Application of cFKBP8-Hsp60 interaction in depression by adjusting mitochondria

Abstract

The invention discloses an application of interaction of cFKBP8-Hsp60 in depression by adjusting mitochondria. Belongs to the technical field of molecular biology and medicine, the interaction between cFKBP8 and Hsp60 is ensured, the key amino acid residue of the interaction between cFKBP8 and Hsp60 is confirmed to be the 13th arginine (cFKBP8 ARG13) of cFKBP8, and the cFKBP8 is overexpressed in SH-SY5Y cells and mouse leading edge cortical neurons by further constructing an expression vector; the results show that the mitochondrial structure and function are damaged, and the mouse shows stress susceptibility, however, the mutation of cFKBP8 ARG13 into alanine alleviates mitochondrial damage and the stress susceptibility of the mouse. Therefore, based on a molecular mechanism of adjusting mitochondria by a cFKBP8-Hsp60 axis, a new thought and a treatment target are provided for the treatment of depression.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of molecular medicine and treatment of neuropsychiatric diseases, and relates to application of cFKBP8-Hsp60 interaction in depression by regulating mitochondria. BACKGROUND

[0002] Depression is a common mental illness with high disability rate. The existing mainstream drugs are mostly targeted at monoamine neurotransmitter system, and have significant defects such as low response rate, slow onset and large side effects, and the clinical needs have not been met.

[0003] Recently, mitochondrial dysfunction is considered as an important pathophysiological mechanism of depression. However, the upstream molecular events driving mitochondrial damage in depression and their key nodes are still unknown, resulting in a severe lack of specific drug targets for directly improving mitochondrial function.

[0004] Although previous studies have found that the protein cFKBP8 encoded by circFKBP8(5S,6) is related to stress susceptibility, it is completely unknown whether and how the protein precisely regulates mitochondrial function, and which functional domain or amino acid site of the protein is the key "druggable" target of depression. Finding and verifying such a target precise to a single amino acid is of great significance for the development of a new generation of antidepressants. SUMMARY

[0005] The purpose of the application is to provide a completely new and functionally verified application of cFKBP8-Hsp60 interaction in depression by regulating mitochondria.

[0006] The technical solution of the application is: the application of cFKBP8-Hsp60 interaction in depression by regulating mitochondria; the cFKBP8 and Hsp60 have interaction, and the interaction plays a key role in regulating mitochondria and promoting stress susceptibility in mice. Further, the key amino acid residues of the cFKBP8-Hsp60 interaction are the 13th arginine of cFKBP8 (cFKBP8 ARG13).

[0007] Further, the cFKBP8 ARG13 is mutated to alanine (cFKBP8 R13A) to destroy the cFKBP8-Hsp60 interaction.

[0008] Further, the regulation of mitochondria includes: (1) increasing the expression amount of cFKBP8 in SH-SY5Y cells damages mitochondria, and increasing the expression amount of cFKBP8 R13A reverses this effect; (2) increasing the expression level of cFKBP8 in mouse prefrontal cortex neurons damages mitochondria, while increasing the expression level of cFKBP8 R13A reverses this effect.

[0009] Further, the damage to mitochondria includes two or more of the following: mitochondrial swelling, ridge rupture or disappearance, reduced matrix electron density, significantly increased proportion of damaged mitochondria, destruction of mitochondrial reticular structure, significantly increased ROS level of cells or mitochondria, and decreased mitochondrial membrane potential.

[0010] Further, the reagent for increasing the expression level of cFKBP8 includes a recombinant vector for overexpressing a cFKBP8 or cFKBP8 R13A gene, wherein the cFKBP8 or cFKBP8 R13A gene is a gene encoding a cFKBP8 or cFKBP8 R13A protein.

[0011] Further, the nucleotide sequence of the cFKBP8 R13A is SEQ ID NO. 1, and the amino acid sequence is SEQ ID NO. 2.

[0012] Further, the recombinant vector for overexpressing the cFKBP8 or cFKBP8 R13A gene includes the cFKBP8 or cFKBP8 R13A gene and an adenovirus-associated vector.

[0013] Further, the use in depression includes that increasing the expression level of cFKBP8 in mouse prefrontal cortex neurons promotes stress susceptibility in mice, while increasing the expression level of cFKBP8 R13A reverses this effect.

[0014] Further, the adenovirus-associated vector includes an rAAV2 / 9 vector.

[0015] Specifically, in the first aspect, the present application first discovers and verifies the interaction between cFKBP8 and Hsp60, and finds that the key amino acid residue of the interaction is cFKBP8 ARG13; specifically comprising: verifying the interaction between cFKBP8 and Hsp60 by immunofluorescence co-localization, co-immunoprecipitation (Co-IP) and GST Pull-down experiment, and finding that cFKBP8 and Hsp60 interact in mitochondria by using Tomm20 labeled mitochondria combined with proximity ligation assay (PLA); then, the key amino acid residues of the combination of the two are predicted by molecular docking, molecular dynamics simulation and computer alanine scanning, and a point mutation expression vector is constructed, and the key amino acid residues of the combination of the two are verified by Co-IP experiment that the key amino acid residues of the combination of the two are cFKBP8 ARG13, and cFKBP8 is combined to the top domain of Hsp60.

[0016] In the second aspect, by constructing overexpression vectors, cFKBP8 and cFKBP8 ARG13 are overexpressed in SH-SY5Y cells, then the mitochondrial morphology is dynamically observed by using MitoTracker Orange probe, the mitochondrial ultrastructure is analyzed by transmission electron microscope, the total level of reactive oxygen species (ROS) in cells and mitochondrial ROS are detected by using DCFH-DA and MitoSOX Red probes respectively, and the change of mitochondrial membrane potential (MMP) is detected by using JC-1 probe; overexpression of cFKBP8 in SH-SY5Y cells leads to destruction of mitochondrial reticular structure, swelling of mitochondria, rupture or disappearance of cristae, decrease of matrix electron density, significant increase of damaged mitochondria, significant increase of ROS or mitochondrial ROS level in cells, and decrease of MMP; however, cFKBP8 ARG13 alleviates the damage of mitochondria.

