Acetylated polypeptide of human DNM1LK711 lysine, specific antibody and application thereof

Abstract

The invention discloses an acetylated polypeptide of human DNM1LK711 lysine, a specific antibody and application of the acetylated polypeptide, and the amino acid sequence of the acetylated polypeptide of the human DNM1LK711 lysine is as shown in SEQ ID NO: 1; the specific antibody can be specifically combined with the acetylated polypeptide of the human DNM1LK711 lysine; the specific antibody is prepared through a coupling reaction of a specific polypeptide sequence modified by acetylation of lysine (DNM1LK711) at the 711th site of DNM1L protein. The invention further finds that the increase of the acetylation level of the K711 site of the DNM1L protein can promote the function enhancement of the DNM1L to cause abnormal fragmentation of mitochondria under injury pathological conditions such as oxidative stress and the like, thereby promoting the attack and progress of oxidative stress mechanism related degenerative diseases. The method has a potential important value for revealing biological influence of acetylation modification in a disease process, so that the method is expected to generate positive influence on development of future disease treatment strategies.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a human DNM1L K711 Lysine acetylated peptides, specific antibodies and their applications. Background Art

[0002] Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Dysfunction of mitochondrial dynamics is believed to be related to the pathogenesis of PD. Recent studies have shown that abnormal mitochondrial fission mediated by DNM1L can induce mitochondrial dysfunction in dopaminergic neurons. However, the exact mechanism behind this association is still unclear. Therefore, in-depth study of the molecular mechanism of dopaminergic neuron death is of great significance for improving the treatment strategy of PD.

[0003] Mitochondrial fission is mainly driven by DNM1L, a member of the GTPase family, which exists in the cytoplasm as a dimer or tetramer. During mitochondrial fission, DNM1L is recruited to the outer membrane of the mitochondria and assembled into a larger oligomeric fission complex, which then causes mitochondrial fission through GTP hydrolysis. Recent studies have confirmed that DNM1L is involved in the regulation of mitochondrial dynamics impairment in a variety of neurodegenerative diseases, including Parkinson's disease. The function of DNM1L is subject to extensive post-translational regulation, such as phosphorylation, acetylation, ubiquitination, and ubiquitination, which may affect the protein stability of DNM1L and mitochondrial function. Due to the relative lack of phosphorylation events in this organelle, acetylation appears to be one of the most common mitochondrial PTMs. Moreover, with the development of acetylation proteomics technology, it has been found that more than 60% of mitochondrial proteins contain acetylation modification sites. Protein acetylation in mitochondria usually leads to loss of organelle integrity and loss of function of oxidative metabolism-related pathways. Importantly, acetylation of multiple mitochondrial proteins is associated with damage to dopaminergic (DA) neurons. In order to identify new therapeutic targets for PD and explore the upstream regulatory mechanism of mitochondrial dysfunction and DA neuron damage under oxidative stress mediated by PD pathogenic factors, we first confirmed the increase in mitochondrial acetylation levels under oxidative stress conditions, performed acetylation mass spectrometry detection and identified DNM1L for the first time. K711 The K711 antibody was used to verify that DNM1L acetylation is involved in the development of diseases. K711Increased acetylation. In addition, existing acetylation antibodies are pan-antibodies, which usually cross-react with proteins at multiple acetylation sites, making it impossible to accurately determine which specific site has undergone acetylation modification. Since different acetylation sites cannot be distinguished, they cannot be used to study the function and biological significance of specific acetylation sites. In molecular biology and disease research, protein acetylation is the key to regulating its function and signal transduction. Site-specific acetylation antibodies can accurately detect specific acetylation sites, revealing their role in physiology and disease, and providing a new perspective for disease diagnosis and treatment. Summary of the invention

[0004] In view of this, the present invention aims to provide a human DNM1L K711 The lysine acetylated polypeptide, specific antibody and application thereof solve the problem that the existing acetylated antibodies are pan-antibodies, cannot distinguish the K711 acetylation site, and cannot be used to study the function and biological significance of specific acetylation sites.

