stimulation-of-cytoplasmic-dna-sensing-pathways-in-vitro-and-in-vivo

Stimulation of Cytoplasmic DNA Sensing Pathways <em>In Vitro </em>and <em>In Vivo</em>

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...

University of Cambridge

The marriage between transducers of cell stress stimuli and their nuclear targets is likely to be achieved in part by some spatial-temporal compartmentalization of the relevant effectors. A candidate compartment for these events is the promyelocytic leukemia nuclear domain (PML-ND), within which are found numerous effectors of damage recognition, repair, and cell death. We predicted that the identification of PML-ND cargo proteins would clarify those biochemical pathways that straddle the recognition of cellular damage and cell fate. We now use mass spectrometry of peptides eluted from PML coprecipitates to demonstrate that the gamma 1 (gamma1) isoform of PLC associates with nuclear PML. Though thought to act primarily in the cytoplasm, we use biochemical fractionation combined with immunocytochemistry to verify the nuclear expression of PLC-gamma1 and its interaction with PML. These are the first data to show an interaction between endogenous levels of a phosphoinositide metabolizing protein and the biophysically labile PML-ND by mass spectrometry and add weight to the view that PML-NDs may act as tumor suppressors by sequestering mitogenic effectors.

Nuclear phospholipase C gamma: punctate distribution and association with the promyelocytic leukemia protein.

&#x53D1;&#x5C55;&#x4E2D;&#x56FD;&#x5BB6;&#x7684; T &#x7EC6;&#x80DE;&#x9047;&#x5230;&#x5916;&#x56F4;&#x81EA;&#x6211;&#x6297;&#x539F;&#x5728;&#x80F8;&#x817A;&#x4E2D;&#x8981;&#x5220;&#x9664; autoreactive &#x514B;&#x9686;&#x3002;&#x76EE;&#x524D;&#x6240;&#x77E5;&#x5728;&#x80F8;&#x817A;&#x9AD3;&#x8D28;&#x4E0A;&#x76AE;&#x7EC6;&#x80DE;&#x4E2D;&#x8868;&#x8FBE;&#xFF0C;&#x5230;&#x81EA;&#x8EAB;&#x514D;&#x75AB;&#x8C03;&#x8282;&#x86CB;&#x767D;&#x8D28; (&#x827E;&#x5C14;&#xFF09;&#xFF0C;&#x8C03;&#x8282;&#x80F8;&#x817A;&#x8F6C;&#x5F55;&#x8FD9;&#x4E9B;&#x5916;&#x56F4;&#x7684;&#x7EC4;&#x7EC7;&#x7279;&#x5F02;&#x6027;&#x6297;&#x539F;&#x7684;&#x5173;&#x952E;&#x4F5C;&#x7528;&#x3002;&#x7A7A;&#x6C14;&#x57FA;&#x56E0;&#x7A81;&#x53D8;&#x4E0E;&#x4E25;&#x91CD;&#x591A;&#x5668;&#x5B98;&#x81EA;&#x8EAB;&#x514D;&#x75AB;&#x6027;&#x7EFC;&#x5408;&#x75C7; &#xFF08;APECED&#xFF09; &#x76F8;&#x5173;&#x8054;&#xFF0C;&#x81EA;&#x8EAB;&#x514D;&#x75AB;&#x6027;&#x6D3B;&#x6027;&#x7A7A;&#x6C14;&#x7F3A;&#x9677;&#x5C0F;&#x9F20;&#x4E2D;&#x7684;&#x6E05;&#x5355;&#x3002;&#x529F;&#x80FD;&#x7A7A;&#x6C14;&#x86CB;&#x767D;&#x8868;&#x793A;&#x4E3A;&#x4E0D;&#x540C;&#x7684;&#x6838; puncta&#xFF0C;&#x867D;&#x7136;&#x6CA1;&#x6709;&#x7ED3;&#x6784;&#x57FA;&#x7840;&#x5B58;&#x5728;&#x89E3;&#x91CA;&#x5176;&#x4E0E;&#x75BE;&#x75C5;&#x7684;&#x76F8;&#x5173;&#x6027;&#x3002;&#x5728;&#x89E3;&#x51B3; APECED &#x7EC6;&#x80DE;&#x751F;&#x7269;&#x5B66;&#x57FA;&#x7840;&#xFF0C;&#x6211;&#x4EEC;&#x53D6;&#x5F97;&#x4E86;&#x610F;&#x5916;&#x7684;&#x53D1;&#x73B0;&#x7A7A;&#x6C14;&#x7A81;&#x53D8;&#x70ED;&#x70B9;&#x5728;&#x4E8E;&#x534A;&#x80F1;&#x6C28;&#x9178;&#x86CB;&#x767D;&#x9176;&#x62DB;&#x8058;&#x57DF;&#x4E2D;&#x3002;&#x5982;&#x4F55;&#x7ED3;&#x5408;&#x540C;&#x6E90;&#x5EFA;&#x6A21;&#x548C;&#x4F53;&#x5916;&#x6570;&#x636E;&#x663E;&#x793A; APECED &#x7A81;&#x53D8;&#x5F71;&#x54CD;&#x6B64;&#x8F6C;&#x5F55;&#x8C03;&#x8282;&#x7684;&#x6D3B;&#x52A8;&#x3002;&#x6211;&#x4EEC;&#x8FD8;&#x63D0;&#x4F9B;&#x7A7A;&#x6C14;&#x7684;&#x5173;&#x8054;&#x4E0E;&#x5168;&#x7403;&#x8F6C;&#x5F55;&#x56E0;&#x5B50;&#xFF0C;&#x53EF;&#x4F5C;&#x4E3A;&#x57FA;&#x7840;&#x7684;&#x8F6C;&#x5F55;&#x8FC7;&#x7A0B;&#x7A7A;&#x6C14;&#x7684;&#x7126;&#x3001; &#x5168;&#x57FA;&#x56E0;&#x7EC4;&#x6539;&#x9020;&#x65B0;&#x578B;&#x4F53;&#x5185;&#x8BC1;&#x636E;&#x3002;

