An in situ detection method for intracellular palmitoylation and its application

Abstract

This invention discloses an in situ detection method for intracellular protein palmitoylation, comprising: (1) obtaining human or animal cells and keeping them viable, and incubating the cells together with a palmitoyl azide-conjugated metabolite; (2) fixing the cells and incubating them sequentially with biotinyne and a fluorescent dye coupled with streptavidin; (3) then observing and photographing the cells treated in step (2) using a fluorescence microscope to determine the original location of intracellular protein palmitoylation. The detection method of this invention does not require cell lysis, can directly reflect the in situ presence of protein palmitoylation within the cell, and can perform semi-quantitative analysis of the corresponding protein palmitoylation level, which can be applied to the assessment of the characteristics and level of intracellular protein palmitoylation. This invention also discloses the application of the aforementioned in situ detection method for intracellular protein palmitoylation in reproductive medicine testing, which has broad application value in the diagnosis of male infertility.

Description

Technical Field

[0001] This invention relates to the field of cell biology technology, and in particular to an in situ detection method for intracellular palmitoylation and its application. Background Technology

[0002] Protein palmitoylation refers to the reversible covalent linking of 16-carbon saturated palmitic acid to cysteine ​​residues in a protein via a thioester. It is an important post-translational modification of proteins (Reference 1: Linder, ME & Deschenes, RJP palmitoylation: policing protein stability and traffic. Nature Reviews Molecular Cell Biology 8, 74-84 (2007); Reference 2: Greaves, J. & Chamberlain, LHDHHC palmitoyl transferases: substrate interactions and (patho)physiology. Trends Biochem Sci. 36, 245-253, doi:10.1016 / j.tibs.2011.01.003 (2011)). Protein palmitoylation plays an important role in cell biology processes, including increasing protein hydrophobicity, regulating protein activity and stability, promoting cell membrane fusion and protein transport and localization; affecting membrane protein conformation and facilitating protein membrane junctions; participating in the regulation of multiple cell signaling pathways, mediating protein-protein, protein-lipid, and other post-translational modifications; and participating in cell signal transduction, including protein phosphorylation.

[0003] Protein palmitoylation is involved in the occurrence or progression of various diseases, including tumors, metabolic disorders, inflammation and infection, and neurological diseases (Reference 3: Ko, P.-J. & Dixon, SJ Protein palmitoylation and cancer. EMBO Reports. 19, doi:10.15252 / embr.201846666 (2018), Reference 4: Qu, M., Zhou, X., Wang, X. & Li, H. Lipid-induced S-palmitoylation as a Vital Regulator of Cell Signaling and Disease Development. Int J Biol Sci. 17, 4223-4237, doi:10.7150 / ijbs.64046 (2021), Reference 5: Dong, D. et al. Palmitoylated GLB1L4 transfers via exosomes to maintain sperm function in ratepididymis.Reproduction(Cambridge,England)161,159-172,doi:10.1530 / REP-20-0423(2021).).

[0004] We previously reported the presence of palmitoylated proteins in sperm, which are associated with sperm maturation and capacitation (Reference 3: Li R, LK, Yang Y, Sun PB, Chen AJ, Ni Y. Palmitoylation of heat shock protein 90 in mouse sperm. Sheng Li Xue Bao. 69, 298-304, doi:10.13294 / j.aps.2017.0034(2017).).

[0005] Methods for detecting protein palmitoylation include radioactive palmitate metabolic labeling, acyl-biotin exchange assay (ABE), and mass spectrometry. However, these methods typically require cell lysis and cannot directly reflect the in-situ presence of palmitoylated proteins within cells, thus hindering research on the functions related to protein palmitoylation.

[0006] Therefore, in situ detection methods for protein palmitoylation are needed to better reveal the physiological characteristics of protein palmitoylation in cells and to deepen the comprehensive understanding of the physiological functions of protein palmitoylation. Summary of the Invention

[0007] This invention provides an in situ detection method for intracellular protein palmitoylation, which can not only detect the original location of intracellular protein palmitoylation in situ, but also perform semi-quantitative analysis based on the degree of protein palmitoylation.

[0008] The technical solution of the present invention is as follows:

[0009] An in situ detection method for intracellular protein palmitoylation includes the following steps:

[0010] (1) Obtain human or animal cells and keep them alive, incubate the cells with azide palmitoyl metabolites; fix the cells after incubation;

[0011] (2) The fixed cells were incubated sequentially with biotinyne and a fluorescent dye coupled with streptavidin.

