RESUMO
The driving forces and conformational pathways leading to amphitropic protein-membrane binding and in some cases also to protein misfolding and aggregation is the subject of intensive research. In this study, a chimeric polypeptide, A-Cage-C, derived from α-Lactalbumin is investigated with the aim of elucidating conformational changes promoting interaction with bilayers. From previous studies, it is known that A-Cage-C causes membrane leakages associated with the sporadic formation of amorphous aggregates on solid-supported bilayers. Here we express and purify double-labelled A-Cage-C and prepare partially deuterated bicelles as a membrane mimicking system. We investigate A-Cage-C in the presence and absence of these bicelles at non-binding (pH 7.0) and binding (pH 4.5) conditions. Using in silico analyses, NMR, conformational clustering, and Molecular Dynamics, we provide tentative insights into the conformations of bound and unbound A-Cage-C. The conformation of each state is dynamic and samples a large amount of overlapping conformational space. We identify one of the clusters as likely representing the binding conformation and conclude tentatively that the unfolding around the central W23 segment and its reorientation may be necessary for full intercalation at binding conditions (pH 4.5). We also see evidence for an overall elongation of A-Cage-C in the presence of model bilayers.
Assuntos
Proteína Oncogênica pp60(v-src)/química , Fragmentos de Peptídeos/química , Peptídeos/química , Lactalbumina/química , Espectroscopia de Ressonância Magnética/métodos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Membranas , Simulação de Dinâmica Molecular , Proteína Oncogênica pp60(v-src)/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeos/metabolismo , Ligação Proteica , Conformação ProteicaRESUMO
Pharmacological chaperones are small compounds that correct the folding of mutant proteins, and represent a promising therapeutic strategy for misfolding diseases. We have performed a screening of 10,000 compounds searching for pharmacological chaperones of tyrosine hydroxylase (TH), the tetrahydrobiopterin (BH4)-dependent enzyme that catalyzes the rate-limiting step in the synthesis of catecholamines. A large number of compounds bound to human TH, isoform 1 (hTH1), but only twelve significantly protected wild-type (hTH1-wt) and mutant TH-R233H (hTH1-p.R202H), associated to the rare neurological disorder TH deficiency (THD), from time-dependent loss of activity. Three of them (named compounds 2, 4 and 5) were subjected to detailed characterization of their functional and molecular effects. Whereas compounds 2 and 4 had a characteristic pharmacological chaperone (stabilizing) effect, compound 5 protected the activity in a higher extent than expected from the low conformational stabilization exerted on hTH1. Compounds 4 and 5 were weak competitive inhibitors with respect to the cofactor BH4 and, as seen by electron paramagnetic resonance, they induced small changes to the first coordination sphere of the catalytic iron. Molecular docking also indicated active-site location with coordination to the iron through a pyrimidine nitrogen atom. Interestingly, compound 5 increased TH activity in cells transiently transfected with either hTH1-wt or the THD associated mutants p.L205P, p.R202H and p.Q381K without affecting the steady-state TH protein levels. This work revealed different mechanisms for the action of pharmacological chaperones and identifies a subtype of compounds that preserve TH activity by weak binding to the catalytic iron. This article is part of a Special Issue entitled: Cofactor-dependent proteins: Evolution, chemical diversity and bio-applications.
Assuntos
Tirosina 3-Mono-Oxigenase/química , Domínio Catalítico , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Simulação de Acoplamento Molecular , Conformação Proteica , Dobramento de Proteína , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
In this manuscript we expand significantly on our earlier communication by investigating the bilayer self-assembly of eight different types of phospholipids in unbiased molecular dynamics (MD) simulations using three widely used all-atom lipid force fields. Irrespective of the underlying force field, the lipids are shown to spontaneously form stable lamellar bilayer structures within 1 microsecond, the majority of which display properties in satisfactory agreement with the experimental data. The lipids self-assemble via the same general mechanism, though at formation rates that differ both between lipid types, force fields and even repeats on the same lipid/force field combination. In addition to zwitterionic phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipids, anionic phosphatidylserine (PS) and phosphatidylglycerol (PG) lipids are represented. To our knowledge this is the first time bilayer self-assembly of phospholipids with negatively charged head groups is demonstrated in all-atom MD simulations.
