reporter-based-growth-assay-for-systematic-analysis-protein

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

automated-delivery-microfabricated-targets-for-intense-laser

Automated Delivery of Microfabricated Targets for Intense Laser Irradiation Experiments

Described is an experimental procedure that enables high-power laser irradiation of microfabricated targets. Targets are brought to the laser focus by a ...

The Hebrew University of Jerusalem

Tel Aviv University

Tel Aviv University Center for Light-Matter Interaction

&#x86CB;&#x767D;&#x8D28;&#x6298;&#x53E0;&#x7684;&#x8D28;&#x91CF;&#x63A7;&#x5236;&#x7F51;&#x7EDC;&#x7684;&#x6B63;&#x5E38;&#x8FD0;&#x4F5C;&#x53D6;&#x51B3;&#x4E8E;&#x7F51;&#x7EDC;&#x7684;&#x80FD;&#x529B;&#x6765;&#x8FA8;&#x660E;&#x5411;&#x672C;&#x673A;&#x7684;&#x56FD;&#x5BB6;&#x5176;&#x86CB;&#x767D;&#x8D28;&#x57FA;&#x4F53;&#x6750;&#x6599;&#x7684;&#x4E0D;&#x540C;&#x7ED3;&#x6784;&#x6270;&#x52A8;&#x3002;&#x68C0;&#x6D4B;&#x7684;&#x7EA0;&#x7F20;&#x56FD;&#x5BB6;&#x5BF9;&#x5355;&#x5143;&#x683C;&#x7684;&#x5BB6;-ostasis &#x7684;&#x4E2D;&#x5FC3;&#x5730;&#x4F4D;&#xFF0C;&#x5C3D;&#x7BA1;&#x5F88;&#x5C11;&#x77E5;&#x7684;&#x7CBE;&#x786E;&#x5E8F;&#x5217;&#x548C;&#x6807;&#x8BB0;&#x4F5C;&#x4E3A;&#x6B63;&#x5728; misfolded &#x86CB;&#x767D;&#x7684;&#x7ED3;&#x6784;&#x7279;&#x5F81;&#x3002;&#x82E5;&#x8981;&#x8C03;&#x67E5;&#x8FD9;&#x4E9B;&#x529F;&#x80FD;&#xFF0C;&#x6211;&#x4EEC;&#x7814;&#x7A76;&#x8981;&#x6C42;&#x4E3A;&#x9000;&#x5316;&#x7684;&#x9175;&#x6BCD;&#x7EBA;&#x9524;&#x86CB;&#x767D;&#x7A81;&#x53D8;&#x4F53; Ndc10p &#x662F;&#x4E00;&#x4E2A;&#x57FA;&#x4F53;&#x7684;&#x86CB;&#x767D;&#x8D28;&#x6298;&#x53E0;&#x7684;&#x8D28;&#x91CF;&#x63A7;&#x5236;&#x901A;&#x8DEF;&#x4ECB;&#x5BFC; E3 (Ub) &#x6CDB;&#x7D20;&#x8FDE;&#x63A5;&#x9176; Doa10p &#x5728;&#x5185;&#x8D28;&#x7F51; &#xFF08;ER&#xFF09; / &#x6838;&#x4FE1;&#x5C01;&#x819C;&#x3002; Ndc10p&#x3002;Ndc10p &#x53D8;&#x79CD;&#x884D;&#x751F;&#x4EA7;&#x54C1;&#xFF0C;&#x91C7;&#x7528;&#x4E00;&#x79CD;&#x53CD;&#x5411;&#x9057;&#x4F20;&#x5B66;&#x65B9;&#x6CD5;&#xFF0C;&#x5206;&#x6790;&#x786E;&#x5B9A;&#x81EA;&#x4E3B;&#x8D28;&#x91CF;&#x63A7;&#x5236;&#x76F8;&#x5173;&#x9000;&#x5316;&#x6BCD;&#x9898;&#x7684;&#x86CB;&#x767D;&#x8D28; C-&#x603B;&#x7AD9;&#x9644;&#x8FD1;&#x3002;&#x8FD9;&#x4E2A;&#x4E3B;&#x9898;&#x7531;&#x7EC4;&#x6210;&#x7684;&#x4E24;&#x4E2A;&#x4E0D;&#x53EF;&#x6216;&#x7F3A;&#x7684;&#x758F;&#x6C34;&#x6027;&#x8981;&#x7D20;&#xFF1A; &#x758F;&#x6C34;&#x6027;&#x548C;&#x8868;&#x9762;&#x7684;&#x4E24;&#x4EB2;&#x6027;&#x87BA;&#x65CB;&#x677E;&#x6563;&#x7684;&#x758F;&#x6C34;&#x6027; C &#x7EC8;&#x7AEF;&#x5C3E;&#x5DF4;&#x3002;&#x7279;&#x5B9A;&#x4E8E;&#x7AD9;&#x70B9;&#x7684;&#x70B9;&#x7A81;&#x53D8;&#x516C;&#x5F00;&#x8FD9;&#x4E9B;&#x5143;&#x7D20;&#xFF0C;&#x89E6;&#x53D1;&#x6CDB;&#x7D20;&#x4ECB;&#x5BFC;&#x548C; Ndc10p &#x7684;&#x70ED;&#x4F11;&#x514B;&#x86CB;&#x767D; 70 &#x4F9D;&#x8D56;&#x966A;&#x62A4;&#x7684;&#x9000;&#x5316;&#x3002;&#x8FD9;&#x4E9B;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x8BC1;&#x5B9E;&#x627F;&#x8BA4;&#x6838;&#x86CB;&#x767D;&#x8D28;&#x7684;&#x672C;&#x673A;&#x7ED3;&#x6784;&#x4E2D;&#x7684;&#x5FAE;&#x5999; perturbation(s) ER &#x7684;&#x8D28;&#x91CF;&#x63A7;&#x5236;&#x7CFB;&#x7EDF;&#x7684;&#x80FD;&#x529B;&#x3002;

