10,594 results on '"Dimerization"'
Search Results
2. Identification of small-molecule allosteric modulators that act as enhancers/disrupters of rhodopsin oligomerization
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Getter, Tamar, Kemp, Albert, Vinberg, Frans, and Palczewski, Krzysztof
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Medicinal and Biomolecular Chemistry ,Chemical Sciences ,Neurosciences ,Eye Disease and Disorders of Vision ,Animals ,Cell Line ,Tumor ,Humans ,Mice ,Protein Multimerization ,Retinal Cone Photoreceptor Cells ,Rhodopsin ,Rod Cell Outer Segment ,G protein–coupled receptor ,dimerization ,high-throughput screening ,phototransduction ,retina ,rhodopsin ,rod photoreceptors ,Biological Sciences ,Medical and Health Sciences ,Biochemistry & Molecular Biology ,Biological sciences ,Biomedical and clinical sciences ,Chemical sciences - Abstract
The elongated cilia of the outer segment of rod and cone photoreceptor cells can contain concentrations of visual pigments of up to 5 mM. The rod visual pigments, G protein-coupled receptors called rhodopsins, have a propensity to self-aggregate, a property conserved among many G protein-coupled receptors. However, the effect of rhodopsin oligomerization on G protein signaling in native cells is less clear. Here, we address this gap in knowledge by studying rod phototransduction. As the rod outer segment is known to adjust its size proportionally to overexpression or reduction of rhodopsin expression, genetic perturbation of rhodopsin cannot be used to resolve this question. Therefore, we turned to high-throughput screening of a diverse library of 50,000 small molecules and used a novel assay for the detection of rhodopsin dimerization. This screen identified nine small molecules that either disrupted or enhanced rhodopsin dimer contacts in vitro. In a subsequent cell-free binding study, we found that all nine compounds decreased intrinsic fluorescence without affecting the overall UV-visible spectrum of rhodopsin, supporting their actions as allosteric modulators. Furthermore, ex vivo electrophysiological recordings revealed that a disruptive, hit compound #7 significantly slowed down the light response kinetics of intact rods, whereas compound #1, an enhancing hit candidate, did not substantially affect the photoresponse kinetics but did cause a significant reduction in light sensitivity. This study provides a monitoring tool for future investigation of the rhodopsin signaling cascade and reports the discovery of new allosteric modulators of rhodopsin dimerization that can also alter rod photoreceptor physiology.
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- 2021
3. Actions and Roles of FSH in Germinative Cells.
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Recchia, Kaiana, Jorge, Amanda, Pessôa, Laís, Botigelli, Ramon, Zugaib, Vanessa, de Souza, Aline, Martins, Daniele, Ambrósio, Carlos, Bressan, Fabiana, and Pieri, Naira
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germ cell line ,gonadotrophin ,reproduction ,Animals ,Dimerization ,Female ,Fertility ,Follicle Stimulating Hormone ,Follicle Stimulating Hormone ,beta Subunit ,Germ Cells ,Gonadotropins ,Granulosa Cells ,Humans ,Male ,Mice ,Ovary ,Pituitary Gland ,Rats ,Receptors ,FSH ,Reproduction ,Sertoli Cells ,Sexual Maturation ,Spermatogenesis ,Spermatogonia - Abstract
Follicle stimulating hormone (FSH) is produced by the pituitary gland in a coordinated hypothalamic-pituitary-gonadal (HPG) axis event, plays important roles in reproduction and germ cell development during different phases of reproductive development (fetal, neonatal, puberty, and adult life), and is consequently essential for fertility. FSH is a heterodimeric glycoprotein hormone of two dissociable subunits, α and β. The FSH β-subunit (FSHβ) function starts upon coupling to its specific receptor: follicle-stimulating hormone receptor (FSHR). FSHRs are localized mainly on the surface of target cells on the testis and ovary (granulosa and Sertoli cells) and have recently been found in testicular stem cells and extra-gonadal tissue. Several reproduction disorders are associated with absent or low FSH secretion, with mutation of the FSH β-subunit or the FSH receptor, and/or its signaling pathways. However, the influence of FSH on germ cells is still poorly understood; some studies have suggested that this hormone also plays a determinant role in the self-renewal of germinative cells and acts to increase undifferentiated spermatogonia proliferation. In addition, in vitro, together with other factors, it assists the process of differentiation of primordial germ cells (PGCLCs) into gametes (oocyte-like and SSCLCs). In this review, we describe relevant research on the influence of FSH on spermatogenesis and folliculogenesis, mainly in the germ cell of humans and other species. The possible roles of FSH in germ cell generation in vitro are also presented.
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- 2021
4. Structures of tweety homolog proteins TTYH2 and TTYH3 reveal a Ca2+-dependent switch from intra- to intermembrane dimerization
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Li, Baobin, Hoel, Christopher M, and Brohawn, Stephen G
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Neurosciences ,1.1 Normal biological development and functioning ,Underpinning research ,Aetiology ,2.1 Biological and endogenous factors ,Neurological ,Generic health relevance ,Animals ,Anoctamins ,Biological Transport ,Calcium ,Cell Adhesion ,Cell Size ,Chloride Channels ,Chronic Pain ,Cryoelectron Microscopy ,Dimerization ,Eukaryota ,Hydrophobic and Hydrophilic Interactions ,Membrane Proteins ,Mice ,Receptor ,EphB2 ,Signal Transduction - Abstract
Tweety homologs (TTYHs) comprise a conserved family of transmembrane proteins found in eukaryotes with three members (TTYH1-3) in vertebrates. They are widely expressed in mammals including at high levels in the nervous system and have been implicated in cancers and other diseases including epilepsy, chronic pain, and viral infections. TTYHs have been reported to form Ca2+- and cell volume-regulated anion channels structurally distinct from any characterized protein family with potential roles in cell adhesion, migration, and developmental signaling. To provide insight into TTYH family structure and function, we determined cryo-EM structures of Mus musculus TTYH2 and TTYH3 in lipid nanodiscs. TTYH2 and TTYH3 adopt a previously unobserved fold which includes an extended extracellular domain with a partially solvent exposed pocket that may be an interaction site for hydrophobic molecules. In the presence of Ca2+, TTYH2 and TTYH3 form homomeric cis-dimers bridged by extracellularly coordinated Ca2+. Strikingly, in the absence of Ca2+, TTYH2 forms trans-dimers that span opposing membranes across a ~130 Å intermembrane space as well as a monomeric state. All TTYH structures lack ion conducting pathways and we do not observe TTYH2-dependent channel activity in cells. We conclude TTYHs are not pore forming subunits of anion channels and their function may involve Ca2+-dependent changes in quaternary structure, interactions with hydrophobic molecules near the extracellular membrane surface, and/or association with additional protein partners.
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- 2021
5. Small sequence variations between two mammalian paralogs of the small GTPase SAR1 underlie functional differences in coat protein complex II assembly
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Melville, David B, Studer, Sean, and Schekman, Randy
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Rare Diseases ,2.1 Biological and endogenous factors ,Underpinning research ,Aetiology ,1.1 Normal biological development and functioning ,Generic health relevance ,Amino Acid Sequence ,Animals ,Binding Sites ,CRISPR-Cas Systems ,Cell Line ,Dimerization ,Gene Editing ,Guanosine Triphosphate ,Humans ,Molecular Dynamics Simulation ,Monomeric GTP-Binding Proteins ,Phylogeny ,Protein Binding ,Protein Conformation ,alpha-Helical ,Recombinant Fusion Proteins ,Sequence Alignment ,Vesicular Transport Proteins ,COPII ,GTPase ,SAR1A ,SAR1B ,SEC23 ,SEC31 ,apolipoprotein ,intracellular trafficking ,membrane trafficking ,secretion ,Chemical Sciences ,Biological Sciences ,Medical and Health Sciences ,Biochemistry & Molecular Biology - Abstract
Vesicles that are coated by coat protein complex II (COPII) are the primary mediators of vesicular traffic from the endoplasmic reticulum to the Golgi apparatus. Secretion-associated Ras-related GTPase 1 (SAR1) is a small GTPase that is part of COPII and, upon GTP binding, recruits the other COPII proteins to the endoplasmic reticulum membrane. Mammals have two SAR1 paralogs that genetic data suggest may have distinct physiological roles, e.g. in lipoprotein secretion in the case of SAR1B. Here we identified two amino acid clusters that have conserved SAR1 paralog-specific sequences. We observed that one cluster is adjacent to the SAR1 GTP-binding pocket and alters the kinetics of GTP exchange. The other cluster is adjacent to the binding site for two COPII components, SEC31 homolog A COPII coat complex component (SEC31) and SEC23. We found that the latter cluster confers to SAR1B a binding preference for SEC23A that is stronger than that of SAR1A for SEC23A. Unlike SAR1B, SAR1A was prone to oligomerize on a membrane surface. SAR1B knockdown caused loss of lipoprotein secretion, overexpression of SAR1B but not of SAR1A could restore secretion, and a divergent cluster adjacent to the SEC31/SEC23-binding site was critical for this SAR1B function. These results highlight that small primary sequence differences between the two mammalian SAR1 paralogs lead to pronounced biochemical differences that significantly affect COPII assembly and identify a specific function for SAR1B in lipoprotein secretion, providing insights into the mechanisms of large cargo secretion that may be relevant for COPII-related diseases.
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- 2020
6. Congenital myasthenic syndrome due to mutations in MUSK suggests that the level of MuSK phosphorylation is crucial for governing synaptic structure
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Cruz, Pedro M Rodríguez, Cossins, Judith, Cheung, Jonathan, Maxwell, Susan, Jayawant, Sandeep, Herbst, Ruth, Waithe, Dominic, Kornev, Alexandr P, Palace, Jacqueline, and Beeson, David
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Biochemistry and Cell Biology ,Biomedical and Clinical Sciences ,Biological Sciences ,Neurosciences ,Pediatric ,Myasthenia Gravis ,Rare Diseases ,Autoimmune Disease ,1.1 Normal biological development and functioning ,Underpinning research ,Neurological ,Adrenergic beta-2 Receptor Agonists ,Alleles ,Amino Acid Substitution ,Animals ,CRISPR-Cas Systems ,Cell Line ,DNA Mutational Analysis ,Female ,Gene Targeting ,Humans ,Mice ,Models ,Molecular ,Molecular Conformation ,Muscle Proteins ,Mutation ,Myasthenic Syndromes ,Congenital ,Pedigree ,Phosphorylation ,Receptor Protein-Tyrosine Kinases ,Receptors ,Cholinergic ,Structure-Activity Relationship ,Synapses ,AChR clustering ,congenital myasthenic syndromes ,dimerization ,muscle-specific kinase ,MuSK phosphorylation ,neuromuscular junction ,receptor tyrosine kinases ,beta 2-adrenergic agonists ,β2-adrenergic agonists ,Genetics ,Clinical Sciences ,Genetics & Heredity ,Clinical sciences - Abstract
MUSK encodes the muscle-specific receptor tyrosine kinase (MuSK), a key component of the agrin-LRP4-MuSK-DOK7 signaling pathway, which is essential for the formation and maintenance of highly specialized synapses between motor neurons and muscle fibers. We report a patient with severe early-onset congenital myasthenic syndrome and two novel missense mutations in MUSK (p.C317R and p.A617V). Functional studies show that MUSK p.C317R, located at the frizzled-like cysteine-rich domain of MuSK, disrupts an integral part of MuSK architecture resulting in ablated MuSK phosphorylation and acetylcholine receptor (AChR) cluster formation. MUSK p.A617V, located at the kinase domain of MuSK, enhances MuSK phosphorylation resulting in anomalous AChR cluster formation. The identification and evidence for pathogenicity of MUSK mutations supported the initiation of treatment with β2-adrenergic agonists with a dramatic improvement of muscle strength in the patient. This work suggests uncharacterized mechanisms in which control of the precise level of MuSK phosphorylation is crucial in governing synaptic structure.
