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PubMed1999

Aminorex, fenfluramine, and chlorphentermine are serotonin transporter substrates. Implications for primary pulmonary hypertension.

BACKGROUND: Coadministration of phentermine and fenfluramine (phen/fen) effectively treats obesity and possibly addictive disorders. The association of fenfluramine and certain other anorexic agents with serious side effects, such as cardiac valvulopathy and primary pulmonary hypertension (PPH), limits the clinical utility of these drugs. Development of new medications that produce neurochemical effects like phen/fen without causing unwanted side effects would be a significant therapeutic breakthrough. METHODS AND RESULTS: We tested the hypothesis that fenfluramine (and other anorexic agents) might increase the risk of PPH through interactions with serotonin (5-HT) transporters. Because 5-HT transporter proteins in the lung and brain are identical, we examined, in rat brain, the effects of selected drugs on 5-HT efflux in vivo and monoamine transporters in vitro as a generalized index of transporter function. Our data show that drugs known or suspected to increase the risk of PPH (eg, aminorex, fenfluramine, and chlorphentermine) are 5-HT transporter substrates, whereas drugs that have not been shown to increase the risk of PPH are less potent in this regard. CONCLUSIONS: We speculate that medications that are 5-HT transporter substrates get translocated into pulmonary cells where, depending on the degree of drug retention, their intrinsic drug toxicity, and individual susceptibility, PPH could develop as a response to high levels of these drugs or metabolites. Emerging evidence suggests that it is possible to develop transporter substrates devoid of adverse side effects. Such medications could have therapeutic application in the management of obesity, drug dependence, depression, and other disorders.

Aminorex↗

PubMed1999

Structure of the small G protein Rap2 in a non-catalytic complex with GTP.

We report a novel crystal form of the small G protein Rap2A in complex with GTP which has no GTPase activity in the crystal. The asymmetric unit contains two complexes which show that a conserved switch I residue, Tyr 32, contributes an extra hydrogen bond to the gamma-phosphate of GTP as compared to related structures with GTP analogs. Since GTP is not hydrolyzed in the crystal, this interaction is unlikely to contribute to the intrinsic GTPase activity. The comparison of other G protein structures to the Rap2-GTP complex suggests that an equivalent interaction is likely to exist in their GTP form, whether unbound or bound to an effector. This interaction has to be released to allow the GAP-activated GTPase, and presumably the intrinsic GTPase activity as well. We also discuss the definition of the flexible regions and their hinges in the light of this structure and the expanding database of G protein structures. We propose that the switch I and switch II undergo either partial or complete disorder-to-order transitions according to their cellular status, thus defining a complex energy landscape comprising more than two conformational states. We observe in addition that the region connecting the switch I and switch II is flexible in Rap2 and other G proteins. This region may be important for protein-protein interactions and possibly behave as a conformational lever arm, as characterized for Arf. Taken together, these observations suggest that the structural mechanisms of small G proteins are significantly driven by entropy-based free energy changes.

Crystallography, X-Ray↗

PubMed1987

Phase transitions and ion currents in a model ferroelectric channel unit.

The hypothesis of ferroelectric electrodiffusion is examined mathematically. A thermodynamic potential, the elastic Gibbs function, written in polynomial form, provides the dielectric equation of state for the model. The other equations of electrodiffusion theory complete the model. This system reduces to a second-order partial differential equation, which is formally solved by the phase-plane method. This solution, applied to the Na channel, leads to a propagating phase-transition wave accompanied by movement of ionic charge. This may be readily interpreted as a transmembrane wave traveling along a ferroelectric unit within, and transporting ions through, the channel. Comparison of the temperature dependence of axonal conduction velocity with that of the spontaneous polarization of Rochelle salt suggests that the Na channel of squid axon contains a ferroelectric unit having a lower Curie point, but decomposing before reaching its upper Curie point. Comparison with data from reconstitution experiments suggests that the ferroelectric unit is a carbohydrate enclosed in an intrinsic protein structure to form a glycoprotein channel. The value experimentally estimated for the surface charge of the Na channel is within the range of spontaneous polarizations of typical ferroelectric crystals. It is argued that the ferroelectric probably is a single crystal of the order-disorder type, which undergoes a first-order transition between a ferroelectric and a paraelectric state during excitational activity. The hypothesis of ferroelectric channel units is consistent with the existence and directionality of the observed "gating" currents.

