isolating-potentiated-hsp104-variants-using-yeast-proteinopathy-models

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...

Perelman School of Medicine at the University of Pennsylvania

The small subunit (SSU) processome is a ribosome biogenesis intermediate that assembles from its subcomplexes onto the pre-18S rRNA with yet unknown order and structure. Here, we investigate the architecture of the UtpB subcomplex of the SSU processome, focusing on the interaction between the half-a-tetratricopeptide repeat (HAT) domain of Utp6 and a specific peptide in Utp21. We present a comprehensive map of the interactions within the UtpB subcomplex and further show that the N-terminal domain of Utp6 interacts with Utp18 while the HAT domain interacts with Utp21. Using a panel of point and deletion mutants of Utp6, we show that an intact HAT domain is essential for efficient pre-rRNA processing and cell growth. Further investigation of the Utp6-Utp21 interaction using both genetic and biophysical methods shows that the HAT domain binds a specific peptide ligand in Utp21, the first example of a HAT domain peptide ligand, with a dissociation constant of 10 muM.

A direct interaction between the Utp6 half-a-tetratricopeptide repeat domain and a specific peptide in Utp21 is essential for efficient pre-rRNA processing.

The design of protein-peptide interactions has a wide array of practical applications and also reveals insight into the basis for molecular recognition. Here, we present the redesign of a tetratricopeptide repeat (TPR) protein scaffold, along with its corresponding peptide ligand. We show that the binding properties of these protein-peptide pairs can be understood, quantitatively, using straightforward chemical considerations. The recognition pairs we have developed are also practically useful for the specific identification of tagged proteins. We demonstrate the facile replacement of these proteins, which we have termed T-Mods (TPR-based recognition module), for antibodies in both detection and purification applications. The new protein-peptide pair has a dissociation constant that is weaker than typical antibody-antigen interactions, yet the recognition pair is highly specific and we have shown that this affinity is sufficient for both Western blotting and affinity purification. Moreover, we demonstrate that this more moderate affinity is actually advantageous for purification applications, because extremely harsh conditions are not required to dissociate the T-Mod-peptide interaction. The results we present are important, not only because they represent a successful application of protein design but also because they help define the properties that should be sought in other scaffolds that are being developed as antibody replacements.

Redesign of a protein-peptide interaction: characterization and applications.

Designer protein modules, which bind specifically to a desired target, have numerous potential applications. One approach to creating such proteins is to construct and screen libraries. Here we present a detailed description of using a split-GFP reassembly assay to screen libraries and identify proteins with novel binding properties. Attractive features of the split-GFP based screen are the absence of false positives and the simplicity, robustness, and ease of automation of the screen. Here, we describe both the construction of a naive protein library, and screening of the library using the split-GFP assay to identify proteins that bind specifically to chosen peptide sequences.

Screening libraries to identify proteins with desired binding activities using a split-GFP reassembly assay.

