CN111514121A - Application of gossypol acetate in preparation of medicines for treating neurodegenerative diseases - Google Patents

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of gossypol acetate in preparation of a medicine for treating neurodegenerative diseases. The invention provides an application of gossypol acetate, which comprises the following steps: gossypol acetate specifically binds to VCP, and interaction of VCP and LC3 and interaction of VCP and mutant mHTT are improved; the gossypol acetate is specifically combined with VCP, so that the formation of VCP, LC3 and mHTT complexes is promoted, and then toxic protein mHTT is degraded by autophagosomes; gossypol acetate improved the mHTT protein-induced disease phenotype in both the huntington cell model and the drosophila disease model. Experiments show that the HD fruit fly model shows obvious reduction of the HTT level of the head after 2 weeks of gossypol acetate administration, and the life cycle and the motor ability of the HD fruit fly model are obviously improved. The gossypol acetate promotes the formation of VCP, LC3 and mHTT complexes by targeting VCP, and accelerates the degradation of toxic protein mHTT through an autophagy pathway; provides a candidate small molecule drug for subsequent HD diseases.

Description

Application of gossypol acetate in preparation of medicines for treating neurodegenerative diseases

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of gossypol acetate in preparation of a medicine for treating neurodegenerative diseases.

Background

In recent years, the prevalence of neurodegenerative diseases (NDD) has been increasing with the aging population. NDD is a type of brain disease with great harmfulness, such as Alzheimer Disease (AD), Parkinson Disease (PD), frontotemporal dementia (FTD), Huntington Disease (HD), and Amyotrophic Lateral Sclerosis (ALS), etc., because abnormally folded proteins cannot be cleared in time due to neuronal damage, and toxicity is generated by accumulating in neurons, thereby causing neuronal cell death. Due to the wide range of such diseases and the lack of effective therapeutic approaches, great challenges remain to be faced clinically. Huntington's Disease (HD) is an important model for the study of neurodegenerative diseases due to its distinct genetic profile. The initiation of HD is caused by the mutation of the Huntington gene (HTT), i.e., the HTT gene with unstable CAG repeat amplification sequence on chromosome 4 of HD patients, and under the healthy state, the HTT gene usually contains 6-35 CAG nucleotide repeats, while in HD patients, the CAG nucleotide repeats usually exceed 40, finally resulting in the excessive extension of the amino-terminal glutamine repeat (polyglutamine, polyQ) of Huntington protein (HTT), and the cytotoxicity caused by accumulation and precipitation of the polyQ mutant Huntington protein (mHTT) in nerve cells is the key of the disease. The clinical manifestations of the disease are complex dyskinetic disorders, the typical features of which include dyskinesia (involuntary movements as dance), cognitive dysfunction (e.g. attention, etc.) and mental disorders (progressive intellectual decline, e.g. apathy). The disease is rare, and is estimated to be 4/10-10/10 thousands of people, the symptoms of young patients are generally severe, mainly myotonia, the dance symptoms of middle-aged patients and intention tremor of patients over 60 years old. The patients of juvenile type are rare, the age of onset is generally middle-aged, and the disease course can be as long as decades. The disease condition of a patient is progressively worsened, generally, the patient will die after 15 to 20 years of illness, and at present, an effective treatment means is lacking clinically.

Autophagy is a highly conserved process of cellular metabolism that degrades a range of substances affecting cellular homeostasis, including damaged organelles, misfolded proteins, and intracellular aggregates. Autophagy is essential to maintain turnover and stability of the various components within the cell, maintaining cellular homeostasis. Autophagy begins with a separation membrane, also known as a phagocytic vesicle, which may be derived from an Endoplasmic Reticulum (ER), golgi, or endosomal-derived lipid bilayer; the phagocytic vesicle is continuously expanded, thereby phagocytosing intracellular substances such as protein aggregates, damaged organelles, ribosomes, etc. into a double-membrane vesicle, called autophagosome; the loaded autophagosome is fused with a lysosome to be mature to form an autophagosome, so that the degradation of the content of the autophagosome by acid protease in the lysosome is promoted; degraded products such as amino acids and other degraded by-products can be transported back into the cytoplasm for re-use in the construction and metabolism of biological macromolecules. Thus, autophagy is considered to be a "circulating factory" of cells. One of the reasons for HD development is due to insufficient clearance from the lysosome pathway by accumulation of mHTT toxic proteins. The specific activation of autophagy and the promotion of mHTT protein degradation are one of effective means for relieving and treating HD diseases.

The Valosin Containing Protein (VCP) is also called p97, and belongs to members of the atpase superfamily (AAA), VCP can combine with various accessory proteins to regulate different biological functions, such as participating in endoplasmic reticulum-related Protein degradation (ERAD), autophagy, apoptosis, ubiquitin-related Protein degradation, nuclear membrane formation, cell cycle regulation, DNA damage repair and other important activities. VCP plays an important role in autophagy-related protein degradation, and the discovery and identification of active natural small molecules, which effectively regulate the functions of the molecules in the autophagy process, are one of the strategies for potentially eliminating accumulated toxic proteins and further alleviating and treating related diseases.

Gossypol (Gossypol) is a polyphenolic compound extracted from cotton seeds, originally used as a dye, but it is photolabile and easily decomposed by light. Subsequent studies found that gossypol had contraceptive effects, but had hypokalemia and the irreversible contraceptive side effects. As research on gossypol progresses, gossypol is gradually found to have more biological effects such as: antiviral, antitumor, antioxidant, antiparasitic, antibacterial, etc. The research shows that gossypol has the effect of activating autophagy, and whether other action modes exist besides the damage to the formation of Bcl-2 and Beclin1 complexes exists in the action mechanism, which is the focus of the research. Gossypol acetate is in the form of acetate of gossypol, has better absorbability, longer action time and fewer side effects, so gossypol acetate is selected in the research.

The invention is based on the theory and discusses the application of gossypol acetate in activating autophagy pathway and treating neurodegenerative diseases (including Huntington's disease).

Disclosure of Invention

The invention aims to provide application of gossypol acetate in preparing a medicament for treating neurodegenerative diseases.

The application of gossypol acetate in preparing the medicament for treating the neurodegenerative diseases provided by the invention is to activate autophagy pathways to play a role through the gossypol acetate, and specifically comprises the following steps:

gossypol acetate specifically binds to VCP, increasing the interaction of VCP with LC 3. LC3 is tubulin light chain 3.

Gossypol acetate specifically binds to VCP, increasing the interaction of VCP with mHTT.

Gossypol acetate specifically binds to VCP, promoting the formation of VCP, LC3 and mHTT complexes, which in turn degrades the toxic protein mHTT by autophagosomes.

The invention also includes the use of gossypol acetate to improve the mHTT protein-induced disease phenotype in huntington cell models and drosophila disease models.

