RESUMEN
Perineuronal nets (PNN), a specialized form of ECM (?), surround numerous neurons in the CNS and allow synaptic connectivity through holes in its structure. We hypothesis that PNNs serve as gatekeepers that guard and protect synaptic territory, and thus may stabilize an engram circuit. We present high-resolution, and 3D EM images of PNN- engulfed neurons showing that synapses occupy the PNN holes, and that invasion of other cellular components are rare. PNN constituents are long-lived and can be eroded faster in an enriched environment, while synaptic proteins have high turnover rate. Preventing PNN erosion by using pharmacological inhibition of PNN-modifying proteases or MMP9 knockout mice allowed normal fear memory acquisition but diminished remote-memory stabilization, supporting the above hypothesis. Significance: In this multidisciplinary work, we challenge the hypothesis that the pattern of holes in the perineuronal nets (PNN) hold the code for very-long-term memories. The scope of this work might lead us closer to the understanding of how we can vividly remember events from childhood to death bed. We postulate that the PNN holes hold the code for the engram. To test this hypothesis, we used three independent experimental strategies; high-resolution 3D electron microscopy, Stable Isotop Labeling in Mammals (SILAM) for proteins longevity, and pharmacologically and genetically interruption of memory consolidation in fear conditioning experiments. All of these experimental results did not dispute the PNN hypothesis.
RESUMEN
Many adult tissues contain postmitotic cells as old as the host organism. The only organelle that does not turn over in these cells is the nucleus, and its maintenance represents a formidable challenge, as it harbors regulatory proteins that persist throughout adulthood. Here we developed strategies to visualize two classes of such long-lived proteins, histones and nucleoporins, to understand the function of protein longevity in nuclear maintenance. Genome-wide mapping of histones revealed specific enrichment of long-lived variants at silent gene loci. Interestingly, nuclear pores are maintained by piecemeal replacement of subunits, resulting in mosaic complexes composed of polypeptides with vastly different ages. In contrast, nondividing quiescent cells remove old nuclear pores in an ESCRT-dependent manner. Our findings reveal distinct molecular strategies of nuclear maintenance, linking lifelong protein persistence to gene regulation and nuclear integrity.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Histonas/metabolismo , Mitosis/fisiología , Poro Nuclear/metabolismo , Animales , Línea Celular , Estudio de Asociación del Genoma Completo , Ratones , Factores de TiempoRESUMEN
Aging is associated with the decline of protein, cell, and organ function. Here, we use an integrated approach to characterize gene expression, bulk translation, and cell biology in the brains and livers of young and old rats. We identify 468 differences in protein abundance between young and old animals. The majority are a consequence of altered translation output, that is, the combined effect of changes in transcript abundance and translation efficiency. In addition, we identify 130 proteins whose overall abundance remains unchanged but whose sub-cellular localization, phosphorylation state, or splice-form varies. While some protein-level differences appear to be a generic property of the rats' chronological age, the majority are specific to one organ. These may be a consequence of the organ's physiology or the chronological age of the cells within the tissue. Taken together, our study provides an initial view of the proteome at the molecular, sub-cellular, and organ level in young and old rats.
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Intracellular proteins with long lifespans have recently been linked to age-dependent defects, ranging from decreased fertility to the functional decline of neurons. Why long-lived proteins exist in metabolically active cellular environments and how they are maintained over time remains poorly understood. Here, we provide a system-wide identification of proteins with exceptional lifespans in the rat brain. These proteins are inefficiently replenished despite being translated robustly throughout adulthood. Using nucleoporins as a paradigm for long-term protein persistence, we found that nuclear pore complexes (NPCs) are maintained over a cell's life through slow but finite exchange of even its most stable subcomplexes. This maintenance is limited, however, as some nucleoporin levels decrease during aging, providing a rationale for the previously observed age-dependent deterioration of NPC function. Our identification of a long-lived proteome reveals cellular components that are at increased risk for damage accumulation, linking long-term protein persistence to the cellular aging process. PAPERCLIP:
Asunto(s)
Encéfalo/citología , Senescencia Celular , Proteínas de Complejo Poro Nuclear/metabolismo , Proteoma/metabolismo , Animales , Encéfalo/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Poro Nuclear/metabolismo , Biosíntesis de Proteínas , RatasRESUMEN
Protein turnover is an effective way of maintaining a functional proteome, as old and potentially damaged polypeptides are destroyed and replaced by newly synthesized copies. An increasing number of intracellular proteins, however, have been identified that evade this turnover process and instead are maintained over a cell's lifetime. This diverse group of long-lived proteins might be particularly prone to accumulation of damage and thus have a crucial role in the functional deterioration of key regulatory processes during ageing.
