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1.
Biochemistry ; 58(6): 590-607, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30489059

RESUMO

Intraneuronal aggregation of TDP-43 is seen in 97% of all amyotrophic lateral sclerosis cases and occurs by a poorly understood mechanism. We developed a simple in vitro model system for the study of full-length TDP-43 aggregation in solution and in protein droplets. We found that soluble, YFP-tagged full-length TDP-43 (yTDP-43) dimers can be produced by refolding in low-salt HEPES buffer; these solutions are stable for several weeks. We found that physiological electrolytes induced reversible aggregation of yTDP-43 into 10-50 nm tufted particles, without amyloid characteristics. The order of aggregation induction potency was K+ < Na+ < Mg2+ < Ca2+, which is the reverse of the Hofmeister series. The kinetics of aggregation were fit to a single-step model, and the apparent rate of aggregation was affected by yTDP-43 and NaCl concentrations. While yTDP-43 alone did not form stable liquid droplets, it partitioned into preformed Ddx4N1 droplets, showing dynamic diffusion behavior consistent with liquid-liquid phase transition, but then aggregated over time. Aggregation of yTDP-43 in droplets also occurred rapidly in response to changes in electrolyte concentrations, mirroring solution behavior. This was accompanied by changes to droplet localization and solvent exchange. Exposure to extracellular-like electrolyte conditions caused rapid aggregation at the droplet periphery. The aggregation behavior of yTDP-43 is controlled by ion-specific effects that occur at physiological concentrations, suggesting a mechanistic role for local electrolyte concentrations in TDP-43 proteinopathies.


Assuntos
Amiloide/química , Proteínas de Ligação a DNA/química , Eletrólitos/farmacologia , Gotículas Lipídicas/efeitos dos fármacos , Agregados Proteicos/efeitos dos fármacos , Amiloide/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Luminescentes/metabolismo
2.
J Vis Exp ; (127)2017 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-28892031

RESUMO

Immunofluorescence is a common method used to visualize subcellular compartments and to determine the localization of specific proteins within a tissue sample. A great hindrance to the acquisition of high quality immunofluorescence images is endogenous autofluorescence of the tissue caused by aging pigments such as lipofuscin or by common sample preparation processes such as aldehyde fixation. This protocol describes how background fluorescence can be greatly reduced through photobleaching using white phosphor light emitting diode (LED) arrays prior to treatment with fluorescent probes. The broad-spectrum emission of white phosphor LEDs allow for bleaching of fluorophores across a range of emission peaks. The photobleaching apparatus can be constructed from off-the-shelf components at very low cost and offers an accessible alternative to commercially available chemical quenchers. A photobleaching pre-treatment of the tissue followed by conventional immunofluorescence staining generates images free of background autofluorescence. Compared to established chemical quenchers which reduced probe as well as background signals, photobleaching treatment had no effect on probe fluorescence intensity while it effectively reduced background and lipofuscin fluorescence. Although photobleaching requires more time for pre-treatment, higher intensity LED arrays may be used to reduce photobleaching time. This simple method can potentially be applied to a variety of tissues, particularly postmitotic tissues that accumulate lipofuscin such as the brain and cardiac or skeletal muscles.


Assuntos
Encéfalo/diagnóstico por imagem , Formaldeído/uso terapêutico , Técnicas Histológicas/métodos , Microscopia de Fluorescência/métodos , Coloração e Rotulagem/métodos , Encéfalo/citologia , Encéfalo/patologia , Humanos
3.
Protein Eng Des Sel ; 30(6): 431-440, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28475686