&#x70ED;&#x4F11;&#x514B;&#x53CD;&#x5E94; (HSR) &#x662F;&#x9AD8;&#x5EA6;&#x4FDD;&#x5B88;&#x7684;&#x4FDD;&#x62A4;&#x673A;&#x5236;&#xFF0C;&#x5B83;&#x542F;&#x7528;&#x8981;&#x7ECF;&#x53D7;&#x4F4F;&#x5404;&#x79CD;&#x73AF;&#x5883;&#x7684;&#x5E94;&#x6FC0;&#x6E90;&#xFF0C;&#x7834;&#x574F;&#x86CB;&#x767D;&#x8D28;&#x7A33;&#x6001; (proteostasis) &#x548C;&#x4FC3;&#x8FDB;&#x86CB;&#x767D;&#x8D28;&#x6298;&#x53E0;&#x7684;&#x5355;&#x5143;&#x683C;&#x3002;&#x6709;&#x4EBA;&#x901A;&#x8FC7;&#x6FC0;&#x6D3B;&#x8F6C;&#x5F55;&#x56E0;&#x5B50;&#x70ED;&#x4F11;&#x514B;&#x56E0;&#x5B50; 1 &#x5F81;&#x6C42; HSR &#x7684;&#x5C0F;&#x5206;&#x5B50;&#x836F;&#x7269;&#x53EF;&#x80FD;&#x6709;&#x52A9;&#x4E8E;&#x51CF;&#x8F7B;&#x86CB;&#x767D;&#x8D28;&#x6298;&#x53E0;&#x548C;&#x805A;&#x96C6;&#x7684;&#x51E0;&#x4E2A;&#x5177;&#x6709;&#x7834;&#x574F;&#x6027;&#x7684;&#x795E;&#x7ECF;&#x9000;&#x884C;&#x6027;&#x75BE;&#x75C5;&#xFF0C;&#x5305;&#x62EC;&#x4EA8;&#x5EF7;&#x987F;&#x75C5; (HD)&#x3002;&#x56FD;&#x9645;&#x9752;&#x5E74;&#x5546;&#x4F1A;&#x7684;&#x8FD9;&#x4E00;&#x95EE;&#x9898;&#xFF0C;&#x5728; Labbadia &#x4E2D;&#x5357;&#x5DE5;&#x4E1A;&#x5927;&#x5B66;&#x4F7F;&#x7528;&#x8111;&#x6E17;&#x900F;&#x70ED;&#x4F11;&#x514B;&#x86CB;&#x767D; 90 &#x6291;&#x5236;&#x5242;&#xFF0C;HSP990&#xFF0C;&#x8BF1;&#x4F7F;&#x5C0F;&#x9F20;&#x6A21;&#x578B;&#x6240;&#x610F;&#x5916; HSR&#xFF0C;&#x4ED6;&#x4EEC;&#x89C2;&#x5BDF;&#x5230; HSP990 &#x8D4B;&#x4E88;&#x53EA;&#x77AC;&#x6001;&#x6539;&#x826F;&#x5B50;&#x96C6;&#x7684;&#x9AD8;&#x6E05;&#x76F8;&#x5173;&#x7684;&#x8868;&#x578B;&#xFF0C;&#x56E0;&#x4E3A;&#x5728;&#x67D3;&#x8272;&#x8D28;&#x5EFA;&#x7B51;&#x6539;&#x5EFA;&#x635F;&#x5BB3;&#x540E;&#x75C5;&#x60C5; HSR&#x3002;&#x8FD9;&#x4E9B;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x8868;&#x660E;&#x534F;&#x540C;&#x7EC4;&#x5408;&#x7597;&#x6CD5;&#xFF0C;&#x540C;&#x65F6;&#x91CA;&#x653E; HSR &#x5E76;&#x9632;&#x6B62;&#x5176;&#x635F;&#x5BB3;&#x53EF;&#x80FD;&#x9700;&#x8981;&#x8FD8;&#x539F;&#x9AD8;&#x6E05; proteostasis&#x3002;

&#x9707;&#x6151;&#xFF1A; &#x91CA;&#x653E;&#x70ED;&#x4F11;&#x514B;&#x53CD;&#x5E94;&#xFF0C;&#x6CBB;&#x7597;&#x4EA8;&#x5EF7;&#x987F;&#x75C5;&#x3002;

The heat shock response (HSR) is a highly conserved protective mechanism that enables cells to withstand diverse environmental stressors that disrupt protein homeostasis (proteostasis) and promote protein misfolding. It has been suggested that small-molecule drugs that elicit the HSR by activating the transcription factor heat shock factor 1 might help mitigate protein misfolding and aggregation in several devastating neurodegenerative disorders, including Huntington disease (HD). In this issue of the JCI, Labbadia et al. use a brain-penetrant Hsp90 inhibitor, HSP990, to induce the HSR in mouse models of HD. Unexpectedly, they observed that HSP990 confers only transient amelioration of a subset of HD-related phenotypes, because alterations in chromatin architecture impair the HSR upon disease progression. These findings suggest that synergistic combination therapies that simultaneously unleash the HSR and prevent its impairment are likely to be needed to restore proteostasis in HD.

Shock and awe: unleashing the heat shock response to treat Huntington disease.