The structural formula of the gossypol acetate is shown as follows:

experiments show that: through enzyme activity detection and protein thermal stability migration experiment,found that gossypol acetate targets a pocket formed by the N-terminal domain of VCP and the D1 domain, and gossypol acetate is found in cells to activate autophagy of cells and reduce Q47 cells and STHdhQ^7/Q111The mHTT level in the cells (HD model cells) can be increased simultaneously with the STHdhQ^7/Q111Cell and survival of iPS-derived neurons in HD patients. Meanwhile, by using in vitro expression of purified VCP protein with a 6 histidine residue label, microtubule-associated protein light chain 3 with a GST label and HTT protein (HTT-Q25/mHTT-Q72) with different numbers of polyglutamine chains and with a 6 histidine residue label, in vitro combination experiments show that gossypol acetate respectively promotes the interaction between VCP and LC3 and the interaction between VCP and mHTT-Q72 by combining VCP, and then degrades toxic protein mHTT through autophagosomes. Finally, the compound is verified to have the effect of improving the mHTT protein-induced disease phenotype in a cell model and a drosophila disease model respectively. The invention not only proves that VCP can be used as a new intervention target for HD diseases, but also provides the application of gossypol acetate in the preparation of medicines for treating neurodegenerative diseases (including HD) by explaining the action mechanism of the gossypol acetate, provides candidate micromolecule medicines for subsequent HD, and simultaneously provides a new reference mode for the research and development of subsequent medicines due to the uniqueness of the action mode of the gossypol acetate.

Drawings

FIG. 1 shows the use of malachite green to measure ATPase activity of VCP proteins, where more than about 700 pools of natural small molecule compounds were screened. Fig. 1 shows the 9 compounds with higher inhibition efficiency on VCP enzyme activity, wherein the inhibition efficiency of gossypol acetate on VCP enzyme activity reaches 80% -90%.

FIG. 2 shows that the inhibition efficiency of the enzyme activity of VCP shows dose dependency with increasing gossypol acetate concentration, and the half-inhibition rate of the enzyme activity of 6XHis-VCP of gossypol acetate is 9.3 + -0.4 μ M.

FIGS. 3-4 show the effect of gossypol acetate on the structure of the VCP protein.

FIG. 3 shows that gossypol acetate bound to VCP does not affect the hexamer formation of the protein function as detected by native polyacrylamide gel electrophoresis.

FIG. 4 shows that gossypol acetate bound to VCP affects the conformation of VCP monomer, resulting in differences in trypsin cleavage pattern (red arrows), with gossypol acetate treatment of histone being more stable, and with gossypol acetate having bands similar to those of gossypol group, with NMS873 histone bands being more stable (NMS-873 is a specific allosteric inhibitor of VCP, IC-873)₅₀At 30nM, its mechanism of action studies showed that NMS-873 interfered with autophagy, thereby inducing cancer cell death).

FIG. 5 shows gossypol acetate in combination with VCP, where K_DThe concentration was 6.9. mu.M.

FIGS. 6-7 show that gossypol acetate improves the thermal stability of VCP proteins. Wherein, under the concentration of 5 MuM gossypol acetate, the protein degradation temperature and the thermal stability T of VCP protein are continuously increased_mThe value is increased from 64.2 +/-0.1 ℃ to 71.1 +/-0.7 ℃.

Fig. 8-12 show that gossypol acetate can bind to the pocket formed by the N-terminal domain and D1 domain of VCP. Wherein:

FIGS. 8-9 show that gossypol acetate alters the thermostability, T, of the VCP-N + D1 fragment protein_mThe value increased from 56.9. + -. 0.1 ℃ to 62.3. + -. 1.6 ℃ but had no effect on the thermal stability of the fragment VCP-N/D2.

FIG. 10 shows that gossypol acetate inhibits ATPase activity of the full-length VCP protein and the fragment VCP-N + D1 protein, but does not affect ATPase activity of the fragment VCP-D1+ D2 protein.

FIG. 11 shows the inhibition efficiency of the 3 molecules except gossypol acetate on the activity of VCP enzyme, which are found in the screening process, on the full-length VCP and truncated protease activity, wherein the 3 molecules can only inhibit the activity of the VCP full-length protein and cannot inhibit the activity of VCP-D1+ D2.

Fig. 12 shows PDB-encoded VCP crystal structure modeling the binding of gossypol acetate to VCP, which binds to the N-terminal domain of VCP protein and the pocket formed by the D1 domain. P < 0.01.

FIGS. 13-17 show the activation of autophagy by gossypol acetate in cells. Wherein:

FIGS. 13-14 show that the autophagy substrate NBR1/NCOA4/p62 showed a decreasing trend and the LC3I type was significantly decreased and the LC3II type was increased for 24 hours after gossypol acetate treatment of the cells, while immunofluorescence confocal setup showed that the level of autophagy substrate (p 62, NCOA4, NDP52) showed a decreasing trend, with increasing gossypol acetate concentration, representing a decreasing red fluorescence spot for p62/NCOA4/NDP52 protein, respectively. Chloroquine (chloroquine), one of the autophagy inhibitors, can block the fusion of autophagosomes with lysosomes, so that autophagy cannot be completed, resulting in the accumulation of autophagy substrates.

FIG. 15 shows that after the cells were treated with NMS873, another VCP inhibitor, DBEQ, for 24 hours, autophagy substrate was increased, autophagy was inhibited, and the level of ubiquitination was higher than that of gossypol acetate.

Fig. 16 shows the influence of gossypol acetate on autophagy flow, and gossypol acetate, rapamycin and chloroquine respectively treat HeLa cell line stably expressing mCherry-EGFP-LC3 for 24 hours, so that gossypol acetate and rapamycin improve autophagy flow level of cells, and chloroquine blocks formation of autophagy flow of cells.

Figure 17 shows that chloroquine blocks gossypol acetate-induced autophagy.

Fig. 18-20 show that gossypol acetate targets VCP to activate autophagy. Wherein:

figure 18 shows inhibition of autophagy, increased autophagy substrate accumulation, increased LC3II, and decreased LC3I following VCP knockdown in Hela cells.

Fig. 19 shows that the expression level of VCP was knocked down in HeLa cells, and after 24 hours of treatment with gossypol acetate, p62 did not significantly decrease in the group with knocked-down VCP, while the level of p62 in the group without VCP knocked down showed a decreasing trend with increasing drug concentration, and gossypol acetate activated autophagy in cells by binding to VCP.

FIG. 20 shows that the expression level of knockdown of Beclin1 in HeLa cells was reduced by p62 in both the control and knockdown Beclin1 groups after 24 hours of gossypol acetate treatment, and still activated autophagy.

FIGS. 21-25 show that gossypol acetate decreased the levels of HTT protein in HD-Q47 cells. Wherein:

FIG. 21 shows IC of gossypol acetate in Q47₅₀About 13.2. mu.M.

Fig. 22 shows that when Q47 cells were treated with gossypol acetate for 24 hours, HTT protein showed a gradient decreasing trend with increasing drug concentration, MW1/2B7 was the antibody pair, intracellular HTT protein levels were measured, MW1 represents pathogenic HTT protein, 2B7 represents total intracellular HTT protein, LC3I decreased, and LC3II increased.

FIG. 23 shows the case where cells were pretreated with chloroquine for 6 hours and then treated with gossypol acetate at various concentrations, autophagy was blocked and the HTT protein was no longer degraded by the autophagy pathway.

FIGS. 24-25 show HTRF assays measuring HTT protein levels in HD cells treated with gossypol acetate, wherein the levels of HTT protein in HD-Q47 and in HD patient-derived pluripotent-derived stem cells decreased. P <0.05, p < 0.001.