RESUMO

In many cases of familial amyotrophic lateral sclerosis (ALS), mutant forms of the Cu,Zn superoxide dismutase protein (SOD1) misfold and aggregate in motor neurons. Monomers of the normally homodimeric SOD1 have been found in patient tissue, presymptomatic mouse models of ALS, and in vitro misfolding assays which suggests that monomerization might be an early step in the pathological SOD1 misfolding pathway. In this study, we targeted the dimer interface with small molecules that might act as chemical chaperones to stabilize the native dimer and prevent downstream misfolding and aggregation. We performed a computational screen with a library of ~4400 drugs and natural compounds that were docked to two pockets around the SOD1 dimer interface. Of the resultant hits, seven were tested for misfolding and aggregation inhibition activity with A4V mutant SOD1. Quercitrin, quercetin-3-ß-d-glucoside (Q3BDG), and, to a markedly lesser extent, epigallocatechin gallate (EGCG) were found to combat misfolding and aggregation induced by hydrogen peroxide, a physiologically relevant stress, as assessed by a gel-based assay and 8-anilinonaphthalene-1-suflonic acid (ANS) fluorescence. Isothermal titration calorimetry (ITC) and a colourimetric assay determined that these molecules directly bind A4V SOD1. Based on these findings, we speculate that quercitrin and Q3BDG may be potential therapeutic inhibitors of misfolding and aggregation in SOD1-associated ALS.


Assuntos
Chaperonas Moleculares/metabolismo , Quercetina/análogos & derivados , Quercetina/metabolismo , Superóxido Dismutase-1/metabolismo , Descoberta de Drogas , Chaperonas Moleculares/química , Simulação de Acoplamento Molecular , Mutação , Dobramento de Proteína , Quercetina/química , Superóxido Dismutase-1/química , Superóxido Dismutase-1/genética
4.
Clin Sci (Lond) ; 131(5): 395-409, 2017 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-28213611

RESUMO

Transthyretin (TTR) amyloidosis (ATTR amyloidosis) is an underdiagnosed and important type of cardiomyopathy and/or polyneuropathy that requires increased awareness within the medical community. Raising awareness among clinicians about this type of neuropathy and lethal form of heart disease is critical for improving earlier diagnosis and the identification of patients for treatment. The following review summarizes current criteria used to diagnose both hereditary and wild-type ATTR (ATTRwt) amyloidosis, tools available to clinicians to improve diagnostic accuracy, available and newly developing therapeutics, as well as a brief biochemical and biophysical background of TTR amyloidogenesis.


Assuntos
Neuropatias Amiloides Familiares/terapia , Neuropatias Amiloides Familiares/diagnóstico , Neuropatias Amiloides Familiares/etiologia , Cardiomiopatias/etiologia , Humanos
5.
Biochemistry ; 56(6): 809-823, 2017 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-28112502

RESUMO

TDP-43 is a dimeric nuclear protein that plays a central role in RNA metabolism. In recent years, this protein has become a focal point of research in the amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) disease spectrum, as pathognomonic inclusions within affected neurons contain post-translationally modified TDP-43. A key question in TDP-43 research involves determining the mechanisms and triggers that cause TDP-43 to form pathological aggregates. This review gives a brief overview of the physiological and pathological roles of TDP-43 and focuses on the structural features of its protein domains and how they may contribute to normal protein function and to disease. A special emphasis is placed on the C-terminal prion-like region thought to be implicated in pathology, as it is where nearly all ALS/FTD-associated mutations reside. Recent structural studies of this domain revealed its crucial role in the formation of phase-separated liquid droplets through a partially populated α-helix. This new discovery provides further support for the theory that liquid droplets such as stress granules may be precursors to pathological aggregates, linking environmental effects such as stress to the potential etiology of the disease. The transition of TDP-43 among soluble, droplet, and aggregate phases and the implications of these transitions for pathological aggregation are summarized and discussed.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Agregação Patológica de Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Humanos , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Transição de Fase , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologia , Conformação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética
7.
Sci Rep ; 6: 25080, 2016 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-27122057

RESUMO

Wild-type and mutant transthyretin (TTR) can misfold and deposit in the heart, peripheral nerves, and other sites causing amyloid disease. Pharmacological chaperones, Tafamidis(®) and diflunisal, inhibit TTR misfolding by stabilizing native tetrameric TTR; however, their minimal effective concentration is in the micromolar range. By immune-targeting sparsely populated TTR misfolding intermediates (i.e. monomers), we achieved fibril inhibition at substoichiometric concentrations. We developed an antibody (misTTR) that targets TTR residues 89-97, an epitope buried in the tetramer but exposed in the monomer. Nanomolar misTTR inhibits fibrillogenesis of misfolded TTR under micromolar concentrations. Pan-specific TTR antibodies do not possess such fibril inhibiting properties. We show that selective targeting of misfolding intermediates is an alternative to native state stabilization and requires substoichiometric concentrations. MisTTR or its derivative may have both diagnostic and therapeutic potential.


