Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and ...

St. Jude Children's Research Hospital

BAK/BAX-mediated mitochondrial outer-membrane permeabilization (MOMP) drives cell death during development and tissue homeostasis from zebrafish to humans. In most cancers, this pathway is inhibited by BCL-2 family antiapoptotic members, which bind and block the action of proapoptotic BCL proteins. We report the 1.5 A crystal structure of calpain-proteolysed BAK, cBAK, to reveal a zinc binding site that regulates its activity via homodimerization. cBAK contains an occluded BH3 peptide binding pocket that binds a BID BH3 peptide only weakly . Nonetheless, cBAK requires activation by truncated BID to induce cytochrome c release in mitochondria isolated from bak/bax double-knockout mouse embryonic fibroblasts. The BAK-mediated MOMP is inhibited by low micromolar zinc levels. This inhibition is alleviated by mutation of the zinc-coordination site in BAK. Our results link directly the antiapoptotic effects of zinc to BAK.

The X-ray structure of a BAK homodimer reveals an inhibitory zinc binding site.

The Ca(2+)-dependent cysteine proteases, calpains, regulate cell migration, cell death, insulin secretion, synaptic function and muscle homeostasis. Their endogenous inhibitor, calpastatin, consists of four inhibitory repeats, each of which neutralizes an activated calpain with exquisite specificity and potency. Despite the physiological importance of this interaction, the structural basis of calpain inhibition by calpastatin is unknown. Here we report the 3.0 A structure of Ca(2+)-bound m-calpain in complex with the first calpastatin repeat, both from rat, revealing the mechanism of exclusive specificity. The structure highlights the complexity of calpain activation by Ca(2+), illustrating key residues in a peripheral domain that serve to stabilize the protease core on Ca(2+) binding. Fully activated calpain binds ten Ca(2+) atoms, resulting in several conformational changes allowing recognition by calpastatin. Calpain inhibition is mediated by the intimate contact with three critical regions of calpastatin. Two regions target the penta-EF-hand domains of calpain and the third occupies the substrate-binding cleft, projecting a loop around the active site thiol to evade proteolysis.

Concerted multi-pronged attack by calpastatin to occlude the catalytic cleft of heterodimeric calpains.

CLL de c&eacute;lulas b/linfoma-2 (BCL-2) y sus parientes constituyen la familia de BCL-2 de prote&iacute;nas, que originalmente se caracterizaron con respecto a su papel en el control de la apoptosis y la integridad de la membrana mitocondrial externa. Observaciones actuales ampl&iacute;an la funci&oacute;n de familia de BCL-2 para incluir numerosas v&iacute;as celulares. Aqu&iacute; vamos a discutir los mecanismos y funciones de la familia de BCL-2 en el contexto de estas v&iacute;as, destacando la integraci&oacute;n compleja y regulaci&oacute;n de la familia de BCL-2 en las decisiones de destino celular.

La reuni&oacute;n de la familia BCL-2.

B &#x7EC6;&#x80DE;&#x642D;&#x5EFA;/&#x6DCB;&#x5DF4;&#x7624;-2 (BCL-2) &#x548C;&#x5176;&#x4EB2;&#x5C5E;&#x7EC4;&#x6210;&#x86CB;&#x767D;&#x8D28;&#xFF0C;&#x6700;&#x521D;&#x5BF9;&#x5B83;&#x4EEC;&#x5728;&#x63A7;&#x5236;&#x7EBF;&#x7C92;&#x4F53;&#x5916;&#x819C;&#x5B8C;&#x6574;&#x6027;&#x548C;&#x7EC6;&#x80DE;&#x51CB;&#x4EA1;&#x7684;&#x4F5C;&#x7528;&#x7279;&#x70B9;&#x662F; bcl-2 &#x7684;&#x5BB6;&#x65CF;&#x3002;&#x5F53;&#x524D;&#x610F;&#x89C1;&#x5C55;&#x5F00; BCL-2 &#x5BB6;&#x5EAD;&#x529F;&#x80FD;&#xFF0C;&#x5305;&#x62EC;&#x8BB8;&#x591A;&#x7EC6;&#x80DE;&#x7684;&#x9014;&#x5F84;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x5C06;&#x8BA8;&#x8BBA;&#x7684;&#x673A;&#x5236;&#x548C;&#x804C;&#x80FD; bcl-2 &#x5BB6;&#x65CF;&#x5728;&#x4E0A;&#x4E0B;&#x6587;&#x4E2D;&#x7684;&#x8FD9;&#x4E9B;&#x9014;&#x5F84;&#xFF0C;&#x7A81;&#x51FA;&#x590D;&#x6742;&#x7684;&#x96C6;&#x6210;&#x548C; bcl-2 &#x5BB6;&#x65CF;&#x7EC6;&#x80DE;&#x547D;&#x8FD0;&#x51B3;&#x5B9A;&#x4E2D;&#x7684;&#x8C03;&#x8282;&#x3002;

BCL-2 &#x5BB6;&#x5EAD;&#x56E2;&#x805A;&#x3002;

B cell CLL/lymphoma-2 (BCL-2) and its relatives comprise the BCL-2 family of proteins, which were originally characterized with respect to their roles in controlling outer mitochondrial membrane integrity and apoptosis. Current observations expand BCL-2 family function to include numerous cellular pathways. Here we will discuss the mechanisms and functions of the BCL-2 family in the context of these pathways, highlighting the complex integration and regulation of the BCL-2 family in cell fate decisions.

The BCL-2 family reunion.

