RESUMO
The functional and structural significance of the intrasubunit disulfide bond in copper-zinc superoxide dismutase (SOD1) was studied by characterizing mutant forms of human SOD1 (hSOD) and yeast SOD1 lacking the disulfide bond. We determined x-ray crystal structures of metal-bound and metal-deficient hC57S SOD1. C57S hSOD1 isolated from yeast contained four zinc ions per protein dimer and was structurally very similar to wild type. The addition of copper to this four-zinc protein gave properly reconstituted 2Cu,2Zn C57S hSOD, and its spectroscopic properties indicated that the coordination geometry of the copper was remarkably similar to that of holo wild type hSOD1. In contrast, the addition of copper and zinc ions to apo C57S human SOD1 failed to give proper reconstitution. Using pulse radiolysis, we determined SOD activities of yeast and human SOD1s lacking disulfide bonds and found that they were enzymatically active at â¼10% of the wild type rate. These results are contrary to earlier reports that the intrasubunit disulfide bonds in SOD1 are essential for SOD activity. Kinetic studies revealed further that the yeast mutant SOD1 had less ionic attraction for superoxide, possibly explaining the lower rates. Saccharomyces cerevisiae cells lacking the sod1 gene do not grow aerobically in the absence of lysine, but expression of C57S SOD1 increased growth to 30-50% of the growth of cells expressing wild type SOD1, supporting that C57S SOD1 retained a significant amount of activity.
Assuntos
Proteínas Mutantes/química , Proteínas de Saccharomyces cerevisiae/química , Superóxido Dismutase/química , Esclerose Lateral Amiotrófica/genética , Apoproteínas/química , Varredura Diferencial de Calorimetria , Dissulfetos/química , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Espectrometria de Massas , Metais/química , Mutação , Estresse Oxidativo , Ligação Proteica , Conformação Proteica , Saccharomyces cerevisiae/química , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria , Superóxidos/química , Zinco/químicaRESUMO
Repetitious patterns enable the auditory system to form prediction models specifying the most likely characteristics of subsequent sounds. Pattern deviations elicit mismatch negativity (MMN), the amplitude of which is modulated by the size of the deviation and confidence in the model. Todd et al. (Neuropsychologia 49:3399-3405, 2011; J Neurophysiol 109:99-105, 2013) demonstrated that a multi-timescale sequence reveals a bias that profoundly distorts the impact of local sound statistics on the MMN amplitude. Two sounds alternate roles as repetitious "standard" and rare "deviant" rapidly (every 0.8 min) or slowly (every 2.4 min). The bias manifests as larger MMN to the sound first encountered as deviant in slow compared to fast changing sequences, but no difference for the sound first encountered as a standard. We propose that the bias is due to how Bayesian priors shape filters of sound relevance. By examining the time-course of change in MMN amplitude we show that the bias manifests immediately after roles change but rapidly disappears thereafter. The bias was reflected in the response to deviant sounds only (not in response to standards), consistent with precision estimates extracted from second order patterns modulating gain differentially for the two sounds. Evoked responses to deviants suggest that pattern extraction and reactivation of priors can operate over tens of minutes or longer. Both MMN and deviant responses establish that: (1) priors are defined by the most proximally encountered probability distribution when one exists but; (2) when no prior exists, one is instantiated by sequence onset characteristics; and (3) priors require context interruption to be updated.
Assuntos
Encéfalo/fisiologia , Potenciais Evocados Auditivos , Estimulação Acústica , Adolescente , Adulto , Teorema de Bayes , Feminino , Humanos , Masculino , Adulto JovemRESUMO
Mismatch negativity (MMN), an evoked response potential elicited when a "deviant" sound violates a regularity in the auditory environment, is integral to auditory scene processing and has been used to demonstrate "primitive intelligence" in auditory short-term memory. Using a new multiple-context and -timescale protocol we show that MMN magnitude displays a context-sensitive modulation depending on changes in the probability of a deviant at multiple temporal scales. We demonstrate a primacy bias causing asymmetric evidence-based modulation of predictions about the environment, and we demonstrate that learning how to learn about deviant probability (meta-learning) induces context-sensitive variation in the accessibility of predictive long-term memory representations that underpin the MMN. The existence of the bias and meta-learning are consistent with automatic attributions of behavioral salience governing relevance-filtering processes operating outside of awareness.
