method-for-identifying-small-molecule-inhibitors-protein-protein

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Feinberg School of Medicine, Northwestern University

Apolipoprotein A-IV (apoA-IV) has myriad functions, including roles as a post-prandial satiety factor and lipid antioxidant. ApoA-IV is expressed in mammalian small intestine and is up-regulated in response to lipid absorption. In newborn swine jejunum, a high fat diet acutely induces a 7-fold increase in apoA-IV expression. To determine whether apoA-IV plays a role in the transport of absorbed lipid, swine apoA-IV was overexpressed in a newborn swine enterocyte cell line, IPEC-1, followed by analysis of the expression of genes related to lipoprotein assembly and lipid transport, as well as quantitation of lipid synthesis and secretion. A full-length swine apoA-IV cDNA was cloned, sequenced, and inserted into a Vp and Rep gene-deficient adeno-associated viral vector, containing the cytomegalovirus immediate early promoter/enhancer and neomycin resistance gene, and was used to transfect IPEC-1 cells. Control cells were transfected with the same vector minus the apoA-IV insert. Using neomycin selection, apoA-IV-overexpressing (+AIV) and control (-AIV) clones were isolated for further study. Both undifferentiated (-D) and differentiated (+D) +AIV cells expressed 40- to 50-fold higher levels of apoA-IV mRNA and both intracellular and secreted apoA-IV protein compared with -AIV cells. Expression of other genes was not affected by apoA-IV overexpression in a manner that would contribute to enhanced lipid secretion. +D +AIV cells secreted 4.9-fold more labeled triacylglycerol (TG), 4.6-fold more labeled cholesteryl ester (CE), and 2-fold more labeled phospholipid (PL) as lipoproteins, mostly in the chylomicron/very low density lipoprotein (VLDL) density range. ApoA-IV overexpression in IPEC-1 cells enhances basolateral TG, CE, and PL secretion in chylomicron/VLDL particles. This enhancement is not associated with up-regulation of other genes involved in lipid transport. ApoA-IV may play a role in facilitating enterocyte lipid transport, particularly in the neonate receiving a diet of high fat breast milk.

Overexpression of apolipoprotein A-IV enhances lipid transport in newborn swine intestinal epithelial cells.

To define the developmental expression of microsomal triglyceride transfer protein (MTP) large subunit mRNA and protein, samples of small intestine and liver were collected from 40-day gestation fetal, 2-day-old newborn, 3-week-old suckling, and 2-month-old weanling swine. In fetal animals, MTP mRNA expression was high in intestine and liver. Postnatally, jejunal expression paralleled the intake of a high-fat breast milk diet and declined after weaning. Ileal expression was comparable with that of jejunum in 2-day-old animals, but declined to low levels afterward. Hepatic expression declined postnatally and remained low. MTP protein expression generally paralleled mRNA expression, except in fetal intestine in which no 97 kDa protein was detected. In 2-day-old piglets, a high-triacylglycerol diet increased jejunal and ileal MTP mRNA levels, as compared to a low-triacylglycerol diet. To test the roles of glucocorticoids and fatty acids in MTP regulation, a newborn swine enterocyte cell line (IPEC-1) was used. Except at day 2 of differentiation, dexamethasone did not influence MTP expression. Fatty acids either up-regulated or down-regulated MTP expression, depending on the specific fatty acid and duration of exposure. Although programmed genetic cues regulate MTP expression during development, clearly the amount and fatty acid composition of dietary lipid also play regulatory roles.

Regulation of MTP expression in developing swine.

Dietary lipid acutely upregulates apolipoprotein (apo) A-IV expression by sevenfold at the pretranslational level in neonatal swine jejunum. To determine the mechanism of this regulation, two-day-old female swine received intraduodenal infusions of low- and high-triacylglycerol (TG) isocaloric diets for 24 h. Nuclear runoff assay confirmed apo A-IV gene transcriptional regulation by the high-TG diet. Footprinting analysis using the swine apo A-IV proximal promoter sequence (+14 to -246 bp) demonstrated three regions protected by the low-TG extracts. Of these three motifs, only ACCTTC showed 100% homology to the human sequence and was further studied. EMSA was performed using probes containing wild-type (WT) and mutant (M) motifs. A shift was noted with the low-TG nuclear extracts with the WT probe but not with the M probe. Excess unlabeled free WT probe competed out the shift, whereas the M probe did not. No significant shift occurred with either probe using high-TG extracts. These results suggest that a repressor protein binds to the ACCTTC motif and becomes unbound during lipid absorption, allowing transcriptional activation of the apo A-IV gene in newborn swine small intestine.

Regulation of apo A-IV transcription by lipid in newborn swine is associated with a promoter DNA-binding protein.