- Published
- 2020
7. Unfolding of monomeric lipoprotein lipase by ANGPTL4: Insight into the regulation of plasma triglyceride metabolism
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Kristensen, Kristian K, Leth-Espensen, Katrine Zinck, Mertens, Haydyn DT, Birrane, Gabriel, Meiyappan, Muthuraman, Olivecrona, Gunilla, Jørgensen, Thomas JD, Young, Stephen G, and Ploug, Michael
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Biochemistry and Cell Biology ,Biological Sciences ,Amino Acid Motifs ,Angiopoietin-Like Protein 4 ,Animals ,Antibodies ,Monoclonal ,Dimerization ,Humans ,Hypertriglyceridemia ,Lipoprotein Lipase ,Protein Unfolding ,Receptors ,Lipoprotein ,Triglycerides ,HDX-MS ,intravascular lipolysis ,lipoprotein lipase ,GPIHBP1 ,surface plasmon resonance - Abstract
The binding of lipoprotein lipase (LPL) to GPIHBP1 focuses the intravascular hydrolysis of triglyceride-rich lipoproteins on the surface of capillary endothelial cells. This process provides essential lipid nutrients for vital tissues (e.g., heart, skeletal muscle, and adipose tissue). Deficiencies in either LPL or GPIHBP1 impair triglyceride hydrolysis, resulting in severe hypertriglyceridemia. The activity of LPL in tissues is regulated by angiopoietin-like proteins 3, 4, and 8 (ANGPTL). Dogma has held that these ANGPTLs inactivate LPL by converting LPL homodimers into monomers, rendering them highly susceptible to spontaneous unfolding and loss of enzymatic activity. Here, we show that binding of an LPL-specific monoclonal antibody (5D2) to the tryptophan-rich lipid-binding loop in the carboxyl terminus of LPL prevents homodimer formation and forces LPL into a monomeric state. Of note, 5D2-bound LPL monomers are as stable as LPL homodimers (i.e., they are not more prone to unfolding), but they remain highly susceptible to ANGPTL4-catalyzed unfolding and inactivation. Binding of GPIHBP1 to LPL alone or to 5D2-bound LPL counteracts ANGPTL4-mediated unfolding of LPL. In conclusion, ANGPTL4-mediated inactivation of LPL, accomplished by catalyzing the unfolding of LPL, does not require the conversion of LPL homodimers into monomers. Thus, our findings necessitate changes to long-standing dogma on mechanisms for LPL inactivation by ANGPTL proteins. At the same time, our findings align well with insights into LPL function from the recent crystal structure of the LPL•GPIHBP1 complex.
- Published
- 2020
8. Rad50 zinc hook functions as a constitutive dimerization module interchangeable with SMC hinge
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Tatebe, Hisashi, Lim, Chew Theng, Konno, Hiroki, Shiozaki, Kazuhiro, Shinohara, Akira, Uchihashi, Takayuki, and Furukohri, Asako
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Underpinning research ,1.1 Normal biological development and functioning ,Generic health relevance ,Acid Anhydride Hydrolases ,Adenosine Triphosphatases ,Animals ,Cell Cycle Proteins ,Chromosomal Proteins ,Non-Histone ,DNA Breaks ,Double-Stranded ,DNA Repair ,DNA-Binding Proteins ,Dimerization ,Escherichia coli ,Homologous Recombination ,Humans ,MRE11 Homologue Protein ,Microscopy ,Atomic Force ,Multiprotein Complexes ,Nuclear Proteins ,Protein Binding ,Protein Conformation ,Saccharomyces cerevisiae ,Saccharomyces cerevisiae Proteins ,Sf9 Cells ,Zinc - Abstract
The human Mre11/Rad50 complex is one of the key factors in genome maintenance pathways. Previous nanoscale imaging by atomic force microscopy (AFM) showed that the ring-like structure of the human Mre11/Rad50 complex transiently opens at the zinc hook of Rad50. However, imaging of the human Mre11/Rad50 complex by high-speed AFM shows that the Rad50 coiled-coil arms are consistently bridged by the dimerized hooks while the Mre11/Rad50 ring opens by disconnecting the head domains; resembling other SMC proteins such as cohesin or condensin. These architectural features are conserved in the yeast and bacterial Mre11/Rad50 complexes. Yeast strains harboring the chimeric Mre11/Rad50 complex containing the SMC hinge of bacterial condensin MukB instead of the RAD50 hook properly functions in DNA repair. We propose that the basic role of the Rad50 hook is similar to that of the SMC hinge, which serves as rather stable dimerization interface.
- Published
- 2020
9. Optimized Vivid-derived Magnets photodimerizers for subcellular optogenetics in mammalian cells
- Author
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Benedetti, Lorena, Marvin, Jonathan S, Falahati, Hanieh, Guillén-Samander, Andres, Looger, Loren L, and De Camilli, Pietro
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Biochemistry and Cell Biology ,Biological Sciences ,Generic health relevance ,Animals ,Biological Transport ,COS Cells ,Chlorocebus aethiops ,Dimerization ,Fungal Proteins ,HeLa Cells ,Humans ,Kinetics ,Light ,Lipid Metabolism ,Mice ,Inbred C57BL ,Optogenetics ,Organelles ,Phosphatidylinositol Phosphates ,Protein Engineering ,Protein Multimerization ,Protein Stability ,Protein Transport ,Hela Cells ,LOV domain ,VAP ,Vivid ,cell biology ,contact sites ,human ,light-dependent dimerizers ,organelle contacts ,Biological sciences ,Biomedical and clinical sciences ,Health sciences - Abstract
Light-inducible dimerization protein modules enable precise temporal and spatial control of biological processes in non-invasive fashion. Among them, Magnets are small modules engineered from the Neurospora crassa photoreceptor Vivid by orthogonalizing the homodimerization interface into complementary heterodimers. Both Magnets components, which are well-tolerated as protein fusion partners, are photoreceptors requiring simultaneous photoactivation to interact, enabling high spatiotemporal confinement of dimerization with a single excitation wavelength. However, Magnets require concatemerization for efficient responses and cell preincubation at 28°C to be functional. Here we overcome these limitations by engineering an optimized Magnets pair requiring neither concatemerization nor low temperature preincubation. We validated these 'enhanced' Magnets (eMags) by using them to rapidly and reversibly recruit proteins to subcellular organelles, to induce organelle contacts, and to reconstitute OSBP-VAP ER-Golgi tethering implicated in phosphatidylinositol-4-phosphate transport and metabolism. eMags represent a very effective tool to optogenetically manipulate physiological processes over whole cells or in small subcellular volumes.
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- 2020
10. Genetic Heterogeneity of BRAF Fusion Kinases in Melanoma Affects Drug Responses
- Author
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Botton, Thomas, Talevich, Eric, Mishra, Vivek Kumar, Zhang, Tongwu, Shain, A Hunter, Berquet, Céline, Gagnon, Alexander, Judson, Robert L, Ballotti, Robert, Ribas, Antoni, Herlyn, Meenhard, Rocchi, Stéphane, Brown, Kevin M, Hayward, Nicholas K, Yeh, Iwei, and Bastian, Boris C
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Biological Sciences ,Cancer ,Development of treatments and therapeutic interventions ,5.1 Pharmaceuticals ,Animals ,Dimerization ,Drug Resistance ,Neoplasm ,Female ,Gene Expression Regulation ,Neoplastic ,Humans ,Intracellular Signaling Peptides and Proteins ,Melanoma ,Mice ,Mice ,Nude ,Mitogen-Activated Protein Kinases ,Oncogene Proteins ,Fusion ,Protein Isoforms ,Protein Kinase Inhibitors ,Proto-Oncogene Proteins B-raf ,RNA Interference ,RNA ,Small Interfering ,Signal Transduction ,Vemurafenib ,ras Proteins ,BRAF fusion ,MEK inhibitor ,RAF inhibitor ,kinase ,melanoma ,paradoxical activation ,pre-clinical ,rearrangement ,sequencing ,translocation ,Biochemistry and Cell Biology ,Medical Physiology ,Biological sciences - Abstract
BRAF fusions are detected in numerous neoplasms, but their clinical management remains unresolved. We identified six melanoma lines harboring BRAF fusions representative of the clinical cases reported in the literature. Their unexpected heterogeneous responses to RAF and MEK inhibitors could be categorized upon specific features of the fusion kinases. Higher expression level correlated with resistance, and fusion partners containing a dimerization domain promoted paradoxical activation of the mitogen-activated protein kinase (MAPK) pathway and hyperproliferation in response to first- and second-generation RAF inhibitors. By contrast, next-generation αC-IN/DFG-OUT RAF inhibitors blunted paradoxical activation across all lines and had their therapeutic efficacy further increased in vitro and in vivo by combination with MEK inhibitors, opening perspectives in the clinical management of tumors harboring BRAF fusions.
- Published
- 2019
11. Structural Basis for Cholesterol Transport-like Activity of the Hedgehog Receptor Patched.
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Zhang, Yunxiao, Bulkley, David P, Xin, Yao, Roberts, Kelsey J, Asarnow, Daniel E, Sharma, Ashutosh, Myers, Benjamin R, Cho, Wonhwa, Cheng, Yifan, and Beachy, Philip A
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Cell Line ,Animals ,Humans ,Mice ,Escherichia coli ,Cholesterol ,Escherichia coli Proteins ,Multidrug Resistance-Associated Proteins ,Recombinant Proteins ,Cryoelectron Microscopy ,Sequence Alignment ,Evolution ,Molecular ,Signal Transduction ,Amino Acid Sequence ,Protein Structure ,Tertiary ,Dimerization ,Hedgehog Proteins ,HEK293 Cells ,Patched-1 Receptor ,Hedgehog ,Hedgehog signaling ,Patched ,RND transporter ,Smoothened ,cholesterol sensor ,cholesterol transport ,cryo-EM ,membrane lipid asymmetry ,1.1 Normal biological development and functioning ,Underpinning research ,Generic health relevance ,Biological Sciences ,Medical and Health Sciences ,Developmental Biology - Abstract
Hedgehog protein signals mediate tissue patterning and maintenance by binding to and inactivating their common receptor Patched, a 12-transmembrane protein that otherwise would suppress the activity of the 7-transmembrane protein Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. A central hydrophobic conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Cholesterol activity in the inner leaflet of the plasma membrane is reduced by PTCH1 expression but rapidly restored by Hedgehog stimulation, suggesting that PTCH1 regulates Smoothened by controlling cholesterol availability.
- Published
- 2018
12. Regulation of Kinase Activity in the Caenorhabditis elegans EGF Receptor, LET-23
- Author
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Liu, Lijun, Thaker, Tarjani M, Freed, Daniel M, Frazier, Nicole, Malhotra, Ketan, Lemmon, Mark A, and Jura, Natalia
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Biochemistry and Cell Biology ,Biological Sciences ,1.1 Normal biological development and functioning ,Underpinning research ,Generic health relevance ,Animals ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Dimerization ,ErbB Receptors ,Phosphorylation ,Phosphotransferases ,Signal Transduction ,EGFR ,ERBB receptors ,HER receptors ,LET-23 ,allosteric kinase activation ,asymmetric dimerization ,kinase activation ,kinase structure ,receptor tyrosine kinase signaling ,Chemical Sciences ,Information and Computing Sciences ,Biophysics ,Biological sciences ,Chemical sciences - Abstract
In the active HER receptor dimers, kinases play distinct roles; one is the catalytically active kinase and the other is its allosteric activator. This specialization enables signaling by the catalytically inactive HER3, which functions exclusively as an allosteric activator upon heterodimerization with other HER receptors. It is unclear whether the allosteric activation mechanism evolved before HER receptors functionally specialized. We determined the crystal structure of the kinase domain of the only EGF receptor in Caenorhabditis elegans, LET-23. Our structure of a non-human EGFR kinase reveals autoinhibitory features conserved in the human counterpart. Strikingly, mutations within the putative allosteric dimer interface abrogate activity of the isolated LET-23 kinase and of the full-length receptor despite these regions being only partially conserved with human EGFR. Our results indicate that ancestral EGFRs have built-in features that poise them for allosteric activation that could facilitate emergence of the catalytically dead, yet functional, orthologs.
- Published
- 2018
13. Protonation state of glutamate 73 regulates the formation of a specific dimeric association of mVDAC1
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Bergdoll, Lucie A, Lerch, Michael T, Patrick, John W, Belardo, Kendrick, Altenbach, Christian, Bisignano, Paola, Laganowsky, Arthur, Grabe, Michael, Hubbell, Wayne L, and Abramson, Jeff
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Analytical Chemistry ,Biochemistry and Cell Biology ,Chemical Sciences ,Biological Sciences ,1.1 Normal biological development and functioning ,Underpinning research ,Algorithms ,Animals ,Glutamates ,Hydrogen-Ion Concentration ,Kinetics ,Mice ,Models ,Molecular ,Mutation ,Protein Conformation ,Protein Multimerization ,Protons ,Voltage-Dependent Anion Channel 1 ,VDAC ,dimerization ,DEER ,native mass spectrometry ,cellular stress - Abstract
The voltage-dependent anion channel (VDAC) is the most abundant protein in the outer mitochondrial membrane and constitutes the primary pathway for the exchange of ions and metabolites between the cytosol and the mitochondria. There is accumulating evidence supporting VDAC's role in mitochondrial metabolic regulation and apoptosis, where VDAC oligomerization has been implicated with these processes. Herein, we report a specific pH-dependent dimerization of murine VDAC1 (mVDAC1) identified by double electron-electron resonance and native mass spectrometry. Intermolecular distances on four singly spin-labeled mVDAC1 mutants were used to generate a model of the low-pH dimer, establishing the presence of residue E73 at the interface. This dimer arrangement is different from any oligomeric state previously described, and it forms as a steep function of pH with an apparent pKa of 7.4. Moreover, the monomer-dimer equilibrium affinity constant was determined using native MS, revealing a nearly eightfold enhancement in dimerization affinity at low pH. Mutation of E73 to either alanine or glutamine severely reduces oligomerization, demonstrating the role of protonated E73 in enhancing dimer formation. Based on these results, and the known importance of E73 in VDAC physiology, VDAC dimerization likely plays a significant role in mitochondrial metabolic regulation and apoptosis in response to cytosolic acidification during cellular stress.