Assuntos
Percepção Auditiva/fisiologia , Variação Contingente Negativa/fisiologia , Potenciais Evocados Auditivos/fisiologia , Aprendizagem/fisiologia , Estimulação Acústica , Adolescente , Adulto , Eletroencefalografia , Feminino , Humanos , Masculino , Tempo de Reação/fisiologiaRESUMO
Mutations in human copper-zinc superoxide dismutase (SOD1) cause an inherited form of the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). Here, we present structures of the pathogenic SOD1 variants D124V and H80R, both of which demonstrate compromised zinc-binding sites. The disruption of the zinc-binding sites in H80R SOD1 leads to conformational changes in loop elements, permitting non-native SOD1-SOD1 interactions that mediate the assembly of these proteins into higher-order filamentous arrays. Analytical ultracentrifugation sedimentation velocity experiments indicate that these SOD1 variants are more prone to monomerization than the wild-type enzyme. Although D124V and H80R SOD1 proteins appear to have fully functional copper-binding sites, inductively coupled plasma mass spectrometery (ICP-MS) and anomalous scattering X-ray diffraction analyses reveal that zinc (not copper) occupies the copper-binding sites in these variants. The absence of copper in these proteins, together with the results of covalent thiol modification experiments in yeast strains with and without the gene encoding the copper chaperone for SOD1 (CCS), suggests that CCS may not fully act on newly translated forms of these polypeptides. Overall, these findings lend support to the hypothesis that immature mutant SOD1 species contribute to toxicity in SOD1-linked ALS.
Assuntos
Esclerose Lateral Amiotrófica , Cobre/metabolismo , Chaperonas Moleculares/metabolismo , Superóxido Dismutase , Zinco/metabolismo , Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/genética , Animais , Sítios de Ligação/genética , Cristalografia por Raios X , Humanos , Camundongos , Camundongos Transgênicos , Chaperonas Moleculares/genética , Mutação , Superóxido Dismutase/química , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Difração de Raios X , Raios XRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease characterized by the destruction of motor neurons in the spinal cord and brain. A subset of ALS cases are linked to dominant mutations in copper-zinc superoxide dismutase (SOD1). The pathogenic SOD1 variants A4V and G93A have been the foci of multiple studies aimed at understanding the molecular basis for SOD1-linked ALS. The A4V variant is responsible for the majority of familial ALS cases in North America, causing rapidly progressing paralysis once symptoms begin and the G93A SOD1 variant is overexpressed in often studied murine models of the disease. Here we report the three-dimensional structures of metal-free A4V and of metal-bound and metal-free G93A SOD1. In the metal-free structures, the metal-binding loop elements are observed to be severely disordered, suggesting that these variants may share mechanisms of aggregation proposed previously for other pathogenic SOD1 proteins.
Assuntos
Substituição de Aminoácidos/genética , Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/genética , Metais , Superóxido Dismutase/química , Superóxido Dismutase/genética , Alanina/genética , Cobre/metabolismo , Cristalografia por Raios X , Variação Genética , Glicina/genética , Humanos , Metais/química , Metais/metabolismo , Ligação Proteica/genética , Processamento de Proteína Pós-Traducional/genética , Estrutura Secundária de Proteína/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Valina/genéticaRESUMO
This paper features two studies confirming a lasting impact of first learning on how subsequent experience is weighted in early relevance-filtering processes. In both studies participants were exposed to sequences of sound that contained a regular pattern on two different timescales. Regular patterning in sound is readily detected by the auditory system and used to form "prediction models" that define the most likely properties of sound to be encountered in a given context. The presence and strength of these prediction models is inferred from changes in automatically elicited components of auditory evoked potentials. Both studies employed sound sequences that contained both a local and longer-term pattern. The local pattern was defined by a regular repeating pure tone occasionally interrupted by a rare deviating tone (p=0.125) that was physically different (a 30msvs. 60ms duration difference in one condition and a 1000Hz vs. 1500Hz frequency difference in the other). The longer-term pattern was defined by the rate at which the two tones alternated probabilities (i.e., the tone that was first rare became common and the tone that was first common became rare). There was no task related to the tones and participants were asked to ignore them while focussing attention on a movie with subtitles. Auditory-evoked potentials revealed long lasting modulatory influences based on whether the tone was initially encountered as rare and unpredictable or common and predictable. The results are interpreted as evidence that probability (or indeed predictability) assigns a differential information-value to the two tones that in turn affects the extent to which prediction models are updated and imposed. These effects are exposed for both common and rare occurrences of the tones. The studies contribute to a body of work that reveals that probabilistic information is not faithfully represented in these early evoked potentials and instead exposes that predictability (or conversely uncertainty) may trigger value-based learning modulations even in task-irrelevant incidental learning.
