recombinant-protein-expression-for-structural-biology-hek-293f

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

University of Leicester

Telomeres are protein-DNA complexes that cap chromosome ends and protect them from being recognized and processed as DNA breaks. Loss of capping function results in genetic instability and loss of cellular viability. The emerging view is that maintenance of an appropriate telomere structure is essential for function. Structural information on telomeric proteins that bind to double and single-stranded telomeric DNA shows that, despite a lack of extensive amino-acid sequence conservation, telomeric DNA recognition occurs via conserved DNA-binding domains. Furthermore, telomeric proteins have multidomain structures and hence are conformationally flexible. A possibility is that telomeric proteins take up different conformations when bound to different partners, providing a simple mechanism for modulating telomere architecture.

Telomere architecture.

Human telomeres consist of tandem arrays of TTAGGG sequence repeats that are specifically bound by two proteins, TRF1 and TRF2. They bind to DNA as preformed homodimers and have the same architecture in which the DNA-binding domains (Dbds) form independent structural units. Despite these similarities, TRF1 and TRF2 have different functions at telomeres. The X-ray crystal structures of both TRF1- and TRF2-Dbds in complex with telomeric DNA (2.0 and 1.8 angstroms resolution, respectively) show that they recognize the same TAGGGTT binding site by means of homeodomains, as does the yeast telomeric protein Rap1p. Two of the three G-C base pairs that characterize telomeric repeats are recognized specifically and an unusually large number of water molecules mediate protein-DNA interactions. The binding of the TRF2-Dbd to the DNA double helix shows no distortions that would account for the promotion of t-loops in which TRF2 has been implicated.

How the human telomeric proteins TRF1 and TRF2 recognize telomeric DNA: a view from high-resolution crystal structures.

Chromatin structure plays a fundamental role in the regulation of nuclear processes such as DNA transcription, replication, recombination, and repair. Despite considerable efforts during three decades, the structure of the 30-nm chromatin fiber remains controversial. To define fiber dimensions accurately, we have produced very long and regularly folded 30-nm fibers from in vitro reconstituted nucleosome arrays containing the linker histone and with increasing nucleosome repeat lengths (10 to 70 bp of linker DNA). EM measurements show that the dimensions of these fully folded fibers do not increase linearly with increasing linker length, a finding that is inconsistent with two-start helix models. Instead, we find that there are two distinct classes of fiber structure, both with unexpectedly high nucleosome density: arrays with 10 to 40 bp of linker DNA all produce fibers with a diameter of 33 nm and 11 nucleosomes per 11 nm, whereas arrays with 50 to 70 bp of linker DNA all produce 44-nm-wide fibers with 15 nucleosomes per 11 nm. Using the physical constraints imposed by these measurements, we have built a model in which tight nucleosome packing is achieved through the interdigitation of nucleosomes from adjacent helical gyres. Importantly, the model closely matches raw image projections of folded chromatin arrays recorded in the solution state by using electron cryo-microscopy.

EM measurements define the dimensions of the "30-nm" chromatin fiber: evidence for a compact, interdigitated structure.

The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) has important roles in adipogenesis and immune response as well as roles in both lipid and carbohydrate metabolism. Although synthetic agonists for PPARgamma are widely used as insulin sensitizers, the identity of the natural ligand(s) for PPARgamma is still not clear. Suggested natural ligands include 15-deoxy-delta12,14-prostaglandin J2 and oxidized fatty acids such as 9-HODE and 13-HODE. Crystal structures of PPARgamma have revealed the mode of recognition for synthetic compounds. Here we report structures of PPARgamma bound to oxidized fatty acids that are likely to be natural ligands for this receptor. These structures reveal that the receptor can (i) simultaneously bind two fatty acids and (ii) couple covalently with conjugated oxo fatty acids. Thermal stability and gene expression analyses suggest that such covalent ligands are particularly effective activators of PPARgamma and thus may serve as potent and biologically relevant ligands.

Structural basis for the activation of PPARgamma by oxidized fatty acids.

Nuclear receptors are arguably the best understood transcriptional regulators. We know a great deal about the mechanisms through which they activate transcription in response to ligand binding and about the mechanisms through which they repress transcription in the absence of ligand. However, endocrine regulation often requires that ligand-bound receptors repress transcription of a subset of genes. An understanding of the mechanism for ligand-induced repression and how this differs from activation has proven elusive. A number of recent studies have directly or indirectly addressed this problem. Yet it seems the more evidence that accumulates, the more complex the mystery becomes.

Negative regulation by nuclear receptors: a plethora of mechanisms.

Eukaryotic transcriptional repressors function by recruiting large coregulatory complexes that target histone deacetylase enzymes to gene promoters and enhancers. Transcriptional repression complexes, assembled by the corepressor NCoR and its homolog SMRT, are crucial in many processes, including development and metabolic physiology. The core repression complex involves the recruitment of three proteins, HDAC3, GPS2 and TBL1, to a highly conserved repression domain within SMRT and NCoR. We have used structural and functional approaches to gain insight into the architecture and biological role of this complex. We report the crystal structure of the tetrameric oligomerization domain of TBL1, which interacts with both SMRT and GPS2, and the NMR structure of the interface complex between GPS2 and SMRT. These structures, together with computational docking, mutagenesis and functional assays, reveal the assembly mechanism and stoichiometry of the corepressor complex.