Animals↗

PubMed2002

CD40 on salivary gland epithelial cells: high constitutive expression by cultured cells from Sjögren's syndrome patients indicating their intrinsic activation.

CD40 has been identified in an expanding list of haematopoietic and non-haematopoietic cells and has received an increased interest based on its role in a variety of cell-mediated responses and its potential to participate in the pathogenesis of chronic inflammatory disorders. Sjögren's syndrome (SS) is an autoimmune exocrinopathy, which is characterized by chronic lymphocytic infiltration of exocrine glands and aberrant activation of epithelial tissues. We studied the expression of CD40 protein in cultured non-neoplastic salivary gland epithelial cell (SGEC) lines as well as in minor SG biopsies obtained from 17 SS patients and 12 controls. Immunocytochemical and flow cytometric analyses had revealed the occurrence of constitutively expressed CD40 molecules on the surface of long-term cultured SGEC lines, which could be further induced by interferon-gamma (IFN-gamma) and IL-1beta cytokines, but not tumour necrosis factor-alpha (TNF-alpha), IL-4, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF) or IFN-alpha. Triggering of SGEC through CD40 enhanced the surface expression of the adhesion molecule intercellular adhesion molecule-1 (ICAM-1)/CD54, but not MHC class I and class II (HLA-DR) molecules. Spontaneous CD40 expression was significantly higher in SGEC lines derived from SS patients, compared with controls (P < 0.001), which is suggestive of their intrinsically activated status. In SG biopsies, CD40 was constitutively expressed by lymphocytes, ductal epithelial cells and endothelial cells but not by other glandular cell types, such as acinar cells, myoepithelial cells and fibroblasts. In addition, CD40L staining was also detected in 30--50% of the infiltrating lymphocytes in the biopsies of SS patients. Our findings indicate the immunoregulatory potential of SGEC and lend further support to a model of intrinsic activation in salivary epithelia in SS, whereby these cells actively participate in the induction and maintenance of lymphocytic infiltrates of patients.

Autoimmune Diseases↗

PubMed2004

Bioengineering of coagulation factor VIII for improved secretion.

Factor VIII (FVIII) functions as a cofactor within the intrinsic pathway of blood coagulation. Quantitative or qualitative deficiencies of FVIII result in the inherited bleeding disorder hemophilia A. Expression of FVIII (domain structure A1-A2-B-A3-C1-C2) in heterologous mammalian systems is 2 to 3 orders of magnitude less efficient compared with other proteins of similar size compromising recombinant FVIII production and gene therapy strategies. FVIII expression is limited by unstable mRNA, interaction with endoplasmic reticulum (ER) chaperones, and a requirement for facilitated ER to Golgi transport through interaction with the mannose-binding lectin LMAN1. Bioengineering strategies can overcome each of these limitations. B-domain-deleted (BDD)-FVIII yields higher mRNA levels, and targeted point mutations within the A1 domain reduce interaction with the ER chaperone immunoglobulin-binding protein. In order to increase ER to Golgi transport we engineered several asparagine-linked oligosaccharides within a short B-domain spacer within BDD-FVIII. A bioengineered FVIII incorporating all of these elements was secreted 15- to 25-fold more efficiently than full-length FVIII both in vitro and in vivo. FVIII bioengineered for improved secretion will significantly increase potential for success in gene therapy strategies for hemophilia A as well as improve recombinant FVIII production in cell culture manufacturing or transgenic animals.

Animals↗

PubMed2001

[Treatment of paroxysmal nocturnal hemoglobinuria (PNH)].