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Senescencia Celular , Proteínas/metabolismo , Proteoma/metabolismo , Homeostasis , Humanos , Biosíntesis de ProteínasRESUMEN
To combat the functional decline of the proteome, cells use the process of protein turnover to replace potentially impaired polypeptides with new functional copies. We found that extremely long-lived proteins (ELLPs) did not turn over in postmitotic cells of the rat central nervous system. These ELLPs were associated with chromatin and the nuclear pore complex, the central transport channels that mediate all molecular trafficking in and out of the nucleus. The longevity of these proteins would be expected to expose them to potentially harmful metabolites, putting them at risk of accumulating damage over extended periods of time. Thus, it is possible that failure to maintain proper levels and functional integrity of ELLPs in nonproliferative cells might contribute to age-related deterioration in cell and tissue function.
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Encéfalo/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Animales , Encéfalo/citología , Senescencia Celular , Cromatina/metabolismo , Femenino , Semivida , Hígado/metabolismo , Mitosis , Proteoma/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Many, perhaps most, proteins, are capable of forming self-propagating, ß-sheet (amyloid) aggregates. Amyloid-like aggregates are found in a wide range of diseases and underlie prion-based inheritance. Despite intense interest in amyloids, structural details have only recently begun to be revealed as advances in biophysical approaches, such as hydrogen-deuterium exchange, X-ray crystallography, solid-state nuclear magnetic resonance (SSNMR), and cryoelectron microscopy (cryoEM), have enabled high-resolution insights into their molecular organization. Initial studies found that despite the highly divergent primary structure of different amyloid-forming proteins, amyloids from different sources share many structural similarities. With higher-resolution information, however, it has become clear that, on the molecular level, amyloids comprise a wide diversity of structures. Particularly surprising has been the finding that identical polypeptides can fold into multiple, distinct amyloid conformations and that this structural diversity can lead to distinct heritable prion states or strains.
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Amiloide/química , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Amiloide/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Microscopía de Fuerza Atómica , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Priones/química , Priones/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Difracción de Rayos XRESUMEN
Prion proteins can adopt multiple infectious strain conformations. Here we investigate how the sequence of a prion protein affects its capacity to propagate specific conformations by exploiting our ability to create two distinct infectious conformations of the yeast [PSI(+)] prion protein Sup35, termed Sc4 and Sc37. PNM2, a G58D point mutant of Sup35 that was originally identified for its dominant interference with prion propagation, leads to rapid, recessive loss of Sc4 but does not interfere with propagation of Sc37. PNM2 destabilizes the amyloid core of Sc37 and causes compensatory effects that slow prion growth but aid prion division and result in robust propagation of Sc37. By contrast, PNM2 does not affect the structure or chaperone-mediated division of Sc4 but interferes with its delivery to daughter cells. Thus, effective delivery of infectious particles during cell division is a crucial and conformation-dependent step in prion inheritance.
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Priones/metabolismo , Saccharomyces cerevisiae/metabolismo , Chaperonas Moleculares/metabolismo , Estructura MolecularRESUMEN
Aggregation-prone proteins often misfold into multiple distinct amyloid conformations that dictate different physiological impacts. Although amyloid formation is triggered by a transient nucleus, the mechanism by which an initial nucleus is formed and allows the protein to form a specific amyloid conformation has been unclear. Here we show that, before fiber formation, the prion domain (Sup35NM, consisting of residues 1-254) of yeast prion Sup35, the [PSI(+)] protein determinant, forms oligomers in a temperature-dependent, reversible manner. Mutational and biophysical analyses revealed that 'non-native' aromatic interactions outside the amyloid core drive oligomer formation by bringing together different Sup35NM monomers, which specifically leads to the formation of highly infectious strain conformations with more limited amyloid cores. Thus, transient non-native interactions in the initial nucleus are pivotal in determining the diversity of amyloid conformations and resulting prion strain phenotypes.