[0017] Thirdly, animal experiments further confirmed that overexpression of cFKBP8 in the anterior limbic cortex of mice promotes mitochondrial damage and stress susceptibility, while overexpression of cFKBP8 ARG13 partially reverses this mitochondrial damage and stress susceptibility. Specifically, this included: constructing an adeno-associated virus expression vector and injecting it into the anterior limbic cortex via stereotactic injection; establishing a two-week short-term chronic unpredictable mild stress (sCUMS) depression model; assessing depressive-like behaviors in mice using the sucrose preference test (SPT), forced swim test (FST), and tail suspension test (TST); evaluating the interaction between cFKBP8-Hsp60 and Hsp60-ClpP using the Co-IP experiment and PLA; observing the ultrastructure of neuronal mitochondria using transmission electron microscopy; and assessing the level of neuronal superoxide anions using dihydroethidium (DHE) staining.

[0018] Beneficial Effects: Compared with existing technologies, this invention has the following significant features: This invention is the first to discover and verify the interaction between cFKBP8 and Hsp60, and uses molecular dynamics simulations, computer alanine scanning prediction, point mutation and Co-IP experiments to verify and identify the key amino acid residue of the cFKBP8-Hsp60 interaction as cFKBP8ARG13; it reveals the mechanism by which the cFKBP8-Hsp60 interaction regulates mitochondria and modulates depressive-like behavior in mice, both in vivo and in vitro; specifically, overexpression of cFKBP8 in SH-SY5Y cells leads to damage to mitochondrial structure and function, while overexpression of cFKBP8 ARG13 significantly alleviates this damage; mice overexpressing cFKBP8 in the anterior marginal cortex exhibit depressive-like behavior earlier, while overexpression of cFKBP8 ARG13 partially reverses this effect; therefore, it provides a target for the development of highly selective and safe antidepressant drugs, and has important theoretical and clinical application significance. Attached Figure Description