&#x66B4;&#x9732;&#x7684;&#x5076;&#x7684;&#x758F;&#x6C34;&#x6027;&#x4FE1;&#x53F7;&#x89E6;&#x53D1;&#x5728;&#x5185;&#x8D28;&#x7F51;/&#x6838;&#x4FE1;&#x5C01; Ndc10 &#x6838;&#x7684;&#x8D28;&#x91CF;&#x63A7;&#x5236;&#x3002;

Proper functioning of the protein-folding quality control network depends on the network's ability to discern diverse structural perturbations to the native states of its protein substrates. Despite the centrality of the detection of misfolded states to cell home-ostasis, very little is known about the exact sequence and structural features that mark a protein as being misfolded. To investigate these features, we studied the requirements for the degradation of the yeast kinetochore protein Ndc10p. Mutant Ndc10p is a substrate of a protein-folding quality control pathway mediated by the E3 ubiquitin (Ub) ligase Doa10p at the endoplasmic reticulum (ER)/nuclear envelope membrane. Analysis of Ndc10p mutant derivatives, employing a reverse genetics approach, identified an autonomous quality control-associated degradation motif near the C-terminus of the protein. This motif is composed of two indispensable hydrophobic elements: a hydrophobic surface of an amphipathic helix and a loosely structured hydrophobic C-terminal tail. Site-specific point mutations expose these elements, triggering ubiquitin-mediated and HSP70 chaperone-dependent degradation of Ndc10p. These findings substantiate the ability of the ER quality control system to recognize subtle perturbation(s) in the native structure of a nuclear protein.

Exposure of bipartite hydrophobic signal triggers nuclear quality control of Ndc10 at the endoplasmic reticulum/nuclear envelope.