Assuntos
Aprendizagem , Percepção da Altura Sonora/fisiologia , Incerteza , Estimulação Acústica , Adolescente , Adulto , Eletroencefalografia , Potenciais Evocados Auditivos/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Psicoacústica , Adulto JovemRESUMO
This paper features two studies confirming a lasting impact of first learning on how subsequent experience is weighted in early relevance-filtering processes. In both studies participants were exposed to sequences of sound that contained a regular pattern on two different timescales. Regular patterning in sound is readily detected by the auditory system and used to form "prediction models" that define the most likely properties of sound to be encountered in a given context. The presence and strength of these prediction models is inferred from changes in automatically elicited components of auditory evoked potentials. Both studies employed sound sequences that contained both a local and longer-term pattern. The local pattern was defined by a regular repeating pure tone occasionally interrupted by a rare deviating tone (p=0.125) that was physically different (a 30msvs. 60ms duration difference in one condition and a 1000Hz vs. 1500Hz frequency difference in the other). The longer-term pattern was defined by the rate at which the two tones alternated probabilities (i.e., the tone that was first rare became common and the tone that was first common became rare). There was no task related to the tones and participants were asked to ignore them while focussing attention on a movie with subtitles. Auditory-evoked potentials revealed long lasting modulatory influences based on whether the tone was initially encountered as rare and unpredictable or common and predictable. The results are interpreted as evidence that probability (or indeed predictability) assigns a differential information-value to the two tones that in turn affects the extent to which prediction models are updated and imposed. These effects are exposed for both common and rare occurrences of the tones. The studies contribute to a body of work that reveals that probabilistic information is not faithfully represented in these early evoked potentials and instead exposes that predictability (or conversely uncertainty) may trigger value-based learning modulations even in task-irrelevant incidental learning.
Assuntos
Estimulação Acústica/métodos , Percepção Auditiva/fisiologia , Potenciais Evocados Auditivos/fisiologia , Aprendizagem/fisiologia , Incerteza , Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Desempenho Psicomotor/fisiologia , Adulto JovemRESUMO
In task-switching paradigms, performance is better when repeating the same task than when alternating between tasks (switch cost) and when repeating a task alone rather than intermixed with another task (mixing cost). These costs remain even after extensive practice and when task cues enable advanced preparation (residual costs). Moreover, residual reaction time mixing cost has been consistently shown to increase with age. Residual switch and mixing costs modulate the amplitude of the stimulus-locked P3b. This mixing effect is disproportionately larger in older adults who also prepare more for and respond more cautiously on these "mixed" repeat trials (Karayanidis et al., 2011). In this paper, we analyze stimulus-locked and response-locked P3 and lateralized readiness potentials to identify whether residual switch and mixing cost arise from the need to control interference at the level of stimulus processing or response processing. Residual mixing cost was associated with control of stimulus-level interference, whereas residual switch cost was also associated with a delay in response selection. In older adults, the disproportionate increase in mixing cost was associated with greater interference at the level of decision-response mapping and response programming for repeat trials in mixed-task blocks. These findings suggest that older adults strategically recruit greater proactive and reactive control to overcome increased susceptibility to post-stimulus interference. This interpretation is consistent with recruitment of compensatory strategies to compensate for reduced repetition benefit rather than an overall decline on cognitive flexibility.
RESUMO
The role in which two tones are first encountered in an unattended oddball sequence affects how deviance detection, reflected by mismatch negativity, treats them later when the roles reverse: a "primacy bias." We tested whether this effect is modulated by previous behavioral relevance assigned to the two tones. To this end, sequences in which the roles of the two tones alternated were preceded by a go/no-go task in which tones were presented with equal probability. Half of the participants were asked to respond to the short sounds, the other half to long sounds. Primacy bias was initially abolished but returned dependent upon the go-stimulus that the participant was assigned. Results demonstrate a long-term impact of prior learning on deviance detection, and that even when prior importance/equivalence is learned, the bias ultimately returns. Results are discussed in terms of persistent go-stimulus specific changes in responsiveness to sound.
