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1.
Plant Cell ; 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31363039

RESUMO

Evolution of the C4 photosynthetic pathway involved in some cases recruitment of housekeeping proteins through gene duplication and their further neofunctionalization. NADP-malic enzyme (ME), the most widespread C4 decarboxylase, has increased its catalytic efficiency and acquired regulatory properties that allowed it to participate in the C4 pathway. Here, we show that regulation of maize C4-NADP-ME activity is much more elaborated than until now indicated. Using mass spectrometry, we identified phosphorylation of the serine 419 (S419) of C4-NADP-ME in protein extracts of maize leaves. The phosphorylation event increases after the light turns on, with a peak at ZT2. Phosphorylation of ZmC4-NADP-ME drastically decreases its activity as shown by the low residual activity of the recombinant phosphomimetic mutant. Analysis of the crystal structure of C4-NADP-ME indicated that S419 is involved in the binding of NADP at the active site. Molecular dynamics simulations and effective binding energy computations indicate a less favorable binding of the cofactor NADP in the phosphomimetic and the phosphorylated variants. We propose that phosphorylation of ZmC4-NADP-ME at S419 during the first hours in the light is a cellular mechanism to fine-tune the enzymatic activity to coordinate the carbon concentration mechanism with the CO2 fixation rate, most probably to avoid CO2 leakiness from bundle sheath cells.

2.
FEBS J ; 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31330084

RESUMO

Human guanylate-binding protein 1 (hGBP1) belongs to the family of dynamin-like proteins and is activated by addition of nucleotides, leading to protein oligomerization and stimulated GTPase activity. In vivo, hGBP1 is post-translationally modified by attachment of a farnesyl group yielding farn-hGBP1. In this study, hydrodynamic differences in farn-hGBP1 and unmodified hGBP1 were investigated using dynamic light scattering (DLS), analytical ultracentrifugation (AUC) and analytical size-exclusion chromatography (SEC). In addition, we performed small-angle X-ray scattering (SAXS) experiments coupled with a SEC setup (SEC-SAXS) to investigate structural properties of nonmodified hGBP1 and farn-hGBP1 in solution. SEC-SAXS measurements revealed that farnesylation keeps hGBP1 in its inactive monomeric and crystal-like conformation in nucleotide-free solution, whereas unmodified hGBP1 forms a monomer-dimer equilibrium both in the inactive ground state in nucleotide-free solution as well as in the activated state that is trapped by addition of the nonhydrolysable GTP analogue GppNHp. Nonmodified hGBP1 is structurally perturbed as compared to farn-hGBP. In particular, GppNHp binding leads to large structural rearrangements and higher conformational flexibility of the monomer and the dimer. Structural changes observed in the nonmodified protein are prerequisites for further oligomer assemblies of farn-hGBP1 that occur in the presence of nucleotides. DATABASE: All SEC-SAXS data, corresponding fits to the data and structural models are deposited in the Small Angle Scattering Biological Data Bank [SASBDB (Nucleic Acids Res, 43, 2015, D357)] with project IDs: SASDEE8, SASDEF8, SASDEG8, SASDEH8, SASDEJ8, SASDEK8, SASDEL8 and SASDEM8.

3.
Molecules ; 24(11)2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31195746

RESUMO

Amyloid-ß peptide (Aß) is an intrinsically disordered protein (IDP) associated with Alzheimer's disease. The structural flexibility and aggregation propensity of Aß pose major challenges for elucidating the interaction between Aß monomers and ligands. All-D-peptides consisting solely of D-enantiomeric amino acid residues are interesting drug candidates that combine high binding specificity with high metabolic stability. Here we characterized the interaction between the 12-residue all-D-peptide D3 and Aß42 monomers, and how the interaction influences Aß42 aggregation. We demonstrate for the first time that D3 binds to Aß42 monomers with submicromolar affinities. These two highly unstructured molecules are able to form complexes with 1:1 and other stoichiometries. Further, D3 at substoichiometric concentrations effectively slows down the ß-sheet formation and Aß42 fibrillation by modulating the nucleation process. The study provides new insights into the molecular mechanism of how D3 affects Aß assemblies and contributes to our knowledge on the interaction between two IDPs.

