RESUMEN
Lysine deacetylases, like histone deacetylases (HDACs) and sirtuins (SIRTs), are involved in many regulatory processes such as control of metabolic pathways, DNA repair, and stress responses. Besides robust deacetylase activity, sirtuin isoforms SIRT2 and SIRT3 also show demyristoylase activity. Interestingly, most of the inhibitors described so far for SIRT2 are not active if myristoylated substrates are used. Activity assays with myristoylated substrates are either complex because of coupling to enzymatic reactions or time-consuming because of discontinuous assay formats. Here we describe sirtuin substrates enabling direct recording of fluorescence changes in a continuous format. Fluorescence of the fatty acylated substrate is different when compared to the deacylated peptide product. Additionally, the dynamic range of the assay could be improved by the addition of bovine serum albumin, which binds the fatty acylated substrate and quenches its fluorescence. The main advantage of the developed activity assay is the native myristoyl residue at the lysine side chain avoiding artifacts resulting from the modified fatty acyl residues used so far for direct fluorescence-based assays. Due to the extraordinary kinetic constants of the new substrates (KM values in the low nM range, specificity constants between 175,000 and 697,000 M-1s-1) it was possible to reliably determine the IC50 and Ki values for different inhibitors in the presence of only 50 pM of SIRT2 using different microtiter plate formats.
Asunto(s)
Sirtuina 3 , Sirtuinas , Sirtuinas/metabolismo , Sirtuina 2/metabolismo , Lisina , Sirtuina 1/metabolismo , Sirtuina 3/metabolismo , Péptidos , ColorantesRESUMEN
Sirtuins are protein deacylases regulating metabolism and stress responses and implicated in aging-related diseases. Modulators of the human sirtuins 1-7 are sought as chemical tools and potential therapeutics, for example, for treatment of cancer. We were able to show that 3-aryl-mercapto-succinylated- and 3-benzyl-mercapto-succinylated peptide derivatives yield selective Sirt5 inhibitors with low nM Ki values. Here, we synthesized and characterized 3-aryl-mercapto-butyrylated peptide derivatives as effective and selective sirtuin 2 inhibitors with KD values in the low nanomolar range. According to kinetic measurements and microscale thermophoresis/surface plasmon resonance experiments, the respective inhibitors bind with the 3-aryl-mercapto moiety in the selectivity pocket of Sirtuin 2, inducing a rearrangement of the active site. In contrast, 3-aryl-mercapto-nonalyl or palmitoyl derivatives are characterized by a switch in the binding mode blocking both the hydrophobic channel by the fatty acyl chain and the nicotinamide pocket by the 3-aryl-mercapto moiety.
Asunto(s)
Sirtuina 2 , Sirtuinas , Dominio Catalítico , Humanos , Lisina/metabolismo , Niacinamida , Péptidos , Sirtuina 2/metabolismo , Sirtuinas/metabolismoRESUMEN
Cyclophilins, enzymes with peptidyl-prolyl cis/trans isomerase activity, are relevant to a large variety of biological processes. The most abundant member of this enzyme family, cyclophilin A, is the cellular receptor of the immunosuppressive drug cyclosporine A (CsA). As a consequence of the pathophysiological role of cyclophilins, particularly in viral infections, there is a broad interest in cyclophilin inhibition devoid of immunosuppressive activity. This Review first gives an introduction into the physiological and pathophysiological roles of cyclophilins. The presentation of non-immunosuppressive cyclophilin inhibitors will commence with drugs based on chemical modifications of CsA. The naturally occurring macrocyclic sanglifehrins have become other lead structures for cyclophilin-inhibiting drugs. Finally, deâ novo designed compounds, whose structures are not derived from or inspired by natural products, will be presented. Relevant synthetic concepts will be discussed, but the focus will also be on biochemical studies, structure-activity relationships, and clinical studies.