La r&#xE9;ponse au choc thermique (HSR) est un m&#xE9;canisme hautement conserv&#xE9; de protection qui permet aux cellules de r&#xE9;sister &#xE0; divers stress environnementaux qui perturbent l&#x26;#39;hom&#xE9;ostasie prot&#xE9;ique (prot&#xE9;ostasie) et de promouvoir mauvais repliement des prot&#xE9;ines. Il a &#xE9;t&#xE9; sugg&#xE9;r&#xE9; que m&#xE9;dicaments &#xE0; petites mol&#xE9;cules qui provoquent le HSR en activant le facteur de transcription de choc thermique facteur 1 pourrait aider &#xE0; att&#xE9;nuer les mauvais repliement des prot&#xE9;ines et de l&#x26;#39;agr&#xE9;gation dans plusieurs maladies neurod&#xE9;g&#xE9;n&#xE9;ratives d&#xE9;vastatrices, y compris la maladie de Huntington (HD). Dans ce num&#xE9;ro de la JCI, Labbadia et al. utiliser un cerveau p&#xE9;n&#xE9;trant Hsp90 inhibiteur, HSP990, pour induire la HSR dans des mod&#xE8;les murins de HD. De fa&#xE7;on inattendue, ils ont observ&#xE9; que HSP990 ne conf&#xE8;re qu&#x26;#39;une am&#xE9;lioration transitoire d&#x26;#39;un sous-ensemble de HD-connexes ph&#xE9;notypes, parce que des modifications dans l&#x26;#39;architecture chromatine nuire &#xE0; la progression de la maladie sur HSR. Ces r&#xE9;sultats sugg&#xE8;rent que les th&#xE9;rapies combinaison synergique qui, simultan&#xE9;ment, de lib&#xE9;rer tout le HSR et d&#x26;#39;emp&#xEA;cher sa d&#xE9;pr&#xE9;ciation sont susceptibles d&#x26;#39;&#xEA;tre n&#xE9;cessaires pour r&#xE9;tablir prot&#xE9;ostasie en HD.

Shock and Awe: Lib&#xE9;rer la r&#xE9;ponse au choc thermique pour traiter la maladie de Huntington

Die Hitze-Schock-Reaktion (HSR) ist eine stark konservierten Schutzmechanismus, der Zellen, diverse Umwelt-Stressoren zu widerstehen, die Protein-Hom&ouml;ostase (Proteostasis) zu st&ouml;ren und zu f&ouml;rdern, Protein misfolding erm&ouml;glicht. Es wurde vermutet, dass kleine-Molek&uuml;l-Medikamente, die die HSR zu entlocken, durch die Aktivierung der Faktor Hitze Schock &Uuml;bertragungfaktor 1 Protein misfolding und Aggregation in mehreren verheerenden Neurodegenerative Erkrankungen, einschlie&szlig;lich der Huntington-Krankheit (HD) zu mildern helfen k&ouml;nnte. In dieser Ausgabe von der JCI Labbadia Et Al. verwenden ein Gehirn-Rostl&ouml;ser Hsp90-Inhibitor, HSP990, der HSR in Maus-Modellen der HD. unerwartet zu veranlassen, sie beobachtet, dass HSP990 nur vor&uuml;bergehende Linderung einer Teilmenge von HD-bezogene Ph&auml;notypen, verleiht, da &Auml;nderungen in der Chromatin-Architektur der HSR bei Fortschreiten der Krankheit beeintr&auml;chtigen. Diese Forschungsergebnisse legen nahe, dass synergistische Kombinationstherapien, die gleichzeitig die HSR zu entfesseln und verhindern ihre Werthaltigkeit Proteostasis in HD wiederherstellen musste sein d&uuml;rften.

Schock und Ehrfurcht: Entfesselung der Hitze-Schock-Antwort Huntington-Krankheit zu behandeln.

La risposta di scossa di calore (HSR) &egrave; un meccanismo protettivo altamente conservato che permette alle cellule di resistere a diversi fattori di stress ambientale che disturbare l&#39;omeostasi della proteina (proteostasis) e promuovere misfolding di proteine. &Egrave; stato suggerito che farmaci piccolo-molecola che suscitano l&#39;HSR attivando il fattore di trascrizione fattore calore shock 1 potrebbero attenuare protein misfolding e aggregazione in diverse patologie neurodegenerative devastante, tra cui la malattia di Huntington (HD). In questo numero della JCI, Labbadia et al. usare un inibitore Hsp90 cervello-liquidi penetranti, HSP990, per indurre il HSR in modelli murini di HD inaspettatamente, hanno osservato che il HSP990 conferisce miglioramento solo temporaneo di un sottoinsieme di fenotipi correlati a HD, perch&eacute; alterazioni nell&#39;architettura della cromatina compromettere l&#39;HSR sulla progressione della malattia. Questi risultati suggeriscono che le terapie di combinazione sinergica che scatena l&#39;HSR e contemporaneamente evitare la compromissione sono suscettibili di essere necessari per ripristinare proteostasis in HD.