&#x80A0;&#x9053;&#x8F7D;&#x8102;&#x86CB;&#x767D; A &#x56DB;&#x8868;&#x8FBE;&#x9AD8;&#x5EA6;&#x53D7;&#x81B3;&#x98DF;&#x8102;&#x80AA;&#x65B0;&#x751F;&#x513F;&#x732A;&#xFF0C;&#x6697;&#x793A;&#x7740;&#x8102;&#x7C7B;&#x5438;&#x6536;&#x7684;&#x4F5C;&#x7528;&#x3002;&#x65B0;&#x751F;&#x513F;&#x732A;&#x80A0;&#x9ECF;&#x819C;&#x4E0A;&#x76AE;&#x7684;&#x963F;&#x62C9;&#x6728;&#x56FE;&#x884C;&#x52A8;&#x7EB2;&#x9886; &#x300B; &#x56DB;&#x672C;&#x6784;&#x8FC7;&#x8868;&#x8FBE;&#x589E;&#x5F3A;&#x57FA;&#x5E95;&#x5916;&#x4FA7;&#x5206;&#x6CCC;&#x7684; TG &#x5BCC;&#x8102;&#x86CB;&#x767D;&#x4E2D;&#x7684;&#x7518;&#x6CB9;&#x4E09;&#x916F; (TG) 4.9-fold (&#xFF0C;S.&#xFF0C;&#x8DEF;&#x9065;&#xFF0C;Y.&#x3001; &#x5B5F;&#xFF0C;S.&#xFF0C;&#x7A0B;&#xFF0C;&#x5341;&#x3001;&#xFF0C;&#x548C;&#x9ED1;&#x8272;&#x3001; D.D.(2002 &#x5E74;) J.Biol.&#x5316;&#x5DE5;&#x5382; 277&#xFF0C;31929 31937)&#x3002;&#x82E5;&#x8981;&#x8C03;&#x67E5;&#x6B64;&#x589E;&#x5F3A;&#x529F;&#x80FD;&#x7684;&#x673A;&#x5236;&#xFF0C;&#x7AE5;&#x5DE5; 1 &#x7EC6;&#x80DE;&#x88AB;&#x67D3;&#x56DB;&#x73AF;&#x7D20;&#x53EF;&#x8C03;&#x7684;&#x8868;&#x8FBE;&#x7CFB;&#x7EDF; &#xFF08;Tet &#x4E0A;&#xFF09;&#x3002;&#x5728;&#x5B75;&#x5316;&#x4E0E;&#x6CB9;&#x9178;&#x7684;&#x5355;&#x5143;&#x683C;&#x4E2D;&#xFF0C;&#x5242;&#x91CF;&#x53CD;&#x5E94;&#x5173;&#x7CFB;&#x6709;&#x4EBA;&#x4E2D;&#x591A;&#x897F;&#x73AF;&#x7D20;&#x6D53;&#x5EA6;&#x548C;&#x57FA;&#x5E95;&#x5916;&#x4FA7;&#x963F;&#x62C9;&#x6728;&#x56FE;&#x884C;&#x52A8;&#x7EB2;&#x9886; &#x300B; &#x56DB;&#x4E0E; TG &#x5206;&#x6CCC;&#x3002;&#x540C;&#x6837;&#x53D7;&#x5230;&#x89C4;&#x7BA1;&#x7684;&#x963F;&#x62C9;&#x6728;&#x56FE;&#x884C;&#x52A8;&#x7EB2;&#x9886; &#x300B; &#x8868;&#x8FBE;-&#x6211;&#x5E76;&#x6CA1;&#x6709;&#x80FD;&#x591F;&#x52A0;&#x5F3A;&#x8102;&#x8D28;&#x5206;&#x6CCC;&#x3002;TG &#x5BCC;&#x8102;&#x86CB;&#x767D;&#x5206;&#x6CCC;&#x591A;&#x897F;&#x73AF;&#x7D20;&#x6CBB;&#x7597;&#x7EC6;&#x80DE;&#x7684;&#x5E73;&#x5747;&#x76F4;&#x5F84;&#x662F;&#x5927;&#x4E8E;&#x4ECE;&#x672A;&#x7ECF;&#x5904;&#x7406;&#x7684;&#x5355;&#x5143;&#x683C; &#xFF08;87.0 &#x4E0E; 53.4 nm nm&#xFF09;&#x3002;&#x57FA;&#x5E95;&#x5916;&#x4FA7; apoB &#x5206;&#x6CCC;&#x51CF;&#x5C11;&#x3002;&#x4F7F;&#x7528;&#x76F8;&#x540C;&#x7684;&#x8868;&#x8FBE;&#x7CFB;&#x7EDF;&#xFF0C;&#x5168;&#x957F;&#x4EBA;&#x7C7B;&#x963F;&#x62C9;&#x6728;&#x56FE;&#x884C;&#x52A8;&#x7EB2;&#x9886; &#x300B; &#x7B2C;&#x56DB; (376 &#x6C28;&#x57FA;&#x9178;&#xFF09; &#xFF1B;&quot;&#x732A;&#x6837;&quot;&#x4EBA;&#x7C7B;&#x963F;&#x62C9;&#x6728;&#x56FE;&#x884C;&#x52A8;&#x7EB2;&#x9886; &#x300B;-&#x56DB;&#xFF0C;&#x7F3A;&#x4E4F; C &#x7EC8;&#x7AEF; EQQQ &#x91CD;&#x590D; (361 &#x6C28;&#x57FA;&#x9178;&#xFF09; &#xFF1B;&#x5E76;&#x8FDB;&#x4E00;&#x6B65;&#x88AB;&#x622A;&#x65AD;&#x4E3A; 343 &#x6C28;&#x57FA;&#x9178;&quot;&#x9E21;&#x6837;&quot;&#x963F;&#x62C9;&#x6728;&#x56FE;&#x884C;&#x52A8;&#x7EB2;&#x9886; &#x300B;-&#x56DB;&#xFF0C;&#x6709;&#x4EBA;&#x5728;&#x7AE5;&#x5DE5; 1 &#x5355;&#x5143;&#x683C;&#x4E2D;&#x3002;&#x589E;&#x52A0;&#x86CB;&#x767D;&#x8D28;&#x5206;&#x6CCC;&#xFF0C;&#x4E0E;&#x8868;&#x793A;&#x5168;&#x957F;&#x4EBA;&#x7C7B;&#x7684;&#x963F;&#x62C9;&#x6728;&#x56FE;&#x884C;&#x52A8;&#x7EB2;&#x9886; &#x300B; &#x7B2C;&#x56DB;&#x5355;&#x5143;&#x683C;&#x6240;&#x663E;&#x793A;&#x7684;&#x5728; TG &#x5206;&#x6CCC; &#xFF1B; &#x589E;&#x52A0;&#x4E86;&#x4E8C;&#x6298;&#x5728;&#x9C9C;&#x660E;&#x7684;&#x5BF9;&#x6BD4;&#xFF0C;&#x7EC6;&#x80DE;&#x8868;&#x8FBE;&#x732A;&#x7C7B;&#x4F3C;&#x4EBA;&#x7C7B;&#x963F;&#x62C9;&#x6728;&#x56FE;&#x884C;&#x52A8;&#x7EB2;&#x9886; &#x300B; &#x56DB;&#x663E;&#x793A;&#x8102;&#x86CB;&#x767D;&#x76F4;&#x5F84;&#x589E;&#x52A0;&#x4E86; 27-fold &#x548C; 25-fold TG &#x5206;&#x6CCC;&#x589E;&#x52A0;&#x3002;&#x5F53;&#x4EBA;&#x7C7B;&#x7684;&#x963F;&#x62C9;&#x6728;&#x56FE;&#x884C;&#x52A8;&#x7EB2;&#x9886; &#x300B; &#x56DB;&#x8FDB;&#x4E00;&#x6B65;&#x88AB;&#x622A;&#x65AD;&#xFF0C;&#x4EE5;&#x4EA7;&#x751F;&#x4E00;&#x79CD;&#x9E21;&#x6837;&#x86CB;&#x767D;&#x8D28;&#x65F6;&#xFF0C;&#x88AB;&#x6291;&#x5236; TG &#x5206;&#x6CCC;&#x3002;&#x6211;&#x4EEC;&#x5F97;&#x51FA;&#x7684;&#x7ED3;&#x8BBA;&#xFF0C;&#x8868;&#x8FBE;&#x7684;&#x732A;&#x963F;&#x62C9;&#x6728;&#x56FE;&#x884C;&#x52A8;&#x7EB2;&#x9886; &#x300B; &#x56DB;&#x589E;&#x5F3A;&#x57FA;&#x5E95;&#x5916;&#x4FA7; TG &#x5206;&#x6CCC;&#x4EE5;&#x5242;&#x91CF;&#x4F9D;&#x8D56;&#x7684;&#x65B9;&#x5F0F;&#x901A;&#x8FC7;&#x589E;&#x52A0;&#x5206;&#x6CCC;&#x8102;&#x86CB;&#x767D;&#x7684;&#x5927;&#x5C0F;&#x3002;&#x8FD9;&#x4E9B;&#x6570;&#x636E;&#x8868;&#x660E;&#x8BE5;&#x533A;&#x57DF;&#x5728;&#x4EBA;&#x7C7B;&#x7684;&#x963F;&#x62C9;&#x6728;&#x56FE;&#x884C;&#x52A8;&#x7EB2;&#x9886; &#x300B; &#x56DB;&#x86CB;&#x767D;&#x8D28;&#x7684;&#x6B8B;&#x7559; 344 &#x5230; 354 &#x662F;&#x5173;&#x952E;&#x80FD;&#x529B;&#x589E;&#x5F3A;&#x8102;&#x8D28;&#x5206;&#x6CCC;&#xFF0C;&#x4E5F;&#x8BB8;&#x901A;&#x8FC7;&#x542F;&#x7528;&#x989D;&#x5916;&#x7684;&#x6838;&#x5FC3; TG &#x6210;&#x4E73;&#x7CDC;&#x5FAE;&#x7C92;&#x9897;&#x7C92;&#x7684;&#x5305;&#x88C5;&#x3002;EQQQ &#x8D44;&#x6E90;&#x5BCC;&#x9976;&#x7684;&#x5730;&#x533A;&#x53EF;&#x80FD;&#x5728;&#x4E73;&#x7CDC;&#x5FAE;&#x7C92;&#x5305;&#x88C5;&#x5728;&#x4EBA;&#x7C7B;&#x4E2D;&#x53D1;&#x6325;&#x6291;&#x5236;&#x6216;&#x8C03;&#x8282;&#x7684;&#x4F5C;&#x7528;&#x3002;