Asunto(s)
Productos Biológicos , Ciclofilinas , Ciclofilina A , Ciclofilinas/química , Ciclosporina/química , Ciclosporina/farmacología , Inmunosupresores/farmacología , Isomerasa de PeptidilprolilRESUMEN
Glucose transporters play an essential role in cancer cell proliferation and survival and have been pursued as promising cancer drug targets. Using microarrays of a library of new macrocycles known as rapafucins, which were inspired by the natural product rapamycin, we screened for new inhibitors of GLUT1. We identified multiple hits from the rapafucin 3D microarray and confirmed one hit as a bona fide GLUT1 ligand, which we named rapaglutinâ A (RgA). We demonstrate that RgA is a potent inhibitor of GLUT1 as well as GLUT3 and GLUT4, with an IC50 value of low nanomolar for GLUT1. RgA was found to inhibit glucose uptake, leading to a decrease in cellular ATP synthesis, activation of AMP-dependent kinase, inhibition of mTOR signaling, and induction of cell-cycle arrest and apoptosis in cancer cells. Moreover, RgA was capable of inhibiting tumor xenografts inâ vivo without obvious side effects. RgA could thus be a new chemical tool to study GLUT function and a promising lead for developing anticancer drugs.
Asunto(s)
Antineoplásicos/química , Proteínas Facilitadoras del Transporte de la Glucosa/antagonistas & inhibidores , Macrólidos/farmacología , Bibliotecas de Moléculas Pequeñas/química , Células A549 , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Células MCF-7 , Macrólidos/química , Estructura Molecular , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Análisis por Matrices de Proteínas , Transducción de Señal , Sirolimus/química , Relación Estructura-Actividad , Serina-Treonina Quinasas TOR/metabolismo , Tacrolimus/química , Proteínas de Unión a TacrolimusRESUMEN
Rapamycin and FK506 are macrocyclic natural products with an extraordinary mode of action, in which they form binary complexes with FK506-binding protein (FKBP) through a shared FKBP-binding domain before forming ternary complexes with their respective targets, mechanistic target of rapamycin (mTOR) and calcineurin, respectively. Inspired by this, we sought to build a rapamycin-like macromolecule library to target new cellular proteins by replacing the effector domain of rapamycin with a combinatorial library of oligopeptides. We developed a robust macrocyclization method using ring-closing metathesis and synthesized a 45,000-compound library of hybrid macrocycles (named rapafucins) using optimized FKBP-binding domains. Screening of the rapafucin library in human cells led to the discovery of rapadocin, an inhibitor of nucleoside uptake. Rapadocin is a potent, isoform-specific and FKBP-dependent inhibitor of the equilibrative nucleoside transporter 1 and is efficacious in an animal model of kidney ischaemia reperfusion injury. Together, these results demonstrate that rapafucins are a new class of chemical probes and drug leads that can expand the repertoire of protein targets well beyond mTOR and calcineurin.
Asunto(s)
Descubrimiento de Drogas/métodos , Macrólidos/química , Macrólidos/metabolismo , Sustancias Protectoras/química , Sustancias Protectoras/metabolismo , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/prevención & control , Animales , Línea Celular , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Proteoma/metabolismo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/prevención & control , Sirolimus/química , Sirolimus/metabolismo , Porcinos , Serina-Treonina Quinasas TOR/química , Serina-Treonina Quinasas TOR/metabolismo , Tacrolimus/química , Tacrolimus/metabolismo , Proteínas de Unión a Tacrolimus/química , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
Clostridioides difficile is the main cause for nosocomial antibiotic associated diarrhea and has become a major burden for the health care systems of industrial countries. Its main virulence factors, the small GTPase glycosylating toxins TcdA and TcdB, are extensively studied. In contrast, the contribution of other factors to development and progression of C. difficile infection (CDI) are only insufficiently understood. Many bacterial peptidyl-prolyl-cis/trans-isomerases (PPIases) have been described in the context of virulence. Among them are the parvulin-type PrsA-like PPIases of Gram-positive bacteria. On this basis, we identified CD630_35000 as the PrsA2 homolog in C. difficile and conducted its enzymatic and phenotypic characterization in order to assess its involvement during C. difficile infection. For this purpose, wild type CdPrsA2 and mutant variants carrying amino acid exchanges mainly in the PPIase domain were recombinantly produced. Recombinant CdPrsA2 showed PPIase activity toward the substrate peptide Ala-Xaa-Pro-Phe with a preference for positively charged amino acids preceding the proline residue. Mutation of conserved residues in its active site pocket impaired the enzymatic activity. A PrsA2 deficient mutant was generated in the C. difficile 630Δerm background using the ClosTron technology. Inactivation of prsA2 resulted in a reduced germination rate in response to taurocholic acid, and in a slight increase in resistance to the secondary bile acids LCA and DCA. Interestingly, in the absence of PrsA2 colonization of mice by C. difficile 630 was significantly reduced. We concluded that CdPrsA2 is an active PPIase that acts as a virulence modulator by influencing crucial processes like sporulation, germination and bile acid resistance resulting in attenuated mice colonization.