[0005] The primary purpose of the present invention is to provide human DNM1L K711 Lysine acetylated peptide, human DNM1L K711 The amino acid sequence of the lysine acetylated polypeptide is shown in SEQ ID NO:1.

[0006] The second invention objective of the present invention is to propose a method that can specifically bind to human DNM1L K711 Antibodies specific for lysine acetylated peptides.

[0007] The third object of the present invention is to provide a method for preparing such an antibody.

[0008] The fourth invention objective of the present invention is to propose the application of such antibody.

[0009] In order to achieve the primary purpose of the present invention, the acetylation modification of the K711 site of the DNM1L protein under oxidative stress conditions was determined for the first time in the early stage by mass spectrometry technology.

[0010] In order to achieve the second invention purpose of the present invention, after obtaining the above data, the present invention has developed a new specific antibody specifically targeting the K711 site of the acetylated DNM1L protein. The specific antibody can specifically bind to human DNM1L K711 Lysine acetylated peptides.

[0011] In order to achieve the third invention objective of the present invention, the technical solution adopted is:

[0012] The specific antibody is produced by coupling the acetylated DNM1L specific polypeptide segment to a carrier protein to stimulate and collect a specific immune response.

[0013] Further, the human DNM1L having the amino acid sequence shown in SEQ ID NO: 1 is used. K711 The conjugate of the lysine acetylated polypeptide and the carrier protein is obtained by immunizing a non-human animal and purifying the serum of the non-human animal (eg, New Zealand white rabbit).

[0014] The specific preparation method is as follows:

[0015] (1) Peptide synthesis:

[0016] Synthetic acetylated modified antigen peptide 1mg; non-modified peptide 1mg.

[0017] The sequence of the acetylated modified antigen polypeptide is: SEDMAQRR-(acetyl)K-EAAD; the sequence of the non-modified polypeptide is SEDMAQRRKEAAD.

[0018] (2) Peptide coupling:

[0019] The peptide was coupled to KLH and used for rabbit immunization.

[0020] (3) Animal immunization:

[0021] Six SPF experimental grade New Zealand white rabbits were selected. After six immunizations, blood was collected three times on the 35th, 47th and 52nd days, 30 mL per rabbit each time, and the supernatant was collected after centrifugation.

[0022] (4) Identification of immune serum:

[0023] The titer and specificity of the serum were tested by ELISA or Dotblot.

[0024] (5) Antibody purification:

[0025] The serum was purified and enriched using protein A affinity column and antigen peptide affinity chromatography column.

[0026] On the other hand, the present invention also discloses the following scheme:

[0027] When the lysine at this site mutates to arginine, the DNM1L protein loses the abnormal division caused by acetylation, thereby protecting the morphological function of mitochondria and promoting cell survival.

[0028] In order to achieve the fourth invention objective of the present invention, the technical solution adopted is:

[0029] The present invention also relates to the application of the antibody in detecting lysine acetylation at the 711th site of DNM1L protein.

[0030] Furthermore, the above-mentioned acetylated-modified specific antibody is used to detect the acetylation level of lysine at position 711 of DNM1L protein in disease models including abnormal mitochondrial fission.

[0031] Preferably, the acetylation of lysine at the 711th site of the DNM1L protein can enhance the function of the DNM1L protein, promote mitochondrial fission and reduce the level of fragmented membrane potential, and impair the function of mitochondria; at the same time, when the site mutates to glutamine, that is, K711Q simulates acetylation, it promotes the effect of reducing the level of mitochondrial fission and fragmented membrane potential, thereby promoting the onset and progression of degenerative diseases related to oxidative stress mechanisms; on the contrary, when the site mutates to arginine, that is, K711R simulates deacetylation, mitochondrial fusion increases, that is, the mitochondrial fragmentation caused by the protection of related diseases is aggravated. Therefore, the acetylation of the 711 site can be used to study the molecular mechanism of diseases related to mitochondrial damage.

