87 results on '"TMD, transmembrane domain"'
Search Results
2. The repertoire of ABC proteins in Clostridioides difficile
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Methinee Pipatthana, Surang Chankhamhaengdecha, Somsak Likhitrattanapisal, Tavan Janvilisri, Matthew Phanchana, Phurt Harnvoravongchai, and Pisut Pongchaikul
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SP, Substrate-binding domain (SBD)-containing protein ,Protein domain ,Biophysics ,ATP-binding cassette transporter ,Computational biology ,Biology ,In silico analysis ,Antimicrobial resistance ,Biochemistry ,Genome ,03 medical and health sciences ,TP, Transmembrane protein ,0302 clinical medicine ,Structural Biology ,Genetics ,NP, Nucleotide-binding domain (NBD)-containing protein ,ABC transporter/protein, ATP-binding cassette transporter/protein ,ECF-type transporter, Energy coupling factor-type transporter ,030304 developmental biology ,ComputingMethodologies_COMPUTERGRAPHICS ,0303 health sciences ,TMD, Transmembrane domain ,SBD, Substrate-binding domain ,Signal transducing adaptor protein ,Computer Science Applications ,Transmembrane domain ,NBD, Nucleotide-binding domain ,Membrane protein ,030220 oncology & carcinogenesis ,Active transporter ,ABC classification ,Function (biology) ,TP248.13-248.65 ,Reference genome ,Biotechnology ,Research Article - Abstract
Graphical abstract, Highlights • ABC transporters transport substrates across membranes driven by ATP hydrolysis. • ABC proteins of C. difficile 630 can be classified into 12 sub-families. • Most NPs are found within sub-families involving in drug export. • Most core NPs in C. difficile are associated with drug efflux system. • ABC proteins in sub-families 3, 6, 7, and 9 may participate in drug resistance., ATP-binding cassette (ABC) transporters belong to one of the largest membrane protein superfamilies, which function in translocating substrates across biological membranes using energy from ATP hydrolysis. Currently, the classification of ABC transporters in Clostridioides difficile is not complete. Therefore, the sequence-function relationship of all ABC proteins encoded within the C. difficile genome was analyzed. Identification of protein domains associated with the ABC system in the C. difficile 630 reference genome revealed 226 domains: 97 nucleotide-binding domains (NBDs), 98 transmembrane domains (TMDs), 30 substrate-binding domains (SBDs), and one domain with features of an adaptor protein. Gene organization and transcriptional unit analyses indicated the presence of 78 ABC systems comprising 28 importers and 50 exporters. Based on NBD sequence similarity, ABC transporters were classified into 12 sub-families according to their substrates. Interestingly, all ABC exporters, accounting for 64% of the total ABC systems, are involved in antibiotic resistance. Based on analysis of ABC proteins from 49 C. difficile strains, the majority of core NBDs are predicted to be involved in multidrug resistance systems, consistent with the ability of this organism to survive exposure to an array of antibiotics. Our findings herein provide another step toward a better understanding of the function and evolutionary relationships of ABC proteins in this pathogen.
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- 2021
3. Structural modeling of a novel membrane-bound globin-coupled sensor in Geobacter sulfurreducens
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Claudio D. Schuster, David Hoogewijs, Sabine Van Doorslaer, Salvador I. Drusin, Luc Moens, Sylvia Dewilde, Catherine Vénien-Bryan, Marcelo A. Martí, Frank Sobott, Dietmar Hammerschmid, Charline Fagnen, Francesca Germani, Institut de minéralogie, de physique des matériaux et de cosmochimie (IMPMC), Muséum national d'Histoire naturelle (MNHN)-Institut de recherche pour le développement [IRD] : UR206-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie et pharmacologie appliquée (LBPA), Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), Université de Fribourg = University of Fribourg (UNIFR), Universidad de Buenos Aires [Buenos Aires] (UBA), Biomedical Sciences, University of Antwerp (UA), University of Antwerp, Department of Physics, and University of Leeds
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DPV, differential pulse voltammetry ,PDE, phosphodiesterase ,Transmembrane-coupled globins ,[SDV]Life Sciences [q-bio] ,SaktrHb, Streptomyces avermitilis truncated hemoglobin-antibiotic monooxygenase ,Biochemistry ,Transmembrane domain ,TmD, Transmembrane domain ,chemistry.chemical_compound ,0302 clinical medicine ,Structural Biology ,Globin-coupled sensor ,RR, resonance Raman ,TD, Transmitter domain ,IM-MS, ion mobility-mass spectrometry ,GsGCS162, GD of GsGCS ,Geobacter sulfurreducens ,Heme ,0303 health sciences ,CeGLB26, Caenorhabditis elegans globin 26 ,biology ,Gb, globin ,CIU, collision-induced unfolding ,CMC, critical micelle concentration ,PccGCS, Pectobacterium carotivorum GCS ,CV, cyclic voltammetry ,Transmembrane protein ,Computer Science Applications ,Chemistry ,GD, globin domain ,030220 oncology & carcinogenesis ,MtTrHbO, Mycobacterium tuberculosis truncated hemoglobin O ,Signal transduction ,Engineering sciences. Technology ,Research Article ,PcMb, Physether catodon myoglobin ,Biotechnology ,AfGcHK, Anaeromyxobacter sp. Fw109-5 GcHK ,BsHemAT, Bacillus subtilis HemAT ,NH4OAc, ammonium acetate ,LmHemAC, Leishmania major HemAC ,CeGLB6, Caenorhabditis elegans globin 6 ,Biophysics ,GGDEF, Gly-Gly-Asp-Glu-Phe motive ,GCS, globin-coupled sensor ,AsFRMF, Ascaris suum FRMF-amide receptor ,SwMb, myoglobin from sperm whale ,GintHb, hemoglobin from Gasterophilus intestinalis ,03 medical and health sciences ,Tetramer ,Genetics ,SHE, standard hydrogen electrode ,BpGReg, Bordetella pertussis Greg ,GsGCS, Geobacter sulfurreducens GCS ,AvGReg, Azotobacter vinilandii Greg ,Globin ,MaPgb, Methanosarcina acetivorans protoglobin ,Biology ,ComputingMethodologies_COMPUTERGRAPHICS ,CeGLB33, Caenorhabditis elegans globin 33 ,030304 developmental biology ,EcDosC, Escherichia coli Dos with DGC activity ,OG, n-octyl-β-d-glucopyranoside ,DDM, n-dodecyl-β-d-maltoside ,biology.organism_classification ,FMRF, H-Phe-Met-Arg-Phe-NH2 neuropeptide ,PsiE, phosphate-starvation-inducible E ,SCE, saturated calomel electrode ,chemistry ,mNgb, mouse neuroglobin ,CCS, collision cross section ,Linker ,TP248.13-248.65 - Abstract
Graphical abstract, Globin-coupled sensors (GCS) usually consist of three domains: a sensor/globin, a linker, and a transmitter domain. The globin domain (GD), activated by ligand binding and/or redox change, induces an intramolecular signal transduction resulting in a response of the transmitter domain. Depending on the nature of the transmitter domain, GCSs can have different activities and functions, including adenylate and di-guanylate cyclase, histidine kinase activity, aerotaxis and/or oxygen sensing function. The gram-negative delta-proteobacterium Geobacter sulfurreducens expresses a protein with a GD covalently linked to a four transmembrane domain, classified, by sequence similarity, as GCS (GsGCS). While its GD is fully characterized, not so its transmembrane domain, which is rarely found in the globin superfamily. In the present work, GsGCS was characterized spectroscopically and by native ion mobility-mass spectrometry in combination with cryo-electron microscopy. Although lacking high resolution, the oligomeric state and the electron density map were valuable for further rational modeling of the full-length GsGCS structure. This model demonstrates that GsGCS forms a transmembrane domain-driven tetramer with minimal contact between the GDs and with the heme groups oriented outward. This organization makes an intramolecular signal transduction less likely. Our results, including the auto-oxidation rate and redox potential, suggest a potential role for GsGCS as redox sensor or in a membrane-bound e−/H+ transfer. As such, GsGCS might act as a player in connecting energy production to the oxidation of organic compounds and metal reduction. Database searches indicate that GDs linked to a four or seven helices transmembrane domain occur more frequently than expected.
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- 2021
4. Structure and function of the vacuolar Ccc1/VIT1 family of iron transporters and its regulation in fungi
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Sergi Puig, María Teresa Martínez-Pastor, David Peris, Raquel Sorribes-Dauden, Ministerio de Ciencia, Innovación y Universidades (España), Generalitat Valenciana, and European Commission
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ISC, Iron-sulfur luster ,CS, Consistency score ,Ccc1 ,Ribosome biogenesis ,Vacuole ,Review Article ,YRE, Yap response elements ,Biochemistry ,Biotecnologia ,0302 clinical medicine ,Structural Biology ,Cg, Candida glabrata ,0303 health sciences ,MAFFT, Multiple Alignment using Fast Fourier Transform ,NRAMP, Natural Resistance-Associated Macrophage Protein ,biology ,VIT1 ,Chemistry ,MBD, Metal-binding domain ,Plants ,Computer Science Applications ,030220 oncology & carcinogenesis ,CRD, Cysteine-rich domain ,Eg, Eucalyptus grandis ,Iron detoxification ,Biotechnology ,CBC, CCAAT-binding core complex ,lcsh:Biotechnology ,Saccharomyces cerevisiae ,VTL, Vacuolar iron transporter-like ,Biophysics ,VIT, Vacuolar iron transporter ,bZIP, basic leucine-zipper ,03 medical and health sciences ,Fongs ,Lipid biosynthesis ,lcsh:TP248.13-248.65 ,Genetics ,Fe, Iron ,Iron transport ,Transcription factor ,030304 developmental biology ,ComputingMethodologies_COMPUTERGRAPHICS ,BLOSUM, BLOcks SUbstitution Matrix ,TMD, Transmembrane domain ,ML, Maximum-likelihood ,Iron regulation ,DNA replication ,Fungi ,biology.organism_classification ,Yeast ,Metabolic pathway ,H, Helix ,Hap, Heme activator protein ,ROS, Reactive oxygen species ,Ferro - Abstract
Iron is an essential micronutrient for most living beings since it participates as a redox active cofactor in many biological processes including cellular respiration, lipid biosynthesis, DNA replication and repair, and ribosome biogenesis and recycling. However, when present in excess, iron can participate in Fenton reactions and generate reactive oxygen species that damage cells at the level of proteins, lipids and nucleic acids. Organisms have developed different molecular strategies to protect themselves against the harmful effects of high concentrations of iron. In the case of fungi and plants, detoxification mainly occurs by importing cytosolic iron into the vacuole through the Ccc1/VIT1 iron transporter. New sequenced genomes and bioinformatic tools are facilitating the functional characterization, evolution and ecological relevance of metabolic pathways and homeostatic networks across the Tree of Life. Sequence analysis shows that Ccc1/VIT1 homologs are widely distributed among organisms with the exception of animals. The recent elucidation of the crystal structure of a Ccc1/VIT1 plant ortholog has enabled the identification of both conserved and species-specific motifs required for its metal transport mechanism. Moreover, recent studies in the yeast Saccharomyces cerevisiae have also revealed that multiple transcription factors including Yap5 and Msn2/Msn4 contribute to the expression of CCC1 in high-iron conditions. Interestingly, Malaysian S. cerevisiae strains express a partially functional Ccc1 protein that renders them sensitive to iron. Different regulatory mechanisms have been described for non-Saccharomycetaceae Ccc1 homologs. The characterization of Ccc1/VIT1 proteins is of high interest in the development of biofortified crops and the protection against microbial-derived diseases., Work in our laboratory is supported by the Spanish Ministry of Science, Innovation and Universities (MICINN) grant BIO2017-87828-C2-1-P, the Regional Government of Valencia “Generalitat Valenciana” grant PROMETEU/2020/014 and FEDER (Fondo Europeo de Desarrollo Regional) funds to SP, and a predoctoral contract from “Generalitat Valenciana” and FEDER funds to RSD. Computations were performed on Tirant III of the Spanish Supercomputing Network (“Servei d'Informàtica de la Universitat de València”) under the project BCV-2018-2-0002 granted to DP.
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- 2020
5. The Sigma-1 receptor is an ER-localized type II membrane protein
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Marina Shenkman, Neeraj Sharma, Nir Ben-Tal, Gerardo Z. Lederkremer, Chaitanya Patel, and Amit Kessel
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TG, thapsigargin ,Strep, streptavidin ,TMD, transmembrane domain ,Endoplasmic Reticulum ,Biochemistry ,ER, endoplasmic reticulum ,membrane topology ,Humans ,Receptors, sigma ,Molecular Biology ,CNX, calnexin ,Calcium signaling ,sulfo-NHS-LC-Biotin, sulfosuccinimidyl-6-(biotinamido)hexanoate ,Sigma-1 receptor ,Chemistry ,Endoplasmic reticulum ,S1R, Sigma-1 receptor ,neurodegeneration ,ER retention ,Cell Biology ,Intracellular Membranes ,MAM, mitochondrion-associated membranes ,Transmembrane protein ,Cell biology ,HEK293 Cells ,cell surface ,Membrane protein ,sigma-1 receptor ,Membrane topology ,Unfolded protein response ,BAP, biotin acceptor peptide ,ER stress ,Research Article - Abstract
The Sigma-1 receptor (S1R) is a transmembrane protein with important roles in cellular homeostasis in normal physiology and in disease. Especially in neurodegenerative diseases, S1R activation has been shown to provide neuroprotection by modulating calcium signaling, mitochondrial function and reducing endoplasmic reticulum (ER) stress. S1R missense mutations are one of the causes of the neurodegenerative Amyotrophic Lateral Sclerosis and distal hereditary motor neuronopathies. Although the S1R has been studied intensively, basic aspects remain controversial, such as S1R topology and whether it reaches the plasma membrane. To address these questions, we have undertaken several approaches. C-terminal tagging with a small biotin-acceptor peptide and BirA biotinylation in cells suggested a type II membrane orientation (cytosolic N-terminus). However, N-terminal tagging gave an equal probability for both possible orientations. This might explain conflicting reports in the literature, as tags may affect the protein topology. Therefore, we studied untagged S1R using a protease protection assay and a glycosylation mapping approach, introducing N-glycosylation sites. Both methods provided unambiguous results showing that the S1R is a type II membrane protein with a short cytosolic N-terminal tail. Assessments of glycan processing, surface fluorescence-activated cell sorting, and cell surface biotinylation indicated ER retention, with insignificant exit to the plasma membrane, in the absence or presence of S1R agonists or of ER stress. These findings may have important implications for S1R-based therapeutic approaches.