RESUMEN
Human Pin1 is a peptidyl prolyl cis/trans isomerase with a unique preference for phosphorylated Ser/Thr-Pro substrate motifs. Here we report that MCM3 (minichromosome maintenance complex component 3) is a novel target of Pin1. MCM3 interacts directly with the WW domain of Pin1. Proline-directed phosphorylation of MCM3 at S112 and T722 are crucial for the interaction with Pin1. MCM3 as a subunit of the minichromosome maintenance heterocomplex MCM2-7 is part of the pre-replication complex responsible for replication licensing and is implicated in the formation of the replicative helicase during progression of replication. Our data suggest that Pin1 coordinates phosphorylation-dependently MCM3 loading onto chromatin and its unloading from chromatin, thereby mediating S phase control.
Asunto(s)
Cromatina/metabolismo , Componente 3 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Peptidilprolil Isomerasa de Interacción con NIMA/química , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Sitios de Unión , Regulación de la Expresión Génica , Células HeLa , Humanos , Componente 3 del Complejo de Mantenimiento de Minicromosoma/química , Componente 3 del Complejo de Mantenimiento de Minicromosoma/genética , Mutación , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Fosforilación , Prolina/metabolismo , Unión Proteica , Fase SRESUMEN
The Bordetella pertussis toxin (PT) is one important virulence factor causing the severe childhood disease whooping cough which still accounted for approximately 63,000 deaths worldwide in children in 2013. PT consists of PTS1, the enzymatically active (A) subunit and a non-covalently linked pentameric binding/transport (B) subunit. After endocytosis, PT takes a retrograde route to the endoplasmic reticulum (ER), where PTS1 is released into the cytosol. In the cytosol, PTS1 ADP-ribosylates inhibitory alpha subunits of trimeric GTP-binding proteins (Giα) leading to increased cAMP levels and disturbed signalling. Here, we show that the cyclophilin (Cyp) isoforms CypA and Cyp40 directly interact with PTS1 in vitro and that Cyp inhibitors cyclosporine A (CsA) and its tailored non-immunosuppressive derivative VK112 both inhibit intoxication of CHO-K1 cells with PT, as analysed in a morphology-based assay. Moreover, in cells treated with PT in the presence of CsA, the amount of ADP-ribosylated Giα was significantly reduced and less PTS1 was detected in the cytosol compared to cells treated with PT only. The results suggest that the uptake of PTS1 into the cytosol requires Cyps. Therefore, CsA/VK112 represent promising candidates for novel therapeutic strategies acting on the toxin level to prevent the severe, life-threatening symptoms caused by PT.
Asunto(s)
Ciclofilinas/antagonistas & inhibidores , Ciclosporina/farmacología , Toxina del Pertussis/toxicidad , Animales , Bordetella pertussis , Células CHO , Cricetulus , Ciclofilinas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes/metabolismoRESUMEN
We demonstrate that fused silica capillaries are suitable for single molecule fluorescence resonance energy transfer (smFRET) measurements at high pressure with an optical quality comparable to the measurement on microscope coverslips. Therefore, we optimized the imaging conditions in a standard square fused silica capillary with an adapted arrangement and evaluated the performance by imaging the focal volume, fluorescence correlation spectroscopy benchmarks, and FRET measurements. We demonstrate single molecule FRET measurements of cold shock protein A unfolding at a pressure up to 2000 bars and show that the unfolded state exhibits an expansion almost independent of pressure.