[0032] Preferably, the application specifically includes the use of the acetylated-modified specific antibody in detecting the lysine acetylation level at site 711 of the DNM1L protein in clinical specimens; determining the correlation between the lysine acetylation level at site 711 of the DNM1L protein and abnormal mitochondrial morphology and function and the ultimate cell or neuron damage in disease models including Parkinson's disease; and guiding the selection of clinical drugs that can inhibit DNM1L acetylation.

[0033] On the other hand, the above-mentioned specific antibody detects the acetylation level of site 711 of DNM1L protein, which can indicate the morphological function of mitochondria.

[0034] Detection of DNM1L in cells or tissues by various antigen-antibody based methods (such as ELISA, WB, IP, IF, IHC, etc.) K711 Develop DNM1L inhibitors K711 A drug for acetylation of the site, and the preparation of monoclonal antibodies or polyclonal antibodies against the acetylation of the site. The application includes that the acetylation site is used as a detection target in diseases including Parkinson's disease to evaluate the morphological function of mitochondria; and is used to guide the development of related drugs that can inhibit DNM1L acetylation and protect abnormal mitochondrial fission.

[0035] By developing the specific antibodies of the present invention, not only can the changes in the levels of acetylation site proteins caused by oxidative stress be detected in disease models, but it also provides a powerful tool for studying mitochondrial function and the molecular mechanisms of oxidative stress-related diseases. This progress will help promote the development of future disease diagnosis and treatment strategies and have a profound impact on related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The K711 site of DNM1L was detected for mass spectrometry;

[0037] Figure 2 DNM1L K711 Mass spectrometry results of lysine acetylated peptides;

[0038] Figure 3 This is the mass spectrometry result of the non-modified peptide of DNM1L (K711ac);

[0039] Figure 4 Dotblot test results of antibody Ab6;

[0040] Figure 5 The specific antibody of the present invention can specifically recognize the K711 site result, and the Western blot detection result of the antibody after HeLa+H2O2 treatment.

[0041] Figure 6 For DNM1L K711 Research results show that acetylation can promote mitochondrial fission;

[0042] Figure 7 For DNM1L K711 Research results show that acetylation promotes mitochondrial function impairment;

[0043] Figure 8 To investigate the changes in the acetylation level of K711 of DNM1L in the classic PD cell model established by using MPP+ to intervene in the SH-SY5Y cell line;

[0044] Fig. 9 To investigate the changes in the acetylation level of K711 of DNM1L in a PD mouse model established using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0046] Commonly used drugs and reagents:

[0047] Most of the drugs and reagents used in this example were purchased from Gibco, Sigma, MCE, Invitrogen, Thermo Fisher Scientific, Millipore, Bio-Rad, Wako, Tsingke Bioengineering and SinoBiological, USA. Most of the antibodies were purchased from Abcam and CellSignaling, USA.

[0048] Embodiment 1:

[0049] This example describes a human DNM1L K711 The acetylated lysine polypeptide has an amino acid sequence as shown in SEQ ID NO: 1.

[0050] SEQ ID NO: 1: SEDMAQRR-(acetyl)K-EAAD.

[0051] The qualitative analysis of the DNM1L protein and K711 acetylation site of this application is as follows:

[0052] After SY5Y cells were treated with H2O2 for oxidative stimulation, cell lysates were collected, and total proteins were separated by SDS-PAGE gel electrophoresis and then stained with Coomassie Brilliant Blue. The gel samples after total protein electrophoresis were subjected to LC-MS / MS analysis.

[0053] The gel samples are digested with a protein endonuclease (usually Trypsin), and then analyzed with LC-MS / MS (nanoLC-QE). Finally, the LCMSMS data are analyzed with mass spectrometry matching software such as MASCOT to obtain qualitative identification information of the target protein polypeptide molecules.

[0054] The specific LC-MS / MS analysis steps are as follows:

[0055] 1) Sample enzymatic hydrolysis:

[0056] After the sample was reduced and alkylated, Trypsin (mass ratio 1:50) was added and enzymatically hydrolyzed at 37°C for 20 hours; the enzymatic hydrolyzate was desalted and freeze-dried, redissolved in 0.1% FA solution, and stored at -20°C for use.