Structural basis for the assembly of the SMRT/NCoR core transcriptional repression machinery.

We previously identified the E3 ubiquitin ligase IDOL as a sterol-dependent regulator of the LDL receptor (LDLR). The molecular pathway underlying IDOL action, however, remains to be determined. Here we report the identification and biochemical and structural characterization of an E2-E3 ubiquitin ligase complex for LDLR degradation. We identified the UBE2D family (UBE2D1-4) as E2 partners for IDOL that support both autoubiquitination and IDOL-dependent ubiquitination of the LDLR in a cell-free system. NMR chemical shift mapping and a 2.1 Å crystal structure of the IDOL RING domain-UBE2D1 complex revealed key interactions between the dimeric IDOL protein and the E2 enzyme. Analysis of the IDOL-UBE2D1 interface also defined the stereochemical basis for the selectivity of IDOL for UBE2Ds over other E2 ligases. Structure-based mutations that inhibit IDOL dimerization or IDOL-UBE2D interaction block IDOL-dependent LDLR ubiquitination and degradation. Furthermore, expression of a dominant-negative UBE2D enzyme inhibits the ability of IDOL to degrade the LDLR in cells. These results identify the IDOL-UBE2D complex as an important determinant of LDLR activity, and provide insight into molecular mechanisms underlying the regulation of cholesterol uptake.

The IDOL-UBE2D complex mediates sterol-dependent degradation of the LDL receptor.

Co-repressor proteins, such as SMRT and NCoR, mediate the repressive activity of unliganded nuclear receptors and other transcription factors. They appear to act as intrinsically disordered "hub proteins" that integrate the activities of a range of transcription factors with a number of histone modifying enzymes. Although these co-repressor proteins are challenging targets for structural studies due to their largely unstructured character, a number of structures have recently been determined of co-repressor interaction regions in complex with their interacting partners. These have yielded considerable insight into the mechanism of assembly of these complexes, the structural basis for the specificity of the interactions and also open opportunities for targeting these interactions therapeutically.

Nuclear hormone receptor co-repressors: structure and function.

E3 &#x6CDB;&#x7D20;&#x8FDE;&#x63A5;&#x9176;&#x7684;&#x5076;&#x50CF; (&#x4F4E;&#x5BC6;&#x5EA6;&#x8102;&#x86CB;&#x767D;&#x53D7;&#x4F53;&#x7684;&#x8BF1;&#x5BFC;&#x578B; degrader) &#x89C4;&#x5B9A;&#x4E86;&#x4F4E;&#x5BC6;&#x5EA6;&#x8102;&#x86CB;&#x767D;&#x53D7;&#x4F53; (LDLR)-&#x4F9D;&#x8D56;&#x80C6;&#x56FA;&#x9187;&#x5438;&#x6536;&#xFF0C;&#x4F46;&#x884C;&#x52A8;&#xFF0C;&#x5176;&#x4E2D;&#x5305;&#x62EC;&#x5176;&#x4E25;&#x683C;&#x7684;&#x7279;&#x5F02;&#x6027;&#x7684;&#x5206;&#x5B50;&#x57FA;&#x7840;&#x53CA;&#x5176;&#x673A;&#x7406;&#x4E86;&#x89E3;&#x751A;&#x5C11;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x5C55;&#x793A;&#x5076;&#x50CF;&#x7528;&#x4E8E;&#x76EE;&#x6807;&#x8BC6;&#x522B;&#x7684; E3 &#x8FDE;&#x63A5;&#x9176;&#x4E4B;&#x95F4;&#x5947;&#x5F02;&#x7684;&#x6218;&#x7565;&#x3002;&#x5C06;&#x5076;&#x50CF;&#x53D1;&#x9175;&#x57DF;&#x76F4;&#x63A5;&#x7ED1;&#x5B9A;&#x5230;&#x8BC6;&#x522B;&#x5E8F;&#x5217;&#x4E2D;&#x7684;&#x8102;&#x86CB;&#x767D;&#x53D7;&#x4F53;&#x7684;&#x7EC6;&#x80DE;&#x8D28;&#x5C3E;&#x5DF4;&#x3002;&#x8FD9;&#x79CD;&#x7269;&#x7406;&#x7684;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x662F;&#x72EC;&#x7ACB;&#x4E8E;&#x5076;&#x50CF;&#x7684;&#x6709;&#x8DA3;&#x65B0;&#x57FA;&#x56E0; (&#x73AF;) &#x57DF; E3 &#x8FDE;&#x63A5;&#x9176;&#x6D3B;&#x6027;&#x548C; autoubiquitination &#x7684;&#x80FD;&#x529B;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x5076;&#x50CF;&#x63A7;&#x5236;&#x901A;&#x8FC7; autoubiquitination &#x7684;&#x72EC;&#x7279;&#x53D1;&#x9175;&#x5B50;&#x57DF;&#x6298;&#x5176;&#x4ED6;&#x53D1;&#x9175;&#x86CB;&#x767D;&#x4E2D;&#x4E0D;&#x5B58;&#x5728;&#x5176;&#x81EA;&#x8EAB;&#x7A33;&#x5B9A;&#x6027;&#x3002;&#x5173;&#x952E;&#x6B8B;&#x7559;&#x5B9A;&#x4E49;&#x7684;&#x5076;&#x50CF; LDLR &#x4E92;&#x52A8;&#x548C;&#x5076;&#x50CF; autoubiquitination &#x529F;&#x80FD;&#x4E0A;&#x662F;&#x5728;&#x4ED6;&#x4EEC;&#x6606;&#x866B;&#x7684;&#x540C;&#x7CFB;&#x7269;&#x5B88;&#x6052;&#x7684;&#x3002;&#x6700;&#x540E;&#xFF0C;&#x6211;&#x4EEC;&#x8BC1;&#x660E;&#x7531;&#x5076;&#x50CF;&#x76EE;&#x6807;&#x8BC6;&#x522B;&#x6D89;&#x53CA;&#x53D1;&#x9175;&#x57DF;&#x3001; &#x819C;&#x78F7;&#x8102;&#x548C;&#x8102;&#x86CB;&#x767D;&#x53D7;&#x4F53;&#x5C3E;&#x4E4B;&#x95F4;&#x7684;&#x4E09;&#x65B9;&#x4E92;&#x52A8;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x6570;&#x636E;&#x6807;&#x8BC6;&#x4F5C;&#x4E3A;&#x8FDB;&#x5316;&#x4FDD;&#x6301;&#x8102;&#x8D28;&#x5438;&#x6536;&#x7684;&#x89C4;&#x7BA1;&#x673A;&#x5236;&#x7684;&#x5076;&#x50CF; LDLR &#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x548C;&#x5EFA;&#x8BAE;&#x8FD9;&#x79CD;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x53EF;&#x80FD;&#x5C31;&#x4F1A;&#x88AB;&#x836F;&#x7406;&#x8C03;&#x8282;&#x8102;&#x8D28;&#x4EE3;&#x8C22;&#x7684;&#x5229;&#x7528;&#x3002;

