Modulation and control of SENP1 phosphorylated modified compound and SIRT3 SUMOylation modified compound and application of SENP1 phosphorylated modified compound and SIRT3 SUMOylation modified compound

A compound and phosphorylation technology, applied in the field of biomedicine, can solve problems such as unclear mitochondrial metabolic process of SIRT3

Inactive Publication Date: 2018-06-12
SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, under physiological and pathological conditions, how to regulate the activity

Method used

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  • Modulation and control of SENP1 phosphorylated modified compound and SIRT3 SUMOylation modified compound and application of SENP1 phosphorylated modified compound and SIRT3 SUMOylation modified compound
  • Modulation and control of SENP1 phosphorylated modified compound and SIRT3 SUMOylation modified compound and application of SENP1 phosphorylated modified compound and SIRT3 SUMOylation modified compound
  • Modulation and control of SENP1 phosphorylated modified compound and SIRT3 SUMOylation modified compound and application of SENP1 phosphorylated modified compound and SIRT3 SUMOylation modified compound

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Example 1 Immunoprecipitation identification of SIRT3 SUMO modification method

[0049] First transfect SIRT3-Flag and HA-SUMO1 into 293T cells to extract mitochondrial components, and then perform immunoprecipitation experiments with M2-Flagaffinity gel (Sigma, A2220) or HA magnetic beads (Thermo, 88836), and use Flag (Sigma, M2) and HA (Sigma, HA-7) antibodies were detected, and a 51kDa SIRT3SUMO modified band was detected ( figure 1 ). Endogenous SIRT3 SUMOylation was detected by extracting mitochondrial fractions from the liver cancer cell line SMMC7721, immunoprecipitating with SIRT3 (Cell Signaling, 5490) antibodies, and then using SIRT3 (Cell Signaling, 5490) and SUMO1 (Abcam, 32058) antibodies, and also detecting SIRT3SUMO modified band to 49kDa size ( figure 1 ). Then, SIRT3-Flag, HA-SUMO1 and RGS-SENP1 plasmids were co-transfected into 293T cells, mitochondrial proteins were extracted and immunoprecipitated with Flag antibody, and the results of Flag and HA...

Embodiment 2

[0071] Embodiment 2 SIRT3SUMO modification site identification method

[0072] Through protein amino acid sequence alignment, the results show that there is a SUMO-modified consensus sequence ψ-K-χ-D around the 288-position lysine of human SIRT3 protein (ψ is a hydrophobic amino acid residue, χ is any amino acid residue ), and this sequence is conserved among different species ( figure 2 ). Use the following pair of primers to mutate the 288th lysine on the SIRT3 plasmid to arginine by PCR method, then co-transfect the SIRT3K288R plasmid and the SUMO1 plasmid into 293T cells, use the SIRT3WT plasmid as a positive control, and pass M2 -Flag affinity gel (Sigma, A2220) or HA magneticbeads (Thermo, 88836) for immunoprecipitation experiments, using Flag (Sigma, M2) and HA (Sigma, HA-7) antibodies for detection, the results show that SIRT3 lysine 288 After mutation, SIRT3 cannot undergo SUMOylation modification, that is, K288 is the SIRT3 SUMOylation modification site ( image ...

Embodiment 3

[0075] Example 3 proves that SIRT3 SUMO modification has an inhibitory effect on the deacetylation level of SIRT3

[0076] Flag-SIRT3WT or Flag-SIRT3K288R expressing cell lines were established by stably transfecting Flag-SIRT3WT or Flag-SIRT3K288R plasmids in endogenous SIRT3 gene silenced liver cancer cells SMMC7721. Immunoprecipitation was carried out by Acetyl-lys (Cell Signaling, 9441) antibody, and then with known SIRT3 deacetylation-modified target protein antibodies such as SOD2 (Abcam, 13534), LCAD (Abcam, 196655), HMGCS2 (Abcam, 137043 ), AceCS2 (Abcam, 66038), the results showed that SIRT3K288R had higher sirtuin activity than wild type ( Figure 5 ).

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Abstract

The invention discloses SUMOylation modification which can be carried out at the K288th site of deacetylase SIRT3 dependent on NAD in a mitochondrion as well as modulation and control of activity of SIRT3; meanwhile, SUMO specific protease 1 (SENP1) can modulate and control SUMOylation modification of the SIRT3, so that related physiological and pathological processes in which SIRT3 participatingof mitochondrion are modulated and controlled; the modulation and control effect of the SENP1 depends on phosphorylated modification of an SENP1S180 site. The invention discloses the influence of a signal modulation and control path of an SENP1-SIRT3 shaft on metabolism of the mitochondrion, the activation of macrophage and T cells, and tumor immunity and tumor growth; therefore, a significance onprevention and treatment of diseases related with mitochondria metabolism and tumor is realized.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to a SENP1 phosphorylation modification compound and a SIRT3 SUMOylation demodification compound and applications thereof, specifically to the phosphorylation modification of the S180 site of SENP1 and the regulation of the SUMO modification of mitochondrial SIRT3 in physiological processes related to cell mitochondrial metabolism. And the role and application in pathology. Background technique [0002] Mitochondria are important energy metabolism and organelles involved in the regulation of cell metabolism in cells. From outside to inside, it can be divided into four regions: adventitia, intermembrane space, intima, and stroma. Under physiological and pathological conditions, the number, shape and intracellular location of mitochondria are dynamically changed, and this change is related to the function of mitochondria. Recent studies have shown that these changes in mitochondria are rela...

Claims

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Application Information

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IPC IPC(8): A61K45/06A61K38/45A61P35/00A61P37/02A61P3/04A61P39/06A61P25/00A61P31/12
Inventor 程金科王田实曹颖贺兼理屠俊左勇郑铨
Owner SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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