FIGS. 26-27 show that gossypol acetate reduces STHdh^Q7/Q111Intracellular HTT protein levels increase cell survival. Wherein:

FIG. 26 shows that the level of Caspase3 in the HD cell control group was significantly higher than that in the HD cell administration group, and compared with the level of Caspase3 in the normal cell group, the signal level in the administration group was also slightly higher than that in the normal group, but both of them were significantly lower than those in the HD cell group in the control group.

FIG. 27 shows the detection of gossypol acetate treatment STHdh by HTRF technique^Q7/Q111A decrease in the level of post-cellular HTT protein. P<0.001。

Figures 28-30 show the case where gossypol acetate increases survival of iPS differentiated neurons reducing HTT protein levels. Wherein:

fig. 28 shows that the number of neurons in the group to which iPS was differentiated, the group to which the HD pathogenic cells of neurons were administered, the HTT knockdown group for HD pathogenic cells, and the group to which normal cells were administered were significantly higher than the control group for HD pathogenic cells (Tuj1 for detection of live nerve cells, red fluorescent spots for Tuj1), and Caspase3 activity of the above three groups was also significantly lower than that of the control group for HD pathogenic cells (green fluorescent spots for Caspase 3).

FIGS. 29 to 30 show that the activity of Caspase3 in the group administered with HD pathogenic cells, the group with HD pathogenic cell HTT knockdown, and the group with normal cells of iPS-derived neurons after iPS treated with gossypol acetate for 48 hours was significantly lower than that in the group with HD pathogenic cells control, and the number of neurons was higher than that in the group with HD pathogenic cells control. P < 0.001.

Fig. 31-32 show that gossypol acetate enhances VCP interaction with LC3 by targeting the N-terminal domain of VCP. Wherein:

figure 31 shows the results of in vitro binding experiments, where VCP interaction with LC3 increased significantly when gossypol acetate concentration reached saturation in the in vitro system.

FIG. 32 shows that gossypol acetate can increase the interaction of LC3 with full-length VCP protein (VCP FL), fragment VCP-N + D1, but not LC3 with fragment VCP-N.

FIGS. 33-35 show that gossypol acetate promotes the interaction of VCP, LC3, and mHTT in vitro. Wherein:

FIG. 33 shows that gossypol acetate promotes the interaction of VCP with mHTT-Q72 in vitro, but does not affect the interaction of VCP with HTT-Q25.

FIG. 34 shows that gossypol acetate does not affect the interaction of mHTT-Q72/HTT-Q25 with LC 3.

Fig. 35 shows that gossypol acetate promotes the interaction between VCP, LC3, mHTT in vitro.

FIGS. 36-38 show that gossypol acetate specifically enhances the interaction of VCP with LC 3. Wherein:

fig. 36 shows that gossypol acetate improved the interaction of VCP with LC3, but did not have a significant effect on the interaction of VCP with P47.

Fig. 37 shows that gossypol acetate increased the interaction of VCP with LC3, and that NMS873 and DBEQ had no significant effect on the interaction of VCP with LC 3.

FIG. 38 shows that gossypol acetate treatment of HD fruit flies carrying the Full-HTT-128Q or Exon1-HTT-72Q genes was effective in reducing levels of HTT protein in the heads of the HD model flies 2 weeks after feeding compared to the control group. P <0.05, p <0.01, p < 0.001.

Figures 39-42 show that gossypol acetate improves survival and motility of HD fruit flies. Wherein:

FIGS. 39-40 show that gossypol acetate increases survival and motility of HD fruit flies carrying the Full-HTT-128Q gene.

FIGS. 41-42 show that gossypol acetate improves survival and motility of HD fruit flies carrying the Exon1-HTT-72Q gene. P <0.001, p < 0.0001.

FIGS. 43-45 show that gossypol acetate improves the ability to recognize and to apply to the environment in AD mice. Wherein:

fig. 43 shows that gossypol acetate improves the ability of AD mice to recognize new and old objects, and AD mice administered for 10 weeks had a preference for new object recognition.

Fig. 44-45 show that AD mice, 10 weeks after dosing, moved more towards the open center and increased distance of movement in the mine experiment. P <0.05, p < 0.01.

Detailed Description

Materials and reagents

1.1 plasmid, vector: pET-28a (+), pcDNA3.1-HisA, pSG5-Flag, pGEX-6P-2.

Host bacteria: escherichia coli XL10, BL21, DH5 α.

Cell line

Cell lines used in the experiments: hela (cervical cancer cells), Q47 (fibroblasts), STHdh^Q7/Q111(CH00096-Q7/Q111) (mouse neural precursor cells), Stable mCherry-EGFP-LC3 HeLa (human cervical cancer cell of LC3 stably expressing mCherry and EGFP fluorescent protein)

STHdh^Q7/Q111The cells were cultured at 33 ℃ with 5% CO₂In a cell incubator, other mammalian cell lines were cultured at 37 ℃ with 5% CO₂In a cell incubator.

Antibodies

Antibodies used in the experiments: anti-GFP tag monoclonal antibody (Clontech), anti-6 XHis tag monoclonal antibody (Senta Cruz), anti-GFP tag monoclonal antibody (Proteintetech), anti-GST tag monoclonal antibody (Senta Cruz), anti-Flag tag monoclonal antibody (Senta Cruz), anti-VCP monoclonal antibody (Proteintech), anti-NBR 1 monoclonal antibody (Senta Cruz), anti-NCOA 4 monoclonal antibody (Senta Cruz), anti-p 62 monoclonal antibody (Proteitech), anti-LC 3 monoclonal antibody (Novus), anti-Lamp 1 monoclonal antibody (Senta Cruz), anti-Ub monoclonal antibody (Abways), anti-NDP 2 monoclonal antibody (Novus), anti-Beclin 1 monoclonal antibody (Proteitech), anti-beta-Tubulin monoclonal antibody (Proteichc), anti-6 XHis tag monoclonal antibody (Senta Cruz), anti-Flag monoclonal antibody (Conta Cruz), anti-VCP 62 monoclonal antibody (Proteitech). All secondary antibodies used in the experiments were purchased from jacksonimanorresearch. The small molecule gossypol acetate used in the experiment was purchased from Doppel Bio Inc.

Preparation of experimental reagent

1.5.1 plasmid construction related reagents

(1) Preparation of LB culture medium

Placing the prepared liquid LB culture medium in a high-pressure moist heat sterilization at 121 ℃ for 15 min;

placing the prepared solid LB culture medium at 121 ℃ for high-pressure moist heat sterilization for 15min, cooling to 55 ℃ (touching with hands), adding the prepared antibiotic according to the proportion of 1:1000, quickly mixing uniformly, pouring into a sterile culture dish in a super clean bench, after solidification, storing at 4 ℃ for use within one month. Liquid medium (1L): 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, and adding deionized water to the mixture until the volume is 1L. Solid medium (1L): 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 15g of agar, and adding deionized water to the mixture until the volume is 1L.

(2) 50 × TAE: 242 g of Tris base is taken, dissolved completely by 800mL of deionized water, 57.1 mL of glacial acetic acid is added, and the volume is adjusted to 1L by 100mL of ethylenediamine tetraacetic acid (0.5 mol/L, pH 8).

