screening-for-amyloid-aggregation-semi-denaturing-detergent-agarose

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

detecting-characterizing-protein-self-assembly-vivo-flow

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

Protein conformational diseases are associated with the aberrant accumulation of amyloid protein aggregates, but whether amyloid formation is cytotoxic or protective is unclear. To address this issue, we investigated a normally benign amyloid formed by the yeast prion [RNQ(+)]. Surprisingly, modest overexpression of Rnq1 protein was deadly, but only when preexisting Rnq1 was in the [RNQ(+)] prion conformation. Molecular chaperones protect against protein aggregation diseases and are generally believed to do so by solubilizing their substrates. The Hsp40 chaperone, Sis1, suppressed Rnq1 proteotoxicity, but instead of blocking Rnq1 protein aggregation, it stimulated conversion of soluble Rnq1 to [RNQ(+)] amyloid. Furthermore, interference with Sis1-mediated [RNQ(+)] amyloid formation exacerbated Rnq1 toxicity. These and other data establish that even subtle changes in the folding homeostasis of an amyloidogenic protein can create a severe proteotoxic gain-of-function phenotype and that chaperone-mediated amyloid assembly can be cytoprotective. The possible relevance of these findings to other phenomena, including prion-driven neurodegenerative diseases and heterokaryon incompatibility in fungi, is discussed.

Chaperone-dependent amyloid assembly protects cells from prion toxicity.

Protein-Konformations&auml;nderungen Krankheiten zugeordnet die aberrante Anh&auml;ufung von Amyloid-Protein-Aggregate, aber es ist unklar, ob Amyloid Bildung zytotoxischen oder sch&uuml;tzende ist. Um dieses Problem zu beheben, untersuchten wir eine Regel gutartige Amyloid gebildet durch die Hefe Prion [RNQ(+)]. &Uuml;berraschenderweise war bescheiden eine &Uuml;berexpression von Rnq1 Protein t&ouml;dlich, aber nur als bereits vorhandenen Rnq1 in der [RNQ(+)]-Prion-Konformation. Molekulare Chaperone gegen Protein Aggregation Krankheiten sch&uuml;tzen und werden im Allgemeinen geglaubt, um dies tun, indem Sie ihre Substrate L&ouml;sungsvermittlung. Das Hsp40 Chaperone, Sis1, unterdr&uuml;ckt Rnq1 Proteotoxicity, aber anstatt zu blockieren Rnq1 Proteinanh&auml;ufung, stimuliert es Umwandlung von l&ouml;slichen Rnq1 in [RNQ(+)]-Amyloid. Dar&uuml;ber hinaus verst&auml;rkt Interferenzen mit Sis1-vermittelte [RNQ(+)] amiloide Bildung Rnq1 Toxizit&auml;t. Diese und andere Daten fest, dass sogar subtile Ver&auml;nderungen in der Faltung Hom&ouml;ostase eines Proteins Amyloidogenic einen schwere Proteotoxic Gain of Function Ph&auml;notyp erstellen k&ouml;nnen und Chaperon-vermittelte Amyloid Assembly Cytoprotective werden kann. M&ouml;gliche Relevanz dieser Ergebnisse auf andere Ph&auml;nomene, einschlie&szlig;lich Prion-gesteuerte Neurodegenerative Krankheiten und Heterokaryon Inkompatibilit&auml;t in Pilzen, wird diskutiert.

Chaperon abh&auml;ngigen Amyloid Versammlung sch&uuml;tzt die Zellen vor Prion-Toxizit&auml;t.

Prions are proteins that convert between structurally and functionally distinct states, one or more of which is transmissible. In yeast, this ability allows them to act as non-Mendelian elements of phenotypic inheritance. To further our understanding of prion biology, we conducted a bioinformatic proteome-wide survey for prionogenic proteins in S. cerevisiae, followed by experimental investigations of 100 prion candidates. We found an unexpected amino acid bias in aggregation-prone candidates and discovered that 19 of these could also form prions. At least one of these prion proteins, Mot3, produces a bona fide prion in its natural context that increases population-level phenotypic heterogeneity. The self-perpetuating states of these proteins present a vast source of heritable phenotypic variation that increases the adaptability of yeast populations to diverse environments.