Assuntos
Bicamadas Lipídicas/química , Fosfolipídeos/química , Simulação de Dinâmica MolecularRESUMO
Sorbitol dehydrogenase inhibitors have been found to prevent, or alleviate, various secondary complications of diabetes mellitus. In the present study, the effects of nucleosides and nucleotides on the rate of sorbitol oxidation catalyzed by the sheep liver enzyme were studied by steady-state kinetics at pH 7.4. Various such compounds, including ATP and the 2'-deoxy-analogues of ATP, ADP and AMP, reversibly inhibit enzyme activity by formation of enzyme-coenzyme-inhibitor ternary complexes. In each case, no deviations from linearity were seen in the double-reciprocal plots using sorbitol or NAD(+) as the varied substrate and there was a linear relationship between inhibitor concentration and the observed inhibitory effects. Sorbitol was docked into a model of the sheep SDH-NAD(+) complex based upon the structure of the human SDH-NAD(+) holoenzyme. The resulting structure of the ternary complex of sheep SDH, NAD(+) and sorbitol (PMDB ID code PM 0078068) shows that the reactive C-2 hydroxyl group of sorbitol is oriented toward the 4'-position of the nicotinamide moiety of the coenzyme, and that the adjacent primary hydroxyl group of sorbitol interacts with the catalytic zinc. The results indicate that the ribose moiety of the inhibitor structures is an important determinant for the observed effects. Specifically, the 2'-position of the ribose ring exerts an effect with respect to inhibitor potency.
Assuntos
L-Iditol 2-Desidrogenase/antagonistas & inibidores , Fígado/enzimologia , Nucleosídeos/química , Nucleotídeos/química , Ovinos/metabolismo , Animais , Ativação Enzimática , Estabilidade EnzimáticaRESUMO
Despite recent improvement in therapy, acute myeloid leukemia (AML) is still associated with high lethality. In the presented study, we analyzed the bioactive compound iodinin (1,6-dihydroxyphenazine 5,10-dioxide) from a marine actinomycetes bacterium for the ability to induce cell death in a range of cell types. Iodinin showed selective toxicity to AML and acute promyelocytic (APL) leukemia cells, with EC50 values for cell death up to 40 times lower for leukemia cells when compared with normal cells. Iodinin also successfully induced cell death in patient-derived leukemia cells or cell lines with features associated with poor prognostic such as FLT3 internal tandem duplications or mutated/deficient p53. The cell death had typical apoptotic morphology, and activation of apoptotic signaling proteins like caspase-3. Molecular modeling suggested that iodinin could intercalate between bases in the DNA in a way similar to the anti-cancer drug daunorubicin (DNR), causing DNA-strand breaks. Iodinin induced apoptosis in several therapy-resistant AML-patient blasts, but to a low degree in peripheral blood leukocytes, and in contrast to DNR, not in rat cardiomyoblasts. The low activity towards normal cell types that are usually affected by anti-leukemia therapy suggests that iodinin and related compounds represent promising structures in the development of anti-cancer therapy.
Assuntos
Actinobacteria/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Leucemia Mieloide , Actinobacteria/química , Adolescente , Adulto , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Daunorrubicina/química , Feminino , Regulação Bacteriana da Expressão Gênica/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Estrutura Molecular , Fenazinas/química , Fenazinas/metabolismo , Fenazinas/farmacologia , Ratos , Adulto JovemRESUMO
GroEL is an ATP dependent molecular chaperone that promotes the folding of a large number of substrate proteins in E. coli. Large-scale conformational transitions occurring during the reaction cycle have been characterized from extensive crystallographic studies. However, the link between the observed conformations and the mechanisms involved in the allosteric response to ATP and the nucleotide-driven reaction cycle are not completely established. Here we describe extensive (in total long) unbiased molecular dynamics (MD) simulations that probe the response of GroEL subunits to ATP binding. We observe nucleotide dependent conformational transitions, and show with multiple 100 ns long simulations that the ligand-induced shift in the conformational populations are intrinsically coded in the structure-dynamics relationship of the protein subunit. Thus, these simulations reveal a stabilization of the equatorial domain upon nucleotide binding and a concomitant "opening" of the subunit, which reaches a conformation close to that observed in the crystal structure of the subunits within the ADP-bound oligomer. Moreover, we identify changes in a set of unique intrasubunit interactions potentially important for the conformational transition.