B cellules CLL/lymphome-2 (BCL-2) et ses parents constituent la famille de BCL-2 des prot&eacute;ines qui ont &eacute;t&eacute; initialement caract&eacute;ris&eacute;s en ce qui concerne leur r&ocirc;le dans le contr&ocirc;le de l&#39;apoptose et l&#39;int&eacute;grit&eacute; de la membrane mitochondriale externe. Les observations actuelles d&eacute;veloppez fonction famille de BCL-2 pour inclure de nombreuses voies cellulaires. Ici nous allons discuter les m&eacute;canismes et les fonctions de la famille BCL-2 dans le contexte de ces voies, en soulignant l&#39;int&eacute;gration complexe et la r&eacute;glementation de la famille BCL-2 dans les d&eacute;cisions de destin cellulaire.

R&eacute;union de la famille BCL-2.

B-Zelle CLL/Lymphom-2 (BCL-2) und seine Verwandten umfassen die Familie BCL-2 Proteine, die urspr&uuml;nglich in Bezug auf ihre Rolle bei der Kontrolle der Integrit&auml;t der mitochondrialen Au&szlig;enmembran und Apoptosis gepr&auml;gt waren. Aktuelle Beobachtungen erweitern Familie BCL-2-Funktion, um zahlreiche zellul&auml;re Wege enthalten. Hier besprechen die Mechanismen und Funktionen der Familie BCL-2 im Zusammenhang mit diesen Bahnen, wir hervorheben, die komplexe Integration und Regulierung der BCL-2 Familie in Zelle Schicksal Entscheidungen.

Die BCL-2 Familie Wiedervereinigung.

B cellula CLL/linfoma-2 (BCL-2) e i suoi parenti comprendono la famiglia BCL-2 di proteine, che sono stati originariamente caratterizzati rispetto al loro ruolo nel controllo della apoptosi e l&#39;integrit&agrave; della membrana mitocondriale esterna. Le osservazioni attuali espandere BCL-2 famiglia funzionano per includere numerose vie cellulari. Qui discuteremo i meccanismi e le funzioni della famiglia BCL-2 nel contesto di questi percorsi, evidenziando l&#39;integrazione complessa e regolamento della famiglia BCL-2 nelle decisioni di destino delle cellule.

La riunione di famiglia BCL-2.

B &#x7D30;&#x80DE; CLL/&#x30EA;&#x30F3;&#x30D1;&#x816B;-2 (BCL 2) &#x3068;&#x305D;&#x306E;&#x89AA;&#x985E; bcl-2 &#x3082;&#x3068;&#x3082;&#x3068;&#x5916;&#x5074;&#x306E;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306E;&#x819C;&#x306E;&#x6574;&#x5408;&#x6027;&#x3068;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x306E;&#x5236;&#x5FA1;&#x306B;&#x304A;&#x3051;&#x308B;&#x305D;&#x306E;&#x5F79;&#x5272;&#x306B;&#x95A2;&#x3057;&#x3066;&#x7279;&#x5FB4;&#x3065;&#x3051;&#x3089;&#x308C;&#x305F;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x69CB;&#x6210;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x73FE;&#x5728;&#x89B3;&#x6E2C; BCL 2 &#x5BB6;&#x65CF;&#x306E;&#x6A5F;&#x80FD;&#x3092;&#x591A;&#x6570;&#x7D30;&#x80DE;&#x30D1;&#x30B9;&#x3092;&#x542B;&#x3080;&#x3088;&#x3046;&#x306B;&#x5C55;&#x958B;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x8907;&#x96D1;&#x306A;&#x7D71;&#x5408;&#x3068;&#x7D30;&#x80DE;&#x904B;&#x547D;&#x6C7A;&#x5B9A;&#x306B;&#x304A;&#x3051;&#x308B; bcl-2 &#x306E;&#x898F;&#x5236;&#x3092;&#x5F37;&#x8ABF;&#x6A5F;&#x69CB;&#x3068;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D4C;&#x8DEF;&#x306E;&#x30B3;&#x30F3;&#x30C6;&#x30AD;&#x30B9;&#x30C8;&#x306B;&#x304A;&#x3051;&#x308B; bcl-2 &#x306E;&#x6A5F;&#x80FD;&#x8AAC;&#x660E;&#x3057;&#x307E;&#x3059;&#x3002;

BCL 2 &#x5BB6;&#x65CF;&#x306E;&#x518D;&#x4F1A;&#x3002;

B &#xC140; CLL/&#xB9BC;&#xD504; &#xC885;-2 (BCL-2)&#xC640; &#xADF8; &#xCE5C;&#xCC99; &#xC6D0;&#xB798; &#xC678;&#xBD80; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC; &#xB9AC;&#xC544; &#xBA64;&#xBE0C;&#xB808;&#xC778; &#xBB34;&#xACB0;&#xC131; &#xBC0F; apoptosis&#xC5D0; &#xADF8;&#xB4E4;&#xC758; &#xC5ED;&#xD560;&#xC5D0; &#xAD00;&#xD558;&#xC5EC; &#xD2B9;&#xC9D5; &#xC774;&#xC5C8;&#xB2E4; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; BCL-2 &#xAC00;&#xC871; &#xAD6C;&#xC131;. &#xD604;&#xC7AC; &#xAD00;&#xCE21; BCL-2 &#xAC00;&#xC871; &#xAE30;&#xB2A5; &#xC218;&#xB9CE;&#xC740; &#xC138;&#xD3EC; &#xACBD;&#xB85C; &#xD3EC;&#xD568; &#xD558;&#xB3C4;&#xB85D; &#xD655;&#xC7A5;. &#xC5EC;&#xAE30; &#xC124;&#xBA85; &#xD569;&#xB2C8;&#xB2E4; &#xBA54;&#xCEE4;&#xB2C8;&#xC998; &#xBC0F; &#xC774;&#xB7EC;&#xD55C; &#xD1B5;&#xB85C;&#xC758; &#xB9E5;&#xB77D;&#xC5D0;&#xC11C; BCL-2 &#xAC00;&#xC871;&#xC758; &#xAE30;&#xB2A5; &#xBCF5;&#xC7A1; &#xD55C; &#xD1B5;&#xD569; &#xBC0F; &#xC138;&#xD3EC; &#xC6B4;&#xBA85; &#xACB0;&#xC815;&#xC758; BCL-2 &#xAC00;&#xC871;&#xC758; &#xADDC;&#xCE59;&#xC744; &#xAC15;&#xC870;.