&#x7A7A;&#x6C14;&#x7684;&#x5361;&#x900F;&#x9732;&#xFF0C;&#x4E2D;&#x592E;&#x5BBD;&#x5BB9;&#x7684;&#x65B0;&#x7ED3;&#x6784;&#x6311;&#x8D77;&#x8F6C;&#x5F55;&#x7684;&#x53EF;&#x5851;&#x6027;&#x3002;

Developing T cells encounter peripheral self-antigens in the thymus in order to delete autoreactive clones. It is now known that the autoimmune regulator protein (AIRE), which is expressed in thymic medullary epithelial cells, plays a key role in regulating the thymic transcription of these peripheral tissue-specific antigens. Mutations in the AIRE gene are associated with a severe multiorgan autoimmune syndrome (APECED), and autoimmune reactivities are manifest in AIRE-deficient mice. Functional AIRE protein is expressed as distinct nuclear puncta, although no structural basis existed to explain their relevance to disease. In addressing the cell biologic basis for APECED, we made the unexpected discovery that an AIRE mutation hot spot lies in a caspase recruitment domain. Combined homology modeling and in vitro data now show how APECED mutations influence the activity of this transcriptional regulator. We also provide novel in vivo evidence for AIRE's association with a global transcription cofactor, which may underlie AIRE's focal, genome-wide, alteration of the transcriptome.

AIRE's CARD revealed, a new structure for central tolerance provokes transcriptional plasticity.

D&#xE9;veloppement des cellules T p&#xE9;riph&#xE9;riques rencontrez des auto-antig&#xE8;nes dans le thymus, afin de supprimer les clones autor&#xE9;actifs. On sait maintenant que la prot&#xE9;ine r&#xE9;gulatrice auto-immune (AIRE), qui est exprim&#xE9;e en cellules &#xE9;pith&#xE9;liales m&#xE9;dullaires thymiques, joue un r&#xF4;le cl&#xE9; dans la r&#xE9;gulation de la transcription du thymus de ces p&#xE9;riph&#xE9;riques tissu-sp&#xE9;cifiques des antig&#xE8;nes. Des mutations dans le g&#xE8;ne AIRE sont associ&#xE9;s &#xE0; un syndrome s&#xE9;v&#xE8;re multivisc&#xE9;rale auto-immune (Apeced), et r&#xE9;activit&#xE9;s auto-immunes se manifestent dans AIRE-souris d&#xE9;ficientes. Fonctionnelle des prot&#xE9;ines AIRE est exprim&#xE9;e en points lacrymaux nucl&#xE9;aire distincte, m&#xEA;me si aucune base structurelle existait pour expliquer leur pertinence &#xE0; la maladie. En abordant la base biologique de la cellule pour APECED, nous avons fait la d&#xE9;couverte inattendue que un endroit chaud AIRE mutation se trouve dans un domaine de recrutement de caspase. Combin&#xE9; mod&#xE9;lisation par homologie et les donn&#xE9;es in vitro montrent maintenant comment les mutations Apeced influencer l&#x26;#39;activit&#xE9; de ce r&#xE9;gulateur transcriptionnel. Nous avons &#xE9;galement fournir de nouvelles preuves in vivo pour l&#x26;#39;association AIRE avec un cofacteur de transcription mondiale, ce qui peut expliquer focale AIRE, le g&#xE9;nome entier, l&#x26;#39;alt&#xE9;ration du transcriptome.