[0012] (3) Then, the cells treated in step (2) were observed and photographed using a fluorescence microscope to determine the original location of protein palmitoylation in the cells.

[0013] Preferably, the in situ detection method for intracellular protein palmitoylation of the present invention further includes: quantifying the cellular fluorescence intensity to determine the relative degree of intracellular protein palmitoylation. This method can be used to evaluate the physiological state and function of cells.

[0014] The in situ detection method for intracellular protein palmitoylation of the present invention can not only detect the original location of protein palmitoylation in situ, but also perform semi-quantitative analysis on the degree of protein palmitoylation to assess cell physiological state and function.

[0015] Preferably, in step (1), the cells are incubated together with azide palmitoyl metabolites, the concentration of which is 20-300 μM.

[0016] Preferably, the azide-conjugated palmitoyl metabolite is Click-iT. TM Palmitic acid azide.

[0017] Furthermore, the incubation time of the cells with the palmitoyl azide metabolite depends on the cell species.

[0018] Preferably, at least one of 4% paraformaldehyde, acetone, methanol and 95% ethanol is used to fix the cells.

[0019] Preferably, in step (2), the fixed cells are incubated with streptomycin / biotin binding blocking buffer before being incubated with biotinyne.

[0020] Incubation with streptomycin / biotin conjugation blocking buffer can eliminate the influence of endogenous biotin, thereby improving the specificity of the test results.

[0021] The streptomycin / biotin binding blocking buffer is Beyotime's biotin detection blocking kit, product brand number P0101.

[0022] Further preferred, the cells were incubated with streptomycin / biotin conjugation blocking buffer for 10-60 min.

[0023] Preferably, the concentration of the fluorescent dye coupled with streptavidin is 0.5-20 μg / mL.

[0024] Preferably, in step (2), the cell nucleus is stained after the cells are incubated with a fluorescent dye coupled with streptavidin.

[0025] Staining the cell nucleus facilitates the observation of fluorescence and intracellular structures.

[0026] The present invention also provides an application of the above-mentioned in situ detection method for intracellular protein palmitoylation in reproductive medicine testing, wherein the cell is sperm.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] The detection method of the present invention does not require cell lysis, can directly reflect the in situ presence of protein palmitoylation in cells, and can perform semi-quantitative analysis of the corresponding palmitoylation level.

[0029] Protein palmitoylation exists in cells and plays an important physiological regulatory role. Determining the intracellular location and level of protein palmitoylation helps to reveal its localization and regulatory functions within cells. This invention not only has broad application prospects in cellular palmitoylation-related research, but also has high application value in assessing sperm physiological status and function, and in the diagnosis of male infertility. Attached Figure Description

[0030] Figure 1 This is a schematic diagram illustrating the principle of the in situ detection method for intracellular palmitoylation of the present invention.

[0031] Figure 2 This is a schematic flowchart of the in situ detection method for intracellular palmitoylation of the present invention;

[0032] Figure 3 This is a comparison chart showing the blocking effect of blocking buffer on endogenous biotin; where (A) represents no blocking buffer used and (B) represents the use of blocking buffer.

[0033] Figure 4The images show the location and fluorescence intensity of palmitoylation of proteins at different time points in mouse sperm during Example 1. (A) shows the location of palmitoylation, with green (Alexa Fluor 488) indicating palmitoylation and blue (DAPI) indicating the cell nucleus. The left side represents 0 minutes, the middle 90 minutes, and the right side 180 minutes. Azide represents palmitoylation labeled with azide, while Azide-free represents the negative control group (no azide added). (B) shows the fluorescence intensity of palmitoylation, analyzed using ImageJ software. The left side represents 0 minutes, the middle 90 minutes, and the right side 180 minutes. Azide represents palmitoylation labeled with azide, while Azide-free represents the negative control group (no azide added). P < 0.05 indicates a statistically significant difference. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0035] like Figure 1 and Figure 2 As shown, an in situ detection method for intracellular palmitoylation of sperm includes the following steps:

[0036] 1) Cell Acquisition

[0037] The cells can be somatic cells or germ cells (sperm cells). Somatic cells can be derived from primary cells digested with enzymes or from cultured passaged cells. If the cells are germ cells (sperm cells), for small animals, sperm can be obtained by dissecting the animal and extracting them from the vas deferens and epididymal tail. For large animals, semen needs to be obtained, seminal plasma removed, and sperm collected. The sperm is then washed with a culture medium to obtain purified sperm. The process should be gentle to avoid damaging the cells and to maintain their viability.