BCL-2 &#xAC00;&#xC871; &#xC0C1;&#xBD09;&#xC785;&#xB2C8;&#xB2E4;.

Interleukin-4 (IL4) is a secreted immunoregulatory cytokine critically involved in host protection from parasitic helminths 1. Reasoning that helminths may have evolved mechanisms to antagonize IL4 to maximize their dispersal, we explored mammalian IL4 evolution.

Diversifying selection and functional analysis of interleukin-4 suggests antagonism-driven evolution at receptor-binding interfaces.

Bcl-2 &#x5BB6;&#x65CF;&#x86CB;&#x767D;&#x8C03;&#x8282;&#x7EC6;&#x80DE;&#x51CB;&#x4EA1;&#x7EBF;&#x7C92;&#x4F53;&#x5916;&#x819C;&#x901A;&#x900F;&#x6027;&#x589E;&#x9AD8; (MOMP) &#x88AB;&#x79F0;&#x4E3A;&#x5173;&#x952E;&#x4E00;&#x6B65;&#x3002;Bcl-2 &#x5BB6;&#x65CF;&#xFF0C;&#x5C0F;&#x7EC4;&#x7684;&#x6210;&#x5458;&#x79F0;&#x4E3A; BH3 &#x53EA;&#x86CB;&#x767D;&#xFF0C;&#x6FC0;&#x6D3B; pro &#x51CB;&#x4EA1;&#x6548;&#x5E94;&#x5668; &#xFF08;Bax &#x548C; Bak&#xFF09;&#xFF0C;&#x542F;&#x52A8; MOMP&#x3002;&#x4ED6;&#x4EEC;&#x8FD9;&#x6837;&#x505A;&#x662F;&#x901A;&#x8FC7;&#x6D88;&#x9664; pro-survival bcl-2 &#x86CB;&#x767D;&#x548C;/&#x6216;&#x76F4;&#x63A5;&#x6FC0;&#x6D3B; Bax/Bak&#x3002;Bim &#x548C;&#x7533;&#x529E;&#x62A5;&#x544A;&#x8BF4;&#x8981;&#x76F4;&#x63A5;&#x6FC0;&#x6D3B;&#x5668; &#xFF1B;&#x4E0D;&#x8FC7;&#xFF0C;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x663E;&#x793A; BH3 &#x80BD;&#x975E; Bim &#x548C;&#x6295;&#x6807;&#x8868;&#x73B0;&#x51FA;&#x4E0D;&#x540C;&#x7A0B;&#x5EA6;&#x7684;&#x76F4;&#x63A5;&#x6FC0;&#x6D3B;&#x7684; Bax &#x6548;&#x5E94;&#x6216; Bak&#xFF0C;&#x5305;&#x62EC; Bmf &#x548C; Noxa BH3s&#x3002;&#x70C8;&#x6027;&#x7684;&#x76F4;&#x63A5;&#x6D3B;&#x5316;&#x5242;&#xFF0C;&#x5982; Bim &#x548C;&#x6295;&#x6807;&#xFF0C;&#x5982;&#x679C;&#x6CA1;&#x6709;&#x6211;&#x4EEC;&#x66F0;&#x65B0;&#x578B;&#x76F4;&#x63A5;&#x6D3B;&#x5316;&#x5242; BH3 &#x914D;&#x4F53;&#x8BF1;&#x5BFC;&#x6548;&#x5E94;&#x4ECB;&#x5BFC;&#x7684;&#x7EC6;&#x80DE;&#x8272;&#x7D20; c &#x7684;&#x91CA;&#x653E;&#x548C;&#x8102;&#x8D28;&#x4F53;&#x900F;&#xFF0C;&#x5373;&#x4F7F;&#x8FD9;&#x4E24;&#x4E2A; Bcl-xl &#x548C; Mcl-1 &#x578B;&#x6297;&#x51CB;&#x4EA1;&#x86CB;&#x767D;&#x88AB;&#x6291;&#x5236;&#x7684;&#x80FD;&#x529B;&#x3002;&#x8FD9;&#x4E9B;&#x8F83;&#x5F31;&#x7684;&#x76F4;&#x63A5;&#x6D3B;&#x5316;&#x5242; BH3 &#x80BD;&#x80FD;&#x591F;&#x5BFC;&#x81F4; MOMP &#x4E0E;&#x76F8;&#x5E94;&#x7684;&#x5168;&#x957F;&#x86CB;&#x767D;&#x8BF1;&#x5BFC;&#x51CB;&#x4EA1;&#x4E2D;&#x7F3A;&#x4E4F; Bim &#x548C;&#x6295;&#x6807;&#x7684;&#x5173;&#x8054;&#x8D77;&#x6765;&#x3002;&#x6211;&#x4EEC;&#x5EFA;&#x8BAE;&#x5728;&#x67D0;&#x4E9B;&#x60C5;&#x51B5;&#x4E0B;&#xFF0C;&#x76F4;&#x63A5;&#x6FC0;&#x6D3B;&#x4EC5; BH3 &#x86CB;&#x767D; Bim &#x548C;&#x6295;&#x6807;&#x4EE5;&#x5916;&#x7684;&#x5176;&#x4ED6;&#x53EF;&#x80FD;&#x5927;&#x5927;&#x6709;&#x52A9;&#x4E8E; MOMP &#x548C;&#x7EC6;&#x80DE;&#x51CB;&#x4EA1;&#x3002;

BH3 &#x57DF;&#x5916; Bim &#x548C;&#x62A5;&#x4EF7;&#x53EF;&#x4EE5;&#x76F4;&#x63A5;&#x6FC0;&#x6D3B; Bax/Bak&#x3002;