&#x4F9D;&#x8D56;&#x4E8E;&#x53D1;&#x9175;&#x7684; E3 &#x8FDE;&#x63A5;&#x9176;&#x8BC6;&#x522B;&#x662F;&#x6709;&#x9488;&#x5BF9;&#x6027;&#x7684;&#x8102;&#x86CB;&#x767D;&#x53D7;&#x4F53;&#x9000;&#x5316;&#x5B88;&#x6052;&#x7684;&#x673A;&#x5236;&#x3002;

The E3 ubiquitin ligase IDOL (inducible degrader of the LDL receptor) regulates LDL receptor (LDLR)-dependent cholesterol uptake, but its mechanism of action, including the molecular basis for its stringent specificity, is poorly understood. Here we show that IDOL uses a singular strategy among E3 ligases for target recognition. The IDOL FERM domain binds directly to a recognition sequence in the cytoplasmic tails of lipoprotein receptors. This physical interaction is independent of IDOL's really interesting new gene (RING) domain E3 ligase activity and its capacity for autoubiquitination. Furthermore, IDOL controls its own stability through autoubiquitination of a unique FERM subdomain fold not present in other FERM proteins. Key residues defining the IDOL-LDLR interaction and IDOL autoubiquitination are functionally conserved in their insect homologs. Finally, we demonstrate that target recognition by IDOL involves a tripartite interaction between the FERM domain, membrane phospholipids, and the lipoprotein receptor tail. Our data identify the IDOL-LDLR interaction as an evolutionarily conserved mechanism for the regulation of lipid uptake and suggest that this interaction could potentially be exploited for the pharmacologic modulation of lipid metabolism.

FERM-dependent E3 ligase recognition is a conserved mechanism for targeted degradation of lipoprotein receptors.

L&#39;ubiquitine ligase E3 IDOL (principe inductible d&eacute;gradant du r&eacute;cepteur LDL) r&eacute;glemente le r&eacute;cepteur des LDL (LDLR)-absorption de cholest&eacute;rol d&eacute;pendant, mais son m&eacute;canisme d&#39;action, y compris la base mol&eacute;culaire de sa sp&eacute;cificit&eacute; stricte, est mal comprise. Nous d&eacute;montrons qu&#39;idole utilise une strat&eacute;gie singuli&egrave;re entre E3 ligases pour la reconnaissance de la cible. Le domaine de l&#39;idole FERM lie directement &agrave; une s&eacute;quence de reconnaissance dans la queue cytoplasmique des r&eacute;cepteurs des lipoprot&eacute;ines. Cette interaction physique est ind&eacute;pendante vraiment nouveau g&egrave;ne (anneau) domaine E3 ligase activit&eacute; int&eacute;ressante de l&#39;idole et sa capacit&eacute; d&#39;autoubiquitination. En outre, IDOL contr&ocirc;le sa propre stabilit&eacute; par le biais d&#39;autoubiquitination d&#39;un pli de sous-domaine unique FERM absent d&#39;autres prot&eacute;ines FERM. R&eacute;sidus cl&eacute;s d&eacute;finissant l&#39;interaction IDOL-LDLR et idole autoubiquitination sont fonctionnellement conserv&eacute;s dans leurs homologues chez les insectes. Finalement, nous d&eacute;montrons que la reconnaissance de la cible de l&#39;idole implique une interaction tripartite entre le domaine FERM, phospholipides membranaires et la queue du r&eacute;cepteur lipoprot&eacute;ine. Nos donn&eacute;es identifient l&#39;interaction IDOL-LDLR comme un m&eacute;canisme &eacute;volutif conserv&eacute;e pour la r&eacute;gulation de l&#39;absorption des lipides et sugg&egrave;rent que cette interaction pourrait potentiellement &ecirc;tre exploit&eacute;e pour la modulation pharmacologique du m&eacute;tabolisme des lipides.