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- 2021
6. Proteomic Analysis of Trichomonas vaginalis Phagolysosome, Lysosomal Targeting, and Unconventional Secretion of Cysteine Peptidases
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Nadine, Zimmann, Petr, Rada, Vojtěch, Žárský, Tamara, Smutná, Kristína, Záhonová, Joel, Dacks, Karel, Harant, Ivan, Hrdý, and Jan, Tachezy
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Proteomics ,CP, cysteine protease ,ST, sucrose-tris buffer ,LAMP, lysosome-associated membrane glycoprotein ,BFA, Brefeldin A ,MPR, mannose 6-phopshate receptor ,SNAP, soluble N-ethylmaleimide-sensitive-factor attachment protein ,DMAP, DNA methyltransferase-associated protein ,TMD, transmembrane domain ,BA, β-amylase ,TEAB, triethylammonium bicarbonate ,Cysteine Proteases ,Phagosomes ,TSP, tetraspanin ,MPR300, mannose 6-phopshate receptor 300 ,mCLCP, mutated cathepsin L-like cysteine peptidase ,CD-MPR, cation-dependent mannose 6-phosphate receptor ,LTS, lysosomal targeting sequence ,LFQ MS, label-free quantitative mass spectrometry ,MRH, mannose 6-phosphate receptor homology ,SNARE, SNAP receptor ,CPBF1, cysteine protease-binding protein family 1 ,TYM, tryptone-yeast extract-maltose medium ,nanoLC-MS, nano reverse-phase liquid chromatography mass spectrometry ,NHS, N-hydroxysuccinimide ,bHX, β-hexosaminidase ,FITC, fluorescein isothiocyanate ,SDC, sodium deoxycholate ,MES, 2-[n-morpholino]-ethanesulfonic acid buffer ,TLCK, tosyl-L-lysyl-chloromethane hydrochloride ,glycosylation ,proteome ,PBS, phosphate-buffered saline ,vATPase, vacuolar ATPase ,CLCP, cathepsin L-like cysteine peptidase ,LGF, large granule fraction ,ER, endoplasmic reticulum ,mannose 6-phosphate receptor ,TCA, trichloracetic acid ,Trichomonas vaginalis ,Humans ,GGA, Golgi-localized, γ-ear-containing, ADP ribosylation factor-binding protein ,Cysteine ,GlcNAc-PT, N-acetylglucosamine-1-phosphotransferase ,s-LTS, lysosomal targeting sequence of soluble protein ,TGN, trans Golgi network ,UCE, uncovering enzyme, N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase ,AP, acid phosphatase ,Research ,M6P, mannose 6-phosphate ,CI-MPR, cation-independent mannose 6-phosphate receptor ,LERP, lysosomal enzyme receptor protein ,EDC, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride ,mBA, mutated β-amylase ,CLQ, chloroquine ,TBSR, Trichomonas beta-sandwich repeat protein ,MS, mass spectrometry ,BBS, borate buffered saline ,phagolysosome ,t-LTS, lysosomal targeting sequence of transmembrane protein ,Lysosomes ,cysteine peptidase ,LF, lactoferrin ,Peptide Hydrolases ,HA, hemagglutinin - Abstract
The lysosome represents a central degradative compartment of eukaryote cells, yet little is known about the biogenesis and function of this organelle in parasitic protists. Whereas the mannose 6-phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide-independent sorting has been reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting and the involvement of lysosomes in hydrolase secretion. The organelles were purified using Percoll and OptiPrep gradient centrifugation and a novel purification protocol based on the phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in a lysosomal proteome of 462 proteins, which were sorted into 21 classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate a dynamic phagolysosomal compartment. Several cysteine proteases such as TvCP2 were previously shown to be secreted. Our experiments showed that secretion of TvCP2 was strongly inhibited by chloroquine, which increases intralysosomal pH, thus indicating that TvCP2 secretion occurs through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified divergent homologues of the M6P receptor TvMPR in the phagolysosomal proteome, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP, which possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, the introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as a recognition marker for lysosomal hydrolases. Our findings provide the first insight into the complexity of T. vaginalis phagolysosomes, their biogenesis, and role in the unconventional secretion of cysteine peptidases., Graphical Abstract, Highlights • Trichomonas vaginalis phagolysosome consist of over 460 proteins. • Lysosomes are involved in secretion of virulence factors such as TvCP2. • N-glycosylation is required for lysosomal protein targeting. • T. vaginalis possesses homologs of mannose 6-phosphate receptor., In Brief Lysosomes represent a central degradative compartment of eukaryotes, yet little is known about biogenesis and function of this organelle in the parasitic protist Trichomonas vaginalis. We analyzed the phagolysosomal proteome that consists of over 460 proteins including important virulence factors. We demonstrated that glycosylation is involved in lysosomal protein targeting in T. vaginalis, which is unprecedented in parasitic protists. In addition to the classical secretory pathway, lysosomes are involved in unconventional protein secretion.
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- 2021
7. Structural basis of NF-κB signaling by the p75 neurotrophin receptor interaction with adaptor protein TRADD through their respective death domains
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Zhen Li, Xianbin Tian, Lilian Kisiswa, Ajeena Ramanujan, Ning Zhang, Carlos F. Ibáñez, Wensu Yuan, Zhi Lin, and Eunice Weiling Sim
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Male ,PEI, polyethylenimine ,0301 basic medicine ,Protein Conformation ,TMD, transmembrane domain ,Receptor, Nerve Growth Factor ,Biochemistry ,RMSD, root mean square deviation ,Neurotrophic factors ,Low-affinity nerve growth factor receptor ,p75 neurotrophin receptor ,Mice, Knockout ,Neurons ,biology ,NF-kappa B ,Signal transducing adaptor protein ,DD, death domain ,TNF Receptor-Associated Death Domain Protein ,Cell biology ,NT, neurotrophin ,Female ,TRADD, TNF receptor associated death domain ,Signal Transduction ,Research Article ,Neurotrophin ,Cell signaling ,death domain ,NGFR, nerve growth factor receptor ,ICD, intracellular domain ,Nerve Tissue Proteins ,Receptors, Nerve Growth Factor ,p75NTR, p75 neurotrophin receptor ,TNFRSF, tumor necrosis factor receptor superfamily ,03 medical and health sciences ,ECD, extracellular cysteine-rich domain ,Animals ,Humans ,cell signaling ,Protein Interaction Domains and Motifs ,protein structure ,NMR, nuclear magnetic resonance ,Molecular Biology ,Death domain ,030102 biochemistry & molecular biology ,ES, embryonic stem ,Cell Biology ,TRADD ,NMR ,Mice, Inbred C57BL ,CGN, cerebellar granule neuron ,030104 developmental biology ,Nerve growth factor ,biology.protein ,sense organs - Abstract
The p75 neurotrophin receptor (p75NTR) is a critical mediator of neuronal death and tissue remodeling and has been implicated in various neurodegenerative diseases and cancers. The death domain (DD) of p75NTR is an intracellular signaling hub and has been shown to interact with diverse adaptor proteins. In breast cancer cells, binding of the adaptor protein TRADD to p75NTR depends on nerve growth factor and promotes cell survival. However, the structural mechanism and functional significance of TRADD recruitment in neuronal p75NTR signaling remain poorly understood. Here we report an NMR structure of the p75NTR-DD and TRADD-DD complex and reveal the mechanism of specific recognition of the TRADD-DD by the p75NTR-DD mainly through electrostatic interactions. Furthermore, we identified spatiotemporal overlap of p75NTR and TRADD expression in developing cerebellar granule neurons (CGNs) at early postnatal stages and discover the physiological relevance of the interaction between TRADD and p75NTR in the regulation of canonical NF-κB signaling and cell survival in CGNs. Our results provide a new structural framework for understanding how the recruitment of TRADD to p75NTR through DD interactions creates a membraneproximal platform, which can be efficiently regulated by various neurotrophic factors through extracellular domains of p75NTR, to propagate downstream signaling in developing neurons.
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- 2021
8. Interaction between the transmembrane domains of neurotrophin receptors p75 and TrkA mediates their reciprocal activation
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Fozia Ahmed, Kalina Hristova, Sergey A. Goncharuk, Andrea Soler-Lopez, Marçal Vilar, Kirill D. Nadezhdin, Taylor P. Light, Konstantin S. Mineev, Alexander S. Arseniev, María Luisa Franco, Ministerio de Economía y Competitividad (España), Generalitat Valenciana, National Institutes of Health (US), Russian Science Foundation, Vilar, Marçal, and Vilar, Marçal [0000-0002-9376-6544]
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Receptor complex ,TMD, transmembrane domain ,Tropomyosin receptor kinase B ,eYFP, enhanced YFP ,Tropomyosin receptor kinase A ,Biochemistry ,mTurq, mTurquoise ,PC12 Cells ,Receptor, Nerve Growth Factor ,Transmembrane domain ,CG, coarse-grained ,FSI, fully quantified spectral imaging ,Receptor ,FGFR3, fibroblast growth factor receptor 3 ,LPR, lipid-to-protein ratio ,NGF ,DMEM, Dulbecco's modified Eagle's medium ,biology ,Chemistry ,TrkA ,neurotrophin ,TrkB ,Cell Differentiation ,transmembrane domain ,POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine ,MD, molecular dynamics ,TNFR, tumor necrosis factor receptor ,Cell biology ,p75ntr ,NT, neurotrophin ,Neurotrophin ,CS, chemical shift ,Research Article ,Protein Binding ,musculoskeletal diseases ,p75 ,animal structures ,Neurogenesis ,ICD, intracellular domain ,NGF, nerve growth factor ,FBS, fetal bovine serum ,Protein Domains ,DPC, dodecylphosphocholine ,TrkA, tyrosine protein kinase receptor A ,Animals ,Receptor, trkA ,Protein kinase A ,Molecular Biology ,LAT, linker for the activation of T-cells ,Cell Biology ,biological factors ,NMR ,Rats ,Nerve growth factor ,nervous system ,biology.protein ,sense organs ,FA, full-atom - Abstract
12 páginas, 6 figuras. CSs fromTrkA-TMD and p75-TMD are deposited in the Biological Magnetic Resonance Data Bank with accession number 25872for TrkA-TMD and 19673 for p75-TMD., The neurotrophin receptors p75 and tyrosine protein kinase receptor A (TrkA) play important roles in the development and survival of the nervous system. Biochemical data suggest that p75 and TrkA reciprocally regulate the activities of each other. For instance, p75 is able to regulate the response of TrkA to lower concentrations of nerve growth factor (NGF), and TrkA promotes shedding of the extracellular domain of p75 by α-secretases in a ligand-dependent manner. The current model suggests that p75 and TrkA are regulated by means of a direct physical interaction; however, the nature of such interaction has been elusive thus far. Here, using NMR in micelles, multiscale molecular dynamics, FRET, and functional studies, we identified and characterized the direct interaction between TrkA and p75 through their respective transmembrane domains (TMDs). Molecular dynamics of p75-TMD mutants suggests that although the interaction between TrkA and p75 TMDs is maintained upon mutation, a specific protein interface is required to facilitate TrkA active homodimerization in the presence of NGF. The same mutations in the TMD protein interface of p75 reduced the activation of TrkA by NGF as well as reducing cell differentiation. In summary, we provide a structural model of the p75-TrkA receptor complex necessary for neuronal development stabilized by TMD interactions., This study was supported bythe Spanish Ministry of Economy and Competitiveness (project BFU2013-42746-P and SAF2017-84096-R), the GeneralitatValenciana Prometeo grant 2018/055 (to M. V.), and the National Institutes of Health GM068619 (to K. H.). NMR studies of TRKA-TM and p75-TM were supported by the Russian Science Founda-tion (grant no. 19-74-30014 to A. S. A.)