Bcl-2 family proteins regulate a critical step in apoptosis referred to as mitochondrial outer membrane permeabilization (MOMP). Members of a subgroup of the Bcl-2 family, known as the BH3-only proteins, activate pro-apoptotic effectors (Bax and Bak) to initiate MOMP. They do so by neutralizing pro-survival Bcl-2 proteins and/or directly activating Bax/Bak. Bim and Bid are reported to be direct activators; however, here we show that BH3 peptides other than Bim and Bid exhibited various degrees of direct activation of the effector Bax or Bak, including Bmf and Noxa BH3s. In the absence of potent direct activators, such as Bim and Bid, we unmasked novel direct activator BH3 ligands capable of inducing effector-mediated cytochrome c release and liposome permeabilization, even when both Bcl-xL- and Mcl-1-type anti-apoptotic proteins were inhibited. The ability of these weaker direct activator BH3 peptides to cause MOMP correlated with that of the corresponding full-length proteins to induce apoptosis in the absence of Bim and Bid. We propose that, in certain contexts, direct activation by BH3-only proteins other than Bim and Bid may significantly contribute to MOMP and apoptosis.

BH3 domains other than Bim and Bid can directly activate Bax/Bak.

Prot&eacute;ines de la famille BCL-2 r&eacute;glementent une &eacute;tape cruciale dans l&#39;apoptose d&eacute;nomm&eacute; perm&eacute;abilisation de la membrane mitochondriale externe (MOMP). Membres d&#39;un sous-groupe de la famille Bcl-2, connu comme les prot&eacute;ines BH3 seulement, activer les effecteurs de pro-apoptotic (Bax et Bak) pour initier MOMP. Ils le font en neutralisant apoptotiques Bcl-2 prot&eacute;ines et/ou en activant directement Bax/Bak. BIM et la soumission sont signal&eacute;s comme &eacute;tant des activateurs directs ; Cependant, ici, nous montrons que BH3 peptides autre que Bim et soumission pr&eacute;sentaient divers degr&eacute;s d&#39;activation directe de l&#39;effecteur Bax ou Bak, y compris le Bmf et Noxa BH3s. En l&#39;absence de puissants activateurs directs, tels que le Bim et de soumission, nous avons d&eacute;masqu&eacute; des ligands de BH3 roman activateur direct capables d&#39;induire la perm&eacute;abilisation induite par l&#39;effecteur du cytochrome c sortie et liposome, m&ecirc;me lorsque les deux prot&eacute;ines anti-apoptotiques de type Bcl-xL et Mcl-1 ont &eacute;t&eacute; inhib&eacute;es. La capacit&eacute; de ces peptides de BH3 activateur direct plus faibles de provoquer MOMP corr&eacute;l&eacute;e &agrave; celle des prot&eacute;ines correspondantes pleine longueur pour induire l&#39;apoptose en l&#39;absence de Bim et la soumission. Nous proposons que, dans certains contextes, activation directe de prot&eacute;ines BH3 seule autre que Bim et la soumission peut-&ecirc;tre contribuer de mani&egrave;re significative &agrave; MOMP et l&#39;apoptose.

BH3 domaines autres que le Bim et la soumission peuvent activer directement Bax/Bak.

BCL-2 Familie Proteine regulieren einen entscheidender Schritt im Apoptosis als mitochondrialen Au&szlig;enmembran Permeabilization (MOMP) bezeichnet. Mitglieder einer Untergruppe der Bcl-2 Familie, bekannt als der BH3-only-Proteine, aktivieren Sie die Pro-apoptotischen Effektoren (Bax und Bak) MOMP zu initiieren. Sie tun dies durch Neutralisierung pro-survival Bcl-2 Proteine und/oder direkt aktivieren Bax/Bak. BIM und Gebot werden berichtet, um direkte Aktivatoren werden; Hier zeigen wir jedoch, dass BH3 Peptide als Bim und Gebot unterschiedlichem direkte Aktivierung der Effektor Bax oder Bak, einschlie&szlig;lich Bmf und Noxa BH3s ausgestellt. In Ermangelung von potenten direkte Aktivatoren, wie Bim und Gebot entlarvt wir neuartige direkte Aktivator BH3 Liganden f&auml;hig induzierende Effektor-vermittelte Cytochrom C Release und Liposomen Permeabilization, auch wenn beide Bcl-xL,- und Mcl-1-Typ Anti-apoptotischen Proteine behindert wurden. Die F&auml;higkeit dieser schw&auml;cheren direkter Aktivator BH3 Peptide, MOMP korreliert mit der entsprechenden Proteine in Apoptosis in Abwesenheit von Bim und Gebot zu veranlassen. Wir schlagen vor, in bestimmten Zusammenh&auml;ngen direkte Aktivierung von BH3-only-Proteine als Bim und Gebot zu MOMP und Apoptosis signifikant beitragen kann.

BH3-Dom&auml;nen als Bim und Gebot k&ouml;nnen Bax/Bak direkt aktiviert werden.

Bcl-2 famiglie proteine regolano un passaggio critico nella apoptosi denominate permeabilization membrana mitocondriale esterna (MOMP). Membri di un sottogruppo della famiglia Bcl-2, conosciuto come le proteine BH3-only, attivare effettori pro-apoptotici (Bax e Bak) di avviare MOMP. Lo fanno cos&igrave; da neutralizzare pro-survival proteine Bcl-2 e/o attivare direttamente Bax/Bak. BIM e Bid sono segnalati per essere attivatori diretti; Tuttavia, qui vi mostriamo che BH3 peptidi diversi Bim e offerta esposto vari gradi di attivazione diretta del effector Bax o Bak, compresi Bmf e Noxa BH3s. In assenza di potenti attivatori diretti, quali Bim e offerta, abbiamo smascherato ligandi di BH3 romanzo diretto attivatore capace di indurre c permeabilization rilascio e liposomi citocromo effettrici-mediata, anche quando entrambe le proteine anti-apoptotic Bcl-xL e Mcl-1-tipo sono state inibite. La capacit&agrave; di questi peptidi di BH3 attivatore diretto pi&ugrave; debole per causare MOMP correlato con quello delle corrispondenti proteine full-length di indurre apoptosi in assenza di Bim e Bid. Proponiamo che, in determinati contesti, attivazione diretta di proteine BH3-only diverso da Bim e offerta pu&ograve; significativamente contribuire alla MOMP e apoptosi.