4.
Nat Plants ; 5(7): 755-765, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31235877

RESUMO

In C4 grasses of agronomical interest, malate shuttled into the bundle sheath cells is decarboxylated mainly by nicotinamide adenine dinucleotide phosphate (NADP)-malic enzyme (C4-NADP-ME). The activity of C4-NADP-ME was optimized by natural selection to efficiently deliver CO2 to Rubisco. During its evolution from a plastidic non-photosynthetic NADP-ME, C4-NADP-ME acquired increased catalytic efficiency, tetrameric structure and pH-dependent inhibition by its substrate malate. Here, we identified specific amino acids important for these C4 adaptions based on strict differential conservation of amino acids, combined with solving the crystal structures of maize and sorghum C4-NADP-ME. Site-directed mutagenesis and structural analyses show that Q503, L544 and E339 are involved in catalytic efficiency; E339 confers pH-dependent regulation by malate, F140 is critical for the stabilization of the oligomeric structure and the N-terminal region is involved in tetramerization. Together, the identified molecular adaptations form the basis for the efficient catalysis and regulation of one of the central biochemical steps in C4 metabolism.

5.
Sci Rep ; 9(1): 54, 2019 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-30631134

RESUMO

Sterile alpha motif (SAM) domains are protein interaction modules that are involved in a diverse range of biological functions such as transcriptional and translational regulation, cellular signalling, and regulation of developmental processes. SH3 domain-containing protein expressed in lymphocytes 1 (SLy1) is involved in immune regulation and contains a SAM domain of unknown function. In this report, the structure of the SLy1 SAM domain was solved and revealed that this SAM domain forms a symmetric homodimer through a novel interface. The interface consists primarily of the two long C-terminal helices, α5 and α5', of the domains packing against each other. The dimerization is characterized by a dissociation constant in the lower micromolar range. A SLy1 SAM domain construct with an extended N-terminus containing five additional amino acids of the SLy1 sequence further increases the stability of the homodimer, making the SLy1 SAM dimer two orders of magnitude more stable than previously studied SAM homodimers, suggesting that the SLy1 SAM dimerization is of functional significance. The SLy1 SAM homodimer contains an exposed mid-loop surface on each monomer, which may provide a scaffold for mediating interactions with other SAM domain-containing proteins via a typical mid-loop-end-helix interface.

6.
Blood ; 132(3): 307-320, 2018 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-29724897

RESUMO

Heat shock protein 90 (HSP90) stabilizes many client proteins, including the BCR-ABL1 oncoprotein. BCR-ABL1 is the hallmark of chronic myeloid leukemia (CML) in which treatment-free remission (TFR) is limited, with clinical and economic consequences. Thus, there is an urgent need for novel therapeutics that synergize with current treatment approaches. Several inhibitors targeting the N-terminal domain of HSP90 are under investigation, but side effects such as induction of the heat shock response (HSR) and toxicity have so far precluded their US Food and Drug Administration approval. We have developed a novel inhibitor (aminoxyrone [AX]) of HSP90 function by targeting HSP90 dimerization via the C-terminal domain. This was achieved by structure-based molecular design, chemical synthesis, and functional preclinical in vitro and in vivo validation using CML cell lines and patient-derived CML cells. AX is a promising potential candidate that induces apoptosis in the leukemic stem cell fraction (CD34+CD38-) as well as the leukemic bulk (CD34+CD38+) of primary CML and in tyrosine kinase inhibitor (TKI)-resistant cells. Furthermore, BCR-ABL1 oncoprotein and related pro-oncogenic cellular responses are downregulated, and targeting the HSP90 C terminus by AX does not induce the HSR in vitro and in vivo. We also probed the potential of AX in other therapy-refractory leukemias. Therefore, AX is the first peptidomimetic C-terminal HSP90 inhibitor with the potential to increase TFR in TKI-sensitive and refractory CML patients and also offers a novel therapeutic option for patients with other types of therapy-refractory leukemia because of its low toxicity profile and lack of HSR.