Cell culture related reagents

(1) Phosphate Buffered Saline (PBS): NaCl 8g, KCl 0.2g, Na₂HPO₄·12H₂O 3.58g，KH₂PO₄Dissolving 0.24g in sterile deionized water, diluting to a constant volume of 1L, adjusting pH to 7.4, sterilizing under high pressure and moist heat, and storing at 4 deg.C for use.

(2) Cell complete medium: adding 50mL of 10% volume fetal bovine serum and 5mL of 100X double-resistant Streptomycin mixed Solution (Penicilin-Streptomycin Solution) into 500mL of DMEM high-sugar medium or 1640 medium, and storing at 4 ℃ for later use.

(3) G418 (100 mg/mL): weighing 2g G418 powder, dissolving in 20mL PBS, filtering with 0.22 μm microporous membrane for sterilization, subpackaging in ultra clean bench, and storing at-20 deg.C.

(4) Preparing DEPC water: absorbing 1mL of DEPC, dissolving in deionized water, fully and uniformly mixing, placing in a fume hood for ventilation overnight, sterilizing by high-pressure moist heat, and storing at 4 ℃ for later use.

(5) 0.1% Alamar Blue solution: 0.5mg of Alamar Blue powder was weighed, dissolved in 50mL of PBS, filtered through a 0.22 μm filter for sterilization, and stored at 4 ℃ after being dispensed.

Cell lysis and protein purification related reagents

(1) PMSF 17.4mg of PMSF powder was weighed and dissolved in 1mL of methanol sufficiently to store the concentration at 100mM and stored at-20 ℃ for later use.

(2) Aprotinin: weighing 1mg of apritinin powder, fully dissolving in 1mL of water, and storing at the storage concentration of 1mg/mL at-20 ℃ for later use.

(3) Leuppeptin: weighing 1mg of Leuteptin powder, dissolving in 2mL of methanol, and storing at-20 ℃ for later use, wherein the storage concentration is 0.5 mg/mL.

(4) Pepstatin A: 1mg of Pepstatin A powder was weighed and dissolved in 2mL of methanol at a storage concentration of 0.5mg/mL and stored at-20 ℃ for further use.

(5) RIPA: 8.76g NaCl and 10g sodium deoxycholate were weighed and dissolved in 10mL10% SDS solution and 10mL1M Tris-HC L (pH 7.2), 1mL Triton-X-100 and 10mL 0.5M EDTA were added, and finally sterile deionized water was added to a constant volume of 1L and stored at room temperature.

(6) Bacterial lysate: 200mM Tris-HCl pH8.0, 50mM NaCl, 5mM MgCl ₂10% by volume of glycerol, 2% Triton-X-100, 20mM imidazole, PMSF(1 mM), lysozyme (1 mg/mL) was added at the time of lysis.

(7) Protein eluent: 200mM Tris-HCl pH8.0, 50mM NaCl, 5mM MgCl ₂10% by volume of glycerol, 20mM imidazole (depending on the nature of the protein, the concentration of imidazole can be adjusted during elution of the protein).

(8) Protein stock solution: 150mM Tris-HCl pH8.0, 100mM KCl, 10% glycerol by volume.

(9) 1M imidazole: imidazole powder was weighed and dissolved well in 200mM Tris-HCl pH8.0, 50mM NaCl, 5mM MgCl2, 10% glycerol solution by volume and stored at 4 ℃ until use.

(10) Coomassie brilliant blue staining solution: 2.5g of Coomassie brilliant blue R250 was weighed out and dissolved in 450mL of methanol, 100mL of glacial acetic acid was added, and finally, deionized water was added to a volume of 1L.

(11) Decoloring liquid: 450mL of methanol and 100mL of acetic acid, and finally the volume is adjusted to 1L by deionized water.

Reagent related to polyacrylamide gel protein electrophoresis

(1) 30% acrylamide gel stock solution: 290g of acrylamide and 10g N, N' -methylene bisacrylamide powder are weighed and dissolved in 900 mL of deionized water, the residue is filtered, the volume is determined to be 1L, and the mixture is stored at 4 ℃ in a dark place.

(2) 10% Ammonium Persulfate (APS): weighing 1g of ammonium persulfate powder, dissolving in 9 mL of deionized water, diluting to 10mL, filtering with 0.45 mu M microporous membrane, and storing at 4 ℃.

(3) 1.5 mol/L Tris (pH = 8.8): 181.6 g of Tris powder is weighed, dissolved in 900 mL of deionized water, the pH value is 8.8, the volume is constant to 1L, and the solution is stored at room temperature.

(4) 1.0 mol/L Tris (pH = 6.8): 121.1 g of Tris powder is dissolved in 900 mL of deionized water, the pH value is 6.8, the volume is constant to 1L, and the solution is stored at room temperature.

(5) 10% SDS: weighing 10g of SDS powder, completely dissolving in 90 mL of deionized water, defoaming, fixing the volume to 100mL, and storing at room temperature.

(6) Preparing SDS-PAGE gel:

the preparation method comprises the following steps of cleaning samples of glass plates with protein glue, loading the two glass plates on a glue preparation frame after the glass plates are dried in the air, uniformly mixing water, 30% polyacrylamide solution, Tris-HCl and SDS according to the ratio of reagents in the following table, finally adding 10% APS and TEMED by using a liquid transfer gun, fully mixing, pouring separation glue into the middle of the glass plates, stopping pouring when the distance from the top of the glass plates is about 1.5cm, immediately adding 1mL of deionized water into the glass plates, and finishing polymerization after about 20 min. At this time, 4% of upper layer concentrated gel is prepared according to the following table, water on the upper layer of the gel is firstly discarded, the water is sucked by absorbent paper, upper layer gel liquid is injected, the sample comb is immediately inserted into the upper layer gel, the gel can be coagulated after being placed at room temperature for about 20min, and the prepared SDS-PAGE gel can be stored for use within one week at 4 ℃.

(7) XTris-Glycine SDS-PAGE gel electrophoresis buffer: weighing 15.15 g Tris, 94 g Glycine, dissolving in 800mL sterile deionized water, adding 50mL 10% SDS solution, diluting to 1L, and storing at room temperature.

(8) Half-dry transfer membrane buffer solution for SDS-PAGE polyacrylamide gel electrophoresis: 14.4 g of Glycine and 3.03 g of Tris base are weighed and dissolved in 800mL of deionized water, 200 mL of methanol is added, the volume is constant to 1L, and the mixture is stored at room temperature.

(9) BN-PAGE gel electrophoresis catholyte (10X): 500mM Tricine, 150mM Tris-HCl pH7.0, 0.2% Coomassie Brilliant blue G250; colorless catholyte: 500mM Tricine, 150mM Tris-HCl pH 7.0.

(10) BN-PAGE gel electrophoresis anolyte (10X): 500mM Bis-Tris-HCl pH 7.0.

(11) BN-PAGE Loading Buffer: 750 mM aminocaproic acid, 5% (W/V) Coomassie Brilliant blue G250.

(12) Ponceau staining solution: weighing 0.5g of ponceau powder, adding 1mL of acetic acid for dissolving, adding sterile deionized water for fixing the volume to 100mL, and storing at room temperature for recycling.