BH3 domini diversi da Bim e Bid possono attivare direttamente Bax/Bak.

Bcl 2 &#x30D5;&#x30A1;&#x30DF;&#x30EA;&#x30FC;&#x306E;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306F;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x5916;&#x819C;&#x900F;&#x904E; (MOMP) &#x3068;&#x3057;&#x3066;&#x547C;&#x3070;&#x308C;&#x308B;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x306E;&#x91CD;&#x8981;&#x306A;&#x624B;&#x9806;&#x3092;&#x898F;&#x5236;&#x3057;&#x307E;&#x3059;&#x3002;Bcl 2 &#x5BB6;&#x65CF;&#x306E;&#x30B5;&#x30D6;&#x30B0;&#x30EB;&#x30FC;&#x30D7;&#x306E;&#x30E1;&#x30F3;&#x30D0;&#x30FC; BH3 &#x3060;&#x3051;&#x86CB;&#x767D;&#x8CEA;&#x3068;&#x3057;&#x3066;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x3001;&#x30D7;&#x30ED; &#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9; &#x30A8;&#x30D5;&#x30A7;&#x30AF;&#x30BF;&#x30FC; (Bax &#x3068; Bak) MOMP &#x3092;&#x958B;&#x59CB;&#x3059;&#x308B;&#x3092;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306B;&#x3057;&#x307E;&#x3059;&#x3002;&#x5F7C;&#x3089;&#x306F;&#x30D7;&#x30ED;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9; Bcl 2 &#x86CB;&#x767D;&#x8CEA;&#x3092;&#x4E2D;&#x548C;&#x304A;&#x3088;&#x3073;/&#x307E;&#x305F;&#x306F;&#x76F4;&#x63A5;&#x30D0;&#x30C3;&#x30AF;&#x30B9;/Bak &#x3092;&#x6D3B;&#x6027;&#x5316;&#x306B;&#x3088;&#x3063;&#x3066;&#x305D;&#x3044;&#x307E;&#x3059;&#x3002;Bim &#x3068;&#x5165;&#x672D;&#x76F4;&#x63A5;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D9;&#x30FC;&#x30BF;&#x30FC;&#x306B;&#x4F1D;&#x3048;&#x3089;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x305F;&#x3060;&#x3057;&#x3001;&#x3053;&#x3053;&#x3067;&#x6211;&#x3005; &#x306F; BH3 &#x30DA;&#x30D7;&#x30C1;&#x30C3;&#x30C9; Bim &#x3068;&#x5165;&#x672D;&#x4EE5;&#x5916;&#x306E;&#x30A8;&#x30D5;&#x30A7;&#x30AF;&#x30BF;&#x30FC; Bax &#x306E;&#x76F4;&#x63A5;&#x6D3B;&#x6027;&#x5316;&#x307E;&#x305F;&#x306F; Bak Bmf Noxa BH3s &#x306A;&#x3069;&#x3001;&#x69D8;&#x3005; &#x306A;&#x7A0B;&#x5EA6;&#x3092;&#x5C55;&#x793A;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;Bim &#x3068;&#x5165;&#x672D;&#x306A;&#x3069;&#x306E;&#x5F37;&#x529B;&#x306A;&#x76F4;&#x63A5;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D9;&#x30FC;&#x30BF;&#x30FC;&#x306E;&#x4E0D;&#x5728;&#x3067;&#x5C0F;&#x8AAC;&#x76F4;&#x63A5;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D9;&#x30FC;&#x30BF;&#x30FC; BH3 &#x914D;&#x4F4D;&#x5B50;&#x3082;&#x4E21;&#x65B9; Bcl-xl &#x3068; Mcl 1 &#x578B;&#x6297;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x86CB;&#x767D;&#x304C;&#x6291;&#x5236;&#x3055;&#x308C;&#x305F;&#x3068;&#x304D;&#x30A8;&#x30D5;&#x30A7;&#x30AF;&#x30BF;&#x30FC;&#x304C;&#x4ECB;&#x5728;&#x3059;&#x308B;&#x30B7;&#x30C8;&#x30AF;&#x30ED;&#x30E0; c &#x30EA;&#x30EA;&#x30FC;&#x30B9;&#x3068;&#x30EA;&#x30DD;&#x30BD;&#x30FC;&#x30E0;&#x306E;&#x900F;&#x904E;&#x3092;&#x8A98;&#x767A;&#x306E;&#x30DE;&#x30B9;&#x30AF;&#x3092;&#x89E3;&#x9664;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;Bim &#x3068;&#x5165;&#x672D;&#x306E;&#x4E0D;&#x5728;&#x3067;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x3092;&#x8A98;&#x767A;&#x3059;&#x308B;&#x5BFE;&#x5FDC;&#x3059;&#x308B;&#x30D5;&#x30EB;&#x30EC;&#x30F3;&#x30B0;&#x30B9;&#x86CB;&#x767D;&#x8CEA;&#x76F8;&#x95A2; MOMP &#x3092;&#x5F15;&#x304D;&#x8D77;&#x3053;&#x3059;&#x3053;&#x308C;&#x3089;&#x5F31;&#x3044;&#x76F4;&#x63A5;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D9;&#x30FC;&#x30BF;&#x30FC; BH3 &#x30DA;&#x30D7;&#x30C1;&#x30C3;&#x30C9;&#x306E;&#x80FD;&#x529B;&#x3002;&#x7279;&#x5B9A;&#x306E;&#x30B3;&#x30F3;&#x30C6;&#x30AD;&#x30B9;&#x30C8;&#x3067;&#x306F;&#x3001;Bim &#x3068;&#x5165;&#x672D;&#x4EE5;&#x5916; BH3 &#x3060;&#x3051;&#x86CB;&#x767D;&#x8CEA;&#x306B;&#x3088;&#x308B;&#x76F4;&#x63A5;&#x6D3B;&#x6027;&#x5316;&#x306F;&#x5927;&#x5E45;&#x306B; MOMP &#x3068;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x306B;&#x8CA2;&#x732E;&#x3059;&#x308B;&#x304B;&#x3082;&#x3057;&#x308C;&#x306A;&#x3044;&#x3053;&#x3068;&#x3092;&#x63D0;&#x6848;&#x3057;&#x307E;&#x3059;&#x3002;