&#x8868;&#x8FBE;&#x7684;&#x8F7D;&#x8102;&#x86CB;&#x767D; A &#x56DB;&#x589E;&#x52A0;&#x4E73;&#x7CDC;&#x5FAE;&#x7C92;&#x7684;&#x5927;&#x5C0F;&#xFF0C;&#x4ECE;&#x800C;&#x589E;&#x5F3A;&#x4E86;&#x7AE5;&#x5DE5; 1 &#x7EC6;&#x80DE;&#x4E2D;&#x7684;&#x8102;&#x8D28;&#x5206;&#x6CCC;&#x3002;

Intestinal apolipoprotein A-IV expression is highly regulated by dietary lipid in newborn swine, suggesting a role in lipid absorption. Constitutive overexpression of apoA-IV in newborn swine enterocytes enhances basolateral secretion of triacylglycerol (TG) in TG-rich lipoproteins 4.9-fold (Lu, S., Yao, Y., Meng, S., Cheng, X., and Black, D. D. (2002) J. Biol. Chem. 277, 31929-31937). To investigate the mechanism of this enhancement, IPEC-1 cells were transfected with a tetracycline-regulatable expression system (Tet-On). In cells incubated with oleic acid, a dose response relationship was observed between medium doxycycline concentration and basolateral apoA-IV and TG secretion. Similarly regulated expression of apoA-I did not enhance lipid secretion. The mean diameter of TG-rich lipoproteins secreted from doxycycline-treated cells was larger than from untreated cells (87.0 nm versus 53.4 nm). Basolateral apoB secretion decreased. Using the same expression system, full-length human apoA-IV (376 amino acids); a "pig-like" human apoA-IV, lacking the C-terminal EQQQ repeats (361 amino acids); and a "chicken-like" apoA-IV, further truncated to 343 amino acids, were expressed in IPEC-1 cells. With increasing protein secretion, cells expressing the full-length human apoA-IV displayed a 2-fold increase in TG secretion; in sharp contrast, cells expressing the pig-like human apoA-IV displayed a 25-fold increase in TG secretion and a 27-fold increase in lipoprotein diameter. When human apoA-IV was further truncated to yield a chicken-like protein, TG secretion was inhibited. We conclude that overexpression of swine apoA-IV enhances basolateral TG secretion in a dose-dependent manner by increasing the size of secreted lipoproteins. These data suggest that the region in the human apoA-IV protein from residues 344 to 354 is critical to its ability to enhance lipid secretion, perhaps by enabling the packaging of additional core TG into chylomicron particles. The EQQQ-rich region may play an inhibitory or modulatory role in chylomicron packaging in humans.

Overexpression of apolipoprotein A-IV enhances lipid secretion in IPEC-1 cells by increasing chylomicron size.

Apolipoprot&eacute;ine intestinale expression A-IV est fortement r&eacute;glement&eacute;e par des lipides alimentaires chez les porcs nouveau-n&eacute;s, ce qui sugg&egrave;re un r&ocirc;le dans l&#39;absorption des lipides. Surexpression constitutive de l&#39;apoA-IV dans les ent&eacute;rocytes de porcs nouveau-n&eacute;s am&eacute;liore la s&eacute;cr&eacute;tion basolat&eacute;rale de triacylglyc&eacute;rol (TG) dans les lipoprot&eacute;ines riches en TG 4,9 (Lu, S., Yao, Y., Meng, S., Cheng, x. et noir, D. D. (2002) J. Biol. Chem. 277, 31929-31937). Pour &eacute;tudier le m&eacute;canisme de cette am&eacute;lioration, les cellules de l&#39;IPEC-1 ont &eacute;t&eacute; transfect&eacute;es avec un syst&egrave;me d&#39;expression t&eacute;tracycline-r&eacute;glementable (Tet-On). Dans les cellules incub&eacute;es avec de l&#39;acide ol&eacute;ique, une relation dose-effet a &eacute;t&eacute; observ&eacute;e entre la concentration moyenne de doxycycline et basolat&eacute;rale apoA-IV et la s&eacute;cr&eacute;tion de TG. R&eacute;glement&eacute;s de m&ecirc;me l&#39;expression de l&#39;apoA-I n&#39;ont pas stimul&eacute; la s&eacute;cr&eacute;tion lipidique. Le diam&egrave;tre moyen des lipoprot&eacute;ines riches en TG s&eacute;cr&eacute;t&eacute;e par les cellules trait&eacute;es doxycycline &eacute;tait sup&eacute;rieur de cellules non trait&eacute;es (87,0 nm contre 53,4 nm). S&eacute;cr&eacute;tion d&#39;apoB basolat&eacute;rales diminue. Utilisant le m&ecirc;me syst&egrave;me d&#39;expression, pleine longueur humaine apoA-IV (376 amino-acides) ; un &laquo; porc-like &raquo; humain apoA-IV, manque le EQQQ C-terminal r&eacute;p&egrave;te (361 aminoacides) ; et un &laquo; poulet-like &raquo; apoA-IV, outre tronqu&eacute; &agrave; 343 acides amin&eacute;s, ont &eacute;t&eacute; exprim&eacute;s dans les cellules de l&#39;IPEC-1. Avec l&#39;augmentation de la s&eacute;cr&eacute;tion de prot&eacute;ines, cellules exprimant l&#39;apoA-IV humaine pleine longueur a affich&eacute; une augmentation de 2 fois dans la s&eacute;cr&eacute;tion de TG. bien au contraire les cellules exprimant le porc comme humain apoA-IV affichent 25 fois une augmentation de s&eacute;cr&eacute;tion de la TG et &eacute;tant une augmentation de diam&egrave;tre de lipoprot&eacute;ines. Lorsque les humain apoA-IV a &eacute;t&eacute; tronqu&eacute;e davantage pour produire une prot&eacute;ine poulet, s&eacute;cr&eacute;tion de TG est inhib&eacute;e. Nous concluons que la surexpression de l&#39;esp&egrave;ce porcine apoA-IV am&eacute;liore la s&eacute;cr&eacute;tion basolat&eacute;rale TG de mani&egrave;re dose-d&eacute;pendante en augmentant la taille des lipoprot&eacute;ines s&eacute;cr&eacute;t&eacute;es. Ces donn&eacute;es sugg&egrave;rent que la r&eacute;gion de la prot&eacute;ine humaine apoA-IV de r&eacute;sidus 344 &agrave; 354 est essentielle &agrave; sa capacit&eacute; &agrave; am&eacute;liorer la s&eacute;cr&eacute;tion lipidique, peut-&ecirc;tre en permettant le conditionnement de base suppl&eacute;mentaire TG en particules de chylomicrons. La r&eacute;gion riche en EQQQ jouerait un r&ocirc;le inhibiteur ou modulateur en emballage de chylomicrons chez l&#39;homme.