Reconnaissance de ligase E3 FERM-d&eacute;pendante est un m&eacute;canisme conserv&eacute; pour d&eacute;gradation cibl&eacute;e des r&eacute;cepteurs des lipoprot&eacute;ines.

Die E3 Ubiquitin Ligase IDOL (durch Induktion Degrader von den LDL-Rezeptor) regelt die LDL-Rezeptor (LDLR)-abh&auml;ngige Cholesterin-Aufnahme, aber seinen Wirkmechanismus, einschlie&szlig;lich die molekulare Grundlage f&uuml;r seine strengen Besonderheit, ist oft nicht ganz verstanden. Hier zeigen wir, dass IDOL eine einzigartige Strategie unter E3-Verkn&uuml;pfung f&uuml;r die Zielerfassung verwendet. Die IDOL FERM-Dom&auml;ne bindet direkt an einer Anerkennung-Sequenz in die zytoplasmatischen Schw&auml;nze von Lipoprotein-Rezeptoren. Diese physische Interaktion ist unabh&auml;ngig von der IDOL wirklich interessante neue (RING) Dom&auml;ne E3 Ligase Genaktivit&auml;t und seine Kapazit&auml;t f&uuml;r Autoubiquitination. Dar&uuml;ber hinaus steuert IDOL eigene Stabilit&auml;t durch Autoubiquitination des einzigartigen FERM Unterdom&auml;ne Falzes in andere FERM-Proteine nicht vorhanden. Schl&uuml;ssel definieren die IDOL-LDLR-Interaktion und IDOL Autoubiquitination R&uuml;ckst&auml;nde sind funktionell in ihren Insekt UCP1 konserviert. Schlie&szlig;lich zeigen wir, dass die Zielerfassung von IDOL eine dreigliedrige Interaktion zwischen der FERM-Dom&auml;ne, Membran-Phospholipiden und der Lipoprotein-Rezeptor-Schwanz umfasst. Unsere Daten identifizieren die IDOL-LDLR-Interaktion als eine evolution&auml;r konservierte Mechanismus f&uuml;r die Regulierung der Lipid-Aufnahme und schlagen vor, da&szlig; diese Interaktion potenziell f&uuml;r die pharmakologische Modulation des Fettstoffwechsels genutzt werden k&ouml;nnten.

FERM-abh&auml;ngige E3 Ligase Anerkennung ist ein konservierten Mechanismus zum gezielten Abbau von Lipoprotein-Rezeptoren.

L&#39;E3 ubiquitina ligasi IDOL (inducibile degrader del recettore LDL) regola il recettore LDL (LDLR)-assorbimento dipendente dal colesterolo, ma il suo meccanismo d&#39;azione, tra cui la base molecolare per la sua specificit&agrave; rigorose, &egrave; capita male. Qui vi mostriamo che idolo utilizza una strategia singolare tra E3 ligasi per il riconoscimento di destinazione. Il dominio FERM IDOL si lega direttamente ad una sequenza di riconoscimento nelle code citoplasmatiche dei recettori delle lipoproteine. Questa interazione fisica &egrave; indipendente davvero interessante nuovo (anello) dominio E3 ligasi attivit&agrave; del gene dell&#39;idolo e la sua capacit&agrave; per autoubiquitination. Inoltre, idolo controlla la propria stabilit&agrave; attraverso autoubiquitination di un&#39;unica piegatura FERM sottodominio non presente in altre proteine FERM. Residui chiave definisce l&#39;interazione LDLR idolo e l&#39;idolo autoubiquitination funzionalmente sono conservate nella loro omologhi dell&#39;insetto. Infine, ci dimostrano che riconoscimento di destinazione da IDOL implica un&#39;interazione tripartita tra il dominio FERM, fosfolipidi di membrana e la coda dei recettori delle lipoproteine. I nostri dati identificano l&#39;interazione IDOL-LDLR come un meccanismo evolutivamente conservato per la regolazione dell&#39;assorbimento dei lipidi e suggeriscono che questa interazione potenzialmente potrebbe essere sfruttata per la modulazione farmacologica del metabolismo lipidico.

Riconoscimento di ligasi E3 FERM-dipendente &egrave; un meccanismo conservato per mirata degradazione dei recettori delle lipoproteine.