Shock and awe: scatenare la risposta di scossa di calore per trattare la malattia di Huntington.

&#x71B1;&#x30B7;&#x30E7;&#x30C3;&#x30AF;&#x5FDC;&#x7B54;&#xFF08;HSR&#xFF09;&#x306F;&#x3001;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x6052;&#x5E38;&#x6027;&#xFF08;proteostasis&#xFF09;&#x3092;&#x7834;&#x58CA;&#x3057;&#x3001;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x30DF;&#x30B9;&#x30D5;&#x30A9;&#x30FC;&#x30EB;&#x30C7;&#x30A3;&#x30F3;&#x30B0;&#x3092;&#x4FC3;&#x200B;&#x200B;&#x9032;&#x3059;&#x308B;&#x591A;&#x69D8;&#x306A;&#x74B0;&#x5883;&#x30B9;&#x30C8;&#x30EC;&#x30B9;&#x306B;&#x8010;&#x3048;&#x308B;&#x305F;&#x3081;&#x306B;&#x7D30;&#x80DE;&#x3092;&#x53EF;&#x80FD;&#x306B;&#x3059;&#x308B;&#x9AD8;&#x5EA6;&#x306B;&#x4FDD;&#x5B58;&#x3055;&#x308C;&#x305F;&#x9632;&#x5FA1;&#x6A5F;&#x69CB;&#x3067;&#x3042;&#x308B;&#x3002;&#x3053;&#x308C;&#x306F;&#x3001;&#x8EE2;&#x5199;&#x56E0;&#x5B50;&#x306E;&#x71B1;&#x30B7;&#x30E7;&#x30C3;&#x30AF;&#x56E0;&#x5B50;1&#x3092;&#x6D3B;&#x6027;&#x5316;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x308A;&#x3001;HSR&#x3092;&#x8A98;&#x767A;&#x3059;&#x308B;&#x4F4E;&#x5206;&#x5B50;&#x85AC;&#x304C;&#x30CF;&#x30F3;&#x30C1;&#x30F3;&#x30C8;&#x30F3;&#x75C5;&#xFF08;HD&#xFF09;&#x3092;&#x542B;&#x3080;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x58CA;&#x6EC5;&#x7684;&#x306A;&#x795E;&#x7D4C;&#x5909;&#x6027;&#x75BE;&#x60A3;&#x3067;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x30DF;&#x30B9;&#x30D5;&#x30A9;&#x30FC;&#x30EB;&#x30C7;&#x30A3;&#x30F3;&#x30B0;&#x3068;&#x51DD;&#x96C6;&#x3092;&#x7DE9;&#x548C;&#x3059;&#x308B;&#x306E;&#x306B;&#x5F79;&#x7ACB;&#x3064;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308B;&#x3053;&#x3068;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3002; JCI&#x3001;&#x30E9;&#x30C3;&#x30D0;&#x30C7;&#x30A3;&#x30A2;&#x3089;&#x306E;&#x3053;&#x306E;&#x554F;&#x984C;&#x3067;&#x306F;&#x3002; HD&#x306E;&#x30DE;&#x30A6;&#x30B9;&#x30E2;&#x30C7;&#x30EB;&#x306B;&#x304A;&#x3044;&#x3066;HSR&#x3092;&#x8A98;&#x5C0E;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3001;&#x8133;&#x6D78;&#x900F;Hsp90&#x306E;&#x963B;&#x5BB3;&#x5264;&#x3001;HSP990&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x4E88;&#x671F;&#x305B;&#x305A;&#x3001;&#x5F7C;&#x3089;&#x306F;&#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3;&#x306E;&#x30A2;&#x30FC;&#x30AD;&#x30C6;&#x30AF;&#x30C1;&#x30E3;&#x306E;&#x5909;&#x5316;&#x306F;&#x3001;&#x75BE;&#x60A3;&#x306E;&#x9032;&#x884C;&#x306B;HSR&#x3092;&#x640D;&#x306A;&#x3046;&#x306E;&#x3067;&#x3001;HSP990&#x306F;&#x3001;HD-&#x95A2;&#x9023;&#x8868;&#x73FE;&#x578B;&#x306E;&#x30B5;&#x30D6;&#x30BB;&#x30C3;&#x30C8;&#x306E;&#x307F;&#x3092;&#x4E00;&#x6642;&#x7684;&#x306A;&#x6539;&#x5584;&#x3092;&#x4E0E;&#x3048;&#x308B;&#x3053;&#x3068;&#x3092;&#x89B3;&#x5BDF;&#x3057;&#x305F;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x77E5;&#x898B;&#x306F;&#x3001;&#x540C;&#x6642;&#x306B;HSR&#x3092;&#x767A;&#x63EE;&#x3057;&#x3001;&#x305D;&#x306E;&#x969C;&#x5BB3;&#x3092;&#x9632;&#x3050;&#x305F;&#x3081;&#x3001;&#x76F8;&#x4E57;&#x7684;&#x306A;&#x4F75;&#x7528;&#x7642;&#x6CD5;&#x306F;&#x3001;HD&#x3067;proteostasis&#x3092;&#x5FA9;&#x5143;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x5FC5;&#x8981;&#x3055;&#x308C;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x9AD8;&#x3044;&#x304A;&#x52E7;&#x3081;&#x3057;&#x307E;&#x3059;&#x3002;