La surexpression de l&#39;apolipoprot&eacute;ine A-IV am&eacute;liore la s&eacute;cr&eacute;tion de lipides dans les cellules de l&#39;IPEC-1 en augmentant la taille des chylomicrons.

Intestinale Apolipoprotein A-IV Ausdruck ist stark von di&auml;tetischen Lipid in Neugeborene Schweine, schl&auml;gt eine Rolle bei der Lipid-Resorption reguliert. Konstitutive &Uuml;berexpression des ApoA-IV in Neugeborene Schweine Enterozyten verbessert Basolateraler Sekretion von Triacylglycerol (TG) im TG-reiche Lipoproteine 4.9-fold (Lu, S., Yao, Y., Meng, S., Cheng, x., und schwarze, D. D. (2002) J. Biol. Chem. 277, 31929-31937). Um den Mechanismus dieser Erweiterung zu untersuchen, wurden die IPEC-1 Zellen mit einem Tetracyclin-dosierbare Expressionssystem transfected (Tet-On). In Zellen inkubiert mit &Ouml;ls&auml;ure wurde eine Dosis-Wirkungs-Beziehung zwischen mittlerer Doxycyclin Konzentration und Basolateraler ApoA-IV TG Sekretion beobachtet. Ebenso reguliert Ausdruck des ApoA-ich nicht Lipid-Sekretion erh&ouml;hen. Der mittlere Durchmesser der TG-reiche Lipoproteine von Doxycyclin-behandelten Zellen abgesondert war gr&ouml;&szlig;er als von unbehandelten Zellen (87.0 nm gegen&uuml;ber 53,4 nm). Basolateraler ApoB Sekretion verringert. Im gleichen Ausdruck System in menschlichen ApoA-IV (376 Aminos&auml;uren); ein &quot;Schwein-artige&quot; menschlicher ApoA-IV fehlt der C-terminalen EQQQ wiederholt (361 Aminos&auml;uren); und ein &quot;Huhn-artige&quot; ApoA-IV, weiter auf 343 Aminos&auml;uren, gek&uuml;rzt in IPEC-1 Zellen ausgedr&uuml;ckt wurden. Mit zunehmender Protein Sekretion, Zellen auszudr&uuml;cken, die in menschliche ApoA-IV wird eine 2-fold Steigerung im TG-Sekretion angezeigt; in scharfem Kontrast angezeigt Zellen zum Ausdruck der Schwein-wie menschlichen ApoA-IV ein 25-fold Anstieg der TG-Sekretion und eine 27-fold Zunahme der Lipoprotein-Durchmesser. Wenn menschliche ApoA-IV weiter abgeschnitten wurde, um ein Huhn-wie Protein zu erhalten, wurde TG Sekretion gehemmt. Wir schlie&szlig;en, dass eine &Uuml;berexpression von Schweinen ApoA-IV verbessert Basolateraler TG-Sekretion eine dosisabh&auml;ngige Weise durch die Erh&ouml;hung der Gr&ouml;&szlig;e der sezernierte Lipoproteine. Diese Daten legen nahe, dass die Region im menschlichen ApoA-IV Protein aus R&uuml;ckst&auml;nden 344-354 entscheidend f&uuml;r seine F&auml;higkeit, Lipid-Sekretion, vielleicht zu verbessern, durch die Aktivierung der Verpackung &uuml;ber zus&auml;tzliche Core TG in Chylomikronen Teilchen. EQQQ-reiche Region kann eine hemmende oder modulierende Rolle in Chylomikronen Verpackung beim Menschen spielen.

Eine &Uuml;berexpression von Apolipoprotein A-IV verbessert Lipid-Sekretion in IPEC-1 Zellen von Chylomikronen-Gr&ouml;&szlig;e erh&ouml;hen.