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- 2021
9. Regulation of a pentameric ligand-gated ion channel by a semiconserved cationic lipid-binding site
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Marijke Brams, Diletta Pasini, Akshay Sridhar, Chris Ulens, Sarah C. R. Lummis, Kumiko Kambara, Rebecca J. Howard, Aujan Mehregan, Erik Lindahl, Daniel Bertrand, Lummis, Sarah [0000-0001-9410-9805], and Apollo - University of Cambridge Repository
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DYNAMICS ,MECHANISM ,0301 basic medicine ,Models, Molecular ,POPG, palmitoyloleoylphosphatidylglycerol ,TMD, transmembrane domain ,DOTAP, dipalmitoyl-3-trimethylammonium-propane ,PG, phosphatidylglycerol ,Crystallography, X-Ray ,Biochemistry ,ACTIVATION ,chemistry.chemical_compound ,PC, phosphatidylcholine ,Xenopus laevis ,Chemistry ,GABAA receptor ,Editors' Pick ,Lipids ,Transmembrane domain ,VSD, voltage-sensor domain ,K+ CHANNEL ,Ligand-gated ion channel ,Protein Structural Elements ,Signal transduction ,site-directed mutagenesis ,Life Sciences & Biomedicine ,Research Article ,Protein Binding ,STRUCTURAL BASIS ,Biochemistry & Molecular Biology ,SOFTWARE NEWS ,Protein subunit ,ICD, intracellular domain ,Cys-loop receptor ,POPC, palmitoyloleoylphosphatidylcholine ,PE, phosphatidylethanolamine ,Cell Line ,03 medical and health sciences ,pLGIC, pentameric ligand-gated ion channel ,Cations ,Animals ,Humans ,Molecular Biology ,POPC ,Ion channel ,G protein-coupled receptor ,GPCR, G-protein-coupled receptor ,Science & Technology ,RECEPTOR ,030102 biochemistry & molecular biology ,RMSD, root-mean-squared deviation ,Cell Biology ,Ligand-Gated Ion Channels ,electrophysiology ,nAChR, nicotinic acetylcholine receptor ,ECD, extracellular domain ,WT, wild-type ,molecular dynamics ,PROTEIN INTERACTIONS ,030104 developmental biology ,GENERAL-ANESTHETICS ,Biophysics ,Oocytes ,ELIC ,pentameric ligand-gated ion channel ,X-RAY-STRUCTURE - Abstract
Pentameric ligand-gated ion channels (pLGICs) are crucial mediators of electrochemical signal transduction in various organisms from bacteria to humans. Lipids play an important role in regulating pLGIC function, yet the structural bases for specific pLGIC-lipid interactions remain poorly understood. The bacterial channel ELIC recapitulates several properties of eukaryotic pLGICs, including activation by the neurotransmitter GABA and binding and modulation by lipids, offering a simplified model system for structure-function relationship studies. In this study, functional effects of noncanonical amino acid substitution of a potential lipid-interacting residue (W206) at the top of the M1-helix, combined with detergent interactions observed in recent X-ray structures, are consistent with this region being the location of a lipid-binding site on the outward face of the ELIC transmembrane domain. Coarse-grained and atomistic molecular dynamics simulations revealed preferential binding of lipids containing a positive charge, particularly involving interactions with residue W206, consistent with cation-π binding. Polar contacts from other regions of the protein, particularly M3 residue Q264, further support lipid binding via headgroup ester linkages. Aromatic residues were identified at analogous sites in a handful of eukaryotic family members, including the human GABAA receptor ε subunit, suggesting conservation of relevant interactions in other evolutionary branches. Further mutagenesis experiments indicated that mutations at this site in ε-containing GABAA receptors can change the apparent affinity of the agonist response to GABA, suggesting a potential role of this site in channel gating. In conclusion, this work details type-specific lipid interactions, which adds to our growing understanding of how lipids modulate pLGICs. ispartof: JOURNAL OF BIOLOGICAL CHEMISTRY vol:297 issue:2 ispartof: location:United States status: published
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- 2021
10. Identification of a clinical compound losmapimod that blocks Lassa virus entry
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Feihu Yan, Ying Guo, Logan Banadyga, Shihua He, Wenjun Zhu, Guo Jiamei, Xiangguo Qiu, Qing Chen, Tang Ke, and Xiaoyu Zhang
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Cyclopropanes ,0301 basic medicine ,MAPK/ERK pathway ,SABV, Sabia virus ,Pyridines ,viruses ,Drug repurposing ,TMD, transmembrane domain ,medicine.disease_cause ,CHPV, Chapare virus ,SI, selectivity index ,p38 Mitogen-Activated Protein Kinases ,α-DG, α-dystroglycan ,Chlorocebus aethiops ,Enzyme Inhibitors ,COPD, chronic obstructive pulmonary diseases ,Lassa fever ,WWAV, Whitewater Arroyo virus ,SSP, stable signal peptide ,chemistry.chemical_classification ,JUNV, Junin virus ,MACV, Machupo virus ,V-ATPase, vacuolar-type H+-ATPase ,Arenavirus ,SlP, site 1 protease ,Entry inhibitor ,GEQ, genome equivalent ,hTfR1, Human transferrin receptor 1 ,LCMV, Lymphocytic choriomeningitis virus ,3. Good health ,CC50, half maximal cytotoxic concentration ,CPE, cytopathic effect ,SSP-GP2 ,VHF, viral hemorrhagic fever ,EC50, half maximal effective concentration ,medicine.drug ,GPC, glycoprotein complex ,030106 microbiology ,Biology ,Antiviral Agents ,Article ,Cell Line ,Viral hemorrhagic fever ,03 medical and health sciences ,Lassa Fever ,GTOV, Guanarito virus ,BSL-4, biosafety level 4 ,Virology ,medicine ,Animals ,Arenaviridae Infections ,Humans ,Lassa virus ,Protein kinase A ,Vero Cells ,LASV, Lassa virus ,Pharmacology ,Losmapimod ,Drug Repositioning ,LAMP1, lysosome-associated membrane protein 1 ,Virus Internalization ,medicine.disease ,LUJV, Lujo virus ,030104 developmental biology ,chemistry ,Glycoprotein ,Viral Fusion Proteins ,CAD, cation amphiphilic drug - Abstract
Lassa virus (LASV) causes Lassa hemorrhagic fever in humans and poses a significant threat to public health in West Africa. Current therapeutic treatments for Lassa fever are limited, making the development of novel countermeasures an urgent priority. In this study, we identified losmapimod, a p38 mitogen-activated protein kinase (MAPK) inhibitor, from 102 screened compounds as an inhibitor of LASV infection. Losmapimod exerted its inhibitory effect against LASV after p38 MAPK down-regulation, and, interestingly, had no effect on other arenaviruses capable of causing viral hemorrhagic fever. Mechanistic studies showed that losmapimod inhibited LASV entry by affecting the stable signal peptide (SSP)-GP2 subunit interface of the LASV glycoprotein, thereby blocking pH-dependent viral fusion. As an aryl heteroaryl bis-carboxyamide derivative, losmapimod represents a novel chemical scaffold with anti-LASV activity, and it provides a new lead structure for the future development of LASV fusion inhibitors., Highlights • Losmapimod was identified as a Lassa virus entry inhibitor following the screening of 102 clinical compounds. • Losmapimod inhibited Lassa virus infection by affecting the SSP-GP2 interface of the Lassa virus glycoprotein. • Losmapimod preferentially inhibits the entry of Lassa virus among arenaviruses known to cause human disease.
- Published
- 2019
11. A Proteomic Study on the Membrane Protein Fraction of T Cells Confirms High Substrate Selectivity for the ER Translocation Inhibitor Cyclotriazadisulfonamide
- Author
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Becky Provinciael, Eberhard Krause, Claudia Rutz, Dominique Schols, Heike Stephanowitz, Joren Stroobants, Eva Pauwels, Enno Hartmann, Kurt Vermeire, and Ralf Schülein
- Subjects
Signal peptide ,Proteomics ,CADA, cyclotriazadisulfonamide ,SILAC, stable isotope labeling by amino acids in cell culture ,T-Lymphocytes ,V5, virus 5 ,cyclotriazadisulfonamide ,ERAD, endoplasmic reticulum–associated degradation ,TMD, transmembrane domain ,Endoplasmic-reticulum-associated protein degradation ,DMSO, dimethyl sulfoxide ,Endoplasmic Reticulum ,ER cotranslational translocation ,ERLEC1, endoplasmic reticulum lectin 1 ,SILAC ,SAS, signal anchor sequence ,Cell Line ,Substrate Specificity ,ER, endoplasmic reticulum ,Stable isotope labeling by amino acids in cell culture ,Humans ,signal peptide ,DNAJC3, DnaJ homolog subfamily C member 3 ,PL, prolactin ,eGFP, enhanced GFP ,Integral membrane protein ,SORT, sortilin ,Inactive Tyrosine-Protein Kinase 7 ,Sulfonamides ,huCD4, human CD4 ,Chemistry ,Endoplasmic reticulum ,Research ,Membrane Proteins ,General Medicine ,HEK293T, human embryonic kidney 293T ,BFP, blue fluorescent protein ,CD4 ,Cell biology ,Membrane protein ,Isotope Labeling ,SP, signal peptide ,DNAJC3 ,tGFP, turbo GFP ,PTK7, inactive tyrosine-protein kinase 7 ,small-molecule inhibitor - Abstract
Cyclotriazadisulfonamide (CADA) inhibits the cotranslational translocation of type I integral membrane protein human CD4 (huCD4) across the endoplasmic reticulum in a signal peptide (SP)–dependent way. Previously, sortilin was identified as a secondary substrate for CADA but showed reduced CADA sensitivity as compared with huCD4. Here, we performed a quantitative proteomic study on the crude membrane fraction of human T-cells to analyze how many proteins are sensitive to CADA. To screen for these proteins, we employed stable isotope labeling by amino acids in cell culture technique in combination with quantitative MS on CADA-treated human T-lymphoid SUP-T1 cells expressing high levels of huCD4. In line with our previous reports, our current proteomic analysis (data available via ProteomeXchange with identifier PXD027712) demonstrated that only a very small subset of proteins is depleted by CADA. Our data also confirmed that cellular expression of both huCD4 and sortilin are affected by CADA treatment of SUP-T1 cells. Furthermore, three additional targets for CADA are identified, namely, endoplasmic reticulum lectin 1 (ERLEC1), inactive tyrosine-protein kinase 7 (PTK7), and DnaJ homolog subfamily C member 3 (DNAJC3). Western blot and flow cytometry analysis of ERLEC1, PTK7, and DNAJC3 protein expression validated susceptibility of these substrates to CADA, although with varying degrees of sensitivity. Additional cell-free in vitro translation/translocation data demonstrated that the new substrates for CADA carry cleavable SPs that are targets for the cotranslational translocation inhibition exerted by CADA. Thus, our quantitative proteomic analysis demonstrates that ERLEC1, PTK7, and DNAJC3 are validated additional substrates of CADA; however, huCD4 remains the most sensitive integral membrane protein for the endoplasmic reticulum translocation inhibitor CADA. Furthermore, to our knowledge, CADA is the first compound that specifically interferes with only a very small subset of SPs and does not affect signal anchor sequences., Graphical Abstract, Highlights • About 3007 proteins quantified in SILAC/MS study on CD4+ T-cells treated with CADA. • Three new targets for CADA were identified: ERLEC1, PTK7, and DNAJC3. • All CADA substrates carry cleavable signal peptides for translocation into ER. • huCD4 remains the most sensitive substrate for the ER translocation inhibitor CADA., In Brief In mammalian cells, one-third of all polypeptides enter the secretory pathway via the ER, driven by specific targeting signals in their sequences, such as N-terminal signal peptides. The synthetic macrocycle CADA was identified as a selective ER translocation inhibitor for huCD4 and SORT. Our current SILAC/MS proteomic survey identified and validated only three additional targets for CADA, thus, confirming the high substrate selectivity of CADA, with the strongest effect on huCD4. Therefore, CADA holds great potential as an immunosuppressive drug.
- Published
- 2021
12. J-domain proteins promote client relay from Hsp70 during tail-anchored membrane protein targeting
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Shu-ou Shan, Yumeng Liu, Hyunju Cho, and Woo Jun Shim
- Subjects
0301 basic medicine ,tail-anchored protein ,NBD, nucleotide binding domain ,Saccharomyces cerevisiae Proteins ,ATPase ,TMD, transmembrane domain ,membrane proteins ,JDP, J-domain protein ,Saccharomyces cerevisiae ,SRP, signal recognition particle ,Endoplasmic Reticulum ,medicine.disease_cause ,Biochemistry ,Hsp70 ,03 medical and health sciences ,Protein targeting ,protein targeting ,medicine ,HSP70 Heat-Shock Proteins ,Molecular Biology ,EV, empty vector ,Signal recognition particle ,Hsp40 ,TPR, tetratricopeptide repeat ,030102 biochemistry & molecular biology ,biology ,SBD, substrate binding domain ,Chemistry ,C-terminus ,Cell Membrane ,GET, guided entry of tail-anchored protein ,Editors' Pick ,Cell Biology ,molecular chaperone ,HSP40 Heat-Shock Proteins ,CTD, C-terminal domain ,SND, SRP-independent ,Cell biology ,Protein Transport ,Tetratricopeptide ,030104 developmental biology ,Membrane protein ,Cyclic nucleotide-binding domain ,biology.protein ,Carrier Proteins ,Biogenesis ,Research Article ,Protein Binding - Abstract
J-domain proteins (JDPs) play essential roles in Hsp70 function by assisting Hsp70 in client trapping and regulating the Hsp70 ATPase cycle. Here, we report that JDPs can further enhance the targeting competence of Hsp70-bound client proteins during tail-anchored protein (TA) biogenesis. In the guided-entry-of-tail-anchored protein pathway in yeast, nascent TAs are captured by cytosolic Hsp70 and sequentially relayed to downstream chaperones, Sgt2 and Get3, for delivery to the ER. We found that two JDPs, Ydj1 and Sis1, function in parallel to support TA targeting to the ER in vivo. Biochemical analyses showed that, while Ydj1 and Sis1 differ in their ability to assist Hsp70 in TA trapping, both JDPs enhance the transfer of Hsp70-bound TAs to Sgt2. The ability of the JDPs to regulate the ATPase cycle of Hsp70 is essential for enhancing the transfer competence of Hsp70-bound TAs in vitro and for supporting TA insertion in vivo. These results demonstrate a role of JDPs in regulating the conformation of Hsp70-bound clients during membrane protein biogenesis.
- Published
- 2021
13. Hydrophilic loop 1 of Presenilin-1 and the APP GxxxG transmembrane motif regulate γ-secretase function in generating Alzheimer-causing Aβ peptides
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Dennis J. Selkoe, Lei Liu, Michael S. Wolfe, and Bianca M. Lauro
- Subjects
0301 basic medicine ,FAD, familial Alzheimer’s disease ,HL-1, hydrophilic loop-1 ,Amyloid beta ,Allosteric regulation ,TMD, transmembrane domain ,γ-secretase ,AD, Alzheimer’s disease ,Biochemistry ,Presenilin ,DMEM, Dulbecco’s modified Eagle’s medium ,Substrate Specificity ,03 medical and health sciences ,Amyloid beta-Protein Precursor ,FBS, fetal bovine serum ,Protein Domains ,APP, amyloid precursor protein ,Alzheimer Disease ,Amyloid precursor protein ,Presenilin-1 ,Humans ,Genetic Predisposition to Disease ,Molecular Biology ,γ-secretase modulator ,Cells, Cultured ,Aβ ,GSM, γ-secretase modulator ,CM, conditioned media ,Amyloid beta-Peptides ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Rational design ,Cell Biology ,Processivity ,Transmembrane protein ,Cell biology ,Aβ, amyloid-beta ,Transmembrane domain ,030104 developmental biology ,PS1, presenilin-1 ,Mutation ,Proteolysis ,biology.protein ,HMW, high-molecular-weight ,Amyloid Precursor Protein Secretases ,Alzheimer’s disease ,Research Article - Abstract
γ-Secretase is responsible for the proteolysis of amyloid precursor protein (APP) into amyloid-beta (Aβ) peptides, which are centrally implicated in the pathogenesis of Alzheimer's disease (AD). The biochemical mechanism of how processing by γ-secretase is regulated, especially as regards the interaction between enzyme and substrate, remains largely unknown. Here, mutagenesis reveals that the hydrophilic loop-1 (HL-1) of presenilin-1 (PS1) is critical for both γ-secretase step-wise cleavages (processivity) and its allosteric modulation by heterocyclic γ-modulatory compounds. Systematic mutagenesis of HL-1, including all of its familial AD mutations and additional engineered variants, and quantification of the resultant Aβ products show that HL-1 is necessary for proper sequential γ-secretase processivity. We identify Y106, L113, and Y115 in HL-1 as key targets for heterocyclic γ-secretase modulators (GSMs) to stimulate processing of pathogenic Aβ peptides. Further, we confirm that the GxxxG domain in the APP transmembrane region functions as a critical substrate motif for γ-secretase processivity: a G29A substitution in APP-C99 mimics the beneficial effects of GSMs. Together, these findings provide a molecular basis for the structural regulation of γ-processivity by enzyme and substrate, facilitating the rational design of new GSMs that lower AD-initiating amyloidogenic Aβ peptides.