7.
Chem Commun (Camb) ; 54(26): 3294-3297, 2018 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-29537428

RESUMO

Pressure can shift the polymer-monomer equilibrium of Aß, increasing pressure first leads to a release of Aß-monomers, surprisingly at pressures higher than 180 MPa repolymerization is induced. By high pressure NMR spectroscopy, differences of partial molar volumes ΔV0 and compressibility factors Δß' of polymerization were determined at different temperatures. The d-enantiomeric peptides RD2 and RD2D3 bind to monomeric Aß with affinities substantially higher than those determined for fibril formation. By reducing the Aß concentration below the critical concentration for polymerization they inhibit the formation of toxic oligomers. Chemical shift perturbation allows the identification of the binding sites. The d-peptides are candidates for drugs preventing Alzheimer's disease. We show that RD2D3 has a positive effect on the cognitive behaviour of transgenic (APPSwDI) mice prone to Alzheimer's disease. The heterodimer complexes have a smaller Stokes radius than Aß alone indicating the recognition of a more compact conformation of Aß identified by high pressure NMR before.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Peptídeos/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Animais , Sítios de Ligação , Dimerização , Humanos , Camundongos , Camundongos Transgênicos , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/uso terapêutico , Ligação Proteica , Estereoisomerismo , Termodinâmica
8.
Int J Biol Macromol ; 113: 631-639, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29476859

RESUMO

The abnormal aggregation of amyloid ß (Aß) peptides in the brain has been recognized as a central event in Alzheimer's disease (AD). Divalent metal ions such as Zn2+ have been shown to be closely involved in modulating Aß self-association. Although the link between Zn2+ dyshomeostasis and brain Aß deposition has been established, the effect of Zn2+ on the aggregation of Aß is still incompletely clarified. By combining analytical ultracentrifugation (AUC), circular dichroism (CD) spectroscopy, thioflavin T (ThT) assay and atomic force microscopy (AFM) imaging, we analyzed the impact of stoichiometric Zn2+ on the aggregation process of Aß42, the main toxic isoform of Aß species in the brain. Aß42 aggregates found in the presence of Zn2+ were smaller in size, non-fibrillary and showed less ß-sheet structures than aggregates formed in absence of Zn2+. AUC showed that Zn2+ was capable of retaining monomeric Aß42 in solution. Zn2+ chelation by EDTA totally reversed the inhibitory effect of Zn2+ on Aß42 fibrillation. Our results provide further evidence that Zn2+ shifts the self-association of Aß42 toward a non-fibrillary pathway by interfering with the aggregation process at multiple levels.


Assuntos
Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Agregados Proteicos/efeitos dos fármacos , Zinco/farmacologia , Humanos , Zinco/química
9.
Sci Rep ; 7(1): 16275, 2017 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-29176708

RESUMO

While amyloid-ß protein (Aß) aggregation into insoluble plaques is one of the pathological hallmarks of Alzheimer's disease (AD), soluble oligomeric Aß has been hypothesized to be responsible for synapse damage, neurodegeneration, learning, and memory deficits in AD. Here, we investigate the in vitro and in vivo efficacy of the D-enantiomeric peptide RD2, a rationally designed derivative of the previously described lead compound D3, which has been developed to efficiently eliminate toxic Aß42 oligomers as a promising treatment strategy for AD. Besides the detailed in vitro characterization of RD2, we also report the results of a treatment study of APP/PS1 mice with RD2. After 28 days of treatment we observed enhancement of cognition and learning behaviour. Analysis on brain plaque load did not reveal significant changes, but a significant reduction of insoluble Aß42. Our findings demonstrate that RD2 was significantly more efficient in Aß oligomer elimination in vitro compared to D3. Enhanced cognition without reduction of plaque pathology in parallel suggests that synaptic malfunction due to Aß oligomers rather than plaque pathology is decisive for disease development and progression. Thus, Aß oligomer elimination by RD2 treatment may be also beneficial for AD patients.

10.
Sci Rep ; 7(1): 2493, 2017 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-28559586

RESUMO

Amyloid ß (Aß) oligomers may play a decisive role in Alzheimer's disease related neurodegeneration, but their structural properties are poorly understood. In this report, sedimentation velocity centrifugation, small angle neutron scattering (SANS) and molecular modelling were used to identify the small oligomeric species formed by the 42 amino acid residue long isoform of Aß (Aß42) in solution, characterized by a sedimentation coefficient of 2.56 S, and a radius of gyration between 2 and 4 nm. The measured sedimentation coefficient is in close agreement with the sedimentation coefficient calculated for Aß42 hexamers using MD simulations at µM concentration. To the best of our knowledge this is the first report detailing the Aß42 oligomeric species by SANS measurements. Our results demonstrate that the smallest detectable species in solution are penta- to hexamers. No evidences for the presence of dimers, trimers or tetramers were found, although the existence of those Aß42 oligomers at measurable quantities had been reported frequently.