Assuntos
Anticorpos/imunologia , Pré-Albumina/imunologia , Pré-Albumina/metabolismo , Dobramento de Proteína , Amiloidose/diagnóstico , Amiloidose/terapia , Animais , Imunoterapia/métodos , Ligação Proteica , Coelhos
9.
Elife ; 42015 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-26418743

RESUMO

Spatially targeted optical microproteomics (STOMP) is a novel proteomics technique for interrogating micron-scale regions of interest (ROIs) in mammalian tissue, with no requirement for genetic manipulation. Methanol or formalin-fixed specimens are stained with fluorescent dyes or antibodies to visualize ROIs, then soaked in solutions containing the photo-tag: 4-benzoylbenzyl-glycyl-hexahistidine. Confocal imaging along with two photon excitation are used to covalently couple photo-tags to all proteins within each ROI, to a resolution of 0.67 µm in the xy-plane and 1.48 µm axially. After tissue solubilization, photo-tagged proteins are isolated and identified by mass spectrometry. As a test case, we examined amyloid plaques in an Alzheimer's disease (AD) mouse model and a post-mortem AD case, confirming known plaque constituents and discovering new ones. STOMP can be applied to various biological samples including cell lines, primary cell cultures, ex vivo specimens, biopsy samples, and fixed post-mortem tissue.


Assuntos
Espectrometria de Massas , Microscopia Confocal/métodos , Doenças Neurodegenerativas/patologia , Imagem Óptica/métodos , Proteínas/análise , Proteômica/métodos , Animais , Camundongos
10.
Acta Neuropathol ; 130(1): 49-61, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25788357

RESUMO

The presence of lower molecular weight species comprising the C-terminal region of TAR DNA-binding protein 43 (TDP-43) is a characteristic of TDP-43 proteinopathy in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Here, we have identified a novel splice variant of TDP-43 that is upregulated in ALS and generates a 35-kDa N-terminally truncated species through use of an alternate translation initiation codon (ATG(Met85)), denoted here as Met(85)-TDP-35. Met(85)-TDP-35 expressed ectopically in human neuroblastoma cells exhibited reduced solubility, cytoplasmic distribution, and aggregation. Furthermore, Met(85)-TDP-35 sequestered full-length TDP-43 from the nucleus to form cytoplasmic aggregates. Expression of Met(85)-TDP-35 in primary motor neurons resulted in the formation of Met(85)-TDP-35-positive cytoplasmic aggregates and motor neuron death. A neo-epitope antibody specific for Met(85)-TDP-35 labeled the 35-kDa lower molecular weight species on immunoblots of urea-soluble extracts from ALS-FTLD disease-affected tissues and co-labeled TDP-43-positive inclusions in ALS spinal cord sections, confirming the physiological relevance of this species. These results show that the 35-kDa low molecular weight species in ALS-FTLD can be generated from an abnormal splicing event and use of a downstream initiation codon and may represent a mechanism by which TDP-43 elicits its pathogenicity.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Morte Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Degeneração Lobar Frontotemporal/metabolismo , Corpos de Inclusão/metabolismo , Neurônios Motores/metabolismo , Idoso , Idoso de 80 Anos ou mais , Processamento Alternativo , Sequência de Aminoácidos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Sequência de Bases , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Citoplasma/metabolismo , Citoplasma/patologia , Proteínas de Ligação a DNA/genética , Feminino , Degeneração Lobar Frontotemporal/genética , Degeneração Lobar Frontotemporal/patologia , Humanos , Corpos de Inclusão/patologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Peso Molecular , Neurônios Motores/patologia , Medula Espinal/metabolismo , Medula Espinal/patologia
11.
Arch Biochem Biophys ; 545: 53-62, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24440310