Intestinale apolipoproteina espressione A-IV &egrave; altamente regolato da lipidi alimentari nel neonato suina, suggerendo un ruolo nell&#39;assorbimento dei lipidi. Costitutiva sovraespressione di apoA-IV nel neonato suina enterociti aumenta la secrezione basolaterale di triacilglicerolo (TG) in lipoproteine ricche di TG 4.9-fold (Lu, S., Yao, Y., Meng, S., Cheng, x. e il nero, D. D. (2002) j Biol Chem. 277, 31929-31937). Per studiare il meccanismo di questo miglioramento, IPEC-1 cellule erano transfected con un sistema di espressione regolabile tetraciclina (Tet-On). In cellule incubate con acido oleico, &egrave; stata osservata una relazione di risposta dose tra concentrazione medio doxiciclina e basolaterale apoA-IV e secrezione di TG. Regolato allo stesso modo espressione di apoA-non ha fatto migliorare la secrezione lipidica. Il diametro medio delle lipoproteine ricche di TG secernuti dalle cellule trattate con doxiciclina &egrave; stata pi&ugrave; grande da cellule non trattate (87,0 nm contro 53,4 nm). Diminuita secrezione apoB basolaterale. Utilizzando lo stesso sistema di espressione, full-length umano apoA-IV (376 aminoacidi); un &quot;maiale-like&quot; umano apoA-IV, manca il C-terminale EQQQ ripete (361 amminoacidi); e un &quot;pollo-come&quot; apoA-IV, ulteriormente troncato a 343 gli amminoacidi, sono stati espressi nelle cellule IPEC-1. Con l&#39;aumento della secrezione di proteine, le cellule che esprimono il full-length umano apoA-IV visualizzato un aumento di 2 volte nella secrezione di TG; in netto contrasto, le cellule che esprimono il maiale-come umano apoA-IV visualizzato un ghiacciamento aumento della secrezione di TG e un 27-fold aumento del diametro delle lipoproteine. Quando umano apoA-IV pi&ugrave; ulteriormente &egrave; stato troncato per produrre una proteina di pollo-come, &egrave; stata inibita secrezione TG. Concludiamo che sovraespressione della suina apoA-IV migliora la secrezione basolaterale TG in modo dose-dipendente aumentando le dimensioni delle lipoproteine secrete. Questi dati suggeriscono che la regione della proteina umana apoA-IV da residui 344-354 &egrave; fondamentale per la sua capacit&agrave; di aumentare la secrezione lipidica, forse consentendo il confezionamento di ulteriori core TG in particelle dei chilomicroni. La regione ricca di EQQQ possa svolgere un ruolo inibitorio o schematizzato nella confezione dei chilomicroni in esseri umani.

Sovraespressione di apolipoproteina A-IV migliora la secrezione dei lipidi nelle cellule IPEC-1 aumentando la dimensione dei chilomicroni.

&#x8178;&#x30A2;&#x30DD;&#x30EA;&#x30DD;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA; A-IV &#x5F0F;&#x9AD8;&#x8102;&#x8CEA;&#x5438;&#x53CE;&#x306B;&#x304A;&#x3051;&#x308B;&#x5F79;&#x5272;&#x3092;&#x793A;&#x5506;&#x3057;&#x3066;&#x3001;&#x65B0;&#x751F;&#x5150;&#x306E;&#x8C5A;&#x306B;&#x98DF;&#x4E8B;&#x8102;&#x8CEA;&#x306B;&#x3088;&#x3063;&#x3066;&#x898F;&#x5236;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;A-IV &#x65B0;&#x751F;&#x5150;&#x8C5A;&#x8178;&#x5185;&#x306E;&#x69CB;&#x6210;&#x306E;&#x904E;&#x5270;&#x767A;&#x73FE;&#x3092;&#x9AD8;&#x3081;&#x308B;&#x57FA;&#x5E95;&#x5206;&#x6CCC;&#x306E; TG &#x30EA;&#x30C3;&#x30C1;&#x30EA;&#x30DD; &#x30C8;&#x30EA;&#x30A2;&#x30B7;&#x30EB;&#x30B0;&#x30EA;&#x30BB; &#x30ED;&#x30FC;&#x30EB; (TG) &#x306E; 4.9-fold (Lu&#x3001;s.&#x3001;&#x516B;&#x5C3E;&#x5E02;&#x8C4A;, &#x5B5F;&#x3001;&#x8061;&#x30FB; &#x30C1;&#x30A7;&#x30F3;&#x3001;X. &#x3068;&#x9ED2;&#x3001;D. D. (2002) J. Biol. chem. 277&#x3001;31929 31937)&#x3002;&#x3053;&#x306E;&#x6A5F;&#x80FD;&#x62E1;&#x5F35;&#x6A5F;&#x69CB;&#x3092;&#x8ABF;&#x3079;&#x308B;&#x305F;&#x3081;&#x306B;&#x3001;IPEC 1 &#x7D30;&#x80DE;&#x30C6;&#x30C8;&#x30E9;&#x30B5;&#x30A4;&#x30AF;&#x30EA;&#x30F3;&#x306B;&#x3088;&#x3063;&#x3066;&#x4FC3;&#x9032;&#x3055;&#x308C;&#x308B;&#x8868;&#x73FE;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x304C;&#x5C0E;&#x5165;&#x3055;&#x308C;&#x305F; (&#x30C6;&#x30C8;&#x4E0A;)&#x3002;&#x30AA;&#x30EC;&#x30A4;&#x30F3;&#x9178;&#x3068;&#x57F9;&#x990A;&#x7D30;&#x80DE;&#x3067;&#x306F;&#x3001;&#x7528;&#x91CF;&#x53CD;&#x5FDC;&#x95A2;&#x4FC2;&#x30DF;&#x30C7;&#x30A3;&#x30A2;&#x30E0; &#x30C9;&#x30AD;&#x30B7;&#x30B5;&#x30A4;&#x30AF;&#x30EA;&#x30F3;&#x306E;&#x6FC3;&#x5EA6;&#x3068;&#x57FA;&#x5E95; A-IV &#x3068; TG &#x5206;&#x6CCC;&#x304C;&#x8A8D;&#x3081;&#x3089;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x540C;&#x69D8;&#x306B;&#x898F;&#x5236;&#x306E;&#x6574;&#x5F62;&#x5F0F;-&#x8102;&#x8CEA;&#x306E;&#x5206;&#x6CCC;&#x3092;&#x4FC3;&#x9032;&#x3057;&#x306A;&#x304B;&#x3063;&#x305F;&#x306F;&#x306A;&#x304F;&#x3002;&#x30C9;&#x30AD;&#x30B7;&#x30B5;&#x30A4;&#x30AF;&#x30EA;&#x30F3;&#x6295;&#x4E0E;&#x7D30;&#x80DE;&#x304B;&#x3089;&#x5206;&#x6CCC;&#x3055;&#x308C;&#x308B; TG &#x30EA;&#x30C3;&#x30C1;&#x30EA;&#x30DD;&#x306E;&#x5E73;&#x5747;&#x76F4;&#x5F84;&#x304B;&#x3089;&#x672A;&#x51E6;&#x7406;&#x7D30;&#x80DE;&#x3088;&#x308A;&#x5927;&#x304D;&#x304B;&#x3063;&#x305F; (87.0 nm 53.4 &#x5BFE; nm)&#x3002;&#x57FA;&#x5E95; apoB &#x5206;&#x6CCC;&#x3092;&#x6E1B;&#x5C11;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x30D5;&#x30EB;&#x30EC;&#x30F3;&#x30B0;&#x30B9;&#x4EBA;&#x9593; A-IV &#xFF08;376 &#x30A2;&#x30DF;&#x30CE;&#x9178;&#xFF09;; &#x540C;&#x3058;&#x8868;&#x73FE;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x3092;&#x4F7F;&#x7528;&#x300C;&#x8C5A;&#x306E;&#x3088;&#x3046;&#x306A;&#x300C;&#x4EBA;&#x9593; C &#x30BF;&#x30FC;&#x30DF;&#x30CA;&#x30EB; EQQQ &#x6B20;&#x3051;&#x3066; A-IV&#x3001;&#xFF08;361 &#x30A2;&#x30DF;&#x30CE;&#x9178;&#xFF09; &#x7E70;&#x308A;&#x8FD4;&#x3055;&#x308C;&#x307E;&#x3059;;&#x300C;&#x30C1;&#x30AD;&#x30F3;&#x306E;&#x3088;&#x3046;&#x306A;&#x300D;&#x3055;&#x3089;&#x306B; 343 &#x30A2;&#x30DF;&#x30CE;&#x9178;&#x306B;&#x5207;&#x308A;&#x6368;&#x3066; A-IV&#x3001;IPEC 1 &#x7D30;&#x80DE;&#x306E;&#x8868;&#x73FE;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x5206;&#x6CCC;&#x5897;&#x52A0;&#x306B;&#x4F34;&#x3044;, &#x30D5;&#x30EB;&#x30EC;&#x30F3;&#x30B0;&#x30B9;&#x4EBA;&#x9593; A-IV &#x767A;&#x73FE;&#x7D30;&#x80DE;&#x306F; TG &#x5206;&#x6CCC;&#x306E; 2 &#x500D;&#x5897;&#x52A0;&#x3092;&#x8868;&#x793A;;&#x5BFE;&#x7167;&#x7684;&#x306B;&#x3001;&#x8C5A;&#x306E;&#x3088;&#x3046;&#x306A;&#x4EBA;&#x9593; A-IV &#x767A;&#x73FE;&#x7D30;&#x80DE;&#x306F; TG &#x5206;&#x6CCC;&#x306E; 25-fold &#x5897;&#x52A0;&#x3068;&#x30EA;&#x30DD;&#x86CB;&#x767D;&#x76F4;&#x5F84;&#x306E; 27-fold &#x5897;&#x52A0;&#x8868;&#x793A;&#x3002;&#x4EBA;&#x9593; A-IV &#x306F;&#x3055;&#x3089;&#x306B;&#x9D8F;&#x306E;&#x3088;&#x3046;&#x306A;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x53CE;&#x91CF;&#x304C;&#x5207;&#x308A;&#x6368;&#x3066;&#x3089;&#x308C;&#x305F;&#x3068;&#x304D;&#x306F;&#x3001;TG &#x5206;&#x6CCC;&#x306F;&#x6291;&#x5236;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x6211;&#x3005; &#x306F;&#x904E;&#x5270;&#x767A;&#x73FE;&#x3092;&#x7DE0;&#x7D50;&#x8C5A;&#x306E; A-IV &#x57FA;&#x5E95; TG &#x5206;&#x6CCC;&#x306F;&#x7528;&#x91CF;&#x4F9D;&#x5B58;&#x7684;&#x306B;&#x5206;&#x6CCC;&#x3055;&#x308C;&#x305F;&#x30EA;&#x30DD;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x30B5;&#x30A4;&#x30BA;&#x3092;&#x5897;&#x3084;&#x3059;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x5411;&#x4E0A;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x6B8B;&#x57FA; 344 &#x306B; 354 &#x304B;&#x3089;&#x4EBA;&#x9593; A-IV &#x86CB;&#x767D;&#x8CEA;&#x306E;&#x5730;&#x57DF;&#x304C;&#x3001;&#x6A5F;&#x80FD;&#x8FFD;&#x52A0;&#x306E;&#x30B3;&#x30A2; TG &#x30E9;&#x30E1;&#x30E9;&#x7C92;&#x5B50;&#x3078;&#x306E;&#x30D1;&#x30C3;&#x30B1;&#x30FC;&#x30B8;&#x5316;&#x3092;&#x6709;&#x52B9;&#x306B;&#x3057;&#x3066;&#x3001;&#x304A;&#x305D;&#x3089;&#x304F;&#x8102;&#x8CEA;&#x306E;&#x5206;&#x6CCC;&#x3092;&#x9AD8;&#x3081;&#x308B;&#x306B;&#x91CD;&#x8981;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x305F;.EQQQ &#x306E;&#x8C4A;&#x5BCC;&#x306A;&#x5730;&#x57DF;&#x963B;&#x5BB3;&#x307E;&#x305F;&#x306F;&#x97F3;&#x306E;&#x5F79;&#x5272;&#x306E;&#x30E9;&#x30E1;&#x30E9;&#x5305;&#x88C5;&#x4EBA;&#x9593;&#x306E;&#x518D;&#x751F;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;