- Published
- 2021
14. Zinc transporter mutations linked to acrodermatitis enteropathica disrupt function and cause mistrafficking
- Author
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Dexin Sui, Chi Zhang, Eziz Kuliyev, and Jian Hu
- Subjects
0301 basic medicine ,acrodermatitis enteropathica ,HRD, helix-rich domain ,TMD, transmembrane domain ,NBD1, nucleotide binding domain 1 ,Biochemistry ,chemistry.chemical_compound ,Loss of Function Mutation ,Missense mutation ,Cation Transport Proteins ,CD, circular dichroism ,LOF, loss-of-function ,ZIP4 ,Chemistry ,ZIP, Zrt-/Irt-like protein ,Acrodermatitis enteropathica ,extracellular domain ,AE, acrodermatitis enteropathica ,Transmembrane protein ,Transmembrane domain ,Zinc ,PCC, Pearson's correlation coefficient ,SNP, single-nucleotide polymorphism ,Intracellular ,Research Article ,Glycosylation ,zinc transporter ,CLSM, confocal laser scanning microscope ,SCD-EDS, spondylocheirodysplastic form of Ehlers–Danlos syndrome ,DMEM, Dulbecco’s modified eagle medium ,03 medical and health sciences ,FBS, fetal bovine serum ,medicine ,Extracellular ,Humans ,CFTR, cystic fibrosis transmembrane conductance regulator ,Amino Acid Sequence ,Molecular Biology ,disease-causing mutation ,CF, cystic fibrosis ,030102 biochemistry & molecular biology ,HEK 293 cells ,Acrodermatitis ,Cell Membrane ,Cell Biology ,medicine.disease ,misfolding ,Molecular biology ,ECD, extracellular domain ,030104 developmental biology ,HEK293 Cells ,mistrafficking ,Carrier Proteins - Abstract
ZIP4 is a representative member of the Zrt-/Irt-like protein (ZIP) transporter family and responsible for zinc uptake from diet. Loss-of-function mutations of human ZIP4 (hZIP4) drastically reduce zinc absorption, causing a life-threatening autosomal recessive disorder, acrodermatitis enteropathica (AE). These mutations occur not only in the conserved transmembrane zinc transport machinery, but also in the extracellular domain (ECD) of hZIP4, which is only present in a fraction of mammalian ZIPs. How these AE-causing ECD mutations lead to ZIP4 malfunction has not be fully clarified. In this work, we characterized all seven confirmed AE-causing missense mutations in hZIP4-ECD and found that the variants exhibited completely abolished zinc transport activity in a cell-based transport assay. Although the variants were able to be expressed in HEK293T cells, they failed to traffic to the cell surface and were largely retained in the ER with immature glycosylation. When the corresponding mutations were introduced in the ECD of ZIP4 from Pteropus Alecto, a close homolog of hZIP4, the variants exhibited structural defects or reduced thermal stability, which likely accounts for intracellular mistrafficking of the AE-associated variants and as such a total loss of zinc uptake activity. This work provides a molecular pathogenic mechanism for AE.
- Published
- 2021
15. SARS-CoV-2 Fusion Peptide has a Greater Membrane Perturbating Effect than SARS-CoV with Highly Specific Dependence on Ca
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Jack H. Freed and Alex L. Lai
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CoV, coronavirus ,MERS, Middle East Respiratory Syndrome ,TMD, transmembrane domain ,Cooperativity ,MOMD, microscopic order but macroscopic disorder ,Cell membrane ,0302 clinical medicine ,SARS, Severe Acute Respiratory Syndrome ,Structural Biology ,POPC, 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine ,FP, fusion peptide ,skin and connective tissue diseases ,CD, circular dichroism ,Peptide sequence ,chemistry.chemical_classification ,0303 health sciences ,Chemistry ,Hydrogen-Ion Concentration ,medicine.anatomical_structure ,POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) ,Severe acute respiratory syndrome-related coronavirus ,Spike Glycoprotein, Coronavirus ,ESR, electron spin resonance ,Thermodynamics ,SUV, small unilamellar vesicle ,Hydrophobic and Hydrophilic Interactions ,Research Article ,ion specificity ,Sequence alignment ,MLV, multilamellar vesicle ,Calorimetry ,Cleavage (embryo) ,ITC, isothermal calorimetry ,03 medical and health sciences ,Viral entry ,medicine ,Amino Acid Sequence ,Binding site ,Molecular Biology ,ComputingMethodologies_COMPUTERGRAPHICS ,ESR ,hydrophobicity ,030304 developmental biology ,Binding Sites ,SARS-CoV-2 ,fungi ,Cell Membrane ,COVID-19 ,ITC ,Virus Internalization ,body regions ,Biophysics ,Calcium ,LUV, large unilamellar vesicle ,Glycoprotein ,Viral Fusion Proteins ,030217 neurology & neurosurgery - Abstract
Graphical abstract, Coronaviruses are a major infectious disease threat, and include the zoonotic-origin human pathogens SARS-CoV-2, SARS-CoV, and MERS-CoV (SARS-2, SARS-1, and MERS). Entry of coronaviruses into host cells is mediated by the spike (S) protein. In our previous ESR studies, the local membrane ordering effect of the fusion peptide (FP) of various viral glycoproteins including the S of SARS-1 and MERS has been consistently observed. We previously determined that the sequence immediately downstream from the S2’ cleavage site is the bona fide SARS-1 FP. In this study, we used sequence alignment to identify the SARS-2 FP, and studied its membrane ordering effect. Although there are only three residue difference, SARS-2 FP induces even greater membrane ordering than SARS-1 FP, possibly due to its greater hydrophobicity. This may be a reason that SARS-2 is better able to infect host cells. In addition, the membrane binding enthalpy for SARS-2 is greater. Both the membrane ordering of SARS-2 and SARS-1 FPs are dependent on Ca2+, but that of SARS-2 shows a greater response to the presence of Ca2+. Both FPs bind two Ca2+ ions as does SARS-1 FP, but the two Ca2+ binding sites of SARS-2 exhibit greater cooperativity. This Ca2+ dependence by the SARS-2 FP is very ion-specific. These results show that Ca2+ is an important regulator that interacts with the SARS-2 FP and thus plays a significant role in SARS-2 viral entry. This could lead to therapeutic solutions that either target the FP-calcium interaction or block the Ca2+ channel.
- Published
- 2020
16. Molecular basis of tail-anchored integral membrane protein recognition by the cochaperone Sgt2
- Author
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Michelle Y. Fry, Shyam M. Saladi, William M. Clemons, and Ku-Feng Lin
- Subjects
0301 basic medicine ,Models, Molecular ,Cellular homeostasis ,TMD, transmembrane domain ,Sti1 ,medicine.disease_cause ,Endoplasmic Reticulum ,Biochemistry ,ERAD, ER-associated degradation ,Protein targeting ,Integral membrane protein ,Heat-Shock Proteins ,Signal recognition particle ,Fungal protein ,Chemistry ,Hop ,GET, guided entry of tail-anchored protein ,SGTA, small glutamine tetratricopeptide repeat protein alpha ,Transmembrane domain ,UBL, ubiquitin-like domain ,Protein Binding ,Research Article ,cochaperones ,Saccharomyces cerevisiae Proteins ,TA, tail-anchored ,Saccharomyces cerevisiae ,Endoplasmic-reticulum-associated protein degradation ,SRP, signal recognition particle ,ER, endoplasmic reticulum ,03 medical and health sciences ,medicine ,protein targeting ,Humans ,HSP70 Heat-Shock Proteins ,HSP90 Heat-Shock Proteins ,tail-anchored proteins ,Molecular Biology ,IMP, integral membrane protein ,TRC, transmembrane recognition complex ,030102 biochemistry & molecular biology ,Ubiquitin ,MLP, mislocalized membrane protein ,C-terminus ,Cell Membrane ,Membrane Proteins ,Cell Biology ,030104 developmental biology ,PMSF, phenylmethylsulfonyl fluoride ,BPA, p-benzoyl-l-phenylalanine ,Biophysics ,Protein Multimerization ,Carrier Proteins ,Molecular Chaperones - Abstract
The targeting and insertion of tail-anchored (TA) integral membrane proteins (IMPs) into the correct membrane is critical for cellular homeostasis. The fungal protein Sgt2, and its human homolog SGTA, is the entry point for clients to the guided entry of tail-anchored protein (GET) pathway, which targets endoplasmic reticulum-bound TA IMPs. Consisting of three structurally independent domains, the C terminus of Sgt2 binds to the hydrophobic transmembrane domain (TMD) of clients. However, the exact binding interface within Sgt2 and molecular details that underlie its binding mechanism and client preference are not known. Here, we reveal the mechanism of Sgt2 binding to hydrophobic clients, including TA IMPs. Through sequence analysis, biophysical characterization, and a series of capture assays, we establish that the Sgt2 C-terminal domain is flexible but conserved and sufficient for client binding. A molecular model for this domain reveals a helical hand forming a hydrophobic groove approximately 15 Å long that is consistent with our observed higher affinity for client TMDs with a hydrophobic face and a minimal length of 11 residues. This work places Sgt2 into a broader family of TPR-containing cochaperone proteins, demonstrating structural and sequence-based similarities to the DP domains in the yeast Hsp90 and Hsp70 coordinating protein, Sti1.
- Published
- 2020
17. Quantitative comparison of ABC membrane protein type I exporter structures in a standardized way
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Bianka Farkas, Georgina Csizmadia, Hedvig Tordai, Zoltán Spagina, and Tamás Hegedűs
- Subjects
0301 basic medicine ,NBD, nucleotide binding domain ,Computer science ,lcsh:Biotechnology ,ICD, intracellular domain ,ABC proteins ,Biophysics ,TMD, transmembrane domain ,Computational biology ,CH, coupling helix ,Type (model theory) ,Biochemistry ,03 medical and health sciences ,Molecular dynamics ,Structural Biology ,lcsh:TP248.13-248.65 ,Membrane proteins ,Genetics ,CFTR, cystic fibrosis transmembrane conductance regulator ,ABC, ATP binding cassette ,Quantitative structural properties ,CG, coarse grained ,Structure comparison ,Membrane insertion ,Continuum electrostatics ,TH, transmembrane helix ,Structure validation ,Computer Science Applications ,COG, center of geometry ,030104 developmental biology ,Membrane protein ,Research Article ,TM, transmembrane ,Biotechnology - Abstract
Summary An increasing number of ABC membrane protein structures are determined by cryo-electron microscopy and X-ray crystallography, consequently identifying differences between their conformations has become an arising issue. Therefore, we propose to define standardized measures for ABC Type I exporter structure characterization. We set conformational vectors, conftors, which describe the relative orientation of domains and can highlight structural differences. In addition, continuum electrostatics calculations were performed to characterize the energetics of membrane insertion illuminating functionally crucial regions. In summary, the proposed metrics contribute to deeper understanding of ABC membrane proteins' structural features, structure validation, and analysis of movements observed in a molecular dynamics trajectory. Moreover, the concept of standardized metrics can be applied not only to ABC membrane protein structures (http://conftors.hegelab.org)., Graphical abstract Unlabelled Image
- Published
- 2018
18. Labeling and measuring stressed mitochondria using a PINK1-based ratiometric fluorescent sensor
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Akira Matsuura, Shunsuke Ishii, Rie Uesugi, and Eisuke Itakura
- Subjects
mitochondrial sensor ,PINK1, PTEN-induced putative kinase 1 ,TMD, transmembrane domain ,Cellular homeostasis ,Mitochondrion ,Biochemistry ,Parkin ,chemistry.chemical_compound ,Chlorocebus aethiops ,Enzyme Stability ,PTEN-induced putative kinase 1 (PINK1) ,Chemistry ,Depolarization ,TERT, telomerase reverse transcriptase ,Mitochondria ,Cell biology ,COS Cells ,OMM, outer mitochondrial membrane ,RTK, receptor tyrosine kinase ,mito-Pain F, mitochondrial PINK1 accumulation index (containing full-length PINK1) ,Research Article ,PMA, phorbol 12-myristate 13-acetate ,mito-Pain, mitochondrial PINK1 accumulation index ,PINK1 ,Carbonyl cyanide m-chlorophenyl hydrazone ,Fluorescence ,CCCP, carbonyl cyanide m-chlorophenyl hydrazone ,ER, endoplasmic reticulum ,Mitochondrial Proteins ,Small Molecule Libraries ,mitochondrial membrane potential ,ROS, reactive oxygen species ,Stress, Physiological ,Animals ,Humans ,DAPI ,Molecular Biology ,MTR, MitoTracker Red ,Fluorescent Dyes ,mitochondrial stress ,KD, kinase dead ,mito-Pain T, truncated mito-Pain ,Cell Biology ,Cytosol ,HEK293 Cells ,MTS, mitochondrial targeting sequence ,Protein Kinases ,DAPI, 4′,6-diamidino-2-phenylindole ,HeLa Cells - Abstract
Mitochondria are essential organelles that carry out a number of pivotal metabolic processes and maintain cellular homeostasis. Mitochondrial dysfunction caused by various stresses is associated with many diseases such as type 2 diabetes, obesity, cancer, heart failure, neurodegenerative disorders, and aging. Therefore, it is important to understand the stimuli that induce mitochondrial stress. However, broad analysis of mitochondrial stress has not been carried out to date. Here, we present a set of fluorescent tools, called mito-Pain (mitochondrial PINK1 accumulation index), which enable the labeling of stressed mitochondria. Mito-Pain uses PTEN-induced putative kinase 1 (PINK1) stabilization on mitochondria and quantifies mitochondrial stress levels by comparison with PINK1-GFP, which is stabilized under mitochondrial stress, and RFP-Omp25, which is constitutively localized on mitochondria. To identify compounds that induce mitochondrial stress, we screened a library of 3374 compounds using mito-Pain and identified 57 compounds as mitochondrial stress inducers. Furthermore, we classified each compound into several categories based on mitochondrial response: depolarization, mitochondrial morphology, or Parkin recruitment. Parkin recruitment to mitochondria was often associated with mitochondrial depolarization and aggregation, suggesting that Parkin is recruited to heavily damaged mitochondria. In addition, many of the compounds led to various mitochondrial morphological changes, including fragmentation, aggregation, elongation, and swelling, with or without Parkin recruitment or mitochondrial depolarization. We also found that several compounds induced an ectopic response of Parkin, leading to the formation of cytosolic puncta dependent on PINK1. Thus, mito-Pain enables the detection of stressed mitochondria under a wide variety of conditions and provides insights into mitochondrial quality control systems.