CARTE AIRE a r&#xE9;v&#xE9;l&#xE9;, une nouvelle structure pour la tol&#xE9;rance centrale Provoque Plasticit&#xE9; de la transcription

Entwickelnde T-Zellen treten periphere selbst-Antigene im Thymus um autoreaktive Klone zu l&ouml;schen. Es ist jetzt bekannt, dass das autoimmune Regulator-Protein (AIRE), das Thymom medull&auml;ren Epithelzellen ausgedr&uuml;ckt wird, eine entscheidende Rolle spielt bei der Regulierung der Thymom Transkription von diesen peripheren Gewebe-spezifischen Antigenen. Mutationen im Gen AIRE ein schweres Syndrom Autoimmun-Syndrom (APECED) zugeordnet sind, und autoimmune Reactivities werden offenbar in AIRE-defizienten M&auml;usen. Funktionale AIRE-Protein wird als unterschiedliche nukleare Puncta, ausgedr&uuml;ckt, obwohl es keine strukturelle Grundlage gab, um ihre Relevanz f&uuml;r die Krankheit zu erkl&auml;ren. Bei der Bew&auml;ltigung der Zelle biologische Grundlage f&uuml;r APECED, machten wir die unerwartete Entdeckung, dass ein Hot-Spot in AIRE Mutation in eine Caspase Recruitment Domain liegt. Kombinierte Homologie Modellierung und zeigen in-vitro-Daten nun wie APECED Mutationen beeinflussen die Aktivit&auml;t dieser transcriptional Regulator. Wir bieten auch neuartige in-vivo Beweis f&uuml;r AIREs Assoziation mit Kofaktor global Transkription, AIREs fokale, genomweiten, Ver&auml;nderung der das Transkriptom zugrunde liegen kann.

AIRE die Karte aufgedeckt, eine neue Struktur f&uuml;r zentrale Toleranz transkriptionelle Plastizit&auml;t provoziert.

In via di sviluppo le cellule T incontro auto-antigeni periferici nel timo al fine di eliminare i cloni autoreattivi. &Egrave; ormai noto che la proteina regolatore autoimmune (AIRE), che &egrave; espressa nelle cellule epiteliali midollari del timo, gioca un ruolo chiave nella regolazione della trascrizione del timo di questi antigeni specifici tessuti periferici. Mutazioni nel gene AIRE sono associate a una grave sindrome autoimmune multiorgano (APECED) e reattivit&agrave; autoimmune si manifestano nei topi deficienti AIRE. Proteina funzionale AIRE &egrave; espressa come distinto puncta nucleare, anche se nessuna base strutturale ha esistito per spiegare la loro rilevanza per la malattia. Nell&#39;affrontare la base biologica delle cellule per APECED, abbiamo fatto l&#39;inattesa scoperta che una mutazione AIRE hot spot si trova in un dominio di reclutamento di caspase. Combinato modellistica di omologia e visualizza ora i dati in vitro come APECED mutazioni influenzano l&#39;attivit&agrave; di questo regolatore di transcriptional. Forniamo anche romanzo prove in vivo per associazione di AIRE con un cofattore trascrizione globale, che pu&ograve; essere alla base di alterazione focale, genome-wide, di AIRE del transcriptome.

SCHEDA di AIRE ha rivelato, una nuova struttura per la tolleranza centrale provoca plasticit&agrave; trascrizionale.