RESUMO

TDP-43 is a nuclear protein whose abnormal aggregates are implicated in ALS and FTLD. Recently, an Asn/Gln rich C-terminal segment of TDP-43 has been shown to produce aggregation in vitro and reproduce most of the protein's pathological hallmarks in cells, but little is known about this segment's structure. Here, CD and 2D heteronuclear NMR spectroscopies provide evidence that peptides corresponding to the wild type and mutated sequences of this segment adopt chiefly disordered conformations that, in the case of the wild type sequence, spontaneously forms a ß-sheet rich oligomer. Moreover, MD simulation provides evidence for a structure consisting of two ß-strands and a well-defined, yet non-canonical structural element. Furthermore, MD simulations of four pathological mutations (Q343R, N345K, G348V and N352S) occurring in this segment predict that all of them could affect this region's structure. In particular, the Q343R variant tends to stabilize disordered conformers, N345K permits the formation of longer, more stable ß-strands, and G348V tends to shorten and destabilize them. Finally, N352S acts to alter the ß-stand register and when S352 is phosphorylated, it induces partial unfolding. Our results provide a better understanding of TDP-43 aggregation process and will be useful to design effectors capable to modulate its progression.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Sequência de Aminoácidos , Esclerose Lateral Amiotrófica/genética , Humanos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Fosforilação , Mutação Puntual , Conformação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína
12.
Neurobiol Dis ; 62: 479-88, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24200866

RESUMO

Mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) are responsible for a subset of amyotrophic lateral sclerosis cases presumably by the acquisition of as yet unknown toxic properties. Additional overexpression of wild-type SOD1 in mutant SOD1 transgenic mice did not improve but rather accelerated the disease course. Recently, it was documented that the presence of wild-type SOD1 (SOD(WT)) reduced the aggregation propensity of mutant SOD1 by the formation of heterodimers between mutant and SOD1(WT) and that these heterodimers displayed at least a similar toxicity in cellular and animal models. In this study we investigated the biochemical and biophysical properties of obligate SOD1 dimers that were connected by a peptide linker. Circular dichroism spectra indicate an increased number of unstructured residues in SOD1 mutants. However, SOD1(WT) stabilized the folding of heterodimers compared to mutant homodimers as evidenced by an increase in resistance against proteolytic degradation. Heterodimerization also reduced the affinity of mutant SOD1 to antibodies detecting misfolded SOD1. In addition, the formation of obligate dimers resulted in a detection of substantial dismutase activity even of the relatively labile SOD1(G85R) mutant. These data indicate that soluble, dismutase-active SOD1 dimers might contribute at least partially to mutant SOD1 toxicity.


Assuntos
Superóxido Dismutase/química , Células HEK293 , Humanos , Mutação , Multimerização Proteica , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1
13.
Arch Biochem Biophys ; 537(1): 62-71, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23811197

RESUMO

The capacity of three designed duodecamer peptides with the low diversity sequence: H1ϕ2I3K4I5D6G7K8ϕ9I10K11H12 where ϕ is His, Phe or Trp, to adopt a ß-hairpin conformation was studied using NMR spectroscopy. Whereas KIAßH, the variant with His at positions two and nine, is disordered, KIAßF, the peptide with Phe at these positions, adopts a small population of ß-hairpin. A high population of ß-hairpin structure was detected for KIAßW, the variant with Trp. Utilizing NMR data, the structure of KIAßW was solved and it reveals a ß-hairpin stabilized by hydrophobic interactions between Ile residues on one face and Trp-Trp and cation-π interactions on the opposite face. Upon adding ATP, these peptides show chemical shift changes indicative of ATP binding. The binding of ATP to KIAßW shows a KD ≈ 20 µM at pH 5, 5 °C and has a 1:1 stoichiometry. The KIAßW-ATP complex was determined using NMR spectroscopy and reveals the adenine ring sandwiched between the two Trp indole rings and that ATP binding induces important conformational changes in His1, Trp2, Lys4, Trp9 and Lys11 in the ß-hairpin. The implications of these results for the hypothetic presence of ß-hairpins and amyloids alongside RNAs on the prebiotic Earth are discussed.


