Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 34
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
PLoS One ; 15(6): e0233967, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32497067

RESUMO

Radiation-induced heart disease presents a significant challenge in the event of an accidental radiation exposure as well as to cancer patients who receive acute doses of irradiation as part of radiation therapy. We utilized the spontaneously hypertensive Wistar-Kyoto rat model, previously shown to demonstrate drug-induced cardiomyopathy, to evaluate the acute and long-term effects of sub-lethal total body gamma irradiation at two, four, and fifty-two weeks. We further examined irreversible oxidative protein carbonylation in the heart immediately following irradiation in the normotensive Wistar-Kyoto rat. Both males and females sustained weight loss and anemic conditions compared to untreated controls over a one-year period as reflected by reduced body weight and low red blood cell count. Increased inflammation was detected by elevated IL-6 serum levels selectively in males at four weeks. Serum cardiac troponin T and I analyses revealed signs of cardiomyopathy at earlier timepoints, but high variability was observed, especially at one year. Echocardiography at two weeks following 5.0Gy treatment revealed a significant decrease in cardiac output in females and a significant decrease in both diastolic and systolic volumes in males. Following 10.0Gy irradiation in the normotensive Wistar-Kyoto rat, the heart tissue showed an increase in total protein oxidative carbonylation accompanied by DNA damage indicated by an increase in γ-H2AX. Using proteomic analyses, we identified several novel proteins which showed a marked difference in carbonylation including those of mitochondrial origin and most notably, cardiac troponin T, one of the key proteins involved in cardiomyocyte contractility. Overall, we present findings of acute oxidative protein damage, DNA damage, cardiac troponin T carbonylation, and long-term cardiomyopathy in the irradiated animals.


Assuntos
Raios gama/efeitos adversos , Coração/efeitos da radiação , Oxirredução/efeitos da radiação , Carbonilação Proteica/efeitos da radiação , Proteínas/química , Animais , Feminino , Masculino , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Irradiação Corporal Total/efeitos adversos
2.
Methods Mol Biol ; 1779: 313-339, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29886541

RESUMO

We detail some of the genetic, biochemical, and physical methods useful in studying amyloids in yeast, particularly the yeast prions. These methods include cytoduction (cytoplasmic mixing), infection of cells with prion amyloids, use of green fluorescent protein fusions with amyloid-forming proteins for cytology, protein purification and amyloid formation, and electron microscopy of filaments.


Assuntos
Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Eletrônica de Transmissão , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
3.
Cold Spring Harb Protoc ; 2017(2)2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28148848

RESUMO

The recognition that certain long-known nonchromosomal genetic elements were actually prions was based not on the specific phenotypic manifestations of those elements, but rather on their unusual genetic properties. Here, we outline methods of prion assay, methods for showing the nonchromosomal inheritance, and methods for determining whether a nonchromosomal trait has the unusual characteristics diagnostic of a prion. Finally, we discuss genetic methods often useful in the study of yeast prions.


Assuntos
Técnicas Genéticas , Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fenótipo
4.
Cold Spring Harb Protoc ; 2017(2)2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28148849

RESUMO

Transfection of yeast with amyloid filaments, made from recombinant protein or prepared from extracts of cells infected with a prion, has become an important method in characterizing yeast prions. Here, we describe a method for transmission of [URE3] with Ure2p amyloid that is based on a previously published protocol for transfection with Sup35p filaments to make cells [PSI+]. This method may be used for other prions by changing just the amyloid source, host strain, and plating medium.


Assuntos
Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Proteínas Priônicas/metabolismo , Príons/genética , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transfecção/métodos , Fenótipo , Saccharomyces cerevisiae/metabolismo
5.
Cold Spring Harb Protoc ; 2017(2)2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28148850

RESUMO

Amyloid fibers are large and extremely stable structures that can resist denaturation by strong anionic detergents, such as sodium dodecyl sulfate or sarkosyl. Here, we present two complementary analytical methods that exploit these properties, enabling the isolation and characterization of amyloid/prion aggregates. The first technique, known as semidenaturating detergent agarose gel electrophoresis, is an immunoblotting technique, conceptually similar to conventional western blotting. It enables the targeted identification of large detergent-resistant protein aggregates using antibodies specific to the protein of interest. The second method, called the technique for amyloid purification and identification, is a nontargeted approach that can isolate amyloid aggregates for analysis by tandem mass spectrometry. The latter approach requires no special genetic tools or antibodies, and can identify amyloid-forming proteins, such as prions, as well as proteins tightly associated with amyloid, from a variety of cell sources.