- Published
- 2018
14. Retinal guanylyl cyclase activating protein 1 forms a functional dimer.
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Lim, Sunghyuk, Roseman, Graham, Peshenko, Igor, Manchala, Grace, Cudia, Diana, Dizhoor, Alexander M, Millhauser, Glenn, and Ames, James B
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Animals ,Cattle ,Spin Labels ,Recombinant Proteins ,Electron Spin Resonance Spectroscopy ,Mutagenesis ,Site-Directed ,Allosteric Regulation ,Amino Acid Sequence ,Amino Acid Motifs ,Protein Conformation ,Dimerization ,Catalysis ,Models ,Molecular ,Guanylate Cyclase-Activating Proteins ,Molecular Docking Simulation ,Models ,Molecular ,Mutagenesis ,Site-Directed ,General Science & Technology - Abstract
Retinal guanylyl cyclases (RetGCs) in vertebrate photoreceptors are regulated by the guanylyl cyclase activator proteins (GCAP1 and GCAP2). Here, we report EPR double electron-electron resonance (DEER) studies on the most ubiquitous GCAP isoform, GCAP1 and site-directed mutagenesis analysis to determine an atomic resolution structural model of a GCAP1 dimer. Nitroxide spin-label probes were introduced at individual GCAP1 residues: T29C, E57C, E133C, and E154C. The intermolecular distance of each spin-label probe (measured by DEER) defined restraints for calculating the GCAP1 dimeric structure by molecular docking. The DEER-derived structural model of the GCAP1 dimer was similar within the experimental error for both the Mg2+-bound activator and Ca2+-bound inhibitor states (RMSD < 2.0 Å). The GCAP1 dimer possesses intermolecular hydrophobic contacts involving the side chain atoms of H19, Y22, F73 and V77. The structural model of the dimer was validated by GCAP1 mutations (H19R, Y22D, F73E, and V77E) at the dimer interface that each abolished protein dimerization. Previous studies have shown that each of these mutants either diminished or completely suppressed the ability of GCAP1 to activate the cyclase. These results suggest that GCAP1 dimerization may affect compartmentalization of GCAP1 in the photoreceptors and/or affect regulation of the cyclase activity.
- Published
- 2018
15. DNA-binding affinity and transcriptional activity of the RelA homodimer of nuclear factor κB are not correlated
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Mulero, Maria Carmen, Huang, De-Bin, Nguyen, H Thien, Wang, Vivien Ya-Fan, Li, Yidan, Biswas, Tapan, and Ghosh, Gourisankar
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Genetics ,Underpinning research ,1.1 Normal biological development and functioning ,Generic health relevance ,Animals ,Base Sequence ,Binding Sites ,Crystallography ,X-Ray ,DNA ,E-Selectin ,Gene Expression Regulation ,Mice ,Models ,Molecular ,Promoter Regions ,Genetic ,Protein Binding ,Protein Domains ,Protein Multimerization ,Transcription Factor RelA ,Transcriptional Activation ,cooperativity ,crystal structure ,dimerization ,DNA transcription ,NF-kB transcription factor ,E-selectin ,RelA ,kB site ,Chemical Sciences ,Biological Sciences ,Medical and Health Sciences ,Biochemistry & Molecular Biology - Abstract
The nuclear factor κB (NF-κB) transcription factor family regulates genes involved in cell proliferation and inflammation. The promoters of these genes often contain NF-κB-binding sites (κB sites) arranged in tandem. How NF-κB activates transcription through these multiple sites is incompletely understood. We report here an X-ray crystal structure of homodimers comprising the RelA DNA-binding domain containing the Rel homology region (RHR) in NF-κB bound to an E-selectin promoter fragment with tandem κB sites. This structure revealed that two dimers bind asymmetrically to the symmetrically arranged κB sites at which multiple cognate contacts between one dimer to the corresponding DNA are broken. Because simultaneous RelA-RHR dimer binding to tandem sites in solution was anti-cooperative, we inferred that asymmetric RelA-RHR binding with fewer contacts likely indicates a dissociative binding mode. We found that both κB sites are essential for reporter gene activation by full-length RelA homodimer, suggesting that dimers facilitate DNA binding to each other even though their stable co-occupation is not promoted. Promoter variants with altered spacing and orientation of tandem κB sites displayed unexpected reporter activities that were not explained by the solution-binding pattern of RelA-RHR. Remarkably, full-length RelA bound all DNAs with a weaker affinity and specificity. Moreover, the transactivation domain played a negative role in DNA binding. These observations suggest that other nuclear factors influence full-length RelA binding to DNA by neutralizing the transactivation domain negative effect. We propose that DNA binding by NF-κB dimers is highly complex and modulated by facilitated association-dissociation processes.
- Published
- 2017
16. Cyclic Hexapeptide Dimers, Antatollamides A and B, from the Ascidian Didemnum molle. A Tryptophan-Derived Auxiliary for l- and d‑Amino Acid Assignments
- Author
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Salib, Mariam N and Molinski, Tadeusz F
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Amino Acids ,Animals ,Cell Proliferation ,Dimerization ,Dose-Response Relationship ,Drug ,Humans ,Molecular Conformation ,Peptides ,Cyclic ,Structure-Activity Relationship ,Tryptophan ,Tumor Cells ,Cultured ,Urochordata ,Medicinal and Biomolecular Chemistry ,Organic Chemistry - Abstract
Two dimerized cyclic hexapeptides, antatollamides A (1) and B (2), were isolated from the colonial ascidian Didemnum molle collected in Pohnpei. The amino acid compositions and sequences were determined by interpretation of MS and 1D and 2D NMR data. Raney Ni reduction of antatollamide A cleaved the dimer to the corresponding monomeric cyclic hexapeptide with replacement of Cys by Ala. The amino acid configuration of 1 was established, after total hydrolysis, by derivatization with a new chiral reagent, (5-fluoro-2,4-dinitrophenyl)-Nα-l-tryptophanamide (FDTA), prepared from l-Trp, followed by LCMS analysis; all amino acids were found to be l-configured except for d-Ala.
- Published
- 2017
17. Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization
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Trease, Andrew J, Capuccino, Juan MV, Contreras, Jorge, Harris, Andrew L, and Sorgen, Paul L
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Biochemistry and Cell Biology ,Medical Physiology ,Biomedical and Clinical Sciences ,Biological Sciences ,Cardiovascular ,Heart Disease ,Underpinning research ,1.1 Normal biological development and functioning ,Amino Acid Sequence ,Animals ,Cell Communication ,Cell Membrane ,Connexins ,Cytoplasm ,Dogs ,Gap Junctions ,HeLa Cells ,Humans ,Ion Channel Gating ,Madin Darby Canine Kidney Cells ,Myocardium ,Phosphorylation ,Phosphotyrosine ,Protein Binding ,Protein Domains ,Protein Multimerization ,Proteolysis ,Signal Transduction ,Gap junctions ,Intercellular communication ,Connexin45 ,Carboxyl terminal domain ,Dimerization ,Protein-protein interactions ,Hela Cells ,Cardiorespiratory Medicine and Haematology ,Cardiovascular System & Hematology ,Biochemistry and cell biology ,Cardiovascular medicine and haematology ,Medical physiology - Abstract
Gap junctions, composed of connexins, mediate electrical coupling and impulse propagation in the working myocardium. In the human heart, the spatio-temporal regulation and distinct functional properties of the three dominant connexins (Cx43, Cx45, and Cx40) suggests non-redundant physiological roles for each isoform. There are substantial differences in gating properties, expression, and trafficking among these isoforms, however, little is known about the determinants of these different phenotypes. To gain insight regarding these determinants, we focused on the carboxyl-terminal (CT) domain because of its importance in channel regulation and large degree of sequence divergence among connexin family members. Using in vitro biophysical experiments, we identified a structural feature unique to Cx45: high affinity (KD~100nM) dimerization between CT domains. In this study, we sought to determine if this dimerization occurs in cells and to identify the biological significance of the dimerization. Using a bimolecular fluorescence complementation assay, we demonstrate that the CT domains dimerize at the plasma membrane. By inhibiting CT dimerization with a mutant construct, we show that CT dimerization is necessary for proper Cx45 membrane localization, turnover, phosphorylation status, and binding to protein partners. Furthermore, CT dimerization is needed for normal intercellular communication and hemichannel activity. Altogether, our results demonstrate that CT dimerization is a structural feature important for correct Cx45 function. This study is significant because discovery of how interactions mediated by the CT domains can be modulated would open the door to strategies to ameliorate the pathological effects of altered connexin regulation in the failing heart.
- Published
- 2017
18. Bioenergetic state regulates innate inflammatory responses through the transcriptional co-repressor CtBP.
- Author
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Shen, Yiguo, Kapfhamer, David, Minnella, Angela M, Kim, Ji-Eun, Won, Seok Joon, Chen, Yanting, Huang, Yong, Low, Ley Hian, Massa, Stephen M, and Swanson, Raymond A
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Microglia ,Macrophages ,Animals ,Mice ,Rats ,NAD ,Alcohol Oxidoreductases ,Glucose ,DNA-Binding Proteins ,NF-kappa B ,Phosphoproteins ,Signal Transduction ,Transcription ,Genetic ,Binding Sites ,Dimerization ,Energy Metabolism ,p300-CBP Transcription Factors ,Immunity ,Innate ,Co-Repressor Proteins ,RAW 264.7 Cells ,Transcription ,Genetic ,Immunity ,Innate ,Nutrition ,Genetics ,1.1 Normal biological development and functioning - Abstract
The innate inflammatory response contributes to secondary injury in brain trauma and other disorders. Metabolic factors such as caloric restriction, ketogenic diet, and hyperglycemia influence the inflammatory response, but how this occurs is unclear. Here, we show that glucose metabolism regulates pro-inflammatory NF-κB transcriptional activity through effects on the cytosolic NADH:NAD+ ratio and the NAD(H) sensitive transcriptional co-repressor CtBP. Reduced glucose availability reduces the NADH:NAD+ ratio, NF-κB transcriptional activity, and pro-inflammatory gene expression in macrophages and microglia. These effects are inhibited by forced elevation of NADH, reduced expression of CtBP, or transfection with an NAD(H) insensitive CtBP, and are replicated by a synthetic peptide that inhibits CtBP dimerization. Changes in the NADH:NAD+ ratio regulate CtBP binding to the acetyltransferase p300, and regulate binding of p300 and the transcription factor NF-κB to pro-inflammatory gene promoters. These findings identify a mechanism by which alterations in cellular glucose metabolism can influence cellular inflammatory responses.Several metabolic factors affect cellular glucose metabolism as well as the innate inflammatory response. Here, the authors show that glucose metabolism regulates pro-inflammatory responses through effects on the cytosolic NADH:NAD+ ratio and the NAD(H)-sensitive transcription co-repressor CtBP.
- Published
- 2017
19. Two transmembrane dimers of the bovine papillomavirus E5 oncoprotein clamp the PDGF β receptor in an active dimeric conformation
- Author
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Karabadzhak, Alexander G, Petti, Lisa M, Barrera, Francisco N, Edwards, Anne PB, Moya-Rodríguez, Andrés, Polikanov, Yury S, Freites, J Alfredo, Tobias, Douglas J, Engelman, Donald M, and DiMaio, Daniel
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Cancer ,Cervical Cancer ,Sexually Transmitted Infections ,Amino Acid Sequence ,Animals ,Cattle ,Cell Line ,Cell Transformation ,Viral ,Dimerization ,Humans ,Membrane Proteins ,Mice ,Molecular Conformation ,Oncogene Proteins ,Viral ,Papillomaviridae ,Papillomavirus Infections ,Protein Multimerization ,Receptor ,Platelet-Derived Growth Factor beta ,transmembrane protein complex ,oncogene ,traptamer ,BPV ,blue native gel electrophoresis - Abstract
The dimeric 44-residue E5 protein of bovine papillomavirus is the smallest known naturally occurring oncoprotein. This transmembrane protein binds to the transmembrane domain (TMD) of the platelet-derived growth factor β receptor (PDGFβR), causing dimerization and activation of the receptor. Here, we use Rosetta membrane modeling and all-atom molecular dynamics simulations in a membrane environment to develop a chemically detailed model of the E5 protein/PDGFβR complex. In this model, an active dimer of the PDGFβR TMD is sandwiched between two dimers of the E5 protein. Biochemical experiments showed that the major PDGFβR TMD complex in mouse cells contains two E5 dimers and that binding the PDGFβR TMD to the E5 protein is necessary and sufficient to recruit both E5 dimers into the complex. These results demonstrate how E5 binding induces receptor dimerization and define a molecular mechanism of receptor activation based on specific interactions between TMDs.