Bon fonctionnement du r&eacute;seau de contr&ocirc;le de la qualit&eacute;-le repliement des prot&eacute;ines d&eacute;pend de la capacit&eacute; du r&eacute;seau &agrave; discerner les diverses perturbations structurelles aux &Eacute;tats de ses substrats de la prot&eacute;ine natives. Malgr&eacute; le caract&egrave;re central de la d&eacute;tection des &Eacute;tats mal repli&eacute;es &agrave; cellule Accueil-ostasis, tr&egrave;s peu est connu sur la s&eacute;quence exacte et les caract&eacute;ristiques structurales qui marquent une prot&eacute;ine comme &eacute;tant pli&eacute;e. Pour &eacute;tudier ces fonctionnalit&eacute;s, nous avons &eacute;tudi&eacute; les exigences pour la d&eacute;gradation de la prot&eacute;ine de kin&eacute;tochore de levure Ndc10p. Ndc10p de Mutant est un substrat d&#39;une voie de contr&ocirc;le de la qualit&eacute;-le repliement des prot&eacute;ines, m&eacute;di&eacute;e par l&#39;ubiquitine (Ub) ligase E3 Doa10p au r&eacute;ticulum endoplasmique (re) / membrane d&#39;enveloppe nucl&eacute;aire. Analyse des d&eacute;riv&eacute;s mutants Ndc10p, utilisant une approche de g&eacute;n&eacute;tique inverse, fait ressortir un motif autonome de d&eacute;gradation associ&eacute;es aux contr&ocirc;les de qualit&eacute; pr&egrave;s de l&#39;extr&eacute;mit&eacute; C-terminale de la prot&eacute;ine. Ce motif est compos&eacute; de deux &eacute;l&eacute;ments hydrophobes indispensables : une surface hydrophobe d&#39;une h&eacute;lice amphipathique et une queue de C-terminale hydrophobe peu structur&eacute;e. Des mutations ponctuelles sp&eacute;cifiques au site exposer ces &eacute;l&eacute;ments, d&eacute;clenchement induite par l&#39;ubiquitine et d&eacute;gradation d&eacute;pendante chaperon HSP70 de Ndc10p. Ces r&eacute;sultats corroborent la capacit&eacute; du syst&egrave;me de contr&ocirc;le de qualit&eacute; ER de reconna&icirc;tre perturbation(s) subtiles dans la structure native d&#39;une prot&eacute;ine nucl&eacute;aire.

Exposition de signal hydrophobe bipartite d&eacute;clenche nucl&eacute;aire contr&ocirc;le de la qualit&eacute; de Ndc10 &agrave; l&#39;enveloppe nucl&eacute;aire/r&eacute;ticulum endoplasmique.

Corretto funzionamento della rete di controllo qualit&agrave; di ripiegamento proteico dipende dalla capacit&agrave; della rete di discernere diverse perturbazioni strutturali agli Stati nativi dei suoi substrati della proteina. Nonostante la centralit&agrave; della rilevazione degli state malpiegate a cella casa-ostasis, molto poco &egrave; conosciuto circa la sequenza esatta e le caratteristiche strutturali che segnano una proteina come essendo denatura. Per indagare su queste caratteristiche, abbiamo studiato i requisiti per la degradazione della proteina cinetocore lievito Ndc10p. Ndc10p mutante &egrave; un substrato di un percorso di controllo di qualit&agrave; ripiegamento proteico mediato dalla ligasi di ubiquitin (Ub) E3 Doa10p al reticolo endoplasmatico (ER) / membrana nucleare. Analisi di mutanti derivati Ndc10p, che impiegano un approccio di genetica inversa, identificato un motivo di degrado autonomo associato al controllo di qualit&agrave; vicino al C-terminale della proteina. Questo motivo &egrave; composto da due elementi indispensabili idrofobici: una superficie idrofoba di un&#39;elica di amphipathic e una scioltamente strutturata coda C-terminale idrofobica. Mutazioni puntiformi site specifiche esporre questi elementi, innescando il ubiquitin-mediato e degradazione dipendente dal chaperone HSP70 di Ndc10p. Questi risultati dimostrare la capacit&agrave; del sistema di controllo qualit&agrave; ER riconoscere perturbation(s) sottile nella struttura nativa di una proteina nucleare.

L&#39;esposizione del segnale idrofobico bipartito innesca nucleare controllo qualit&agrave; di Ndc10 presso la busta del reticolo endoplasmatico/nucleare.

