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ELife May 2020Approximately 25% of eukaryotic genes code for integral membrane proteins that are assembled at the endoplasmic reticulum. An abundant and widely conserved multi-protein...
Approximately 25% of eukaryotic genes code for integral membrane proteins that are assembled at the endoplasmic reticulum. An abundant and widely conserved multi-protein complex termed EMC has been implicated in membrane protein biogenesis, but its mechanism of action is poorly understood. Here, we define the composition and architecture of human EMC using biochemical assays, crystallography of individual subunits, site-specific photocrosslinking, and cryo-EM reconstruction. Our results suggest that EMC's cytosolic domain contains a large, moderately hydrophobic vestibule that can bind a substrate's transmembrane domain (TMD). The cytosolic vestibule leads into a lumenally-sealed, lipid-exposed intramembrane groove large enough to accommodate a single substrate TMD. A gap between the cytosolic vestibule and intramembrane groove provides a potential path for substrate egress from EMC. These findings suggest how EMC facilitates energy-independent membrane insertion of TMDs, explain why only short lumenal domains are translocated by EMC, and constrain models of EMC's proposed chaperone function.
Topics: Cytosol; Endoplasmic Reticulum; HEK293 Cells; Humans; Hydrophobic and Hydrophilic Interactions; Membrane Proteins; Protein Domains; SEC Translocation Channels
PubMed: 32459176
DOI: 10.7554/eLife.57887 -
Yakugaku Zasshi : Journal of the... 2022Cytochrome P450 (P450, CYP) and uridine 5'-diphospho-glucuronosyltransferase (UGT) are major drug-metabolizing enzymes known to catalyze substrate oxidation and...
Cytochrome P450 (P450, CYP) and uridine 5'-diphospho-glucuronosyltransferase (UGT) are major drug-metabolizing enzymes known to catalyze substrate oxidation and glucuronidation, respectively. Both enzymes are located within the endoplasmic reticulum (ER) membrane; however, their membrane topologies differ, with P450 facing cytosol and a major part of UGTs located on the luminal side. Because of the large differences in the reactions that they catalyze and their membrane topologies, it had been believed that P450 and UGT function separately. However, some chemicals are oxidized by P450 and undergo further conjugation by UGT. Therefore, it is important to consider that P450 and UGT may form a complex within the ER membrane and regulate each other's function through this interaction. To prove this hypothesis, we constructed a co-expression system for CYP3A4, P450 reductase, and UGT2B7/1A9 with a baculovirus-insect cell expression system. This system allowed us to compare CYP3A4 activity in the presence and absence of UGT2B7/1A9 co-expression and revealed that the UGTs suppress CYP3A4 activity. The suppressive effect of UGT2B7 was not limited to the enzymatic activity of CYP3A4 but also extended to the entire catalytic cycle, which may be resulted from the inhibition of substrate-binding to the P450. Analysis using UGT mutants indicated that one of the hydrophobic regions within the luminal portion of the UGT interacts with CYP3A4. Furthermore, we suggested that UGT1A suppresses CYP3A activity in vivo by treating rats with dexamethasone. Thus, the functional interactions between P450 and UGT would advance our understanding of the large inter-individual differences in drug-metabolizing capacity.
Topics: Animals; Rats; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Cytosol; Glucuronosyltransferase; Microsomes, Liver; Protein Binding
PubMed: 36328446
DOI: 10.1248/yakushi.22-00124 -
PloS One 2012The pantothenate kinases (PanK) catalyze the first and the rate-limiting step in coenzyme A (CoA) biosynthesis and regulate the amount of CoA in tissues by differential...