- Published
- 2017
20. Protease-activated receptor-4 and purinergic receptor P2Y12 dimerize, co-internalize, and activate Akt signaling via endosomal recruitment of β-arrestin
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Smith, Thomas H, Li, Julia G, Dores, Michael R, and Trejo, JoAnn
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Biochemistry and Cell Biology ,Biomedical and Clinical Sciences ,Biological Sciences ,Cardiovascular ,Amino Acid Substitution ,Animals ,Bioluminescence Resonance Energy Transfer Techniques ,COS Cells ,Cell Line ,Tumor ,Chlorocebus aethiops ,Endocytosis ,Endosomes ,Humans ,Immunoprecipitation ,Microscopy ,Fluorescence ,Protein Multimerization ,Protein Transport ,Proto-Oncogene Proteins c-akt ,Receptor ,PAR-1 ,Receptors ,Purinergic P2Y12 ,Receptors ,Thrombin ,Recombinant Fusion Proteins ,Signal Transduction ,beta-Arrestin 2 ,ADP ,Akt PKB ,G protein-coupled receptor ,arrestin ,dimerization ,endosome ,thrombin ,Chemical Sciences ,Medical and Health Sciences ,Biochemistry & Molecular Biology ,Biological sciences ,Biomedical and clinical sciences ,Chemical sciences - Abstract
Vascular inflammation and thrombosis require the concerted actions of several different agonists, many of which act on G protein-coupled receptors (GPCRs). GPCR dimerization is a well-established phenomenon that can alter protomer function. In platelets and other cell types, protease-activated receptor-4 (PAR4) has been shown to dimerize with the purinergic receptor P2Y12 to coordinate β-arrestin-mediated Akt signaling, an important mediator of integrin activation. However, the mechanism by which the PAR4-P2Y12 dimer controls β-arrestin-dependent Akt signaling is not known. We now report that PAR4 and P2Y12 heterodimer internalization is required for β-arrestin recruitment to endosomes and Akt signaling. Using bioluminescence resonance energy transfer, immunofluorescence microscopy, and co-immunoprecipitation in cells expressing receptors exogenously and endogenously, we demonstrate that PAR4 and P2Y12 specifically interact and form dimers expressed at the cell surface. We also found that activation of PAR4 but not of P2Y12 drives internalization of the PAR4-P2Y12 heterodimer. Remarkably, activated PAR4 internalization was required for recruitment of β-arrestin to endocytic vesicles, which was dependent on co-expression of P2Y12. Interestingly, stimulation of the PAR4-P2Y12 heterodimer promotes β-arrestin and Akt co-localization to intracellular vesicles. Moreover, activated PAR4-P2Y12 internalization is required for sustained Akt activation. Thus, internalization of the PAR4-P2Y12 heterodimer is necessary for β-arrestin recruitment to endosomes and Akt signaling and lays the foundation for examining whether blockade of PAR4 internalization reduces integrin and platelet activation.
- Published
- 2017
21. Differential effects of the dynein-regulatory factor Lissencephaly-1 on processive dynein-dynactin motility
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Gutierrez, Pedro A, Ackermann, Bryce E, Vershinin, Michael, and McKenney, Richard J
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Biochemistry and Cell Biology ,Biological Sciences ,1.1 Normal biological development and functioning ,Underpinning research ,Generic health relevance ,1-Alkyl-2-acetylglycerophosphocholine Esterase ,Animals ,Dimerization ,Dynactin Complex ,Dyneins ,Humans ,Mice ,Microscopy ,Fluorescence ,Microtubule-Associated Proteins ,Microtubules ,Mutagenesis ,Site-Directed ,Mutation ,Nerve Tissue Proteins ,Peptide Fragments ,Protein Interaction Domains and Motifs ,Protein Multimerization ,Rats ,Recombinant Fusion Proteins ,Sf9 Cells ,Spodoptera ,Sus scrofa ,Tubulin ,cytoskeleton ,dynein ,microtubule ,molecular motor ,single-molecule biophysics ,Chemical Sciences ,Medical and Health Sciences ,Biochemistry & Molecular Biology ,Biological sciences ,Biomedical and clinical sciences ,Chemical sciences - Abstract
Cytoplasmic dynein is the primary minus-end-directed microtubule motor protein in animal cells, performing a wide range of motile activities, including transport of vesicular cargos, mRNAs, viruses, and proteins. Lissencephaly-1 (LIS1) is a highly conserved dynein-regulatory factor that binds directly to the dynein motor domain, uncoupling the enzymatic and mechanical cycles of the motor and stalling dynein on the microtubule track. Dynactin, another ubiquitous dynein-regulatory factor, releases dynein from an autoinhibited state, leading to a dramatic increase in fast, processive dynein motility. How these opposing activities are integrated to control dynein motility is unknown. Here, we used fluorescence single-molecule microscopy to study the interaction of LIS1 with the processive dynein-dynactin-BicD2N (DDB) complex. Surprisingly, in contrast to the prevailing model for LIS1 function established in the context of dynein alone, we found that binding of LIS1 to DDB does not strongly disrupt processive motility. Motile DDB complexes bound up to two LIS1 dimers, and mutational analysis suggested that LIS1 binds directly to the dynein motor domains during DDB movement. Interestingly, LIS1 enhanced DDB velocity in a concentration-dependent manner, in contrast to observations of the effect of LIS1 on the motility of isolated dynein. Thus, LIS1 exerts concentration-dependent effects on dynein motility and can synergize with dynactin to enhance processive dynein movement. Our results suggest that the effect of LIS1 on dynein motility depends on both LIS1 concentration and the presence of other regulatory factors such as dynactin and may provide new insights into the mechanism of LIS1 haploinsufficiency in the neurodevelopmental disorder lissencephaly.
- Published
- 2017
22. Epigallocatechin gallate has pleiotropic effects on transmembrane signaling by altering the embedding of transmembrane domains
- Author
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Ye, Feng, Yang, Chansik, Kim, Jiyoon, MacNevin, Christopher J, Hahn, Klaus M, Park, Dongeun, Ginsberg, Mark H, and Kim, Chungho
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Biochemistry and Cell Biology ,Biological Sciences ,Complementary and Integrative Health ,Nutrition ,Amino Acid Substitution ,Animals ,Antioxidants ,CHO Cells ,Catechin ,Cricetulus ,Dietary Supplements ,Dimerization ,ErbB Receptors ,Humans ,Integrin alpha2 ,Integrin beta3 ,Ligands ,Lipid Bilayers ,Models ,Molecular ,Mutation ,Peptide Fragments ,Platelet Glycoprotein GPIIb-IIIa Complex ,Protein Interaction Domains and Motifs ,Recombinant Fusion Proteins ,Signal Transduction ,Talin ,epidermal growth factor receptor ,integrin ,polyphenol ,talin ,transmembrane domain ,Chemical Sciences ,Medical and Health Sciences ,Biochemistry & Molecular Biology ,Biological sciences ,Biomedical and clinical sciences ,Chemical sciences - Abstract
Epigallocatechin gallate (EGCG) is the principal bioactive ingredient in green tea and has been reported to have many health benefits. EGCG influences multiple signal transduction pathways related to human diseases, including redox, inflammation, cell cycle, and cell adhesion pathways. However, the molecular mechanisms of these varying effects are unclear, limiting further development and utilization of EGCG as a pharmaceutical compound. Here, we examined the effect of EGCG on two representative transmembrane signaling receptors, integrinαIIbβ3 and epidermal growth factor receptor (EGFR). We report that EGCG inhibits talin-induced integrin αIIbβ3 activation, but it activates αIIbβ3 in the absence of talin both in a purified system and in cells. This apparent paradox was explained by the fact that the activation state of αIIbβ3 is tightly regulated by the topology of β3 transmembrane domain (TMD); increases or decreases in TMD embedding can activate integrins. Talin increases the embedding of integrin β3 TMD, resulting in integrin activation, whereas we observed here that EGCG decreases the embedding, thus opposing talin-induced integrin activation. In the absence of talin, EGCG decreases the TMD embedding, which can also disrupt the integrin α-β TMD interaction, leading to integrin activation. EGCG exhibited similar paradoxical behavior in EGFR signaling. EGCG alters the topology of EGFR TMD and activates the receptor in the absence of EGF, but inhibits EGF-induced EGFR activation. Thus, this widely ingested polyphenol exhibits pleiotropic effects on transmembrane signaling by modifying the topology of TMDs.
- Published
- 2017
23. Amino-terminal domains of kainate receptors determine the differential dependence on Neto auxiliary subunits for trafficking
- Author
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Sheng, Nengyin, Shi, Yun Stone, and Nicoll, Roger A
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Biochemistry and Cell Biology ,Biomedical and Clinical Sciences ,Biological Sciences ,Mental Health ,Neurosciences ,Underpinning research ,1.1 Normal biological development and functioning ,Neurological ,Amino Acid Substitution ,Animals ,CA1 Region ,Hippocampal ,Dimerization ,LDL-Receptor Related Proteins ,Membrane Proteins ,Mice ,Miniature Postsynaptic Potentials ,Organ Culture Techniques ,Patch-Clamp Techniques ,Protein Domains ,Protein Interaction Maps ,Protein Transport ,Pyramidal Cells ,Rats ,Receptors ,Kainic Acid ,Receptors ,N-Methyl-D-Aspartate ,Recombinant Proteins ,Structure-Activity Relationship ,Synaptic Transmission ,kainate receptor ,amino-terminal domain ,Neto proteins ,synaptic trafficking - Abstract
The kainate receptor (KAR), a subtype of glutamate receptor, mediates excitatory synaptic responses at a subset of glutamatergic synapses. However, the molecular mechanisms underlying the trafficking of its different subunits are poorly understood. Here we use the CA1 hippocampal pyramidal cell, which lacks KAR-mediated synaptic currents, as a null background to determine the minimal requirements for the extrasynaptic and synaptic expression of the GluK2 subunit. We find that the GluK2 receptor itself, in contrast to GluK1, traffics to the neuronal surface and synapse efficiently and the auxiliary subunits Neto1 and Neto2 caused no further enhancement of these two trafficking processes. However, the regulation of GluK2 biophysical properties by Neto proteins is the same as that of GluK1. We further determine that it is the amino-terminal domains (ATDs) of GluK1 and GluK2 that control the strikingly different trafficking properties between these two receptors. Moreover, the ATDs are critical for synaptic expression of heteromeric receptors at mossy fiber-CA3 synapses and also mediate the differential dependence on Neto proteins for surface and synaptic trafficking of GluK1 and GluK2. These results highlight the fundamental differences between the two major KAR subunits and their interplay with Neto auxiliary proteins.
- Published
- 2017
24. In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits
- Author
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Mühleip, Alexander W, Dewar, Caroline E, Schnaufer, Achim, Kühlbrandt, Werner, and Davies, Karen M
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1.1 Normal biological development and functioning ,Underpinning research ,Adenosine Triphosphate ,Amino Acid Sequence ,Animals ,Catalysis ,Catalytic Domain ,Consensus Sequence ,Dimerization ,Euglena gracilis ,Mitochondria ,Models ,Molecular ,Protein Conformation ,Proton-Translocating ATPases ,Protozoan Proteins ,Rotation ,Sequence Alignment ,Sequence Homology ,Amino Acid ,Trypanosoma brucei brucei ,electron cryotomography ,mitochondrial ATP synthase ,rotary catalysis ,subtomogram averaging ,trypanosome - Abstract
We used electron cryotomography and subtomogram averaging to determine the in situ structures of mitochondrial ATP synthase dimers from two organisms belonging to the phylum euglenozoa: Trypanosoma brucei, a lethal human parasite, and Euglena gracilis, a photosynthetic protist. At a resolution of 32.5 Å and 27.5 Å, respectively, the two structures clearly exhibit a noncanonical F1 head, in which the catalytic (αβ)3 assembly forms a triangular pyramid rather than the pseudo-sixfold ring arrangement typical of all other ATP synthases investigated so far. Fitting of known X-ray structures reveals that this unusual geometry results from a phylum-specific cleavage of the α subunit, in which the C-terminal αC fragments are displaced by ∼20 Å and rotated by ∼30° from their expected positions. In this location, the αC fragment is unable to form the conserved catalytic interface that was thought to be essential for ATP synthesis, and cannot convert γ-subunit rotation into the conformational changes implicit in rotary catalysis. The new arrangement of catalytic subunits suggests that the mechanism of ATP generation by rotary ATPases is less strictly conserved than has been generally assumed. The ATP synthases of these organisms present a unique model system for discerning the individual contributions of the α and β subunits to the fundamental process of ATP synthesis.