&#x9069;&#x5207;&#x306B;&#x6A5F;&#x80FD;&#x3059;&#x308B;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x6298;&#x308A;&#x7573;&#x307F;&#x306E;&#x54C1;&#x8CEA;&#x7BA1;&#x7406;&#x30CD;&#x30C3;&#x30C8;&#x30EF;&#x30FC;&#x30AF;&#x306E;&#x591A;&#x69D8;&#x306A;&#x69CB;&#x9020;&#x306E;&#x6442;&#x52D5;&#x306F;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x57FA;&#x677F;&#x306E;&#x30CD;&#x30A4;&#x30C6;&#x30A3;&#x30D6;&#x72B6;&#x614B;&#x306B;&#x8B58;&#x5225;&#x30CD;&#x30C3;&#x30C8;&#x30EF;&#x30FC;&#x30AF;&#x306E;&#x80FD;&#x529B;&#x306B;&#x4F9D;&#x5B58;&#x3057;&#x307E;&#x3059;&#x3002;&#x30BB;&#x30EB;&#x306E;&#x5BB6;-ostasis &#x5909;&#x6027;&#x72B6;&#x614B;&#x306E;&#x691C;&#x51FA;&#x306E;&#x4E2D;&#x5FC3;&#x306B;&#x3082;&#x304B;&#x304B;&#x308F;&#x3089;&#x305A;&#x3001;&#x975E;&#x5E38;&#x306B;&#x5C11;&#x3057;&#x306E;&#x6B63;&#x78BA;&#x306A;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x3068;&#x30DF;&#x30B9;&#x30D5;&#x30A9;&#x30FC;&#x30EB;&#x30C9;&#x3055;&#x308C;&#x3066;&#x3068;&#x3057;&#x3066;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x3092;&#x30DE;&#x30FC;&#x30AF;&#x69CB;&#x9020;&#x306E;&#x6A5F;&#x80FD;&#x306B;&#x3064;&#x3044;&#x3066;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x9175;&#x6BCD;&#x52D5;&#x539F;&#x4F53;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA; Ndc10p&#x3002; &#x5909;&#x7570;&#x4F53; Ndc10p &#x306E;&#x5206;&#x89E3;&#x304C; E3 &#xFF08;Ub&#xFF09; &#x30E6;&#x30D3;&#x30AD;&#x30C1;&#x30F3;&#x30EA; Doa10p &#x3067;&#x5C0F;&#x80DE;&#x4F53; &#xFF08;ER&#xFF09; &#x3092;&#x5A92;&#x4ECB;&#x3068;&#x3057;&#x305F;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x6298;&#x308A;&#x7573;&#x307F;&#x306E;&#x54C1;&#x8CEA;&#x5236;&#x5FA1;&#x7D4C;&#x8DEF;&#x306E;&#x57FA;&#x677F;&#x306F;&#x3053;&#x308C;&#x3089;&#x306E;&#x6A5F;&#x80FD;&#x3092;&#x8ABF;&#x3079;&#x308B;&#x306B;&#x306F;&#x3001;&#x6211;&#x3005; &#x306E;&#x8981;&#x4EF6;&#x7814;&#x7A76;/&#x6838;&#x819C;&#x819C;&#x3002;&#x89E3;&#x6790;&#x3001;&#x9006;&#x907A;&#x4F1D;&#x5B66;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x3092;&#x63A1;&#x7528; Ndc10p &#x5909;&#x7570;&#x8A98;&#x5C0E;&#x4F53;&#x306E;&#x81EA;&#x5F8B;&#x7684;&#x306A;&#x54C1;&#x8CEA;&#x7BA1;&#x7406;&#x95A2;&#x9023;&#x5206;&#x89E3;&#x30E2;&#x30C1;&#x30FC;&#x30D5;&#x306F;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E; C &#x672B;&#x7AEF;&#x8FD1;&#x304F;&#x8B58;&#x5225;&#x3002;&#x3053;&#x306E;&#x30E2;&#x30C1;&#x30FC;&#x30D5;&#x306F;&#x3001;2 &#x3064;&#x306E;&#x4E0D;&#x53EF;&#x6B20;&#x306A;&#x758E;&#x6C34;&#x6027;&#x306E;&#x8981;&#x7D20;&#x3067;&#x69CB;&#x6210;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;: &#x4E21;&#x89AA;&#x5A92;&#x6027;&#x30D8;&#x30EA;&#x30C3;&#x30AF;&#x30B9;&#x3068;&#x3001;&#x7DE9;&#x3084;&#x304B;&#x306B;&#x69CB;&#x9020;&#x5316;&#x758E;&#x6C34;&#x6027; C &#x672B;&#x7AEF;&#x306E;&#x758E;&#x6C34;&#x6027;&#x8868;&#x9762;&#x3002;&#x30B5;&#x30A4;&#x30C8;&#x56FA;&#x6709;&#x306E;&#x70B9;&#x7A81;&#x7136;&#x5909;&#x7570;&#x306F;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x30E6;&#x30D3;&#x30AD;&#x30C1;&#x30F3;&#x3092;&#x4ECB;&#x3057;&#x305F;&#x30C8;&#x30EA;&#x30AC;&#x30FC;&#x8981;&#x7D20;&#x3068; HSP70 &#x30B7;&#x30E3;&#x30DA;&#x30ED;&#x30F3;&#x4F9D;&#x5B58;&#x6027;&#x52A3;&#x5316; Ndc10p &#x306E;&#x516C;&#x958B;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x8ABF;&#x67FB;&#x7D50;&#x679C;&#x306F;&#x3001;&#x5FAE;&#x5999;&#x306A; perturbation(s) &#x6838;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x30CD;&#x30A4;&#x30C6;&#x30A3;&#x30D6;&#x69CB;&#x9020;&#x3092;&#x8A8D;&#x8B58;&#x3059;&#x308B;&#x3088;&#x3046;&#x306B;&#x5C0F;&#x80DE;&#x4F53;&#x54C1;&#x8CEA;&#x7BA1;&#x7406;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x306E;&#x80FD;&#x529B;&#x3092;&#x7ACB;&#x8A3C;&#x3057;&#x307E;&#x3059;&#x3002;