Asunto(s)
Proteínas y Péptidos de Choque por Frío/química , Transferencia Resonante de Energía de Fluorescencia , Presión , Desplegamiento ProteicoRESUMEN
Binary enterotoxins Clostridium (C.) botulinum C2 toxin, C. perfringens iota toxin and C. difficile toxin CDT are composed of a transport (B) and a separate non-linked enzyme (A) component. Their B-components mediate endocytic uptake into mammalian cells and subsequently transport of the A-components from acidic endosomes into the cytosol, where the latter ADP-ribosylate G-actin resulting in cell rounding and cell death causing clinical symptoms. Protein folding enzymes, including Hsp90 and peptidyl-prolyl cis/trans isomerases facilitate transport of the A-components across endosomal membranes. Here, we identified Hsp70 as a novel host cell factor specifically interacting with A-components of C2, iota and CDT toxins to facilitate their transport into the cell cytosol. Pharmacological Hsp70-inhibition specifically prevented pH-dependent trans-membrane transport of A-components into the cytosol thereby protecting living cells and stem cell-derived human miniguts from intoxication. Thus, Hsp70-inhibition might lead to development of novel therapeutic strategies to treat diseases associated with bacterial ADP-ribosylating toxins.
Asunto(s)
ADP Ribosa Transferasas/metabolismo , Bacterias/metabolismo , Enterotoxinas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Animales , Chlorocebus aethiops , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Humanos , Concentración de Iones de Hidrógeno , Unión Proteica , Células VeroRESUMEN
Diphtheria toxin kills human cells because it delivers its enzyme domain DTA into their cytosol where it inhibits protein synthesis. After receptor-mediated uptake of the toxin, DTA translocates from acidic endosomes into the cytosol, which might be assisted by host cell factors. Here we investigated the role of Hsp90 and its co-chaperones during the uptake of native diphtheria toxin into human cells and identified the components of the Hsp90 machinery including Hsp90, Hsp70, Cyp40 and the FK506 binding proteins FKBP51 and FKBP52 as DTA binding partners. Moreover, pharmacological inhibition of the chaperone activity of Hsp90 and Hsp70 and of the peptidyl-prolyl cis/trans isomerase (PPIase) activity of Cyps and FKBPs protected cells from intoxication with diphtheria toxin and inhibited the pH-dependent trans-membrane transport of DTA into the cytosol. In conclusion, these host cell factors facilitate toxin uptake into human cells, which might lead to development of novel therapeutic strategies against diphtheria.
Asunto(s)
Toxina Diftérica/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Animales , Células CHO , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Cricetulus , Citosol/metabolismo , Toxina Diftérica/toxicidad , Activación Enzimática/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Concentración de Iones de Hidrógeno , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Unión Proteica , Transporte de Proteínas , ProteolisisRESUMEN
Cylophilins (Cyps) belong to the ubiquitously distributed enzyme class of peptidyl prolyl cis/trans isomerases (EC5.2.1.8), which are foldases capable of accelerating slow steps in the refolding of denatured proteins. At least 20 different Cyp isoenzymes are broadly distributed among all organs and cellular compartments in humans. Extracellularly localized Cyps came into the scientific focus recently because of their involvement in the control of inflammatory diseases, as well as viral and bacterial infections. However, detailed insights into Cyp functions are often hampered by the lack of sensitive detection methods. We present an improved method for affinity purification and detection of Cyp in biotic samples in this manuscript. The procedure takes advantage of two novel cyclosporine A derivatives. Derivative 1 was used to capture Cyps from the sample while derivative 2 was applied for selective release from the affinity matrix. Using this approach, eight different Cyp (CypA, CypB, CypC, Cyp40 (PPID), CypE, CypD (PPIF), CypH, and CypL1) were unambiguously detected in healthy human blood plasma. Moreover, extracellular CypA was found to be partially modified by Nε acetylation on residues Lys44, Lys133, Lys155, as well as Nα acetylation at the N-terminal Val residue. Nα acetylation of Ser2 residue was also found for Cyp40.