11.
PLoS One ; 12(5): e0177024, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28472107

RESUMO

Ethylenediaminetetraacetic acid (EDTA) is widely used in the life sciences as chelating ligand of metal ions. However, formation of supramolecular EDTA aggregates at pH > 8 has been reported, which may lead to artifactual assay results. When applied as a buffer component at pH ≈ 10 in differential scanning fluorimetry (TSA) using SYPRO Orange as fluorescent dye, we observed a sharp change in fluorescence intensity about 20°C lower than expected for the investigated protein. We hypothesized that this change results from SYPRO Orange/EDTA interactions. TSA experiments in the presence of SYPRO Orange using solutions that contain EDTA-Na+ but no protein were performed. The TSA experiments provide evidence that suggests that at pH > 9, EDTA4- interacts with SYPRO Orange in a temperature-dependent manner, leading to a fluorescence signal yielding a "denaturation temperature" of ~68°C. Titrating Ca2+ to SYPRO Orange and EDTA solutions quenched fluorescence. Ethylene glycol tetraacetic acid (EGTA) behaved similarly to EDTA. Analytical ultracentrifugation corroborated the formation of EDTA aggregates. Molecular dynamics simulations of free diffusion of EDTA-Na+ and SYPRO Orange of in total 27 µs suggested the first structural model of EDTA aggregates in which U-shaped EDTA4- arrange in an inverse bilayer-like manner, exposing ethylene moieties to the solvent, with which SYPRO Orange interacts. We conclude that EDTA aggregates induce a SYPRO Orange-based fluorescence in TSA. These results make it relevant to ascertain that future TSA results are not influenced by interference between EDTA, or EDTA-related molecules, and the fluorescent dye.


Assuntos
Ácido Edético/química , Corantes Fluorescentes/química , Dicroísmo Circular , Fluorescência , Fluorometria/métodos , Simulação de Dinâmica Molecular , Estrutura Molecular , Proteínas/química
12.
J Biol Chem ; 292(23): 9583-9598, 2017 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-28438837

RESUMO

Aggregation of specific proteins in the brains of patients with chronic mental illness as a result of disruptions in proteostasis is an emerging theme in the study of schizophrenia in particular. Proteins including DISC1 (disrupted in schizophrenia 1) and dysbindin-1B are found in insoluble forms within brain homogenates from such patients. We recently identified TRIOBP-1 (Trio-binding protein 1, also known as Tara) to be another such protein through an epitope discovery and proteomics approach by comparing post-mortem brain material from schizophrenia patients and control individuals. We hypothesized that this was likely to occur as a result of a specific subcellular process and that it, therefore, should be possible to identify a region of the TRIOBP-1 protein that is essential for its aggregation to occur. Here, we probe the domain organization of TRIOBP-1, finding it to possess two distinct coiled-coil domains: the central and C-terminal domains. The central domain inhibits the depolymerization of F-actin and is also responsible for oligomerization of TRIOBP-1. Along with an N-terminal pleckstrin homology domain, the central domain affects neurite outgrowth. In neuroblastoma cells it was found that the aggregation propensity of TRIOBP-1 arises from its central domain, with a short "linker" region narrowed to within amino acids 324-348, between its first two coiled coils, as essential for the formation of TRIOBP-1 aggregates. TRIOBP-1 aggregation, therefore, appears to occur through one or more specific cellular mechanisms, which therefore have the potential to be of physiological relevance for the biological process underlying the development of chronic mental illness.