Paroxysmal nocturnal hemoglobinuria is an acquired clonal disorder of the hematopoietic stem cell in which intravascular hemolysis is due to an intrinsic defect in the membrane of red cells that makes them increasingly susceptible to lysis by complement. The phenotypic hallmark of PNH cells is an absence or marked deficiency of GPI-anchored proteins such as CD 59+, CD 55+ and others which normally protect cells from the action of complement. PHN is closely associated with aplastic anemia. Some degree of bone marrow failure is always present. Management of PNH is complicated by a highly variable clinical picture and course. Some patients have severe anemia aggravated by hemolytic crises and associated thromboses. Bone marrow failure is accompanied with frequent infections and hemorrhagic manifestations due to thrombocytopenia. With the exception of marrow transplantation, no definite therapy is available. In the exceptional circumstance in which the patient has a syngeneic twin, bone marrow transplantation is the most appropriate therapy for severe PNH because of absence of graft-versus-host disease. In general syngeneic transplantation without preconditioning has been unsuccessful because abnormal hematopoiesis returns. Allogeneic bone marrow transplantation has been used, but the transplant-associated morbidity and mortality are high due mainly to the fatal graft-versus-host disease and severe posttransplant marrow failure. Use of an unrelated donor transplant has to be considered as contraindicated. PNH is associated with striking predisposition to intravascular thrombosis which often involves the portal system or the brain. Fatal thromboses account for about 40-50% of all deaths in patients with PNH. The etiology of the thrombophilia in PNH is not fully clarified. Anticoagulation or thrombolytic therapy is required for treatment of venous thrombosis, the latter vena cava. Prophylactic anticoagulation in patients without contraindications such as severe thrombocytopenia seems to be justified. However, whether such therapy may be efficacious in reducing the incidence of thromboses or affect survival is conjectural. PNH patients have varying degree of platelet activation and some authors suggest that antiplatelet therapy might be efficacious in reducing the incidence and severity of venous thrombosis in PNH. Pregnancy is hazardous. Female patients should avoid the use of oral contraceptives. Pregnant patients require combined care of an experienced hematologist and obstetrician specialized in the management of high-risk pregnancies.

Anemia, Aplastic↗

PubMed1999

Genetic evidence of an accessory activity required specifically for cubilin brush-border expression and intrinsic factor-cobalamin absorption.

Cubilin is a high molecular weight multiligand receptor that mediates intestinal absorption of intrinsic factor-cobalamin and selective protein reabsorption in renal tubules. The genetic basis of selective intestinal cobalamin malabsorption with proteinuria was investigated in a canine model closely resembling human Imerslund-Gräsbeck syndrome caused by cubilin mutations. Canine CUBN cDNA was cloned and sequenced, showing high identity with human and rat CUBN cDNAs. An intragenic CUBN marker was identified in the canine family and used to test the hypothesis of genetic linkage of the disease and CUBN loci. Linkage was rejected, indicating that the canine disorder resembling Imerslund-Gräsbeck syndrome is caused by defect of a gene product other than cubilin. These results imply that there may be locus heterogeneity among human kindreds with selective intestinal cobalamin malabsorption and proteinuria and that normal brush-border expression of cubilin requires the activity of an accessory protein.

Amino Acid Sequence↗

PubMed2005

Regulation of monoamine transporters: influence of psychostimulants and therapeutic antidepressants.

Synaptic neurotransmission in the central nervous system (CNS) requires the precise control of the duration and the magnitude of neurotransmitter action at specific molecular targets. At the molecular level, neurotransmitter signaling is dynamically regulated by a diverse set of macromolecules including biosynthetic enzymes, secretory proteins, ion channels, pre- and postsynaptic receptors and transporters. Monoamines, 5-hydroxytryptamine or serotonin (5-HT), norepinephrine (NE), and dopamine (DA) play an important modulatory role in the CNS and are involved in numerous physiological functions and pathological conditions. Presynaptic plasma membrane transporters for 5-HT (SERT), NE (NET), and DA (DAT), respectively, control synaptic actions of these monoamines by rapidly clearing the released amine. Monoamine transporters are the sites of action for widely used antidepressants and are high affinity molecular targets for drugs of abuse including cocaine, amphetamine, and 3,4-methylenedioxymetamphetamine (MDMA) "Ecstasy." Monoamine transporters also serve as molecular gateways for neurotoxins. Emerging evidence indicates that regulation of transporter function and surface expression can be rapidly modulated by "intrinsic" transporter activity itself, and antidepressant and psychostimulant drugs that block monoamine transport have a profound effect on transporter regulation. Therefore, disregulations in the functioning of monoamine transporters may underlie many disorders of transmitter imbalance such as depression, attention deficit hyperactivity disorder, and schizophrenia. This review integrates recent progress in understanding the molecular mechanisms of monoamine transporter regulation, in particular, posttranscriptional regulation by phosphorylation and trafficking linked to cellular protein kinases, protein phosphatases, and transporter interacting proteins. The review also discusses the possible role of psychostimulants and antidepressants in influencing monoamine transport regulation.