&#x958B;&#x767A;T&#x7D30;&#x80DE;&#x306F;&#x81EA;&#x5DF1;&#x53CD;&#x5FDC;&#x6027;&#x30AF;&#x30ED;&#x30FC;&#x30F3;&#x3092;&#x524A;&#x9664;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3001;&#x80F8;&#x817A;&#x3067;&#x306E;&#x672B;&#x68A2;&#x81EA;&#x5DF1;&#x6297;&#x539F;&#x306B;&#x906D;&#x9047;&#x3059;&#x308B;&#x3002;&#x305D;&#x308C;&#x306F;&#x4ECA;&#x80F8;&#x817A;&#x306E;&#x9AC4;&#x8CEA;&#x4E0A;&#x76AE;&#x7D30;&#x80DE;&#x306B;&#x767A;&#x73FE;&#x3057;&#x3066;&#x3044;&#x308B;&#x81EA;&#x5DF1;&#x514D;&#x75AB;&#x30EC;&#x30AE;&#x30E5;&#x30EC;&#x30FC;&#x30BF;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#xFF08;AIRE&#xFF09;&#x306F;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x672B;&#x68A2;&#x7D44;&#x7E54;&#x7279;&#x7570;&#x7684;&#x6297;&#x539F;&#x306E;&#x80F8;&#x817A;&#x306E;&#x8EE2;&#x5199;&#x306E;&#x8ABF;&#x7BC0;&#x306B;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3057;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x304C;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x3002; AIRE&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x5909;&#x7570;&#x306F;&#x3001;&#x91CD;&#x5EA6;&#x306E;&#x591A;&#x81D3;&#x5668;&#x81EA;&#x5DF1;&#x514D;&#x75AB;&#x75C7;&#x5019;&#x7FA4;&#xFF08;APECED&#xFF09;&#x3001;&#x304A;&#x3088;&#x3073;&#x81EA;&#x5DF1;&#x514D;&#x75AB;&#x53CD;&#x5FDC;&#x6027;&#x30DE;&#x30CB;&#x30D5;&#x30A7;&#x30B9;&#x30C8;&#x306F;AIRE&#x6B20;&#x640D;&#x30DE;&#x30A6;&#x30B9;&#x3067;&#x3042;&#x308B;&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x69CB;&#x9020;&#x7684;&#x306A;&#x6839;&#x62E0;&#x306F;&#x3001;&#x75C5;&#x6C17;&#x3068;&#x306E;&#x95A2;&#x9023;&#x6027;&#x3092;&#x8AAC;&#x660E;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x5B58;&#x5728;&#x3057;&#x3066;&#x3044;&#x307E;&#x305B;&#x3093;&#x304C;&#x3001;&#x6A5F;&#x80FD;AIRE&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306F;&#x3001;&#x7570;&#x306A;&#x308B;&#x6838;&#x306E;&#x6D99;&#x70B9;&#x3068;&#x3057;&#x3066;&#x8868;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002; APECED&#x306E;&#x305F;&#x3081;&#x306E;&#x7D30;&#x80DE;&#x751F;&#x7269;&#x5B66;&#x7684;&#x57FA;&#x790E;&#x3092;&#x30A2;&#x30C9;&#x30EC;&#x30C3;&#x30B7;&#x30F3;&#x30B0;&#x3067;&#x200B;&#x200B;&#x306F;&#x3001;AIRE&#x306E;&#x5909;&#x7570;&#x30DB;&#x30C3;&#x30C8;&#x30B9;&#x30DD;&#x30C3;&#x30C8;&#x306F;&#x3001;&#x30AB;&#x30B9;&#x30D1;&#x30FC;&#x30BC;&#x52D5;&#x54E1;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x4E88;&#x671F;&#x3057;&#x306A;&#x3044;&#x767A;&#x898B;&#x3092;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x30DB;&#x30E2;&#x30ED;&#x30B8;&#x30FC;&#x30E2;&#x30C7;&#x30EA;&#x30F3;&#x30B0;&#x3092;&#x7D44;&#x307F;&#x5408;&#x308F;&#x305B;&#x3066;&#x3001;&#x30A4;&#x30F3;&#x30D3;&#x30C8;&#x30ED;&#x3067;&#x30C7;&#x30FC;&#x30BF;&#x304C;APECED&#x5909;&#x7570;&#x306F;&#x3001;&#x3053;&#x306E;&#x8EE2;&#x5199;&#x8ABF;&#x7BC0;&#x56E0;&#x5B50;&#x306E;&#x6D3B;&#x6027;&#x306B;&#x5F71;&#x97FF;&#x3092;&#x53CA;&#x307C;&#x3059;&#x65B9;&#x6CD5;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x307E;&#x305F;&#x3001;AIRE&#x306E;&#x7126;&#x70B9;&#x306F;&#x3001;&#x30B2;&#x30CE;&#x30E0;&#x30EF;&#x30A4;&#x30C9;&#x3001;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30AF;&#x30EA;&#x30D7;&#x30C8;&#x30FC;&#x30E0;&#x306E;&#x5909;&#x5316;&#x306E;&#x6839;&#x5E95;&#x306B;&#x3042;&#x308B;&#x3053;&#x3068;&#x304C;&#x4E16;&#x754C;&#x7684;&#x306A;&#x8EE2;&#x5199;&#x56E0;&#x5B50;&#x3068;&#x306E;AIRE&#x306E;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x306E;&#x305F;&#x3081;&#x306B;in vivo&#x3067;&#x306E;&#x8A3C;&#x62E0;&#x3067;&#x5C0F;&#x8AAC;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

AIRE&#x306E;&#x30AB;&#x30FC;&#x30C9;&#x306F;&#x3001;&#x30BB;&#x30F3;&#x30C8;&#x30E9;&#x30EB;&#x30C8;&#x30EC;&#x30E9;&#x30F3;&#x30B9;&#x306E;&#x305F;&#x3081;&#x306E;&#x65B0;&#x3057;&#x3044;&#x69CB;&#x9020;&#x304C;&#x8EE2;&#x5199;&#x53EF;&#x5851;&#x6027;&#x3092;&#x8A98;&#x767A;&#x3059;&#x308B;&#x3001;&#x660E;&#x3089;&#x304B;&#x306B;