A systematic survey identifies prions and illuminates sequence features of prionogenic proteins.

Prionen sind Proteine, die Konvertierung zwischen strukturell und funktionell verschiedenen Staaten, mindestens eine davon ist &uuml;bertragbar. In der Hefe kann diese F&auml;higkeit als non-Mendelian Elemente der ph&auml;notypischen Vererbung zu agieren. Um unser Verst&auml;ndnis der Prion Biologie zu f&ouml;rdern, haben wir eine Bioinformatic Proteom-weite Umfrage f&uuml;r Prionogenic Proteine in S. Cerevisiae, gefolgt von experimentellen Untersuchungen von 100 Prion-Kandidaten durchgef&uuml;hrt. Wir fanden ein unerwarteter Aminos&auml;ure-Vorurteil in Aggregation neigende Kandidaten und entdeckt, dass 19 dieser auch Prionen bilden k&ouml;nnte. Mindestens eines dieser Prionproteine, Mot3, produziert ein gutgl&auml;ubiger Prion in seinem nat&uuml;rlichen Kontext, der Bev&ouml;lkerung auf ph&auml;notypischen Heterogenit&auml;t erh&ouml;ht. Die Selbstl&auml;ufer Staaten dieser Proteine pr&auml;sentieren eine gro&szlig;e Quelle der vererbbare ph&auml;notypische Variation, die die Anpassungsf&auml;higkeit von Hefe Bev&ouml;lkerungsteilen in verschiedenen Umgebungen erh&ouml;ht.

Eine systematische Untersuchung Prionen identifiziert und leuchtet Sequenz Merkmale der Prionogenic Proteine.

Prions are fascinating but often misunderstood protein aggregation phenomena. The traditional association of the mammalian prion protein with disease has overshadowed a potentially more interesting attribute of prions: their ability to create protein-based molecular memories. In fungi, prions alter the relationship between genotype and phenotype in a heritable way that diversifies clonal populations. Recent findings in yeast indicate that prions might be much more common than previously realized. Moreover, prion-driven phenotypic diversity increases under stress, and can be amplified by the dynamic maturation of prion-initiating states. In this article, we suggest that these qualities allow prions to act as 'bet-hedging' devices that facilitate the adaptation of yeasts to stressful environments, and might speed the evolution of new traits.

Prions, protein homeostasis, and phenotypic diversity.

Prionen sind faszinierend, aber oft missverstandenen Protein Aggregation Ph&auml;nomene. Die traditionelle Verbindung von S&auml;ugetier-Prionproteins mit Krankheit hat ein potentiell interessanter-Attribut von Prionen &uuml;berschattet: ihre F&auml;higkeit, Protein-basierte Molekulare Erinnerungen zu erstellen. In Pilzen ver&auml;ndern Prionen die Beziehung zwischen Genotyp und Ph&auml;notyp vererbbare gewisserma&szlig;en, die klonale Populationen diversifiziert. Neuere Befunde in der Hefe zeigen, dass Prionen sehr viel h&auml;ufiger als bisher realisiert werden k&ouml;nnte. Dar&uuml;ber hinaus Prion-gesteuerte ph&auml;notypische Vielfalt unter Stress erh&ouml;ht und verst&auml;rkt werden kann, durch die dynamische Reifung der Prion-Einleitung Staaten. In diesem Artikel wird empfohlen, diese Qualit&auml;ten erlauben Prionen als &#39;Wette Hedge Accounting&#39; Ger&auml;te handeln, die Erleichterung der Anpassung der Hefen zu stressig Umgebungen und unter Umst&auml;nden beschleunigt die Entwicklung von neuen Merkmalen.

Prionen, Protein-Hom&ouml;ostase und ph&auml;notypische Vielfalt.