E3 &#x30E6;&#x30D3;&#x30AD;&#x30C1;&#x30F3;&#x30EA; &#x30AC;&#x30FC;&#x30BC; &#x30A2;&#x30A4;&#x30C9;&#x30EB; (LDL &#x53D7;&#x5BB9;&#x4F53;&#x8A98;&#x5C0E;&#x6027;&#x5FAE;&#x751F;&#x7269;) &#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B; LDL &#x53D7;&#x5BB9;&#x4F53; (LDLR)-&#x4F9D;&#x5B58;&#x30B3;&#x30EC;&#x30B9;&#x30C6;&#x30ED;&#x30FC;&#x30EB;&#x5438;&#x53CE;&#x304C;&#x53B3;&#x683C;&#x306A;&#x7279;&#x7570;&#x6027;&#x306E;&#x5206;&#x5B50;&#x57FA;&#x76E4;&#x3092;&#x542B;&#x3080;&#x30A2;&#x30AF;&#x30B7;&#x30E7;&#x30F3;&#x306E;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x306F;&#x3088;&#x304F;&#x7406;&#x89E3;&#x3055;&#x308C;&#x3066;&#x3002;&#x3053;&#x3053;&#x3067;&#x30A2;&#x30A4;&#x30C9;&#x30EB; &#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x8A8D;&#x8B58;&#x306E;&#x305F;&#x3081;&#x306E;&#x7279;&#x7570;&#x6226;&#x7565; E3 &#x30EA;&#x30AC;&#x30FC;&#x30BC;&#x306E;&#x9593;&#x3067;&#x4F7F;&#x7528;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x30A2;&#x30A4;&#x30C9;&#x30EB; FERM &#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x306F;&#x8A8D;&#x8B58;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9; &#x30EA;&#x30DD;&#x86CB;&#x767D;&#x53D7;&#x5BB9;&#x4F53;&#x306E;&#x7D30;&#x80DE;&#x8CEA;&#x30C6;&#x30FC;&#x30EB;&#x306B;&#x76F4;&#x63A5;&#x30D0;&#x30A4;&#x30F3;&#x30C9;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x7269;&#x7406;&#x7684;&#x306A;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306F;&#x3001;&#x30A2;&#x30A4;&#x30C9;&#x30EB;&#x306E;&#x672C;&#x5F53;&#x306B;&#x8208;&#x5473;&#x6DF1;&#x3044;&#x65B0;&#x3057;&#x3044;&#x907A;&#x4F1D;&#x5B50; (&#x30EA;&#x30F3;&#x30B0;) &#x30C9;&#x30E1;&#x30A4;&#x30F3; E3 &#x30EA;&#x30AC;&#x30FC;&#x30BC;&#x6D3B;&#x52D5;&#x3068;&#x305D;&#x306E;&#x5BB9;&#x91CF; autoubiquitination &#x306E;&#x305F;&#x3081;&#x306E;&#x72EC;&#x7ACB;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x30A2;&#x30A4;&#x30C9;&#x30EB;&#x4ED6; FERM &#x86CB;&#x767D;&#x8CEA;&#x306E;&#x5B58;&#x5728;&#x3057;&#x306A;&#x3044;&#x4E00;&#x610F; FERM &#x30B5;&#x30D6;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x500D;&#x306E; autoubiquitination &#x3092;&#x901A;&#x3058;&#x3066;&#x3001;&#x72EC;&#x81EA;&#x306E;&#x5B89;&#x5B9A;&#x5236;&#x5FA1;&#x3057;&#x307E;&#x3059;&#x3002;&#x30A2;&#x30A4;&#x30C9;&#x30EB; LDLR &#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3068;&#x30A2;&#x30A4;&#x30C9;&#x30EB;&#x306E; autoubiquitination &#x3092;&#x5B9A;&#x7FA9;&#x3059;&#x308B;&#x30AD;&#x30FC;&#x6B8B;&#x57FA;&#x306F;&#x3001;&#x6A5F;&#x80FD;&#x7684;&#x3001;&#x6606;&#x866B;&#x306E;&#x540C;&#x65CF;&#x4F53;&#x3067;&#x4FDD;&#x5B58;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x6700;&#x5F8C;&#x306B;&#x3001;&#x6211;&#x3005; &#x306E;&#x30A2;&#x30A4;&#x30C9;&#x30EB;&#x306B;&#x3088;&#x308B;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x8A8D;&#x8B58; FERM &#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x3001;&#x819C;&#x30EA;&#x30F3;&#x8102;&#x8CEA;&#x304A;&#x3088;&#x3073;&#x30EA;&#x30DD;&#x86CB;&#x767D;&#x53D7;&#x5BB9;&#x4F53;&#x5C3E;&#x3068;&#x306E;&#x4E09;&#x8005;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3092;&#x4F34;&#x3044;&#x307E;&#x3059;&#x3092;&#x30C7;&#x30E2;&#x30F3;&#x30B9;&#x30C8;&#x30EC;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3057;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x306E;&#x30C7;&#x30FC;&#x30BF;&#x306F;&#x30A2;&#x30A4;&#x30C9;&#x30EB; LDLR &#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3068;&#x3057;&#x3066;&#x306E;&#x8102;&#x8CEA;&#x6442;&#x53D6;&#x91CF;&#x306E;&#x8ABF;&#x7BC0;&#x306E;&#x9032;&#x5316;&#x7684;&#x4FDD;&#x5B58;&#x306E;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x8B58;&#x5225;&#x3057;&#x3001;&#x3053;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x304C;&#x8102;&#x8CEA;&#x4EE3;&#x8B1D;&#x306E;&#x85AC;&#x7406;&#x5B66;&#x7684;&#x5909;&#x8ABF;&#x306E;&#x6F5C;&#x5728;&#x7684;&#x60AA;&#x7528;&#x3055;&#x308C;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;