Animals↗

PubMed2005

Factor XI deficiency database: an interactive web database of mutations, phenotypes, and structural analysis tools.

Factor XI (FXI) is the zymogen of a serine protease enzyme in the intrinsic pathway of blood coagulation and is an important factor in the creation of a stable fibrin clot. Deficiency of FXI leads to an injury-related bleeding disorder and is remarkable for the lack of correlation between bleeding symptoms and FXI coagulant activity (FXI:C). The FXI protein is composed of five domains: four tandem repeat domains of approximately 80 residues known as Apple (Ap) domains, and the catalytic serine protease (Sp) domain. A total of 65 mutations throughout the FXI gene (F11) have been reported in FXI deficient patients. An interactive web database of these mutations has been created (www.FactorXI.org) that integrates the phenotypic data with genetic data and structural homology models for the five FXI domains. The database provides a central repository for all reported genetic alterations within F11. With the use of recently developed visualization tools, each mutation can be highlighted on the structural models of the FXI domains together with an appropriate survey of patient data, such as FXI:C levels and FXI antigen levels. The database also enables new F11 mutations to be interpreted. The interactive design of this database will lead to a more comprehensive comparative understanding of the genetic factors that influence bleeding risk.

DNA Mutational Analysis↗

PubMed2001

Function and regulation of monoamine transporters: focus on the norepinephrine transporter.

Presynaptic norepinephrine transporters (NETs) mediate the rapid clearance of norepinephrine from synaptic spaces. NET is a member of the Na+ and Cl- -coupled neurotransmitter transporter gene family, which also includes the serotonin and dopamine transporters. Recent studies reveal that these transporter molecules might be a dynamic component of synaptic plasticity, rather than a constitutive determinant of neurotransmitter levels in synaptic spaces. Recognition that cellular signaling molecules and transporter ligands, including cocaine, amphetamines, and antidepressants, can modify transporter intrinsic activity, trafficking, phosphorylation, and protein levels suggests opportunities for revealing unknown mechanisms of drug action. Control of these properties of transporter function may allow for the development of new strategies to modulate monoaminergic neurotransmission and identify regulatory pathways that may be compromised in psychiatric, neurologic, and neurodegenerative disorders.

Journal Article↗

PubMed1989

[Membrane receptors and G regulatory proteins].

The article summarizes new findings concerning membranous receptor-effector complexes that are stimulated by various extracellular signals like hormones, neurotransmitters, growth factors, etc. Central role in this transmembranous signalling process play so-called G regulatory proteins (they bind GTP), that ensure the activation-deactivation processes between receptor and effector (enzymes or ion channels). The examples of intracellular signals ("second messengers") are cyclic AMP, inositol-trisphosphate, diacylglycerol or the accumulated ions (e. g. Ca2+). G regulatory proteins are composed of three subunits: receptor activation leads to their dissociation and the released alpha subunit with bound GTP activates the appropriate effector. Its inactivation occurs after GTP degradation by the intrinsic GTPase activity of alpha subunit which then associates with beta gamma subunits. New findings concerning the regulatory role of G proteins enable the explanation of mutual relations between various membranous receptor-effector complexes and further they also serve as basis for the explanation of some clinical disorders that are called "receptor diseases".

Animals↗

PubMed2006

The bunyavirus nucleocapsid protein is an RNA chaperone: possible roles in viral RNA panhandle formation and genome replication.