Protein aggregates are associated with a variety of debilitating human diseases, but they can have functional roles as well. Both pathological and nonpathological protein aggregates display tremendous diversity, with substantial differences in aggregate size, morphology, and structure. Among the different aggregation types, amyloids are particularly remarkable, because of their high degree of order and their ability to form self-perpetuating conformational states. Amyloids form the structural basis for a group of proteins called prions, which have the ability to generate new phenotypes by a simple switch in protein conformation that does not involve changes in the sequence of the DNA. Although protein aggregates are notoriously difficult to study, recent technological developments and, in particular, the use of yeast prions as model systems, have been very instrumental in understanding fundamental aspects of aggregation. Here, we provide a range of biochemical, cell biological and yeast genetic methods that are currently used in our laboratory to study protein aggregation and the formation of amyloids and prions.

Biochemical, cell biological, and genetic assays to analyze amyloid and prion aggregation in yeast.

Protein-Aggregate sind mit einer Vielzahl von schw&auml;chenden Krankheiten des Menschen verbunden, aber funktionale Rollen auch haben k&ouml;nnen. Beide pathologische und nonpathological Protein-Aggregate zeigen enorme Vielfalt, mit erheblichen Unterschieden in der aggregierten Gr&ouml;&szlig;e, Morphologie und Struktur. Unter den verschiedenen Aggregation-Arten sind amyloide besonders bemerkenswert, wegen ihrer hohen Grad an Ordnung und ihre F&auml;higkeit, Selbstl&auml;ufer Konformations&auml;nderungen Staaten zu bilden. Amyloide bilden die strukturelle Grundlage f&uuml;r eine Gruppe von Proteinen, genannt Prionen, die haben die F&auml;higkeit zu neue Ph&auml;notypen durch einen einfachen Schalter in Protein Konformation zu generieren, die keine &Auml;nderungen in der Sequenz der DNA verbunden ist. Obwohl Protein-Aggregate notorisch schwierig sind zu studieren, die j&uuml;ngste technologische Entwicklungen und, insbesondere die Verwendung von Hefe Prionen als Modellsysteme, waren sehr grundlegende Aspekte der Aggregation zu verstehen. Hier bieten wir eine Reihe von biochemischen, Zelle biologischen und Hefe genetische Methoden, die derzeit in unserem Labor verwendet werden, um Proteinanh&auml;ufung zu studieren und die Bildung von amyloide und Prionen.

Biochemische, Zelle biologische und genetische Proben Amyloid und Prion Aggregation in der Hefe zu analysieren.

Prionen sind eine ungew&ouml;hnliche Form der Epigenetik: ihre stabile Vererbung und komplexe Ph&auml;notypen durch Proteinfaltung anstatt Nukleins&auml;ure-assoziierte Ver&auml;nderungen zustande kommen. Mit intimen Verbindungen zum Protein-Hom&ouml;ostase und eine bemerkenswerte Empfindlichkeit zu betonen sind Prionen einen robusten Mechanismus, der Umwelt Extreme mit den Erwerb und die Vererbung von neuen Merkmalen verkn&uuml;pft.

Epigenetics im Extremfall: Prionen und dem Erbe der &ouml;kologisch erworbenen Merkmale.

Prions are an unusual form of epigenetics: Their stable inheritance and complex phenotypes come about through protein folding rather than nucleic acid-associated changes. With intimate ties to protein homeostasis and a remarkable sensitivity to stress, prions are a robust mechanism that links environmental extremes with the acquisition and inheritance of new traits.

Epigenetics in the extreme: prions and the inheritance of environmentally acquired traits.

Proteins of all kinds can self-assemble into highly ordered Î²-sheet aggregates known as amyloid fibrils, important both biologically and clinically. However, the specific molecular structure of a fibril can vary dramatically depending on sequence and environmental conditions, and mutations can drastically alter amyloid function and pathogenicity. Experimental structure determination has proven extremely difficult with only a handful of NMR-based models proposed, suggesting a need for computational methods.

A method for probing the mutational landscape of amyloid structure.