[0019] Figure 1This is a schematic diagram illustrating the interaction between cFKBP8 and Hsp60 in an embodiment of the present invention. Figures AD show the interaction between exogenous cFKBP8 and Hsp60 detected by Co-IP in HEK293 and SH-SY5Y cells after co-transfection with a Flag-tagged cFKBP8 plasmid and a HA-tagged Hsp60 plasmid (n=3). Figures EF show the interaction between endogenous cFKBP8 and Hsp60 in monkey brain DLPFC detected by Co-IP (n=1). Figure G shows the immunofluorescence staining of cFKBP8 (red) and Hsp60 (green) in SH-SY5Y cells, with DAPI (blue) representing the cell nucleus; the scale bar is 20 μm. Figure H shows the quantitative analysis of immunofluorescence colocalization of cFKBP8 and Hsp60 using Pearson's correlation coefficient and Mander's overlap coefficient. R-values ​​(ranging from -1 to 1) show the spatial distribution correlation between the two, with 1 indicating complete colocalization and 0 indicating no correlation; Mander's coefficients (ranging from 0 to 1) assess the degree of fluorescence signal overlap, with 1 indicating complete overlap and 0 indicating no overlap; among them, the M1 coefficient (0.693) reflects the overlap ratio between the cFKBP8 red signal and the Hsp60 green signal, while the M2 coefficient (1) characterizes the distribution of the Hsp60 green signal in the cFKBP8 red signal, n=3; Figure I shows the distribution of cFKBP8 (red) and Hsp60 (green) fluorescence signals in a specific region of the cell; the horizontal axis represents the linear distance (μm) along the selected cell region, and the vertical axis represents the relative fluorescence intensity (au), n=3; Figure J shows the GST pull-down verification of the direct interaction between cFKBP8 and Hsp60; Figure K shows the interaction between cFKBP8 and Hsp60 in mitochondria in SH-SY5Y cells detected by adjacent linkage technology (PLA), where Tomm20 is used as a mitochondrial-specific localization marker, and the scale bar is 20μm; Figure 2 This is a schematic diagram of molecular dynamics simulation of cFKBP8 and Hsp60 within 100 ns in an embodiment of the present invention; wherein, Figure A is the RMSD change of the cFKBP8 and Hsp60 protein backbone; Figure B is the RMSF of cFKBP8 and Hsp60 protein residues; Figure C is the Rg of the cFKBP8-Hsp60 complex; Figure D is the number of hydrogen bonds formed between cFKBP8 and Hsp60; Figure E is the number of atomic contacts between cFKBP8 and Hsp60; Figure F is the Gibbs free energy morphology diagram, with red to blue representing free energy from high to low. Figure 3This is an identification diagram of key residues at the cFKBP8 interface in the cFKBP8 interaction of cFKBP8 and Hsp60 in this embodiment of the invention; wherein, Figure A is a conformational diagram of the cFKBP8-Hsp60 protein complex at 100 ns in molecular dynamics simulation; Figure B is the residue energy decomposition analysis of cFKBP8; Figure C is the alanine scan results of ARG7, ARG13, ARG15, ARG24, ARG79, and ARG101, which have low average energy values ​​in the residue energy decomposition analysis of cFKBP8; Figures D and E are the empty control plasmid of cFKBP8, the wild-type cFKBP8 plasmid with Flag tag (Flag-cFKBP8 WT), the cFKBP8 mutant plasmid with Flag tag mutated to ALA at position 13 (Flag-cFKBP8 R13A), and the cFKBP8 mutant plasmid with Flag tag mutated to ALA at position 15 (Flag-cFKBP8 R13A). The cFKBP8 mutant plasmids (R15A), (Flag-cFKBP8 R24A), and (Flag-cFKBP8 3A), which mutate ARG at position 24 to ALA, and (Flag-cFKBP8 3A), which mutate ARG at positions 13, 15, and 24 to ALA, were co-transfected with Hsp60 plasmids (HA-tagged) into HEK293 cells. The interaction between cFKBP8 and its mutant cFKBP8 with Hsp60 was analyzed by Co-IP and Western blotting techniques, with β-actin as an internal control. Figure 4 Figure 1 shows the identification of key residues at the Hsp60 interface in the cFKBP8-Hsp60 interaction in this embodiment of the invention. Figure A is the residue energy decomposition analysis of Hsp60; Figure B is the computed alanine scan result of residues with low average energy values ​​in the Hsp60 residue energy decomposition analysis; Figure C shows the protein-protein interaction detected by Co-IP after co-transfection of Hsp60 empty control, wild-type Hsp60 plasmid with HA tag (HA-Hsp60 WT), a series of single-point mutant plasmids (R429A, D100A, E384A, E388A, E548A, E107A, E392A), and a seven-site joint mutant plasmid (HA-Hsp60 7A) with a cFKBP8 plasmid with a Flag tag in HEK293 cells, with β-actin as an internal control; Figure D is a schematic diagram of the Hsp60 domain. Figure 5This is a schematic diagram illustrating the regulatory effect of overexpression of cFKBP8 or cFKBP8 R13A on mitochondria in SH-SY5Y cells in this embodiment of the invention. In Figure A1, SH-SY5Y cells were transfected with Flag-labeled cFKBP8 WT, cFKBP8 R13A, cFKBP8 3A, or an empty control plasmid for 48 hours. Figure A shows mitochondrial morphology using the MitoTracker Orange fluorescent probe, with a scale bar of 10 μm. Figure B shows the average branch length of mitochondria calculated using the Image J plugin Mitochondria Analyzer, n=60 cells. Figures C and D show the ultrastructure of mitochondria assessed by transmission electron microscopy and the quantitative analysis of the proportion of damaged mitochondria, with red arrows pointing to mitochondria, a scale bar of 500 nm, and n=3. Figure E shows the effect of using the DCFH-DA fluorescent probe and MitoSOX. Red staining was used to assess the levels of cellular ROS and mitochondrial ROS, with a scale bar of 20 μm. Figure F shows the detection of mitochondrial membrane potential by JC-1 staining, with a scale bar of 20 μm. Figure G shows the quantitative analysis of cellular ROS fluorescence intensity detected by the DCFH-DA probe, n=3. Figure H shows the quantitative analysis of mitochondrial ROS fluorescence intensity detected by MitoSOXRed staining, n=3. Figure I shows the quantification of mitochondrial membrane potential by measuring the ratio of red to green fluorescence intensity, n=3. P* < 0.05, P** < 0.01, P*** < 0.001, and P**** < 0.0001. Data are expressed as mean ± standard error (Mean ± SEM). Figure 6Figure 1 is a schematic diagram illustrating the regulation of neuronal mitochondria and depressive-like behavior by overexpression of cFKBP8 or cFKBP8 R13A in the anterior limbic cortex of mice in this invention. Figure A is a schematic diagram of experimental groups and a timeline of experimental design. Figure B is a representative image of successful expression of rAAV virus EGFP in the anterior limbic cortex of mice, with a scale bar of 200µm. Figure C is the analysis of saccharide preference rate in SPT, n≥12. Figure D is the statistics of immobility time in FST, n≥11. Figure E is the statistics of immobility time in TST, n≥12. Figure F is a transmission electron microscope image of the ultrastructure of mitochondria in each group of neurons, with a scale bar of 500nm. Figure G is a quantitative analysis of the proportion of mitochondrial damage, n=3. Figure H is a DHE-stained image showing the superoxide anion (red fluorescence) level in EGFP-positive neurons (green) in the anterior limbic cortex, with a scale bar of 20µm. µm; Figure I shows the quantitative analysis of DHE fluorescence intensity in EGFP-positive neurons, n=3; Figure J shows the experimental group diagram and experimental design timeline; Figure K shows the sugar water preference rate analysis in SPT, n≥12; Figure L shows the FST immobility time statistics, n≥12; Figure M shows the TST immobility time statistics, n≥12; Figure N shows the ultrastructural changes of mitochondria in neurons of each group in transmission electron microscopy, scale bar is 500nm; Figure O shows the quantitative analysis of mitochondrial damage ratio, n=3; Figure P shows the superoxide anion (red fluorescence) level in EGFP-positive neurons (green) in the anterior marginal cortex with DHE staining, scale bar is 20µm; Figure Q shows the quantitative analysis of DHE fluorescence intensity in EGFP-positive neurons, n=3; P * < 0.05, P ** < 0.01, P*** < 0.001 and P **** < 0.0001, data are expressed as mean ± standard error (Mean ± SEM). Detailed Implementation

[0020] The specific technical solution of the present invention will be further described in detail below with reference to specific examples.