(13) 10XPBS storage solution: 121.2 g of Tris-base and 90g of NaCl are weighed and dissolved in 900 mL of sterilized deionized water, the pH value is adjusted to 7.4, the volume is constant to 1L, and the mixture is stored at room temperature.

(14) 1 XPBST: diluting 10XPBS into 1XPBS by deionized water, adding Tween20 into the solution at a ratio of 1:1000, fully mixing the solution uniformly, and storing the solution at room temperature.

(15) Western Blot blocking solution (5% skim milk solution): 2.5g of skim milk powder was weighed out and dissolved in 50mL of 1XPBST and stored at 4 ℃ for later use on the same day.

(16) 5% BSA: 0.5g BSA powder was dissolved in 10mL 1XPBST and stored at 4 ℃ for the same day.

(17) 25%/75% ethanol solution: 250/750mL of absolute ethyl alcohol is measured, sterile deionized water is added to the absolute volume of 1L, and the mixture is stored at room temperature.

(18) 6X SDS-PAGE loading buffer: 25g SDS and 30mg bromophenol blue were weighed, dissolved in 87.5mL 1M Tris-HCl (pH 6.8), 100mL glycerol was added, 12.5mL beta-mercaptoethanol was added in a fume hood, and finally sterile deionized water was added to a constant volume of 250mL and stored at room temperature.

(19) Striping Buffer: 20mL of 10% SDS solution was measured, 6.2mL of 1M Tris-HCl (pH 6.8) was added, 0.7mL of beta-mercaptoethanol was added in a fume hood, and finally sterile deionized water was added to a constant volume of 100mL and stored at room temperature.

Preparation of small molecule reagents

(1) Gossypol acetate stock (50 mM): 2.9mg of gossypol acetate powder was weighed out and dissolved well in 100. mu.L of DMSO at a stock concentration of 50mM and stored at-20 ℃.

(2) Chloroquine (chloroquine, 100 mM): chloroquine powder was weighed, dissolved in sterile deionized water, filtered through a 0.22 μm filter for sterilization, stored at a concentration of 100mM, and stored at-20 ℃.

(3) NMS873 (20 mM): 10.4mg NMS873 powder was weighed out and dissolved in 1mL DMSO at a stock concentration of 20mM and stored at-20 ℃.

(4) DBEQ (20 mM): 6.8mg DBEQ powder was weighed out and dissolved in 1mL DMSO at a stock concentration of 20mM and stored at-20 ℃.

(5) Rapamycin (Rapamycin, 50 mM): 45.7mg of Rapamycin powder was weighed out and dissolved in 1mL of DMSO at a stock concentration of 50mM and stored at-20 ℃.

(6) Kanamycin (Kanamycin, 50 mg/mL): 1g of Kanamycin was weighed, dissolved in 20mL of deionized water, sterilized by filtration through a 0.22 μm filter, stored at a concentration of 50mg/mL, aliquoted through 1.5mL sterilized EP tubing and stored at-20 ℃. The processes of filtering and subpackaging antibiotics are finished in a super clean bench.

(7) Ampicillin (Ampicillin, 100 mg/mL): 1g Ampicillin was weighed, dissolved in 10mL deionized water, filtered through a 0.22 μm filter for sterilization, stored at a concentration of 100mg/mL, dispensed through 1.5mL sterilized EP tube and stored at-20 ℃. The processes of filtering and subpackaging antibiotics are finished in a super clean bench.

(8) Isopropylthio- β -D-galactoside (IPTG, 1M): 2.38g of IPTG were weighed, dissolved in 10mL of sterile deionized water, filtered through a 0.22 μ M filter membrane to sterilize at a storage concentration of 1M, aliquoted in 1.5mL sterile EP tubes and stored at-20 ℃. The filtering and subpackaging process is finished in a super clean bench.

(9) G418 (100 mg/mL): the powder 2g G418 was weighed, dissolved in 20mL PBS, sterile filtered through a 0.22 μm filter and dispensed into 1.5mL sterile EP tubes and stored at-20 ℃.

(10) EDTA solution (1M): 37.2g of EDTA are weighed into 70mL of sterile deionized water, stirred well and the pH is adjusted to 8.0 using NaOH. Finally, the volume is adjusted to 100mL by using sterile deionized water. Storing at room temperature.

Preparation of immunofluorescence related reagents

(1) A permeabilization solution: 100mM Tris-HCl, pH7.5, 50mM NaCl, 1% Triton X-100

(2) Cooling methanol: the methanol is pre-cooled for 5 hours at the temperature of minus 20 ℃.

Preparation of related reagent for enzyme activity detection

(1) Malachite green working solution: 0.081 percent of W/V solution of malachite green storage solution is prepared in advance in sterile deionized water respectively, 2.3 percent of W/V solution of polyvinyl alcohol storage solution is dissolved in the sterile deionized water, and 5.7 percent of W/V solution of ammonium molybdate tetrahydrate is dissolved in 6Mol HCl; the three stock solutions were mixed with sterile deionized water according to 2: 1: 1: 2, storing at 4 deg.C, and taking care that the malachite green working solution is prepared and used preferably one day ahead of time.

(2) ATPase activity detection System 1 XBuffer: 0.017% Triton X-100, 100mM Tris-HCl pH7.4, 20mM KCl, 6 mM MgCl₂。

(3) Potassium hydrogen phosphate solution (50 mM): 0.34g of anhydrous potassium hydrogen phosphate powder was weighed, dissolved in 50mL of sterile deionized water, and stored at room temperature.

(4) Trisodium citrate stop solution: 34% W/V, trisodium citrate powder was weighed and dissolved in sterile deionized water.

(5) ATP-Mg stock (100 mM): ATP powder was weighed and dissolved in 1.7.7 (2) 1 XBuffer.

(6) ATP-Mg working solution: 100mM ATP-Mg stock solution was diluted to 2.5mM with 1XBuffer in 1.7.7 (2) as the working solution.

Computer analysis software

2.1 bioinformatic pathways

NCBI HTTp:// www.ncbi.nlm.nih.gov; and acquiring gene information.

BLAT, HTTp:// ucsc.genome.edu/BLAT; and (5) aligning the sequences.

RCSB HTTp:// www.pdb.org/pdb/home/home.do; protein structure information.

Computer analysis software

2.2.1 Bioelectrophoresis gel imaging and scanning analysis software

Smart View 3.0；

UVP Gelworks ID Advanced Version 2.51。

Primer design and sequence alignment software

CE Design V1.04;

Serial Cloner;

Gene Snap。

Image processing software

Adobe photoshop CS2；

Powerpoint

ImageJ 1.46r

2.2.4 data analysis and statistics software

Microsoft office series software Word, Excel, PowerPoint; GraphPad prism 6

3 Experimental methods

3.1 construction of VCP expression plasmid

3.1.1 obtaining the Gene of interest

pET28a (+) -VCP recombinant primers were designed by using CE Design V1.04 software, and the VCP was cloned into prokaryotic expression plasmid vector pET28a (+) by gene editing using pSG5-Flag-VCP as a template. Firstly, VCP target gene is obtained through PCR reaction. And (3) PCR reaction system: template (50 ng/. mu.L) 1. mu.L, forward primer (10 mM) 1. mu.L, reverse primer (10 mM) 1. mu.L, PCR enzyme 1. mu.L, 10 Xbuffer 5. mu.L, and sterile deionized water to make up to 50. mu.L. And (3) PCR reaction steps: pre-denaturation at 95 ℃ for 30 sec; denaturation at 95 ℃ for 30 sec; annealing at 60 ℃ for 30 sec; extending for 1min at 72 ℃; cooling at 12 ℃.