- Published
- 2021
19. Human and viral membrane–associated E3 ubiquitin ligases MARCH1 and MIR2 recognize different features of CD86 to downregulate surface expression
- Author
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Melissa J. Call, Alan F. Rubin, Xinyu Wu, Matthew E. Call, Julie V. Nguyen, Stephen Wilcox, and Raphael Trenker
- Subjects
0301 basic medicine ,TMD, transmembrane domain ,Biochemistry ,JM, juxtamembrane ,MIR, modulator of immune recognition ,deep mutational scanning ,membrane protein ,MFI, mean fluorescence intensity ,ICAM-1, intercellular adhesion molecule 1 ,biology ,Chemistry ,membrane-associated E3 ubiquitin ligase ,Acquired immune system ,Cell biology ,Protein Transport ,Transmembrane domain ,IRES, internal ribosome entry site ,Kaposi's sarcoma virus ,Research Article ,KSHV, Kaposi's sarcoma herpesvirus ,Ubiquitin-Protein Ligases ,DOX, doxycycline ,Antigen presentation ,Protein domain ,Down-Regulation ,chemical and pharmacologic phenomena ,protein–protein interactions ,Major histocompatibility complex ,Viral Proteins ,03 medical and health sciences ,Protein Domains ,Humans ,DMS, deep mutational scanning ,MHC, major histocompatibility complex ,Molecular Biology ,UMI, unique molecular identifier ,CD86 ,030102 biochemistry & molecular biology ,HLA, human leukocyte antigen ,Cell Membrane ,immune regulation ,MARCH, membrane-associated RING-CH ,Cell Biology ,Viral membrane ,HEK293 Cells ,030104 developmental biology ,Membrane protein ,Mutation ,PolyVal TMD, PolyValine TM domain ,biology.protein ,B7-2 Antigen ,HeLa Cells ,TM, transmembrane - Abstract
Immune-stimulatory ligands, such as major histocompatibility complex molecules and the T-cell costimulatory ligand CD86, are central to productive immunity. Endogenous mammalian membrane-associated RING-CHs (MARCH) act on these and other targets to regulate antigen presentation and activation of adaptive immunity, whereas virus-encoded homologs target the same molecules to evade immune responses. Substrate specificity is encoded in or near the membrane-embedded domains of MARCHs and the proteins they regulate, but the exact sequences that distinguish substrates from nonsubstrates are poorly understood. Here, we examined the requirements for recognition of the costimulatory ligand CD86 by two different MARCH-family proteins, human MARCH1 and Kaposi's sarcoma herpesvirus modulator of immune recognition 2 (MIR2), using deep mutational scanning. We identified a highly specific recognition surface in the hydrophobic core of the CD86 transmembrane (TM) domain (TMD) that is required for recognition by MARCH1 and prominently features a proline at position 254. In contrast, MIR2 requires no specific sequences in the CD86 TMD but relies primarily on an aspartic acid at position 244 in the CD86 extracellular juxtamembrane region. Surprisingly, MIR2 recognized CD86 with a TMD composed entirely of valine, whereas many different single amino acid substitutions in the context of the native TM sequence conferred MIR2 resistance. These results show that the human and viral proteins evolved completely different recognition modes for the same substrate. That some TM sequences are incompatible with MIR2 activity, even when no specific recognition motif is required, suggests a more complicated mechanism of immune modulation via CD86 than was previously appreciated.
- Published
- 2021
20. Voltage-gated and background K+ channel subunits expressed by the bushy cells of the rat cochlear nucleus
- Author
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Pál, Balázs, Pór, Ágnes, Pocsai, Krisztina, Szücs, Géza, and Rusznák, Zoltán
- Subjects
- *
BLOOD plasma , *CEREBROSPINAL fluid , *SPINAL cord , *BLOOD proteins , *MEMBRANE proteins - Abstract
Abstract: Bushy cells of the ventral cochlear nucleus produce a single, short latency action potential at the beginning of long depolarisations. In the present work an immunochemical survey was performed to detect the presence of K+ channel subunits which may contribute to the specific membrane properties of the bushy cells. The immunocytochemical experiments conducted on enzymatically isolated bushy cells indicated positive immunolabelling for several subunits known to be responsible for the genesis of rapidly inactivating K+ currents. Bushy cells showed strong expression of Kv3.4, 4.2 and 4.3 subunits, with the lack of Kv1.4 specific immunoreaction. The Kv3.4-specific immunoreaction had a specific, patchy appearance. Bushy cells also expressed various members of the Kv1 subunit family, most notably Kv1.1, 1.2, 1.3 and 1.6. Weak positivity could be observed for Kv3.2 subunits. The positive immunolabelling for Kv3.4, Kv4.2 and Kv4.3 was confirmed in free-floating tissue slices. Voltage-clamp experiments performed on positively identified bushy cells in brain slices corroborated the presence and activity of Kv3.4 and Kv4.2/4.3 containing K+ channels. Bushy cell showed strong immunopositivity for TASK-1 channels too. The results presented in this work indicate that bushy cells possess several types of voltage-gated K+ channel subunits whose activity may contribute to the membrane properties and firing characteristics of these neurones. [Copyright &y& Elsevier]
- Published
- 2005
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21. Mistargeted MRPΔF728 mutant is rescued by intracellular GSH
- Author
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Buyse, Frédéric, Vandenbranden, Michel, and Ruysschaert, Jean-Marie
- Subjects
- *
INFRARED spectroscopy , *SPECTRUM analysis , *CELL membranes , *CYSTIC fibrosis , *LUNG diseases - Abstract
Abstract: The most common cystic fibrosis-causing mutation is the deletion of the widely conserved phenylalanine 508 (ΔF508) of CFTR. The mutant is unable to fold correctly and to transit to the plasma membrane. MRP1 belongs to the same subfamily of ABC proteins as CFTR and confers resistance to a wide range of chemotherapeutic drugs. By analogy, phenylalanine 728 was deleted in MRP1. Our results shown that MRPΔF728 is correctly targeted to the plasma membrane, actively transports doxorubicin (DOX) and vincristine (VCR) and shares a structure identical to MRP1. Intracellular GSH depletion however results in a mistargeted mutant that is retained into the cytoplasm, while in the same conditions wild-type MRP1 is correctly routed to the plasma membrane. The GSH-protein complex could adopt a stable conformation protected against proteolytic degradation and correctly targeted to the plasma membrane. [Copyright &y& Elsevier]
- Published
- 2004
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- View/download PDF
22. Computer simulations of membrane proteins
- Author
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Ash, Walter L., Zlomislic, Marian R., Oloo, Eliud O., and Tieleman, D. Peter
- Subjects
- *
MEMBRANE proteins , *BIOMOLECULES , *MOLECULAR dynamics , *MICROSCOPY - Abstract
Computer simulations are rapidly becoming a standard tool to study the structure and dynamics of lipids and membrane proteins. Increasing computer capacity allows unbiased simulations of lipid and membrane-active peptides. With the increasing number of high-resolution structures of membrane proteins, which also enables homology modelling of more structures, a wide range of membrane proteins can now be simulated over time spans that capture essential biological processes. Longer time scales are accessible by special computational methods. We review recent progress in simulations of membrane proteins. [Copyright &y& Elsevier]
- Published
- 2004
- Full Text
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23. Identification and expression of a mouse muscle-specific CTL1 gene
- Author
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Yuan, Zongfei, Wagner, Laura, Poloumienko, Arkadi, and Bakovic, Marica
- Subjects
- *
GENETICS , *SURGERY , *MEDICINE , *GENES - Abstract
In this study, a mouse gene and cDNA encoding for a novel skeletal muscle-specific choline transporter-like protein 1 (mCTL1) were identified and mCTL1 mRNA and protein expression characterized. The mCTL1 cDNA is 2888-bp long; consisting of a 653-amino-acid open-reading frame, 8–11 putative transmembrane domains, three N-glycosylation sites and seven protein kinase C phosphorylation sites. The mCTL1 gene is localized to chromosome 4B2, at 182 kb in length, and encoded by 17 exons. Although the mCTL1 mRNA was expressed in several mouse tissues such as muscle, brain, heart and testis, the protein analyses of multiple tissues and membrane vesicles reveal that mCTL1 is exclusively expressed in skeletal muscle. Expression of His-tagged mCTL1 in Cos-7 cells produces an increase in saturable choline uptake that is sensitive to a Na+-ion gradient, ethanolamine and the Ca2+-channel blocker verapamil, and insensitive to low concentrations of hemicholinium-3. [Copyright &y& Elsevier]
- Published
- 2004
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24. Structural understanding of efflux-mediated drug resistance: potential routes to efflux inhibition
- Author
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McKeegan, Kenneth S, Borges-Walmsley, M Ines, and Walmsley, Adrian R
- Subjects
- *
ANTINEOPLASTIC agents , *MULTIDRUG resistance , *DRUG resistance , *CANCER treatment , *CELLS - Abstract
The active efflux of cytotoxic drugs mediated by multidrug transporters is the basis of multidrug resistance in prokaryotic and eukaryotic cells. Individual multidrug transporters can be extremely versatile, often exhibiting a staggering range of substrate specificity that can negate the effects of clinically relevant therapies. The effective treatment of bacterial, fungal and protozoan infections, along with certain cancer treatments, has been compromised by the presence of multidrug transporters. Traditionally, advances in the understanding of multidrug transporters have been made through biochemical analyses; more recently, however, fundamental advances have been made with the elucidation of several three dimensional structures of representative multidrug pumps. Biochemical and structural analysis of multidrug pumps could lead to the development of novel ‘anti-efflux’ therapies. [Copyright &y& Elsevier]
- Published
- 2004
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25. Regulation of exocytosis in neurons and neuroendocrine cells
- Author
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An, Seong and Zenisek, David
- Subjects
- *
NEURONS , *MOLECULES , *EXOCYTOSIS , *FUSION (Phase transformation) , *NERVOUS system - Abstract
Neurons communicate with one another through the release of molecules from synaptic vesicles and large dense core granules through the process of exocytosis. During exocytosis, molecules are released to the extracellular space through a fusion pore, which can either dilate, resulting in full fusion, or close, resulting in incomplete exocytosis, often referred to as ‘kiss and run’ exocytosis. Recently, there has been much interest in the regulation of this process in both neurons and neuroendocrine cells. There has been much recent work that addresses the existence of incomplete exocytosis in neurons and neuroendocrine cells, as well as recent work probing the molecular components and modulation of the fusion pore. [Copyright &y& Elsevier]
- Published
- 2004
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26. A Highly Unusual Palindromic Transmembrane Helical Hairpin Formed by SARS Coronavirus E Protein
- Author
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Arbely, Eyal, Khattari, Ziad, Brotons, Guillaume, Akkawi, Mutaz, Salditt, Tim, and Arkin, Isaiah T.
- Subjects
- *
MEMBRANE proteins , *AQUAPORINS , *HEPATITIS viruses , *HEPATITIS D virus - Abstract
The agent responsible for the recent severe acute respiratory syndrome (SARS) outbreak is a previously unidentified coronavirus. While there is a wealth of epidemiological studies, little if any molecular characterization of SARS coronavirus (SCoV) proteins has been carried out. Here we describe the molecular characterization of SCoV E protein, a critical component of the virus responsible for virion envelope morphogenesis. We conclusively show that SCoV E protein contains an unusually short, palindromic transmembrane helical hairpin around a previously unidentified pseudo-center of symmetry, a structural feature which seems to be unique to SCoV. The hairpin deforms lipid bilayers by way of increasing their curvature, providing for the first time a molecular explanation of E protein''s pivotal role in viral budding. The molecular understanding of this critical component of SCoV may represent the beginning of a concerted effort aimed at inhibiting its function, and consequently, viral infectivity. [Copyright &y& Elsevier]
- Published
- 2004
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27. The mitochondrial ABC transporter Atm1p functions as a homodimer
- Author
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Chloupková, Maja, Reaves, Scott K., LeBard, Linda M., and Koeller, David M.