[0021] Example 1: Verification of the interaction between cFKBP8 and Hsp60; This invention first confirmed the interaction between cFKBP8 and Hsp60 using immunofluorescence co-localization, Co-IP, PLA, and GST pull-down. Then, in SH-SY5Y cells, Tomm20-labeled mitochondria combined with PLA technology showed an endogenous interaction between cFKBP8 and Hsp60 in mitochondria; the steps are as follows: I. The HEK293 and SH-SY5Y cell lines used in this invention were purchased from the Chinese Academy of Sciences Cell Bank, and pcDNA 3.1 Zeo(+)-Flag-cFKBP8 expression vector (hereinafter referred to as Flag-cFKBP8 plasmid) and pcDNA 3.1-HA-Hsp60 expression vector (hereinafter referred to as HA-Hsp60 plasmid) were constructed. First, cFKBP8 and Hsp60 were overexpressed in HEK293 and SH-SY5Y cells, respectively. Co-IP was used to verify that cFKBP8 and Hsp60 did indeed interact. Then, Co-IP was used to confirm that endogenous cFKBP8 and Hsp60 in the dorsolateral prefrontal cortex (DLPFC) of monkey brain interacted. Figure 1 AF); the steps are as follows: 1. HEK293 and SH-SY5Y cells were co-transfected with Flag-cFKBP8 plasmid and HA-Hsp60 plasmid, respectively. (1) HEK293 cells were transfected using ExFect Transfection Reagent (Vazyme, T101): (a) When the cell density reaches 60%, the ultraviolet sterilization bench is disinfected for 30 min, and the culture medium is warmed to 37°C. (b) Preparation of transfection complex: tube A: 1 mL Opti-MEM + 60 μL ExFect; tube B: 1 mL Opti-MEM + 30 μg plasmid (15 μg added to each tube for total transfection). After mixing tubes A and B separately, drop the liquid from tube A into tube B and mix well. Incubate at room temperature for 15 min. (c) After changing the cell culture medium, add the complex, change the medium after 6 hours, and collect the sample for testing after 48 hours; (2) SH-SY5Y cells were transfected using Lipofectamine™ 3000 transfection reagent (Thermo Fisher, L3000015): (a) When the cell density reaches 60%, the ultraviolet sterilization bench is disinfected for 30 min, and the culture medium is warmed to 37°C. (b) Preparation of transfection complex: Set up two tubes, A and B. Add 750 μL of Opti-MEM to each tube. Add 45 μL of Lipo3000 to tube A and mix well. Add 15 μg of plasmid to tube B (7.5 μg for co-transfection). Mix well and then add 30 μL of P3000 and mix again. Drop the liquid from tube A into tube B and let it stand at room temperature for 15 min. (c) After changing the cell culture medium, add the complex, and collect the sample for testing after 48 hours; (3) The non-human primate samples used in this invention were obtained from naturally deceased crab-eating macaques, and the protein samples of the DLPFC brain region were donated by relevant personnel. 2. Co-IP and Western blot verification of the interaction between cFKBP8 and Hsp60 (1) The steps for preparing Co-IP samples are as follows: (a) Preparation of protein lysis buffer: Prepare lysis buffer by adding 1 mL Pierce IP Lysis Buffer (Thermo Fisher, 87787) + 10 μL 100 mM PMSF (Beyotime, ST507) and store on ice for later use; (b) Cell protein extraction: Discard the culture medium, wash twice with pre-cooled PBS, add freshly prepared lysis buffer, gently shake to ensure complete coverage, quickly collect with a cell scraper into a 1.5 mL EP tube, vortex for 30 s every 5 min, lyse on ice for 30 min, centrifuge at 12000 rpm for 30 min at 4 °C, aspirate the supernatant into an empty EP tube, and retain 10 μL of supernatant for protein concentration determination; Tissue protein extraction: Weigh the tissue, add 100 mg of tissue to 1 mL of lysis buffer into a 2 mL EP tube, place on ice, add one 3 mm zirconia grinding bead to each tube, grind in a pre-cooled grinding bowl at 60 Hz for 10 times, 60 s / time, with a 5 s interval, and lyse on ice for 1 h; (c) Protein quantification was performed using the Pierce BCA protein quantification kit (Thermo Fisher, 23227): BCA reagent A and B were mixed at a ratio of 50:1 (200 μL / well), and the standards were diluted according to Table 1. Two replicates were set for each concentration. The sample to be tested was diluted 1:10 (4.5 μL sample + 40.5 μL double-distilled water). 200 μL of working solution and 20 μL of standard / sample were added to each well. The mixture was incubated at 37°C in the dark for 30 min. The absorbance at 562 nm was read using a microplate reader. The protein concentration was calculated according to the standard curve. (d) Input sample preparation: Take 150 μL of protein sample, add 5× protein loading buffer (Beyotime, P0015L) at a ratio of 4:1, mix well, boil in a metal bath at 100℃ for 10 min, and store at -20℃ for later use. (e) IP sample preparation: The remaining protein sample was diluted with PBS to 1 μg / μL, 500 μg / tube; (f) Antibody incubation: Add Flag antibody (Medical & Biological Laboratories Co., Ltd, M185-3L) or HA antibody (Proteintech, 51064-2-AP) or cFKBP8 antibody (Huaan Biotechnology) or control rabbit IgG (Aibotek, AC005) / mouse IgG (Aibotek, AC011) to the tube at a ratio of 1:500. Vortex to mix and centrifuge, then incubate overnight at 4°C. (g) Incubation with agarose beads (SANTA CRUZ, sc-2003): Add 20 μL to each tube and incubate overnight at 4°C by rotation; (h) Washing and boiling: Centrifuge the sample at 2500 rpm at 4℃ for 5 min, discard the supernatant, add 1 mL PBS, centrifuge again, discard the supernatant, repeat 4 times, after discarding the supernatant for the last time, add 40 μL 1×SDS protein loading buffer (Beyotime, P0287-10 ml), boil in a metal bath at 100℃ for 10 min, cool on ice and store at -20℃; Table 1. Standard ratios for BCA protein concentration determination *Pipette the mixed solution from the tube (2) Protein gel electrophoresis: Take out the 4-20% gradient precast gel (GenScript, M42015C), remove the bottom protective film and fix it in the electrophoresis tank, add 1×MOPS electrophoresis buffer (GenScript, M00138) and vertically remove the comb; thaw the pre-melted sample and 180kDa pre-stained marker (Vazyme, MP201) on ice and accurately add the sample to the sample well; connect the electrodes correctly, first electrophore at 80V constant voltage until the marker bands are clearly separated, then switch to 120V constant voltage to continue electrophoresis, and adjust the total time according to the molecular weight of the target protein; (3) Transfer and blocking: Prepare and pre-cool 1× transfer buffer in advance, cut PVDF membrane (4.5×8cm) and activate with methanol for later use; soak sponge / filter paper in the tray, separate the pre-coated gel and rinse in transfer buffer, assemble the transfer clamp in sequence (sponge→filter paper→PVDF membrane→gel→filter paper→sponge), remove air bubbles with roller and insert into the transfer tank, add pre-cooled buffer; transfer at a constant current of 300mA in an ice bath, the time is adjusted according to the molecular weight of the protein; after the transfer is completed, wash the PVDF membrane with TBST for 5 min and block with 5% skim milk at room temperature for 1.5 hours; (4) Antibody incubation and exposure: After blocking, wash the membrane 3 times with TBST for 10 min each time, and incubate the primary antibody diluted with 3% BSA overnight on a shaker at 4°C; after washing with TBST, dilute the secondary antibody with 5% BSA at a ratio of 1:5000 and incubate at room temperature for 1 h; after washing with TBST again, mix ECL luminescent solution at a ratio of 1:1 to evenly cover the membrane surface, collect signals with a chemiluminescence imager, and analyze the gray values ​​of the bands with ImageJ. Figure 1 AF indicates that cFKBP8 and Hsp60 have both exogenous and endogenous interactions.