- Published
- 2017
25. Studies on the Detection, Expression, Glycosylation, Dimerization, and Ligand Binding Properties of Mouse Siglec-E*
- Author
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Siddiqui, Shoib, Schwarz, Flavio, Springer, Stevan, Khedri, Zahra, Yu, Hai, Deng, Lingquan, Verhagen, Andrea, Naito-Matsui, Yuko, Jiang, Weiping, Kim, Daniel, Zhou, Jie, Ding, Beibei, Chen, Xi, Varki, Nissi, and Varki, Ajit
- Subjects
Biochemistry and Cell Biology ,Biological Sciences ,1.1 Normal biological development and functioning ,Underpinning research ,Inflammatory and immune system ,Amino Acid Substitution ,Animals ,Antibodies ,Antigens ,CD ,Antigens ,Differentiation ,B-Lymphocyte ,Dendritic Cells ,Gene Expression Regulation ,Glycosylation ,Humans ,Macrophages ,Mice ,Mice ,Knockout ,Monocytes ,Mutagenesis ,Mutation ,Missense ,Neutrophils ,Protein Multimerization ,Protein Tyrosine Phosphatase ,Non-Receptor Type 6 ,Rats ,Rats ,Inbred Lew ,Sialic Acid Binding Immunoglobulin-like Lectins ,Siglec-9 ,Siglec-E ,cell signaling ,dimerization ,flow cytometry ,monoclonal antibody ,sialic acid ,Chemical Sciences ,Medical and Health Sciences ,Biochemistry & Molecular Biology ,Biological sciences ,Biomedical and clinical sciences ,Chemical sciences - Abstract
CD33-related Siglecs are a family of proteins widely expressed on innate immune cells. Binding of sialylated glycans or other ligands triggers signals that inhibit or activate inflammation. Immunomodulation by Siglecs has been extensively studied, but relationships between structure and functions are poorly explored. Here we present new data relating to the structure and function of Siglec-E, the major CD33-related Siglec expressed on mouse neutrophils, monocytes, macrophages, and dendritic cells. We generated nine new rat monoclonal antibodies specific to mouse Siglec-E, with no cross-reactivity to Siglec-F. Although all antibodies detected Siglec-E on transfected human HEK-293T cells, only two reacted with mouse bone marrow neutrophils by flow cytometry and on spleen sections by immunohistochemistry. Moreover, whereas all antibodies recognized Siglec-E-Fc on immunoblots, binding was dependent on intact disulfide bonds and N-glycans, and only two antibodies recognized native Siglec-E within spleen lysates. Thus, we further investigated the impact of Siglec-E homodimerization. Homology-based structural modeling predicted a cysteine residue (Cys-298) in position to form a disulfide bridge between two Siglec-E polypeptides. Mutagenesis of Cys-298 confirmed its role in dimerization. In keeping with the high level of 9-O-acetylation found in mice, sialoglycan array studies indicate that this modification has complex effects on recognition by Siglec-E, in relationship to the underlying structures. However, we found no differences in phosphorylation or SHP-1 recruitment between dimeric and monomeric Siglec-E expressed on HEK293A cells. Phylogenomic analyses predicted that only some human and mouse Siglecs form disulfide-linked dimers. Notably, Siglec-9, the functionally equivalent human paralog of Siglec-E, occurs as a monomer.
- Published
- 2017
26. Challenges and Opportunities in Protease-Activated Receptor Drug Development.
- Author
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Hamilton, Justin R and Trejo, JoAnn
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Animals ,Humans ,Oligopeptides ,Receptors ,Proteinase-Activated ,Signal Transduction ,Drug Discovery ,biased signaling ,dimerization ,endothelial ,platelet ,thrombin ,Pharmacology & Pharmacy ,Medical and Health Sciences ,Biological Sciences - Abstract
Protease-activated receptors (PARs) are a unique class of G protein-coupled receptors (GPCRs) that transduce cellular responses to extracellular proteases. PARs have important functions in the vasculature, inflammation, and cancer and are important drug targets. A unique feature of PARs is their irreversible proteolytic mechanism of activation that results in the generation of a tethered ligand that cannot diffuse away. Despite the fact that GPCRs have proved to be the most successful class of druggable targets, the development of agents that target PARs specifically has been challenging. As a consequence, researchers have taken a remarkable diversity of approaches to develop pharmacological entities that modulate PAR function. Here, we present an overview of the diversity of therapeutic agents that have been developed against PARs. We further discuss PAR biased signaling and the influence of receptor compartmentalization, posttranslational modifications, and dimerization, which are important considerations for drug development.
- Published
- 2017
27. Dynamic conformational changes in the rhesus TRIM5α dimer dictate the potency of HIV-1 restriction
- Author
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Lamichhane, Rajan, Mukherjee, Santanu, Smolin, Nikolai, Pauszek, Raymond F, Bradley, Margret, Sastri, Jaya, Robia, Seth L, Millar, David, and Campbell, Edward M
- Subjects
Medical Microbiology ,Biomedical and Clinical Sciences ,HIV/AIDS ,Amino Acid Sequence ,Animals ,Carrier Proteins ,Cell Line ,Dimerization ,Disease Models ,Animal ,HIV Infections ,HIV-1 ,Humans ,Macaca mulatta ,Mutation ,Protein Conformation ,Single molecule FRET ,TRIM5alpha ,Molecular dynamics simulation ,smFRET ,Restriction factor ,Coiled coil ,Dimer ,Tripartite Motif ,Biological Sciences ,Agricultural and Veterinary Sciences ,Medical and Health Sciences ,Virology ,Agricultural ,veterinary and food sciences ,Biological sciences ,Biomedical and clinical sciences - Abstract
The TRIM5α protein from rhesus macaques (rhTRIM5α) mediates a potent inhibition of HIV-1 infection via a mechanism that involves the abortive disassembly of the viral core. We have demonstrated that alpha-helical elements within the Linker 2 (L2) region, which lies between the SPRY domain and the Coiled-Coil domain, influence the potency of restriction. Here, we utilize single-molecule FRET analysis to reveal that the L2 region of the TRIM5α dimer undergoes dynamic conformational changes, which results in the displacement of L2 regions by 25 angstroms relative to each other. Analysis of restriction enhancing or abrogating mutations in the L2 region reveal that restriction defective mutants are unable to undergo dynamic conformational changes and do not assume compact, alpha-helical conformations in the L2 region. These data suggest a model in which conformational changes in the L2 region mediate displacement of CA bound SPRY domains to induce the destabilization of assembled capsid during restriction.
- Published
- 2017
28. The synthetic diazonamide DZ-2384 has distinct effects on microtubule curvature and dynamics without neurotoxicity
- Author
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Wieczorek, Michal, Tcherkezian, Joseph, Bernier, Cynthia, Prota, Andrea E, Chaaban, Sami, Rolland, Yannève, Godbout, Claude, Hancock, Mark A, Arezzo, Joseph C, Ocal, Ozhan, Rocha, Cecilia, Olieric, Natacha, Hall, Anita, Ding, Hui, Bramoullé, Alexandre, Annis, Matthew G, Zogopoulos, George, Harran, Patrick G, Wilkie, Thomas M, Brekken, Rolf A, Siegel, Peter M, Steinmetz, Michel O, Shore, Gordon C, Brouhard, Gary J, and Roulston, Anne
- Subjects
Neurosciences ,Cancer ,Neurodegenerative ,5.1 Pharmaceuticals ,Development of treatments and therapeutic interventions ,Animals ,Antineoplastic Agents ,Cell Line ,Tumor ,Crystallography ,X-Ray ,Dimerization ,Genomics ,Humans ,Lactams ,Macrocyclic ,Mice ,Microscopy ,Electron ,Microtubules ,Mitosis ,Neoplasm Transplantation ,Neurons ,Oxazoles ,Tubulin ,Vinblastine ,Vinca Alkaloids ,Vinorelbine ,Biological Sciences ,Medical and Health Sciences - Abstract
Microtubule-targeting agents (MTAs) are widely used anticancer agents, but toxicities such as neuropathy limit their clinical use. MTAs bind to and alter the stability of microtubules, causing cell death in mitosis. We describe DZ-2384, a preclinical compound that exhibits potent antitumor activity in models of multiple cancer types. It has an unusually high safety margin and lacks neurotoxicity in rats at effective plasma concentrations. DZ-2384 binds the vinca domain of tubulin in a distinct way, imparting structurally and functionally different effects on microtubule dynamics compared to other vinca-binding compounds. X-ray crystallography and electron microscopy studies demonstrate that DZ-2384 causes straightening of curved protofilaments, an effect proposed to favor polymerization of tubulin. Both DZ-2384 and the vinca alkaloid vinorelbine inhibit microtubule growth rate; however, DZ-2384 increases the rescue frequency and preserves the microtubule network in nonmitotic cells and in primary neurons. This differential modulation of tubulin results in a potent MTA therapeutic with enhanced safety.
- Published
- 2016
29. Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart DISTINCT FUNCTIONAL PROPERTIES AND ISOFORM-SELECTIVE INHIBITORS*
- Author
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Habeck, Michael, Tokhtaeva, Elmira, Nadav, Yotam, Ben Zeev, Efrat, Ferris, Sean P, Kaufman, Randal J, Bab-Dinitz, Elizabeta, Kaplan, Jack H, Dada, Laura A, Farfel, Zvi, Tal, Daniel M, Katz, Adriana, Sachs, George, Vagin, Olga, and Karlish, Steven JD
- Subjects
Medical Physiology ,Biomedical and Clinical Sciences ,Heart Disease ,Cardiovascular ,Adenosine Triphosphatases ,Animals ,Cation Transport Proteins ,Cell Adhesion Molecules ,Neuronal ,Dimerization ,Enzyme Inhibitors ,Humans ,Isoenzymes ,Kinetics ,Mice ,Myocardium ,Potassium ,Sodium ,Sodium-Potassium-Exchanging ATPase ,Na+/K+-ATPase ,alpha2beta2 ,cardiomyocyte ,excitation-contraction coupling ,isoform assembly ,isoform-selective inhibitors ,kinetics ,medicinal chemistry ,molecular modeling ,Chemical Sciences ,Biological Sciences ,Medical and Health Sciences ,Biochemistry & Molecular Biology ,Biological sciences ,Biomedical and clinical sciences ,Chemical sciences - Abstract
The Na,K-ATPase α2 subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca2+, whereas α1 has a more conventional role of maintaining ion homeostasis. The β subunit differentially regulates maturation, trafficking, and activity of α-β heterodimers. It is not known whether the distinct role of α2 in the heart is related to selective assembly with a particular one of the three β isoforms. We show here by immunofluorescence and co-immunoprecipitation that α2 is preferentially expressed with β2 in T-tubules of cardiac myocytes, forming α2β2 heterodimers. We have expressed human α1β1, α2β1, α2β2, and α2β3 in Pichia pastoris, purified the complexes, and compared their functional properties. α2β2 and α2β3 differ significantly from both α2β1 and α1β1 in having a higher K0.5K+ and lower K0.5Na+ for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K+ and shift of the E1P-E2P conformational equilibrium toward E1P. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of α2β2 and α2β3 over α1β1 (range 22-33-fold). Molecular modeling suggests a possible basis for isoform selectivity. The preferential assembly, specific T-tubular localization, and low K+ affinity of α2β2 could allow an acute response to raised ambient K+ concentrations in physiological conditions and explain the importance of α2β2 for cardiac muscle contractility. The high sensitivity of α2β2 to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of α2β2-selective digoxin derivatives for reducing cardiotoxicity.
- Published
- 2016
30. GluA1 signal peptide determines the spatial assembly of heteromeric AMPA receptors
- Author
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He, Xue-Yan, Li, Yan-Jun, Kalyanaraman, Chakrapani, Qiu, Li-Li, Chen, Chen, Xiao, Qi, Liu, Wen-Xue, Zhang, Wei, Yang, Jian-Jun, Chen, Guiquan, Jacobson, Matthew P, and Shi, Yun Stone
- Subjects
Neurosciences ,1.1 Normal biological development and functioning ,Underpinning research ,Animals ,Brain ,Dimerization ,Humans ,Protein Conformation ,Protein Sorting Signals ,Rats ,Receptors ,AMPA ,Synaptic Transmission ,AMPA receptors ,signal peptide ,spatial assembly ,stoichiometry - Abstract
AMPA-type glutamate receptors (AMPARs) mediate fast excitatory neurotransmission and predominantly assemble as heterotetramers in the brain. Recently, the crystal structures of homotetrameric GluA2 demonstrated that AMPARs are assembled with two pairs of conformationally distinct subunits, in a dimer of dimers formation. However, the structure of heteromeric AMPARs remains unclear. Guided by the GluA2 structure, we performed cysteine mutant cross-linking experiments in full-length GluA1/A2, aiming to draw the heteromeric AMPAR architecture. We found that the amino-terminal domains determine the first level of heterodimer formation. When the dimers further assemble into tetramers, GluA1 and GluA2 subunits have preferred positions, possessing a 1-2-1-2 spatial assembly. By swapping the critical sequences, we surprisingly found that the spatial assembly pattern is controlled by the excisable signal peptides. Replacements with an unrelated GluK2 signal peptide demonstrated that GluA1 signal peptide plays a critical role in determining the spatial priority. Our study thus uncovers the spatial assembly of an important type of glutamate receptors in the brain and reveals a novel function of signal peptides.