- Subjects
- *
IRON in the body , *CYSTIC fibrosis , *GENETIC disorders , *LUNG diseases - Abstract
The ATP-binding cassette (ABC) transporters constitute one of the largest families of proteins in evolution. The ATM1 gene of the yeast Saccharomyces cerevisiae encodes an ABC protein, which is localized to the mitochondrial inner membrane. A deletion of ATM1 results in the accumulation of up to a 30-fold excess of mitochondrial iron, loss of mitochondrial cytochromes and abnormalities of cytosolic iron metabolism. In this study, we have evaluated the role of conserved sequence elements in Atm1p in its function and dimerization in vivo. We report that conserved residues in the Walker A and B motifs of the nucleotide binding domain, which are required for ATP binding and hydrolysis, are essential for Atm1p function. In addition, we provide evidence that ATP binding is important for Atm1p dimerization. [Copyright &y& Elsevier]
- Published
- 2004
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28. Expression and biochemical analysis of the entire HIV-2 gp41 ectodomain: determinants of stability map to N- and C-terminal sequences outside the 6-helix bundle core
- Author
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Lay, Chan-Sien, Wilson, Kirilee A., Kobe, Bostjan, Kemp, Bruce E., Drummer, Heidi E., and Poumbourios, Pantelis
- Subjects
- *
HIV , *CELL membranes , *ESCHERICHIA coli , *ESCHERICHIA - Abstract
The folding of HIV gp41 into a 6-helix bundle drives virus-cell membrane fusion. To examine the structural relationship between the 6-helix bundle core domain and other regions of gp41, we expressed in Escherichia coli, the entire ectodomain of HIV-2ST gp41 as a soluble, trimeric maltose-binding protein (MBP)/gp41 chimera. Limiting proteolysis indicated that the Cys-591–Cys-597 disulfide-bonded region is outside a core domain comprising two peptides, Thr-529–Trp-589 and Val-604–Ser-666. A biochemical examination of MBP/gp41 chimeras encompassing these core peptides indicated that the N-terminal polar segment, 521–528, and C-terminal membrane-proximal segment, 658–666, cooperate in stabilizing the ectodomain. A functional interaction between sequences outside the gp41 core may contribute energy to membrane fusion. [Copyright &y& Elsevier]
- Published
- 2004
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29. Nucleotide Dependent Monomer/Dimer Equilibrium of OpuAA, the Nucleotide-binding Protein of the Osmotically Regulated ABC Transporter OpuA from Bacillus subtilis
- Author
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Horn, Carsten, Bremer, Erhard, and Schmitt, Lutz
- Subjects
- *
BACILLUS subtilis , *NUCLEOTIDES , *GLYCINE - Abstract
The OpuA system of Bacillus subtilis is a member of the substrate-binding-protein-dependent ABC transporter superfamily and serves for the uptake of the compatible solute glycine betaine under hyperosmotic growth conditions. Here, we have characterized the nucleotide-binding protein (OpuAA) of the B. subtilis OpuA transporter in vitro. OpuAA was overexpressed heterologously in Escherichia coli as a hexahistidine tag fusion protein and purified to homogeneity by affinity and size exclusion chromatography (SEC). Dynamic monomer/dimer equilibrium was observed for OpuAA, and the KD value was determined to be 6 μM. Under high ionic strength assay conditions, the monomer/dimer interconversion was diminished, which enabled separation of both species by SEC and separate analysis of both monomeric and dimeric OpuAA. In the presence of 1 M NaCl, monomeric OpuAA showed a basal ATPase activity (KM=0.45 mM; k2=2.3 min−1), whereas dimeric OpuAA showed little ATPase activity under this condition. The addition of nucleotides influenced the monomer/dimer ratio of OpuAA, demonstrating different oligomeric states during its catalytic cycle. The monomer was the preferred species under post-hydrolysis conditions (e.g. ADP/Mg2+), whereas the dimer dominated the nucleotide-free and ATP-bound states. The affinity and stoichiometry of monomeric or dimeric OpuAA/ATP complexes were determined by means of the fluorescent ATP-analog TNP–ATP. One molecule of TNP–ATP was bound in the monomeric state and two TNP–ATP molecules were detected in the dimeric state of OpuAA. Binding of TNP–ADP/Mg2+ to dimeric OpuAA induced a conformational change that led to the decay of the dimer. On the basis of our data, we propose a model that couples changes in the oligomeric state of OpuAA with ATP hydrolysis. [Copyright &y& Elsevier]
- Published
- 2003
- Full Text
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30. Mechanisms of membrane permeabilization by picornavirus 2B viroporin
- Author
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Nieva, José L., Agirre, Aitziber, Nir, Shlomo, and Carrasco, Luis
- Subjects
- *
PICORNAVIRUSES , *RNA viruses , *CELL membranes , *BIOMOLECULES - Abstract
Cell infection by picornaviruses leads to membrane permeabilization. Recent evidence suggests the involvement of the non-structural protein 2B in this process. We have recently reported the detection of 2B porin-like activity in isolated membrane–protein systems that lack other cell components. According to data derived from these model membranes, four self-aggregated 2B monomers (i.e. tetramers) would be sufficient to permeabilize a single lipid vesicle, allowing the free diffusion of solutes under ca. 1000 Da. Our findings also support a role for lipids in protein oligomerization and subsequent pore opening. The lipid dependence of these processes points to negatively charged cytofacial surfaces as 2B cell membrane targets. [Copyright &y& Elsevier]
- Published
- 2003
- Full Text
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31. Cell surface expression of functional hepatitis C virus E1 and E2 glycoproteins
- Author
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Drummer, Heidi E., Maerz, Anne, and Poumbourios, Pantelis
- Subjects
- *
HEPATITIS C virus , *CHEMILUMINESCENCE - Abstract
Hepatitis C virus (HCV) glycoproteins E1 and E2 are believed to be retained in the endoplasmic reticulum (ER) or cis-Golgi compartment via retention signals located in their transmembrane domains. Here we describe the detection of E1 and E2 at the surface of transiently transfected HEK 293T and Huh7 cells. Surface-localized E1E2 heterodimers presented exclusively as non-covalently associated complexes. Surface-expressed E2 contained trans-Golgi modified complex/hybrid type carbohydrate and migrated diffusely between 70 and 90 kDa while intracellular E1 and E2 existed as high mannose 35 kDa and 70 kDa precursors, respectively. In addition, surface-localized E1E2 heterodimers were incorporated into E1E2-pseudotyped HIV-1 particles that were competent for entry into Huh7 cells. These studies suggest that functional HCV glycoproteins are not retained exclusively in the ER and transit through the secretory pathway. [Copyright &y& Elsevier]
- Published
- 2003
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32. Mass Spectrometry and Site-directed Mutagenesis Identify Several Autophosphorylated Residues Required for the Activity of PrkC, a Ser/Thr Kinase from Bacillus subtilis
- Author
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Madec, Edwige, Stensballe, Allan, Kjellström, Sven, Cladière, Lionel, Obuchowski, Michal, Jensen, Ole Nørregaard, and Séror, Simone J.
- Subjects
- *
MUTAGENESIS , *MASS spectrometry - Abstract
We have shown recently that PrkC, which is involved in developmental processes in Bacillus subtilis, is a Ser/Thr kinase with features of the receptor kinase family of eukaryotic Hanks kinases. In this study, we expressed and purified from Escherichia coli the cytoplasmic domain of PrkC containing the kinase and a short juxtamembrane region. This fragment, which we designate PrkCc, undergoes autophosphorylation in E. coli. PrkCc is further autophosphorylated in vitro, apparently through a trans-kinase, intermolecular reaction. PrkC also displays kinase activity with myelin basic protein. Using high mass accuracy electrospray tandem mass spectrometry (LC-MS/MS) and nanoelectrospray tandem mass spectrometry, we identified seven phosphorylated threonine and one serine residue in PrkCc. All the corresponding residues were replaced by systematic site-directed mutagenesis and the purified mutant proteins were tested for in vitro kinase activity. Single and multiple replacement of four threonine residues, clustered between residues 162 and 167 in a putative activation loop, substantially reduced kinase activity and the effect was clearly additive. Replacement of the other three threonine residues, clustered between residues 290 and 320, had relatively little effect on activity. In contrast, substitution of Ser214, which is conserved in closely related receptor kinase-like bacterial proteins, independently affected activity and may represent a novel regulatory mechanism. When projected onto a 3D structure of PrkC modelled on the structure of known Hanks kinases, the first cluster of phospho-threonine residues falls precisely in the activation loop, controlling the access of substrate and ATP to the catalytic site of many eukaryotic receptor kinases, whereas the second cluster is located in the juxtamembrane region. These results indicate that regulation of PrkC kinase activity (and presumably autophosphorylation) includes a conserved activation loop mechanism. The juxtamembrane phospho-threonine residues may be essential, for example for the recruitment of other proteins necessary for a PrkC signalling cascade or for coupling to other signalling pathways. This is the first structure–function analysis of a bacterial receptor-like kinase of the Hanks family. [Copyright &y& Elsevier]
- Published
- 2003
- Full Text
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33. Crystal Structure of the Nucleotide-binding Domain of the ABC-transporter Haemolysin B: Identification of a Variable Region Within ABC Helical Domains
- Author
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Schmitt, Lutz, Benabdelhak, Houssain, Blight, Mark A., Holland, I. Barry, and Stubbs, Milton T.
- Subjects
- *
ESCHERICHIA coli , *BACTERIA - Abstract
The ABC-transporter haemolysin B is a central component of the secretion machinery that translocates the toxin, haemolysin A, in a Sec-independent fashion across both membranes of E. coli. Here, we report the X-ray crystal structure of the nucleotide-binding domain (NBD) of HlyB. The molecule shares the common overall architecture of ABC-transporter NBDs. However, the last three residues of the Walker A motif adopt a 310 helical conformation, stabilized by a bound anion. In consequence, this results in an unusual interaction between the Walker A lysine residue and the Walker B glutamate residue. As these residues are normally required to be available for ATP binding, for catalysis and for dimer formation of ABC domains, we suggest that this conformation may represent a latent monomeric form of the NBD. Surprisingly, comparison of available NBD structures revealed a structurally diverse region (SDR) of about 30 residues within the helical arm II domain, unique to each of the eight NBDs analyzed. As this region interacts with the transmembrane part of ABC-transporters, the SDR helps to explain the selectivity and/or targeting of different NBDs to their cognate transmembrane domains. [Copyright &y& Elsevier]
- Published
- 2003
- Full Text
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34. Structure of MsbA from Vibrio cholera: A Multidrug Resistance ABC Transporter Homolog in a Closed Conformation
- Author
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Chang, Geoffrey
- Subjects
- *
POLYETHYLENE glycol , *CANCER treatment - Abstract
The spread of multidrug resistance (MDR) is a world health crisis that presents a significant challenge to the treatment of cancer and infection. MDR can be caused by a group of ABC (MDR-ABC) transporters that move hydrophobic drug molecules and lipids across the cell membrane. To gain insight into the conformational changes these transporters undergo when flipping hydrophobic substrates across the lipid bilayer, we have determined the structure of the lipid flippase MsbA from Vibrio cholera (VC-MsbA) to 3.8 A˚. Structural comparison of VC-MsbA to MsbA from Escherichia coli reveals that the transporters share a structurally conserved core of transmembrane α-helices, but differ in the relative orientations of their nucleotide-binding domains (NBD). The transmembrane domain of VC-MsbA is captured in a closed conformation and the structure supports a “power stroke” model of transporter dynamics where opposing NBDs associate upon ATP binding. The separation of the α and β domains of the NBD suggests the possibility that their association could make them competent to bind ATP and gives further insight into the structural basis for catalytic regulation. [Copyright &y& Elsevier]
- Published
- 2003
- Full Text
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35. A Specific Interaction Between the NBD of the ABC-transporter HlyB and a C-Terminal Fragment of its Transport Substrate Haemolysin A
- Author
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Benabdelhak, Houssain, Kiontke, Stephan, Horn, Carsten, Ernst, Robert, Blight, Mark A., Holland, I. Barry, and Schmitt, Lutz
- Subjects
- *
ESCHERICHIA coli , *TRANSFERASES - Abstract
A member of the family of RTX toxins, Escherichia coli haemolysin A, is secreted from Gram-negative bacteria. It carries a C-terminal secretion signal of approximately 50 residues, targeting the protein to the secretion or translocation complex, in which the ABC-transporter HlyB is a central element. We have purified the nucleotide-binding domain of HlyB (HlyB–NBD) and a C-terminal 23 kDa fragment of HlyA plus the His-tag (HlyA1), which contains the secretion sequence. Employing surface plasmon resonance, we were able to demonstrate that the HlyB–NBD and HlyA1 interact with a
KD of approximately 4 μM. No interaction was detected between the HlyA fragment and unrelated NBDs, OpuAA, involved in import of osmoprotectants, and human TAP1–NBD, involved in the export of antigenic peptides. Moreover, a truncated version of HlyA1, lacking the secretion signal, failed to interact with the HlyB–NBD. In addition, we showed that ATP accelerated the dissociation of the HlyB–NBD/HlyA1 complex. Taking these results together, we propose a model for an early stage of initiation of secretion in vivo, in which the NBD of HlyB, specifically recognizes the C terminus of the transport substrate, HlyA, and where secretion is initiated by subsequent displacement of HlyA from HlyB by ATP. [Copyright &y& Elsevier]- Published
- 2003
- Full Text
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36. Expression of syntaxin 1C, an alternative splice variant of HPC-1/syntaxin 1A, is enhanced by phorbol-ester stimulation in astroglioma: participation of the PKC signaling pathway1<FN ID="FN1"><NO>1</NO>The nucleotide sequences of human HPC-1/syntaxin 1A and syntaxin 1C cDNA have been deposited in the DDBJ nucleotide sequence database under accession numbers D37932 and AB086954M, respectively.</FN>
- Author
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Nakayama, Takahiro, Mikoshiba, Katsuhiko, Yamamori, Tetsuo, and Akagawa, Kimio
- Subjects
- *
NEUROTRANSMITTERS , *ASTROCYTOMAS - Abstract
Syntaxin 1C is an alternative splice variant of HPC-1/syntaxin 1A; the latter participates in neurotransmitter release and is assigned to the gene domain responsible for Williams’ syndrome (WS). It is expressed in the soluble fraction extracted from human astroglioma cell lines T98G and U87MG. Quantitative immunoblot and indirect immunofluorescence analyses revealed that the expression of syntaxin 1C was upregulated by phorbol 12-myristate 13-acetate (PMA), but not by forskolin. A protein kinase C (PKC) inhibitor suppressed this enhancement. These results suggest that syntaxin 1C expression is regulated via the PKC signal pathway. This is the first report of a signal transduction system that directly affects the expression of syntaxin protein. [Copyright &y& Elsevier]
- Published
- 2003
- Full Text
- View/download PDF
37. Functional cysteine-less subunits of the transporter associated with antigen processing (TAP1 and TAP2) by de novo gene assembly
- Author
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Heintke, Susanne, Chen, Min, Ritz, Ulrike, Lankat-Buttgereit, Brigitte, Koch, Joachim, Abele, Rupert, Seliger, Barbara, and Tampé, Robert
- Subjects
- *
PEPTIDES , *CHEMILUMINESCENCE - Abstract
Within the adaptive immune system the transporter associated with antigen processing (TAP) plays a pivotal role in loading of peptides onto major histocompatibility (MHC) class I molecules. As a central tool to investigate the structure and function of the TAP complex, we created cysteine-less human TAP subunits by de novo gene synthesis, replacing all 19 cysteines in TAP1 and TAP2. After expression in TAP-deficient human fibroblasts, cysteine-less TAP1 and TAP2 are functional with respect to adenosine triphosphate (ATP)-dependent peptide transport and inhibition by ICP47 from herpes simplex virus. Cysteine-less TAP1 and TAP2 restore maturation and intracellular trafficking of MHC class I molecules to the cell surface. [Copyright &y& Elsevier]
- Published
- 2003
- Full Text
- View/download PDF
38. Thermodynamics of Peptide Binding to the Transporter Associated with Antigen Processing (TAP)
- Author
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Neumann, Lars, Abele, Rupert, and Tampé, Robert
- Subjects
- *
ADENOSINE triphosphate , *ANTIGENS - Abstract
The ATP-binding cassette (ABC) transporter TAP plays an essential role in antigen processing and immune response to infected or malignant cells. TAP translocates proteasomal degradation products from the cytosol into the endoplasmic reticulum, where MHC class I molecules are loaded with these peptides. Kinetically stable peptide–MHC complexes are transported to the cell surface for inspection by cytotoxic T lymphocytes. The transport cycle of TAP is initiated by peptide binding, which is responsible for peptide selection and for stimulation of ATP-hydrolysis and subsequent translocation. Here we have analysed the driving forces for the formation of the peptide–TAP complex by kinetic and thermodynamic methods. First, the apparent peptide association and dissociation rates were determined at various temperatures. Strikingly, very high activation energies for apparent association (Eaass=106 kJ mol−1) and dissociation (Eadiss=80 kJ mol−1) of the peptide–TAP complex were found. Next, the temperature-dependence of the peptide affinity constants was investigated by equilibrium-binding assays. Along with calculations of free enthalpy ΔG, enthalpy ΔH and entropy ΔS, a large positive change in heat capacity was resolved (ΔC°=23 kJ mol−1 K−1), indicating a fundamental structural reorganization of the TAP complex upon peptide binding. The inspection of the conformational entropy reveals that approximately one-fourth of all TAP residues is rearranged. These thermodynamic studies indicate that at physiological temperature, peptide binding is endothermic and driven by entropy. [Copyright &y& Elsevier]
- Published
- 2002
- Full Text
- View/download PDF
39. Molecular cloning and functional expression of a proline transporter from Manduca sexta
- Author
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Sandhu, Sumandeep K., Ross, Linda S., and Gill, Sarjeet S.