Assuntos
Percepção Auditiva/fisiologia , Potenciais Evocados Auditivos/fisiologia , Estimulação Acústica , Adolescente , Adulto , Feminino , Humanos , Masculino , Desempenho Psicomotor/fisiologia , Adulto JovemRESUMO
Repetitive patterning facilitates inferences about likely properties of sound to follow. Mismatch negativity (MMN) occurs when sound fails to match an inference. Smaller MMN in schizophrenia indexes deficient gain control (difference in utilizing a limited dynamic range). Although it is clear that this group has a lower limit to MMN size, this study addressed whether smaller MMN indicates impaired perceptual inference. MMN was elicited to four deviants in two sequences: one in which occurrence was random and one in which it was paired. Despite smaller MMN, persons with schizophrenia are equally able to reduce MMN size evoked by a deviant when its occurrence is cued. Results also expose alterations in the evoked response to repeated sounds that appear to be exacerbations of age-related amplitude decline. Since these anomalies impact the computed MMN, they highlight the need to identify all contributions to limits in gain control in schizophrenia.
Assuntos
Antecipação Psicológica/fisiologia , Psicologia do Esquizofrênico , Estimulação Acústica , Adulto , Cognição/fisiologia , Feminino , Humanos , Masculino , Testes Neuropsicológicos , Escalas de Wechsler , Adulto JovemRESUMO
Pattern learning facilitates prediction about upcoming events. Within the auditory system such predictions can be studied by examining effects on a component of the auditory-evoked potential known as mismatch negativity (MMN). MMN is elicited when sound does not conform to the characteristics inferred from statistical probabilities derived from the recent past. Stable patterning in sequences elevates confidence in automatically generated perceptual inferences about what sound should come next and when. MMN amplitude should be larger when sequence is highly stable compared to when it is more volatile. This expectation has been tested using a multi-timescale paradigm. In this study, two sounds of different duration alternate roles as a predictable repetitive "standard" and rare MMN-eliciting "deviation." The paradigm consists of sound sequences that differ in the rate at which the roles of two tones alternate, varying from slowly changing (high stability) to rapidly alternating (low stability). Previous studies using this paradigm discovered a "primacy bias" affecting how stability in patterning impacts MMN amplitude. The primacy bias refers to the observation that the effect of longer-term stability within sequences only appears to impact MMN to the sound first encountered as deviant (the sound that is rare when the sequence commences). This study determines whether this order-driven bias generalizes to sequences that contain two tones differing in pitch. By manipulating (within-subjects) the order in which sounds are encountered as deviants the data demonstrate the two defining characteristics of primacy bias: (1) sequence stability only ever impacts MMN amplitude to the first-deviant sound; and (2) within higher stability sequences, MMN is significantly larger when a sound is the first compared to when it is the second deviant. The results are consistent with a general order-driven bias exerting modulating effects on MMN amplitude over a longer timescale.
RESUMO
Cued-trials task-switching paradigms have been used extensively to examine ageing-related changes in cognitive control. Many studies report an increase in mixing cost (i.e., cost of repeating the same task in a single-task vs. a mixed-task block) and a less reliable increase in switch cost (i.e., cost of switching vs. repeating tasks in a mixed-task block) in old as compared to young adults. However, there is substantial variability between studies in the emergence and size of age effects on mixing and/or switch cost. In this study, we examined variation in mixing cost and switch cost as a function of task practice and preparation interval across the adult lifespan (18-79 years) using a paradigm that promotes advance preparation and reduces cue encoding differences between switch and repeat trials. Both preparation interval and task practice modulated mixing cost and switch cost-but task practice mediated the effects of preparation interval and age differentially for mixing cost and switch cost. Mixing cost was consistently larger in older participants, reduced with preparation and varied little with task practice. In contrast, the effect of preparation interval on switch cost varied with task practice. Reduction in switch cost with preparation interval emerged in younger participants by the second practice session and even later in older participants. When fully practiced, older participants showed greater mixing cost but less switch cost than younger participants. Age effects on both mixing cost and switch cost were mediated by changes in processing of repeat trials, indicative of reduced differentiation between switch and repeat trials in mixed-task blocks. This is consistent with reduced cognitive flexibility with increasing age.