- Published
- 2016
31. Oligomerization of the microtubule‐associated protein tau is mediated by its N‐terminal sequences: implications for normal and pathological tau action
- Author
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Feinstein, H Eric, Benbow, Sarah J, LaPointe, Nichole E, Patel, Nirav, Ramachandran, Srinivasan, Do, Thanh D, Gaylord, Michelle R, Huskey, Noelle E, Dressler, Nicolette, Korff, Megan, Quon, Brady, Cantrell, Kristi Lazar, Bowers, Michael T, Lal, Ratnesh, and Feinstein, Stuart C
- Subjects
Biochemistry and Cell Biology ,Biological Sciences ,Dementia ,Brain Disorders ,Acquired Cognitive Impairment ,Alzheimer's Disease ,Aging ,Alzheimer's Disease including Alzheimer's Disease Related Dementias (AD/ADRD) ,Neurosciences ,Neurodegenerative ,1.1 Normal biological development and functioning ,Neurological ,Amino Acid Sequence ,Animals ,Dimerization ,Humans ,Mass Spectrometry ,Microscopy ,Atomic Force ,Microtubules ,Models ,Biological ,Peptides ,Protein Binding ,tau Proteins ,Alzheimer's ,atomic force microscopy ,Ferguson analysis ,ion mobility-mass spectrometry ,microtubule dynamics ,oligomerization ,Neurology & Neurosurgery ,Biochemistry and cell biology - Abstract
Despite extensive structure-function analyses, the molecular mechanisms of normal and pathological tau action remain poorly understood. How does the C-terminal microtubule-binding region regulate microtubule dynamics and bundling? In what biophysical form does tau transfer trans-synaptically from one neuron to another, promoting neurodegeneration and dementia? Previous biochemical/biophysical work led to the hypothesis that tau can dimerize via electrostatic interactions between two N-terminal 'projection domains' aligned in an anti-parallel fashion, generating a multivalent complex capable of interacting with multiple tubulin subunits. We sought to test this dimerization model directly. Native gel analyses of full-length tau and deletion constructs demonstrate that the N-terminal region leads to multiple bands, consistent with oligomerization. Ferguson analyses of native gels indicate that an N-terminal fragment (tau(45-230) ) assembles into heptamers/octamers. Ferguson analyses of denaturing gels demonstrates that tau(45-230) can dimerize even in sodium dodecyl sulfate. Atomic force microscopy reveals multiple levels of oligomerization by both full-length tau and tau(45-230) . Finally, ion mobility-mass spectrometric analyses of tau(106-144) , a small peptide containing the core of the hypothesized dimerization region, also demonstrate oligomerization. Thus, multiple independent strategies demonstrate that the N-terminal region of tau can mediate higher order oligomerization, which may have important implications for both normal and pathological tau action. The microtubule-associated protein tau is essential for neuronal development and maintenance, but is also central to Alzheimer's and related dementias. Unfortunately, the molecular mechanisms underlying normal and pathological tau action remain poorly understood. Here, we demonstrate that tau can homo-oligomerize, providing novel mechanistic models for normal tau action (promoting microtubule growth and bundling, suppressing microtubule shortening) and pathological tau action (poisoning of oligomeric complexes).
- Published
- 2016
32. Paired octamer rings of retinoschisin suggest a junctional model for cell–cell adhesion in the retina
- Author
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Tolun, Gökhan, Vijayasarathy, Camasamudram, Huang, Rick, Zeng, Yong, Li, Yan, Steven, Alasdair C, Sieving, Paul A, and Heymann, J Bernard
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Eye Disease and Disorders of Vision ,Neurosciences ,Underpinning research ,1.1 Normal biological development and functioning ,Eye ,Amino Acid Sequence ,Animals ,Cell Adhesion ,Cell Adhesion Molecules ,Computer Simulation ,Dimerization ,Eye Proteins ,Intercellular Junctions ,Mice ,Models ,Biological ,Models ,Molecular ,Multiprotein Complexes ,Protein Conformation ,Retina ,retinoschisin ,X-linked retinoschisis ,discoidin domain ,cryo-electron microscopy ,single particle analysis - Abstract
Retinoschisin (RS1) is involved in cell-cell junctions in the retina, but is unique among known cell-adhesion proteins in that it is a soluble secreted protein. Loss-of-function mutations in RS1 lead to early vision impairment in young males, called X-linked retinoschisis. The disease is characterized by separation of inner retinal layers and disruption of synaptic signaling. Using cryo-electron microscopy, we report the structure at 4.1 Å, revealing double octamer rings not observed before. Each subunit is composed of a discoidin domain and a small N-terminal (RS1) domain. The RS1 domains occupy the centers of the rings, but are not required for ring formation and are less clearly defined, suggesting mobility. We determined the structure of the discoidin rings, consistent with known intramolecular and intermolecular disulfides. The interfaces internal to and between rings feature residues implicated in X-linked retinoschisis, indicating the importance of correct assembly. Based on this structure, we propose that RS1 couples neighboring membranes together through octamer-octamer contacts, perhaps modulated by interactions with other membrane components.
- Published
- 2016
33. Signaling via the NFκB system
- Author
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Mitchell, Simon, Vargas, Jesse, and Hoffmann, Alexander
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Biochemistry and Cell Biology ,Biological Sciences ,Animals ,Dimerization ,Humans ,I-kappa B Kinase ,Inflammation ,Models ,Biological ,NF-kappa B ,Neoplasms ,Oxidative Stress ,Protein Processing ,Post-Translational ,Signal Transduction ,Clinical Sciences ,Other Medical and Health Sciences ,Biochemistry and cell biology - Abstract
The nuclear factor kappa B (NFκB) family of transcription factors is a key regulator of immune development, immune responses, inflammation, and cancer. The NFκB signaling system (defined by the interactions between NFκB dimers, IκB regulators, and IKK complexes) is responsive to a number of stimuli, and upon ligand-receptor engagement, distinct cellular outcomes, appropriate to the specific signal received, are set into motion. After almost three decades of study, many signaling mechanisms are well understood, rendering them amenable to mathematical modeling, which can reveal deeper insights about the regulatory design principles. While other reviews have focused on upstream, receptor proximal signaling (Hayden MS, Ghosh S. Signaling to NF-κB. Genes Dev 2004, 18:2195-2224; Verstrepen L, Bekaert T, Chau TL, Tavernier J, Chariot A, Beyaert R. TLR-4, IL-1R and TNF-R signaling to NF-κB: variations on a common theme. Cell Mol Life Sci 2008, 65:2964-2978), and advances through computational modeling (Basak S, Behar M, Hoffmann A. Lessons from mathematically modeling the NF-κB pathway. Immunol Rev 2012, 246:221-238; Williams R, Timmis J, Qwarnstrom E. Computational models of the NF-KB signalling pathway. Computation 2014, 2:131), in this review we aim to summarize the current understanding of the NFκB signaling system itself, the molecular mechanisms, and systems properties that are key to its diverse biological functions, and we discuss remaining questions in the field. WIREs Syst Biol Med 2016, 8:227-241. doi: 10.1002/wsbm.1331 For further resources related to this article, please visit the WIREs website.
- Published
- 2016
34. Biochemistry and Biology of GDF11 and Myostatin
- Author
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Walker, Ryan G, Poggioli, Tommaso, Katsimpardi, Lida, Buchanan, Sean M, Oh, Juhyun, Wattrus, Sam, Heidecker, Bettina, Fong, Yick W, Rubin, Lee L, Ganz, Peter, Thompson, Thomas B, Wagers, Amy J, and Lee, Richard T
- Subjects
Medical Physiology ,Biomedical and Clinical Sciences ,Heart Disease ,Cardiovascular ,Aging ,1.1 Normal biological development and functioning ,Underpinning research ,Musculoskeletal ,Adult ,Amino Acid Sequence ,Animals ,Bone Morphogenetic Proteins ,Brain ,Dimerization ,Female ,Follistatin ,Follistatin-Related Proteins ,Growth Differentiation Factors ,Heart ,Heart Diseases ,Humans ,Male ,Mice ,Models ,Molecular ,Molecular Sequence Data ,Muscles ,Myocardium ,Myostatin ,Organ Specificity ,Protein Conformation ,Protein Structure ,Tertiary ,Rats ,Sequence Alignment ,Sequence Homology ,Amino Acid ,Signal Transduction ,Structure-Activity Relationship ,muscle ,heart disease ,myocardium ,ligands ,Cardiorespiratory Medicine and Haematology ,Clinical Sciences ,Cardiovascular System & Hematology ,Cardiovascular medicine and haematology ,Clinical sciences - Abstract
Growth differentiation factor 11 (GDF11) and myostatin (or GDF8) are closely related members of the transforming growth factor β superfamily and are often perceived to serve similar or overlapping roles. Yet, despite commonalities in protein sequence, receptor utilization and signaling, accumulating evidence suggests that these 2 ligands can have distinct functions in many situations. GDF11 is essential for mammalian development and has been suggested to regulate aging of multiple tissues, whereas myostatin is a well-described negative regulator of postnatal skeletal and cardiac muscle mass and modulates metabolic processes. In this review, we discuss the biochemical regulation of GDF11 and myostatin and their functions in the heart, skeletal muscle, and brain. We also highlight recent clinical findings with respect to a potential role for GDF11 and/or myostatin in humans with heart disease. Finally, we address key outstanding questions related to GDF11 and myostatin dynamics and signaling during development, growth, and aging.
- Published
- 2016
35. Fibroblast growth factor 2 dimer with superagonist in vitro activity improves granulation tissue formation during wound healing
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Decker, Caitlin G, Wang, Yu, Paluck, Samantha J, Shen, Lu, Loo, Joseph A, Levine, Alex J, Miller, Lloyd S, and Maynard, Heather D
- Subjects
Animals ,Blood Vessels ,Blotting ,Western ,Cell Movement ,Cross-Linking Reagents ,Dermis ,Diabetes Mellitus ,Experimental ,Fibroblast Growth Factor 2 ,Fibroblasts ,Granulation Tissue ,Human Umbilical Vein Endothelial Cells ,Male ,Mice ,Neovascularization ,Physiologic ,Polyethylene Glycols ,Protein Multimerization ,Wound Healing ,Wound healing ,Diabetic wounds ,Growth factor ,PEGylation ,Dimerization ,Angiogenesis - Abstract
Site-specific chemical dimerization of fibroblast growth factor 2 (FGF2) with the optimal linker length resulted in a FGF2 homodimer with improved granulation tissue formation and blood vessel formation at exceptionally low concentrations. Homodimers of FGF2 were synthesized through site-specific linkages to both ends of different molecular weight poly(ethylene glycols) (PEGs). The optimal linker length was determined by screening dimer-induced metabolic activity of human dermal fibroblasts and found to be that closest to the inter-cysteine distance, 70 Å, corresponding to 2 kDa PEG. A straightforward analysis of the kinetics of second ligand binding as a function of tether length showed that, as the polymerization index (the number of monomer repeat units in the polymer, N) of the tether decreases, the mean time for second ligand capture decreases as ∼N(3/2), leading to an enhancement of the number of doubly bound ligands in steady-state for a given (tethered) ligand concentration. FGF2-PEG2k-FGF2 induced greater fibroblast metabolic activity than FGF2 alone, all other dimers, and all monoconjugates, at each concentration tested, with the greatest difference observed at low (0.1 ng/mL) concentration. FGF2-PEG2k-FGF2 further exhibited superior activity compared to FGF2 for both metabolic activity and migration in human umbilical vein endothelial cells, as well as improved angiogenesis in a coculture model in vitro. Efficacy in an in vivo wound healing model was assessed in diabetic mice. FGF2-PEG2k-FGF2 increased granulation tissue and blood vessel density in the wound bed compared to FGF2. The results suggest that this rationally designed construct may be useful for improving the fibroblast matrix formation and angiogenesis in chronic wound healing.
- Published
- 2016
36. Disruption of Rhodopsin Dimerization with Synthetic Peptides Targeting an Interaction Interface.
- Author
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Jastrzebska, Beata, Chen, Yuanyuan, Orban, Tivadar, Jin, Hui, Hofmann, Lukas, and Palczewski, Krzysztof
- Subjects
G protein ,G protein-coupled receptor (GPCR) ,oligomerization ,receptor ,receptor structure-function ,Amino Acid Sequence ,Animals ,Dimerization ,GTP-Binding Proteins ,HEK293 Cells ,Humans ,Mice ,Molecular Sequence Data ,Peptides ,Rhodopsin - Abstract
Although homo- and heterodimerizations of G protein-coupled receptors (GPCRs) are well documented, GPCR monomers are able to assemble in different ways, thus causing variations in the interactive interface between receptor monomers among different GPCRs. Moreover, the functional consequences of this phenomenon, which remain to be clarified, could be specific for different GPCRs. Synthetic peptides derived from transmembrane (TM) domains can interact with a full-length GPCR, blocking dimer formation and affecting its function. Here we used peptides corresponding to TM helices of bovine rhodopsin (Rho) to investigate the Rho dimer interface and functional consequences of its disruption. Incubation of Rho with TM1, TM2, TM4, and TM5 peptides in rod outer segment (ROS) membranes shifted the resulting detergent-solubilized protein migration through a gel filtration column toward smaller molecular masses with a reduced propensity for dimer formation in a cross-linking reaction. Binding of these TM peptides to Rho was characterized by both mass spectrometry and a label-free assay from which dissociation constants were calculated. A BRET (bioluminescence resonance energy transfer) assay revealed that the physical interaction between Rho molecules expressed in membranes of living cells was blocked by the same four TM peptides identified in our in vitro experiments. Although disruption of the Rho dimer/oligomer had no effect on the rates of G protein activation, binding of Gt to the activated receptor stabilized the dimer. However, TM peptide-induced disruption of dimer/oligomer decreased receptor stability, suggesting that Rho supramolecular organization could be essential for ROS stabilization and receptor trafficking.