Assuntos
Amiloide/análise , Amiloide/isolamento & purificação , Príons/análise , Príons/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Saccharomyces cerevisiae/química , Eletroforese em Gel de Ágar , Immunoblotting , Desnaturação Proteica , Espectrometria de Massas em Tandem
6.
Cold Spring Harb Protoc ; 2017(2)2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28148884

RESUMO

Infectious proteins (prions) are usually self-templating filamentous protein polymers (amyloids). Yeast prions are genes composed of protein and, like the multiple alleles of DNA-based genes, can have an array of "variants," each a distinct self-propagating amyloid conformation. Like the lethal mammalian prions and amyloid diseases, yeast prions may be lethal, or only mildly detrimental, and show an array of phenotypes depending on the protein involved and the prion variant. Yeast prions are models for both rare mammalian prion diseases and for several very common amyloidoses such as Alzheimer's disease, type 2 diabetes, and Parkinson's disease. Here, we describe their detection and characterization using genetic, cell biological, biochemical, and physical methods.


Assuntos
Amiloide/genética , Amiloide/metabolismo , Modelos Biológicos , Príons/genética , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
7.
Pharm Res ; 34(4): 765-779, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28150167

RESUMO

PURPOSE: Protein carbonylation is an irreversible modification of Lys, Arg, Thr and Pro amino acids under conditions of oxidative stress. Previous studies have reported specific carbonylated residues in purified recombinant albumins, albeit with a lack of agreement between the studies. Currently, structural factors that determine site-specific protein carbonylation are not well understood. METHODS: In this study, we utilized metal-catalyzed oxidizing conditions to generate carbonylation in recombinant human serum albumin (HSA) and granulocyte-colony stimulating factor (G-CSF), two proteins with distinct metal-binding abilities. To estimate predictability of HSA carbonylation sites, the same oxidative reaction was repeated based on the previously reported conditions. For G-CSF, oxidative conditions were gradually adjusted to achieve substantial levels of protein carbonylation. Corresponding accumulation of specific oxidized residues was identified and confirmed with high-resolution mass spectrometry. RESULTS: Our HSA dataset contained 55 carbonylated residues and showed a significant overlap with the previously published pooled data, indicating a certain level of carbonylation site specificity for albumins. Oxidation of G-CSF under multiple oxidative conditions consistently showed a highly specific carbonylation at position Pro45. We also detected a previously unreported, oxidation-induced cleavage site in G-CSF between His44 and Pro45, which might be attributed to a presence of a potential metal-binding site near residue Pro45. CONCLUSIONS: Our results show distinct patterns of protein carbonylation for HSA and G-CSF. Thus, specificity of protein carbonylation induced by metal-catalyzed oxidation is protein dependent and might be predicted by availability of transition metal binding site(s) within the protein.


Assuntos
Fator Estimulador de Colônias de Granulócitos/química , Metais/química , Carbonilação Proteica , Albumina Sérica/química , Aminoácidos/química , Sítios de Ligação , Biocatálise , Humanos , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química
8.
PLoS One ; 11(12): e0168283, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28030582