Assuntos
Envelhecimento/fisiologia , Atenção/fisiologia , Cognição/fisiologia , Prática Psicológica , Desempenho Psicomotor/fisiologia , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tempo de Reação/fisiologiaRESUMO
Task-switching paradigms produce a highly consistent age-related increase in mixing cost [longer response time (RT) on repeat trials in mixed-task than single-task blocks] but a less consistent age effect on switch cost (longer RT on switch than repeat trials in mixed-task blocks). We use two approaches to examine the adult lifespan trajectory of control processes contributing to mixing cost and switch cost: latent variables derived from an evidence accumulation model of choice, and event-related potentials (ERP) that temporally differentiate proactive (cue-driven) and reactive (target-driven) control processes. Under highly practiced and prepared task conditions, aging was associated with increasing RT mixing cost but reducing RT switch cost. Both effects were largely due to the same cause: an age effect for mixed-repeat trials. In terms of latent variables, increasing age was associated with slower non-decision processes, slower rate of evidence accumulation about the target, and higher response criterion. Age effects on mixing costs were evident only on response criterion, the amount of evidence required to trigger a decision, whereas age effects on switch cost were present for all three latent variables. ERPs showed age-related increases in preparation for mixed-repeat trials, anticipatory attention, and post-target interference. Cue-locked ERPs that are linked to proactive control were associated with early emergence of age differences in response criterion. These results are consistent with age effects on strategic processes controlling decision caution. Consistent with an age-related decline in cognitive flexibility, younger adults flexibly adjusted response criterion from trial-to-trial on mixed-task blocks, whereas older adults maintained a high criterion for all trials.
RESUMO
Mutations in the gene encoding human copper-zinc superoxide dismutase (SOD1) cause a dominant form of the progressive neurodegenerative disease amyotrophic lateral sclerosis. Transgenic mice expressing the human G85R SOD1 variant develop paralytic symptoms concomitant with the appearance of SOD1-enriched proteinaceous inclusions in their neural tissues. The process(es) through which misfolding or aggregation of G85R SOD1 induces motor neuron toxicity is not understood. Here we present structures of the human G85R SOD1 variant determined by single crystal x-ray diffraction. Alterations in structure of the metal-binding loop elements relative to the wild type enzyme suggest a molecular basis for the metal ion deficiency of the G85R SOD1 protein observed in the central nervous system of transgenic mice and in purified recombinant G85R SOD1. These findings support the notion that metal-deficient and/or disulfide-reduced mutant SOD1 species contribute to toxicity in SOD1-linked amyotrophic lateral sclerosis.
Assuntos
Substituição de Aminoácidos , Esclerose Lateral Amiotrófica/enzimologia , Dobramento de Proteína , Superóxido Dismutase/química , Esclerose Lateral Amiotrófica/genética , Animais , Sítios de Ligação , Cristalografia por Raios X , Humanos , Camundongos , Camundongos Transgênicos , Neurônios Motores/enzimologia , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1RESUMO
Mutations in Cu,Zn superoxide dismutase (SOD1) can cause amyotrophic lateral sclerosis (ALS) through mechanisms proposed to involve SOD1 misfolding, but the intracellular factors that modulate folding and stability of SOD1 are largely unknown. By using yeast and mammalian expression systems, we demonstrate here that SOD1 stability is governed by post-translational modification factors that target the SOD1 disulfide. Oxidation of the human SOD1 disulfide in vivo was found to involve both the copper chaperone for SOD1 (CCS) and the CCS-independent pathway for copper activation. When both copper pathways were blocked, wild type SOD1 stably accumulated in yeast cells with a reduced disulfide, whereas ALS SOD1 mutants A4V, G93A, and G37R were degraded. We describe here an unprecedented role for the thiol oxidoreductase glutaredoxin in reducing the SOD1 disulfide and destabilizing ALS mutants. Specifically, the major cytosolic glutaredoxin of yeast was seen to reduce the intramolecular disulfide of ALS SOD1 mutant A4V SOD1 in vivo and in vitro. By comparison, glutaredoxin was less reactive toward the disulfide of wild type SOD1. The apo-form of A4V SOD1 was highly reactive with glutaredoxin but not SOD1 containing both copper and zinc. Glutaredoxin therefore preferentially targets the immature form of ALS mutant SOD1 lacking metal co-factors. Overall, these studies implicate a critical balance between cellular reductants such as glutaredoxin and copper activation pathways in controlling the disulfide and stability of SOD1 in vivo.