- Published
- 2015
37. Mn(II) Binding and Subsequent Oxidation by the Multicopper Oxidase MnxG Investigated by Electron Paramagnetic Resonance Spectroscopy
- Author
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Tao, Lizhi, Stich, Troy A, Butterfield, Cristina N, Romano, Christine A, Spiro, Thomas G, Tebo, Bradley M, Casey, William H, and Britt, R David
- Subjects
Inorganic Chemistry ,Chemical Sciences ,Animals ,Bacillus ,Cattle ,Dimerization ,Electron Spin Resonance Spectroscopy ,Kinetics ,Manganese ,Oxidation-Reduction ,Oxides ,Oxidoreductases ,Protein Binding ,General Chemistry ,Chemical sciences ,Engineering - Abstract
The dynamics of manganese solid formation (as MnOx) by the multicopper oxidase (MCO)-containing Mnx protein complex were examined by electron paramagnetic resonance (EPR) spectroscopy. Continuous-wave (CW) EPR spectra of samples of Mnx, prepared in atmosphere and then reacted with Mn(II) for times ranging from 7 to 600 s, indicate rapid oxidation of the substrate manganese (with two-phase pseudo-first-order kinetics modeled using rate coefficients of: k(1obs) = 0.205 ± 0.001 s(-1) and k(2obs) = 0.019 ± 0.001 s(-1)). This process occurs on approximately the same time scale as in vitro solid MnOx formation when there is a large excess of Mn(II). We also found CW and pulse EPR spectroscopic evidence for at least three classes of Mn(II)-containing species in the reaction mixtures: (i) aqueous Mn(II), (ii) a specifically bound mononuclear Mn(II) ion coordinated to the Mnx complex by one nitrogenous ligand, and (iii) a weakly exchange-coupled dimeric Mn(II) species. These findings provide new insights into the molecular mechanism of manganese mineralization.
- Published
- 2015
38. Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway
- Author
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Nan, Xiaolin, Tamgüney, Tanja M, Collisson, Eric A, Lin, Li-Jung, Pitt, Cameron, Galeas, Jacqueline, Lewis, Sophia, Gray, Joe W, McCormick, Frank, and Chu, Steven
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Biochemistry and Cell Biology ,Biological Sciences ,Cancer ,2.1 Biological and endogenous factors ,Animals ,Cell Line ,Cricetinae ,Dimerization ,Enzyme Activation ,Guanosine Triphosphate ,MAP Kinase Signaling System ,Mitogen-Activated Protein Kinases ,ras Proteins ,Ras dimer ,MAPK signaling ,cancer ,single molecule imaging ,superresolution microscopy - Abstract
Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRas(G12D), a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRas(G12D) is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.
- Published
- 2015
39. A Structural Perspective on the Regulation of the Epidermal Growth Factor Receptor
- Author
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Kovacs, Erika, Zorn, Julie Anne, Huang, Yongjian, Barros, Tiago, and Kuriyan, John
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Cancer ,1.1 Normal biological development and functioning ,Underpinning research ,Animals ,Dimerization ,Epidermal Growth Factor ,ErbB Receptors ,Humans ,Protein Binding ,Protein Structure ,Tertiary ,receptor tyrosine kinase ,ligand-induced dimerization ,asymmetric dimer ,oncogenic mutations ,transmembrane coupling ,Biological Sciences ,Medical and Health Sciences ,Biochemistry & Molecular Biology - Abstract
The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays a critical role in the pathogenesis of many cancers. The structure of intact forms of this receptor has yet to be determined, but intense investigations of fragments of the receptor have provided a detailed view of its activation mechanism, which we review here. Ligand binding converts the receptor to a dimeric form, in which contacts are restricted to the receptor itself, allowing heterodimerization of the four EGFR family members without direct ligand involvement. Activation of the receptor depends on the formation of an asymmetric dimer of kinase domains, in which one kinase domain allosterically activates the other. Coupling between the extracellular and intracellular domains may involve a switch between alternative crossings of the transmembrane helices, which form dimeric structures. We also discuss how receptor regulation is compromised by oncogenic mutations and the structural basis for negative cooperativity in ligand binding.
- Published
- 2015
40. Regulation of Bone Morphogenetic Protein 9 (BMP9) by Redox-dependent Proteolysis*
- Author
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Wei, Zhenquan, Salmon, Richard M, Upton, Paul D, Morrell, Nicholas W, and Li, Wei
- Subjects
Biochemistry and Cell Biology ,Biomedical and Clinical Sciences ,Biological Sciences ,Underpinning research ,1.1 Normal biological development and functioning ,Animals ,Cell Line ,Crystallization ,Crystallography ,X-Ray ,DNA ,Complementary ,Dimerization ,Disulfides ,Endothelial Cells ,Gene Expression Regulation ,Growth Differentiation Factor 2 ,Growth Differentiation Factors ,Humans ,Ligands ,Mice ,Oxidation-Reduction ,Phenotype ,Proteolysis ,Recombinant Proteins ,Signal Transduction ,Bone Morphogenetic Protein ,Cell Signaling ,Crystal Structure ,Disulfide ,Endothelial Cell ,Redox Regulation ,Chemical Sciences ,Medical and Health Sciences ,Biochemistry & Molecular Biology ,Biological sciences ,Biomedical and clinical sciences ,Chemical sciences - Abstract
BMP9, a member of the TGFβ superfamily, is a homodimer that forms a signaling complex with two type I and two type II receptors. Signaling through high-affinity activin receptor-like kinase 1 (ALK1) in endothelial cells, circulating BMP9 acts as a vascular quiescence factor, maintaining endothelial homeostasis. BMP9 is also the most potent BMP for inducing osteogenic signaling in mesenchymal stem cells in vitro and promoting bone formation in vivo. This activity requires ALK1, the lower affinity type I receptor ALK2, and higher concentrations of BMP9. In adults, BMP9 is constitutively expressed in hepatocytes and secreted into the circulation. Optimum concentrations of BMP9 are essential to maintain the highly specific endothelial-protective function. Factors regulating BMP9 stability and activity remain unknown. Here, we showed by chromatography and a 1.9 Å crystal structure that stable BMP9 dimers could form either with (D-form) or without (M-form) an intermolecular disulfide bond. Although both forms of BMP9 were capable of binding to the prodomain and ALK1, the M-form demonstrated less sustained induction of Smad1/5/8 phosphorylation. The two forms could be converted into each other by changing the redox potential, and this redox switch caused a major alteration in BMP9 stability. The M-form displayed greater susceptibility to redox-dependent cleavage by proteases present in serum. This study provides a mechanism for the regulation of circulating BMP9 concentrations and may provide new rationales for approaches to modify BMP9 levels for therapeutic purposes.
- Published
- 2014
41. Four Levels of Hierarchical Organization, Including Noncovalent Chainmail, Brace the Mature Tumor Herpesvirus Capsid against Pressurization
- Author
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Zhou, Z Hong, Hui, Wong Hoi, Shah, Sanket, Jih, Jonathan, O’Connor, Christine M, Sherman, Michael B, Kedes, Dean H, and Schein, Stan
- Subjects
Biochemistry and Cell Biology ,Biological Sciences ,Cancer ,Amino Acid Sequence ,Animals ,Capsid ,Capsid Proteins ,Cryoelectron Microscopy ,Dimerization ,Models ,Molecular ,Molecular Sequence Data ,Protein Structure ,Tertiary ,Rhadinovirus ,Chemical Sciences ,Information and Computing Sciences ,Biophysics ,Biological sciences ,Chemical sciences - Abstract
Like many double-stranded DNA viruses, tumor gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus withstand high internal pressure. Bacteriophage HK97 uses covalent chainmail for this purpose, but how this is achieved noncovalently in the much larger gammaherpesvirus capsid is unknown. Our cryoelectron microscopy structure of a gammaherpesvirus capsid reveals a hierarchy of four levels of organization: (1) Within a hexon capsomer, each monomer of the major capsid protein (MCP), 1,378 amino acids and six domains, interacts with its neighboring MCPs at four sites. (2) Neighboring capsomers are linked in pairs by MCP dimerization domains and in groups of three by heterotrimeric triplex proteins. (3) Small (∼280 amino acids) HK97-like domains in MCP monomers alternate with triplex heterotrimers to form a belt that encircles each capsomer. (4) One hundred sixty-two belts concatenate to form noncovalent chainmail. The triplex heterotrimer orchestrates all four levels and likely drives maturation to an angular capsid that can withstand pressurization.
- Published
- 2014
42. Molecular architecture of mammalian nitric oxide synthases
- Author
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Campbell, Melody G, Smith, Brian C, Potter, Clinton S, Carragher, Bridget, and Marletta, Michael A
- Subjects
Neurosciences ,Underpinning research ,1.1 Normal biological development and functioning ,Generic health relevance ,Animals ,Calmodulin ,Crystallization ,Dimerization ,Holoenzymes ,Humans ,Imaging ,Three-Dimensional ,Mammals ,Mice ,Microscopy ,Electron ,Transmission ,Nitric Oxide ,Nitric Oxide Synthase Type I ,Nitric Oxide Synthase Type II ,Nitric Oxide Synthase Type III ,Oxidoreductases ,Protein Binding ,Protein Structure ,Secondary ,Protein Structure ,Tertiary ,Rats ,heme ,flavin ,electron microscopy ,conformational heterogeneity - Abstract
NOSs are homodimeric multidomain enzymes responsible for producing NO. In mammals, NO acts as an intercellular messenger in a variety of signaling reactions, as well as a cytotoxin in the innate immune response. Mammals possess three NOS isoforms--inducible, endothelial, and neuronal NOS--that are composed of an N-terminal oxidase domain and a C-terminal reductase domain. Calmodulin (CaM) activates NO synthesis by binding to the helical region connecting these two domains. Although crystal structures of isolated domains have been reported, no structure is available for full-length NOS. We used high-throughput single-particle EM to obtain the structures and higher-order domain organization of all three NOS holoenzymes. The structures of inducible, endothelial, and neuronal NOS with and without CaM bound are similar, consisting of a dimerized oxidase domain flanked by two separated reductase domains. NOS isoforms adopt many conformations enabled by three flexible linkers. These conformations represent snapshots of the continuous electron transfer pathway from the reductase domain to the oxidase domain, which reveal that only a single reductase domain participates in electron transfer at a time, and that CaM activates NOS by constraining rotational motions and by directly binding to the oxidase domain. Direct visualization of these large conformational changes induced during electron transfer provides significant insight into the molecular underpinnings governing NO formation.
- Published
- 2014
43. γδ T cells recognize the insulin B:9-23 peptide antigen when it is dimerized through thiol oxidation.
- Author
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Aydintug, M, Zhang, Li, Wang, Chao, Liang, Dongchun, Wands, J, Michels, Aaron, Hirsch, Brooke, Day, Brian, Zhang, Gongyi, Sun, Deming, Eisenbarth, George, OBrien, Rebecca, and Born, Willi
- Subjects
Autoimmune diabetes ,Autoreactivity ,Gamma delta T cells ,Insulin ,Oxidation ,T Cell Receptor ,Animals ,Antigens ,Diabetes Mellitus ,Type 1 ,Dimerization ,Female ,Insulin ,Mice ,Mice ,Inbred NOD ,Oxidation-Reduction ,Peptide Fragments ,Receptors ,Antigen ,T-Cell ,gamma-delta ,Sulfhydryl Compounds - Abstract
The insulin peptide B:9-23 is a natural antigen in the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D). In addition to αβ T cells and B cells, γδ T cells recognize the peptide and infiltrate the pancreatic islets where the peptide is produced within β cells. The peptide contains a cysteine in position 19 (Cys19), which is required for the γδ but not the αβ T cell response, and a tyrosine in position 16 (Tyr16), which is required for both. A peptide-specific mAb, tested along with the T cells, required neither of the two amino acids to bind the B:9-23 peptide. We found that γδ T cells require Cys19 because they recognize the peptide antigen in an oxidized state, in which the Cys19 thiols of two peptide molecules form a disulfide bond, creating a soluble homo-dimer. In contrast, αβ T cells recognize the peptide antigen as a reduced monomer, in complex with the MHCII molecule I-A(g7). Unlike the unstructured monomeric B:9-23 peptide, the γδ-stimulatory homo-dimer adopts a distinct secondary structure in solution, which differs from the secondary structure of the corresponding portion of the native insulin molecule. Tyr16 is required for this adopted structure of the dimerized insulin peptide as well as for the γδ response to it. This observation is consistent with the notion that γδ T cell recognition depends on the secondary structure of the dimerized insulin B:9-23 antigen.