&#x885D;&#x6483;&#x3068;&#x754F;&#x6016;&#xFF1A;&#x30CF;&#x30F3;&#x30C1;&#x30F3;&#x30C8;&#x30F3;&#x75C5;&#x3092;&#x6CBB;&#x7642;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3001;&#x71B1;&#x30B7;&#x30E7;&#x30C3;&#x30AF;&#x5FDC;&#x7B54;&#x3092;&#x89E3;&#x304D;&#x653E;&#x3064;

&#xC5F4; &#xCDA9;&#xACA9; &#xC751;&#xB2F5; (HSR)&#xB294; &#xC138;&#xD3EC; &#xB2E8;&#xBC31;&#xC9C8; &#xD56D;&#xC0C1;&#xC131; (proteostasis) &#xD558; &#xACE0; &#xB2E8;&#xBC31;&#xC9C8; misfolding&#xB97C; &#xD64D;&#xBCF4; &#xD558;&#xB294; &#xB2E4;&#xC591; &#xD55C; &#xD658;&#xACBD; &#xC2A4;&#xD2B8;&#xB808;&#xC2A4;&#xB97C; &#xACAC;&#xB51C; &#xC218; &#xC788;&#xB3C4;&#xB85D; &#xB9E4;&#xC6B0; &#xBCF4;&#xC874;&#xB41C; &#xBCF4;&#xD638; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC785;&#xB2C8;&#xB2E4;. &#xC804;&#xC0AC; &#xC778;&#xC790; &#xC5F4; &#xC1FC;&#xD06C; &#xC694;&#xC18C; 1&#xC744; &#xD65C;&#xC131;&#xD654; &#xD558; &#xC5EC; HSR&#xB97C; &#xC720;&#xB3C4; &#xD558;&#xB294; &#xC791;&#xC740; &#xBD84;&#xC790; &#xC57D;&#xBB3C; &#xB2E8;&#xBC31;&#xC9C8; misfolding &#xBC0F; &#xC9D1;&#xACC4; &#xD5CC;&#xD305;&#xD134; &#xBCD1; (HD)&#xB97C; &#xD3EC;&#xD568; &#xD558; &#xC5EC; &#xC5EC;&#xB7EC; &#xAC00;&#xC9C0; &#xCE58;&#xBA85;&#xC801;&#xC778; &#xC2E0;&#xACBD; &#xD1F4;&#xD589; &#xC131; &#xC7A5;&#xC560;&#xC5D0; &#xB3C4;&#xC6C0;&#xC774; &#xB420; &#xC81C;&#xC548; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774; &#xBB38;&#xC81C;&#xB294; JCI&#xC758; Labbadia &#xADF8; &#xC678; &#xC5EC;&#xB7EC;&#xBD84; &#xB1CC; penetrant Hsp90 &#xC5B5;&#xC81C;&#xC81C;, HSP990, HD. &#xC608;&#xAE30;&#xCE58; &#xC54A;&#xAC8C;&#xC758; &#xB9C8;&#xC6B0;&#xC2A4; &#xBAA8;&#xB378;&#xC5D0;&#xC11C; HSR &#xC720;&#xB3C4;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC;, &#xADF8;&#xB4E4;&#xC740; &#xAD00;&#xCC30; hsp990&#xC758; HD &#xAD00;&#xB828; &#xACE0;&#xAE30;, &#xD558;&#xC704; &#xC9D1;&#xD569;&#xB9CC; &#xACFC;&#xB3C4; &#xAC1C;&#xB7C9; &#xC218; &#xC5EC; chromatin &#xC544;&#xD0A4;&#xD14D;&#xCC98; &#xBCC0;&#xACBD; &#xC9C8;&#xBCD1;&#xC758; &#xC9C4;&#xD589;&#xC5D0; &#xB530;&#xB77C; HSR&#xC5D0; &#xC545;&#xC601;&#xD5A5;. &#xC774; &#xBC1C;&#xACAC; &#xB3D9;&#xC2DC;&#xC5D0; HSR&#xB97C; &#xBC1C;&#xD718; &#xD558; &#xACE0; &#xADF8; &#xC7A5;&#xC560;&#xB97C; &#xBC29;&#xC9C0; &#xD558;&#xB294; &#xACF5;&#xB3D9; &#xC131; &#xC870;&#xD569; &#xC694;&#xBC95; hd&#xC5D0;&#xC11C; proteostasis&#xB97C; &#xBCF5;&#xC6D0; &#xD558;&#xB294; &#xB370; &#xD544;&#xC694;&#xD55C; &#xAC00;&#xB2A5;&#xC131;&#xC774; &#xC788;&#xB2E4;&#xB294; &#xAC83;&#xC774; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xCDA9;&#xACA9;&#xACFC; &#xACF5;&#xD3EC;: &#xD5CC;&#xD305;&#xD134; &#xBCD1; &#xCE58;&#xB8CC;&#xC5D0; &#xB300; &#xD55C; &#xC5F4; &#xCDA9;&#xACA9; &#xC751;&#xB2F5;&#xC744; &#xCE69;&#xB2C8;&#xB2E4;.