Asunto(s)
Ciclofilinas/sangre , Ciclosporina/sangre , Proteoma/genética , Proteómica , Acetilación , Cromatografía Líquida de Alta Presión , Ciclofilinas/clasificación , Ciclosporina/clasificación , HumanosRESUMEN
Sirtuins are NAD(+) dependent lysine deacylases involved in many regulatory processes such as control of metabolic pathways, DNA repair and stress response. Modulators of sirtuin activity are required as tools for uncovering the biological function of these enzymes and as potential therapeutic agents. Systematic discovery of such modulators is hampered by the lack of direct and continuous activity assays. The present study describes a novel continuous assay based on the increase of a fluorescence signal subsequent to sirtuin mediated removal of a fluorescent acyl chain from a modified TNFα-derived peptide. This substrate is well recognized by human sirtuins 1-6 and represents the best sirtuin 2 substrate described so far with a kcat/KM-value of 176 000 M(-1)s(-1). These extraordinary substrate properties allow the first determination of Ki-values for the specific Sirt2 inhibitory peptide S2iL5 (600 nM) and for the quasi-universal sirtuin inhibitor peptide thioxo myristoyl TNFα (80 nM).
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/aislamiento & purificación , Sirtuinas/antagonistas & inhibidores , Sirtuinas/análisis , HumanosRESUMEN
AIM: To characterize the biochemical features of the newest member of cyclophilin family of peptidyl-prolyl cis/trans-isomerases (PPIases), cyclophilin J (CYPJ). MATERIALS AND METHODS: PPIase assays were performed on purified hCYPJ and its mutated variants. The substrate specificity, half-maximal inhibitory concentration (IC50) of cyclosporin A (CsA) inhibition and circular dichroism (CD) spectrum of CYPJ were measured. Mercury pathway profiling luciferase assays were also performed. RESULTS: The catalytic number/Michaelis constant (kcat/KM) value of CYPJ was 9.5×10(4) s(-1)M(-1). CYPJ additionally catalyzed norleucine-proline, isoleucine-proline and glutamine-proline peptides compared to CYPA and Escherichia coli PPIases. CYPJ was inhibited by CsA in a dose-dependent manner with IC50 of 12.1±0.9 µM. The CD spectrum of CYPJ was similar to CYPA. CYPJ significantly up-regulated the transcription of E-box, E2F, retinoblastoma (Rb), p53, activator protein 1 (AP1), NF-κB and phospho-cAMP response element (CRE) cis-response element in 293T cells. CONCLUSION: CYPJ structurally resembles CYPA. It is sensitive to inhibition by CsA and plays a role in regulating cell growth, proliferation, and apoptosis.
Asunto(s)
Ciclofilinas/metabolismo , Catálisis , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Ciclofilinas/antagonistas & inhibidores , Ciclofilinas/genética , Ciclosporina/metabolismo , Ciclosporina/farmacología , Relación Dosis-Respuesta a Droga , Factores de Transcripción E2F/genética , Factores de Transcripción E2F/metabolismo , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Células HEK293 , Humanos , Cinética , Mutación , FN-kappa B/genética , FN-kappa B/metabolismo , Unión Proteica , Proteínas Recombinantes/metabolismo , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Especificidad por Sustrato , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Transcripción Genética , Transfección , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Cyclophilins interact directly with the Alzheimer's disease peptide Aß (amyloid ß-peptide) and are therefore involved in the early stages of Alzheimer's disease. Aß binding to CypD (cyclophilin D) induces dysfunction of human mitochondria. We found that both CypD and CypA suppress in vitro fibril formation of Aß(1-40) at substoichiometric concentrations when present early in the aggregation process. The prototypic inhibitor CsA (cyclosporin A) of both cyclophilins as well as the new water-soluble MM258 derivative prevented this suppression. A SPOT peptide array approach and NMR titration experiments confirmed binding of Aß(1-40) to the catalytic site of CypD mainly via residues Lys(16)-Glu(22) The peptide Aß(16-20) representing this section showed submicromolar IC50 values for the peptidyl prolyl cis-trans isomerase activity of CypD and CypA and low-micromolar KD values in ITC experiments. Chemical cross-linking and NMR-detected hydrogen-deuterium exchange experiments revealed a shift in the populations of small Aß(1-40) oligomers towards the monomeric species, which we investigated in the present study as being the main process of prevention of Aß fibril formation by cyclophilins.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Ciclofilina A/metabolismo , Ciclofilinas/metabolismo , Fragmentos de Péptidos/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Peptidil-Prolil Isomerasa F , Ciclosporina/farmacología , Activación Enzimática/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Mitocondrias/metabolismoRESUMEN
