LU500145B1 - A Method for Studying Acetylation Sites of Autophagy-related Proteins in Mature Adipocytes - Google Patents
A Method for Studying Acetylation Sites of Autophagy-related Proteins in Mature Adipocytes Download PDFInfo
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- LU500145B1 LU500145B1 LU500145A LU500145A LU500145B1 LU 500145 B1 LU500145 B1 LU 500145B1 LU 500145 A LU500145 A LU 500145A LU 500145 A LU500145 A LU 500145A LU 500145 B1 LU500145 B1 LU 500145B1
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Abstract
A method for studying acetylation sites of autophagy-related proteins in mature adipocytes is disclosed in the invention, relating to the technical field of wine formulations. It further comprises the following steps of step I, determining whether there is acetylation modification of autophagy-related protein A. Specifically, culturing adipocytes and inducing them to differentiate into mature adipocytes, and then treating them with deacetylase inhibitors. The method for studying the acetylation site of autophagy-related proteins in mature adipocytes is to identify the acetylation site based on prediction and the specific location of the acetylation modification site can be obtained. The relocation and evaluation of acetylation modification sites makes the identification of acetylation modification sites more accurate and credible, which can greatly improve the credibility of search results. Subsequently, the functional study of acetylation sites is carried out by virus infection, so the time consumed in the research experiment can be shortened.
Description
DESCRIPTION A Method for Studying Acetylation Sites of Autophagy-related Proteins in Mature Adipocytes
TECHNICAL FIELD The invention relates to the technical field of wine formulations, in particular to a method for studying acetylation sites of autophagy-related proteins in mature adipocytes.
BACKGROUND Protein acetylation is the process of adding acetyl groups to protein lysine residues under the action of acetyltransferase. It is a mechanism for cells to control gene expression, protein activity or physiological processes. Acetylation is a post-translational modification method of protein that is necessary to ensure protein activity and has a highly regulatory effect. It can occur in core histones, nearly 40 transcription factors and more than 30 other protein targets, from bacteria to humans. Protein acetylation not only plays a key role in nuclear function, but also has an important regulatory role in various cytoplasmic metabolism, involving cytoskeletal dynamics, energy metabolism, endocytosis and autophagy, and even including transmembrane signal transduction. The identification of acylation sites will be the basis for understanding the molecular mechanism of acetylation. The study of acetylation sites of autophagy-related proteins in adipocytes is of great significance in biology and is a further embodiment of human cognition of life. Therefore, a variety of acetylation sites of autophagy-related proteins in adipocytes have gradually appeared in the biological circles. However, most of the current methods for studying the acetylation sites of autophagy-related proteins in adipocytes are directly staining the adipocytes, and then observing through microscopes and other equipment. However, these methods not only failed to determine whether there is acetylation modification in adipocytes, but also failed to predict the acetylation site, resulting in the blindness in the research process of protein acetylation sites, which easily wastes the valuable time of researchers, and at the same time 1t also reduces the credibility of the experimental results. Therefore, a method for studying the acetylation sites of autophagy- related proteins in mature adipocytes is urgently needed to solve the above problems.
SUMMARY (1) Technical problems to be solved In view of the shortcomings of the prior art, the present invention provides a method for studying the acetylation sites of autophagy-related proteins in mature adipocytes, which solves the problem that most of the current methods for studying autophagy-related protein acetylation are directly staining the adipocytes, and then observing through microscopes and other equipment. Not only is there a lack of judgment on whether there is acetylation modification in adipocytes, but also a lack of prediction of acetylation sites, which leads to much blindness in the research process of protein acetylation sites. In addition, it is easy to waste the valuable time of researchers, and the credibility of the experimental results is reduced as well. (2) Technical solution In order to achieve the above objectives, the present invention adopts a method for studying acetylation sites of autophagy-related proteins in mature adipocytes, which includes the following steps.
Step I.
Determining whether there is acetylation modification of autophagy-related protein A.
Culturing adipocytes and inducing them to differentiate into mature adipocytes, and then treating them with deacetylase inhibitors.