[0022] II. In SH-SY5Y cells, the co-localization of cFKBP8 and Hsp60 was detected by immunofluorescence co-localization assay; the steps are as follows: (1) Immunofluorescence staining: (a) Cell fixation: The treated cells were washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 15 min, and then washed with PBS for 5 min each time, for a total of 3 times. (b) Washing and permeabilization: Permeabilize with 0.2% PBST for 15 min, then wash twice with PBS; (c) Blocking: 5% BSA prepared with 0.2% PBST, blocked at room temperature for 1.5 h; (d) Primary antibody incubation: Use 1% BSA diluted with 0.2% PBST solution as the primary antibody dilution solution. Mix cFKBP8 and Hsp60 antibody stock solutions together according to the antibody usage ratio to prepare the primary antibody incubation solution and incubate overnight at 4°C. (e) Secondary antibody incubation: Wash 3 times with PBS, add a mixture of goat anti-mouse fluorescent secondary antibody Alexa Fluor 488 (1:250) and goat anti-rabbit fluorescent secondary antibody Alexa Fluor 594 (1:250) under the dark, and incubate at room temperature for 1 h; (f) Mounting and imaging: Wash with PBS 3 times, 15 min each time, mount with DAPI, and observe by confocal microscope; (2) Colocation analysis: Quantitative analysis was performed using ImageJ software and its plugins, including Pearson's correlation coefficient (R) and Mander's overlap coefficient (R1, R2). Figure 1 GH showed a Pearson's R value of 0.93, indicating a high correlation between the fluorescence signals of cFKBP8 and Hsp60; a Mander's R1 value of 0.693, indicating 69.3% overlap between the cFKBP8 and Hsp60 signals; and a Mander's R2 value of 1, indicating that the Hsp60 signal is completely distributed within the cFKBP8 signal region. Figure 1Line plot analysis showed that the fluorescence intensities of cFKBP8 (red curve) and Hsp60 (green curve) highly overlapped in multiple regions, further confirming their close spatial relationship. These results indicate that cFKBP8 and Hsp60 have significant co-localization in SH-SY5Y cells, suggesting that they may jointly participate in specific biological processes within the cells. III. GST Pull-down experiment to verify the direct interaction between cFKBP8 and Hsp60; the steps are as follows: (1) The recombinant fusion purified proteins GST-Hsp60 and His-cFKBP8 were commissioned to a bioengineering company in Wuhan; the GST pull-down experiment was performed using the GST pull-down kit (Jinkairui, JKR23011), and the experimental steps are as follows: (a) Pretreatment of magnetic beads: Take 30 μL of GSH magnetic beads and wash twice with 0.5 mL of pre-cooled washing buffer; (b) Bait protein binding: Experimental group: magnetic beads + 200 μg GST-Hsp60, control group: magnetic beads + 200 μg GST, add incubation buffer to 1 mL, incubate at room temperature for 2 h; (c) Interacting protein binding: Add 200 μg His-cFKBP8 to each group, add incubation buffer to 1 mL, incubate overnight at 4°C, and wash 3 times; (d) Elution: Add 80 μL of elution buffer to each tube, incubate in a 100℃ metal bath for 10 min, centrifuge at 12000 rpm for 5 min, collect the supernatant, add 20 μL of 5× loading buffer, and incubate in a 100℃ metal bath for 10 min. (e) Input sample preparation: Input control: 50 μg GST + 50 μg His-cFKBP8; Input experiment: 50 μg GST-Hsp60 + 50 μg His-cFKBP8, each with 1 / 4 volume of 5× loading buffer, 100℃ metal bath for 10 min, and stored at 4℃ for later use. (2) Western blot detection after GST pull-down The proteins in each group were subjected to protein electrophoresis, transfer to a membrane, blocking, antibody incubation, and exposure, with the specific steps being consistent with those described above. Figure 1 The results showed that a specific immunoblot band of His-cFKBP8 protein was detected in the GST-Hsp60 proteome, but not in the GST negative control group; this result indicates that there is a specific direct physical interaction between cFKBP8 and Hsp60. IV. In SH-SY5Y cells, the interaction between endogenous cFKBP8 and Hsp60 was detected by PLA, and mitochondria were simultaneously labeled with Tomm20 immunofluorescence to determine whether the interaction between the two was enriched in mitochondria; the steps are as follows: (1) PLA was generated using the Duolink® In Situ Red mouse / rabbit starter kit (Sigma Aldrich, DUO92101), and the steps are as follows: (a) Reagent preparation: Dissolve washing buffer A and B powder in 1000 mL of double-distilled water to prepare 1× solution; then, dilute 1× washing buffer B at 1:100 to prepare 0.01× solution and store at 4℃ for later use. (b) Cell treatment: Take 50 mL of 1× washing buffer A, B and 0.01× washing buffer B and incubate at room temperature for 1 h; at the same time, fix the cells in the confocal dish with 4% paraformaldehyde and perform membrane perforation treatment. (c) Blocking: Cover cells with Duolink® blocking solution and incubate at 37°C for 60 min. Do not dry. (d) Primary antibody incubation: Dilute cFKBP8 antibody (1:100) and Hsp60 antibody (1:1000) with Duolink® antibody diluent and incubate overnight at 4°C; (e) PLA probe incubation: After washing, add a mixture of 8 μL PLA probe, 8 μL MINUS PLA probe and 241 μL diluent, and incubate at 37°C for 60 min. (f) Ligation reaction: After washing, add a mixture of 1 μL ligase and 39 μL 1× ligation buffer, and incubate at 37°C for 30 min; (g) Amplification reaction: After washing, add 1 μL of polymerase mixture and 79 μL of 1× amplification buffer mixture, and incubate at 37°C in the dark for 100 min; (h) Final washing: Wash twice with 1× washing buffer B for 10 min each time, and then wash with 0.01× washing buffer B for 1 min. (2) Tomm20 was immunofluorescence stained according to the standard method. The ratio of Tomm20 primary antibody (Proteintech, 11802-1-AP) was 1:1000, and the ratio of fluorescent secondary antibody Alexa Fluor 488 was 1:250. Figure 1The results showed that cFKBP8 and Hsp60 generated significant PLA fluorescence signals in SH-SY5Y cells, and these signals highly overlapped with Tomm20-labeled mitochondria, indicating that cFKBP8 and Hsp60 have an endogenous interaction in mitochondria. This invention, by optimizing the PLA experimental procedure and combining it with Tomm20 co-localization labeling, can efficiently detect the interaction between cFKBP8 and Hsp60 and their distribution in mitochondria, providing a reliable technical solution for studying protein interaction mechanisms.