&#xAC1C;&#xBC1C; T &#xC138;&#xD3EC; autoreactive &#xD074;&#xB860;&#xC744; &#xC0AD;&#xC81C; &#xD558;&#xB824;&#xBA74; thymus&#xC5D0;&#xC11C; &#xC8FC;&#xBCC0; &#xC790;&#xAE30; &#xD56D; &#xC6D0; &#xBC1C;&#xC0DD;. &#xC9C0;&#xAE08; &#xC54C;&#xB824;&#xC838; thymic &#xACE8; &#xC218; &#xC0C1;&#xD53C; &#xC138;&#xD3EC; &#xB2E8;&#xC704;&#xB85C; &#xBA74;&#xC5ED; &#xB808; &#xADE4; &#xB808;&#xC774; &#xD130; &#xB2E8;&#xBC31;&#xC9C8; (&#xB974;), &#xC774;&#xB7EC;&#xD55C; &#xC8FC;&#xBCC0; &#xC870;&#xC9C1;&#xC758; &#xD2B9;&#xC815; &#xD56D; &#xC6D0;&#xC758; thymic &#xC804;&#xC0AC; &#xC870;&#xC808;&#xC5D0; &#xC911;&#xC694; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xD55C;&#xB2E4;. AIRE &#xC720;&#xC804;&#xC790;&#xC758; &#xBCC0;&#xC774; &#xC2EC;&#xD55C; multiorgan &#xC790;&#xAC00; &#xBA74;&#xC5ED; &#xC99D;&#xD6C4;&#xAD70; (APECED) &#xBC0F; &#xBA74;&#xC5ED; reactivities &#xB41C; &#xB974; &#xBD88;&#xCDA9;&#xBD84; &#xD55C; &#xC950;&#xC5D0;&#xC11C; &#xB9E4;&#xB2C8;&#xD398;&#xC2A4;&#xD2B8;. &#xAE30;&#xB2A5; &#xB974; &#xB2E8;&#xBC31;&#xC9C8; &#xAD6C;&#xC870; &#xAE30;&#xC900; &#xC9C8;&#xBCD1;&#xC758; &#xAD00;&#xB828;&#xC131;&#xC744; &#xC124;&#xBA85; &#xD558;&#xAE30; &#xC704;&#xD574; &#xC874;&#xC7AC; &#xD558;&#xC9C0;&#xB9CC; &#xB69C;&#xB837;&#xD55C; &#xD575; puncta&#xB85C; &#xD45C;&#xC2DC; &#xB429;&#xB2C8;&#xB2E4;. APECED&#xC5D0; &#xB300; &#xD55C; &#xC140; &#xC0DD;&#xBB3C; &#xD559;&#xC801; &#xAE30;&#xCD08;&#xB97C; &#xD574;&#xACB0;, &#xC6B0;&#xB9AC;&#xB294; &#xB974; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xD56B; &#xC2A4;&#xD3FF; caspase &#xBAA8;&#xC9D1; &#xB3C4;&#xBA54;&#xC778;&#xC5D0; &#xB193;&#xC5EC; &#xC608;&#xAE30;&#xCE58; &#xC54A;&#xC740; &#xBC1C;&#xACAC;&#xC744; &#xD588;&#xB2E4;. &#xD638;&#xBAB0;&#xB85C;&#xC9C0; &#xBAA8;&#xB378;&#xB9C1; &#xBC0F; &#xCCB4; &#xC678; &#xB370;&#xC774;&#xD130; &#xC9C0;&#xAE08; &#xC1FC;&#xB97C; &#xC5B4;&#xB5BB;&#xAC8C; &#xACB0;&#xD569; APECED &#xB3CC;&#xC5F0;&#xBCC0;&#xC774;&#xC774; transcriptional &#xB808; &#xADE4; &#xB808;&#xC774; &#xD130;&#xC758; &#xD65C;&#xB3D9;&#xC5D0; &#xC601;&#xD5A5;&#xC744;. &#xC6B0;&#xB9AC;&#xB294; &#xB610;&#xD55C; &#xB974;&#xC758; &#xCD08;&#xC810;, &#xAC8C;&#xB188; &#xC804;&#xCCB4; &#xBCC0;&#xACBD; &#xD569;&#xB2C8;&#xB2E4; transcriptome &#xAE30;&#xCD08; &#xC218; &#xC788;&#xB294; &#xAE00;&#xB85C;&#xBC8C; &#xC804;&#xC0AC; &#xACF5;&#xB3D9; &#xC778;&#xC790;&#xC640; &#xB974;&#xC758; &#xD611;&#xD68C;&#xC5D0; &#xB300; &#xD55C; &#xC18C;&#xC124; vivo&#xC5D0;&#xC11C; &#xC99D;&#xAC70;&#xB97C; &#xC81C;&#xACF5;.

&#xC5D0; &#xB974;&#xC758; &#xCE74;&#xB4DC; &#xACF5;&#xAC1C;, &#xC911;&#xC559; &#xACF5;&#xCC28;&#xC5D0; &#xB300; &#xD55C; &#xC0C8;&#xB85C;&#xC6B4; &#xAD6C;&#xC870; transcriptional &#xC18C;&#xC131;&#xC744; &#xBD88;&#xB7EC; &#xC77C;&#xC73C;&#xD0A4;.