FERM &#x4F9D;&#x5B58; E3 &#x30EA;&#x30AC;&#x30FC;&#x30BC;&#x8A8D;&#x8B58;&#x5BFE;&#x8C61;&#x306E;&#x7279;&#x6027;&#x52A3;&#x5316;&#x30EA;&#x30DD;&#x86CB;&#x767D;&#x53D7;&#x5BB9;&#x4F53;&#x306E;&#x4FDD;&#x5B58;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3067;&#x3059;&#x3002;

E3 &#xC720;&#xBE44;&#xD034;&#xD2F4; &#xB9AC;&#xAC00; &#xC6B0;&#xC0C1; (LDL &#xC218;&#xC6A9; &#xCCB4;&#xC758; &#xC720;&#xB3C4;&#xD560; &#xC218; &#xC788;&#xB294; degrader) &#xC870;&#xC808; LDL &#xC218;&#xC6A9; &#xCCB4; (LDLR)-&#xC885;&#xC18D; &#xCF5C;&#xB808;&#xC2A4;&#xD14C;&#xB864; &#xC774;&#xD574; &#xD558;&#xC9C0;&#xB9CC; &#xADF8;&#xAC83;&#xC758; &#xC5C4;&#xACA9;&#xD55C; &#xD2B9;&#xC774;&#xC131;&#xC5D0; &#xB300; &#xD55C; &#xBD84;&#xC790; &#xAE30;&#xCD08;&#xB97C; &#xD3EC;&#xD568; &#xD558; &#xC5EC; &#xB3D9;&#xC791;&#xC758; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC740; &#xC81C;&#xB300;&#xB85C; &#xC774;&#xD574;. &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC;&#xAC00; &#xC544;&#xC774;&#xB3CC; E3 ligases &#xAC00;&#xC6B4;&#xB370; &#xB2E8; &#xC218; &#xC804;&#xB7B5;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xB300;&#xC0C1; &#xC778;&#xC2DD;&#xC5D0; &#xB300; &#xD55C; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. &#xC544;&#xC774;&#xB3CC; &#xBCF4;&#xD5D8;&#xC774; &#xB3C4;&#xBA54;&#xC778; &#xC778;&#xC2DD; &#xC2DC;&#xD000;&#xC2A4; &#xB2E8;&#xBC31; &#xC218;&#xC6A9; &#xCCB4;&#xC758; &#xC138;&#xD3EC;&#xC9C8; &#xAF2C;&#xB9AC;&#xC5D0; &#xC9C1;&#xC811; &#xBC14;&#xC778;&#xB529;&#xB429;&#xB2C8;&#xB2E4;. &#xC774; &#xBB3C;&#xB9AC;&#xC801;&#xC73C;&#xB85C; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC740; &#xC544;&#xC774;&#xB3CC;&#xC758; &#xC815;&#xB9D0; &#xC7AC;&#xBBF8; &#xC788;&#xB294; &#xC0C8;&#xB85C;&#xC6B4; &#xC720;&#xC804;&#xC790; (&#xBC18;&#xC9C0;) &#xB3C4;&#xBA54;&#xC778; E3 &#xB9AC;&#xAC00; &#xD65C;&#xB3D9; &#xBC0F; autoubiquitination&#xC5D0; &#xB300; &#xD55C; &#xC6A9;&#xB7C9;&#xC5D0; &#xAD00;&#xACC4; &#xC5C6;&#xC774;. &#xB610;&#xD55C;, &#xC544;&#xC774;&#xB3CC; &#xB3C5;&#xD2B9;&#xD55C; &#xBCF4;&#xD5D8;&#xC774; &#xD558;&#xC704; &#xB3C4;&#xBA54;&#xC778; &#xBC30; &#xB2E4;&#xB978; &#xBCF4;&#xD5D8;&#xC774; &#xB2E8;&#xBC31;&#xC9C8;&#xC5D0; &#xC5C6;&#xB294; autoubiquitination &#xD1B5;&#xD574; &#xC548;&#xC815;&#xC131;&#xC744; &#xC81C;&#xC5B4;&#xD569;&#xB2C8;&#xB2E4;. &#xC544;&#xC774;&#xB3CC;-LDLR &#xC0C1;&#xD638; &#xC791;&#xC6A9; &#xBC0F; &#xC6B0;&#xC0C1; autoubiquitination &#xC815;&#xC758; &#xD0A4; &#xC794;&#xB958;&#xBB3C;&#xC740; &#xAE30;&#xB2A5;&#xC801;&#xC73C;&#xB85C; &#xADF8;&#xB4E4;&#xC758; &#xACE4;&#xCDA9; homologs&#xC5D0; &#xBCF4;&#xC874; &#xB429;&#xB2C8;&#xB2E4;. &#xB9C8;&#xC9C0;&#xB9C9;&#xC73C;&#xB85C;, &#xC544;&#xC774;&#xB3CC;&#xC5D0;&#xC758; &#xD55C; &#xB300;&#xC0C1; &#xC778;&#xC2DD; &#xBCF4;&#xD5D8;&#xC774; &#xB3C4;&#xBA54;&#xC778;, &#xB9C9; &#xC778;&#xC9C0;&#xC9C8;&#xACFC; &#xB2E8;&#xBC31; &#xC218;&#xC6A9; &#xCCB4; &#xAF2C;&#xB9AC; &#xC0AC;&#xC774; 3 &#xC790;&#xAC04; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC744; &#xD3EC;&#xD568; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xC758; &#xB370;&#xC774;&#xD130; &#xC9C0;&#xC9C8; &#xD1B5;&#xD48D; &#xAD00;&#xC758; &#xADDC;&#xCE59;&#xC5D0; &#xB300; &#xD55C; &#xC9C4;&#xD654; &#xC801;&#xC73C;&#xB85C; &#xBCF4;&#xC874;&#xB41C; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC73C;&#xB85C; &#xC544;&#xC774;&#xB3CC;-LDLR &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC744; &#xC2DD;&#xBCC4; &#xD558; &#xACE0;&#xC774; &#xC0C1;&#xD638; &#xC791;&#xC6A9; &#xC9C0;&#xC9C8; &#xB300;&#xC0AC;&#xC758; pharmacologic &#xBCC0;&#xC870;&#xC5D0; &#xB300; &#xD55C; &#xC7A0;&#xC7AC;&#xC801;&#xC73C;&#xB85C; &#xC545;&#xC6A9; &#xB420; &#xC218; &#xC788;&#xB294; &#xAC83;&#xC774; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xBCF4;&#xD5D8;&#xC774; &#xC885;&#xC18D; E3 &#xB9AC;&#xAC00; &#xC778;&#xC2DD; &#xB2E8;&#xBC31; &#xC218;&#xC6A9; &#xCCB4;&#xC758; &#xD0C0;&#xAC9F;&#xB41C; &#xC800;&#xD558;&#xC5D0; &#xB300; &#xD55C; &#xBCF4;&#xC874;&#xB41C; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC785;&#xB2C8;&#xB2E4;.