BH3 &#x30C9;&#x30E1;&#x30A4;&#x30F3; Bim &#x3068;&#x5165;&#x672D;&#x4EE5;&#x5916;&#x306F;&#x76F4;&#x63A5;&#x30D0;&#x30C3;&#x30AF;&#x30B9;/Bak &#x3092;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306B;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;

Bcl-2 &#xAC00;&#xC871; &#xB2E8;&#xBC31;&#xC9C8; &#xC870;&#xC808; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544; &#xBC14;&#xAE65; &#xB9C9; permeabilization (MOMP) &#xB77C;&#xACE0; &#xD558;&#xB294; apoptosis&#xC5D0; &#xC911;&#xC694; &#xD55C; &#xB2E8;&#xACC4;. Bcl-2 &#xAC00;&#xC871;&#xC758; &#xD558;&#xC704; &#xADF8;&#xB8F9;&#xC758; &#xBA64;&#xBC84; &#xD504;&#xB85C; apoptotic &#xC774;&#xD399;&#xD130; (Bax&#xC640; &#xBC15;) MOMP &#xC2DC;&#xC791; &#xD65C;&#xC131;&#xD654; BH3 &#xC804;&#xC6A9; &#xB2E8;&#xBC31;&#xC9C8;&#xC73C;&#xB85C; &#xC54C;&#xB824;&#xC9C4;. &#xADF8;&#xB4E4;&#xC740; pro-survival bcl-2 &#xB2E8;&#xBC31;&#xC9C8;&#xC744; &#xC911;&#xD654; &#xB610;&#xB294; &#xC9C1;&#xC811; Bax/&#xC774;&#xBA85;&#xBC15;&#xC744; &#xD65C;&#xC131;&#xD654; &#xD558; &#xC5EC; &#xADF8;&#xB807;&#xAC8C;. Bim &#xBC0F; &#xC785;&#xCC30;&#xC740; &#xBCF4;&#xACE0; &#xC9C1;&#xC811; &#xD65C;&#xC131; &#xC81C;; &#xADF8;&#xB7EC;&#xB098;, &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC;&#xAC00; Bim &#xBC0F; &#xC785;&#xCC30; BH3 &#xD3A9; &#xD2F0; &#xB4DC; &#xC774;&#xD399;&#xD130; Bax&#xC758; &#xC9C1;&#xC811; &#xD65C;&#xC131;&#xD654; &#xB610;&#xB294; &#xBC15; Noxa BH3s Bmf &#xB4F1;&#xC758; &#xB2E4;&#xC591; &#xD55C;&#xB3C4; &#xC804;&#xC2DC; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. Bim &#xBC0F; &#xC785;&#xCC30;, &#xAC19;&#xC740; &#xAC15;&#xB825;&#xD55C; &#xC9C1;&#xC811; &#xD65C;&#xC131; &#xC81C; &#xC5C6;&#xC774; &#xC6B0;&#xB9AC; &#xC18C;&#xC124;&#xC744; &#xC9C1;&#xC811; &#xD65C;&#xC131; BH3 ligands &#xB450; Bcl xL-&#xBC0F; Mcl-1 &#xD615; &#xC548;&#xD2F0;-apoptotic &#xB2E8;&#xBC31;&#xC9C8; &#xC800;&#xD574; &#xB41C; &#xACBD;&#xC6B0;&#xC5D0; &#xC774;&#xD399;&#xD130; &#xC911;&#xC7AC; &#xC2DC; &#xD1A0; &#xD06C;&#xB86C; c&#xC744; &#xB9B4;&#xB9AC;&#xC2A4; liposome permeabilization, &#xC720;&#xB3C4; &#xB9C8;&#xC2A4;&#xD06C;. Bim &#xBC0F; &#xC785;&#xCC30;&#xC774; &#xC5C6;&#xC744; &#xACBD;&#xC6B0;&#xC5D0; apoptosis&#xB97C; &#xC720;&#xB3C4; &#xD558;&#xAE30; &#xC704;&#xD574; &#xD574;&#xB2F9; &#xC804;&#xCCB4; &#xAE38;&#xC774; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xC0C1;&#xD638; &#xC5F0;&#xAD00; MOMP &#xBC1C;&#xC0DD;&#xD560; &#xC774;&#xB7EC;&#xD55C; &#xC57D;&#xD55C; &#xD65C;&#xC131;&#xAE30;&#xB97C; &#xC9C1;&#xC811; BH3 &#xD3A9; &#xD2F0; &#xB4DC;&#xC758; &#xB2A5;&#xB825;. &#xC6B0;&#xB9AC;&#xB294;, &#xD2B9;&#xC815; &#xC0C1;&#xD669;&#xC5D0;&#xC11C; Bim &#xBC0F; &#xC785;&#xCC30; BH3 &#xC804;&#xC6A9; &#xB2E8;&#xBC31;&#xC9C8;&#xC5D0; &#xC758;&#xD574; &#xC9C1;&#xC811; &#xD65C;&#xC131;&#xD654; &#xD06C;&#xAC8C; &#xAE30;&#xC5EC;&#xD560; &#xC218; MOMP &#xACE0; apoptosis&#xB97C; &#xC81C;&#xC548; &#xD569;&#xB2C8;&#xB2E4;.