Assuntos
Chaperonina 60/química , Nucleotídeos/metabolismo , Subunidades Proteicas/química , Sítios de Ligação , Chaperonina 60/metabolismo , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Nucleotídeos/química , Conformação Proteica , Subunidades Proteicas/metabolismoRESUMO
Mutant superoxide dismutase 1 (SOD1) may form cyclic structures due to its greater instability from aberrant demetallization and oxidation of cysteine bonds. This cyclic structure may allow SOD1 to form ion channels on membranes such as the mitochondrial membrane, causing imbalances in the concentration of intracellular ions as a potential mechanism for the progressive neuron death involved in amyotrophic lateral sclerosis (ALS). Using docking programs within modeling software, models of mutant SOD1 dimers and eventually ring oligomers were constructed based on known descriptions of such structures in addition to information on the orientation of the models associated with a membrane. The resulting structure consists of a ring of four demetallated mutant SOD1 dimers with cross-linked disulfide bonds. Stability of the octamer model was supported by the molecular dynamics simulations. Further analysis of the octamer model indicated that its inner- and outer-pore diameters were stable, matching the dimensions of known SOD1 ion channels.
Assuntos
Esclerose Lateral Amiotrófica , Superóxido Dismutase , Esclerose Lateral Amiotrófica/genética , Cisteína/química , Dissulfetos/química , Humanos , Mutação , Superóxido Dismutase/química , Superóxido Dismutase/genética , Superóxido Dismutase-1/química , Superóxido Dismutase-1/genéticaRESUMO
Vesicular monoamine transporter 2 (VMAT2) is responsible for packing monoamine neurotransmitters into synaptic vesicles for storage and subsequent neurotransmission. VMAT2 inhibitors are approved for symptomatic treatment of tardive dyskinesia and Huntington's chorea, but despite being much-studied inhibitors their exact binding site and mechanism behind binding and inhibition of monoamine transport are not known. Here we report the identification of several approved drugs, notably ß2-adrenergic agonists salmeterol, vilanterol and formoterol, ß2-adrenergic antagonist carvedilol and the atypical antipsychotic ziprasidone as inhibitors of rat VMAT2. Further, plausible binding modes of the established VMAT2 inhibitors reserpine and tetrabenazine and hit compounds salmeterol and ziprasidone were identified using molecular dynamics simulations and functional assays using VMAT2 wild-type and mutants. Our findings show VMAT2 as a potential off-target of treatments with several approved drugs in use today and can also provide important first steps in both drug repurposing and therapy development targeting VMAT2 function.
Assuntos
Antipsicóticos , Animais , Ratos , Agonistas Adrenérgicos , Antipsicóticos/farmacologia , Piperazinas , Proteínas Vesiculares de Transporte de Monoamina/genética , Antagonistas Adrenérgicos/farmacologiaRESUMO
Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of dopamine (DA) and other catecholamines, and its dysfunction leads to DA deficiency and parkinsonisms. Inhibition by catecholamines and reactivation by S40 phosphorylation are key regulatory mechanisms of TH activity and conformational stability. We used Cryo-EM to determine the structures of full-length human TH without and with DA, and the structure of S40 phosphorylated TH, complemented with biophysical and biochemical characterizations and molecular dynamics simulations. TH presents a tetrameric structure with dimerized regulatory domains that are separated 15 Å from the catalytic domains. Upon DA binding, a 20-residue α-helix in the flexible N-terminal tail of the regulatory domain is fixed in the active site, blocking it, while S40-phosphorylation forces its egress. The structures reveal the molecular basis of the inhibitory and stabilizing effects of DA and its counteraction by S40-phosphorylation, key regulatory mechanisms for homeostasis of DA and TH.