Desarrollo de las c&#xE9;lulas T perif&#xE9;ricas encuentran autoant&#xED;genos en el timo con el fin de eliminar clones autorreactivos. Ahora se sabe que la prote&#xED;na regulador autoinmune (AIRE), que se expresa en c&#xE9;lulas epiteliales t&#xED;micas medulares, juega un papel clave en la regulaci&#xF3;n de la transcripci&#xF3;n del timo de estos perif&#xE9;ricos tejidos espec&#xED;ficos de ant&#xED;genos. Las mutaciones en el gen AIRE est&#xE1;n asociados con un severo s&#xED;ndrome multiorg&#xE1;nico autoinmune (APECED) y reactividades autoinmunes se manifiestan en ratones con deficiencia de AIRE. Funcional de prote&#xED;nas AIRE se expresa en puntos lagrimales nuclear clara, aunque no exist&#xED;a una base estructural para explicar su importancia a la enfermedad. Al abordar la base biol&#xF3;gica de c&#xE9;lulas de APECED, hicimos el descubrimiento inesperado de que un punto de mutaci&#xF3;n AIRE caliente se encuentra en un dominio de reclutamiento de caspasas. Combinado homolog&#xED;a de modelado y los datos in vitro muestran ahora c&#xF3;mo las mutaciones APECED influir en la actividad de este regulador de la transcripci&#xF3;n. Tambi&#xE9;n ofrecemos la novela de pruebas in vivo para la asociaci&#xF3;n AIRE con un cofactor global de transcripci&#xF3;n, que puede ser la base central de AIRE, en todo el genoma, la alteraci&#xF3;n del transcriptoma.

TARJETA DE AIRE Revelada una nueva estructura para la Tolerancia central Provoca Plasticidad transcripcional

When thymic epithelia begin to synthesize peripheral tissue antigens such as insulin, we are seeing the result of autoimmune regulator (AIRE) activity and the workings of central tolerance. AIRE is an extraordinary protein that repatterns the transcriptome of medullary thymic epithelia (mTECs) to produce a stroma decorated with peripheral self-peptides. These peptidic arrays are used to purge self-reactive T cells, thereby averting autoimmunity. We now propose that an inherently cytotoxic event such as global chromatin modification paves the way for AIRE action. This injury stimulus might impose temporal restrictions for the T-cell education process and is endured, at least transiently, by the unique cellular environment provided by the medullary thymic epithelia.

Death in the AIRE.

Nuclear domains of promyelocytic leukemia protein (PML) are known to act as signaling nodes in many cellular processes. Although the impact of PML expression in driving cell fate decisions for injured cells is well established, the function of PML in the context of tissue development is less well understood. Here, the in vivo role of PML in developmental processes in the murine mammary gland has been investigated. Data are presented showing that expression of PML is tightly regulated by three members of the Stat family of transcription factors that orchestrate the functional development of the mammary secretory epithelium during pregnancy. Developmental phenotypes were also discovered in the virgin and pregnant Pml null mouse, typified by aberrant differentiation of mammary epithelia with reduced ductal and alveolar development. PML depletion was also found to disturb the balance of two distinct luminal progenitor populations. Overall, it is shown that PML is required for cell lineage determination in bi-potent luminal progenitor cells and that the precise regulation of PML expression is required for functional differentiation of alveolar cells.

PML depletion disrupts normal mammary gland development and skews the composition of the mammary luminal cell progenitor pool.

Vaccinia virus (VACV) uses microtubules for export of virions to the cell surface and this process requires the viral protein F12. Here we show that F12 has structural similarity to kinesin light chain (KLC), a subunit of the kinesin-1 motor that binds cargo. F12 and KLC share similar size, pI, hydropathy and cargo-binding tetratricopeptide repeats (TPRs). Moreover, molecular modeling of F12 TPRs upon the crystal structure of KLC2 TPRs showed a striking conservation of structure. We also identified multiple TPRs in VACV proteins E2 and A36. Data presented demonstrate that F12 is critical for recruitment of kinesin-1 to virions and that a conserved tryptophan and aspartic acid (WD) motif, which is conserved in the kinesin-1-binding sequence (KBS) of the neuronal protein calsyntenin/alcadein and several other cellular kinesin-1 binding proteins, is essential for kinesin-1 recruitment and virion transport. In contrast, mutation of WD motifs in protein A36 revealed they were not required for kinesin-1 recruitment or IEV transport. This report of a viral KLC-like protein containing a KBS that is conserved in several cellular proteins advances our understanding of how VACV recruits the kinesin motor to virions, and exemplifies how viruses use molecular mimicry of cellular components to their advantage.

Vaccinia protein F12 has structural similarity to kinesin light chain and contains a motor binding motif required for virion export.

The IκB kinase (IKK) complex regulates activation of NF-κB, a critical transcription factor in mediating inflammatory and immune responses. Not surprisingly, therefore, many viruses seek to inhibit NF-κB activation. The vaccinia virus B14 protein contributes to virus virulence by binding to the IKKβ subunit of the IKK complex and preventing NF-κB activation in response to pro-inflammatory stimuli. Previous crystallographic studies showed that the B14 protein has a Bcl-2-like fold and forms homodimers in the crystal. However, multi-angle light scattering indicated that B14 is in monomer-dimer equilibrium in solution. This transient self-association suggested that the hydrophobic dimerization interface of B14 might also mediate its interaction with IKKβ, and this was investigated by introducing amino acid substitutions on the dimer interface. One mutant (Y35E) was entirely monomeric but still co-immunoprecipitated with IKKβ and blocked both NF-κB nuclear translocation and NF-κB-dependent gene expression. Therefore, B14 homodimerization is nonessential for binding and inhibition of IKKβ. In contrast, a second monomeric mutant (F130K) neither bound IKKβ nor inhibited NF-κB-dependent gene expression, demonstrating that this residue is required for the B14-IKKβ interaction. Thus, the dimerization and IKKβ-binding interfaces overlap and lie on a surface used for protein-protein interactions in many viral and cellular Bcl-2-like proteins.