Obtaining pET28a (+) vector by enzyme digestion

An enzyme digestion reaction system: pET28a (+) (4. mu.g), EcoRI Enzyme 1. mu.L, BamHI Enzyme 1. mu.L, 10 Xbuffer 5. mu.L, and finally sterile deionized water was added to make up to 50. mu.L.

The enzyme digestion system is incubated for 30min at 37 ℃, and then agarose gel with proper concentration is selected according to the size of the vector fragment for agarose gel electrophoresis. In general, a relatively low concentration of agarose gel is used to separate DNA fragments of high relative molecular mass, while a high concentration of agarose gel is used to separate DNA fragments of low relative molecular mass. DNA fragments smaller than 0.5kb, which can be separated on a 1.2-1.5% agarose gel; DNA fragments larger than 10kb, which can be used in 0.3-0.7% gel; when the DNA fragment is in between, 0.8-1.0% agarose gel may be used.

Weighing 1g of agarose solid by using a balance, putting the agarose solid into a 500mL glass bottle, adding 100mL of 1XTAE, putting the glass bottle in a microwave oven, heating the glass bottle with high fire until the agarose solid is boiled, completely melting and uniformly mixing the agarose solid, cooling the glass bottle to 50-60 ℃, adding Gel Red dye according to the using ratio (1: 40000), fully and uniformly mixing the agarose solid and the Gel solid, carefully pouring the mixture into a rubber plate inserted with comb teeth for about 40 minutes, and after agarose Gel is solidified, putting the agarose Gel into an electrophoresis tank filled with 1X electrophoresis buffer solution for spotting. Adding 10XLoading Buffer into the sample to be loaded, mixing uniformly, starting sample application, and adjusting the parameters of an electrophoresis apparatus to perform electrophoresis after the sample loading is finished. Constant voltage electrophoresis is adopted, 50V is used for a small-size electrophoresis apparatus, 100V is used for a medium-size electrophoresis apparatus, and 150V is used for a large-size electrophoresis apparatus. After the electrophoresis of the sample is finished, the cleavage result is observed in a gel imaging system.

And (3) recovering the enzyme-digested product, recovering the required target strip according to the method of the multifunctional agarose gel recovery kit, and detecting the concentration by using a NanoDrop2000 ultramicro spectrophotometer. Note that the formulated agarose gel needs to be used on the same day.

Recombinant ligation of target Gene to vector

A recombination reaction system: after the enzyme digestion, the carrier, PCR product, Fusion enzyme1 μ L, 2 Xbuffer solution 5 μ L, finally add sterile deionized water to make up to 10 μ L.

And (3) carrying out enzyme digestion on the recovered vector and the PCR product according to the ratio of 1: 3 (ratio by mass of vector to PCR product) was added to a fresh sterile 1.5mL EP tube, placed at 37 ℃ and ligated for 30 min. After completion, the ligation product was allowed to stand on ice to await further transformation.

Ligation product conversion

First, 50. mu.L of competent cell DH 5. alpha. was placed on ice using a 1.5mL sterile EP tube, 1. mu.L of the above ligation product was added, mixed well, incubated on ice for 30min, heat-shocked at 42 ℃ for 90sec, placed on ice for 1min, and then 400. mu.L of LB was added. Placing on a floating plate, placing on a shaking table at 37 ℃, centrifuging after 1 hour, rotating at 3000rpm for 5min, discarding 200 mu of supernatant, uniformly mixing the residual bacterial liquid, coating on an LB plate with corresponding resistance, and culturing in a constant-temperature bacterial incubator at 37 ℃. After 20 hours or so, the growth of the monoclone is observed.

And (3) when the corresponding monoclonal antibody of the strain grows up, selecting the monoclonal antibody, putting the monoclonal antibody into 3mL of LB culture medium with corresponding resistance, putting the LB culture medium into a shaker at 37 ℃, shaking the bacteria for about 15 hours, centrifuging at 5000rpm for 5min, collecting the bacteria, extracting the plasmids by using a small extraction kit of the plasmids, detecting the concentration by using NanoDrop2000, and sending plasmid samples out for sequencing to ensure the correct sequence of the plasmids.

Construction of prokaryotic expression plasmid for expression of VCP truncated protein

3.2.1 obtaining the target Gene

The primers for the VCP mutant were designed, and the VCP prokaryotic expression mutant was constructed using pET28a (+) -VCP wild type as a template according to the following procedure. Obtaining the target plasmid, and carrying out PCR reaction: template (50 ng/. mu.L) 1. mu.L, forward primer (10 mM) 1. mu.L, reverse primer (10 mM) 1. mu.L, PCR enzyme 1. mu.L, 10 Xbuffer 5. mu.L, and sterile deionized water to make up to 50. mu.L.

And (3) PCR reaction steps: pre-denaturation at 95 ℃ for 30 sec; denaturation at 95 ℃ for 30 sec; annealing at 60 ℃ for 30 sec; extending for 1min at 72 ℃; cooling at 12 ℃.

And after the PCR is finished, recovering the PCR product according to the steps of the multifunctional glue recovery kit. NanoDrop2000 for nucleic acid concentration.

Enzyme digestion of PCR product

DpnI digestion reaction system: PCR recovered product 2. mu.g, DpnI Enzyme 1. mu.L, 10 Xbuffer 5. mu.L, and finally added sterile deionized water to make up to 50. mu.L.

The enzyme digestion system is incubated for 30min at 37 ℃, and after the DpnI enzyme digestion is finished, the enzyme digestion product is placed on ice to be converted. DpnI recognizes and cleaves the GATC sequence of adenine N6 (N6-methyaddenine) in the double helix strand of DNA. The template plasmid in PCR is extracted from Escherichia coli DH5 alpha, so the template plasmid has DpnI recognition site, and the plasmid produced in PCR process has no methylation theoretically, so the DpnI is added after PCR is finished, the template plasmid is cut by enzyme, and the new synthesized target product is remained.

Conversion of the cleavage products

The product after the above enzyme digestion is transformed by sucking 1 uL. 50 μ L of DH5 α cells were first harvested from a 1.5mL sterile EP tube, placed on ice, 1 μ L of the cleaved product was added, mixed well, incubated on ice for 30min, heat shocked at 42 ℃ for 90sec, placed on ice for 1min, and then 400 μ LLB was added. Placing on a floating plate, placing on a shaking table at 37 ℃, centrifuging after 1 hour, rotating at 3000rpm for 5min, discarding 200 mu L of supernatant, uniformly mixing the rest bacterial liquid, coating an LB plate, and culturing in a constant-temperature bacterial incubator at 37 ℃. After 20 hours or so, the growth of the monoclone is observed.