- Published
- 2014
44. On guanidinium and cellular uptake.
- Author
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Wexselblatt, Ezequiel, Esko, Jeffrey, and Tor, Yitzhak
- Subjects
Animals ,Biological Transport ,Cations ,Cell Culture Techniques ,Cell Membrane ,Crystallography ,X-Ray ,Dimerization ,Guanidine ,HeLa Cells ,Humans ,Hydrogen Bonding ,Molecular Structure - Abstract
Guanidinium-rich scaffolds facilitate cellular translocation and delivery of bioactive cargos through biological barriers. Although impressive uptake has been demonstrated for nonoligomeric and nonpept(o)idic guanidinylated scaffolds in cell cultures and animal models, the fundamental understanding of these processes is lacking. Charge pairing and hydrogen bonding with cell surface counterparts have been proposed, but their exact role remains putative. The impact of the number and spatial relationships of the guanidinium groups on delivery and organelle/organ localization is yet to be established.
- Published
- 2014
45. The upstream conserved regions (UCRs) mediate homo- and hetero-oligomerization of type 4 cyclic nucleotide phosphodiesterases (PDE4s).
- Author
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Xie, Moses, Blackman, Brigitte, Scheitrum, Colleen, Mika, Delphine, Blanchard, Elise, Lei, Tao, Conti, Marco, and Richter, Wito
- Subjects
Biochemistry and Cell Biology ,Biological Sciences ,1.1 Normal biological development and functioning ,Underpinning research ,Amino Acid Sequence ,Animals ,COS Cells ,Chlorocebus aethiops ,Conserved Sequence ,Cyclic AMP-Dependent Protein Kinases ,Cyclic Nucleotide Phosphodiesterases ,Type 4 ,Cytosol ,Dimerization ,Enzyme Activation ,HEK293 Cells ,Humans ,Isoenzymes ,Luminescent Proteins ,Molecular Weight ,Phosphorylation ,Protein Interaction Domains and Motifs ,Protein Multimerization ,Protein Processing ,Post-Translational ,Rats ,Recombinant Fusion Proteins ,Recombinant Proteins ,cAMP ,cyclic nucleotide phosphodiesterase 4 ,dimer ,oligomerization ,phosphodiesterase ,Chemical Sciences ,Medical and Health Sciences ,Biochemistry & Molecular Biology ,Biochemistry and cell biology - Abstract
PDE4s (type 4 cyclic nucleotide phosphodiesterases) are divided into long and short forms by the presence or absence of conserved N-terminal domains termed UCRs (upstream conserved regions). We have shown previously that PDE4D2, a short variant, is a monomer, whereas PDE4D3, a long variant, is a dimer. In the present study, we have determined the apparent molecular masses of various long and short PDE4 variants by size-exclusion chromatography and sucrose density-gradient centrifugation. Our results indicate that dimerization is a conserved property of all long PDE4 forms, whereas short forms are monomers. Dimerization is mediated by the UCR domains. Given their high sequence conservation, the UCR domains mediate not only homo-oligomerization, but also hetero-oligomerization of distinct PDE4 long forms as detected by co-immunoprecipitation assays and FRET microscopy. Endogenous PDE4 hetero-oligomers are, however, low in abundance compared with homo-dimers, revealing the presence of mechanisms that predispose PDE4s towards homo-oligomerization. Oligomerization is a prerequisite for the regulatory properties of the PDE4 long forms, such as their PKA (protein kinase A)-dependent activation, but is not necessary for PDE4 protein-protein interactions. As a result, individual PDE4 protomers may independently mediate protein-protein interactions, providing a mechanism whereby PDE4s contribute to the assembly of macromolecular signalling complexes.
- Published
- 2014
46. Nitric oxide synthase domain interfaces regulate electron transfer and calmodulin activation
- Author
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Smith, Brian C, Underbakke, Eric S, Kulp, Daniel W, Schief, William R, and Marletta, Michael A
- Subjects
1.1 Normal biological development and functioning ,Underpinning research ,Generic health relevance ,Animals ,Calmodulin ,Deuterium Exchange Measurement ,Dimerization ,Electron Transport ,Electrophoresis ,Polyacrylamide Gel ,Flavin Mononucleotide ,Flavin-Adenine Dinucleotide ,Fluorescence ,Heme ,Mass Spectrometry ,Models ,Molecular ,Nitric Oxide Synthase Type II ,Protein Conformation ,Species Specificity ,iNOS ,NO signaling ,flavin ,hemoprotein - Abstract
Nitric oxide (NO) produced by NO synthase (NOS) participates in diverse physiological processes such as vasodilation, neurotransmission, and the innate immune response. Mammalian NOS isoforms are homodimers composed of two domains connected by an intervening calmodulin-binding region. The N-terminal oxidase domain binds heme and tetrahydrobiopterin and the arginine substrate. The C-terminal reductase domain binds FAD and FMN and the cosubstrate NADPH. Although several high-resolution structures of individual NOS domains have been reported, a structure of a NOS holoenzyme has remained elusive. Determination of the higher-order domain architecture of NOS is essential to elucidate the molecular underpinnings of NO formation. In particular, the pathway of electron transfer from FMN to heme, and the mechanism through which calmodulin activates this electron transfer, are largely unknown. In this report, hydrogen-deuterium exchange mass spectrometry was used to map critical NOS interaction surfaces. Direct interactions between the heme domain, the FMN subdomain, and calmodulin were observed. These interaction surfaces were confirmed by kinetic studies of site-specific interface mutants. Integration of the hydrogen-deuterium exchange mass spectrometry results with computational docking resulted in models of the NOS heme and FMN subdomain bound to calmodulin. These models suggest a pathway for electron transfer from FMN to heme and a mechanism for calmodulin activation of this critical step.
- Published
- 2013
47. Allosteric Activation of Functionally Asymmetric RAF Kinase Dimers
- Author
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Hu, Jiancheng, Stites, Edward C, Yu, Haiyang, Germino, Elizabeth A, Meharena, Hiruy S, Stork, Philip JS, Kornev, Alexandr P, Taylor, Susan S, and Shaw, Andrey S
- Subjects
Biochemistry and Cell Biology ,Biological Sciences ,Cancer ,1.1 Normal biological development and functioning ,Underpinning research ,Allosteric Regulation ,Amino Acid Motifs ,Amino Acid Sequence ,Animals ,Cell Line ,Dimerization ,Enzyme Activation ,Humans ,Mice ,Models ,Molecular ,Molecular Sequence Data ,Mutation ,Phosphorylation ,Protein Conformation ,Protein Kinases ,Proto-Oncogene Proteins B-raf ,Proto-Oncogene Proteins c-raf ,Sequence Alignment ,Tryptophan ,raf Kinases ,Medical and Health Sciences ,Developmental Biology ,Biological sciences ,Biomedical and clinical sciences - Abstract
Although RAF kinases are critical for controlling cell growth, their mechanism of activation is incompletely understood. Recently, dimerization was shown to be important for activation. Here we show that the dimer is functionally asymmetric with one kinase functioning as an activator to stimulate activity of the partner, receiver kinase. The activator kinase did not require kinase activity but did require N-terminal phosphorylation that functioned allosterically to induce cis-autophosphorylation of the receiver kinase. Based on modeling of the hydrophobic spine assembly, we also engineered a constitutively active mutant that was independent of Ras, dimerization, and activation-loop phosphorylation. As N-terminal phosphorylation of BRAF is constitutive, BRAF initially functions to activate CRAF. N-terminal phosphorylation of CRAF was dependent on MEK, suggesting a feedback mechanism and explaining a key difference between BRAF and CRAF. Our work illuminates distinct steps in RAF activation that function to assemble the active conformation of the RAF kinase.
- Published
- 2013
48. Challenges and Opportunities in Protease-Activated Receptor Drug Development
- Author
-
Hamilton, Justin R and Trejo, JoAnn
- Subjects
Pharmacology and Pharmaceutical Sciences ,Biomedical and Clinical Sciences ,Cancer ,Animals ,Drug Discovery ,Humans ,Oligopeptides ,Receptors ,Proteinase-Activated ,Signal Transduction ,thrombin ,platelet ,endothelial ,biased signaling ,dimerization ,Biological Sciences ,Medical and Health Sciences ,Pharmacology & Pharmacy ,Pharmacology and pharmaceutical sciences - Abstract
Protease-activated receptors (PARs) are a unique class of G protein-coupled receptors (GPCRs) that transduce cellular responses to extracellular proteases. PARs have important functions in the vasculature, inflammation, and cancer and are important drug targets. A unique feature of PARs is their irreversible proteolytic mechanism of activation that results in the generation of a tethered ligand that cannot diffuse away. Despite the fact that GPCRs have proved to be the most successful class of druggable targets, the development of agents that target PARs specifically has been challenging. As a consequence, researchers have taken a remarkable diversity of approaches to develop pharmacological entities that modulate PAR function. Here, we present an overview of the diversity of therapeutic agents that have been developed against PARs. We further discuss PAR biased signaling and the influence of receptor compartmentalization, posttranslational modifications, and dimerization, which are important considerations for drug development.
- Published
- 2013
49. Structure of the arginine methyltransferase PRMT5-MEP50 reveals a mechanism for substrate specificity.
- Author
-
Ho, Meng-Chiao, Wilczek, Carola, Bonanno, Jeffrey B, Xing, Li, Seznec, Janina, Matsui, Tsutomu, Carter, Lester G, Onikubo, Takashi, Kumar, P Rajesh, Chan, Man K, Brenowitz, Michael, Cheng, R Holland, Reimer, Ulf, Almo, Steven C, and Shechter, David
- Subjects
Animals ,Xenopus laevis ,Xenopus Proteins ,Chromosomal Proteins ,Non-Histone ,Catalytic Domain ,Protein Conformation ,Substrate Specificity ,Dimerization ,Models ,Molecular ,Protein-Arginine N-Methyltransferases ,Chromosomal Proteins ,Non-Histone ,Models ,Molecular ,General Science & Technology - Abstract
The arginine methyltransferase PRMT5-MEP50 is required for embryogenesis and is misregulated in many cancers. PRMT5 targets a wide variety of substrates, including histone proteins involved in specifying an epigenetic code. However, the mechanism by which PRMT5 utilizes MEP50 to discriminate substrates and to specifically methylate target arginines is unclear. To test a model in which MEP50 is critical for substrate recognition and orientation, we determined the crystal structure of Xenopus laevis PRMT5-MEP50 complexed with S-adenosylhomocysteine (SAH). PRMT5-MEP50 forms an unusual tetramer of heterodimers with substantial surface negative charge. MEP50 is required for PRMT5-catalyzed histone H2A and H4 methyltransferase activity and binds substrates independently. The PRMT5 catalytic site is oriented towards the cross-dimer paired MEP50. Histone peptide arrays and solution assays demonstrate that PRMT5-MEP50 activity is inhibited by substrate phosphorylation and enhanced by substrate acetylation. Electron microscopy and reconstruction showed substrate centered on MEP50. These data support a mechanism in which MEP50 binds substrate and stimulates PRMT5 activity modulated by substrate post-translational modifications.
- Published
- 2013
50. SUMOylation Silences Heterodimeric TASK Potassium Channels Containing K2P1 Subunits in Cerebellar Granule Neurons
- Author
-
Plant, Leigh D, Zuniga, Leandro, Araki, Dan, Marks, Jeremy D, and Goldstein, Steve AN
- Subjects
Neurosciences ,Underpinning research ,1.1 Normal biological development and functioning ,Neurological ,Animals ,Blotting ,Western ,Cerebellum ,Dimerization ,Fluorescence Resonance Energy Transfer ,Halothane ,In Situ Hybridization ,Membrane Potentials ,Mice ,Microscopy ,Confocal ,Multiprotein Complexes ,Nerve Tissue Proteins ,Neurons ,Potassium ,Potassium Channels ,Tandem Pore Domain ,Rats ,Sumoylation ,Biochemistry and Cell Biology - Abstract
The standing outward K(+) current (IKso) governs the response of cerebellar granule neurons to natural and medicinal stimuli including volatile anesthetics. We showed that SUMOylation silenced half of IKso at the surface of cerebellar granule neurons because the underlying channels were heterodimeric assemblies of K2P1, a subunit subject to SUMOylation, and the TASK (two-P domain, acid-sensitive K(+)) channel subunits K2P3 or K2P9. The heterodimeric channels comprised the acid-sensitive portion of IKso and mediated its response to halothane. We anticipate that SUMOylation also influences sensation and homeostatic mechanisms in mammals through TASK channels formed with K2P1.
- Published
- 2012
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