may contribute to the evolution and progression of autoimmune-mediated neurological diseases positive for SynI autoantibodies.

A channel profile report of the unusual K+ channel KtrB.

KtrAB is a key player in bacterial K+ uptake required for K+ homeostasis and osmoadaptation. The system is unique in structure and function. It consists of the K+-translocating channel subunit KtrB, which forms a dimer in the membrane, and the soluble regulatory subunit KtrA, which attaches to the cytoplasmic side of the dimer as an octameric ring conferring Na+ and ATP dependency to the system. Unlike most K+ channels, KtrB lacks the highly conserved T(X)GYG selectivity filter sequence. Instead, only a single glycine residue is found in each pore loop, which raises the question of how selective the ion channel is. Here, we characterized the KtrB subunit from the Gram-negative pathogen Vibrio alginolyticus by isothermal titration calorimetry, solid-supported membrane-based electrophysiology, whole-cell K+ uptake, and ACMA-based transport assays. We found that, despite its simple selectivity filter, KtrB selectively binds K+ with micromolar affinity. Rb+ and Cs+ bind with millimolar affinities. However, only K+ and the poorly binding Na+ are efficiently translocated, based on size exclusion by the gating loop. Importantly, the physiologically required K+ over Na+ selectivity is provided by the channel's high affinity for potassium, which interestingly results from the presence of the sodium ions themselves. In the presence of the KtrA subunit, sodium ions further decrease the Michaelis-Menten constant for K+ uptake from milli- to micromolar concentrations and increase the Vmax, suggesting that Na+ also facilitates channel gating. In conclusion, high binding affinity and facilitated K+ gating allow KtrAB to function as a selective K+ channel.

Secondary structure of caveolins: a mini review.

Caveolae are 50-100 nm invaginations found within the plasma membrane of cells. Caveolae are involved in many processes that are essential for homeostasis, most notably endocytosis, mechano-protection, and signal transduction. Within these invaginations, the most important proteins are caveolins, which in addition to participating in the aforementioned processes are structural proteins responsible for caveolae biogenesis. When caveolin is misregulated or mutated, many disease states can arise which include muscular dystrophy, cancers, and heart disease. Unlike most integral membrane proteins, caveolin does not have a transmembrane orientation; instead, it is postulated to adopt an unusual topography where both the N- and C-termini lie on the cytoplasmic side of the membrane, and the hydrophobic span adopts an intramembrane loop conformation. While knowledge concerning the biology of caveolin has progressed apace, fundamental structural information has proven more difficult to obtain. In this mini-review, we curate as well as critically assess the structural data that have been obtained on caveolins to date in order to build a robust and compelling model of the caveolin secondary structure.

Role of Endoplasmic Reticulum-Associated Proteins in Flavivirus Replication and Assembly Complexes.

Flavivirus replication in host cells requires the formation of replication and assembly complexes on the cytoplasmic side of the endoplasmic reticulum (ER) membrane. These complexes consist of an ER membrane, viral proteins, and host proteins. Genome-wide investigations have identified a number of ER multiprotein complexes as vital factors for flavivirus replication. The detailed mechanisms of the role of ER complexes in flavivirus replication are still largely elusive. This review highlights the fact that the ER multiprotein complexes are crucial for the formation of flavivirus replication and assembly complexes, and the ER complexes could be considered as a target for developing successful broad-spectrum anti-flavivirus drugs.

Mechanisms of nuclear mRNA export: A structural perspective.

Export of mRNA from the nucleus to the cytoplasm is a critical process for all eukaryotic gene expression. As mRNA is synthesized, it is packaged with a myriad of RNA-binding proteins to form ribonucleoprotein particles (mRNPs). For each step in the processes of maturation and export, mRNPs must have the correct complement of proteins. Much of the mRNA export pathway revolves around the heterodimeric export receptor yeast Mex67•Mtr2/human NXF1•NXT1, which is recruited to signal the completion of nuclear mRNP assembly, mediates mRNP targeting/translocation through the nuclear pore complex (NPC), and is displaced at the cytoplasmic side of the NPC to release the mRNP into the cytoplasm. Directionality of the transport is governed by at least two DEAD-box ATPases, yeast Sub2/human UAP56 in the nucleus and yeast Dbp5/human DDX19 at the cytoplasmic side of the NPC, which respectively mediate the association and dissociation of Mex67•Mtr2/NXF1•NXT1 onto the mRNP. Here we review recent progress from structural studies of key constituents in different steps of nuclear mRNA export. These findings have laid the foundation for further studies to obtain a comprehensive mechanistic view of the mRNA export pathway.

NMDA receptor C-terminal signaling in development, plasticity, and disease.