[0023] Example 2: Identification of the cFKBP8-Hsp60 interaction interface; In this example, the key amino acid residues for the binding of the two were identified by combining molecular docking, molecular dynamics simulation, computer alanine scanning, and point mutagenesis experiments; the steps are as follows: I. Prediction of key residues for the interaction between cFKBP8 and Hsp60.

[0024] (1) Homology analysis of the cFKBP8 protein sequence was performed based on the SWISS-MODEL platform, and the 10 proteins with the highest homology were selected as templates; (2) The best prediction model, Model-01, is selected based on the Global Model Quality Estimation (GMQE) score for subsequent calculations; (3) Obtain the Hsp60 crystal structure (PDB ID: 7AZP) from the PDB database; (4) Use AutoDockTools-1.5.7 to perform structural optimization treatments such as dehydration and hydrogenation on cFKBP8 and Hsp60; (5) Protein-protein docking was performed using the GRAMM server; (6) The composite material was further optimized using AutoDockTools-1.5.7 after docking; (7) Based on the docking results, simulate the force field using GROMACS 5.0 software and CHARMM36. (8) Construct a periodic boundary condition system and add ions to neutralize the charge; (9) Minimize energy using the steepest gradient method; (10) Perform 50,000 balancing steps each under the NVT and NPT ensembles; (11) Finally, a 100ns molecular dynamics simulation was performed at 300K with a time step of 2.0fs. The coordinates were saved once every fs for analysis. (12) Using the MM-PBSA method, based on 100ns molecular dynamics simulation trajectory data, calculate the binding free energy of the cFKBP8-Hsp60 complex; (13) The equilibrium trajectory in the last 20 ns of the molecular dynamics simulation was calculated by residue energy decomposition to predict the key residues for the interaction between cFKBP8 and Hsp60. (14) The alanine scan calculation module using the gmx_MMPBSA method uses the GROMACS toolkit to mutate the key residues predicted by molecular dynamics simulation to alanine and calculate the binding free energy. II. Co-IP verification of key residues involved in the interaction between cFKBP8 and Hsp60; plasmid construction was outsourced to a biotechnology company in Suzhou. (1) Construct cFKBP8 mutant plasmids based on prediction results: Design and construct the following Flag-labeled cFKBP8 mutant plasmids: Flag-cFKBP8 R13A (ARG13 mutated to ALA), Flag-cFKBP8 R15A (ARG15 mutated to ALA), Flag-cFKBP8 R24A (ARG24 mutated to ALA), Flag-cFKBP8 3A (R13A / R15A / R24A triple mutant); The above mutant plasmids were co-transfected into HEK293 cells with HA-labeled Hsp60 wild-type plasmids, and the proteins were extracted after 48 h. Co-IP was used for verification, and the experimental steps were the same as described above. (2) Construct Hsp60 mutant plasmids based on prediction results: Design and construct the following HA-labeled Hsp60 mutant plasmids: HA-Hsp60 R429A (ARG429 mutated to ALA), HA-Hsp60 D100A (ASP100 mutated to ALA), HA-Hsp60 E384A (GLU384 mutated to ALA), HA-Hsp60 E388A (GLU388 mutated to ALA), HA-Hsp60 E548A (GLU548 mutated to ALA), HA-Hsp60 E107A (GLU107 mutated to ALA), HA-Hsp60 E392A (GLU392 mutated to ALA) and HA-Hsp60 7A (seven-site full mutation); The above 8 plasmids were co-transfected into HEK293 cells with Flag-labeled cFKBP8 wild-type plasmid, and Co-IP was used for verification. The experimental steps were the same as described above. Figure 2AF is a parameter analysis of molecular dynamics simulations, including root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), hydrogen bonds, atomic contact number, and Gibbs free energy morphology. All of these indicate that the cFKBP8-Hsp60 complex reaches a stable binding state within 80-100 ns. MMPBSA calculations show that the cFKBP8-Hsp60 binding free energy TOTAL = -49.13 kcal / mol, indicating strong binding. Figure 3 A); Residue energy decomposition calculations show that ARG7 / 13 / 15 / 24 / 79 / 101 may be key residues on the cFKBP8 interface ( Figure 3 B), where the alanine mutation in ARG13 / 15 / 24 results in ΔΔH > 0.5 kcal / mol ( Figure 3 C); Co-IP experiments showed that the cFKBP8 R13A and cFKBP8 3A mutants significantly disrupted the interaction between cFKBP8 and Hsp60, confirming that ARG13 on cFKBP8 is a key residue for their interaction. Figure 3 D and E); Residue energy decomposition calculations of Hsp60 in the last 20 ns of molecular dynamics simulations revealed that 20 residues in Hsp60 significantly contribute to the binding free energy, including: ARG197, ARG405, ASP179, ASP196, ASP329, ASP330, ASP387, ASP395, ASP52, ASP61, ASP76, GLU360, GLU362, GLU364, GLU368, GLU41, GLU524, GLU528, GLU83, and GLU84. Figure 4 A); The alanine scan results for these 20 residues showed that ARG405 (complete sequence ARG429), ASP76 (complete sequence ASP100), GLU360 (complete sequence GLU384), GLU364 (complete sequence GLU388), GLU524 (complete sequence GLU548), GLU83 (complete sequence GLU107), and GLU368 (complete sequence GLU392) had ΔΔH > 0.5 kcal / mol. Figure 4 B); Co-IP experimental results showed that single-point mutations and seven-site mutations in GLU384, GLU388, and GLU392 all significantly reduced the binding of cFKBP8 to Hsp60. Figure 4 C); GLU384, GLU388, and GLU392 sites are located precisely in the apical domain of Hsp60 (Figure 4 D).