- Subjects
- *
MOLECULAR cloning , *PROLINE - Abstract
We report the molecular cloning of a L-proline transporter, MasPROT cDNA and its splice variants MasPROT.16 and MasPROT.2 from the central nervous system of Manduca sexta. Sequence analysis revealed that MasPROT belongs to a family of high affinity Na+/Cl− dependent neurotransmitter transporters. The deduced amino acid (aa) sequence of 556 aa having an estimated molecular mass of 58.9 kDa is predicted to have 12 putative transmembrane domains (TMD) and a characteristic large extracellular loop between TMD3 and TMD4. Sequence comparison to other members of the family indicates that it falls into the glycine–proline transporter subfamily. Transiently expressed MasPROT cDNA in Xenopus oocytes exclusively transported proline. Northern analysis shows that it is expressed predominantly in central nervous system, however, low levels are present in midgut, hindgut and Malpighian tubules. Two mRNA transcripts of sizes 3.6 and 8 Kb were found in all tissues except hindgut, where only a smaller transcript exists. RT–PCR and Southern blot analysis revealed the presence of MasPROT transcripts in flight muscles but not in leg muscles. Our preliminary data suggests that this transporter is an insect homologue of mammalian proline transporters. MasPROT.16 is a short splice variant encoding for 174 amino acids and shares 138 amino acids from the N terminus of MasPROT. MasPROT.2 is a long splice variant that contains six introns that coincide precisely with the previously mapped exon/intron boundaries of the members of this superfamily. [Copyright &y& Elsevier]
- Published
- 2002
- Full Text
- View/download PDF
40. Influence of hydrophobic mismatch and palmitoylation on the association of transmembrane α-helical peptides with detergent-resistant membranes
- Author
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van Duyl, Bianca Y., Rijkers, Dirk T.S., de Kruijff, Ben, and Killian, J. Antoinette
- Subjects
- *
PROTEINS , *BIOLOGICAL membranes , *LIQUIDS , *LIPIDS - Abstract
The aim of this study was to gain insight into the mechanism through which transmembrane proteins are targeted to liquid ordered (Lo) phase domains or rafts. This was investigated by analyzing the Triton X-100 resistance of designed transmembrane peptides in model membranes of 1,2-dioleoyl-sn-glycero-3-phosphocholine, sphingomyelin and cholesterol (1/1/1, molar ratio), which contain both Lo phase domains and fluid bilayers. By using peptides with one or two palmitate chains covalently linked to their N-terminus or with variable hydrophobic lengths, the roles of protein palmitoylation and of mismatch between the transmembrane segment of the protein and the bilayer thickness, respectively, were investigated. The results show that neither hydrophobic matching nor palmitoylation is sufficient for partitioning of peptides into Lo phase domains. It is concluded that the Lo phase itself, due to the tight packing of the lipids, constitutes an unfavorable environment for accommodation of protein transmembrane segments. [Copyright &y& Elsevier]
- Published
- 2002
- Full Text
- View/download PDF
41. cDNA cloning and characterization of tobacco ABC transporter: NtPDR1 is a novel elicitor-responsive gene1<FN ID="FN1"><NO>1</NO>The nucleotide sequence reported in this paper has been submitted to the DDBJ, EMBL and GenBank nucleotide sequence databases with the accession number AB075550.</FN>
- Author
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Sasabe, Michiko, Toyoda, Kazuhiro, Shiraishi, Tomonori, Inagaki, Yoshishige, and Ichinose, Yuki
- Subjects
- *
MULTIDRUG resistance , *TOBACCO - Abstract
We isolated an INF1 elicitin-inducible cDNA encoding a pleiotropic drug resistance (PDR)-type ATP-binding cassette (ABC) transporter homolog (NtPDR1) in suspension-cultured tobacco Bright Yellow-2 (BY-2) cells by application of differential display PCR. The NtPDR1 (Nicotiana tabacum PDR protein 1) gene also encodes a 162 kDa protein that includes two putative hydrophilic domains containing the ABC signature motif and two putative hydrophobic domains. Expression of the NtPDR1 gene was rapidly and strongly activated by treatment of BY-2 cells with INF1 elicitin. Further, treatment of BY-2 cells with flagellin, a bacterial proteinaceous hypersensitive reaction elicitor, or yeast extract, a general elicitor, also induced NtPDR1 gene expression. These results indicate that NtPDR1 may be involved in the general defense response in tobacco. This is the first report that microbial elicitors induce the expression of a plant ABC transporter gene. [Copyright &y& Elsevier]
- Published
- 2002
- Full Text
- View/download PDF
42. FAD-linked mutations in presenilin 1 alter the length of Aβ peptides derived from βAPP transmembrane domain mutants
- Author
-
Murphy, M. Paul, Uljon, Sacha N., Golde, Todd E., and Wang, Rong
- Subjects
- *
AMYLOID beta-protein precursor , *ALZHEIMER'S disease - Abstract
γ-Secretase is an enzymatic activity responsible for the final cleavage of the amyloid precursor protein leading to the production of the amyloid β-peptide (Aβ). γ-Secretase is likely an aspartyl protease, since its activity can be inhibited by both pepstatin and active-site directed aspartyl protease inhibitors. Recent work has indicated that presenilins 1 and 2 may actually be the γ-secretase enzymes. Presenilin (PS) mutations, which lead to an increase in the production of a longer form of Aβ, are also the most common cause of familial Alzheimer’s disease (FAD). Therefore, in an attempt to better characterize the substrate preferences of γ-secretase, we performed experiments to determine how FAD-linked mutations in PS1 would affect the generation of Aβ peptides from full length precursor substrates that we have previously demonstrated to be proteolytically cleaved at alternative sites and/or by enzymatic activities that are pharmacologically distinct. Presenilin mutations increased the production of Aβ peptides from sites distal to the primary cleavage site (‘longer’ peptides) and in several cases also decreased production of ‘shorter’ peptides. These results support a model in which the FAD-linked mutants subtly alter the conformation of the γ-secretase complex to favor the production of long Aβ. [Copyright &y& Elsevier]
- Published
- 2002
- Full Text
- View/download PDF
43. The diversity of ACBD proteins - From lipid binding to protein modulators and organelle tethers
- Author
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Frans A. Kuypers, Markus Islinger, Suzan Kors, Eric Soupene, Michael Schrader, Tim P. Levine, and Joseph L. Costello
- Subjects
0301 basic medicine ,Models, Molecular ,Protein Conformation ,FFAT motif ,TMD, transmembrane domain ,chemistry.chemical_compound ,FFAT, two phenylalanines (FF) in an acidic tract ,0302 clinical medicine ,Neoplasms ,Acyl-CoA binding domain containing protein ,Pathogen host interaction ,Phylogeny ,Membrane contact sites ,Phylogenetic tree ,Peroxisome ,GOLD, Golgi dynamic ,Cell biology ,Binding domain ,MCS, membrane contact site ,Coenzyme A ,POMC, pro-opiomelanocortin ,Biology ,Article ,Evolution, Molecular ,Fungal Proteins ,ACBD, acyl-CoA binding domain containing protein ,ER, endoplasmic reticulum ,03 medical and health sciences ,Protein Domains ,VAP, vesicle-associated membrane protein (VAMP)–associated protein ,Organelle ,Peroxisomes ,Animals ,Molecular Biology ,ACBP, acyl-CoA binding protein ,RO, replication organelle ,Fungi ,Lipid metabolism ,ACB, acyl-CoA binding domain ,Cell Biology ,biology.organism_classification ,Lipid Metabolism ,VLCFA, very long-chain fatty acid ,PTS, peroxisomal targeting signal ,030104 developmental biology ,chemistry ,GABA, gamma-aminobutyric acid ,LCFA, long-chain fatty acid ,Carrier Proteins ,MTS, mitochondrial targeting sequence ,030217 neurology & neurosurgery ,Function (biology) ,Bacteria - Abstract
Members of the large multigene family of acyl-CoA binding domain containing proteins (ACBDs) share a conserved motif required for binding of Coenzyme A esterified fatty acids of various chain length. These proteins are present in the three kingdoms of life, and despite their predicted roles in cellular lipid metabolism, knowledge about the precise functions of many ACBD proteins remains scarce. Interestingly, several ACBD proteins are now suggested to function at organelle contact sites, and are recognized as host interaction proteins for different pathogens including viruses and bacteria. Here, we present a thorough phylogenetic analysis of the ACBD family and discuss their structure and evolution. We summarize recent findings on the various functions of animal and fungal ACBDs with particular focus on peroxisomes, the role of ACBD proteins at organelle membranes, and their increasing recognition as targets for pathogens., Graphical abstract Unlabelled Image, Highlights • Acyl-CoA binding domain-containing proteins (ACBDs) can be found among eukaryotes and prokaryotes. • Additional protein interaction domains are a characteristic of most extended ACBDs. • A set of a small ACBD and an ACBD with a C-terminal ankyrin-repeat motif is found in all eukaryote branches. • Fungi and metazoans contain genes for ACBD5/ATG37 but the proteins do not appear to possess conserved functions • Several extended metazoan ACBDs are hijacked by viruses in order to facilitate effective viral replication.
- Published
- 2019
44. The C99 domain of the amyloid precursor protein resides in the disordered membrane phase
- Author
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Yelena Peskova, Ajit Tiwari, Arina Hadziselimovic, Ricardo Capone, James M. Hutchison, Charles R. Sanders, and Anne K. Kenworthy
- Subjects
0301 basic medicine ,DRM, detergent-resistant membrane ,TMD, transmembrane domain ,amyloid precursor protein ,TfR, transferrin receptor ,confocal microscopy ,BACE1, β-site amyloid precursor protein cleaving enzyme 1 ,Cleavage (embryo) ,Biochemistry ,gamma secretase ,v, number of GPMVs measured ,Amyloid beta-Protein Precursor ,03 medical and health sciences ,Membrane Microdomains ,Protein Domains ,APP, amyloid precursor protein ,Lo, liquid ordered ,Amyloid precursor protein ,Humans ,Molecular Biology ,Lipid raft ,Gamma secretase ,Ld, liquid disordered ,Amyloid beta-Peptides ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Vesicle ,membrane bilayer ,Cell Membrane ,Cell Biology ,Raft ,Aβ, amyloid-β ,GPMV, giant plasma membrane vesicle ,Transmembrane protein ,lipid raft ,Transmembrane domain ,Cholesterol ,030104 developmental biology ,GUV, giant unilamellar vesicle ,Mutation ,biology.protein ,Biophysics ,Amyloid Precursor Protein Secretases ,Alzheimer’s disease ,Research Article ,HeLa Cells - Abstract
Processing of the amyloid precursor protein (APP) via the amyloidogenic pathway is associated with the etiology of Alzheimer's disease. The cleavage of APP by β-secretase to generate the transmembrane 99-residue C-terminal fragment (C99) and subsequent processing of C99 by γ-secretase to yield amyloid-β (Aβ) peptides are essential steps in this pathway. Biochemical evidence suggests that amyloidogenic processing of C99 occurs in cholesterol- and sphingolipid-enriched liquid-ordered phase membrane rafts. However, direct evidence that C99 preferentially associates with these rafts has remained elusive. Here, we tested this by quantifying the affinity of C99-GFP for raft domains in cell-derived giant plasma membrane vesicles (GPMVs). We found that C99 was essentially excluded from ordered domains in vesicles from HeLa cells, undifferentiated SH-SY5Y cells, or SH-SY5Y-derived neurons; instead, ∼90% of C99 partitioned into disordered domains. The strong association of C99 with disordered domains occurred independently of its cholesterol-binding activity or homodimerization, or of the presence of the familial Alzheimer disease Arctic mutation (APP E693G). Finally, through biochemical studies we confirmed previous results, which showed that C99 is processed in the plasma membrane by α-secretase, in addition to the well-known γ-secretase. These findings suggest that C99 itself lacks an intrinsic affinity for raft domains, implying that either i) amyloidogenic processing of the protein occurs in disordered regions of the membrane, ii) processing involves a marginal subpopulation of C99 found in rafts, or iii) as-yet-unidentified protein-protein interactions with C99 in living cells drive this protein into membrane rafts to promote its cleavage therein.
