Use of Ell as an E3 ubiquitin ligase

A sequence and protein technology, applied in the application field of ELL as E3 ubiquitin ligase, can solve the problems of non-expression and inactivation

Active Publication Date: 2019-03-05
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Of note, point mutations in FBW7 in human cancers can result in its inactivation and likely non-expression (Farrell and Ears 2014)

Method used

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  • Use of Ell as an E3 ubiquitin ligase
  • Use of Ell as an E3 ubiquitin ligase
  • Use of Ell as an E3 ubiquitin ligase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1, ELL promotes c-Myc degradation

[0078] The amino acid sequence of protein c-Myc is sequence 2, and the nucleotide sequence of its coding gene is sequence 1;

[0079] The amino acid sequence of protein ELL is sequence 4, and the nucleotide sequence of its coding gene is sequence 3.

[0080] The above sequence was artificially synthesized.

[0081] 1. ELL reduces the expression of exogenous Myc

[0082] The plasmid HA-c-Myc (WT) expressing c-Myc is a plasmid obtained by inserting the c-Myc coding gene into the XbaI and BamHI sites of the pCMV-HA vector (Clontech);

[0083] The plasmid HA-ELL expressing ELL is a plasmid obtained by inserting the coding gene of ELL into the SalI and KpnI sites of the pCMV-HA vector;

[0084] Plasmid HA-empty is the pCMV-HA vector.

[0085] Plasmids HA-ELL and HA-empty were co-transfected with HA-c-Myc(WT) at a mass ratio of 1:1 to HEK293 cells. On the second day after transfection, the supernatant was collected for Western ...

Embodiment 2

[0102] Example 2, ELL acts as an E3 ubiquitin ligase to degrade c-Myc protein

[0103] 1. ELL can enhance the ubiquitination of c-Myc

[0104] In order to verify the type of protein degradation caused by ELL, inhibitors were used, including chloroquine (lysosomal proteolysis inhibitor, Chlq) (Sigma, 87111906), ammonium chloride (lysosomal proteolysis inhibitor, NH 4 CL) (Sinopharm, 12125-02-9), AICAR (autophagy inhibitor) (Cayman, 2627-69-2) and MG132 (proteasome inhibitor) (Sigma, M8699) to determine whether they can block ELL-mediated degradation of c-Myc.

[0105] Plasmid HA-ELL, plasmid HA-ELL and plasmid HA-c-Myc were transfected into HEK293 cells respectively. On the second day after transfection, Chlq and AICAR were added to the cell culture medium respectively, and the final concentrations of Chlq and AICAR reached 20mM, continue to culture for 6 hours, collect the supernatant for Western blot detection, the antibody used is shown in the figure. Take no inhibitor as...

Embodiment 3

[0175] Example 3, ELL inhibits the tumor growth of colon xenografts

[0176] The HT116 cells transfected with pHAGE-control, the HT116 cells transfected with pHAGE-ELL, and the HT116 cells transfected with pHAGE-ELL (C595A) in Example 2 were subjected to transplantation tumor growth experiments: 15 male nude mice were randomly divided into 3 groups , 5 in each group, three groups of nude mice were subcutaneously injected with the above three different cell lines, and the amount of cells injected into each nude mouse was 2×10 6 indivual. From the third week, the length, width, and height of the tumor were measured weekly and the tumor volume was calculated using V=π.abc / 6 (Flatz et al. 2010). After 6 weeks, the nude mice were sacrificed, the weight of the tumor was measured and the corresponding gene expression was analyzed. Lung tissues were analyzed histologically after HE staining.

[0177] Tumor volume results such as Image 6 A and 6B, the tumor growth rate of HCT116 c...

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Abstract

The present invention discloses the application of ELL as an E3 ubiquitin ligase, and the present invention provides the application of ELL protein or its truncated body in at least one of the following 1)-6): 1) preparation of degraded c-Myc protein products; 2 ) as E3 ubiquitinase ligase; 3) preparing a product for inhibiting tumor cell proliferation; 4) preparing a product for inhibiting tumor formation; 5) preparing for identification or assisting in identifying whether the tissue to be tested is a tumor tissue product; Test whether the patient is a tumor patient product. The experiment of the present invention proves that the present invention finds that ELL binds to c-Myc and induces it to be degraded by proteasome. Both in vivo and in vitro studies show that ELL has the characteristics of E3 ubiquitin ligase, and ELL can inhibit tumor cell proliferation and tumor formation, It can also be used as a molecular marker for clinical diagnosis of tumors, as a new type of c-Myc E3 ubiquitin ligase, and can be used to prepare tumor-inhibiting drugs or kits, as well as kits for diagnosing tumors.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an application of ELL as an E3 ubiquitin ligase. Background technique [0002] The gene product of ELL was first identified as a translocation fusion (MLL-ELL) with the MLL gene in acute myeloid leukemia (AML) (Thirman et al. 1994). Further biochemical studies found that ELL is a transcription elongation factor. In vitro experiments (In vitro), it can increase the transcription elongation activity of RNA polymerase II. Later in vivo studies (Invivo) revealed that it is associated with transcriptionally active sites of genes (Eissenberg et al. 2002, Shilatifard et al. 1996). ELL is part of two distinct elongation complexes, the super elongation complex (SEC) and the small elongation complex (LEC) (Hu et al. 2013, Luo et al. 2012, Smith and Shilatifard 2013). As one of the most active forms of the positive transcriptional elongation factor (P-TEFb), the super elongation complex (SEC...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00A61K38/43A61P35/00G01N33/574G01N33/573
CPCA61K38/00C12N9/93C12Y603/02019G01N33/573G01N33/57419G01N2333/90
Inventor 肖武汉陈瑜姬伟周迟
Owner INST OF AQUATIC LIFE ACAD SINICA
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