The NMDA subtype of ionotropic glutamate receptor is a sophisticated integrator and transducer of information. NMDAR-mediated signals control diverse processes across the life course, including synaptogenesis and synaptic plasticity, as well as contribute to excitotoxic processes in neurological disorders. At the basic biophysical level, the NMDAR is a coincidence detector, requiring the co-presence of agonist, co-agonist, and membrane depolarization in order to open. However, the NMDAR is not merely a conduit for ions to flow through; it is linked on the cytoplasmic side to a large network of signaling and scaffolding proteins, primarily via the C-terminal domain of NMDAR GluN2 subunits. These physical interactions help to organize the signaling cascades downstream of NMDAR activation. Notably, the NMDAR does not come in a single form: the subunit composition of the NMDAR, particularly the GluN2 subunit subtype (GluN2A–D), influences the biophysical properties of the channel. Moreover, a growing number of studies have illuminated the extent to which GluN2 C-terminal interactions vary according to GluN2 subtype and how this impacts on the processes that NMDAR activity controls. We will review recent advances, controversies, and outstanding questions in this active area of research.

Steady state analysis of influx and transbilayer distribution of ergosterol in the yeast plasma membrane

BackgroundThe transbilayer sterol distribution between both plasma membrane (PM) leaflets has long been debated. Recent studies in mammalian cells and in yeast show that the majority of sterol resides in the inner PM leaflet. Since sterol flip-flop in model membranes is rapid and energy-independent, a mechanistic understanding for net enrichment of sterol in one leaflet is lacking. Import of ergosterol in yeast can take place via the ABC transporters Aus1/Pdr11 under anaerobic growth conditions, eventually followed by rapid non-vesicular sterol transport to the endoplasmic reticulum (ER). Little is known about how these transport steps are dynamically coordinated.MethodsHere, a kinetic steady state model is presented which considers sterol import via Aus1/Pdr11, sterol flip-flop across the PM, bi-molecular complex formation and intracellular sterol release followed by eventual transport to and esterification of sterol in the ER. The steady state flux is calculated, and a thermodynamic analysis of feasibility is presented.ResultsIt is shown that the steady state sterol flux across the PM can be entirely controlled by irreversible sterol import via Aus1/Pdr11. The transbilayer sterol flux at steady state is a non-linear function of the chemical potential difference of sterol between both leaflets. Non-vesicular release of sterol on the cytoplasmic side of the PM lowers the attainable sterol enrichment in the inner leaflet. Including complex formation of sterol with phospholipids or proteins can explain several puzzling experimental observations; 1) rapid sterol flip-flop across the PM despite net sterol enrichment in one leaflet, 2) a pronounced steady state sterol gradient between PM and ER despite fast non-vesicular sterol exchange between both compartments and 3) a non-linear dependence of ER sterol on ergosterol abundance in the PM.ConclusionsA steady state model is presented that can account for the observed sterol asymmetry in the yeast PM, the strong sterol gradient between PM and ER and threshold-like expansion of ER sterol for increasing sterol influx into the PM. The model also provides new insight into selective uptake of cholesterol and its homeostasis in mammalian cells, and it provides testable predictions for future experiments.

Imaging within single NPCs reveals NXF1's role in mRNA export on the cytoplasmic side of the pore.

Translocation of mRNA through the nuclear pore complex (NPC) requires interactions with different NPC regions. To determine the interactions that are crucial for effective mRNA export in living cells, we examined mRNA export within individual pores by applying various types of mRNA export blocks that stalled mRNPs at different stages of transition. Focusing on the major mRNA export factor NXF1, we found that initial mRNP binding to the NPC did not require NXF1 in the NPC, whereas release into the cytoplasm did. NXF1 localization in the NPC did not require RNA or RNA binding. Superresolution microscopy showed that NXF1 consistently occupied positions on the cytoplasmic side of the NPC. Interactions with specific nucleoporins were pinpointed using FLIM-FRET for measuring protein-protein interactions inside single NPCs, showing that Dbp5 helicase activity of mRNA release is conserved in yeast and humans. Altogether, we find that specific interactions on the cytoplasmic side of the NPC are fundamental for the directional flow of mRNA export.

A one-gate elevator mechanism for the human neutral amino acid transporter ASCT2

The human Alanine Serine Cysteine Transporter 2 (ASCT2) is a neutral amino acid exchanger that belongs to the solute carrier family 1 (SLC1A). SLC1A structures have revealed an elevator-type mechanism, in which the substrate is translocated across the cell membrane by a large displacement of the transport domain, whereas a small movement of hairpin 2 (HP2) gates the extracellular access to the substrate-binding site. However, it has remained unclear how substrate binding and release is gated on the cytoplasmic side. Here, we present an inward-open structure of the human ASCT2, revealing a hitherto elusive SLC1A conformation. Strikingly, the same structural element (HP2) serves as a gate in the inward-facing as in the outward-facing state. The structures reveal that SLC1A transporters work as one-gate elevators. Unassigned densities near the gate and surrounding the scaffold domain, may represent potential allosteric binding sites, which could guide the design of lipidic-inhibitors for anticancer therapy.