Assuntos
Cobre/metabolismo , Dissulfetos/química , Oxirredutases/fisiologia , Superóxido Dismutase/metabolismo , Cobre/química , Fibroblastos/metabolismo , Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica , Glutarredoxinas , Humanos , Mutação , Oxirredutases/química , Oxirredutases/metabolismo , Oxigênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Compostos de Sulfidrila/químicaRESUMO
Mutations in copper-zinc superoxide dismutase cause the neurodegenerative disease amyotrophic lateral sclerosis. Many of the mutant proteins have increased turnover in vivo and decreased thermal stability. Here we show that purified, metal-free superoxide dismutases are degraded in vitro by purified 20 S proteasome in the absence of ATP and without ubiquitinylation, whereas their metal-bound counterparts are not. The rate of degradation by the proteasome varied among the mutants studied, and the rate correlated with the in vivo half-life. The monomeric forms of both mutant and wild-type superoxide dismutase are particularly susceptible to degradation by the proteasome. Exposure of hydrophobic regions as a consequence of decreased thermal stability may allow the proteasome to recognize these molecules as non-native.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Superóxido Dismutase/genética , Trifosfato de Adenosina/química , Esclerose Lateral Amiotrófica/genética , Animais , Dissulfetos/química , Temperatura Alta , Metais/química , Peptídeos/química , Complexo de Endopeptidases do Proteassoma/química , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase/fisiologia , Superóxido Dismutase-1 , Temperatura , Fatores de Tempo , Ubiquitina/químicaRESUMO
Beta2microglobulin (beta2m) is the major protein component of the fibrillar amyloid deposits isolated from patients diagnosed with dialysis-related amyloidosis (DRA). While investigating the molecular mechanism of amyloid fibril formation by beta2m, we found that the beta2m C-terminal peptide of 28 residues (cbeta2m) itself forms amyloid fibrils. When viewed by electron microscopy, cbeta2m aggregates appear as elongated unbranched fibers, the morphology typical for amyloids. Cbeta2m fibers stain with Congo red and show apple-green birefringence in polarized light, characteristic of amyloids. The observation that the beta2m C-terminal fragment readily forms amyloid fibrils implies that beta2m amyloid fibril formation proceeds via interactions of amyloid forming segments, which become exposed when the beta2m subunit is partially unfolded.
Assuntos
Aminoácidos/química , Amiloide/metabolismo , Microglobulina beta-2/química , Sequência de Aminoácidos , Aminoácidos/metabolismo , Amiloide/biossíntese , Amiloide/ultraestrutura , Birrefringência , Dicroísmo Circular , Vermelho Congo/química , Escherichia coli/genética , Escherichia coli/metabolismo , Luz , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/ultraestrutura , Espalhamento de Radiação , Espectrofotometria/métodos , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismo , Microglobulina beta-2/ultraestruturaRESUMO
Because copper is both an essential cofactor and a toxic metal, different strategies have evolved to appropriately regulate its homeostasis as a function of changing environmental copper levels. In this report, we describe a metallochaperone-like protein from Schizosaccharomyces pombe that maintains the delicate balance between essentiality and toxicity. This protein, designated Pccs, has four distinct domains. SOD activity assays reveal that the first three domains of Pccs are necessary and sufficient to deliver copper to its target, copper-zinc superoxide dismutase (SOD1). Pccs domain IV, which is absent in Saccharomyces cerevisiae CCS1, contains seventeen cysteine residues, eight pairs of which are in a potential metal coordination arrangement, Cys-Cys. We show that S. cerevisiae ace1Delta mutant cells expressing the full-length Pccs molecule are resistant to copper toxicity. Furthermore, we demonstrate that the Pccs domain IV enhances copper resistance of the ace1Delta cells by an order of magnitude compared with that observed in the same strain expressing a pccs+ I-II-III allele encoding Pccs domains I-III. We consistently found that S. pombe cells disrupted in the pccs+ gene exhibit an increased sensitivity to copper and cadmium. Furthermore, we demonstrate that overexpression of pccs+ is associated with increased copper resistance in fission yeast cells. Taken together, our findings suggest that Pccs activates apo-SOD1 under copper-limiting conditions through the use of its first three domains and protects cells against metal ion toxicity via its fourth domain.