La ligasa de ubiquitina E3 IDOL (probi&oacute;ticos inducible del receptor LDL) regula el receptor de LDL (LDLR)-absorci&oacute;n de colesterol dependiente, pero su mecanismo de acci&oacute;n, incluyendo las bases moleculares de su especificidad estricta, es mal entendida. Aqu&iacute; mostramos que &iacute;dolo utiliza una singular estrategia entre E3 ligasas para reconocimiento de destino. El dominio de FERM &iacute;dolo se une directamente a una secuencia de reconocimiento en las colas citopl&aacute;smicas de los receptores de lipoprote&iacute;nas. Esta interacci&oacute;n f&iacute;sica es independiente realmente interesante nuevo (anillo) dominio E3 ligase de la actividad de los genes del &iacute;dolo y su capacidad para autoubiquitination. Adem&aacute;s, el &iacute;dolo controla su propia estabilidad a trav&eacute;s de autoubiquitination de un pliegue de subdominio FERM &uacute;nico no est&aacute; presente en otras prote&iacute;nas FERM. Residuos claves que definen la interacci&oacute;n del &iacute;dolo-LDLR y autoubiquitination IDOL funcionalmente se conservan en sus hom&oacute;logos insectos. Finalmente, demostramos que el reconocimiento objetivo por &iacute;dolo implica una interacci&oacute;n tripartita entre el dominio FERM, fosfol&iacute;pidos de la membrana y la cola de receptor de la lipoprote&iacute;na. Nuestros datos identifican la interacci&oacute;n del &iacute;dolo-LDLR como un mecanismo evolutivamente conservado para la regulaci&oacute;n de la absorci&oacute;n de l&iacute;pidos y sugieren que esta interacci&oacute;n potencialmente podr&iacute;a ser aprovechada para la modulaci&oacute;n farmacol&oacute;gica del metabolismo lip&iacute;dico.

Reconocimiento de ligasa E3 FERM-dependiente es un mecanismo conservado para degradaci&oacute;n espec&iacute;fica de receptores de lipoprote&iacute;nas.