When the monoclone of the strain is visible by naked eyes, selecting a monoclone strain with a proper size, putting the monoclone strain into 3mL of LB culture medium with corresponding resistance, placing the mixture into a shaker at 37 ℃, shaking the bacteria for about 15 hours, centrifuging the mixture for 5min at 5000rpm, collecting the bacteria, extracting the plasmids according to the steps of a small-amount extraction kit of the plasmids, detecting the concentration of the plasmids, and sequencing the samples to ensure that the plasmid sequence is correct.

Construction of pET28a (+) -Flag-VCP prokaryotic expression plasmid

3.3.1 obtaining the Gene of interest

Recombinant primers were designed and a prokaryotic expression plasmid of VCP was constructed using the wild type of pET28a (+) -VCP as a template, according to the following procedure. Obtaining the target plasmid, and carrying out PCR reaction: template (50 ng/. mu.L) 1. mu.L, forward primer (10 mM) 1. mu.L, reverse primer (10 mM) 1. mu.L, PCR enzyme 1. mu.L, 10 Xbuffer 5. mu.L, and sterile deionized water to make up to 50. mu.L.

The vector was digested to obtain pET28a (+) and the 6XHis sequence was excised

An enzyme digestion reaction system: pET28a (+) (4. mu.g), Xhol Enzyme 1. mu.L, BamHI Enzyme 1. mu.L, 10 Xbuffer 5. mu.L, and finally water was added to make up to 50. mu.L. Agarose gel electrophoresis and gel recovery, see experimental methods 3.1.2 for details.

Recombination and product ligation

The steps of recombination and connection of the target gene and the vector after enzyme digestion and transformation of the connection product are described in experimental formulas 3.1.3 and 3.1.4.

Construction of pGEX-6P-2-P47 prokaryotic expression plasmid

Designing a recombinant primer by taking pGEX-6P-2 as a vector, taking pSG5-Flag-P47 wild type as a template, and carrying out the following steps: obtaining a target plasmid, carrying out enzyme digestion on a vector, connecting a target gene with the enzyme digestion vector, transforming the plasmid, and gradually constructing a pGEX-6P-2-P47 prokaryotic expression plasmid. See experimental method 3.2 for details of the procedure. All primers are shown in Table 1.

Protein purification

3.5.1 plasmid transformation and mycoprotein Induction

pET28a (+) -VCP prokaryotic expression plasmid is transformed into BL21 competent cell (prokaryotic expression strain special for protein expression), and the transformation procedure is detailed in experimental method 3.1.4. Selecting a monoclonal strain, placing the monoclonal strain in an LB liquid culture medium with corresponding antibiotics, shaking the strain, adding 0.5mM IPTG into a super clean bench when the OD value reaches 0.8-1, inducing for 20 hours at 18 ℃, centrifuging for 10min at 5000rpm, and collecting the strain.

Protein purification

And (3) cracking the thallus, adding a cracking solution, and fully blowing and beating the thallus until the thallus is completely blown away. Placed on ice and lysed for half an hour (lysate: 100mM Tris-HCl, pH7.5, 500mM KCl, 5mM MgCl)₂2% Triton-X-100, 10% glycerol, adding 1mg/mL lysozyme and PMSF when cracking bacteria). The ultrasonicator fully cracks the bacteria (the parameters are power 200 w, ultrasonic 5s, intermittent 5s, circulation 30 times or so, the liquid to be cracked becomes no more viscous or clear or semitransparent, and the ultrasonic cracking time is properly prolonged or shortened according to the difference of bacterial strains, the quantity of the bacteria or the freezing condition of the bacteria). The lysis process cannot be excessive, otherwise the white foam produced by lysis would denature the protein.

After the cleavage, the mixture was centrifuged at 10000rpm and 4 ℃ for 30min, and the mixture was carefully trimmed. At this time, the desired protein was in the supernatant, which was added to Ni-NTA Resin that had been pre-washed with lysate, incubated at 4 ℃ for 1 hour, first with an eluent containing 30mM imidazole (100 mM Tris-HCl, pH7.5, 500mM KCl, 5mM MgCl)₂10% glycerol) was washed 3 times with Ni-NTAResin, centrifuged at 4 ℃ and 2000rpm for 3min, and the supernatant was discarded to remove non-specifically bound contaminating proteins. Finally, the target protein was eluted from the Ni-NTA Resin with an eluent containing 50/75/150/200mM imidazole.

Coomassie brilliant blue staining (CBS)

And (4) carrying out SDS-PAGE gel electrophoresis to detect whether the target protein is eluted. Respectively taking 50uL of elution samples, adding 10uL of 6X sample loading buffer solution into each sample, carrying out water bath at 100 ℃ for 10min, carefully adding the prepared protein samples into a pore groove of SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel by using a sample loading needle, carrying out electrophoresis at constant voltage of 90V for about 30min, allowing the protein samples to enter the separation gel at the lower layer, then reducing the voltage to 120V, continuing the electrophoresis at constant voltage, and finishing the electrophoresis until bromophenol blue completely runs out of the separation gel.

After protein electrophoresis is finished, putting protein glue into a staining solution, namely a Coomassie brilliant blue staining solution, starting staining, placing in a horizontal shaking table, standing for 1 hour at room temperature, abandoning the staining solution, washing with deionized water, adding a destaining solution, placing in the shaking table, standing for 1 hour at room temperature, abandoning the destaining solution, adding a new destaining solution (uniformly mixing the prepared destaining solution and the deionized water according to a volume ratio of 1: 1), destaining overnight, and allowing a clear strip to be visible the next day.

Protein desalting, concentration and preservation

And (4) selecting a relatively pure elution component with high protein elution concentration according to the protein glue decolorization condition, and desalting the protein. Protein was desalted and excess imidazole was removed from the solution according to the protocol for protein desalting column. The method comprises the steps of firstly cleaning a protein desalting column with 25mL of deionized water, then washing with 25mL of protein storage solution, adding 2.5mL of protein sample to be desalted after liquid in the desalting column completely flows out, immediately adding the protein storage solution into the column after the sample completely flows out, and collecting 3.5mL of liquid flowing out of the desalting column at the moment, wherein the protein is in the 3.5mL of solution. To increase the concentration of the desired protein, an ultracentrifuge concentration column was used, at 5000rpm, 4 ℃, centrifuged (the length of centrifugation was determined according to the protein concentration volume, and appropriately lengthened or shortened), and the sample remaining in the centrifuge tube was collected. Protein concentration was measured using BCA kit and proteins were dispensed into 1.5mL EP tubes, stored in 50mM Tris-HCl pH7.5, 100mM KCl, 10% glycerol solution, and placed at-80 ℃ until use. The protein desalting process is carried out in a cold storage, so that the protein is prevented from being degraded in the purification process.

Regeneration

The Ni-NTA Reasin can be repeatedly recycled and regenerated, and the process needs to wash the beads by using various different solutions, and comprises the following specific steps: washing the beads with 2 column volumes of regeneration solution (6M guanidine hydrochloride, 0.2M acetic acid); washing with 5 column volumes of water; wash with 3 column volumes of 2% SDS; washing with 3 column volumes of 2% SDS solution; washing with 1 column volume of 25% ethanol solution; washing with 1 column volume of 50% ethanol; washing with 1 column volume of 75% ethanol; washing with 5 column volumes of 100% ethanol; washing with 1 column volume of 75% ethanol; washing with 1 column volume of 50% ethanol; washing with 1 column volume of 25% ethanol; washing with 1 column volume of water; washing with 5 column volumes of 100mM EDTA, pH8.0 for 10min (chelating to remove excess Ni ions); washing with 5 column volumes of water; binding was performed with 2 column volumes of 100mM SiO 4 for 30min at room temperature (after this step the beads were allowed to stand at 4 ℃ for the next use).