The pantothenate kinases (PanK) catalyze the first and the rate-limiting step in coenzyme A (CoA) biosynthesis and regulate the amount of CoA in tissues by differential isoform expression and allosteric interaction with metabolic ligands. The four human and mouse PanK proteins share a homologous carboxy-terminal catalytic domain, but differ in their amino-termini. These unique termini direct the isoforms to different subcellular compartments. PanK1α isoforms were exclusively nuclear, with preferential association with the granular component of the nucleolus during interphase. PanK1α also associated with the perichromosomal region in condensing chromosomes during mitosis. The PanK1β and PanK3 isoforms were cytosolic, with a portion of PanK1β associated with clathrin-associated vesicles and recycling endosomes. Human PanK2, known to associate with mitochondria, was specifically localized to the intermembrane space. Human PanK2 was also detected in the nucleus, and functional nuclear localization and export signals were identified and experimentally confirmed. Nuclear PanK2 trafficked from the nucleus to the mitochondria, but not in the other direction, and was absent from the nucleus during G2 phase of the cell cycle. The localization of human PanK2 in these two compartments was in sharp contrast to mouse PanK2, which was exclusively cytosolic. These data demonstrate that PanK isoforms are differentially compartmentalized allowing them to sense CoA homeostasis in different cellular compartments and enable interaction with regulatory ligands produced in these same locations.
Topics: Active Transport, Cell Nucleus; Amino Acid Sequence; Animals; Cell Compartmentation; Cell Nucleolus; Chromosomes, Mammalian; Clathrin; Cytosol; Endosomes; Fatty Acids, Unsaturated; Humans; Isoenzymes; Mammals; Mice; Mitochondria; Mitosis; Molecular Sequence Data; Mutagenesis; Nuclear Export Signals; Nuclear Localization Signals; Phosphotransferases (Alcohol Group Acceptor); Sequence Alignment
PubMed: 23152917
DOI: 10.1371/journal.pone.0049509 -
Cellular Physiology and Biochemistry :... 2016The 8-aminoquinoline tafenoquine has been shown to be effective against Plasmodia, Leishmania and Trypanosoma. The substance is at least in part effective by triggering...
BACKGROUND/AIMS
The 8-aminoquinoline tafenoquine has been shown to be effective against Plasmodia, Leishmania and Trypanosoma. The substance is at least in part effective by triggering apoptosis of the parasites. Similar to apoptosis, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the regulation of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, zVAD sensitive caspases, SB203580 sensitive p38 kinase, staurosporine sensitive protein kinase C as well as D4476 sensitive casein kinase. The present study explored, whether tafenoquine induces eryptosis and aimed to possibly identify cellular mechanisms involved.
METHODS
Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) dependent fluorescence, and ceramide abundance utilizing specific antibodies.
RESULTS
A 48 hours exposure of human erythrocytes to tafenoquine (500 ng/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly increased Fluo3-fluorescence, and significantly increased DCFDA fluorescence. Tafenoquine did not significantly modify ceramide abundance. The effect of tafenoquine on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. The effect of tafenoquine on annexin-V-binding was not significantly blunted by zVAD (10 µM), SB203580 (2 µM) or staurosporine (1 µM). The effect of tafenoquine on annexin-V-binding was significantly blunted but not abolished by D4476 (10 µM).
CONCLUSIONS
Tafenoquine triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to stimulation of Ca2+ entry, oxidative stress and possibly activation of casein kinase.
Topics: Aminoquinolines; Calcium; Cytosol; Eryptosis; Humans; Oxidative Stress; Phosphatidylserines; Reactive Oxygen Species
PubMed: 27855400
DOI: 10.1159/000452514 -
Proceedings of the National Academy of... Jan 2016Protein patterns are known to adapt to cell shape and serve as spatial templates that choreograph downstream processes like cell polarity or cell division. However, how...
Protein patterns are known to adapt to cell shape and serve as spatial templates that choreograph downstream processes like cell polarity or cell division. However, how can pattern-forming proteins sense and respond to the geometry of a cell, and what mechanistic principles underlie pattern formation? Current models invoke mechanisms based on dynamic instabilities arising from nonlinear interactions between proteins but neglect the influence of the spatial geometry itself. Here, we show that patterns can emerge as a direct result of adaptation to cell geometry, in the absence of dynamical instability. We present a generic reaction module that allows protein densities robustly to adapt to the symmetry of the spatial geometry. The key component is an NTPase protein that cycles between nucleotide-dependent membrane-bound and cytosolic states. For elongated cells, we find that the protein dynamics generically leads to a bipolar pattern, which vanishes as the geometry becomes spherically symmetrical. We show that such a reaction module facilitates universal adaptation to cell geometry by sensing the local ratio of membrane area to cytosolic volume. This sensing mechanism is controlled by the membrane affinities of the different states. We apply the theory to explain AtMinD bipolar patterns in [Formula: see text] EcMinDE Escherichia coli. Due to its generic nature, the mechanism could also serve as a hitherto-unrecognized spatial template in many other bacterial systems. Moreover, the robustness of the mechanism enables self-organized optimization of protein patterns by evolutionary processes. Finally, the proposed module can be used to establish geometry-sensitive protein gradients in synthetic biological systems.