&#x30A2;&#x30DD;&#x30EA;&#x30DD;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA; A-IV &#x306E;&#x904E;&#x5270;&#x767A;&#x73FE; IPEC 1 &#x7D30;&#x80DE;&#x5185;&#x306E;&#x8102;&#x8CEA;&#x306E;&#x5206;&#x6CCC;&#x30E9;&#x30E1;&#x30E9; &#x30B5;&#x30A4;&#x30BA;&#x3092;&#x5897;&#x3084;&#x3059;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x5F37;&#x5316;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;

&#xC7A5; apolipoprotein A IV &#xC2DD; &#xB192;&#xC740; &#xC2DD;&#xC774; &#xC9C0;&#xC9C8; &#xC9C0;&#xC9C8; &#xD761;&#xC218; &#xD558;&#xB294; &#xC5ED;&#xD560;&#xC744; &#xC81C;&#xC548; &#xD558;&#xB294; &#xC2E0;&#xC0DD;&#xC544; &#xB3FC;&#xC9C0;&#xC5D0; &#xC758;&#xD574; &#xADDC;&#xC81C; &#xB429;&#xB2C8;&#xB2E4;. &#xAC13; &#xD0DC;&#xC5B4;&#xB09C; &#xB3FC;&#xC9C0; enterocytes&#xC5D0; apoA-4&#xC758; &#xC81C;&#xC815; overexpression 4.9-fold TG &#xD48D;&#xBD80;&#xD55C; &#xC9C0; &#xB2E8;&#xBC31;&#xC9C8;&#xC5D0; triacylglycerol (TG)&#xC758; basolateral &#xBD84; &#xBE44; &#xD5A5;&#xC0C1; (Lu, &#xBBF8;, &#xC57C; &#xC624;, Y., &#xBA69;, S., &#xCCC9;, &#xC5D1;&#xC2A4;, &#xADF8;&#xB9AC;&#xACE0; &#xBE14;&#xB799;, &#xB514; &#xB514; (2002) J. Biol. &#xD654;&#xD559; 277, 31929 31937). &#xC774; &#xD5A5;&#xC0C1; &#xD558;&#xB294; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC744; &#xC870;&#xC0AC;, IPEC 1 &#xC140; &#xD56D;&#xC0DD;&#xBB3C;&#xC9C8; regulatable &#xC2DD; &#xC2DC;&#xC2A4;&#xD15C;&#xACFC; &#xD398; &#xD588;&#xB2E4; (Tet&#xC5D0;). &#xC62C;&#xB808;&#xC0B0;&#xACFC; &#xC778; &#xD050;&#xBCA0;&#xC774; &#xD305; &#xD558;&#xB294; &#xC140;&#xC5D0; &#xC911;&#xAC04; &#xC544;&#xBAA9;&#xC2DC;&#xC2E4;&#xB9B0;&#xACFC; &#xB18D;&#xB3C4;, basolateral apoA IV &#xBC0F; TG &#xBD84; &#xBE44; &#xC0AC;&#xC774; &#xBCF5;&#xC6A9;&#xB7C9; &#xC751;&#xB2F5; &#xAD00;&#xACC4; &#xAD00;&#xCC30; &#xB418;&#xC5C8;&#xB2E4;. &#xB9C8;&#xCC2C;&#xAC00;&#xC9C0;&#xB85C; apoA &#xD45C;&#xD604;&#xC758; &#xADDC;&#xC81C;-&#xC9C0;&#xC9C8; &#xBD84; &#xBE44;&#xB97C; &#xAC15;&#xD654; &#xD558;&#xC9C0; &#xC54A;&#xC558;&#xB2E4;. Doxycycline &#xCE58;&#xB8CC; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xBD84; &#xBE44; &#xD558;&#xB294; TG &#xD48D;&#xBD80;&#xD55C; &#xC9C0; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xD3C9;&#xADE0; &#xC9C1;&#xACBD;&#xC740; &#xCE58;&#xB8CC; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xBCF4;&#xB2E4; &#xD070; (87.0 53.4 &#xB300; &#xB274; &#xBA55;&#xC2DC;&#xCF54; &#xB274; &#xBA55;&#xC2DC;&#xCF54;). Basolateral apoB &#xBD84; &#xBE44; &#xAC10;&#xC18C;. &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xB3D9;&#xC77C;&#xD55C; &#xC2DD; &#xC2DC;&#xC2A4;&#xD15C;, &#xC804;&#xCCB4; &#xAE38;&#xC774; &#xC778;&#xAC04;&#xC758; apoA IV (376 &#xC544;&#xBBF8;&#xB178;&#xC0B0;); &quot;&#xB3FC;&#xC9C0; &#xAC19;&#xC740;&quot; &#xC778;&#xAC04;&#xC758; apoA-IV&#xB97C;, C &#xD130;&#xBBF8;&#xB110; EQQQ &#xBD80;&#xC871; &#xD55C; &#xBC18;&#xBCF5; (361 &#xC544;&#xBBF8;&#xB178;&#xC0B0;); &#xADF8;&#xB9AC;&#xACE0;&#xB294; &quot;&#xCE58;&#xD0A8; &#xAC19;&#xC740;&quot; apoA-4, 343 &#xC544;&#xBBF8;&#xB178;&#xC0B0;, &#xC798;&#xB9BD;&#xB2C8;&#xB2E4; &#xCD94;&#xAC00; IPEC 1 &#xC140;&#xC5D0; &#xD45C;&#xD604; &#xD588;&#xB2E4;. &#xB2E8;&#xBC31;&#xC9C8; &#xBD84; &#xBE44; &#xC99D;&#xAC00; &#xD568;&#xAED8; &#xC804;&#xCCB4; &#xAE38;&#xC774; &#xC778;&#xAC04;&#xC758; apoA IV&#xB97C; &#xD45C;&#xD604; &#xD558;&#xB294; &#xC138;&#xD3EC;&#xB294; TG &#xBD84; &#xBE44;; 2-fold &#xC99D;&#xAC00; &#xD45C;&#xC2DC; &#xC608;&#xB9AC;&#xD55C; &#xB300;&#xC870;&#xC5D0;&#xC11C; &#xB3FC;&#xC9C0; &#xAC19;&#xC740; &#xC778;&#xAC04; apoA-IV&#xB97C; &#xD45C;&#xD604; &#xD558;&#xB294; &#xC140; TG &#xBD84; &#xBE44;&#xC758; 25-fold &#xC99D;&#xAC00; &#xB2E8;&#xBC31; &#xC9C1;&#xACBD;&#xC5D0;&#xC11C; 27-fold &#xC99D;&#xAC00; &#xD45C;&#xC2DC; &#xB429;&#xB2C8;&#xB2E4;. &#xC778;&#xAC04;&#xC758; apoA IV &#xB2ED;&#xACE0;&#xAE30; &#xAC19;&#xC740; &#xB2E8;&#xBC31;&#xC9C8;&#xC744; &#xC5BB;&#xC744; &#xCD94;&#xAC00; &#xC798;&#xB838;&#xC2B5;&#xB2C8;&#xB2E4;, TG &#xBD84; &#xBE44; &#xC800;&#xD574; &#xD588;&#xB2E4;. &#xC6B0;&#xB9AC;&#xAC00; &#xACB0;&#xB860;&#xC744; &#xADF8; overexpression &#xB3FC;&#xC9C0; apoA IV &#xD5A5;&#xC0C1; basolateral TG &#xBD84; &#xBE44; &#xBCF5;&#xC6A9;&#xB7C9; &#xC758;&#xC874; &#xBC29;&#xC2DD;&#xC5D0;&#xC11C; &#xBE44; &#xC9C0; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xD06C;&#xAE30;&#xB97C; &#xB298;&#xB9AC;&#xBA74;. &#xC774;&#xB7EC;&#xD55C; &#xB370;&#xC774;&#xD130; &#xC794;&#xB958;&#xBB3C; 344 354&#xC5D0;&#xC11C;&#xC5D0;&#xC11C; &#xC778;&#xAC04;&#xC758; apoA IV &#xB2E8;&#xBC31;&#xC9C8;&#xC5D0;&#xC11C; &#xC9C0;&#xC5ED; &#xCD94;&#xAC00; &#xCF54;&#xC5B4; TG chylomicron &#xC785;&#xC790;&#xB85C; &#xD3EC;&#xC7A5;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC544;&#xB9C8;&#xB3C4; &#xC9C0;&#xC9C8; &#xBD84; &#xBE44;&#xB97C; &#xAC15;&#xD654; &#xD558;&#xB294; &#xB2A5;&#xB825;&#xC744; &#xC911;&#xC694; &#xD55C; &#xC81C;&#xC548;. EQQQ &#xD48D;&#xBD80;&#xD55C; &#xC9C0;&#xC5ED; &#xC778;&#xAC04;&#xC5D0;&#xC11C; chylomicron &#xD3EC;&#xC7A5;&#xC5D0;&#xC11C; &#xAE08;&#xC9C0; &#xB610;&#xB294; modulatory &#xC5ED;&#xD560;&#xC744; &#xC7AC;&#xC0DD;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

Apolipoprotein A 4&#xC758; overexpression chylomicron &#xD06C;&#xAE30;&#xB97C; &#xB298;&#xB824; IPEC-1 &#xC138;&#xD3EC;&#xC5D0; &#xC9C0;&#xC9C8; &#xBD84; &#xBE44;&#xB97C; &#xD5A5;&#xC0C1; &#xC2DC;&#xD0B5;&#xB2C8;&#xB2E4;.