Assuntos
Actinas , Proteínas dos Microfilamentos , Agregação Patológica de Proteínas , Actinas/química , Actinas/genética , Actinas/metabolismo , Linhagem Celular Tumoral , Humanos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Domínios de Homologia à Plecstrina , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo
13.
J Biol Chem ; 292(16): 6468-6477, 2017 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-28249940

RESUMO

Disrupted in Schizophrenia 1 (DISC1) is a scaffolding protein of significant importance for neurodevelopment and a prominent candidate protein in the pathology of major mental illness. DISC1 modulates a number of critical neuronal signaling pathways through protein-protein interactions; however, the mechanism by which this occurs and how DISC1 causes mental illness is unclear, partly because knowledge of the structure of DISC1 is lacking. A lack of homology with known proteins has hindered attempts to define its domain composition. Here, we employed the high-throughput Expression of Soluble Proteins by Random Incremental Truncation (ESPRIT) technique to identify discretely folded regions of human DISC1 via solubility assessment of tens of thousands of fragments of recombinant DISC1. We identified four novel structured regions, named D, I, S, and C, at amino acids 257-383, 539-655, 635-738, and 691-836, respectively. One region (D) is located in a DISC1 section previously predicted to be unstructured. All regions encompass coiled-coil or α-helical structures, and three are involved in DISC1 oligomerization. Crucially, three of these domains would be lost or disrupted by a chromosomal translocation event after amino acid 597, which has been strongly linked to major mental illness. Furthermore, we observed that a known illness-related frameshift mutation after amino acid 807 causes the C region to form aberrantly multimeric and aggregated complexes with an unstable secondary structure. This newly revealed domain architecture of DISC1, therefore, provides a powerful framework for understanding the critical role of this protein in a variety of devastating mental illnesses.


Assuntos
Mutação , Proteínas do Tecido Nervoso/química , Transtornos Psicóticos/genética , Esquizofrenia/genética , Mutação da Fase de Leitura , Humanos , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Desnaturação Proteica , Domínios Proteicos , Dobramento de Proteína , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Transdução de Sinais
14.
J Biol Chem ; 291(43): 22806-22818, 2016 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-27551038

RESUMO

Chlamydiae sp. are obligate intracellular pathogens that cause a variety of diseases in humans. The adhesion of Chlamydiae to the eukaryotic host cell is a pivotal step in pathogenesis. The adhesin family of polymorphic membrane proteins (Pmp) in Chlamydia pneumoniae consists of 21 members. Pmp21 binds to the epidermal growth factor receptor (EGFR). Pmps contain large numbers of FXXN (where X is any amino acid) and GGA(I/L/V) motifs. At least two of these motifs are crucial for adhesion by certain Pmp21 fragments. Here we describe how the two FXXN motifs in Pmp21-D (D-Wt), a domain of Pmp21, influence its self-interaction, folding, and adhesive capacities. Refolded D-Wt molecules form oligomers with high sedimentation values (8-85 S). These oligomers take the form of elongated protofibrils, which exhibit Thioflavin T fluorescence, like the amyloid protein fragment ß42. A mutant version of Pmp21-D (D-Mt), with FXXN motifs replaced by SXXV, shows a markedly reduced capacity to form oligomers. Secondary-structure assays revealed that monomers of both variants exist predominantly as random coils, whereas the oligomers form predominantly ß-sheets. Adhesion studies revealed that oligomers of D-Wt (D-Wt-O) mediate significantly enhanced binding to human epithelial cells relative to D-Mt-O and monomeric protein species. Moreover, D-Wt-O binds EGFR more efficiently than D-Wt monomers. Importantly, pretreatment of human cells with D-Wt-O reduces infectivity upon subsequent challenge with C. pneumoniae more effectively than all other protein species. Hence, the FXXN motif in D-Wt induces the formation of ß-sheet-rich oligomeric protofibrils, which are important for adhesion to, and subsequent infection of human cells.


Assuntos
Adesinas Bacterianas/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Chlamydophila pneumoniae/metabolismo , Multimerização Proteica/fisiologia , Adesinas Bacterianas/genética , Motivos de Aminoácidos , Substituição de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Chlamydophila pneumoniae/genética , Receptores ErbB/genética , Humanos , Mutação de Sentido Incorreto , Ligação Proteica
15.
ACS Nano ; 10(8): 7582-97, 2016 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-27404114