Topics: Cell Polarity; Cytosol; Models, Biological; Protein Conformation; Proteins
PubMed: 26739566
DOI: 10.1073/pnas.1515191113 -
The Journal of Biological Chemistry Sep 2000
Review
Topics: Animals; Cytosol; Glycosylation; Humans; Proteins
PubMed: 10924527
DOI: 10.1074/jbc.R000010200 -
ACS Nano Mar 2017Genome editing through the delivery of CRISPR/Cas9-ribonucleoprotein (Cas9-RNP) reduces unwanted gene targeting and avoids integrational mutagenesis that can occur...
Genome editing through the delivery of CRISPR/Cas9-ribonucleoprotein (Cas9-RNP) reduces unwanted gene targeting and avoids integrational mutagenesis that can occur through gene delivery strategies. Direct and efficient delivery of Cas9-RNP into the cytosol followed by translocation to the nucleus remains a challenge. Here, we report a remarkably highly efficient (∼90%) direct cytoplasmic/nuclear delivery of Cas9 protein complexed with a guide RNA (sgRNA) through the coengineering of Cas9 protein and carrier nanoparticles. This construct provides effective (∼30%) gene editing efficiency and opens up opportunities in studying genome dynamics.
Topics: CRISPR-Cas Systems; Cytosol; Gene Editing; Gene Transfer Techniques; Protein Engineering; Ribonucleoproteins
PubMed: 28129503
DOI: 10.1021/acsnano.6b07600 -
Chemical Research in Toxicology Nov 20121,3-Butadiene (BD) is an air pollutant whose toxicity and carcinogenicity have been considered primarily mediated by its reactive metabolites, 3,4-epoxy-1-butene and...
1,3-Butadiene (BD) is an air pollutant whose toxicity and carcinogenicity have been considered primarily mediated by its reactive metabolites, 3,4-epoxy-1-butene and 1,2,3,4-diepoxybutane, formed in liver and extrahepatic tissues by cytochromes P450s. A possible alternative metabolic pathway in bone marrow and immune cells is the conversion of BD to the chlorinated allylic alcohol 1-chloro-2-hydroxy-3-butene (CHB) by myeloperoxidase in the presence of hydrogen peroxide and chloride ion. In the present study, we investigated the in vitro bioactivation of CHB by alcohol dehydrogenases (ADH) under in vitro physiological conditions (pH 7.4, 37 °C). The results provide clear evidence for CHB being converted to 1-chloro-3-buten-2-one (CBO) by purified horse liver ADH and rat liver cytosol. CBO readily reacted with glutathione (GSH) under assay conditions to form three products: two CBO-mono-GSH conjugates [1-chloro-4-(S-glutathionyl)butan-2-one (3) and 1-(S-glutathionyl)-3-buten-2-one (4)] and one CBO-di-GSH conjugate [1,4-bis(S-glutathionyl)butan-2-one (5)]. CHB bioactivation and the ratios of the three GSH conjugates formed were dependent upon incubation time, GSH and CHB concentrations, and the presence of ADH or rat liver cytosol. The ADH enzymatic reaction followed Michaelis-Menten kinetics with a K(m) at 3.5 mM and a k(cat) at 0.033 s(-1). After CBO was incubated with freshly isolated mouse erythrocytes, globin dimers were detected using SDS-PAGE and silver staining, providing evidence that CBO can act as a protein cross-linking agent. Collectively, the results provide clear evidence for CHB bioactivation by ADH and rat liver cytosol to yield CBO. The bifunctional alkylating ability of CBO suggests that it may play a role in BD toxicity and/or carcinogenicity.