Mapping the IkappaB kinase beta (IKKbeta)-binding interface of the B14 protein, a vaccinia virus inhibitor of IKKbeta-mediated activation of nuclear factor kappaB.

Vaccinia virus (VACV) strain Western Reserve protein C4 has been characterized and its function and contribution to virus virulence assessed. Bioinformatic analysis showed that C4 is conserved in six orthopoxvirus species and shares 43 % amino acid identity with VACV protein C16, a known virulence factor. A recombinant VACV expressing a C-terminally tagged version of C4 showed that, like C16, this 37 kDa protein is expressed early during infection and localizes to both the cytoplasm and the nucleus. Functional assays using a firefly luciferase reporter plasmid under the control of a nuclear factor kappa B (NF-κB)-dependent promoter demonstrated that C4 inhibits NF-κB activation at, or downstream of, the inhibitor of kappa kinase (IKK) complex. Consistent with this, C4 inhibited interleukin-1β-induced translocation of p65 into the nucleus. A VACV lacking the C4L gene (vΔC4) showed no significant differences from wild-type virus in growth kinetics or spread in cell culture, but had reduced virulence in a murine intranasal model of infection. vΔC4-infected mice exhibited fewer symptoms, lost less weight and recovered 7 days earlier than animals infected with control viruses expressing C4. Furthermore, bronchoalveolar lavage fluid from vΔC4-infected mice had increased cell numbers at day 5 post-infection, which correlated with reduced lung virus titres from this time onward. C4 represents the ninth VACV protein to inhibit NF-κB activation and remarkably, in every case examined, loss of each protein individually caused an alteration in virus virulence, despite the presence of other NF-κB inhibitors.

Vaccinia virus protein C4 inhibits NF-κB activation and promotes virus virulence.

Innate immunity is the first immunological defence against pathogens. During virus infection detection of nucleic acids is crucial for the inflammatory response. Here we identify DNA-dependent protein kinase (DNA-PK) as a DNA sensor that activates innate immunity. We show that DNA-PK acts as a pattern recognition receptor, binding cytoplasmic DNA and triggering the transcription of type I interferon (IFN), cytokine and chemokine genes in a manner dependent on IFN regulatory factor 3 (IRF-3), TANK-binding kinase 1 (TBK1) and stimulator of interferon genes (STING). Both cells and mice lacking DNA-PKcs show attenuated cytokine responses to both DNA and DNA viruses but not to RNA or RNA virus infection. DNA-PK has well-established functions in the DNA repair and V(D)J recombination, hence loss of DNA-PK leads to severe combined immunodeficiency (SCID). However, we now define a novel anti-microbial function for DNA-PK, a finding with implications for host defence, vaccine development and autoimmunity.DOI:http://dx.doi.org/10.7554/eLife.00047.001.

DNA-PK is a DNA sensor for IRF-3-dependent innate immunity.

The transcription factor NF-κB is essential for immune responses against pathogens and its activation requires the phosphorylation, ubiquitination and proteasomal degradation of IκBα. Here we describe an inhibitor of NF-κB from vaccinia virus that has a closely related counterpart in variola virus, the cause of smallpox, and mechanistic similarity with the HIV protein Vpu. Protein A49 blocks NF-κB activation by molecular mimicry and contains a motif conserved in IκBα which, in IκBα, is phosphorylated by IKKβ causing ubiquitination and degradation. Like IκBα, A49 binds the E3 ligase β-TrCP, thereby preventing ubiquitination and degradation of IκBα. Consequently, A49 stabilised phosphorylated IκBα (p-IκBα) and its interaction with p65, so preventing p65 nuclear translocation. Serine-to-alanine mutagenesis within the IκBα-like motif of A49 abolished β-TrCP binding, stabilisation of p-IκBα and inhibition of NF-κB activation. Remarkably, despite encoding nine other inhibitors of NF-κB, a VACV lacking A49 showed reduced virulence in vivo.

Poxvirus targeting of E3 ligase β-TrCP by molecular mimicry: a mechanism to inhibit NF-κB activation and promote immune evasion and virulence.