[0057] 2) Mass spectrometry analysis:

[0058] Solution A is a 0.1% formic acid in water, and solution B is a 0.1% formic acid in acetonitrile (84% acetonitrile). After the column is equilibrated with 95% solution A, the sample is loaded onto the Trap column by an automatic sampler. 1H gradient (For complex sample)

[0059] 3) Data analysis:

[0060] The raw file of mass spectrometry test was searched against the corresponding database (as shown in Table 1) using Mascot2.2 software, and finally the identified protein results were obtained.

[0061] Table 1: Database search parameters

[0062]

[0063]

[0064] In summary, the results of sample identification are as follows: sp|O00429|DNM1L_HUMAN Dynamin-1-likeprotein OS=Homo sapiens OX=9606GN=DNM1LPE=1SV=2, etc.

[0065] In addition, it should be noted that the DNM1L protein described in the present application can also be referred to as Drp1 protein.

[0066] like Figure 1 As shown, the results of acetylation mass spectrometry analysis showed that a new lysine acetylation modification appeared at the K711 site of the DRP1 protein, and its specific peptide number (or unique peptide count) was the largest among all the modifications we observed.

[0067] Embodiment 2:

[0068] This example provides a human DNM1L that can specifically bind to the human DNM1L described in Example 1. K711 A method for preparing a specific antibody for a lysine acetylated polypeptide comprises the following steps:

[0069] 1) Peptide synthesis:

[0070] Peptide 1: DNM1L (K711ac) acetylated peptide SEDMAQRR-(acetyl)K-EAAD, 1 mg;

[0071] Peptide 2: Non-modified peptide SEDMAQRRKEAAD, 1 mg.

[0072] according to Figure 2 and Figure 3 It can be seen that the sequences of the two peptides are accurate. The vertical axis represents the intensity of the ion peak, and the horizontal axis represents the ratio of mass to charge. The difference between the measured mass and the theoretical mass of the two peptides is within 10ppm, and the sequence is correct.

[0073] 2) Peptide coupling:

[0074] The polypeptide is coupled to KLH and used for immunization of non-human animals (rabbits, specifically New Zealand white rabbits, are used in this example).

[0075] 3) Animal immunization:

[0076] This example uses 6 SPF experimental New Zealand white rabbits (of course, in actual use, other non-human animals can also be used, not limited to rabbits), dilute the immunogen with physiological saline, and then mix it with the corresponding adjuvant in a 1:1 ratio. The antigen and adjuvant are completely mixed to form a stable emulsion. The antigen mixture is extracted with a syringe and injected into two points under the skin of the shoulders and two points in the muscles of the hind legs of the rabbit. Each area is about 1 / 4 volume of the immunogen. After 6 immunizations, blood is drawn 3 times on the 35th, 47th, and 52nd days, 30mL / rabbit each time, and the supernatant is taken after centrifugation.

[0077] 4) Identification of immune serum:

[0078] In the ELISA experiment, in a 96-well ELISA plate coated with 50ug / well of antigen-modified peptide and control peptide, the antibody was added at different dilution ratios for incubation (e.g. 1:54K, 1:162K). Then, enzyme-labeled secondary antibody and TMB substrate were applied to detect the binding of peptide and antibody by color development. The ELISA positive (OD450>1.0) antibody dilution of Ab6 antibody was greater than 54,000, and the signal was 10 times stronger than that of negative non-modified peptide. The results of Ab6 antibody ELISA test are shown in Table 2.

[0079] Table 2: Ab6 antibody ELISA test results

[0080]

[0081] Dotblot detection was used to fix the modified peptides and non-modified peptides in gradients of 1ng, 4ng, 16ng, and 64ng on the PVDF membrane. After the membrane surface was dried, it was blocked at room temperature for 60 minutes, and the antibody was added for incubation. Then, enzyme-labeled secondary antibodies and chemiluminescent substrates were added to detect the binding of the peptide and the antibody. The serum with positive test results was further purified. Figure 4 It can be seen that the Ab6 antibody has a positive detection limit of 4 ng and the signal is more than 10 times stronger than that of the unmodified peptide.