Intestinal apolipoprotein expresi&oacute;n A-IV est&aacute; altamente regulada por l&iacute;pidos diet&eacute;ticos en los cerdos reci&eacute;n nacidos, sugiriendo un papel en la absorci&oacute;n de los l&iacute;pidos. Sobreexpresi&oacute;n constitutiva de apoA-IV en enterocitos de cerdos reci&eacute;n nacidos mejora secreci&oacute;n basolateral de triacilglicerol (TG) en lipoprote&iacute;nas ricas en TG 4.9-fold (Lu, S., Yao, Y., Meng, S., Cheng, x. y negro, D. D. (2002) J. Biol Chem 277, 31929-31937). Para investigar el mecanismo de esta mejora, las c&eacute;lulas IPEC-1 fueron transfectadas con un sistema de expresi&oacute;n tetraciclina-regulable (Tet-On). En las c&eacute;lulas incubadas con &aacute;cido oleico, se observ&oacute; una relaci&oacute;n entre dosis y respuesta entre concentraci&oacute;n media doxiciclina y la basolateral apoA-IV y la secreci&oacute;n de TG. Igualmente regula la expresi&oacute;n de apoA-no mejoran la secreci&oacute;n de l&iacute;pidos. El di&aacute;metro medio de lipoprote&iacute;nas ricas en TG secretada de c&eacute;lulas tratadas con doxiciclina era m&aacute;s grande de las c&eacute;lulas no tratadas (87.0 nm frente a 53.4 nm). Disminuci&oacute;n de la secreci&oacute;n de apoB basolateral. Utilizando el mismo sistema de expresi&oacute;n, apoA-IV humana integral (376 amino&aacute;cidos); un &quot;cerdo-como&quot; humano apoA-IV, falta el EQQQ c-terminal repeticiones (361 amino&aacute;cidos); y un &quot;pollo-como&quot; apoA-IV, m&aacute;s se truncan a 343 amino&aacute;cidos, se expresaron en las c&eacute;lulas IPEC-1. Con el aumento de la secreci&oacute;n de la prote&iacute;na, las c&eacute;lulas que expresan la apoA-IV humano integral muestran un doble aumento en la secreci&oacute;n de TG; en contraste, las c&eacute;lulas que expresan el cerdo-como humano apoA-IV muestran un 25-veces aumento en la secreci&oacute;n de TG y un doblez aumento de di&aacute;metro de la lipoprote&iacute;na. Cuando humano apoA-IV fue truncado m&aacute;s para producir una prote&iacute;na como pollo, secreci&oacute;n de TG fue inhibida. Concluimos que la sobreexpresi&oacute;n de cerdos apoA-IV mejora basolateral TG secreci&oacute;n en forma dosis dependiente por aumentar el tama&ntilde;o de las lipoprote&iacute;nas secretadas. Estos datos sugieren que la regi&oacute;n en la prote&iacute;na humana apoA-IV de residuos 344 a 354 es cr&iacute;tica para su capacidad para aumentar la secreci&oacute;n de l&iacute;pidos, quiz&aacute;s habilitando el embalaje de base adicional TG en part&iacute;culas de quilomicrones. La regi&oacute;n de EQQQ-ricos puede desempe&ntilde;ar un papel inhibitorio o modulador en los empaques de quilomicrones en seres humanos.

Sobreexpresi&oacute;n de apolipoprote&iacute;na A-IV aumenta la secreci&oacute;n de l&iacute;pidos en las c&eacute;lulas IPEC-1 incrementando el tama&ntilde;o de quilomicrones.

The effects of ovarian hormone on neuronal growth and function are well known. However, equally important, but often neglected, are ovarian hormone effects on glia. Our in vivo and in vitro studies show that estradiol modifies both neuronal growth and glial activity and these effects are tightly linked. Estradiol stimulates neurite growth and the release of the glial apolipoprotein E (apoE) in culture studies. Estradiol-stimulated neurite growth in these cultures requires apoE. Estradiol replacement in ovariectomized mice transiently increases the expression of apoE, the low density lipoprotein receptor related protein (LRP) and synaptophysin throughout the brain. Continuous estradiol replacement over two months loses effect on apoE, LRP, and synaptophysin and suppresses reactive gliosis. Estrous cycle variation of glial activation (GFAP) and apoE are not identical. We propose that estradiol (and other ovarian hormones) functions as a zeitgeber to co-ordinate neuronal-glial interactions. Co-ordination assures temporally appropriate excitatory and inhibitory interactions between glia and neurons. With aging and the loss of ovarian cyclicity, some of this co-ordination must be diminished. These observations present significant clinical implications. Approaches to hormone therapy (HT), for diminishing the risk of chronic neurological diseases, need to consider the temporal nature of ovarian hormones in brain repair and plasticity. Moreover, approaches must consider apoE genotype. The neuroprotective effects of HT in numerous chronic age-related diseases may represent effective co-ordination of repair processes rather than direct disease-specific actions. Moreover, the role of glial-derived proteins in neuroprotection should not be ignored.

Estradiol regulation of astroglia and apolipoprotein E: an important role in neuronal regeneration.

Numerous epidemiology studies have shown protective effects of hormone therapy (HT) on chronic neurological diseases. We have proposed that some of the neuroprotective effects of estrogen are mediated by apolipoprotein E (apoE). Polymorphisms of receptors for apoE modify the risk for dementia. To our knowledge, no reports exist showing CNS effects of estrogen replacement on members of the low-density lipoprotein receptor family. The current study focused on the effect of estradiol-17beta (E2) replacement on protein expression of two members of the receptor family, the low-density lipoprotein receptor (LDL-r) and low-density lipoprotein receptor related protein (LRP) in ovariectomized mice. Five days of E2 replacement significantly increased LRP expression in the hippocampus, olfactory bulb and neocortex but not in cerebellum. In contrast, E2 treatment decreased LDL-r protein expression in olfactory bulb. HT modification of both apoE and LRP could have wide-spread effects on cellular function given LRP's manifold signaling functions.

Estradiol replacement increases the low-density lipoprotein receptor related protein (LRP) in the mouse brain.

To understand a possible role for transient potential receptor vanilloid 1 (TRPV1) ion channels in sumatriptan relief of pain mediated by trigeminal nociceptors.

Xiangying Cheng

Davee Department of Neurology and Clinical Neurosciences

Feinberg School of Medicine, Northwestern University

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Xiangying Cheng

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

Feinberg School of Medicine, Northwestern University

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Davee Department of Neurology and Clinical Neurosciences