Finally, immunoprecipitation (IP) is used to determine whether there is acetylation modification of protein A.
Step II.
Using mass spectrometry or software to predict possible acetylation sites of protein A.
Step III.
Acetylation site identification.
Tool cell: Using 293T cells to identify acetylation sites and construct plasmids with mutations in acetylation sites; transfecting 293T cells, and observing the changes in the acetylation level of protein A after site mutations by IP method.
Mature adipocytes: Constructing a lentivirus with mutation of acetylation sites and using it to infect mature adipocytes; then observing the changes in the acetylation level of protein A after site mutations by IP method.
Step IV.
Study on the function of acetylation sites.
The functional study of autophagy-related protein A is to study its effect on autophagy, and follow-up research on each pathway is based on different animal models and diseases.
Using overexpression lentivirus of autophagy-related protein A and overexpression lentivirus with acetylation site mutations to infect mature adipocytes respectively.
Observing the changes of autophagosomes with transmission electron microscope.
Carrying out GFP-LC3 lentivirus infection simultaneously.
Then observing the punctate aggregation of GFP-LC3 with a confocal fluorescence microscope, and detecting the expression changes of autophagy marker protein LC3 by Western blot.
After treatment with hydroxychloroquine, the expression changes of autophagy marker protein LC3 are detected by Western blot again, that is, adopting autophagy flux method to detect the effect on autophagy.
Preferably, the adipocyte adopts 3T3-L1 cell line.
Preferably, nicotinamide (NAM) and trichostatin A (TSA) are used as deacetylase inhibitors.
Preferably, the IP method uses protein A antibodies to precipitate mature adipocytes treated with deacetylase inhibitors, and then hybridizes with universal acetylation antibody to determine whether protein A has acetylated modification.
Preferably, when predicting the possible acetylation sites of protein A, the prokaryotic protein acetylation data collected from UniProt, CPLM and NCBI protein databases are used as a reference.
Preferably, the software prediction uses MATLAB software and prediction software platform ProAcePred consturcted by C# programming language, wherein the prediction software platform ProAcePred only requires the user to submit at least one prokaryotic protein sequence, and then it can automatically provide information about the potential acetylation site of the protein, thereby achieving high-throughput prediction of the protein A acetylation sites.
Preferably, the mass spectrometry is the use of the open source free software ProteoWizard to convert the raw data collected by mass spectrum into visual mgf format data. (3) Beneficial effects The beneficial effects of the present invention are now shown as follows.
1. The method for studying the acetylation sites of autophagy-related proteins in mature adipocytes uses mass spectrometry or software to predict the possible acetylation sites of protein À, and the resulting predicted data maps are then compared with the prokaryotic protein acetylation data collected from protein databases such as UniProt, CPLM and NCBI, so the main existing regions of possible acetylation sites of protein A can be further determined. It is possible to quickly, efficiently and accurately obtain the possible acetylation modification site information of protein A without cumbersome gene mutation techniques. It further identifies the acetylation sites on the basis of prediction and obtains the specific position of the acetylation modification site. By relocating and evaluating the acetylation modification site, the identification of the acetylation modification site is more accurate and credible. Therefore, the credibility of the search results can be greatly improved. Subsequently, the method of virus infection is used to study the function of the acetylation sites, which can shorten the time consumed by the research experiment to a certain extent. Moreover, the method is reliable and has good repeatability.
2. In the method for studying the acetylation sites of autophagy-related proteins in mature adipocytes, deacetylase inhibitors that can interfere with the function of protein A deacetylase and increase the acetylation degree of protein A in the cell are used. By using
IP, a method of using antibody-specific reaction to purify and enrich the target protein A, the resulting precipitate can be used to determine whether there is acetylation modification in the protein A.
3. Based on this method, through identification of acetylation sites, after mature adipocytes are infected by lentivirus, IP is used to observe the changes in the acetylation level of protein A after site mutations, and the transfection test of tool cells is used as a reference. Then the identification results of the acetylation site can be obtained. Western blot is used to detect the expression changes of the autophagy marker protein LC3 twice, so the autophagy flux method can be used to detect the effect on autophagy.