Cellular RNA chaperones are crucial for the genesis of correctly folded functional RNAs. Using several complementary in vitro assays we find that the bunyavirus nucleocapsid protein (N) is an RNA chaperone. In the Bunyaviridae genomic RNA is in stable "panhandle" formation that arises through the hydrogen bonding of the terminal nucleotides of the RNA. The RNA chaperone function of N facilitates panhandle formation even though the termini are separated by >2 kb. RNA panhandle formation is likely driven by the exceptionally high base-pairing specificity of the terminal nucleotides as evidenced by P-num analysis. N protein can nonspecifically dissociate RNA duplexes. In addition, following panhandle formation, the RNA chaperone activity of N also appears to be involved in dissociation of the RNA panhandle and remains in association with the 5' terminus of the viral RNA following dissociation. Thus, N likely functions in the initiation of genome replication to allow efficient initiation of RNA synthesis by the viral polymerase. The RNA chaperone activity of N may be facilitated by an intrinsically disordered domain that catalyzes RNA unfolding driven by reciprocal entropy transfer. These observations highlight the essential features that are probably common to all RNA chaperones in which the role of the chaperone is to nonspecifically dissociate higher order structure and formation of functional higher order structure may often be predicted by RNA P-num value. The data also highlight features of N that are probably specifically important during replication of bunyavirus RNA.

Amino Acid Sequence↗

PubMed2006

Synphilin isoforms and the search for a cellular model of lewy body formation in Parkinson's disease.

A common finding in many neurodegenerative diseases is the presence of inclusion bodies made of aggregated proteins in neurons of affected brain regions. In Parkinson's disease, the inclusion bodies are referred to as Lewy bodies and their main component is alpha-synuclein. Although many studies have suggested that inclusion bodies may be cell protective, it is still not clear whether Lewy bodies promote or inhibit dopaminergic cell death in Parkinson's disease. Synphilin-1 interacts with alpha-synuclein and is present in Lewy bodies. Accumulation of ubiquitylated synphilin-1 leads to massive formation of inclusion bodies, which resemble Lewy bodies by their ability to recruit alpha-synuclein. We have recently isolated an isoform of synphilin-1, synphilin-1A, that spontaneously aggregates in cells, and is present in detergent-insoluble fractions of brain protein samples from alpha-synucleinopathy patients. Synphilin-1A displays marked neuronal toxicity and, upon proteasome inhibition, accumulates into ubiquitylated inclusions with concomitant reduction of its intrinsic toxicity. The fact that alpha-synuclein interacts with synphilin-1A, and is recruited to synphilin-1A inclusion bodies in neurons together with synphilin-1, further indicates that synphilin-1A cell model is relevant for research on Parkinson's disease. Synphilin-1A cell model may help provide important insights regarding the role of inclusion bodies in Parkinson's disease and other neurodegenerative disorders.

Animals↗

PubMed1994

Ehlers-Danlos syndrome type IV. Clinical and molecular aspects and guidelines for diagnosis and management.

The clinical, biochemical and molecular characteristics of Ehlers-Danlos syndrome type IV are reviewed. The numerous clinical manifestations result from the generalized and excessive tissue fragility characteristic for this disorder. The variability in cutaneous involvement may hamper recognition of the condition and requires careful clinical examination. Clinical diagnosis can be confirmed by demonstration of a collagen type III defect. The severity of the phenotype appears to be related mainly to the intrinsic stability of mutant type III collagen protein and its disturbing effect on normal collagen fibrillogenesis in the extracellular matrix.

Collagen↗

PubMed1999

Receptor-mediated endocytosis of cobalamin (vitamin B12).

Dietary cobalamin (Cbl) (vitamin B12) is utilized as methyl-Cbl and the coenzyme 5'-deoxyadenosyl Cbl by cells of the body that have the enzymes methionine synthase and methyl malonyl CoA mutase, which convert homocysteine to methionine and methyl malonyl CoA to succinyl CoA, respectively. Prior to conversions and utilizations as the active alkyl forms of Cbl, dietary Cbl is absorbed and transported across cellular plasma membranes by two receptor-mediated events. First, dietary and biliary Cbl bound to gastric intrinsic factor (IF) presented apically to the ileal absorptive enterocytes is transported to the circulation by receptor-mediated endocytosis via apically expressed IF-Cbl receptor. Second, Cbl bound to plasma transcobalamin (TC) II is taken up from the circulation by all cells via a TC II receptor expressed in the plasma membrane of these cells, and in polarized cells via a TC II receptor expressed in the basolateral membranes. This review updates recent work and focuses on (a) the molecular and cellular aspects of Cbl binding protein ligands, IF and TC II, and their cell-surface receptors, IF-Cbl receptor and TC II receptor; (b) the cellular sorting pathways of internalized Cbl bound to IF and TC II in polarized epithelial cells; and (c) the absorption and transport disorders that cause Cbl deficiency.