Topics: Alcohol Dehydrogenase; Alkylation; Animals; Butanols; Butanones; Cytosol; Erythrocytes; Glutathione; Horses; Liver; Molecular Structure; Rats
PubMed: 23110628
DOI: 10.1021/tx300369b -
British Journal of Pharmacology Oct 19901. Using front-surface fluorometry with fura-2-loaded porcine coronary arterial strips, we simultaneously measured effects of a Ca2+ antagonist, diltiazem, on cytosolic...
1. Using front-surface fluorometry with fura-2-loaded porcine coronary arterial strips, we simultaneously measured effects of a Ca2+ antagonist, diltiazem, on cytosolic Ca2+ concentrations [( Ca2+]i) and on tension development. 2. In the presence of extracellular Ca2+ (1.25 mM), histamine concentration-dependently induced abrupt (the first component) and then sustained (the second component) elevations of [Ca2+]i. In the absence of extracellular Ca2+, histamine induced transient elevations of [Ca2+]i, and the time course was similar to that of the first component observed in the presence of extracellular Ca2+. Histamine caused a greater contraction for a given change in [Ca2+]i than did potassium, at [Ca2+]i over 300 nM. 3. Diltiazem, 10(-8)M to 10(-5)M, concentration-dependently inhibited the second component of [Ca2+]i elevation and tension development induced by histamine (10(-5) M). Only at higher concentrations (over 10(-5) M) did diltiazem inhibit the first component of increases in [Ca2+]i and tension development induced by histamine, both in the presence and absence of extracellular Ca2+. 4. Diltiazem (10(-6) M) inhibited increases in [Ca2+]i and tension development induced by cumulative applications of extracellular Ca2+ during K(+)-depolarization. The curve of [Ca2+]i against tension of these Ca2(+)-induced contractions obtained in diltiazem-treated strips overlapped with that obtained in untreated strips. This suggests that diltiazem has no direct effects on contractile elements. 5. In contrast, the histamine-induced Ca2(+)-tension curve (second component) was shifted in parallel to the left by diltiazem. 6. We conclude that diltiazem, at therapeutic concentrations, specifically inhibits extracellular Ca2+- dependent increases in [Ca2 +]i, with no effects on the release of Ca2 + from intracellular store sites or on Ca2 +-sensitivity of the contractile elements involved in the contractions induced by elevations of [Ca2 +]i.
Topics: Animals; Calcium; Coronary Vessels; Cytosol; Diltiazem; Drug Interactions; Female; Fluorometry; Fura-2; Histamine; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth, Vascular; Swine
PubMed: 2257435
DOI: 10.1111/j.1476-5381.1990.tb12700.x -
ELife Dec 2021Neural activity has been implicated in the motility and outgrowth of glial cell processes throughout the central nervous system. Here, we explore this phenomenon in...
Neural activity has been implicated in the motility and outgrowth of glial cell processes throughout the central nervous system. Here, we explore this phenomenon in Müller glia, which are specialized radial astroglia that are the predominant glial type of the vertebrate retina. Müller glia extend fine filopodia-like processes into retinal synaptic layers, in similar fashion to brain astrocytes and radial glia that exhibit perisynaptic processes. Using two-photon volumetric imaging, we found that during the second postnatal week, Müller glial processes were highly dynamic, with rapid extensions and retractions that were mediated by cytoskeletal rearrangements. During this same stage of development, retinal waves led to increases in cytosolic calcium within Müller glial lateral processes and stalks. These regions comprised distinct calcium compartments, distinguished by variable participation in waves, timing, and sensitivity to an M1 muscarinic acetylcholine receptor antagonist. However, we found that motility of lateral processes was unaffected by the presence of pharmacological agents that enhanced or blocked wave-associated calcium transients. Finally, we found that mice lacking normal cholinergic waves in the first postnatal week also exhibited normal Müller glial process morphology. Hence, outgrowth of Müller glial lateral processes into synaptic layers is determined by factors that are independent of neuronal activity.
Topics: Animals; Calcium; Cell Physiological Phenomena; Cytosol; Ependymoglial Cells; Female; Male; Mice; Mice, Inbred C57BL; Retina; Synaptic Transmission
PubMed: 34913435
DOI: 10.7554/eLife.73202