DESCRIPTION OF THE INVENTION The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the field without creative work shall fall within the protection scope of the present invention. The present invention provides a method for studying acetylation sites of autophagy- related proteins in mature adipocytes, which includes the following steps. Step I. Determining whether there is acetylation modification of autophagy-related protein A. Culturing adipocytes and inducing them to differentiate into mature adipocytes, and then treating them with deacetylase inhibitors, wherein the deacetylase inhibitors can be NAM and TSA. Finally, IP is used to determine whether there is acetylation modification of protein A.
The IP method uses protein A antibodies to precipitate mature adipocytes treated with deacetylase inhibitors, and then hybridizes with universal acetylation antibody to determine whether protein A has acetylated modification.
Step Il.
Using mass spectrometry or software to predict possible acetylation sites of protein A.
The mass spectrometry is the use of the open source free software ProteoWizard to convert the raw data collected by mass spectrum into visual mgf format data.
Step III.
Acetylation site identification.
Tool cell: Using 293T cells to identify acetylation sites and construct plasmids with mutations in acetylation sites; transfecting 293T cells, and observing the changes in the acetylation level of protein A after site mutations by IP method.
When predicting the possible acetylation sites of protein A, the prokaryotic protein acetylation data collected from UniProt, CPLM and NCBI protein databases are used as a reference.
Besides, the software prediction uses MATLAB software and prediction software platform ProAcePred consturcted by C# programming language, wherein the prediction software platform ProAcePred only requires the user to submit at least one prokaryotic protein sequence, and then it can automatically provide information about the potential acetylation site of the protein, thereby achieving high-throughput prediction of the protein A acetylation sites.
Mature adipocytes: Constructing a lentivirus with mutation of acetylation sites and using it to infect mature adipocytes; then observing the changes in the acetylation level of protein A after site mutations by IP method.
Step IV.
Study on the function of acetylation sites.
The functional study of autophagy-related protein A 1s to study 1ts effect on autophagy, and follow-up research on each pathway is based on different animal models and diseases.
Using overexpression lentivirus of autophagy-related protein A and overexpression lentivirus with acetylation site mutations to infect mature adipocytes respectively.
Observing the changes of autophagosomes with transmission electron microscope.
Carrying out GFP-LC3 lentivirus infection simultaneously.
Then observing the punctate aggregation of GFP-LC3 with a confocal fluorescence microscope, and detecting the expression changes of autophagy marker protein LC3 by Western blot.
After treatment with hydroxychloroquine, the expression changes of autophagy marker protein LC3 are detected by Western blot again, that is, adopting autophagy flux method to detect the effect on autophagy.
Embodiment 1 The identification steps of protein acetylation modification sites based on mass spectrometric data from LTQ Orbitrap XL mass spectrometer, Thermo Company are as follows.
In Embodiment 1, the format of the data collected by the LTQ Orbitrap XL mass spectrometer is “. RAW”, and the open source free software ProteoWizard can be used directly to convert the data into standard mgf format files.
Then performing MASCOT and pFind database search and relocating the acetylation modification site.
Finally, automatically exporting credibility evaluation results and spectrogram in batches.
Using Embodiment 1, the data collected by LTQ Orbitrap XL mass spectrometer from Thermo Company is used to evaluate acetylation sites, so as to obtain acetylation sites with high credibility, which effectively eliminates the false positive probability brought by database search software.
Embodiment 2 Experimental procedure for studying the correlation between protein acetylation and tumor development.
In this Embodiment 2, the clinical samples of breast cancer patients are detected, and the results show that the acetylation level of hsp90 at K292 site in cancer patient tissues is significantly higher than that in normal tissues.
Using ack292-hsp90 antibody to detect hsp90 acetylation level during tumor development in mice.
Then MDA-MB-231 breast cancer cells cultured overnight in 10% FBS medium are digested and diluted into a cell suspension containing 2x10 cells/ml.
Balb/c nude mice are inoculated with 2x 10° MDA-MB-231 cells.