Bim &#xBC0F; &#xC785;&#xCC30;&#xAC00; &#xC544;&#xB2CC; BH3 &#xB3C4;&#xBA54;&#xC778; &#xC9C1;&#xC811; Bax/&#xC774;&#xBA85;&#xBC15;&#xC744; &#xD65C;&#xC131;&#xD654;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

Prote&iacute;nas familias BCL-2 regulan un paso cr&iacute;tico en la apoptosis contemplados como la permeabilizaci&oacute;n de la membrana mitocondrial externa (MOMP). Los miembros de un subgrupo de la familia de Bcl-2, conocido como las prote&iacute;nas s&oacute;lo BH3, activar efectores pro-apopt&oacute;tica Bax y Bak para iniciar MOMP. Lo hacen mediante neutralizaci&oacute;n Pro-supervivencia prote&iacute;nas Bcl-2 o activar directamente Bax/Bak. BIM y Bid se divulgan para ser activadores directos; sin embargo, aqu&iacute; mostramos que BH3 p&eacute;ptidos diferentes Bim y oferta exhibieron diversos grados de activaci&oacute;n directa de la unidad de efectos Bax o Bak, Bmf como Noxa BH3s. En ausencia de potentes activadores directos, como Bim y oferta, hemos desenmascarado ligandos de BH3 novela activador directo capaces de inducir la permeabilizaci&oacute;n efectora mediada por el citocromo c liberaci&oacute;n y liposomas, aun cuando ambas prote&iacute;nas de anti-apopt&oacute;tica Bcl-xL y Mcl-1-tipo fueron inhibidos. La capacidad de estos p&eacute;ptidos de BH3 activador directo m&aacute;s d&eacute;biles para causar MOMP correlacionada con la de las correspondientes prote&iacute;nas de larga duraci&oacute;n inducir apoptosis en la ausencia de Bim y oferta. Proponemos que, en determinados contextos, activaci&oacute;n directa por las prote&iacute;nas s&oacute;lo BH3 distintos Bim y oferta puede contribuir significativamente a MOMP y apoptosis.

Dominios BH3 distintos Bim y oferta pueden activar directamente Bax/Bak.

During apoptosis, the BCL-2 protein family controls mitochondrial outer membrane permeabilization (MOMP), but the dynamics of this regulation remain controversial. We employed chimeric proteins composed of exogenous BH3 domains inserted into a tBID backbone that can activate the proapoptotic effectors BAX and BAK to permeabilize membranes without being universally sequestered by all antiapoptotic BCL-2 proteins. We thus identified two "modes" whereby prosurvival BCL-2 proteins can block MOMP, by sequestering direct-activator BH3-only proteins ("MODE 1") or by binding active BAX and BAK ("MODE 2"). Notably, we found that MODE 1 sequestration is less efficient and more easily derepressed to promote MOMP than MODE 2. Further, MODE 2 sequestration prevents mitochondrial fusion. We provide a unified model of BCL-2 family function that helps to explain otherwise paradoxical observations relating to MOMP, apoptosis, and mitochondrial dynamics.

A unified model of mammalian BCL-2 protein family interactions at the mitochondria.

The BCL-2-family protein BAK is responsible for mitochondrial outer-membrane permeabilization (MOMP), which leads to apoptosis. The BCL-2 homology 3 (BH3)-only protein BID activates BAK to perform this function. We report the NMR solution structure of the human BID BH3-BAK complex, which identified the activation site at the canonical BH3-binding groove of BAK. Mutating the BAK BH1 in the groove prevented activation and MOMP but not the binding of BID. BAK BH3 mutations allowed BID binding and activation but blunted function by blocking BAK oligomerization. BAK activation follows a 'hit-and-run' mechanism whereby BID dissociates from the trigger site, which allows BAK oligomerization at an overlapping interface. In contrast, the BH3-only proteins NOXA and BAD are predicted to clash with the trigger site and are not activators of BAK. These findings provide insights into the early stages of BAK activation.

BID-induced structural changes in BAK promote apoptosis.

Active metabolism regulates oocyte cell death via calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated phosphorylation of caspase-2, but the link between metabolic activity and CaMKII is poorly understood. Here we identify coenzyme A (CoA) as the key metabolic signal that inhibits Xenopus laevis oocyte apoptosis by directly activating CaMKII. We found that CoA directly binds to the CaMKII regulatory domain in the absence of Ca(2+) to activate CaMKII in a calmodulin-dependent manner. Furthermore, we show that CoA inhibits apoptosis not only in X. laevis oocytes but also in Murine oocytes. These findings uncover a direct mechanism of CaMKII regulation by metabolism and further highlight the importance of metabolism in preserving oocyte viability.

Metabolic activation of CaMKII by coenzyme A.

During apoptotic cell death, cellular stress signals converge at the mitochondria to induce mitochondrial outer-membrane permeabilization (MOMP) through B cell lymphoma-2 (BCL-2) family proteins and their effectors. BCL-2 proteins function through protein-protein interactions, the mechanisms and structural aspects of which are only now being uncovered. Recently, the elucidation of the dynamic features underlying their function has highlighted their structural plasticity and the consequent complex thermodynamic landscape governing their protein-protein interactions. These studies show that canonical interactions involve a conserved, hydrophobic groove, whereas non-canonical interactions function allosterically outside the groove. We review the latest structural advances in understanding the interactions and functions of mammalian BCL-2 family members, and discuss new opportunities to modulate these proteins in health and disease. 