Proteine aller Art k&ouml;nnen in self-assemble hoch &beta;-Blatt Aggregate bekannt als Amyloid-Fibrillen, wichtig, sowohl biologisch als auch klinisch bestellt. Jedoch die spezifische Molekulare Struktur des eine Fibrille kann erheblich variieren, je nach Sequenz und Umweltbedingungen und Mutationen k&ouml;nnen drastisch ver&auml;ndern, Amyloid-Funktion und Pathogenit&auml;t. Experimentelle Struktur Bestimmung bew&auml;hrt &auml;u&szlig;erst schwierig, mit nur einer Handvoll von NMR-basierte Modelle vorgeschlagen, Bedarf f&uuml;r einen Berechnungsmethoden.

Eine Methode zur &Uuml;berpr&uuml;fung der Mutationszucht Landschaft des Amyloid-Struktur.

Sequences rich in glutamine (Q) and asparagine (N) residues often fail to fold at the monomer level. This, coupled to their unusual hydrogen-bonding abilities, provides the driving force to switch between disordered monomers and amyloids. Such transitions govern processes as diverse as human protein-folding diseases, bacterial biofilm assembly, and the inheritance of yeast prions (protein-based genetic elements). A systematic survey of prion-forming domains suggested that Q and N residues have distinct effects on amyloid formation. Here, we use cell biological, biochemical, and computational techniques to compare Q/N-rich protein variants, replacing Ns with Qs and Qs with Ns. We find that the two residues have strong and opposing effects: N richness promotes assembly of benign self-templating amyloids; Q richness promotes formation of toxic nonamyloid conformers. Molecular simulations focusing on intrinsic folding differences between Qs and Ns suggest that their different behaviors are due to the enhanced turn-forming propensity of Ns over Qs.

Opposing effects of glutamine and asparagine govern prion formation by intrinsically disordered proteins.

Sequenzen, die reich an Glutamin (Q) und R&uuml;ckst&auml;nde von Asparagin (N) oft nicht auf der Ebene der Monomer Falten. Dies, gekoppelt mit ihrer ungew&ouml;hnlichen Wasserstoff-Bindung-F&auml;higkeiten, die treibende Kraft zum Wechseln zwischen ungeordneten Monomere und amyloide bietet. Solche &Uuml;berg&auml;nge Regeln Prozesse so vielf&auml;ltig wie Krankheiten beim Menschen-Proteinfaltung, bakterielle Biofilm-Assembly und die Vererbung von Hefe Prionen (proteinhaltige genetischen Elementen). Eine systematische Untersuchung der Bildung von Prion Dom&auml;nen vorgeschlagen, dass R&uuml;ckst&auml;nde von Q und N unterschiedliche Auswirkungen auf Amyloid Bildung haben. Hier verwenden wir Zelle biologische, biochemische und computational Techniken, um Q/N-reichen Protein-Varianten zu vergleichen, Qs und Qs mit Ns Ns ersetzen. Wir finden, dass die zwei R&uuml;ckst&auml;nde starke und gegens&auml;tzliche Wirkungen haben: N Reichtum f&ouml;rdert Versammlung des gutartigen selbst-Vorlagen enthalten; Q Reichtum f&ouml;rdert Bildung von toxischen nonamyloid Konformere. Molekulare Simulationen auf systeminterne Faltreifen Unterschiede zwischen Qs und Ns schlagen vor, da&szlig; ihre unterschiedlichen Verhaltensweisen durch die erweiterten Zuges-bildenden Neigung des Ns &uuml;ber Qs sind.

Gegenl&auml;ufige Effekte von Glutamin und Asparagin Regeln Prion-Bildung durch intrinsisch ungeordneten Proteine.