RESUMEN
Many amyloid inhibitors resemble molecules that form chemical aggregates, which are known to inhibit many proteins. Eight known chemical aggregators inhibited amyloid formation of the yeast and mouse prion proteins Sup35 and recMoPrP in a manner characteristic of colloidal inhibition. Similarly, three known anti-amyloid molecules inhibited beta-lactamase in a detergent-dependent manner, which suggests that they too form colloidal aggregates. The colloids localized to preformed fibers and prevented new fiber formation in electron micrographs. They also blocked infection of yeast cells with Sup35 prions, which suggests that colloidal inhibition may be relevant in more biological milieus.
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Acetofenonas/farmacología , Benzopiranos/farmacología , Clioquinol/farmacología , Rojo Congo/farmacología , Flavanonas/farmacología , Fenolftaleínas/farmacología , Ftalimidas/farmacología , Priones/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Saccharomyces cerevisiae/metabolismo , Acetofenonas/química , Animales , Benzopiranos/química , Clioquinol/química , Rojo Congo/química , Detergentes/química , Flavanonas/química , Ratones , Microscopía Electrónica de Transmisión/métodos , Estructura Molecular , Peso Molecular , Tamaño de la Partícula , Factores de Terminación de Péptidos , Fenolftaleínas/química , Ftalimidas/química , Priones/química , Priones/metabolismo , Priones/farmacocinética , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/farmacocinética , Sensibilidad y Especificidad , Relación Estructura-Actividad , Inhibidores de beta-Lactamasas , beta-Lactamasas/químicaRESUMEN
Among the many surprises to arise from studies of prion biology, perhaps the most unexpected is the strain phenomenon whereby a single protein can misfold into structurally distinct, infectious states that cause distinguishable phenotypes. Similarly, proteins can adopt a spectrum of conformations in non-infectious diseases of protein folding; some are toxic and others are well tolerated. However, our understanding of the structural differences underlying prion strains and how these differences alter their physiological impact remains limited. Here we use a combination of solution NMR, amide hydrogen/deuterium (H/D) exchange and mutagenesis to study the structural differences between two strain conformations, termed Sc4 and Sc37 (ref. 5), of the yeast Sup35 prion. We find that these two strains have an overlapping amyloid core spanning most of the Gln/Asn-rich first 40 amino acids that is highly protected from H/D exchange and very sensitive to mutation. These features indicate that the cores are composed of tightly packed beta-sheets possibly resembling 'steric zipper' structures revealed by X-ray crystallography of Sup35-derived peptides. The stable structure is greatly expanded in the Sc37 conformation to encompass the first 70 amino acids, revealing why this strain shows increased fibre stability and decreased ability to undergo chaperone-mediated replication. Our findings establish that prion strains involve large-scale conformational differences and provide a structural basis for understanding a broad range of functional studies, including how conformational changes alter the physiological impact of prion strains.
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Priones/química , Priones/clasificación , Pliegue de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/clasificación , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Mutagénesis , Mutación , Resonancia Magnética Nuclear Biomolecular , Factores de Terminación de Péptidos , Fenotipo , Priones/genética , Priones/patogenicidad , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
A principle that has emerged from studies of protein aggregation is that proteins typically can misfold into a range of different aggregated forms. Moreover, the phenotypic and pathological consequences of protein aggregation depend critically on the specific misfolded form. A striking example of this is the prion strain phenomenon, in which prion particles composed of the same protein cause distinct heritable states. Accumulating evidence from yeast prions such as [PSI+] and mammalian prions argues that differences in the prion conformation underlie prion strain variants. Nonetheless, it remains poorly understood why changes in the conformation of misfolded proteins alter their physiological effects. Here we present and experimentally validate an analytical model describing how [PSI+] strain phenotypes arise from the dynamic interaction among the effects of prion dilution, competition for a limited pool of soluble protein, and conformation-dependent differences in prion growth and division rates. Analysis of three distinct prion conformations of yeast Sup35 (the [PSI+] protein determinant) and their in vivo phenotypes reveals that the Sup35 amyloid causing the strongest phenotype surprisingly shows the slowest growth. This slow growth, however, is more than compensated for by an increased brittleness that promotes prion division. The propensity of aggregates to undergo breakage, thereby generating new seeds, probably represents a key determinant of their physiological impact for both infectious (prion) and non-infectious amyloids.