Assuntos
Dopamina/farmacologia , Inibidores Enzimáticos/farmacologia , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , Tirosina 3-Mono-Oxigenase/química , Sequência de Aminoácidos , Domínio Catalítico , Catecolaminas/metabolismo , Microscopia Crioeletrônica , Dopamina/química , Dopamina/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Modelos Moleculares , Fosforilação , Ligação Proteica , Domínios Proteicos , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The first part of the catalytic cycle of the pterin-dependent, dioxygen-using nonheme-iron aromatic amino acid hydroxylases, leading to the Fe(IV)=O hydroxylating intermediate, has been investigated by means of density functional theory. The starting structure in the present investigation is the water-free Fe-O(2) complex cluster model that represents the catalytically competent form of the enzymes. A model for this structure was obtained in a previous study of water-ligand dissociation from the hexacoordinate model complex of the X-ray crystal structure of the catalytic domain of phenylalanine hydroxylase in complex with the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) (PAH-Fe(II)-BH(4)). The O-O bond rupture and two-electron oxidation of the cofactor are found to take place via a Fe-O-O-BH(4) bridge structure that is formed in consecutive radical reactions involving a superoxide ion, O(2)(-). The overall effective free-energy barrier to formation of the Fe(IV)=O species is calculated to be 13.9â kcal mol(-1), less than 2â kcal mol(-1) lower than that derived from experiment. The rate-limiting step is associated with a one-electron transfer from the cofactor to dioxygen, whereas the spin inversion needed to arrive at the quintet state in which the O-O bond cleavage is finalized, essentially proceeds without activation.
Assuntos
Aminoácidos Aromáticos/química , Biopterinas/análogos & derivados , Coenzimas/química , Compostos Ferrosos/química , Ferro/química , Oxigenases de Função Mista/química , Pterinas/química , Aminoácidos Aromáticos/metabolismo , Biopterinas/química , Biopterinas/metabolismo , Catálise , Coenzimas/metabolismo , Cristalografia por Raios X , Compostos Ferrosos/metabolismo , Hidroxilação , Ligantes , Oxigenases de Função Mista/metabolismo , Estrutura Molecular , OxirreduçãoRESUMO
Background: Clinically, corticosteroids are used mainly for their immune-modulatory properties but are also known to influence mood. Despite evidence of a role in regulating tryptophan hydroxylases (TPH), key enzymes in serotonin biosynthesis, a direct action of corticosteroids on these enzymes has not been systematically investigated. Methodology & results: Corticosteroid effects on TPHs were tested using an in vitro assay. The compound with the strongest modulatory effect, beclomethasone dipropionate, activated TPH1 and TPH2 with low micromolar potency. Thermostability assays suggested a stabilizing mechanism, and computational docking indicated that beclomethasone dipropionate interacts with the TPH active site. Conclusion: Beclomethasone dipropionate is a stabilizer of TPHs, acting as a pharmacological chaperone. Our findings may inspire further development of steroid scaffolds as putative antidepressant drugs.
Assuntos
Corticosteroides/farmacologia , Triptofano Hidroxilase/metabolismo , Corticosteroides/síntese química , Corticosteroides/química , Domínio Catalítico/efeitos dos fármacos , Estabilidade Enzimática , Humanos , Conformação Molecular , Simulação de Acoplamento MolecularRESUMO
Tyrosine hydroxylase (TH) catalyses the (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4)-dependent conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-Dopa), which is the rate-limiting step in the synthesis of dopamine and other catecholamine neurotransmitters and hormones. Dysfunctional mutant TH causes tyrosine hydroxylase deficiency (THD), characterized by symptoms ranging from mild l-Dopa responsive dystonia to severe neuropathy. THD-associated mutations often present misfolding and a propensity to aggregate, characteristics that can also be manifested by dysregulated wild-type TH. TH - and subsequently dopamine - is also reduced in Parkinson's disease (PD) due to the selective death of dopaminergic neurons. Thus, TH is a target for stabilizing small molecular weight compounds that can function as pharmacological chaperones, restoring enzyme folding and function. In this work we carried out a screening of a compound library with 1280 approved drugs and we identified levalbuterol, a beta2-adrenergic agonist that is broadly used in asthma treatment, as an interesting validated binder of human TH. Levalbuterol stabilized TH with reduced affinity compared to dopamine, the end-product and regulatory feedback inhibitor of TH, but without compromising enzymatic activity. Moreover, levalbuterol also delays the formation of TH aggregates and makes the enzyme less sensitive to dopamine, effects that could contribute to ameliorate disorders related to TH, such as THD and PD.