La respuesta de choque t&#xE9;rmico (RSS) es un mecanismo altamente conservado de protecci&#xF3;n que permite a las c&#xE9;lulas para soportar diversos factores de estr&#xE9;s ambientales que alteran la homeostasis de prote&#xED;nas (Proteostasis) y promover el mal plegamiento de prote&#xED;nas. Se ha sugerido que los f&#xE1;rmacos de peque&#xF1;as mol&#xE9;culas que provocan la reacci&#xF3;n de hipersensibilidad mediante la activaci&#xF3;n del factor de transcripci&#xF3;n factor de choque t&#xE9;rmico 1 podr&#xED;a ayudar a mitigar el mal plegamiento y agregaci&#xF3;n de prote&#xED;nas en varios trastornos devastadores neurodegenerativas, entre ellas la enfermedad de Huntington (HD). En esta edici&#xF3;n de la JCI, Labbadia et al. utilizar un cerebro penetrante Hsp90 inhibidor, HSP990, para inducir la reacci&#xF3;n de hipersensibilidad en modelos de rat&#xF3;n de alta definici&#xF3;n. Inesperadamente, se observ&#xF3; que HSP990 s&#xF3;lo confiere la mejor&#xED;a transitoria de un subconjunto de HD-fenotipos relacionados, debido a alteraciones en la arquitectura de la cromatina poner en peligro el HSR al avance de la enfermedad. Estos hallazgos sugieren que las terapias de combinaci&#xF3;n sin&#xE9;rgica que a la vez desencadenan la reacci&#xF3;n de hipersensibilidad y evitar su deterioro es probable que sean necesarias para restablecer Proteostasis en HD.

Conmoci&#xF3;n y Pavor: Liberar la respuesta al estr&#xE9;s de tratar la enfermedad de Huntington

It is not understood how Hsp104, a hexameric AAA+ ATPase from yeast, disaggregates diverse structures, including stress-induced aggregates, prions, and α-synuclein conformers connected to Parkinson disease. Here, we establish that Hsp104 hexamers adapt different mechanisms of intersubunit collaboration to disaggregate stress-induced aggregates versus amyloid. To resolve disordered aggregates, Hsp104 subunits collaborate noncooperatively via probabilistic substrate binding and ATP hydrolysis. To disaggregate amyloid, several subunits cooperatively engage substrate and hydrolyze ATP. Importantly, Hsp104 variants with impaired intersubunit communication dissolve disordered aggregates, but not amyloid. Unexpectedly, prokaryotic ClpB subunits collaborate differently than Hsp104 and couple probabilistic substrate binding to cooperative ATP hydrolysis, which enhances disordered aggregate dissolution but sensitizes ClpB to inhibition and diminishes amyloid disaggregation. Finally, we establish that Hsp104 hexamers deploy more subunits to disaggregate Sup35 prion strains with more stable "cross-β" cores. Thus, operational plasticity enables Hsp104 to robustly dissolve amyloid and nonamyloid clients, which impose distinct mechanical demands.