RESUMO

Mitochondrial dysregulation is closely associated with excessive reactive oxygen species (ROS) production. Altered redox homeostasis has been implicated in the onset of several diseases including cancer. Mitochondrial DNA (mtDNA) and proteins are particularly sensitive to ROS as they are in close proximity to the respiratory chain (RC). Mitoquinone (MitoQ), a mitochondria-targeted redox agent, selectively damages breast cancer cells possibly through damage induced via enhanced ROS production. However, the effects of MitoQ and other triphenylphosphonium (TPP+) conjugated agents on cancer mitochondrial homeostasis remain unknown. The primary objective of this study was to determine the impact of mitochondria-targeted agent [(MTAs) conjugated to TPP+: mitoTEMPOL, mitoquinone and mitochromanol-acetate] on mitochondrial physiology and mtDNA integrity in breast (MDA-MB-231) and lung (H23) cancer cells. The integrity of the mtDNA was assessed by quantifying the degree of mtDNA fragmentation and copy number, as well as by measuring mitochondrial proteins essential to mtDNA stability and maintenance (TFAM, SSBP1, TWINKLE, POLG and POLRMT). Mitochondrial status was evaluated by measuring superoxide production, mitochondrial membrane depolarization, oxygen consumption, extracellular acidification and mRNA or protein levels of the RC complexes along with TCA cycle activity. In this study, we demonstrated that all investigated MTAs impair mitochondrial health and decrease mtDNA integrity in MDA-MB-231 and H23 cells. However, differences in the degree of mitochondrial damage and mtDNA degradation suggest unique properties among each MTA that may be cell line, dose and time dependent. Collectively, our study indicates the potential for TPP+ conjugated molecules to impair breast and lung cancer cells by targeting mitochondrial homeostasis.


Assuntos
Neoplasias da Mama/patologia , Neoplasias Pulmonares/patologia , Mitocôndrias/genética , Mitocôndrias/patologia , Compostos Organofosforados/farmacologia , Carcinoma de Pequenas Células do Pulmão/patologia , Superóxidos/metabolismo , Ubiquinona/análogos & derivados , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , DNA Mitocondrial/genética , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredução , Estresse Oxidativo , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Ubiquinona/farmacologia
9.
Pharm Res ; 33(2): 526-39, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26499343

RESUMO

PURPOSE: Ascorbic acid has been considered as a potential radical scavenging excipient for pharmaceutical formulations. However, under certain circumstances, ascorbic acid can generate reactive oxygen species via redox cycling. The objective of this study was to investigate ascorbic acid-induced oxidative carbonylation of therapeutic proteins and correlate the increase in carbonylation with protein aggregation. METHODS: An optimized ELISA for quantifying carbonyl levels was used to compare the oxidizing potentials of ascorbic acid and hydrogen peroxide by testing four pharmaceutically-relevant proteins (human serum albumin, immunoglobulin G, granulocyte-colony stimulating factor and calcitonin). Several transition metals at micromolar concentrations were evaluated for their ability to enhance ascorbic acid-induced protein carbonylation. Protein aggregation under oxidative conditions, with or without free radical scavengers, was measured by aggregate binding fluorescent dye and confirmed by microfluidic imaging. RESULTS: Addition of ascorbic acid alone resulted in higher increases in carbonylation than addition of hydrogen peroxide. The presence of trace amounts (>75 ppb) of copper enhanced oxidative effects of ascorbic acid, whereas other tested metals did not comparably promote oxidation. During oxidation, protein destabilization indicated by loss of the full-length protein, positively correlated with the increase in protein aggregation. However, levels of aggregation did not always correlate with the levels of protein carbonylation. At comparable carbonylation levels, addition of copper produced greater protein destabilization and aggregation than addition of iron. CONCLUSIONS: The results strongly suggest that ascorbic acid with traces of metals, especially copper, can promote therapeutic protein carbonylation and potentially aggregation. At similar carbonylation levels, some oxidative conditions may lead to greater protein destabilization than others.