[0025] Example 3: In this example, cFKBP8, cFKBP8 R13A, and cFKBP8 3A were overexpressed in SH-SY5Y cells. MitoTracker Orange probe was used to observe mitochondrial morphology, transmission electron microscopy was used to assess mitochondrial ultrastructure, DCFH-DA and MitoSOX Red probes were used to detect intracellular total ROS levels and mitochondrial superoxide anion levels, respectively, and JC-1 probe was used to detect changes in mitochondrial membrane potential. These experiments revealed that cFKBP8 induces mitochondrial structural and functional damage by binding to Hsp60. The steps are as follows: The nucleotide sequence of cFKBP8 R13A is as shown in SEQ ID NO. 1, and the amino acid sequence is as shown in SEQ ID NO. 2; SEQ ID NO. 1: 5'-GCTAGCATGCTCACGGGGCAGGGAGCGCGTGGCCCTGGCCAACGCCAAGCGGGAGTGCGGCAACGCCCACTACCAGCGGGCGGACTTCGTCCTGGCCGCCAACTCCTACGACCTCGCCATCAAGGCTATCACCTCCAGCGCCAAAGTGGACATGACGTTCGAGGAGGAGGCACAGCTCCTGCAGTTGAAGGTGAAGTGTCTGAACAACCTGGCGGCCTCGCAGCTGAAG CTCGACCACTACCGCGCAGCCCTGCGCTCCTGCAGCCTTGTGCTGGAGCACCAGCCAGACAACATCAAGGCTCTCTTCCGCAAGGGCAAGGAGCCCATACATCCCCCCGCACGCGGCCCTGTGCCTGGAGGTGACCCTGAAGACGGCTGTGGACGGGCCGACTACAAAGACCATGACGGTGATTATAAAAGATCATGACATCGATTACAAGGATGACGATGACAAGTGA-3' SEQ ID NO.2:MLTGQERVALANAKRECGNAHYQRADFVLAANSYDLAIKAITSSAKVDMTFEEEAQLLQLKVKCLNNLAASQLKLDHYRAALRSCSLVLEHQPDNIKALFRKGKEPIHPPARGPVPGGDPEDGCGRA 1. Transfect SH-SY5Y cells with the empty control plasmid and the Flag-labeled cFKBP8 WT, cFKBP8R13A, and cFKBP8 3A plasmids, respectively, following the same transfection steps as described above; 2. Observe mitochondrial morphology using the MitoTracker Orange probe. (1) Dissolve PK Mito Orange powder (Puhai Jingshan, PKMO-1) in 20 μL DMSO to prepare a 250 μM stock solution; (2) Take the culture medium preheated to 37°C and add it to the mother liquor at a ratio of 1:2000 to prepare the working solution. Prepare and use immediately. (3) Remove the original culture medium, add PK Mito working solution, and incubate at 37°C for 15 min; (4) Discard the staining solution, wash once with preheated medium, add Hoechst 33342 (Thermo Fisher, R37165) to stain the nuclei, and incubate at room temperature for 5 min; (5) Take pictures under a confocal microscope; 3. Use the DCFH-DA probe to detect the total intracellular ROS level. (1) Dilute the fluorescent probe DCFH-DA (Solepro, CA1410) to 10 μmol / L using serum-free culture medium at a ratio of 1:1000; (2) Discard the original culture medium, add 500 μL of probe working solution, and incubate at 37°C in the dark for 20 min, shaking gently 3-5 times during the period; (3) Wash three times with serum-free medium, and add Hoechst 33342 to stain the nuclei for 5 min on the last wash; (4) Observe using a confocal microscope; 4. Detection of mitochondrial superoxide anion levels using the MitoSOX Red probe. (1) Preparation of stock solution: Dissolve 50 μg of MitoSOX Red powder (MCE, HY-D1055) in 13 μL of DMSO to prepare a 5 mM stock solution; (2) Add 1 μL of the stock solution to 1 mL of serum-free culture medium to prepare a 2 μM working solution. Note that it should be prepared and used immediately. (3) Discard the culture medium, add 500 μL of working solution, and incubate at 37°C for 15 min; (4) Wash the serum-free culture medium 3 times, add Hoechst 33342 for 5 min for the last time, observe with confocal microscopy and take pictures immediately for detection; 5. Use the JC-1 probe to detect mitochondrial membrane potential (1) Preparation of staining working solution: Dilute 50 μL of 200× JC-1 dye into 8 mL of ultrapure water, sonicate in a water bath at 37℃ for 15 min until completely dissolved, and then add 2 mL of 5× staining buffer. (2) Replace the old culture medium with 1 mL of fresh culture medium, then add 1 mL of JC-1 working solution and incubate at 37°C for 20 min; (3) Wash twice with pre-cooled 1×JC-1 buffer, then add 2 mL of fresh culture medium; (4) Add Hoechst 33342 staining solution, incubate for 5 minutes, and then immediately observe and photograph using a confocal microscope; 6. Transmission electron microscopy analysis of mitochondrial ultrastructure (1) After digesting the cells with trypsin, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend in 3% glutaraldehyde fixative diluted 1:5, and let stand at 4℃ for 5 min. (2) Transfer to a 1.5 mL EP tube and centrifuge at 12000 rpm for 10 min; (3) Discard the supernatant, add fresh 3% glutaraldehyde fixative, fix overnight at 4°C, and send to a biotechnology company in Chengdu for testing; The mitochondrial morphology and ultrastructure of the cFKBP8 WT group showed abnormal changes. First, the mitochondria were highly fragmented or punctate, accompanied by mild swelling and disruption of the network structure. Second, the mitochondria swelled, lost matrix, and had broken or disappeared cristae. Finally, the mitochondria exhibited significant functional impairment, manifested by significantly increased cellular ROS and mitochondrial superoxide anion levels and decreased mitochondrial membrane potential. However, overexpression of cFKBP8 R13A or cFKBP8 R13A alleviated the above-mentioned mitochondrial structural and functional abnormalities. Figure 5 ).