RESUMO

Therapeutically active small molecules represent promising nonimmunogenic alternatives to antibodies for specifically targeting disease-relevant receptors. However, a potential drawback compared to antibody-antigen interactions may be the lower affinity of small molecules toward receptors. Here, we overcome this low-affinity problem by coating the surface of nanoparticles (NPs) with multiple ligands. Specifically, we explored the use of gold and platinum nanoparticles to increase the binding affinity of Aß-specific small molecules to inhibit Aß peptide aggregation into fibrils in vitro. The interactions of bare NPs, free ligands, and NP-bound ligands with Aß are comprehensively studied via physicochemical methods (spectroscopy, microscopy, immunologic tests) and cell assays. Reduction of thioflavin T fluorescence, as an indicator for ß-sheet content, and inhibition of cellular Aß excretion are even more effective with NP-bound ligands than with the free ligands. The results from this study may have implications in the development of therapeutics for treating Alzheimer's disease.


Assuntos
Peptídeos beta-Amiloides/química , Ligantes , Nanopartículas Metálicas , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/terapia , Ouro , Fragmentos de Peptídeos
16.
ACS Chem Neurosci ; 7(8): 1088-96, 2016 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-27240424

RESUMO

Alzheimer's disease (AD) is a neurodegenerative disorder and the most common type of dementia. Until now, there is no curative therapy available. Previously, we selected the amyloid-beta (Aß) targeting peptide D3 consisting of 12 d-enantiomeric amino acid residues by mirror image phage display as a potential drug candidate for the treatment of AD. In the current approach, we investigated the optimization potential of linear D3 with free C-terminus (D3COOH) by chemical modifications. First, the impact of the net charge was investigated and second, cyclization was introduced which is a well-known tool for the optimization of peptides for enhanced target affinity. Following this strategy, three D3 derivatives in addition to D3COOH were designed: C-terminally amidated linear D3 (D3CONH2), cyclic D3 (cD3), and cyclic D3 with an additional arginine residue (cD3r) to maintain the net charge of linear D3CONH2. These four compounds were compared to each other according to their binding affinities to Aß(1-42), their efficacy to eliminate cytotoxic oligomers, and consequently their potency to neutralize Aß(1-42) oligomer induced neurotoxicity. D3CONH2 and cD3r versions with equally increased net charge showed superior properties over D3COOH and cD3, respectively. The cyclic versions showed superior properties compared to their linear version with equal net charge, suggesting cD3r to be the most efficient compound among these four. Indeed, treatment of the transgenic AD mouse model Tg-SwDI with cD3r significantly enhanced spatial memory and cognition of these animals as revealed by water maze performance. Therefore, charge increase and cyclization imply suitable modification steps for an optimization approach of the Aß targeting compound D3.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Peptídeos Cíclicos/química , Peptídeos Cíclicos/uso terapêutico , Doença de Alzheimer/complicações , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/ultraestrutura , Precursor de Proteína beta-Amiloide/genética , Animais , Linhagem Celular Tumoral , Transtornos Cognitivos/tratamento farmacológico , Transtornos Cognitivos/etiologia , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Conformação Molecular/efeitos dos fármacos , Mutação/genética , Neuroblastoma/patologia , Oligopeptídeos/química , Fragmentos de Peptídeos/ultraestrutura , Peptídeos Cíclicos/metabolismo , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Placa Amiloide/ultraestrutura , Ligação Proteica/efeitos dos fármacos , Estereoisomerismo
17.
Chembiochem ; 17(8): 657-76, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26910367

RESUMO

The deposition of amyloid in brain tissue in the context of neurodegenerative diseases involves the formation of intermediate species-termed oligomers-of lower molecular mass and with structures that deviate from those of mature amyloid fibrils. Because these oligomers are thought to be primarily responsible for the subsequent disease pathogenesis, the elucidation of their structure is of enormous interest. Nevertheless, because of the high aggregation propensity and the polydispersity of oligomeric species formed by the proteins or peptides in question, the preparation of appropriate samples for high-resolution structural methods has proven to be rather difficult. This is why theoretical approaches have been of particular importance in gaining insights into possible oligomeric structures for some time. Only recently has it been possible to achieve some progress with regard to the experimentally based structural characterization of defined oligomeric species. Here we discuss how theory and experiment are used to determine oligomer structures and what can be done to improve the integration of the two disciplines.