Assuntos
Dopamina/química , Levalbuterol/química , Agregados Proteicos , Dobramento de Proteína , Tirosina 3-Mono-Oxigenase/química , Distúrbios Distônicos/congênito , Distúrbios Distônicos/enzimologia , Distúrbios Distônicos/genética , Humanos , Tirosina 3-Mono-Oxigenase/genéticaRESUMO
Phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of L-Phe to L-Tyr. Dysfunctional PAH results in phenylketonuria and mammalian PAH is therefore highly regulated and displays positive cooperativity for L-Phe (Hill coefficient (h)=2). L-Phe does not bind to the regulatory ACT domain in full-length tetrameric human PAH and cooperativity is elicited by homotropic binding to the catalytic site (Thórólfsson et al. in Biochemistry 41:7573-7585, 2002). PAH from Caenorhabditis elegans (cePAH) is devoid of cooperativity for L-Phe (h=0.9), and, as shown in this work, structural analysis reveal an additional L-Phe binding site at the regulatory domain of full-length cePAH. This site involves the GA(S)L/ISRP motifs, which are also found in ACT domains of other L-Phe binding proteins, such as prephenate dehydratase. Isothermal titration calorimetry further demonstrated 2 binding sites per subunit for cePAH versus ~1 for hPAH. Steric occlusion of the regulatory site, notably by residues Lys215/Tyr216 from the adjacent catalytic domain, appears to hinder regulatory binding in full-length hPAH. Accordingly, the humanized mutant Q215K/N216Y of cePAH binds ~1.4 L-Phe/subunit. This mutant also displays high catalytic activity and certain positive cooperativity for L-Phe (h=1.4). Our results support that the acquisition of positive cooperativity in mammalian forms of PAH is accompanied by a closure of the regulatory L: -Phe binding site. Concomitantly, the function of the regulatory ACT domain appears to be adapted from amino acid binding to serving the communication of conformational changes among catalytic subunits.
Assuntos
Caenorhabditis elegans/enzimologia , Fenilalanina Hidroxilase/química , Fenilalanina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Biocatálise , Calorimetria , Dicroísmo Circular , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fenilalanina Hidroxilase/genética , Fenilalanina Hidroxilase/metabolismo , Alinhamento de Sequência , EstereoisomerismoRESUMO
Aim: Tryptophan hydroxylase 1 (TPH1) catalyzes serotonin synthesis in peripheral tissues. Selective TPH1 inhibitors may be useful for treating disorders related to serotonin dysregulation. Results & methodology: Screening using a thermal shift assay for TPH1 binders yielded Compound 1 (2-(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one), which showed high potency (50% inhibition at 98 ± 30 nM) and selectivity for inhibiting TPH over related aromatic amino acid hydroxylases in enzyme activity assays. Structure-activity relationships studies revealed several analogs of 1 showing comparable potency. Kinetic studies suggested a noncompetitive mode of action of 1, with regards to tryptophan and tetrahydrobiopterin. Computational docking studies and live cell assays were also performed. Conclusion: This TPH1 inhibitor scaffold may be useful for developing new therapeutics for treating elevated peripheral serotonin.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Nervos Periféricos/efeitos dos fármacos , Serotonina/biossíntese , Tiazóis/farmacologia , Triptofano Hidroxilase/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Nervos Periféricos/metabolismo , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/química , Triptofano Hidroxilase/metabolismoRESUMO
Serotonin (5-HT) is a hormone and neurotransmitter that modulates neural activity as well as a wide range of other physiological processes including cardiovascular function, bowel motility, and platelet aggregation. 5-HT synthesis is catalyzed by tryptophan hydroxylase (TPH) which exists as two distinct isoforms; TPH1 and TPH2, which are responsible for peripheral and central 5-HT, respectively. Due to the implication of 5-HT in a number of pathologies, including depression, anxiety, autism, sexual dysfunction, irritable bowel syndrome, inflammatory bowel disease, and carcinoid syndrome, there has been a growing interest in finding modulators of these enzymes in recent years. We thus performed high-throughput screening (HTS) using a fluorescence-based thermal shift assay (DSF) to search the Prestwick Chemical Library containing 1,280 compounds, mostly FDA-approved drugs, for TPH1 binders. We here report the identification of omeprazole, a proton pump inhibitor, as an inhibitor of TPH1 and TPH2 