Assuntos
Trifosfato de Adenosina/química , Modelos Químicos , Modelos Moleculares , Peptídeos/química , Motivos de Aminoácidos , Simulação por Computador , Conformação Proteica
14.
PLoS One ; 8(5): e63047, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23675452

RESUMO

Susceptibility of a particular species to prion disease is affected by small differences in the sequence of PrP and correlates with the propensity of its PrP to assume the ß-state. A helix-cap motif in the ß2-α2-loop of native α-helical rabbit PrP, a resistant species, contains sequence differences that influence intra- and interspecies transmission. To determine the effect the helix-cap motif on ß-state refolding propensity, we mutated S170N, S174N, and S170N/S174N of the rabbit PrP helix-cap to resemble that of hamster PrP and conversely, N170S, N174S, and N170S/N174S of hamster PrP to resemble the helix-cap of rabbit PrP. High-resolution crystal structures (1.45-1.6 Å) revealed that these mutations ablate hydrogen-bonding interactions within the helix-cap motif in rabbit PrP(C). They also alter the ß-state-misfolding propensity of PrP; the serine mutations in hamster PrP decrease the propensity up to 35%, whereas the asparagine mutations in rabbit PrP increase it up to 42%. Rapid dilution of rabbit and hamster into ß-state buffer conditions causes quick conversion to ß-state monomers. Kinetic monitoring using size-exclusion chromatography showed that the monomer population decreases exponentially mirrored by an increase in an octameric species. The monomer-octamer transition rates are faster for hamster than for rabbit PrP. The N170S/N174S mutant of hamster PrP has a smaller octamer component at the endpoint compared to the wild-type, whereas the kinetics of octamer formation in mutant and wild-type rabbit PrP are comparable. These findings demonstrate that the sequence of the ß2-α2 helix-cap affects refolding to the ß-state and subsequently, may influence susceptibility to prion disease.


Assuntos
Príons/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Asparagina/química , Dicroísmo Circular , Cricetinae , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Doenças Priônicas/etiologia , Príons/genética , Conformação Proteica , Dobramento de Proteína/efeitos dos fármacos , Estrutura Secundária de Proteína , Coelhos , Alinhamento de Sequência , Serina/química , Ureia/farmacologia
15.
Proteins ; 81(8): 1285-303, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23508986

RESUMO

Enormous strides have been made in the last 100 years to extend human life expectancy and to combat the major infectious diseases. Today, the major challenges for medical science are age-related diseases, including cancer, heart disease, lung disease, renal disease, and late-onset neurodegenerative disease. Of these, only the neurodegenerative diseases represent a class of disease so poorly understood that no general strategies for prevention or treatment exist. These diseases, which include Alzheimer's disease, Parkinson's disease, Huntington's disease, the transmissible spongiform encephalopathies, and amyotrophic lateral sclerosis (ALS), are generally fatal and incurable. The first section of this review summarizes the diversity and common features of the late-onset neurodegenerative diseases, with a particular focus on protein misfolding and aggregation-a recurring theme in the molecular pathology. The second section focuses on the particular case of ALS, a late-onset neurodegenerative disease characterized by the death of central nervous system motor neurons, leading to paralysis and patient death. Of the 10% of ALS cases that show familial inheritance (familial ALS), the largest subset is caused by mutations in the SOD1 gene, encoding the Cu, Zn superoxide dismutase (SOD1). The unusual kinetic stability of SOD1 has provided a unique opportunity for detailed structural characterization of conformational states potentially involved in SOD1-associated ALS. This review discusses past studies exploring the stability, folding, and misfolding behavior of SOD1, as well as the therapeutic possibilities of using detailed knowledge of misfolding pathways to target the molecular mechanisms underlying ALS and other neurodegenerative diseases.