Two weeks after inoculation with tumor cells (the tumor is about 100mm°), mouse tumor samples are collected.
The ack292-hsp90 antibody is then used to detect the acetylation level of hsp90 in K292 site in mouse tumor samples.
The results show that the expression levels of CDC37 and active CDC37 during the development of breast cancer do not change with the development of the tumor.
Similarly, the expression level of hsp90 does not change significantly.
However, the expression level of hsp90 acetylated at K292 site in tumor tissues continues to increase with the continuous development of tumors.
The results show that hsp90 acetylated at K292 is positively correlated with the development of tumors.
The specific embodiments described above further describe the purpose, technical solutions and beneficial effects of the present invention in detail.
It should be understood that the above descriptions are only specific embodiments of the present invention and are not intended to limit the present invention.
Any modification, equivalent replacement, improvement and the like, made within the spirit and principle of the present invention, should be included in the protection scope of the present invention.
Claims (7)
1. A method for studying acetylation sites of autophagy-related proteins in mature adipocytes, characterized by comprising the following steps: Step I; determining whether there is acetylation modification of autophagy-related protein A; culturing adipocytes and inducing them to differentiate into mature adipocytes, and then treating them with deacetylase inhibitors; finally, IP is used to determine whether there is acetylation modification of protein A; Step II; using mass spectrometry or software to predict possible acetylation sites of protein A; Step III; acetylation site identification; tool cell: using 293T cells to identify acetylation sites and construct plasmids with mutations in acetylation sites; transfecting 293T cells, and observing the changes in the acetylation level of protein A after site mutations by IP method; mature adipocytes: constructing a lentivirus with mutation of acetylation sites and using it to infect mature adipocytes; then observing the changes in the acetylation level of protein A after site mutations by IP method; Step IV; study on the function of acetylation sites; the functional study of autophagy-related protein A is to study its effect on autophagy, and follow-up research on each pathway is based on different animal models and diseases; using overexpression lentivirus of autophagy-related protein A and overexpression lentivirus with acetylation site mutations to infect mature adipocytes respectively;
observing the changes of autophagosomes with transmission electron microscope; carrying out GFP-LC3 lentivirus infection simultaneously; then observing the punctate aggregation of GFP-LC3 with a confocal fluorescence microscope, and detecting the expression changes of autophagy marker protein LC3 by Western blot; after treatment with hydroxychloroquine, the expression changes of autophagy marker protein LC3 are detected by western blot again, that is, adopting autophagy flux method to detect the effect on autophagy.
2. The method for studying acetylation sites of autophagy-related proteins in mature adipocytes as stated in Claim 1, characterized in that the adipocyte adopts 3T3-L1 cell line.
3. The method for studying acetylation sites of autophagy-related proteins in mature adipocytes as stated in Claim 1, characterized in that NAM and TSA are used as deacetylase inhibitors.
4. The method for studying acetylation sites of autophagy-related proteins in mature adipocytes as stated in Claim 1, characterized in that the IP method uses protein A antibodies to precipitate mature adipocytes treated with deacetylase inhibitors, and then hybridizes with universal acetylation antibody to determine whether protein A has acetylated modification.
5. The method for studying acetylation sites of autophagy-related proteins in mature adipocytes as stated in Claim 1, characterized in that when predicting the possible acetylation sites of protein A, the prokaryotic protein acetylation data collected from UniProt, CPLM and NCBI protein databases are used as a reference.
6. The method for studying acetylation sites of autophagy-related proteins in mature adipocytes as stated in Claim 1, characterized in that the software prediction uses MATLAB software and prediction software platform ProAcePred consturcted by C# programming language, wherein the prediction software platform ProAcePred only requires the user to submit at least one prokaryotic protein sequence, and then it can automatically provide information about the potential acetylation site of the protein, thereby achieving high-throughput prediction of the protein A acetylation sites.
7. The method for studying acetylation sites of autophagy-related proteins in mature adipocytes as stated in Claim 1, characterized in that the mass spectrometry is the use of the open source free software ProteoWizard to convert the raw data collected by mass spectrum into visual mgf format data.
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