Many players in BCL-2 family affairs.

B-cell lymphoma 2 (BCL-2) family proteins mediate mitochondrial apoptosis by regulating mitochondrial outer membrane permeabilization (MOMP), which leads to the activation of the downstream caspase cascade to execute apoptosis. The pro-apoptotic and anti-apoptotic BCL-2 proteins function through protein-protein interactions in soluble and membrane-associated states. How soluble BCL-2 proteins interact is well understood. Anti-apoptotic proteins, such as BCL-2 and BCL-xL, and the pro-apoptotic effectors of MOMP, including BAK and BAX, interact with pro-apoptotic BCL-2 homology 3 (BH3)-only proteins similarly. Whereas anti-apoptotic BCL-2 proteins tightly bind all the BH3-only proteins to block apoptosis initiation, the effector BCL-2 proteins are potently triggered by specific BH3-only proteins to undergo conformational changes, membrane association and insertion, oligomerization, and pore formation. The anti-apoptotic BCL-2 proteins also inhibit the activated effectors. p53 is a direct BAX activator inhibited by BCL-xL, defining a prototype non-canonical modulator of BCL-2 proteins-mediated MOMP. How BCL-2 proteins cooperate in the presence of membranes remains poorly understood, impeding our understanding of MOMP and apoptosis. Here, we highlight the latest structural views of MOMP by BCL-2 proteins.

Discoveries and controversies in BCL-2 protein-mediated apoptosis.

Necroptosis is a cell death pathway regulated by the receptor interacting protein kinase 3 (RIPK3) and the mixed lineage kinase domain-like (MLKL) pseudokinase. How MLKL executes plasma membrane rupture upon phosphorylation by RIPK3 remains controversial. Here, we characterize the hierarchical transduction of structural changes in MLKL that culminate in necroptosis. The MLKL brace, proximal to the N-terminal helix bundle (NB), is involved in oligomerization to facilitate plasma membrane targeting through the low-affinity binding of NB to phosphorylated inositol polar head groups of phosphatidylinositol phosphate (PIP) phospholipids. At the membrane, the NB undergoes a "rolling over" mechanism to expose additional higher-affinity PIP-binding sites responsible for robust association to the membrane and displacement of the brace from the NB. PI(4,5)P2 is the preferred PIP-binding partner. We investigate the specific association of MLKL with PIPs and subsequent structural changes during necroptosis. 

Sequential Engagement of Distinct MLKL Phosphatidylinositol-Binding Sites Executes Necroptosis.

The mitochondrial pathway of apoptosis is initiated by mitochondrial outer membrane permeabilization (MOMP). The BCL-2 family effectors BAX and BAK are thought to be absolutely required for this process. Here, we report that BCL-2 ovarian killer (BOK) is a bona fide yet unconventional effector of MOMP that can trigger apoptosis in the absence of both BAX and BAK. However, unlike the canonical effectors, BOK appears to be constitutively active and unresponsive to antagonistic effects of the antiapoptotic BCL-2 proteins. Rather, BOK is controlled at the level of protein stability by components of the endoplasmic reticulum (ER)-associated degradation pathway. BOK is ubiquitylated by the AMFR/gp78 E3 ubiquitin ligase complex and targeted for proteasomal degradation in a VCP/p97-dependent manner, which allows survival of the cell. When proteasome function, VCP, or gp78 activity is compromised, BOK is stabilized to induce MOMP and apoptosis independently of other BCL-2 proteins.

BOK Is a Non-canonical BCL-2 Family Effector of Apoptosis Regulated by ER-Associated Degradation.

The effector B cell lymphoma-2 (BCL-2) protein BCL-2 ovarian killer (BOK) induces mitochondrial outer membrane permeabilization (MOMP) to initiate apoptosis upon inhibition of the proteasome. How BOK mediates MOMP is mechanistically unknown. The NMR structure of the BCL-2 core of human BOK reveals a conserved architecture with an atypical hydrophobic groove that undergoes conformational exchange. Remarkably, the BCL-2 core of BOK spontaneously associates with purified mitochondria to release cytochrome c in MOMP assays. Alanine substitution of a unique glycine in helix α1 stabilizes BOK, as shown by thermal shift and urea denaturation analyses, and significantly inhibits MOMP, liposome permeabilization, and cell death. Activated BID does not activate WT BOK or the stabilized alanine mutant to promote cell death. We propose that BOK-mediated membrane permeabilization is governed in part by its unique metastability of the hydrophobic groove and helix α1 and not through activation by BH3 ligands.

Intrinsic Instability of BOK Enables Membrane Permeabilization in Apoptosis.

Necroptosis is an important form of lytic cell death triggered by injury and infection, but whether mixed lineage kinase domain-like (MLKL) is sufficient to execute this pathway is unknown. In a genetic selection for human cell mutants defective for MLKL-dependent necroptosis, we identified mutations in IPMK and ITPK1, which encode inositol phosphate (IP) kinases that regulate the IP code of soluble molecules. We show that IP kinases are essential for necroptosis triggered by death receptor activation, herpesvirus infection, or a pro-necrotic MLKL mutant. In IP kinase mutant cells, MLKL failed to oligomerize and localize to membranes despite proper receptor-interacting protein kinase-3 (RIPK3)-dependent phosphorylation. We demonstrate that necroptosis requires IP-specific kinase activity and that a highly phosphorylated product, but not a lowly phosphorylated precursor, potently displaces the MLKL auto-inhibitory brace region. These observations reveal control of MLKL-mediated necroptosis by a metabolite and identify a key molecular mechanism underlying regulated cell death.

Tudor Moldoveanu

Department of Structural Biology,
Department of Chemical Biology and Therapeutics

St. Jude Children's Research Hospital

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Tudor Moldoveanu

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

St. Jude Children's Research Hospital

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Department of Structural Biology,
Department of Chemical Biology and Therapeutics