Assuntos
Amiloide/química , Simulação de Dinâmica Molecular , Amiloide/metabolismo , Humanos , Doenças Neurodegenerativas/metabolismo , Ligação Proteica , Conformação Proteica
18.
PLoS One ; 11(2): e0150267, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26919121

RESUMO

Small proteins like amyloid beta (Aß) monomers are related to neurodegenerative disorders by aggregation to insoluble fibrils. Small angle neutron scattering (SANS) is a nondestructive method to observe the aggregation process in solution. We show that SANS is able to resolve monomers of small molecular weight like Aß for aggregation studies. We examine Aß monomers after prolonged storing in d-hexafluoroisopropanol (dHFIP) by using SANS and dynamic light scattering (DLS). We determined the radius of gyration from SANS as 1.0±0.1 nm for Aß1-40 and 1.6±0.1 nm for Aß1-42 in agreement with 3D NMR structures in similar solvents suggesting a solvent surface layer with 5% increased density. After initial dissolution in dHFIP Aß aggregates sediment with a major component of pure monomers showing a hydrodynamic radius of 1.8±0.3 nm for Aß1-40 and 3.2±0.4 nm for Aß1-42 including a surface layer of dHFIP solvent molecules.


Assuntos
Peptídeos beta-Amiloides/análise , Difração de Nêutrons , Fragmentos de Peptídeos/análise , Espalhamento a Baixo Ângulo , Peptídeos beta-Amiloides/química , Humanos , Hidrodinâmica , Ligações de Hidrogênio , Modelos Químicos , Modelos Moleculares , Fragmentos de Peptídeos/química , Propanóis , Agregação Patológica de Proteínas , Conformação Proteica , Solventes
19.
PLoS One ; 10(12): e0143634, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26624888

RESUMO

Nucleophosmin (NPM1, also known as B23, numatrin or NO38) is a pentameric RNA-binding protein with RNA and protein chaperon functions. NPM1 has increasingly emerged as a potential cellular factor that directly associates with viral proteins; however, the significance of these interactions in each case is still not clear. In this study, we have investigated the physical interaction of NPM1 with both human immunodeficiency virus type 1 (HIV-1) Rev and Herpes Simplex virus type 1 (HSV-1) US11, two functionally homologous proteins. Both viral proteins show, in mechanistically different modes, high affinity for a binding site on the N-terminal oligomerization domain of NPM1. Rev, additionally, exhibits low-affinity for the central histone-binding domain of NPM1. We also showed that the proapoptotic cyclic peptide CIGB-300 specifically binds to NPM1 oligomerization domain and blocks its association with Rev and US11. Moreover, HIV-1 virus production was significantly reduced in the cells treated with CIGB-300. Results of this study suggest that targeting NPM1 may represent a useful approach for antiviral intervention.


Assuntos
Fenômenos Biofísicos , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Animais , Células COS , Cercopithecus aethiops , HIV-1 , Células HeLa , Humanos , Modelos Moleculares , Proteínas Nucleares/química , Peptídeos Cíclicos/metabolismo , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas de Ligação a RNA/química , Proteínas Virais/química , Produtos do Gene rev do Vírus da Imunodeficiência Humana/química
20.
Sci Rep ; 5: 13222, 2015 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-26394756

RESUMO

Strong evidence exists for a central role of amyloid ß-protein (Aß) oligomers in the pathogenesis of Alzheimer's disease. We have developed a fast, reliable and robust in vitro assay, termed QIAD, to quantify the effect of any compound on the Aß aggregate size distribution. Applying QIAD, we studied the effect of homotaurine, scyllo-inositol, EGCG, the benzofuran derivative KMS88009, ZAß3W, the D-enantiomeric peptide D3 and its tandem version D3D3 on Aß aggregation. The predictive power of the assay for in vivo efficacy is demonstrated by comparing the oligomer elimination efficiency of D3 and D3D3 with their treatment effects in animal models of Alzheimer´s disease.


Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/metabolismo , Agregação Patológica de Proteínas/tratamento farmacológico , Animais , Proteínas de Transporte/farmacologia , Catequina/análogos & derivados , Catequina/farmacologia , Modelos Animais de Doenças , Ferredoxina-NADP Redutase/farmacologia , Humanos , Inositol/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Oligopeptídeos/farmacologia , Taurina/análogos & derivados , Taurina/farmacologia
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