[0038] 2) Cellular uptake of azide-bound palmitoyl metabolites

[0039] The obtained cells were incubated with azide-palmitoyl metabolites for varying times to allow the palmitoyl azide to enter the cells. The incubation time was adjusted according to experimental needs. The cells were then washed with culture medium after the incubation period.

[0040] 3) Cell fixation

[0041] Protein immobilization allows the palmitoyl (azidide-labeled) groups bound to intracellular protein cysteine ​​residues to be fixed in situ within the cell. Simultaneously, the cells are coated onto pre-treated, non-detachable special glass slides.

[0042] 4) Streptomycin / Biotin conjugation blocking buffer reaction

[0043] Since subsequent operations will react with biotinyne, to avoid the influence of endogenous biotin on the results, an additional step involving a streptomycin / biotin binding blocking buffer is added to eliminate the influence of endogenous biotin and improve the specificity of the results. Figure 3 As shown.

[0044] 5) Reaction with biotinyne

[0045] Based on the detection principle, the azide reacts with biotinyne to form a binding compound, providing conditions for subsequent fluorescent labeling.

[0046] 6) Streptavidin reaction coupled with fluorescently labeled probe

[0047] The reaction of palmitoylated biotin with a fluorescently labeled probe to streptavidin is facilitated by fluorescence microscopy.

[0048] 7) Fluorescence microscopy observation

[0049] To facilitate observation of fluorescence and intracellular structures, observation can be performed simultaneously using nuclear dyes.

[0050] 8) Semi-quantitative fluorescence intensity

[0051] Images obtained from fluorescence microscopy are quantitatively analyzed using software.

[0052] Example 1

[0053] Taking the detection of palmitoylation of intracellular proteins in mouse sperm as an example:

[0054] 1) Take male ICR mice older than 8 weeks, euthanize them with CO2, dissect them to remove the vas deferens and epididymal tail, remove fat and blood vessels, place them in a U-shaped culture dish, add HTF (human oviduct fluid) culture medium containing 0.3% BSA (bovine serum albumin) preheated at 37°C, and cut them up to release sperm.

[0055] 2) Let stand for 15 minutes, then transfer the sperm suspension to a 15mL centrifuge tube and centrifuge immediately (500g, 10 seconds). Transfer the supernatant sperm suspension to a new 5mL centrifuge tube, add an appropriate amount of HTF (containing 0.3% BSA) to resuspend, and adjust the concentration to 10×10⁻⁶. 6 / mL, the sperm suspension volume is 4mL.

[0056] 3) Take 2 mL of sperm suspension as a blank control group, without adding Click-iT. TM Palmitate azide (catalog number, C10265; Invitrogen, USA), the remaining 2 mL of sperm suspension was added to 2 μL of Click-iT TM Palmitic acid azide (to a final concentration of 50 μM) was incubated in the dark.

[0057] 4) At incubation times of 0 minutes, 90 minutes, and 180 minutes, 400 μL of sperm suspension containing azide was taken, regrouped, and labeled; the blank control group was treated in the same way.

[0058] 5) Centrifuge each sperm sample at 500g for 15 minutes at 4°C; discard the supernatant, retain the bottom precipitate, add 500μL of pre-cooled PBS at 4°C, centrifuge at 500g for 10 minutes twice at 4°C. Discard the supernatant, retain the bottom precipitate.

[0059] 6) Fix sperm cells with 4% paraformaldehyde for 30 minutes.

[0060] 7) Centrifuge the fixed sperm at 500g for 5 minutes, discard the supernatant, keep the bottom precipitate, add 500μL PBS to resuspend, wash twice, and add 400μL PBS to resuspend.

[0061] 8) Using an immunohistochemistry pen (BC004, Beijing Baishayi Technology Co., Ltd.), draw two equal-sized rectangles on a The Silane-Prep slide (S4651-72EA, Sigma, Aldrich, MO, USA). Add 5 μL of sample smear to each rectangle, spreading it evenly. Observe the sperm density under a microscope. Adjust the sperm density as needed, and allow the liquid to dry naturally. Then, perform the following procedures within each rectangle.

[0062] 9) Cover completely with 400 μL of 0.25% Triton X-100 and allow to infiltrate at room temperature for 15 minutes.