with low micromolar potency and high selectivity over the other aromatic amino acid hydroxylases. The S-enantiomer of omeprazole, esomeprazole, has recently also been described as an inhibitor of monoamine oxidase-A (MAO-A), the main enzyme responsible for 5-HT degradation, albeit with lower potency compared to the effect on TPH1 and TPH2. In order to investigate the net effect of simultaneous inhibition of TPH and MAO-A in vivo, we administered high-dose (100 mg/kg) omeprazole to CD-1 mice for 4 days, after which the animals were subjected to the tail suspension test. Finally, central (whole brain) and peripheral (serum) 5-HT content was measured using liquid chromatography-mass spectrometry (LC-MS). Omeprazole treatment significantly increased 5-HT concentrations, both in brain and in serum, and reduced the time spent immobile in the tail suspension test relative to vehicle control. Thus, the MAO-A inhibition afforded by high-dose omeprazole appears to overcome the opposing effect on 5-HT produced by inhibition of TPH1 and TPH2. Further modification of proton pump inhibitor scaffolds may yield more selective modulators of 5-HT metabolism.
RESUMO
The metabolic network of sphingolipids plays important roles in cancer biology. Prominent sphingolipids include ceramides and sphingosine-1-phosphate that regulate multiple aspects of growth, apoptosis, and cellular signaling. Although a significant number of enzymatic regulators of the sphingolipid pathway have been described in detail, many remained poorly characterized. Here we applied a patient-derived systemic approach to identify and molecularly define progestin and adipoQ receptor family member IV (PAQR4) as a Golgi-localized ceramidase. PAQR4 was approximately 5-fold upregulated in breast cancer compared with matched control tissue and its overexpression correlated with disease-specific survival rates in breast cancer. Induction of PAQR4 in breast tumors was found to be subtype-independent and correlated with increased ceramidase activity. These findings establish PAQR4 as Golgi-localized ceramidase required for cellular growth in breast cancer. SIGNIFICANCE: Induction of and cellular dependency on de novo sphingolipid synthesis via PAQR4 highlights a central vulnerability in breast cancer that may serve as a viable therapeutic target.
Assuntos
Neoplasias da Mama/metabolismo , Ceramidases/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Animais , Apoptose/fisiologia , Neoplasias da Mama/patologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Xenoenxertos , Humanos , Lisofosfolipídeos/metabolismo , Células MCF-7 , Camundongos Endogâmicos NOD , Camundongos SCID , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Regulação para CimaRESUMO
Chromatin post-translational modifications are thought to be important for epigenetic effects on gene expression. Methylation of histone N-terminal tail lysine residues constitutes one of many such modifications, executed by families of histone lysine methyltransferase (HKMTase). One such protein is ASHH2 from the flowering plant Arabidopsis thaliana, equipped with the interaction domain, CW, and the HKMTase domain, SET. The CW domain of ASHH2 is a selective binder of monomethylation at lysine 4 on histone H3 (H3K4me1) and likely helps the enzyme dock correctly onto chromatin sites. The study of CW and related interaction domains has so far been emphasizing lock-key models, missing important aspects of histone-tail CW interactions. We here present an analysis of the ASHH2 CW-H3K4me1 complex using NMR and molecular dynamics, as well as mutation and affinity studies of flexible coils. ß-augmentation and rearrangement of coils coincide with changes in the flexibility of the complex, in particular the η1, η3 and C-terminal coils, but also in the ß1 and ß2 strands and the C-terminal part of the ligand. Furthermore, we show that mutating residues with outlier dynamic behaviour affect the complex binding affinity despite these not being in direct contact with the ligand. Overall, the binding process is consistent with conformational selection. We propose that this binding mechanism presents an advantage when searching for the correct post-translational modification state among the highly modified and flexible histone tails, and also that the binding shifts the catalytic SET domain towards the nucleosome. DATABASES: Structural data are available in the PDB database under the accession code 6QXZ. Resonance assignments for CW42 in its apo- and holo-forms are available in the BMRB database under the accession code 27251.