Vaccinia virus (VACV) expresses many proteins that are non-essential for virus replication but promote virulence by inhibiting components of the host immune response to infection. These immunomodulators include a family of proteins that have, or are predicted to have, a structure related to the B-cell lymphoma (Bcl)-2 protein. Five members of the VACV Bcl-2 family (N1, B14, A52, F1 and K7) have had their crystal structure solved, others have been characterized and a function assigned (C6, A46), and others are predicted to be Bcl-2 proteins but are uncharacterized hitherto (N2, B22, C1). Data presented here show that N2 is a nuclear protein that is expressed early during infection and inhibits the activation of interferon regulatory factor (IRF)3. Consistent with its nuclear localization, N2 inhibits IRF3 downstream of the TANK-binding kinase (TBK)-1 and after IRF3 translocation into the nucleus. A mutant VACV strain Western Reserve lacking the N2L gene (vΔN2) showed normal replication and spread in cultured cells compared to wild-type parental (vN2) and revertant (vN2-rev) viruses, but was attenuated in two murine models of infection. After intranasal infection, the vΔN2 mutant induced lower weight loss and signs of illness, and virus was cleared more rapidly from the infected tissue. In the intradermal model of infection, vΔN2 induced smaller lesions that were resolved more rapidly. In summary, the N2 protein is an intracellular virulence factor that inhibits IRF3 activity in the nucleus.

Vaccinia virus protein N2 is a nuclear IRF3 inhibitor that promotes virulence.

Viruses have evolved sophisticated strategies to exploit host cell function for their benefit. Here we show that under physiologically normal oxygen levels (normoxia) vaccinia virus (VACV) infection leads to a rapid stabilization of hypoxia-inducible factor (HIF)-1α, its translocation into the nucleus and the activation of HIF-responsive genes, such as vascular endothelial growth factor (VEGF), glucose transporter-1, and pyruvate dehydrogenase kinase-1. HIF-1α stabilization is mediated by VACV protein C16 that binds the human oxygen sensing enzyme prolyl-hydroxylase domain containing protein (PHD)2 and thereby inhibits PHD2-dependent hydroxylation of HIF-1α. The binding between C16 and PHD2 is direct and specific, and ectopic expression of C16 alone induces transcription of HIF-1α responsive genes. Conversely, a VACV strain lacking the gene for C16, C16L, is unable to induce HIF-1α stabilization. Interestingly, the N-terminal region of C16 is predicted to have a PHD2-like structural fold but lacks the catalytic active site residues of PHDs. The induction of a hypoxic response by VACV is reminiscent of the biochemical consequences of solid tumor formation, and illustrates a poxvirus strategy for manipulation of cellular gene expression and biochemistry.

A mechanism for induction of a hypoxic response by vaccinia virus.

Virus infection of mammalian cells is sensed by pattern recognition receptors and leads to an innate immune response that restricts virus replication and induces adaptive immunity. In response, viruses have evolved many countermeasures that enable them to replicate and be transmitted to new hosts, despite the host innate immune response. Poxviruses, such as vaccinia virus (VACV), have large DNA genomes and encode many proteins that are dedicated to host immune evasion. Some of these proteins are secreted from the infected cell, where they bind and neutralize complement factors, interferons, cytokines and chemokines. Other VACV proteins function inside cells to inhibit apoptosis or signalling pathways that lead to the production of interferons and pro-inflammatory cytokines and chemokines. In this review, these VACV immunomodulatory proteins are described and the potential to create more immunogenic VACV strains by manipulation of the gene encoding these proteins is discussed.

Vaccinia virus immune evasion: mechanisms, virulence and immunogenicity.

The innate immune system is critical in the response to infection by pathogens and it is activated by pattern recognition receptors (PRRs) binding to pathogen associated molecular patterns (PAMPs). During viral infection, the direct recognition of the viral nucleic acids, such as the genomes of DNA viruses, is very important for activation of innate immunity. Recently, DNA-dependent protein kinase (DNA-PK), a heterotrimeric complex consisting of the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs was identified as a cytoplasmic PRR for DNA that is important for the innate immune response to intracellular DNA and DNA virus infection. Here we show that vaccinia virus (VACV) has evolved to inhibit this function of DNA-PK by expression of a highly conserved protein called C16, which was known to contribute to virulence but by an unknown mechanism. Data presented show that C16 binds directly to the Ku heterodimer and thereby inhibits the innate immune response to DNA in fibroblasts, characterised by the decreased production of cytokines and chemokines. Mechanistically, C16 acts by blocking DNA-PK binding to DNA, which correlates with reduced DNA-PK-dependent DNA sensing. The C-terminal region of C16 is sufficient for binding Ku and this activity is conserved in the variola virus (VARV) orthologue of C16. In contrast, deletion of 5 amino acids in this domain is enough to knockout this function from the attenuated vaccine strain modified vaccinia virus Ankara (MVA). In vivo a VACV mutant lacking C16 induced higher levels of cytokines and chemokines early after infection compared to control viruses, confirming the role of this virulence factor in attenuating the innate immune response. Overall this study describes the inhibition of DNA-PK-dependent DNA sensing by a poxvirus protein, adding to the evidence that DNA-PK is a critical component of innate immunity to DNA viruses.

Brian J. Ferguson

Department of Pathology

University of Cambridge

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

Brian J. Ferguson

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

University of Cambridge

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

Department of Pathology