Assuntos
Ácido Ascórbico/farmacologia , Excipientes/farmacologia , Sequestradores de Radicais Livres/farmacologia , Oxidantes/farmacologia , Agregados Proteicos/efeitos dos fármacos , Carbonilação Proteica/efeitos dos fármacos , Proteínas/química , Animais , Cobre/química , Humanos , Oxirredução/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Coelhos , Salmão
10.
Am J Pathol ; 185(10): 2641-52, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26435412

RESUMO

Traumatic brain injury affects the whole body in addition to the direct impact on the brain. The systemic response to trauma is associated with the hepatic acute-phase response. To further characterize this response, we performed controlled cortical impact injury on male mice and determined the expression of serum amyloid A1 (SAA1), an apolipoprotein, induced at the early stages of the acute-phase response in liver and plasma. After cortical impact injury, induction of SAA1 was detectable in plasma at 6 hours post-injury and in liver at 1 day post-injury, followed by gradual diminution over time. In the liver, cortical impact injury increased neutrophil and macrophage infiltration, apoptosis, and expression of mRNA encoding the chemokines CXCL1 and CXCL10. An increase in angiotensin II AT1 receptor mRNA at 3 days post-injury was also observed. Administration of the AT1 receptor antagonist telmisartan 1 hour post-injury significantly decreased liver SAA1 levels and CXCL10 mRNA expression, but did not affect CXCL1 expression or the number of apoptotic cells or infiltrating leukocytes. To our knowledge, this is the first study to demonstrate that SAA1 is induced in the liver after traumatic brain injury and that telmisartan prevents this response. Elucidating the molecular pathogenesis of the liver after brain injury will assist in understanding the efficacy of therapeutic approaches to brain injury.


Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Benzimidazóis/farmacologia , Benzoatos/farmacologia , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/metabolismo , Fígado/efeitos dos fármacos , Proteína Amiloide A Sérica/metabolismo , Reação de Fase Aguda/metabolismo , Animais , Lesões Encefálicas/patologia , Quimiocina CXCL1/metabolismo , Quimiocina CXCL10/metabolismo , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Telmisartan
11.
PLoS One ; 10(8): e0136362, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26317359

RESUMO

Intracellular protein aggregation is the hallmark of several neurodegenerative diseases. Aggregates formed by polyglutamine (polyQ)-expanded proteins, such as Huntingtin, adopt amyloid-like structures that are resistant to denaturation. We used a novel purification strategy to isolate aggregates formed by human Huntingtin N-terminal fragments with expanded polyQ tracts from both yeast and mammalian (PC-12) cells. Using mass spectrometry we identified the protein species that are trapped within these polyQ aggregates. We found that proteins with very long intrinsically-disordered (ID) domains (≥ 100 amino acids) and RNA-binding proteins were disproportionately recruited into aggregates. The removal of the ID domains from selected proteins was sufficient to eliminate their recruitment into polyQ aggregates. We also observed that several neurodegenerative disease-linked proteins were reproducibly trapped within the polyQ aggregates purified from mammalian cells. Many of these proteins have large ID domains and are found in neuronal inclusions in their respective diseases. Our study indicates that neurodegenerative disease-associated proteins are particularly vulnerable to recruitment into polyQ aggregates via their ID domains. Also, the high frequency of ID domains in RNA-binding proteins may explain why RNA-binding proteins are frequently found in pathological inclusions in various neurodegenerative diseases.


Assuntos
Proteínas do Tecido Nervoso/metabolismo , Peptídeos/metabolismo , Agregados Proteicos , Proteínas de Ligação a RNA/metabolismo , Animais , Humanos , Proteína Huntingtina , Proteínas do Tecido Nervoso/genética , Células PC12 , Peptídeos/genética , Proteínas de Ligação a RNA/genética , Ratos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
12.
Prion ; 7(6): 464-8, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24366087

RESUMO

The capacity to polymerize into amyloid fibrils is common to many proteins. While some proteins naturally form these fibrils to serve functional roles, amyloid is usually associated with pathogenic processes in which specific proteins aberrantly aggregate within cells or tissues. Though the contribution of amyloid fibrils to actual disease pathogenesis is not always clear, one possibility is that the titration of essential proteins from solution into aggregates contributes to the cellular degeneration common to many amyloid diseases. Using mammalian and yeast model systems, we recently showed that the common biophysical properties of amyloid aggregates--including strong resistance to dissolution--enable stringent purification and identification of both amyloid-forming and amyloid-associated proteins directly from cells. Strikingly, many proteins that were previously implicated in formation or clearance of intracellular aggregates, including several stress granule components, were found to co-aggregate with amyloid formed by a polyglutamine-expanded huntingtin fragment. This direct evaluation of proteins within aggregates can help identify new amyloid-forming proteins, as well as proteins that can indirectly contribute to disease mechanisms.