[0082] 5) Antibody purification:

[0083] The serum was purified and enriched using protein A affinity column and antigen peptide affinity chromatography column.

[0084] After the serum is filtered through a filter, it is loaded onto a well-balanced protein A chromatography column; the column is washed with PBS buffer solution, and then eluted with 150mM glycine buffer. The eluate is collected and adjusted to pH 7 by adding a neutralizing buffer solution. The crude IgG obtained after protein A purification is further loaded onto a well-balanced antigen polypeptide affinity chromatography column to specifically enrich the target antibody. Finally, the target antibody is loaded onto a non-modified affinity chromatography column, and the effluent is directly collected to remove non-specific antibody components.

[0085] 6) Western blot (WB) detection:

[0086] After cell treatment, collect the cells. Prepare protein samples on ice. Discard the culture medium, wash with PBS three times, scrape the cells in the culture dish into a 1.5ml Eppendorf (EP) tube; discard the supernatant after centrifugation. Add cell lysis buffer, lyse on ice for 30 minutes, and sonicate once at 30% power of the ultrasonic disruptor for 5 seconds each time. Then centrifuge the cell lysate at 4℃13,500rpm for 10 minutes. Take the cell lysate, add 5×Loading Buffer, boil in a boiling metal bath for 5 minutes, and place at -20℃ for western blot analysis.

[0087] according to Figure 5 It can be seen that in the Hela cell line, DNM1L K711 Antibodies, DNM1L by immunoblotting K711 The antibody can specifically recognize the K711 site, and its expression increases under hydrogen peroxide treatment conditions. The above shows that Ab6 antibody can recognize endogenous DNM1L (K711ac) by WB, and the band size is accurate.

[0088] Embodiment 3:

[0089] This example describes DNM1L K711 Related research on acetylation promoting mitochondrial fission:

[0090] Dilute MitoTracker Red CMXRos with buffer to a final concentration of 50 nM. For staining of adherent cells, aspirate the culture medium and add preheated (37°C) MitoTracker probe dilution. Incubate at 37°C in the dark for 30 min, discard the staining solution, and replace with fresh preheated buffer. Immediately use confocal FV3000 to acquire images. Figure 6It can be seen that the statistical results of the aspect ratio reflect the normal degree of mitochondrial morphology. The closer the aspect ratio is to 1, the more fragmented the mitochondria are. That is, by mutating the lysine at position 711 to glutamine, i.e. K711Q, to simulate acetylation, and mutating the position to arginine, i.e. K711R, to simulate deacetylation, two plasmids were established, and after transfection into the Hela cell line, mitochondrial morphology staining with Mitotracker was performed to determine that simulated acetylation produced an effect of promoting mitochondrial fission and fragmentation; K711R simulated deacetylation, and mitochondrial fusion increased. That is, double verification confirmed that DNM1L K711 Acetylation can promote mitochondrial fission.

[0091] Embodiment 4:

[0092] This example describes DNM1L K711 Related research on acetylation promoting mitochondrial function damage:

[0093] Use HBSS buffer to dilute TMRE dye to a final concentration of 10nM and incubate at 37℃ in the dark for 30min. After washing away excess dye with PBS, quickly acquire images on the confocal FV3000. TMRE dye can bind to the negative charge inside the mitochondria, causing fluorescence quenching, and this quenching is potential-dependent, that is, if the mitochondria are normal, the fluorescence intensity is high, and if the mitochondria are fragmented and damaged, the fluorescence intensity is low. Figure 7 It can be seen that after transfection of K711Q and K711R plasmids into Hela cell lines, mitochondrial function, i.e., membrane potential, was stained with TMRE, and it was determined that simulated acetylation promoted the decrease of mitochondrial membrane potential, while K711R did not produce a decrease in membrane potential, which confirmed that DNM1L K711 Acetylation promotes mitochondrial dysfunction.