[0063] 10) Wash the sample with 3% BSA for 10 minutes each time, for a total of 3 times.

[0064] 11) Cover completely with 400 μL of 1X streptomycin / biotin binding blocking buffer and incubate at room temperature for 30 minutes.

[0065] 12) Wash the sample with 3% BSA for 10 minutes each time, for a total of 3 times.

[0066] 13) Cover completely with 400 μL of 1X Biotin blocking buffer and incubate at room temperature for 30 minutes.

[0067] 14) Prepare Click cell reaction buffer (catalog number C10269, Invitrogen, USA) according to the instructions of the cell reaction buffer kit, add biotinyne (catalog number B10185, Invitrogen, USA) to a final concentration of 5 μM, and use it within 15 minutes after preparation.

[0068] 15) Cover with 100 μL Click cell reaction buffer and incubate at room temperature for 30 minutes.

[0069] 16) Wash the sample with 3% BSA for 10 minutes each time, for a total of 3 times.

[0070] 17) Covers Alexa Fluor formulations of 200μL. TM Staining was performed using 488 streptavidin conjugate (catalog number S11223, Invitrogen, USA) at a concentration of 1 μg / mL, and incubated at room temperature in the dark for 1 hour.

[0071] 18) Wash three times with PBS, five minutes each time.

[0072] 19) Cover with 200 μL of 0.5 μg / mL DAPI solution (to stain cell nuclei) and incubate for 5 minutes.

[0073] 20) Wash three times with PBS, five minutes each time.

[0074] 21) After air drying, observe and photograph the image using a fluorescence microscope (Nikon 80i, Japan). The excitation wavelength of the filter was 495 nm, and the emission wavelength was 519 nm.

[0075] 22) The location of protein palmitoylation was determined based on the fluorescence characteristics of the mice. Green (Alexa Fluor 488) indicates the presence of protein palmitoylation, and blue (DAPI) indicates the cell nucleus. Fluorescence intensity was analyzed using ImageJ software. Palmitoylated proteins labeled with green fluorescence could be observed, such as... Figure 4 As shown in Figure A. Fluorescence intensity can be quantified, such as... Figure 4 As shown in B.

[0076] The embodiments described above provide a detailed explanation of the technical solutions and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, and equivalent substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for in situ detection of intracellular protein palmitoylation, comprising, The method comprises the following steps: (1) obtaining human or animal cells and keeping them alive, incubating the cells with azide palmitoyl metabolites, and fixing the cells after the incubation is completed; (2) sequentially incubating the fixed cells with biotin alkyne and fluorescent dye coupled with streptavidin; (3) observing and photographing the cells treated in step (2) by fluorescence microscopy to determine the original position of protein palmitoylation in the cells.

2. The method for in situ detection of intracellular protein palmitoylation according to claim 1, wherein, The method further comprises: Quantifying the fluorescence intensity of the cells to determine the relative degree of protein palmitoylation in the cells.

3. The method for in situ detection of intracellular protein palmitoylation according to claim 1, wherein, In step (1), the cells are incubated with azide palmitoyl metabolites, and the concentration of the azide palmitoyl metabolites is 20-300 μM.

4. The method for in situ detection of intracellular protein palmitoylation according to claim 1 or 3, wherein The azide palmitoyl metabolite bound is Click-iT TM Palmitic acid azide.

5. The method for in situ detection of intracellular protein palmitoylation according to claim 1 or 3, wherein The cells are fixed by using at least one of 4% paraformaldehyde, acetone, methanol and 95% ethanol.

6. The method of in situ detection of intracellular protein palmitoylation of claim 1, wherein, In step (2), the fixed cells are incubated with streptavidin / biotin binding blocking buffer before being incubated with biotin alkyne.

7. The method for in situ detection of intracellular protein palmitoylation according to claim 6, wherein, The cells are incubated with the streptavidin / biotin binding blocking buffer for 10-60 min.

8. The method of in situ detection of intracellular protein palmitoylation of claim 1, wherein, The concentration of the fluorescent dye coupled with streptavidin is 0.5-20 μg / mL.

9. The method of in situ detection of intracellular protein palmitoylation of claim 1, wherein, In step (2), the nucleus of the cells is stained after the cells are incubated with the fluorescent dye coupled with streptavidin.

10. Use of a method for in situ detection of intracellular protein palmitoylation according to any one of claims 1 to 9 in the detection of reproductive medicine, characterized in that, The cells are sperm.

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