Assuntos
Doenças Neurodegenerativas/patologia , Dobramento de Proteína , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Humanos , Modelos Moleculares , Mutação , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Superóxido Dismutase/análise , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1
16.
Arch Biochem Biophys ; 528(2): 118-26, 2012 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-23022061

RESUMO

An Arg-rich peptide called RIA7; sequence ac-ARAAAAAIRAIAAIIRAGGY-am, tetramerizes to form a well folded, four helix X-bundle protein. The Arg side chains are solvent exposed and the hydrophobic core is composed of the side chains from some Alas, all the Iles and the C-terminal Tyr. Since Gly, Ala and Ile, and in lesser amounts Arg and Tyr have been reported to form under putative prebiotic Earth conditions, it is plausible that RIA7-like peptides might have formed on the primitive Earth and interacted with RNAs. The interaction of RIA7 with two RNAs was tested and the formation of insoluble aggregates was observed. These results contrast with previous studies of a Lys-rich variant, called KIA7, which promotes the cleavage of RNAs. Their close structural similarity makes RIA7 and KIA7 an excellent system to compare the relative contributions of Arg and Lys to protein conformational stability. NMR-monitored hydrogen/deuterium exchange measurements and CD-monitored thermal denaturation experiments performed at different peptide and salt concentrations reveal that the conformational stabilities of RIA7 and KIA7 are practically the same. This finding has relevance for protein engineering as Lys is frequently replaced by Arg to improve ligand binding and membrane association and penetration.


Assuntos
Peptídeos/química , Sequência de Aminoácidos , Arginina/química , Sequência de Bases , Dicroísmo Circular , Medição da Troca de Deutério , Planeta Terra , Lisina/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação de Ácido Nucleico , Peptídeos/metabolismo , Dobramento de Proteína , Multimerização Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , RNA/química , RNA/metabolismo , Espectrometria de Fluorescência , Termodinâmica
17.
J Neurosci ; 32(26): 8791-9, 2012 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-22745481

RESUMO

There is increasing evidence that toxicity of mutant superoxide dismutase-1 (SOD1) in amyotrophic lateral sclerosis (ALS) is linked to its propensity to misfold and to aggregate. Immunotargeting of differently folded states of SOD1 has provided therapeutic benefit in mutant SOD1 transgenic mice. The specific region(s) of the SOD1 protein to which these immunization approaches target are, however, unknown. In contrast, we have previously shown, using a specific antibody [SOD1 exposed dimer interface (SEDI) antibody], that the dimer interface of SOD1 is abnormally exposed both in mutant SOD1 transgenic mice and in familial ALS cases associated with mutations in the SOD1 gene (fALS1). Here, we show the beneficial effects of an active immunization strategy using the SEDI antigenic peptide displayed on a branched peptide dendrimer to target monomer/misfolded in SOD1(G37R) and SOD1(G93A) mutant SOD1 transgenic mice. Immunization delayed disease onset and extended disease duration, with survival times increased by an average of 40 d in SOD1(G37R) mice. Importantly, this immunization strategy favored a Th2 immune response, thereby precluding deleterious neuroinflammatory effects. Furthermore, the beneficial effects of immunization correlated with a reduction in accumulation of both monomer/misfolded and oligomeric SOD1 species in the spinal cord, the intended targets of the immunization strategy. Our results support that SOD1 misfolding/aggregation plays a central role in SOD1-linked ALS pathogenesis and identifies monomeric/misfolded SOD1 as a therapeutic target for SOD1-related ALS.


Assuntos
Esclerose Lateral Amiotrófica/imunologia , Dobramento de Proteína , Superóxido Dismutase/imunologia , Vacinação/métodos , Fatores Etários , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/fisiopatologia , Análise de Variância , Animais , Anticorpos/metabolismo , Axônios/patologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Regulação da Expressão Gênica/imunologia , Gliose/etiologia , Gliose/genética , Gliose/prevenção & controle , Humanos , Imunoprecipitação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/genética , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Mutação/genética , Fragmentos de Peptídeos/imunologia , Desempenho Psicomotor/fisiologia , Medula Espinal/citologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1
18.
Structure ; 20(7): 1141-53, 2012 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-22632832

RESUMO

The E3 ligases recruit substrate proteins for targeted ubiquitylation. Recent insights into the mechanisms of ubiquitylation demonstrate that E3 ligases can possess active regulatory properties beyond those of a simple assembly scaffold. Here, we describe the dimeric structure of the E3 ligase adaptor protein SPOP (speckle-type POZ protein) in complex with the N-terminal domain of Cul3 at 2.4 Å resolution. We find that SPOP forms large oligomers that can form heteromeric species with the closely related paralog SPOPL. In combination, SPOP and SPOPL (SPOP-like) form a molecular rheostat that can fine-tune E3 ubiquitin ligase activity by affecting the oligomeric state of the E3 complex. We propose that adaptor protein self-assembly provides a graded level of regulation of the SPOP/Cul3 E3 ligase toward its multiple protein substrates.