- Published
- 2021
45. ATP-binding cassette transporters mediate differential biosynthesis of glycosphingolipid species
- Author
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Clifford A. Lingwood, Monique Budani, and Christiane Auray-Blais
- Subjects
B3GALT1, β-1,3-galactosyltransferase 1 ,LacCer ,GCS, GlcCer synthase ,TMD, transmembrane domain ,ATP-binding cassette transporter ,Biochemistry ,GLTP, glycolipid transfer protein ,NC, negative control ,chemistry.chemical_compound ,Endocrinology ,Tumor Cells, Cultured ,Lc3, lactotriaosylceramide ,LCS, LacCer synthase ,music.instrument ,glycosphingolipid ,biology ,ATP synthase ,Glycosphingolipid ,FAPP2, phosphatidylinositol-four-phosphate adapter protein 2 ,glucosylceramide flippase ,photoprobes ,lipids (amino acids, peptides, and proteins) ,ABC transporter ,Research Article ,GlcCer synthase ,GSL, glycosphingolipid ,Ceramide ,GlcCer, glucosylceramide ,metabolic channeling ,Cer, ceramide ,QD415-436 ,GlcCer pools ,Glycosphingolipids ,Lactosylceramide ,Biosynthesis ,Glycosyltransferase ,metabolic labeling ,Humans ,music ,KD, knockdown ,Transporter ,Cell Biology ,chemistry ,siRNA, small interfering RNA ,biology.protein ,LacCer, lactosylceramide ,GSL anabolism ,ATP-Binding Cassette Transporters ,B4GALT6, β-1,4-galactosyltransferase 6 ,NBD, nitrobenzo-2-oxa-1,3-diazole ,ST3GAL5, β-galactoside α-2,3-sialyltransferase 5, GM3 synthase - Abstract
The cytosolic-oriented glucosylceramide (GlcCer) synthase is enigmatic, requiring nascent GlcCer translocation to the luminal Golgi membrane to access glycosphingolipid (GSL) anabolic glycosyltransferases. The mechanism by which GlcCer is flipped remains unclear. To investigate the role of GlcCer-binding partners in this process, we previously made cleavable, biotinylated, photoreactive GlcCer analogs in which the reactive nitrene was closely apposed to the GlcCer head group, while maintaining a C16-acyl chain. GlcCer-binding protein specificity was validated for both photoprobes. Using one probe, XLB, here we identified ATP-binding cassette (ABC) transporters ABCA3, ABCB4, and ABCB10 as unfractionated microsomal GlcCer-binding proteins in DU-145 prostate tumor cells. siRNA knockdown (KD) of these transporters differentially blocked GSL synthesis assessed in toto and via metabolic labeling. KD of ABCA3 reduced acid/neutral GSL levels, but increased those of LacCer, while KD of ABCB4 preferentially reduced neutral GSL levels, and KD of ABCB10 reduced levels of both neutral and acidic GSLs. Depletion of ABCA12, implicated in GlcCer transport, preferentially decreased neutral GSL levels, while ABCB1 KD preferentially reduced gangliosides, but increased neutral GSL Gb3. These results imply that multiple ABC transporters may provide distinct but overlapping GlcCer and LacCer pools within the Golgi lumen for anabolism of different GSL series by metabolic channeling. Differential ABC family member usage may fine-tune GSL biosynthesis depending on cell/tissue type. We conclude that ABC transporters provide a new tool for the regulation of GSL biosynthesis and serve as potential targets to reduce selected GSL species/subsets in diseases in which GSLs are dysregulated.
- Published
- 2021
46. Monomeric bile acids modulate the ATPase activity of detergent-solubilized ABCB4/MDR3
- Author
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Lutz Schmitt, Sander H. J. Smits, and Tim Kroll
- Subjects
0301 basic medicine ,G/T/DCA, glyco-/tauro-/deoxycholic acid ,NBD, nucleotide binding domain ,critical micelle concentration ,TMD, transmembrane domain ,030204 cardiovascular system & hematology ,MDR3, multidrug-resistant protein 3 ,ATPase activity ,Biochemistry ,PC, phosphatidylcholine ,chemistry.chemical_compound ,0302 clinical medicine ,Endocrinology ,Chenodeoxycholic acid ,ABC, ATP binding cassette ,LCA, lithocholic acid ,TCDCA, taurochenodeoxycholic acid ,CA, cholic acid ,CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate ,Bile acid ,TLCA ,Deoxycholic acid ,ABC transporter ,Taurolithocholic acid ,Research Article ,medicine.drug_class ,TCA, taurocholic acid ,Taurochenodeoxycholic acid ,QD415-436 ,CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate ,digestive system ,UDCA, ursodeoxycholic acid ,CDCA, chenodeoxycholic acid ,DOPC, 1,2-dioleoyl-sn-glycero-3- phosphocholine ,Bile Acids and Salts ,03 medical and health sciences ,medicine ,Nor-UDCA ,BAM, bile acid mixture ,G/T/UDCA, glyco/-tauro-/ursodeoxycholic acid ,BSEP, bile salt export pump ,bile acids ,G/T/CA, glyco-/tauro-/cholic acid ,Cholic acid ,cholesterol ,Cell Biology ,Taurocholic acid ,Bile Salt Export Pump ,NTCP, sodium taurocholate transporting peptide ,G/T/CDCA, glyco-/tauro-/chenodeoxycholic acid ,030104 developmental biology ,DCA, deoxycholic acid ,chemistry ,TLCA, taurolithocholic acid ,ABCB4/MDR3 - Abstract
ABCB4, also called multidrug-resistant protein 3 (MDR3), is an ATP binding cassette transporter located in the canalicular membrane of hepatocytes that specifically translocates phosphatidylcholine (PC) lipids from the cytoplasmic to the extracellular leaflet. Due to the harsh detergent effect of bile acids, PC lipids provided by ABCB4 are extracted into the bile. While it is well known that bile acids are the major extractor of PC lipids from the membrane into bile, it is unknown whether only PC lipid extraction is improved or whether bile acids also have a direct effect on ABCB4. Using in vitro experiments, we investigated the modulation of ATP hydrolysis of ABC by different bile acids commonly present in humans. We demonstrated that all tested bile acids stimulated ATPase activity except for taurolithocholic acid, which inhibited ATPase activity due to its hydrophobic nature. Additionally, we observed a nearly linear correlation between the critical micelle concentration and maximal stimulation by each bile acid, and that this modulation was maintained in the presence of PC lipids. This study revealed a large effect of 24-nor-ursodeoxycholic acid, suggesting a distinct mode of regulation of ATPase activity compared with other bile acids. In addition, it sheds light on the molecular cross talk of canalicular ABC transporters of the human liver.
- Published
- 2021
47. Familial Alzheimer’s disease mutations in amyloid protein precursor alter proteolysis by γ-secretase to increase amyloid β-peptides of ≥45 residues
- Author
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Michael S. Wolfe, Sujan Devkota, and Todd D. Williams
- Subjects
0301 basic medicine ,amyloid β-protein precursor (APP) ,Amyloid ,Proteolysis ,Mutant ,TMD, transmembrane domain ,Peptide ,amyloid β-protein (Aβ) ,CHO Cells ,Tandem mass spectrometry ,Biochemistry ,intramembrane proteolysis ,HEK, human embryonic kidney ,Amyloid beta-Protein Precursor ,03 medical and health sciences ,Cricetulus ,MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight ,Protein Domains ,Alzheimer Disease ,Cricetinae ,medicine ,Animals ,Humans ,Molecular Biology ,mass spectrometry ,FAD, familial Alzheimer's disease ,chemistry.chemical_classification ,LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry ,DDM, n-dodecyl-β-D-maltoside ,Amyloid beta-Peptides ,Aβ, amyloid β-protein ,030102 biochemistry & molecular biology ,medicine.diagnostic_test ,Tetrapeptide ,Chemistry ,pathogenesis ,HEK 293 cells ,Cell Biology ,APP, amyloid β-protein precursor ,Q-TOF, quadrupole time-of-flight ,AICD, APP intracellular domain ,Transmembrane domain ,030104 developmental biology ,MS, mass spectrometry ,Mutation ,Amyloid Precursor Protein Secretases ,Research Article - Abstract
Production of amyloid β-protein (Aβ) is carried out by the membrane-embedded γ-secretase complex. Mutations in the transmembrane domain of amyloid β-protein precursor (APP) associated with early-onset familial Alzheimer's disease (FAD) can alter the ratio of aggregation-prone 42-residue Aβ (Aβ42) to 40-residue Aβ (Aβ40). However, APP substrate is proteolyzed processively by γ-secretase along two pathways: Aβ49→Aβ46→Aβ43→Aβ40 and Aβ48→Aβ45→Aβ42→Aβ38. Effects of FAD mutations on each proteolytic step are unknown, largely due to difficulties in detecting and quantifying longer Aβ peptides. To address this, we carried out systematic and quantitative analyses of all tri- and tetrapeptide coproducts from proteolysis of wild-type and 14 FAD-mutant APP substrates by purified γ-secretase. These small peptides, including FAD-mutant forms, were detected by tandem mass spectrometry and quantified by establishing concentration curves for each of 32 standards. APP intracellular domain (AICD) coproducts were quantified by immunoblot, and the ratio of AICD products corresponding to Aβ48 and Aβ49 was determined by mass spectrometry. Levels of individual Aβ peptides were determined by subtracting levels of peptide coproducts associated with degradation from those associated with production. This method was validated for Aβ40 and Aβ42 by specific ELISAs and production of equimolar levels of Aβ and AICD. Not all mutant substrates led to increased Aβ42/40. However, all 14 disease-causing mutations led to inefficient processing of longer forms of Aβ ≥ 45 residues. In addition, the effects of certain mutations provided insight into the mechanism of processive proteolysis: intermediate Aβ peptides apparently remain bound for subsequent trimming and are not released and reassociated.
- Published
- 2021
48. Double J-domain piloting of an Hsp70 substrate
- Author
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Stefan G.D. Rüdiger and Júlia Aragonès Pedrola
- Subjects
0301 basic medicine ,translocation ,TMD, transmembrane domain ,Endoplasmic Reticulum ,medicine.disease_cause ,Biochemistry ,Hsp70 ,Substrate Specificity ,ER, endoplasmic reticulum ,Editors' Pick Highlight ,03 medical and health sciences ,Protein Domains ,protein folding ,J-domain proteins ,Protein targeting ,protein targeting ,medicine ,HSP70 Heat-Shock Proteins ,tail-anchored proteins ,Sgt2, small glutamine-rich tetratricopeptide repeat-containing protein 2 ,Endoplasmic Reticulum/metabolism ,Molecular Biology ,Protein maturation ,proteostasis ,JDPs, J-domain proteins ,030102 biochemistry & molecular biology ,Hsp70, heat shock 70 kDa protein ,Chemistry ,Endoplasmic reticulum ,molecular chaperones ,HSP70 Heat-Shock Proteins/metabolism ,Cell Biology ,Cell biology ,Transmembrane domain ,030104 developmental biology ,Proteostasis ,GET, guided-entry-of-tail-anchored protein ,ATPase cycle ,Protein folding ,Biogenesis ,TAs, tail-anchored proteins ,Protein Binding - Abstract
Heat shock 70 kDa protein (Hsp70) chaperones play a crucial role in the biogenesis of tail-anchored proteins (TAs), starting a downstream cascade to the endoplasmic reticulum (ER) via the guided-entry-of-tail-anchored protein (GET) pathway. J-domain proteins (JDPs) are generally known to assist Hsp70s, but their specific role in TA targeting remains unclear. Cho et al. now identify two separate functions for JDPs in the process, in the initial capture of the TA and the transfer into the GET pathway. These data suggest that several Hsp70 cycles could be involved at distinct steps during protein maturation.
- Published
- 2021
49. Mutational insights into the envelope protein of SARS-CoV-2
- Author
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M. Rafiul Islam, M. Anwar Hossain, Israt Dilruba Mishu, Md. Mizanur Rahaman, Israt Islam, Munawar Sultana, M. Shaminur Rahman, and M. Nazmul Hoque
- Subjects
0301 basic medicine ,nt, nucleotide ,SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2 ,S, spike ,Mutant ,aa, amino acid ,TMD, transmembrane domain ,E, envelope ,Biology ,Genome ,Article ,Envelope protein ,Transmembrane domain ,03 medical and health sciences ,0302 clinical medicine ,NP, non-polar ,M, membrane ,Genetics ,Nucleotide ,Gene ,chemistry.chemical_classification ,PC, positively charged ,SARS-CoV-2 ,NC, negatively charged ,Triple cysteine motif ,CTD, C-terminal domain ,Amino acid ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,Glycoprotein ,Mutations ,Cysteine ,N, nucleocapsid - Abstract
The ongoing mutations in the structural proteins of SARS-CoV-2 are the major impediment for prevention and control of the COVID-19 disease. Presently we focused on evolution of the envelope (E) protein, one of the most enigmatic and less studied protein among the four structural proteins (S, E, M and N) associated with multitude of immunopathological functions of SARS-CoV-2. In the present study, we comprehensively analyzed 81,818 high quality E protein sequences of SARS-CoV-2 globally available in the GISAID database as of 20 August 2020. Compared to Wuhan reference strain, our mutational analysis explored only 1.2 % (982/81818) mutant strains undergoing a total of 115 unique amino acid (aa) substitutions in the E protein, highlighting the fact that most (98.8 %) of the E protein of SARS-CoV-2 strains are highly conserved. Moreover, we found 58.77 % (134 of 228) nucleotides (nt) positions of SARS-CoV-2 E gene encountering a total of 176 unique nt-level mutations globally, which may affect the efficacy of real time RT-PCR-based molecular detection of COVID-19. Importantly, higher aa variations observed in the C-terminal domain (CTD) of the E protein, particularly at Ser55-Phe56, Arg69 and the C-terminal end (DLLV: 72–75) may alter the binding of SARS-CoV-2 Envelope protein to tight junction-associated PALS1 and thus could play a key role in COVID-19 pathogenesis. Furthermore, this study revealed the V25A mutation in the transmembrane domain which is a key factor for the homopentameric conformation of E protein. Our analysis also observed a triple cysteine motif harboring mutation (L39M, A41S, A41V, C43F, C43R, C43S, C44Y, N45R) which may hinder the binding of E protein with spike glycoprotein. These results therefore suggest the continuous monitoring of the structural proteins including the envelope protein of SARS-CoV-2 since the number of genome sequences from across the world are continuously increasing., Highlights • We found 1.2 % and 58.77% of 81,818 strains possessing amino acid and nucleotide mutations, respectively, in SARS-CoV-2 E protein. • NTD, TMD, and CTD domains of E protein had aa substitutions at 7, 25, and 31 sites, respectively. • A total of 115 unique aa mutations were observed in 63 (84%) sites of the primary structure of E protein.
- Published
- 2020
50. Modulating effects of RAMPs on signaling profiles of the glucagon receptor family.
- Author
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Shao L, Chen Y, Zhang S, Zhang Z, Cao Y, Yang D, and Wang MW
- Abstract
Receptor activity-modulating proteins (RAMPs) are accessory molecules that form complexes with specific G protein-coupled receptors (GPCRs) and modulate their functions. It is established that RAMP interacts with the glucagon receptor family of GPCRs but the underlying mechanism is poorly understood. In this study, we used a bioluminescence resonance energy transfer (BRET) approach to comprehensively investigate such interactions. In conjunction with cAMP accumulation, G α
q activation and β -arrestin1/2 recruitment assays, we not only verified the GPCR-RAMP pairs previously reported, but also identified new patterns of GPCR-RAMP interaction. While RAMP1 was able to modify the three signaling events elicited by both glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), and RAMP2 mainly affected β -arrestin1/2 recruitment by GCGR, GLP-1R and glucagon-like peptide-2 receptor, RAMP3 showed a widespread negative impact on all the family members except for growth hormone-releasing hormone receptor covering the three pathways. Our results suggest that RAMP modulates both G protein dependent and independent signal transduction among the glucagon receptor family members in a receptor-specific manner. Mapping such interactions provides new insights into the role of RAMP in ligand recognition and receptor activation., (© 2022 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.)- Published
- 2022
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