The self-templating conformations of yeast prion proteins act as epigenetic elements of inheritance. Yeast prions might provide a mechanism for generating heritable phenotypic diversity that promotes survival in fluctuating environments and the evolution of new traits. However, this hypothesis is highly controversial. Prions that create new traits have not been found in wild strains, leading to the perception that they are rare 'diseases' of laboratory cultivation. Here we biochemically test approximately 700 wild strains of Saccharomyces for [PSI(+)] or [MOT3(+)], and find these prions in many. They conferred diverse phenotypes that were frequently beneficial under selective conditions. Simple meiotic re-assortment of the variation harboured within a strain readily fixed one such trait, making it robust and prion-independent. Finally, we genetically screened for unknown prion elements. Fully one-third of wild strains harboured them. These, too, created diverse, often beneficial phenotypes. Thus, prions broadly govern heritable traits in nature, in a manner that could profoundly expand adaptive opportunities.

Prions are a common mechanism for phenotypic inheritance in wild yeasts.

Die selbst-Vorlagen-Konformationen Hefe Prion-Proteine wirken als epigenetische Elemente der Vererbung. Hefe Prionen k&ouml;nnte bieten einen Mechanismus zur Erzeugung von vererbbare ph&auml;notypische Vielfalt, die &uuml;berleben in Umgebungen mit schwankenden und die Entwicklung der neuen Charakterz&uuml;ge f&ouml;rdert. Diese Hypothese ist jedoch h&ouml;chst umstritten. Prionen, die neue Eigenschaften erstellen wurden in wilden St&auml;mmen, f&uuml;hrt zu der Wahrnehmung, dass sie seltene Krankheiten der Labor-Anbau sind nicht gefunden. Hier testen wir biochemisch rund 700 wilde St&auml;mme von Saccharomyces f&uuml;r [PSI(+)] oder [MOT3(+)], und diese Prionen in vielen zu finden. Sie &uuml;bertragen verschiedene Ph&auml;notypen, die h&auml;ufig unter selektiven Bedingungen vorteilhaft waren. Einfache meiotischen Re-assortment der Variation innerhalb einer Sorte leicht mancher behoben, ein Merkmal, robust und Prion-unabh&auml;ngig machen. Schlie&szlig;lich sehen wir genetisch f&uuml;r unbekannte Prion-Elemente. Ein Drittel der wilden St&auml;mme mancher voll sie. Diese, erstellt auch unterschiedliche, oft vorteilhafte Ph&auml;notypen. So Regeln Prionen weitgehend vererbbaren Merkmale in der Natur, in einer Weise, die zutiefst adaptive M&ouml;glichkeiten erweitern k&ouml;nnte.

Prionen sind ein gemeinsamer Mechanismus f&uuml;r die ph&auml;notypische Vererbung in wilden Hefen.

Remarkably simple proteins play outsize roles in the execution of developmental complexity within biological systems. Sequence information determines structure and hence function, so how do low complexity sequences fulfill their functions? Recent discoveries are raising the curtain on a new dimension of the sequence-structure paradigm. In it, function derives not from the structures of individual proteins, but instead, from dynamic material properties of entire ensembles of the proteins acting in unison through phase changes. These phases include liquids, one-dimensional crystals, and - as elaborated herein - even glasses. The peculiar thermodynamics of glass-like protein assemblies, in particular, illuminate new principles of information flow through and, at times, orthogonal to the central dogma of molecular biology.

A glass menagerie of low complexity sequences.

Diverse animal and plant viruses block the re-infection of host cells by the same or highly similar viruses through superinfection exclusion (SIE), a widely observed, yet poorly understood phenomenon. Here we demonstrate that SIE of turnip crinkle virus (TCV) is exclusively determined by p28, one of the two replication proteins encoded by this virus. p28 expressed from a TCV replicon exerts strong SIE to a different TCV replicon. Transiently expressed p28, delivered simultaneously with, or ahead of, a TCV replicon, largely recapitulates this repressive activity. Interestingly, p28-mediated SIE is dramatically enhanced by C-terminally fused epitope tags or fluorescent proteins, but weakened by N-terminal modifications, and it inversely correlates with the ability of p28 to complement the replication of a p28-defective TCV replicon. Strikingly, p28 in SIE-positive cells forms large, mobile punctate inclusions that trans-aggregate a non-coalescing, SIE-defective, yet replication-competent p28 mutant. These results support a model postulating that TCV SIE is caused by the formation of multimeric p28 complexes capable of intercepting fresh p28 monomers translated from superinfector genomes, thereby abolishing superinfector replication. This model could prove to be applicable to other RNA viruses, and offer novel targets for antiviral therapy.