Assuntos
Amiloide/análise , Amiloide/metabolismo , Animais , Humanos , Peptídeos/análise , Peptídeos/metabolismo , Agregados Proteicos , Agregação Patológica de Proteínas/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo
13.
J Biol Chem ; 288(38): 27100-27111, 2013 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-23926098

RESUMO

The formation of amyloid aggregates is implicated both as a primary cause of cellular degeneration in multiple human diseases and as a functional mechanism for providing extraordinary strength to large protein assemblies. The recent identification and characterization of several amyloid proteins from diverse organisms argues that the amyloid phenomenon is widespread in nature. Yet identifying new amyloid-forming proteins usually requires a priori knowledge of specific candidates. Amyloid fibers can resist heat, pressure, proteolysis, and denaturation by reagents such as urea or sodium dodecyl sulfate. Here we show that these properties can be exploited to identify naturally occurring amyloid-forming proteins directly from cell lysates. This proteomic-based approach utilizes a novel purification of amyloid aggregates followed by identification by mass spectrometry without the requirement for special genetic tools. We have validated this technique by blind identification of three amyloid-based yeast prions from laboratory and wild strains and disease-related polyglutamine proteins expressed in both yeast and mammalian cells. Furthermore, we found that polyglutamine aggregates specifically recruit some stress granule components, revealing a possible mechanism of toxicity. Therefore, core amyloid-forming proteins as well as strongly associated proteins can be identified directly from cells of diverse origin.


Assuntos
Amiloide , Peptídeos , Príons , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Amiloide/genética , Amiloide/metabolismo , Animais , Humanos , Células PC12 , Peptídeos/genética , Peptídeos/metabolismo , Príons/genética , Príons/metabolismo , Proteômica/métodos , Ratos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Dodecilsulfato de Sódio/química , Ureia/química
14.
Mol Microbiol ; 86(6): 1531-47, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23078282

RESUMO

Many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) are linked to the accumulation of specific protein aggregates in affected regions of the nervous system. SOD1, TDP-43, FUS and optineurin (OPTN) proteins were identified to form intraneuronal inclusions in ALS patients. In addition, mutations in OPTN are associated with both ALS and glaucoma. As the pathological role of OPTN in neuronal degeneration remains unresolved, we created a yeast model to study its potential for aggregation and toxicity. We observed that both wild type and disease-associated mutants of OPTN form toxic non-amyloid aggregates in yeast. Similar to reported cell culture and mouse models, the OPTN E50K mutant shows enhanced toxicity in yeast, implying a conserved gain-of-function mechanism. Furthermore, OPTN shows a unique aggregation pattern compared to other disease-related proteins in yeast. OPTN aggregates colocalize only partially with the insoluble protein deposit (IPOD) site markers, but coincide perfectly with the prion seed-reducing protein Btn2 and several other aggregation-prone proteins, suggesting that protein aggregates are not limited to a single IPOD site. Importantly, changes in the Btn2p level modify OPTN toxicity and aggregation. This study generates a mechanistic framework for investigating how OPTN may trigger pathological changes in ALS and other OPTN-linked neurodegenerative disorders.