[0026] Example 4: In this example, recombinant adeno-associated virus was injected into the anterior limbic cortex of wild-type C57BL / 6J male mice using stereotactic brain imaging. Figure 6As shown, wild-type cFKBP8 and cFKBP8 R13A (with arginine mutated to alanine at position 13 of cFKBP8) were specifically overexpressed in the anterior limbic cortex neurons of mice. Two weeks later, the mice were subjected to a two-week chronic unpredictable mild stress (sCUMS). Subsequently, the depressive-like behavior of the mice was assessed using the sucrose preference test (SPT), forced swimming test (FST), and tail suspension test (TST). The ultrastructure of neuronal mitochondria was observed by transmission electron microscopy, and the level of neuronal superoxide anion was assessed by dihydroethidium (DHE) staining. The results showed that mice overexpressing wild-type cFKBP8 exhibited stress susceptibility, neuronal mitochondrial ultrastructural damage, and increased superoxide anion levels, while overexpression of cFKBP8 R13A alleviated this damage and stress susceptibility.

Claims

1. cFKBP8-Hsp60 interaction plays a role in depression by regulating mitochondria.

2. The application according to claim 1, characterized in that, The key amino acid residue for the interaction between cFKBP8 and Hsp60 is arginine at position 13 of cFKBP8; that is: cFKBP8 ARG13.

3. The application according to claim 2, characterized in that, The cFKBP8 ARG13 mutation to cFKBP8 R13A disrupts the cFKBP8-Hsp60 interaction.

4. The application according to claim 1, characterized in that, The regulation of mitochondria includes: (1) Increasing the expression level of cFKBP8 in SH-SY5Y cells damages mitochondria, while increasing the expression level of cFKBP8 R13A reverses this effect; (2) Increasing the expression level of cFKBP8 in mouse anterior limbic cortex neurons damages mitochondria, while increasing the expression level of cFKBP8 R13A reverses this effect.

5. The application according to claim 1, characterized in that, The damage to mitochondria includes: mitochondrial swelling, cristae breakage or disappearance, reduced matrix electron density, increased proportion of damaged mitochondria, disruption of mitochondrial network structure, increased cellular or mitochondrial ROS levels, and decreased mitochondrial membrane potential.

6. The application according to claim 1, characterized in that, The reagent for increasing the expression level of cFKBP8 includes a recombinant vector that overexpresses the cFKBP8 or cFKBP8 R13A gene, wherein the cFKBP8 or cFKBP8 R13A gene is the gene encoding the cFKBP8 or cFKBP8R13A protein.

7. The application according to claim 6, characterized in that, The nucleotide sequence of cFKBP8 R13A is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO.

2.

8. The application according to claim 6, characterized in that, The recombinant vector overexpressing the cFKBP8 or cFKBP8 R13A gene includes the cFKBP8 or cFKBP8 R13A gene and an adenovirus-associated vector.

9. The application according to claim 1, characterized in that, The application in depression involves increasing the expression of cFKBP8 in mouse anterior limbic cortex neurons to promote stress susceptibility in mice, while increasing the expression of cFKBP8 R13A reverses this effect.

10. The application according to claim 9, characterized in that, The adenovirus-associated vectors include the rAAV2 / 9 vector.

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