A self-perpetuating repressive state of a viral replication protein blocks superinfection by the same virus.

Protein self-assemblies modulate protein activities over biological timescales that can exceed the lifetimes of the proteins or even the cells that harbor them. We hypothesized that these timescales relate to kinetic barriers inherent to the nucleation of ordered phases. To investigate nucleation barriers in living cells, we developed distributed amphifluoric FRET (DAmFRET). DAmFRET exploits a photoconvertible fluorophore, heterogeneous expression, and large cell numbers to quantify via flow cytometry the extent of a protein's self-assembly as a function of cellular concentration. We show that kinetic barriers limit the nucleation of ordered self-assemblies and that the persistence of the barriers with respect to concentration relates to structure. Supersaturation resulting from sequence-encoded nucleation barriers gave rise to prion behavior and enabled a prion-forming protein, Sup35 PrD, to partition into dynamic intracellular condensates or to form toxic aggregates. Our results suggest that nucleation barriers govern cytoplasmic inheritance, subcellular organization, and proteotoxicity.

Quantifying Nucleation In Vivo Reveals the Physical Basis of Prion-like Phase Behavior.

CARD9 and CARD11 drive immune cell activation by nucleating Bcl10 polymerization, but are held in an autoinhibited state prior to stimulation. Here, we elucidate the structural basis for this autoinhibition by determining the structure of a region of CARD9 that includes an extensive interface between its caspase recruitment domain (CARD) and coiled-coil domain. We demonstrate, for both CARD9 and CARD11, that disruption of this interface leads to hyperactivation in cells and to the formation of Bcl10-templating filaments in vitro, illuminating the mechanism of action of numerous oncogenic mutations of CARD11. These structural insights enable us to characterize two similar, yet distinct, mechanisms by which autoinhibition is relieved in the course of canonical CARD9 or CARD11 activation. We also dissect the molecular determinants of helical template assembly by solving the structure of the CARD9 filament. Taken together, these findings delineate the structural mechanisms of inhibition and activation within this protein family.

ArticleTotal : 2	Year
Publication title Cited by 187	2008
Publication title Cited by 16	2019

Article	Year
Chaperone-dependent amyloid assembly protects cells from prion toxicity. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 18480252	2008
A systematic survey identifies prions and illuminates sequence features of prionogenic proteins. Cell\| PubMed ID: 19345193	2009
Prions, protein homeostasis, and phenotypic diversity. Trends in cell biology\| PubMed ID: 20071174	2010
Biochemical, cell biological, and genetic assays to analyze amyloid and prion aggregation in yeast. Methods in enzymology\| PubMed ID: 20946833	2010
Epigenetics in the extreme: prions and the inheritance of environmentally acquired traits. Science (New York, N.Y.)\| PubMed ID: 21030648	2010
A method for probing the mutational landscape of amyloid structure. Bioinformatics (Oxford, England)\| PubMed ID: 21685090	2011
Opposing effects of glutamine and asparagine govern prion formation by intrinsically disordered proteins. Molecular cell\| PubMed ID: 21726811	2011
Prions are a common mechanism for phenotypic inheritance in wild yeasts. Nature\| PubMed ID: 22337056	2012
A glass menagerie of low complexity sequences. Current opinion in structural biology\| PubMed ID: 27258703	2016
A self-perpetuating repressive state of a viral replication protein blocks superinfection by the same virus. PLoS pathogens\| PubMed ID: 28267773	2017
Quantifying Nucleation In Vivo Reveals the Physical Basis of Prion-like Phase Behavior. Molecular cell\| PubMed ID: 29979963	2018
Structures of autoinhibited and polymerized forms of CARD9 reveal mechanisms of CARD9 and CARD11 activation. Nature communications\| PubMed ID: 31296852	2019

Randal Halfmann

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