Assuntos
Desnaturação Proteica , Multimerização Proteica , Fator de Transcrição TFIIIA/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Humanos , Proteínas de Membrana Transportadoras , Proteínas Mutantes/metabolismo , Proteínas Mutantes/toxicidade , Mutação de Sentido Incorreto , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIIIA/toxicidade
15.
Proc Natl Acad Sci U S A ; 109(40): E2683-90, 2012 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-22949655

RESUMO

Even deadly prions may be widespread in nature if they spread by infection faster than they kill off their hosts. The yeast prions [PSI+] and [URE3] (amyloids of Sup35p and Ure2p) were not found in 70 wild strains, while [PIN+] (amyloid of Rnq1p) was found in ∼16% of the same population. Yeast prion infection occurs only by mating, balancing the detrimental effects of carrying the prion. We estimated the frequency of outcross mating as about 1% of mitotic doublings from the known detriment of carrying the 2-µm DNA plasmid (∼1%) and its frequency in wild populations (38/70). We also estimated the fraction of total matings that are outcross matings (∼23-46%) from the fraction of heterozygosity at the highly polymorphic RNQ1 locus (∼46%). These results show that the detriment of carrying even the mildest forms of [PSI+], [URE3], or [PIN+] is greater than 1%. We find that Rnq1p polymorphisms in wild strains include several premature stop codon alleles that cannot propagate [PIN+] from the reference allele and others with several small deletions and point mutations which show a small transmission barrier. Wild strains carrying [PIN+] are far more likely to be heterozygous at RNQ1 and other loci than are [pin-] strains, probably reflecting its being a sexually transmitted disease. Because sequence differences are known to block prion propagation or ameliorate its pathogenic effects, we hypothesize that polymorphism of RNQ1 was selected to protect cells from detrimental effects of the [PIN+] prion.


Assuntos
Amiloide/genética , Evolução Biológica , Plasmídeos/genética , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sexo , Leveduras/genética , Amiloide/metabolismo , Sequência de Bases , Genética Populacional , Dados de Sequência Molecular , Mutação/genética , Príons/genética , Reprodução/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Seleção Genética , Análise de Sequência de DNA , Leveduras/metabolismo
16.
Methods Mol Biol ; 849: 321-46, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22528100

RESUMO

We detail some of the genetic, biochemical, and physical methods useful in studying amyloids in yeast, particularly the yeast prions. These methods include cytoduction (cytoplasmic mixing), infection of cells with prion amyloids, use of green fluorescent protein fusions with amyloid-forming proteins for cytology, protein purification and amyloid formation, and electron microscopy of filaments.


Assuntos
Amiloide/química , Amiloide/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Amiloide/genética , Amiloide/isolamento & purificação , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Transporte Biológico , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Endopeptidase K/metabolismo , Fusão Gênica , Vetores Genéticos/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Fenótipo , Príons/química , Príons/genética , Príons/isolamento & purificação , Príons/metabolismo , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteólise , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Transformação Genética , Uracila/metabolismo
17.
Prion ; 5(4): 250-7, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22052354

RESUMO

In recent years there have been several reports of human neurodegenerative diseases that involve protein misfolding being modeled in the yeast Saccharomyces cerevisiae. This review summarizes recent advances in understanding the specific mechanisms underlying intracellular neuronal pathology during Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD), including SOD1, TDP-43 and FUS protein inclusions and the potential of these proteins to be involved in pathogenic prion-like mechanisms. More specifically, we focus on findings from yeast systems that offer tremendous possibilities for screening for genetic and chemical modifiers of disease-related proteotoxicity.


Assuntos
Amiloide/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Degeneração Lobar Frontotemporal/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Amiloide/química , Esclerose Lateral Amiotrófica/patologia , Proteínas de Ligação a DNA , Degeneração Lobar Frontotemporal/patologia , Humanos , Modelos Moleculares , Príons/química , Proteína FUS de Ligação a RNA/química , Proteína FUS de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1
18.
Protein Cell ; 2(3): 223-36, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21452073