Alkaloids Purified from Aristotelia chilensis Inhibit the Human α3β4 Nicotinic Acetylcholine Receptor with Higher Potencies Compared with the Human α4β2 and α7 Subtypes.

The alkaloids aristoteline (1), aristoquinoline (2), and aristone (3) were purified from the leaves of the Maqui tree Aristotelia chilensis and chemically characterized by NMR spectroscopy. The pharmacological activity of these natural compounds was evaluated on human (h) α3β4, α4β2, and α7 nicotinic acetylcholine receptors (AChRs) by Ca2+ influx measurements. The results suggest that these alkaloids do not have agonistic, but inhibitory, activity on each receptor subtype. The obtained IC50 values indicate the following receptor selectivity: hα3β4 > hα4β2 ≫ hα7. In the particular case of hα3β4 AChRs, 1 (0.40 ± 0.20 μM) and 2 (0.96 ± 0.38 μM) show higher potencies compared with 3 (167 ± 3 μM). Molecular docking and structure-activity relationship results indicate that ligand lipophilicity is important for the interaction with the luminal site located close to the cytoplasmic side of the hα3β4 ion channel between positions -2' and -4'. Compound 1 could be used as a molecular scaffold for the development of more potent noncompetitive inhibitors with higher selectivity for the hα3β4 AChR that could serve for novel addiction and depression therapies.

Proton uptake mechanism in bacteriorhodopsin captured by serial synchrotron crystallography.

Conformational dynamics are essential for proteins to function. We adapted time-resolved serial crystallography developed at x-ray lasers to visualize protein motions using synchrotrons. We recorded the structural changes in the light-driven proton-pump bacteriorhodopsin over 200 milliseconds in time. The snapshot from the first 5 milliseconds after photoactivation shows structural changes associated with proton release at a quality comparable to that of previous x-ray laser experiments. From 10 to 15 milliseconds onwards, we observe large additional structural rearrangements up to 9 angstroms on the cytoplasmic side. Rotation of leucine-93 and phenylalanine-219 opens a hydrophobic barrier, leading to the formation of a water chain connecting the intracellular aspartic acid-96 with the retinal Schiff base. The formation of this proton wire recharges the membrane pump with a proton for the next cycle.

Coro2b, a podocyte protein downregulated in human diabetic nephropathy, is involved in the development of protamine sulphate-induced foot process effacement

Podocytes have an important role in the pathogenesis of diabetic nephropathy (DN). Podocyte foot process effacement, mediated largely by the actin-based cytoskeleton of foot processes, is commonly detected in DN and is believed to be a key pathogenic event in the development of proteinuria. In this study, we identified coronin 2b (Coro2b), a member of known actin-regulating proteins, the coronins, as a highly podocyte-enriched molecule located at the cytoplasmic side of the apical plasma membrane. Studies in human renal biopsies show that glomerular Coro2b expression is significantly down-regulated in patients with DN. Studies in knockout mice indicate that Coro2b is not required for the development or maintenance of the glomerular filtration barrier. Moreover, inactivation of Coro2b specifically in podocytes does not affect the outcome of nephropathy in a streptozotocin-induced diabetes model. However, Coro2b seems to modulate the reorganization of foot processes under pathological conditions as Coro2b knockout podocytes are partially protected from protamine sulfate perfusion-induced foot process effacement. Taken together, our study suggests a role for Coro2b in the pathogenesis of glomerulopathies. Further studies regarding the involvement of Coro2b in podocyte health and diseases are warranted.

CpHMD-Then-QM/MM Identification of the Amino Acids Responsible for the Anabaena Sensory Rhodopsin pH-Dependent Electronic Absorption Spectrum.

Anabaena Sensory Rhodopsin (ASR), a microbial photoactive protein featuring the retinal chromophore in two different conformations, exhibits a pH-dependent electronic absorption spectrum. Using the recently developed CpHMD-then-QM/MM multiscale protocol applied to ASR embedded in a membrane model, the pH-induced changes in its maximum absorption wavelength have been reproduced and analyzed. While the acidic tiny red-shift is essentially correlated with the deprotonation of an aspartic acid located on the ASR extracellular side, the larger blue-shift experimentally reported at pH values larger than 5 involves a cluster of titrating residues sitting on the cytoplasmic side. The ASR pH-dependent spectrum is the consequence of the competitive stabilization of retinal ground and excited states by the protein electrostatic potential.