&#x9732;&#x51FA;&#x4E8C;&#x90E8;&#x758E;&#x6C34;&#x6027;&#x4FE1;&#x53F7;&#x306E; Ndc10 &#x5C0F;&#x80DE;&#x4F53;/&#x6838;&#x5C01;&#x7B52;&#x3067;&#x306E;&#x539F;&#x5B50;&#x529B;&#x306E;&#x54C1;&#x8CEA;&#x7BA1;&#x7406;&#x3092;&#x30C8;&#x30EA;&#x30AC;&#x30FC;&#x3057;&#x307E;&#x3059;&#x3002;

&#xB2E8;&#xBC31;&#xC9C8; &#xD3F4;&#xB529; &#xD488;&#xC9C8; &#xAD00;&#xB9AC; &#xB124;&#xD2B8;&#xC6CC;&#xD06C;&#xC758; &#xC801;&#xC808; &#xD55C; &#xAE30;&#xB2A5; &#xB2E4;&#xC591; &#xD55C; &#xAD6C;&#xC870;&#xC801; &#xC12D; &#xB2E8;&#xBC31;&#xC9C8; &#xAE30;&#xD310;&#xC758; &#xAE30;&#xBCF8; &#xC0C1;&#xD0DC;&#xB97C; &#xBD84;&#xBCC4; &#xD558;&#xB294; &#xB124;&#xD2B8;&#xC6CC;&#xD06C;&#xC758; &#xB2A5;&#xB825;&#xC5D0; &#xB530;&#xB77C; &#xB2EC;&#xB77C; &#xC9D1;&#xB2C8;&#xB2E4;. Misfolded &#xC0C1;&#xD0DC; &#xC140; &#xD648;-ostasis&#xB97C; &#xAC10;&#xC9C0;&#xC758; &#xC911;&#xC2EC;&#xC801; &#xC704;&#xCE58;&#xC5D0;&#xB3C4; &#xBD88;&#xAD6C; &#xD558; &#xACE0; &#xAC70;&#xC758; &#xC815;&#xD655;&#xD55C; &#xC21C;&#xC11C;&#xC640; &#xD45C;&#xC2DC; misfolded &#xB418;&#xB85C; &#xB2E8;&#xBC31;&#xC9C8; &#xAD6C;&#xC870; &#xAE30;&#xB2A5;&#xC5D0; &#xB300; &#xD55C; &#xC54C;&#xB824;&#xC838; &#xC788;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xAE30;&#xB2A5;&#xC744; &#xC870;&#xC0AC; &#xD558;&#xAE30; &#xC704;&#xD574; &#xC6B0;&#xB9AC; &#xACF5;&#xBD80; &#xC694;&#xAD6C; &#xD6A8; &#xBAA8; kinetochore &#xB2E8;&#xBC31;&#xC9C8; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; Ndc10p Ndc10p.&#xC758; &#xC800;&#xD558;&#xB294; &#xB2E8;&#xBC31;&#xC9C8; &#xD3F4;&#xB529; &#xD488;&#xC9C8; &#xAD00;&#xB9AC; &#xD1B5;&#xB85C; E3 &#xC720;&#xBE44;&#xD034;&#xD2F4; (Ub) &#xB9AC;&#xAC00; Doa10p &#xBC14;&#xC778;&#xB529;&#xACFC; &#xADF8;&#xBB3C; (ER)&#xC5D0; &#xC758;&#xD574; &#xC911;&#xC7AC;&#xC758; &#xAE30;&#xD310; / &#xD575; &#xB9C9;. Ndc10p &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xD30C;&#xC0DD; &#xC0C1;&#xD488;, &#xB9AC;&#xBC84;&#xC2A4; &#xC720;&#xC804;&#xD559; &#xC811;&#xADFC;&#xBC95;&#xC744; &#xCC44;&#xC6A9;&#xC758; &#xBD84;&#xC11D; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; C &#xB9D0;&#xB2E8; &#xADFC;&#xCC98; &#xC790;&#xC728;&#xC801;&#xC778; &#xD488;&#xC9C8; &#xAD00;&#xB9AC; &#xAD00;&#xB828; &#xC800;&#xD558; &#xBAA8;&#xD2F0;&#xBE0C;&#xB97C; &#xC2DD;&#xBCC4; &#xD569;&#xB2C8;&#xB2E4;. &#xC774; &#xC8FC;&#xC81C;&#xB294; &#xB450; &#xD544;&#xC218; &#xC18C;&#xC218; &#xC131; &#xC694;&#xC18C;&#xB85C; &#xAD6C;&#xC131;: amphipathic &#xB098;&#xC120; &#xACE0; &#xB290;&#xC2A8;&#xD558;&#xAC8C; &#xAD6C;&#xC870;&#xD654; &#xB41C; &#xC18C;&#xC218; &#xC131; C &#xD130;&#xBBF8;&#xB110; &#xAF2C;&#xB9AC;&#xC758; &#xC18C;&#xC218; &#xC131; &#xD45C;&#xBA74;. &#xC0AC;&#xC774;&#xD2B8;&#xBCC4; &#xC810; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xB178;&#xCD9C; &#xC774;&#xB7EC;&#xD55C; &#xC694;&#xC18C;, &#xC720;&#xBE44;&#xD034;&#xD2F4; &#xC911;&#xC7AC; &#xD2B8;&#xB9AC;&#xAC70;&#xB9C1; &#xBC0F; ndc10p&#xC758; HSP70 &#xBCF4;&#xD638;&#xC790; &#xC885;&#xC18D; &#xC800;&#xD558;. &#xC774;&#xB7EC;&#xD55C; &#xC5F0;&#xAD6C; &#xACB0;&#xACFC; &#xC785;&#xC99D; &#xD575; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xAE30;&#xBCF8; &#xAD6C;&#xC870;&#xC5D0; &#xBBF8;&#xBB18;&#xD55C; perturbation(s) &#xC778;&#xC2DD; &#xD558;&#xB294; &#xC751;&#xAE09;&#xC2E4; &#xD488;&#xC9C8; &#xAD00;&#xB9AC; &#xC2DC;&#xC2A4;&#xD15C;&#xC758; &#xAE30;&#xB2A5;.

&#xC774; &#xBD84; &#xC18C;&#xC218; &#xC131; &#xC2E0;&#xD638; &#xB178;&#xCD9C; Ndc10 &#xBC14;&#xC778;&#xB529;&#xACFC; &#xADF8;&#xBB3C; &#xD575; &#xBD09;&#xD22C;&#xC5D0;&#xC758; &#xC6D0;&#xC790;&#xB825; &#xD488;&#xC9C8; &#xAD00;&#xB9AC;&#xB97C; &#xD2B8;&#xB9AC;&#xAC70;&#xD569;&#xB2C8;&#xB2E4;.

