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A key protein numb for hepatic and intestinal cholesterol absorption and its use

A cholesterol-using technology, applied in the field of Numb protein in the absorption of cholesterol, can solve the problem of high energy consumption

Active Publication Date: 2017-02-22
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although cholesterol can be synthesized de novo from simple acetyl-CoA in the body, this process consumes a lot of energy

Method used

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  • A key protein numb for hepatic and intestinal cholesterol absorption and its use
  • A key protein numb for hepatic and intestinal cholesterol absorption and its use
  • A key protein numb for hepatic and intestinal cholesterol absorption and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0187] Example 1 Deletion or mutation of NPC1L1 YVNxxF affects its endocytosis

[0188] 1.1 Transfection of plasmids with different amino acid deletions at the C-terminus of NPC1L1, and observe the changes in the localization of NPC1L1 in cells. The result is as figure 1 As shown in A-B, when the NPC1L1 protein lacks 17 amino acids, after cholesterol administration, NPC1L1 localizes the same as the wild type, mainly in the endocytic cycle complex. But when amino acids 22 and 27 were deleted, NPC1L1 endocytosis was slowed when cholesterol was administered and it remained localized at the plasma membrane. Thus, amino acids 1306-1311 play an important role in the cellular localization of NPC1L1.

[0189] 1.2 Mutate each amino acid at position 1306-1311 of NPC1L1 protein to alanine, transfect CRL1601 cells, 24 hours later, fix the cells with 4% PFA for 15 minutes, permeabilize the cells with 0.2% triton X100, stain with fluorescent antibodies, and confocal after mounting Observ...

Embodiment 2

[0191]Example 2 Effect of Y1306 mutation of NPC1L1 protein on NPC1L1 endocytosis and cholesterol absorption

[0192] Using cells stably expressing wild-type NPC1L1 and Y1306 mutant NPC1L1, first extract intracellular cholesterol, and then deliver cholesterol for different time periods to observe the speed of NPC1L1 endocytosis and cholesterol absorption of the two cells. It can be seen that the wild-type NPC1L1 quickly endocytized into the cells after being given cholesterol, and most of them entered the cells at 60 minutes. At the same time, cholesterol also entered the cells along with NPC1L1 during this process. However, the endocytosis of NPC1L1 after the Y1306 mutation was significantly inhibited, and most of the NPC1L1 remained on the cell membrane when cholesterol was administered for 60 minutes. Similarly, the endocytosis of cholesterol was also inhibited ( figure 2 A, B). This result indicates that NPC1L1 Y1306 mutation will lead to the reduction of NPC1L1 and chole...

Embodiment 3

[0193] Example 3 The effect of the mutation of NPC1L1 protein Y1306 on the absorption of bile cholesterol by NPC1L1 in the liver

[0194] Methods: Adenovirus was injected into the tail vein of mice, and the ability of NPC1L1 to absorb cholesterol in bile was observed by adenovirus-mediated overexpression of NPC1L1. On the fourth day after the injection of adenovirus, the mice were sacrificed, the expression of NPC1L1 in various tissues of the mice was observed, and the cholesterol absorption of the mice was analyzed.

[0195] The result is as image 3 As shown in A-C, NPC1L1 protein is mainly expressed in the liver. There is almost no expression in other major tissues, and the expression levels of NPC1L1WT and Y1306A point mutations are also comparable. In the liver, wild-type NPC1L1 is mainly expressed on the surface of the bile canaliculus, and has a good co-localization with Marker Mrp2 on the surface of the bile canaliculus. However, after the NPC1l1Y1306A point mutatio...

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Abstract

The invention discloses a key protein Numb for cholesterol absorption in liver and intestine and an application thereof. Particularly, disclosed are an application of a Numb protein inhibitor in regulating cholesterol absorption and an application of an inhibitor with combined Numb protein with NPC1L1 protein. The invention further discloses a NPC1L1 mutant protein which can't be combined with Numb, and thus the cholesterol absorption mediated by the NPC1L2 protein can be reduced.

Description

technical field [0001] The present invention relates to the field of biomolecules, in particular to the role of Numb protein in cholesterol absorption. Background technique [0002] Cholesterol is a class of small sterol molecules with the basic structure of cyclopentane polyhydrophenanthrene, which widely exists in various organisms. It is a key component of biofilm structure, regulating membrane fluidity and phase transition. Cholesterol is also the only precursor raw material for the synthesis of bile acids and sterol hormones, which are necessary for life. In particular, the biological functions of cell membranes include many important cell signal transduction pathways, cell activities such as phagocytosis, and cell membrane metabolism. Cholesterol is required Participation. [0003] The amount of cholesterol in the body is strictly regulated, and abnormal cholesterol levels can be very harmful. A large number of studies have shown that high levels of cholesterol in t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/395A61K48/00A61K45/00A61P3/06C12Q1/68C12Q1/02G01N33/68G01N33/53C07K14/47C12N15/12C12N15/85C12N5/10C07K19/00
Inventor 宋保亮李培山缪红华张莹钰
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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