Operational plasticity enables hsp104 to disaggregate diverse amyloid and nonamyloid clients.

There are no therapies that reverse the proteotoxic misfolding events that underpin fatal neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). Hsp104, a conserved hexameric AAA+ protein from yeast, solubilizes disordered aggregates and amyloid but has no metazoan homolog and only limited activity against human neurodegenerative disease proteins. Here, we reprogram Hsp104 to rescue TDP-43, FUS, and α-synuclein proteotoxicity by mutating single residues in helix 1, 2, or 3 of the middle domain or the small domain of nucleotide-binding domain 1. Potentiated Hsp104 variants enhance aggregate dissolution, restore proper protein localization, suppress proteotoxicity, and in a C. elegans PD model attenuate dopaminergic neurodegeneration. Potentiating mutations reconfigure how Hsp104 subunits collaborate, desensitize Hsp104 to inhibition, obviate any requirement for Hsp70, and enhance ATPase, translocation, and unfoldase activity. Our work establishes that disease-associated aggregates and amyloid are tractable targets and that enhanced disaggregases can restore proteostasis and mitigate neurodegeneration.

Potentiated Hsp104 variants antagonize diverse proteotoxic misfolding events.

Aberrant protein folding is severely problematic and manifests in numerous disorders, including amyotrophic lateral sclerosis (ALS), Parkinson disease (PD), Huntington disease (HD), and Alzheimer disease (AD). Patients with each of these disorders are characterized by the accumulation of mislocalized protein deposits. Treatments for these disorders remain palliative, and no available therapeutics eliminate the underlying toxic conformers. An intriguing approach to reverse deleterious protein misfolding is to upregulate chaperones to restore proteostasis. We recently reported our work to re-engineer a prion disaggregase from yeast, Hsp104, to reverse protein misfolding implicated in human disease. These potentiated Hsp104 variants suppress TDP-43, FUS, and α-synuclein toxicity in yeast, eliminate aggregates, reverse cellular mislocalization, and suppress dopaminergic neurodegeneration in an animal model of PD. Here, we discuss this work and its context, as well as approaches for further developing potentiated Hsp104 variants for application in reversing protein-misfolding disorders.

Reversing deleterious protein aggregation with re-engineered protein disaggregases.

Protein misfolding is implicated in numerous lethal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and Parkinson disease (PD). There are no therapies that reverse these protein-misfolding events. We aim to apply Hsp104, a hexameric AAA+ protein from yeast, to target misfolded conformers for reactivation. Hsp104 solubilizes disordered aggregates and amyloid, but has limited activity against human neurodegenerative disease proteins. Thus, we have previously engineered potentiated Hsp104 variants that suppress aggregation, proteotoxicity and restore proper protein localization of ALS and PD proteins in Saccharomyces cerevisiae, and mitigate neurodegeneration in an animal PD model. Here, we establish that potentiated Hsp104 variants possess broad substrate specificity and, in yeast, suppress toxicity and aggregation induced by wild-type TDP-43, FUS and α-synuclein, as well as missense mutant versions of these proteins that cause neurodegenerative disease. Potentiated Hsp104 variants also rescue toxicity and aggregation of TAF15 but not EWSR1, two RNA-binding proteins with a prion-like domain that are connected with the development of ALS and frontotemporal dementia. Thus, potentiated Hsp104 variants are not entirely non-specific. Indeed, they do not unfold just any natively folded protein. Rather, potentiated Hsp104 variants are finely tuned to unfold proteins bearing short unstructured tracts that are not recognized by wild-type Hsp104. Our studies establish the broad utility of potentiated Hsp104 variants.

Meredith E. Jackrel

Department of Biochemistry and Biophysics

Perelman School of Medicine at the University of Pennsylvania

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Meredith E. Jackrel

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry and Biophysics

Perelman School of Medicine at the University of Pennsylvania

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Department of Biochemistry and Biophysics