[0094] Embodiment 5:

[0095] This example records the changes in the acetylation level of K711 of DNM1L in a Parkinson's (PD) cell model:

[0096] SH-SY5Y cells with 70% density were treated with 2 mM MPP+ for 24 h, and the cells were collected and prepared for WB detection. Figure 8 It can be seen that the acetylation level of K711 of DNM1L increased in the classic Parkinson's disease (PD) cell model established by intervening SH-SY5Y cell line with MPP+.

[0097] Embodiment 6:

[0098] This example records the changes in the acetylation level of K711 of DNM1L in a Parkinson's mouse model (MPTP):

[0099] MPTP was diluted in physiological saline and injected into the abdominal cavity of C57 mice (about 0.5-1 cm to the side of the intersection of the line connecting the two thigh roots and the midline of the abdomen) at a dose of 25 mg / kg for five consecutive days. The SNc brain tissue of the mice was sampled and subjected to WB detection. Fig. 9 It can be seen that the acetylation level of K711 of DNM1L was increased in the mouse model established by MPTP.

[0100] In summary, the present invention first uses mass spectrometry technology to identify the lysine 711 of DNM1L protein under oxidative stress conditions (DNM1L K711 ) acetylation modification, and found that the increase in the acetylation level of the K711 site of the DNM1L protein can promote the enhancement of DNM1L function and promote abnormal mitochondrial fission. Subsequently, an antibody for the K711 acetylation site of DNM1L was developed, which can specifically detect DNM1L K711 The acetylation level of the DNM1LK711 site can be detected by antigen-antibody-based methods (such as ELISA, WB, IP, IF, IHC, etc.), providing a potential tool for studying the molecular mechanisms of diseases related to mitochondrial function and oxidative stress pathological conditions; thus, it can be used to develop drugs that can inhibit DNM1L K711 In addition, the present invention has potential important value in revealing the biological impact of acetylation modification in the disease process, and is expected to have a positive impact on the development of future disease diagnosis and treatment strategies.

[0101] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A human DNM1L K711 A lysine acetylated polypeptide, characterized in that Human DNM1L K711 The amino acid sequence of the lysine acetylated polypeptide is shown in SEQ ID NO:

1.

2. A specific antibody, characterized in that: The specific antibody can specifically bind to the human DNM1L described in claim 1 K711 Lysine acetylated peptides.

3. The antibody according to claim 2, characterized in that The antibody is through the lysine 711 of DNM1L protein (DNM1L K711 ) is prepared by a coupling reaction of a specific polypeptide sequence modified by acetylation.

4. The antibody according to claim 2, characterized in that The antibody is a polyclonal antibody; the polyclonal antibody is a human DNM1L antibody using the amino acid sequence shown in SEQ ID NO: 1 K711 The conjugate of lysine acetylated polypeptide and carrier protein is obtained by immunizing non-human animals and purifying the serum extracted from the non-human animals.

5. The antibody according to claim 3, characterized in that When the lysine at this site mutates to arginine, the DNM1L protein loses the abnormal division caused by acetylation, thereby protecting the morphological function of mitochondria and promoting cell survival.

6. Use of the antibody according to any one of claims 2 to 4 in detecting lysine acetylation at position 711 of DNM1L protein.

7. Use of the antibody according to any one of claims 2 to 4 in diagnosing diseases related to abnormal mitochondrial fission.

8. The use according to claim 7, characterized in that: The invention also includes the use of the acetylated specific antibody in detecting the lysine acetylation level at position 711 of the DNM1L protein in clinical specimens; Determine the application of the lysine acetylation level at position 711 of DNM1L protein and the correlation between abnormal mitochondrial morphology and function and the ultimate cell or neuronal damage in relevant disease models including Parkinson's disease; and for guiding the selection of clinical drugs that can inhibit DNM1L acetylation.

9. The use according to claim 7, characterized in that: The antibody is used to indicate the morphology and function of mitochondria.

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