Assuntos
Arabidopsis/enzimologia , Histona-Lisina N-Metiltransferase/química , Histonas/química , Sítios de Ligação , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Processamento de Proteína Pós-TraducionalRESUMO
Hassallidins are cyclic glycolipopeptides produced by cyanobacteria and other prokaryotes. The hassallidin structure consists of a peptide ring of eight amino acids where a fatty acid chain, additional amino acids, and sugar moieties are attached. Hassallidins show antifungal activity against several opportunistic human pathogenic fungi, but does not harbor antibacterial effects. However, they have not been studied on mammalian cells, and the mechanism of action is unknown. We purified hassallidin D from cultured cyanobacterium Anabaena sp. UHCC 0258 and characterized its effect on mammalian and fungal cells. Ultrastructural analysis showed that hassallidin D disrupts cell membranes, causing a lytic/necrotic cell death with rapid presence of disintegrated outer membrane, accompanied by internalization of small molecules such as propidium iodide into the cells. Furthermore, artificial liposomal membrane assay showed that hassallidin D selectively targets sterol-containing membranes. Finally, in silico membrane modeling allowed us to study the interaction between hassallidin D and membranes in detail, and confirm the role of cholesterol for hassallidin-insertion into the membrane. This study demonstrates the mechanism of action of the natural compound hassallidin, and gives further insight into how bioactive lipopeptide metabolites selectively target eukaryotic cell membranes.
Assuntos
Antifúngicos/metabolismo , Antineoplásicos/metabolismo , Glicolipídeos/metabolismo , Glicopeptídeos/metabolismo , Lipopeptídeos/metabolismo , Lipídeos de Membrana/metabolismo , Esteróis/metabolismo , Anabaena/química , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Glicopeptídeos/isolamento & purificação , Glicopeptídeos/farmacologia , Humanos , Lipopeptídeos/isolamento & purificação , Lipopeptídeos/farmacologia , Membranas Mitocondriais/efeitos dos fármacosRESUMO
PURPOSE: To clinically and genetically characterise a second family with dominant ARL3-related retinitis pigmentosa due to a specific ARL3 missense variant, p.(Tyr90Cys). METHODS: Clinical examination included optical coherence tomography, electroretinography, and ultra-wide field retinal imaging with autofluorescence. Retrospective data were collected from the registry of inherited retinal diseases at Oslo university hospital. DNA was analysed by whole-exome sequencing and Sanger sequencing. The ARL3 missense variant was visualized in a 3D-protein structure. RESULTS: The phenotype was non-syndromic retinitis pigmentosa with cataract associated with early onset of decreased central vision and central retinal thinning. Sanger sequencing confirmed the presence of a de novo ARL3 missense variant p.(Tyr90Cys) in the index patient and his affected son. We did not find any other cases with rare ARL3 variants in a cohort of 431 patients with retinitis pigmentosa-like disease. By visualizing Tyr90 in the 3D protein structure, it seems to play an important role in packing of the α/ß structure of ADP-ribosylation factor-like 3 (ARL3). When changing Tyr90 to cysteine, we observe a loss of interactions in the core of the α/ß structure that is likely to affect folding and stability of ARL3. CONCLUSION: Our study confirms that the ARL3 missense variant p.(Tyr90Cys) causes retinitis pigmentosa. In 2016, Strom et al. reported the exact same variant in a mother and two children with RP, labelled ?RP83 in the OMIM database. Now the questionmark can be removed, and ARL3 should be added to the list of genes that may cause non-syndromic dominant retinitis pigmentosa.