Amino Acid Sequence↗

PubMed2004

Characterization of peptides corresponding to the seven transmembrane domains of human adenosine A2a receptor.

Human adenosine A(2)a receptor is a member of the G-protein-coupled receptor (GPCR) superfamily of seven-helix transmembrane (TM) proteins. To test general models for membrane-protein folding and to identify specific features of folding and assembly for this representative member of an important and poorly understood class of proteins, we synthesized peptides corresponding to its seven TM domains. We assessed the ability of the peptides to insert into micelles and vesicles and measured secondary structure for each peptide in aqueous and membrane-mimetic environments. CD spectra indicate that each of the seven TM peptides form thermally stable, independent alpha-helical structures in both micelles and vesicles. The helical content of the peptides depends on the nature of the membrane-mimetic environment. Four of the peptides (TM3, TM4, TM5, and TM7) exhibit very high-helical structure, near the predicted maximum for their TM segments. The TM1 peptide also adopts relatively high alpha-helical structures. In contrast, two of peptides, TM2 and TM6, display low alpha helicity. Similarly, the ability of the peptides to insert into membrane-mimetic environments, assayed by intrinsic tryptophan fluorescence and fluorescence quenching, varied markedly. Most peptides exhibit higher alpha helicity in anionic sodium dodecyl sulfate than in neutral dodecyl-beta-D-maltoside micelles, and TM2 was disordered in zwiterionic DMPC but was alpha-helical in negatively charged DMPC/DMPG vesicles. These findings strongly suggest that electrostatic interactions between lipids and peptides control the insertion of the peptides and may be involved in membrane-protein-folding events. The measured helical content of these TM domains does not correlate with the predicted helicity based on amino acid sequence, pointing out that, while hydrophobic interactions can be a major determinant for folding of TM peptides, other factors, such as electrostatic interactions or helix-helix interactions, may play significant roles for specific TM domains. Our results represent a comprehensive analysis of helical propensities for a human GPCR and support models for membrane-protein folding in which interactions between TM domains are required for proper insertion and folding of some TM helix domains. The tendency of some peptides to self-associate, especially in aqueous environments, underscores the need to prevent improper interactions during folding and refolding of membrane proteins in vivo and in vitro.

Amino Acid Sequence↗

PubMed2006

Alternative O-GlcNAcylation/O-phosphorylation of Ser16 induce different conformational disturbances to the N terminus of murine estrogen receptor beta.

Serine and threonine residues in many proteins can be modified by either phosphorylation or GlcNAcylation. To investigate the mechanism of O-GlcNAc and O-phosphate's reciprocal roles in modulating the degradation and activity of murine estrogen receptor beta (mER-beta), the conformational changes induced by O-GlcNAcylation and O-phosphorylation of Ser(16) in 17-mer model peptides corresponding to the N-terminal intrinsically disordered (ID) region of mER-beta were studied by NMR techniques, circular dichroism (CD), and molecular dynamics simulations. Our results suggest that O-phosphorylation discourages the turn formation in the S(15)STG(18) fragment. In contrast, O-GlcNAcylation promotes turn formation in this region. Thus, we postulate that the different changes of the local structure in the N-terminal S(15)STG(18) fragment of mER-beta caused by O-phosphate or O-GlcNAc modification might lead to the disturbances to the dynamic ensembles of the ID region of mER-beta, which is related to its modulatory activity.

Acetylglucosamine↗

PubMed2005

Probing conformational disorder in neurotensin by two-dimensional solid-state NMR and comparison to molecular dynamics simulations.

An approach is introduced to characterize conformational ensembles of intrinsically unstructured peptides on the atomic level using two-dimensional solid-state NMR data and their combination with molecular dynamics simulations. For neurotensin, a peptide that binds with high affinity to a G-protein coupled receptor, this method permits the investigation of the changes in conformational preferences of a neurotransmitter transferred from a frozen aqueous solution via a lipid model phase to the receptor-bound form. The results speak against a conformational pre-organization of the ligand in detergents in which the receptor has been shown to be functional. Further extensions to the study of protein folding are possible.

Detergents↗