Assuntos
Cobre/metabolismo , Chaperonas Moleculares/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/metabolismo , Alelos , Motivos de Aminoácidos , Sequência de Aminoácidos , Transporte Biológico , Cádmio/química , Cobre/química , Cisteína/química , Relação Dose-Resposta a Droga , Proteínas de Fluorescência Verde , Humanos , Íons , Proteínas Luminescentes/metabolismo , Chaperonas Moleculares/química , Dados de Sequência Molecular , Mutação , Fenótipo , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Homologia de Sequência de Aminoácidos , Prata/química , Superóxido Dismutase/química , Superóxido Dismutase-1 , Temperatura , Fatores de Tempo , Zinco/químicaRESUMO
The dissociation of apo- and metal-bound human copper-zinc superoxide dismutase (SOD1) dimers induced by the chaotrope guanidine hydrochloride (GdnHCl) or the reductant Tris(2-carboxyethyl)phosphine (TCEP) has been analyzed using analytical ultracentrifugation. Global fitting of sedimentation equilibrium data under native solution conditions (without GdnHCl or TCEP) demonstrate that both the apo- and metal-bound forms of SOD1 are stable dimers. Sedimentation velocity experiments show that apo-SOD1 dimers dissociate cooperatively over the range 0.5-1.0 M GdnHCl. In contrast, metal-bound SOD1 dimers possess a more compact shape and dissociate at significantly higher GdnHCl concentrations (2.0-3.0 M). Reduction of the intrasubunit disulfide bond within each SOD1 subunit by 5-10 mM TCEP promotes dissociation of apo-SOD1 dimers, whereas the metal-bound enzyme remains a stable dimer under these conditions. The Cys-57 --> Ser mutant of SOD1, a protein incapable of forming the intrasubunit disulfide bond, sediments as a monomer in the absence of metal ions and as a dimer when metals are bound. Taken together, these data indicate that the stability imparted to the human SOD1 dimer by metal binding and the formation of the intrasubunit disulfide bond are mediated by independent molecular mechanisms. By combining the sedimentation data with previous crystallographic results, a molecular explanation is provided for the existence of different SOD1 macromolecular shapes and multiple SOD1 dimeric species with different stabilities.
Assuntos
Dimerização , Estabilidade Enzimática/efeitos dos fármacos , Guanidina/farmacologia , Fosfinas/farmacologia , Superóxido Dismutase/química , Superóxido Dismutase/genética , Fenômenos Químicos , Físico-Química , Cristalização , Dissulfetos/química , Eletroforese em Gel de Poliacrilamida , Humanos , Indicadores e Reagentes , Modelos Moleculares , Estrutura Molecular , Oxirredução , Superóxido Dismutase-1 , UltracentrifugaçãoRESUMO
More than 90 point mutations in human CuZn superoxide dismutase lead to the development of familial amyotrophic lateral sclerosis, known also as motor neuron disease. A growing body of evidence suggests that a subset of mutations located close to the dimeric interface can lead to a major destabilization of the mutant enzymes. We have determined the crystal structures of the Ala4Val (A4V) and Ile113Thr (I113T) mutants to 1.9 and 1.6 A, respectively. In the A4V structure, small changes at the dimer interface result in a substantial reorientation of the two monomers. This effect is also seen in the case of the I113T crystal structure, but to a smaller extent. X-ray solution scattering data show that in the solution state, both of the mutants undergo a more pronounced conformational change compared with wild-type superoxide dismutase (SOD) than that observed in the A4V crystal structure. Shape reconstructions from the x-ray scattering data illustrate the nature of this destabilization. Comparison of these scattering data with those for bovine CuZn SOD measured at different temperatures shows that an analogous change in the scattering profile occurs for the bovine enzyme in the temperature range of 70-80 degrees C. These results demonstrate that the A4V and I113T mutants are substantially destabilized in comparison with wild-type SOD1, and it is possible that the pathogenic properties of this subset of familial amyotrophic lateral sclerosis mutants are at least in part due to this destabilization.