Hsp70 family proteins are folding helper proteins involved in a wide variety of cellular pathways. Members of this family interact with key factors in signal transduction, transcription, cell-cycle control, and stress response. Here, we developed the first Hsp70 low molecular weight inhibitor specifically targeting the peptide binding site of human Hsp70. After demonstrating that the inhibitor modulates the Hsp70 function in the cell, we used the inhibitor to show for the first time that the stress-inducible chaperone Hsp70 functions as molecular component for entry of a bacterial protein toxin into mammalian cells. Pharmacological inhibition of Hsp70 protected cells from intoxication with the binary actin ADP-ribosylating iota toxin from Clostridium perfringens, the prototype of a family of enterotoxins from pathogenic Clostridia and inhibited translocation of its enzyme component across cell membranes into the cytosol. This finding offers a starting point for novel therapeutic strategies against certain bacterial toxins.
Asunto(s)
ADP Ribosa Transferasas/toxicidad , Toxinas Bacterianas/toxicidad , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Apoptosis , Sitios de Unión/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Células HEK293 , Proteínas HSP70 de Choque Térmico/metabolismo , Células HT29 , Células HeLa , Humanos , Células VeroRESUMEN
Alzheimer's disease (AD) is a fatal neurodegenerative disorder in humans and the main cause of dementia in aging societies. The disease is characterized by the aberrant formation of ß-amyloid (Aß) peptide oligomers and fibrils. These structures may damage the brain and give rise to cerebral amyloid angiopathy, neuronal dysfunction, and cellular toxicity. Although the connection between AD and Aß fibrillation is extensively documented, much is still unknown about the formation of these Aß aggregates and their structures at the molecular level. Here, we combined electron cryomicroscopy, 3D reconstruction, and integrative structural modeling methods to determine the molecular architecture of a fibril formed by Aß(1-42), a particularly pathogenic variant of Aß peptide. Our model reveals that the individual layers of the Aß fibril are formed by peptide dimers with face-to-face packing. The two peptides forming the dimer possess identical tilde-shaped conformations and interact with each other by packing of their hydrophobic C-terminal ß-strands. The peptide C termini are located close to the main fibril axis, where they produce a hydrophobic core and are surrounded by the structurally more flexible and charged segments of the peptide N termini. The observed molecular architecture is compatible with the general chemical properties of Aß peptide and provides a structural basis for various biological observations that illuminate the molecular underpinnings of AD. Moreover, the structure provides direct evidence for a steric zipper within a fibril formed by full-length Aß peptide.
Asunto(s)
Péptidos beta-Amiloides/ultraestructura , Amiloide/ultraestructura , Microscopía por Crioelectrón , Fragmentos de Péptidos/ultraestructura , Péptidos/química , Multimerización de Proteína , Secuencia de Aminoácidos , Amiloide/química , Péptidos beta-Amiloides/química , Mapeo Epitopo , Procesamiento de Imagen Asistido por Computador , Fragmentos Fab de Inmunoglobulinas/química , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Estructura Secundaria de ProteínaRESUMEN
BACKGROUND: Peptidyl prolyl cis/trans isomerases (PPIases) assist the folding and restructuring of client proteins by catalysis of the slow rotational motion of peptide bonds preceding a proline residue. Catalysis is performed by relatively small, distinct protein domains of 10 to 18kDa for all PPIase families. PPIases are involved in a wide variety of physiological and pathophysiological processes like signal transduction, cell differentiation, apoptosis as well as viral, bacterial and parasitic infection. SCOPE OF REVIEW: There are multidomain PPIases consisting of one to up to four catalytic domains of the respective PPIase family supplemented by N- or C-terminal extensions. This review examines the biochemical and functional properties of the members of the PPIase class of enzymes which contain additional protein domains with defined biochemical functions. MAJOR CONCLUSIONS: The versatile domain architecture of multidomain PPIases is important for the control of enzyme specificity and organelle-specific targeting, the establishment of molecular connections and hence the coordination of PPIase functions across the cellular network. GENERAL SIGNIFICANCE: Accessory domains covalently linked to a PPIase domain supply an additional layer of control to the catalysis of prolyl isomerization in specific client proteins. Understanding these control mechanisms will provide new insights into the physiological mode of action of the multidomain PPIases and their ability to form therapeutic targets. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.