Histone deacetylase enzymes (HDACs) are emerging cancer drug targets. They regulate gene expression by removing acetyl groups from lysine residues in histone tails, resulting in chromatin condensation. The enzymatic activity of most class I HDACs requires recruitment into multi-subunit co-repressor complexes, which are in turn recruited to chromatin by repressive transcription factors. Here we report the structure of a complex between an HDAC and a co-repressor, namely, human HDAC3 with the deacetylase activation domain (DAD) from the human SMRT co-repressor (also known as NCOR2). The structure reveals two remarkable features. First, the SMRT-DAD undergoes a large structural rearrangement on forming the complex. Second, there is an essential inositol tetraphosphate molecule--D-myo-inositol-(1,4,5,6)-tetrakisphosphate (Ins(1,4,5,6)P(4))--acting as an 'intermolecular glue' between the two proteins. Assembly of the complex is clearly dependent on the Ins(1,4,5,6)P(4), which may act as a regulator--potentially explaining why inositol phosphates and their kinases have been found to act as transcriptional regulators. This mechanism for the activation of HDAC3 appears to be conserved in class I HDACs from yeast to humans, and opens the way to novel therapeutic opportunities.

Structure of HDAC3 bound to co-repressor and inositol tetraphosphate.

Formation of the death-inducing signaling complex (DISC) is a critical step in death receptor-mediated apoptosis, yet the mechanisms underlying assembly of this key multiprotein complex remain unclear. Using quantitative mass spectrometry, we have delineated the stoichiometry of the native TRAIL DISC. While current models suggest that core DISC components are present at a ratio of 1:1, our data indicate that FADD is substoichiometric relative to TRAIL-Rs or DED-only proteins; strikingly, there is up to 9-fold more caspase-8 than FADD in the DISC. Using structural modeling, we propose an alternative DISC model in which procaspase-8 molecules interact sequentially, via their DED domains, to form a caspase-activating chain. Mutating key interacting residues in procaspase-8 DED2 abrogates DED chain formation in cells and disrupts TRAIL/CD95 DISC-mediated procaspase-8 activation in a functional DISC reconstitution model. This provides direct experimental evidence for a DISC model in which DED chain assembly drives caspase-8 dimerization/activation, thereby triggering cell death.

A death effector domain chain DISC model reveals a crucial role for caspase-8 chain assembly in mediating apoptotic cell death.

Class I histone deacetylases (HDAC1, HDAC2, and HDAC3) are recruited by cognate corepressor proteins into specific transcriptional repression complexes that target HDAC activity to chromatin resulting in chromatin condensation and transcriptional silencing. We previously reported the structure of HDAC3 in complex with the SMRT corepressor. This structure revealed the presence of inositol-tetraphosphate [Ins(1,4,5,6)P4] at the interface of the two proteins. It was previously unclear whether the role of Ins(1,4,5,6)P4 is to act as a structural cofactor or a regulator of HDAC3 activity. Here we report the structure of HDAC1 in complex with MTA1 from the NuRD complex. The ELM2-SANT domains from MTA1 wrap completely around HDAC1 occupying both sides of the active site such that the adjacent BAH domain is ideally positioned to recruit nucleosomes to the active site of the enzyme. Functional assays of both the HDAC1 and HDAC3 complexes reveal that Ins(1,4,5,6)P4 is a bona fide conserved regulator of class I HDAC complexes.

Class I HDACs share a common mechanism of regulation by inositol phosphates.

Nuclear receptors are transcription factors that regulate gene expression through the ligand-controlled recruitment of a diverse group of proteins known as coregulators. Most nuclear receptor coregulators function in large multi-protein complexes that modify chromatin and thereby regulate the transcription of target genes. Structural and functional studies are beginning to reveal how these complexes are assembled bringing together multiple functionalities that mediate: recruitment to specific genomic loci through interaction with transcription factors; recruitment of enzymatic activities that either modify or remodel chromatin and targeting the complexes to their chromatin substrate. These activities are regulated by post-translational modifications, alternative splicing and small signalling molecules. This review focuses on our current understanding of coregulator complexes and aims to highlight the common principles that are beginning to emerge.

An evolving understanding of nuclear receptor coregulator proteins.

BCL6 is a transcriptional repressor that is over-expressed due to chromosomal translocations, or other abnormalities, in ∼40% of diffuse large B-cell lymphoma. BCL6 interacts with co-repressor, SMRT, and this is essential for its role in lymphomas. Peptide or small molecule inhibitors, which prevent the association of SMRT with BCL6, inhibit transcriptional repression and cause apoptosis of lymphoma cells in vitro and in vivo. In order to discover compounds, which have the potential to be developed into BCL6 inhibitors, we screened a natural product library. The ansamycin antibiotic, rifamycin SV, inhibited BCL6 transcriptional repression and NMR spectroscopy confirmed a direct interaction between rifamycin SV and BCL6. To further determine the characteristics of compounds binding to BCL6-POZ we analyzed four other members of this family and showed that rifabutin, bound most strongly. An X-ray crystal structure of the rifabutin-BCL6 complex revealed that rifabutin occupies a partly non-polar pocket making interactions with tyrosine58, asparagine21 and arginine24 of the BCL6-POZ domain. Importantly these residues are also important for the interaction of BLC6 with SMRT. This work demonstrates a unique approach to developing a structure activity relationship for a compound that will form the basis of a therapeutically useful BCL6 inhibitor.

Louise Fairall

Department of Biochemistry

University of Leicester

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

Louise Fairall

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

University of Leicester

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

Department of Biochemistry