Assuntos
Proteínas Culina/química , Proteínas Nucleares/química , Proteínas Repressoras/química , Ubiquitina/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Proteínas Culina/genética , Proteínas Culina/metabolismo , Escherichia coli , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Plasmídeos , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação
19.
J Mol Biol ; 421(4-5): 631-52, 2012 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-22542526

RESUMO

Among the diseases of protein misfolding, amyotrophic lateral sclerosis (ALS) is unusual in that the proteinaceous neuronal inclusions that are the hallmark of the disease have neither the classic fibrillar appearance of amyloid by transmission electron microscopy nor the affinity for the dye Congo red that is a defining feature of amyloid. Mutations in the Cu, Zn superoxide dismutase (SOD1) cause the largest subset of inherited ALS cases. The mechanism by which this highly stable enzyme misfolds to form non-amyloid aggregates is currently poorly understood, as are the stresses that initiate misfolding. The oxidative damage hypothesis proposes that SOD1's normal free radical scavenger role puts it at risk of oxidative damage and that it is this damage that triggers the misfolding primed by mutation. Here, we present evidence that hydrogen peroxide treatment, which generates free radical species at the SOD1 active site, causes oxidative damage to active-site histidine residues, leading to major structural changes and non-amyloid aggregation similar to that seen in ALS. Time-resolved measurements of release of bound metal ligands, exposure of hydrophobic surface area, and alterations in the SOD1 proton NMR spectrum have allowed us to model the early structural changes occurring as SOD1 misfolds, prior to aggregation. ALS-causing SOD1 mutations apparently alter this pathway by increasing exposure of buried epitopes in misfolded species populated at endpoint. We have identified a well-populated early misfolding intermediate that could serve as a target for therapies designed to block downstream misfolding and aggregation events and thereby treat SOD1-associated ALS.


Assuntos
Dobramento de Proteína , Superóxido Dismutase/metabolismo , Esclerose Lateral Amiotrófica/fisiopatologia , Humanos , Peróxido de Hidrogênio/metabolismo , Modelos Químicos , Modelos Moleculares , Oxirredução , Desnaturação Proteica , Multimerização Proteica , Superóxido Dismutase-1
20.
Eur Biophys J ; 41(1): 63-72, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22038076

RESUMO

The first life on Earth is believed to have been based on RNA, but might have taken advantage of amino acids and short peptides which form readily under conditions like those of the primitive Earth. We have shown that simple peptides adopt specifically folded four-helix bundle structures that can recognize and cleave RNA. Here, to explore the limits of conformational specificity, we characterize a simpler peptide composed of just Lys, Ile, Ala, and Gly called KIA7I. Using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations, we find kinks in the helices of KIA7I and multiple C-terminal conformations. These results suggest that the C-terminal Ile residue does not completely occupy the hydrophobic pocket that is filled by aromatic side-chains in well-folded KIA7 variants. The capacity of arenes to fill this cavity was tested. Using NMR, we show that benzene and phenol can bind KIA7I, but do not bind the well-folded variant KIA7W or hen egg white lysozyme. Benzene also binds Aß(1-40), a mostly disordered polypeptide implicated in Alzheimer's disease. 8-Anilinonaphthalene-1-sulfonate (ANS) fluorescence is further enhanced in the presence of both KIA7I and arenes relative to KIA7I alone. This ANS fluorescence enhancement is stronger for smaller and less polar arenes and less ordered KIA variants. These results suggest that arenes are not confined to the pocket, but penetrate and loosen the hydrophobic core of KIA7I.


Assuntos
Aminoácidos , Benzeno/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Fenol/metabolismo , Conformação Proteica , Alanina , Glicina , Isoleucina , Lisina , Simulação de Dinâmica Molecular , Espectrometria de Fluorescência
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