Funcionamiento de la red de control de calidad de plegamiento de la prote&iacute;na depende de la capacidad de la red discernir diversas perturbaciones estructurales a los Estados nativos de sus sustratos de prote&iacute;na. A pesar de la importancia de la detecci&oacute;n de Estados mal plegadas para celular home-ostasis, muy poco se sabe sobre la secuencia exacta y caracter&iacute;sticas estructurales que marcan una prote&iacute;na como ser mal. Para investigar estas caracter&iacute;sticas, estudiamos los requisitos para la degradaci&oacute;n de la prote&iacute;na del cinetocoro de levadura Ndc10p. mutante Ndc10p es un sustrato de una v&iacute;a de control de calidad de plegamiento de la prote&iacute;na mediada por la ligasa de ubiquitina (Ub) E3 Doa10p en el ret&iacute;culo endopl&aacute;smico (ER) / membrana de envoltura nuclear. An&aacute;lisis de derivados mutantes Ndc10p, con un enfoque de la gen&eacute;tica reversa, identific&oacute; un motivo de degradaci&oacute;n asociados de control de calidad aut&oacute;noma cerca el c-terminal de la prote&iacute;na. Este motivo se compone de dos elementos hidr&oacute;fobos indispensables: una superficie de una h&eacute;lice de anfip&aacute;ticos y una cola hidrof&oacute;bica libremente estructurada del c-terminal. Site-specific mutaciones de punto exponer estos elementos, la activaci&oacute;n mediada por la ubiquitina y degradaci&oacute;n de HSP70 acompa&ntilde;ante dependiente de Ndc10p. Estos resultados corroborar la capacidad del sistema de control de calidad de ER para reconocer perturbation(s) sutiles en la estructura nativa de una prote&iacute;na nuclear.

Exposici&oacute;n de se&ntilde;al hidrof&oacute;bico bipartita desencadena nuclear control de calidad de Ndc10 en la envoltura nuclear ret&iacute;culo endopl&aacute;smico.

Endoplasmic reticulum (ER) stress develops when the ER is overloaded with too many proteins to fold. This elicits a signaling pathway called the unfolded protein response. The unfolded protein response is physiologically required for the terminal development of B cells into antibody-secreting plasma cells. Ring Box Protein 1 (RBX1) is a 14-kDa protein necessary for ubiquitin ligation activity of the multimeric cullin ring ubiquitin ligases (CRLs). As RBX1 is shared by a large number of CRLs, alterations in its activity may lead to global changes in protein stability. We discovered that RBX1 is cleaved in the course of LPS-induced plasma cell differentiation and in multiple myeloma cell lines upon induction of pharmacological ER stress. The cleavage is executed by several caspase proteases that cleave RBX1 eight amino acids from the N terminus. To address the possible implication of RBX1 cleavage for CRL activity, we replaced the endogenous RBX1 homolog of the yeast Saccharomyces cerevisiae, Roc1, with the wild type or the N-terminal Δ8 mutant human RBX1. We show that yeast expressing the cleaved RBX1 are hypersensitive to ER stress and are impaired in CRL-mediated ubiquitination and degradation. We propose a model by which N-terminal cleavage of RBX1 impairs its activity and promotes susceptibility to ER stress induction.

Endoplasmic reticulum stress induces a caspase-dependent N-terminal cleavage of RBX1 protein in B cells.

Protein elimination by the ubiquitin-proteasome system requires the presence of a cis-acting degradation signal. Efforts to discern degradation signals of misfolded proteasome substrates thus far revealed a general mechanism whereby the exposure of cryptic hydrophobic motifs provides a degradation determinant. We have previously characterized such a determinant, employing the yeast kinetochore protein Ndc10 as a model substrate. Ndc10 is essentially a stable protein that is rapidly degraded upon exposure of a hydrophobic motif located at the C-terminal region. The degradation motif comprises two distinct and essential elements: DegA, encompassing two amphipathic helices, and DegB, a hydrophobic sequence within the loosely structured C-terminal tail of Ndc10. Here we show that the hydrophobic nature of DegB is irrelevant for the ubiquitylation of substrates containing the Ndc10 degradation motif, but is essential for proteasomal degradation. Mutant DegB, in which the hydrophobic sequence was disrupted, acted as a dominant degradation inhibitory element when expressed at the C-terminal regions of ubiquitin-dependent and -independent substrates of the 26S proteasome. This mutant stabilized substrates in both yeast and mammalian cells, indicative of a modular recognition moiety. The dominant function of the mutant DegB provides a powerful experimental tool for evaluating the physiological implications of stabilization of specific proteasome substrates in intact cells and for studying the associated pathological effects.

Itamar Cohen

Department of Biological Chemistry

The Hebrew University of Jerusalem

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Automated Delivery of Microfabricated Targets for Intense Laser Irradiation Experiments

Itamar Cohen

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The Hebrew University of Jerusalem

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Automated Delivery of Microfabricated Targets for Intense Laser Irradiation Experiments

Department of Biological Chemistry