Asunto(s)
Isomerasa de Peptidilprolil/química , Animales , Humanos , Isomerasa de Peptidilprolil/metabolismo , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin and Clostridium difficile CDT belong to the family of binary actin ADP-ribosylating toxins and are composed of a binding/translocation component and a separate enzyme component. The enzyme components ADP-ribosylate G-actin in the cytosol of target cells resulting in depolymerization of F-actin, cell rounding and cell death. The binding/translocation components bind to their cell receptors and form complexes with the respective enzyme components. After receptor-mediated endocytosis, the binding/translocation components form pores in membranes of acidified endosomes and the enzyme components translocate through these pores into the cytosol. This step is facilitated by the host cell chaperone heat shock protein 90 and peptidyl-prolyl cis/trans isomerases including cyclophilin A. Here, we demonstrate that a large isoform of cyclophilin A, the multi-domain enzyme cyclophilin 40 (Cyp40), binds to the enzyme components C2I, Ia and CDTa in vitro. Isothermal titration calorimetry revealed a direct binding to C2I with a calculated affinity of 101 nM and to Ia with an affinity of 1.01 µM. Closer investigation for the prototypic C2I revealed that binding to Cyp40 did not depend on its ADP-ribosyltransferase activity but was stronger for unfolded C2I. The interaction of C2I with Cyp40 was also demonstrated in lysates from C2-treated cells by pull-down. Treatment of cells with a non-immunosuppressive cyclosporine A derivative, which still binds to and inhibits the peptidyl-prolyl cis/trans isomerase activity of cyclophilins, protected cells from intoxication with C2, iota and CDT toxins, offering an attractive approach for development of novel therapeutic strategies against binary actin ADP-ribosylating toxins.
Asunto(s)
ADP Ribosa Transferasas/antagonistas & inhibidores , Proteínas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Botulínicas/antagonistas & inhibidores , Ciclofilinas/antagonistas & inhibidores , ADP Ribosa Transferasas/metabolismo , ADP Ribosa Transferasas/toxicidad , Actinas/metabolismo , Adenosina Difosfato/metabolismo , Animales , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidad , Toxinas Botulínicas/metabolismo , Toxinas Botulínicas/toxicidad , Chlorocebus aethiops , Ciclofilinas/metabolismo , Ciclosporina/farmacología , Células HeLa , Humanos , Transporte de Proteínas/efectos de los fármacos , Células VeroRESUMEN
The cyclophilin family of peptidyl prolyl cis/trans isomerases includes several isoforms found to be secreted in response to different stimuli, thus existing both in the interior and the exterior of cells. The extracellular fractions of the cyclophilins CypA and CypB are involved in the control of cell-cell communication. By binding to the cell membrane receptor CD147 and cell surface heparans they elicit a variety of intracellular signaling cascades involved in inflammatory processes. Increased levels of cyclophilins in inflammatory tissues and body fluids are considered as an inflammatory response to injury. Thus, the extracellular portion of cyclophilins probably plays an important role in human diseases associated with acute or chronic inflammation like rheumatoid arthritis, sepsis, asthma and cardiovascular diseases. Specific inhibition of the cyclophilins in the extracellular space may open an effective therapeutic approach for treating inflammatory diseases.