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by the premature loss of motor neurons. While the underlying cellular mechanisms of neuron degeneration are unknown, the cytoplasmic aggregation of several proteins is associated with sporadic and familial forms of the disease. Both wild-type and mutant forms of the RNA-binding proteins FUS and TDP-43 accumulate in cytoplasmic inclusions in the neurons of ALS patients. It is not known if these so-called proteinopathies are due to a loss of function or a gain of toxicity resulting from the formation of cytoplasmic aggregates. Here we present a model of FUS toxicity using the yeast Saccharomyces cerevisiae in which toxicity is associated with greater expression and accumulation of FUS in cytoplasmic aggregates. We find that FUS and TDP-43 have a high propensity for co-aggregation, unlike the aggregation patterns of several other aggregation-prone proteins. Moreover, the biophysical properties of FUS aggregates in yeast are distinctly different from many amyloidogenic proteins, suggesting they are not composed of amyloid.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Multimerização Proteica , Proteína FUS de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Esclerose Lateral Amiotrófica/patologia , Proliferação de Células/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Detergentes/farmacologia , Humanos , Cinética , Peptídeos/metabolismo , Príons/química , Príons/metabolismo , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Transporte Proteico , Proteína FUS de Ligação a RNA/química , Proteína FUS de Ligação a RNA/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
19.
J Mol Biol ; 409(2): 263-77, 2011 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-21497604

RESUMO

Intracellular fibril formation by Ure2p produces the non-Mendelian genetic element [URE3] in Saccharomyces cerevisiae, making Ure2p a prion protein. We show that solid-state NMR spectra of full-length Ure2p fibrils, seeded with infectious prions from a specific [URE3] strain and labeled with uniformly (15)N-(13)C-enriched Ile, include strong, sharp signals from Ile residues in the globular C-terminal domain (CTD) with both helical and nonhelical (13)C chemical shifts. Treatment with proteinase K eliminates these CTD signals, leaving only nonhelical signals from the Gln-rich and Asn-rich N-terminal segment, which are also observed in the solid-state NMR spectra of Ile-labeled fibrils formed by residues 1-89 of Ure2p. Thus, the N-terminal segment, or "prion domain" (PD), forms the fibril core, while CTD units are located outside the core. We additionally show that, after proteinase K treatment, Ile-labeled Ure2p fibrils formed without prion seeding exhibit a broader set of solid-state NMR signals than do prion-seeded fibrils, consistent with the idea that structural variations within the PD core account for prion strains. Measurements of (13)C-(13)C magnetic dipole-dipole couplings among (13)C-labeled Ile carbonyl sites in full-length Ure2p fibrils support an in-register parallel ß-sheet structure for the PD core of Ure2p fibrils. Finally, we show that a model in which CTD units are attached rigidly to the parallel ß-sheet core is consistent with steric constraints.


Assuntos
Amiloide/química , Glutationa Peroxidase/química , Príons/química , Proteínas de Saccharomyces cerevisiae/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
20.
Genetics ; 188(2): 339-48, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21467567

RESUMO

[URE3] is an amyloid-based prion of Ure2p, a regulator of nitrogen catabolism in Saccharomyces cerevisiae. The Ure2p of the human pathogen Candida albicans can also be a prion in S. cerevisiae. We find that overproduction of the disaggregating chaperone, Hsp104, increases the frequency of de novo [URE3] prion formation by the Ure2p of S. cerevisiae and that of C. albicans. This stimulation is strongly dependent on the presence of the [PIN(+)] prion, known from previous work to enhance [URE3] prion generation. Our data suggest that transient Hsp104 overproduction enhances prion generation through persistent effects on Rnq1 amyloid, as well as during overproduction by disassembly of amorphous Ure2 aggregates (generated during Ure2p overproduction), driving the aggregation toward the amyloid pathway. Overproduction of other major cytosolic chaperones of the Hsp70 and Hsp40 families (Ssa1p, Sse1p, and Ydj1p) inhibit prion formation, whereas another yeast Hsp40, Sis1p, modulates the effects of Hsp104p on both prion induction and prion curing in a prion-specific manner. The same factor may both enhance de novo prion generation and destabilize existing prion variants, suggesting that prion variants may be selected by changes in the chaperone network.


Assuntos
Glutationa Peroxidase/metabolismo , Proteínas de Choque Térmico/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Western Blotting , Candida albicans/genética , Candida albicans/metabolismo , Epigênese Genética , Glutationa Peroxidase/genética , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/genética , Modelos Genéticos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Príons/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Transformação Genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...