Ultra-separation concentration tube recovery

Washing the ultrafiltration tube with deionized water for 2 times, adding 5mL deionized water, centrifuging at room temperature and 5000rpm for 5min, adding 50% ethanol into the tube after centrifuging, and storing in a refrigerator at 4 deg.C for next use.

Protein desalting column recovery

After the protein desalination is finished, washing the desalting column with 25mL of deionized water, then washing the desalting column with 20mL of 25% ethanol, filling the desalting column with 25% ethanol, and storing at 4 ℃ for recycling.

Protein purification

GST-LC3 prokaryotic expression plasmid obtained from Shanghai drug of Chinese academy of sciences, BL21 protein expression strain transformation, induction, lysis steps are detailed in 2.1, and GST-beads are used to capture GST-LC3 protein in lysate during the protein purification process, and the GST-beads are incubated for 1 hour at 4 ℃. GST-LC3 protein was finally eluted from GST-beads with an eluent containing 20mM glutathione, and the eluted protein was desalted, concentrated, and assayed for protein concentration by BCA, as described in Experimental method 2.4.5. The protein was stored in 50mM Tris-HCl pH7.5, 100mM KCl, 10% glycerol solution, and placed at-80 ℃ until use.

Protein purification

HTT-Q25/mHTT-Q72 prokaryotic expression plasmid, BL21 protein expression strain transformation, induction and cracking steps are detailed in 3.5.2, His-beads are used for grabbing His-HTT-Q25/mHTT-Q72 protein in a lysate, and 200mM imidazole eluent (100 mM Tris-HCl pH7.5, 500mM KCl, 5mM MgCl) is finally used_2,10% glycerol) to elute the protein of interest from Ni-NTAResin. Desalting protein, concentrating, and measuring protein concentration by BCA (burst cutting edge), wherein the steps are detailed in 2.4.5. Stored in 50mM Tris-HClpH 7.5,100mM KCl, 10% glycerol solution, and placed at-80 ℃ until ready for use.

Protein purification

The prokaryotic expression plasmid (the vector cuts off the nucleic acid fragment expressing 6 XHis) of the pET28a (+) -Flag-VCP obtained by construction is transformed into BL21 competent cells. Plasmid transformation, induction and lysis are carried out in the following steps of 3.5, Flag-beads capture Flag-VCP protein in lysate, 10mM polypeptide-containing eluent elutes the Flag-VCP protein from the Flag-beads, the eluted protein is desalted, concentrated and BCA is used for measuring the protein concentration, the steps are carried out in 3.5.4, 50mM Tris-HClpH 7.5,100mM KCl and 10% glycerol solution and placed at-80 ℃ for standby.

Protein purification

The obtained pGEX-6P-2-P47 prokaryotic expression plasmid is constructed and transformed into BL21 competent cells. Plasmid transformation, induction, lysis, as detailed in Experimental method 3.5.2, GST-P47 protein in lysate is captured by GST-beads, GST-LC3 protein is eluted from GST-beads by eluent containing 20mM glutathione, eluted protein is desalted, concentrated, and protein concentration is measured by BCA as detailed in Experimental method 3.5.4. The protein was stored in 50mM Tris-HCl pH7.5, 100mM KCl, 10% glycerol solution, and placed at-80 ℃ until use.

ATP enzyme activity screening and sex detection method

The VCP protein has ATPase activity, can catalyze ATP to be hydrolyzed into ADP and phosphate ions, and the catalytic rate of the protein can be calculated by detecting the release condition of phosphate. Phosphate radical ions can form phosphomolybdic heteropoly acid precipitate with ammonium molybdate, and the phosphomolybdic heteropoly acid precipitate is complexed with malachite green molecules under the acidic condition to form soluble complex, and the maximum absorption wavelength of the complex is near 620 nm. The screening and detection methods of the VCP enzyme activity are the same, and the malachite green colorimetric method is adopted to detect the release rate of the inorganic phosphorus.

Drawing phosphoric acid standard curve

Phosphorus ion standard gradient Pi (μ M): 0, 1, 5,10, 20, 50, 100, 200, 300, 500;

phosphorus ion standard reaction system (25 μ L): 12.5 mu L of 2X buffer solution and 1 mu L of phosphate ion standard, and finally adding sterile phosphate radical-free ionized water to make up to 25 uL.

A high-concentration Potassium hydrogen phosphate solution (50 mM) was prepared, and a phosphorus ion gradient standard was prepared according to the table. And (3) detecting OD620 values of different phosphorus ion concentrations by using the malachite green working solution, and drawing a corresponding phosphorus ion standard curve.

Screening for enzymatic Activity

ATPase activity reaction System: 12.5 mu L of 2X buffer solution, 1 mu L of VCP protein, 1 mu M of micromolecule/DMSO, 0.25 mu L of ATP-Mg working solution, and finally adding sterile phosphate radical-free ionized water to make up to 25 uL.

Each reaction system contains 40 mu M compounds and 1 mu M6 XHis-VCP wild type proteins, the solution system is ATP enzyme activity reaction buffer solution, the volume of each reaction is 25 mu l, all reactants except ATP are firstly prepared into mixed solution, ATP is added and mixed uniformly after all preparation work is finished, the reaction solution is immediately added into a transparent 96-well plate by a MultiDrop automatic liquid separator, the mixture is incubated on ice for 30 minutes, so that the compounds and the proteins can be fully combined, and then the mixture is placed at 37 ℃ for reaction for 1.5 hours. And after the reaction is finished, adding 80 mu l/hole of malachite green working solution by using a Multidrop automatic liquid separator, then immediately adding 10 mu l of 34% sodium citrate, uniformly mixing, placing at 37 ℃ for reaction for 15min, and detecting the absorbance at the wavelength of 620nm on an EnSpire multifunctional enzyme labeling instrument. The mean value of the signal of the samples without VCP protein was used as background value and the mean value of the signal of the samples of the DMSO group was used as control.

Enzyme activity detection

The reaction system for enzyme activity detection is 3.10.2, and small molecule and protein are incubated on ice for 30min, and ATP-Mg is added²⁺The working solution was incubated at 37 ℃ for 1.5 hours. Then 80 mu L of malachite green working solution is added, 10 mu L of 34% sodium citrate is immediately added to stop the hydrolysis reaction of ATP, the mixture is incubated for 15min at 37 ℃, the absorbance at the wavelength of 620nm is detected on an EnSpire multifunctional microplate reader, and finally, GraphPad Prism 6 is used for data analysis. In the process, all reagents do not contain phosphorus ions, otherwise, phosphorus ion detection is influenced. The malachite green working solution is not recommended to be prepared for use immediately, and is preferably prepared at least one day ahead, so that the effect is optimal.

Isothermal Titration Calorimetry (Isotermal Titration Calorimetry, ITC)

The instrument was cleaned before the experiment was started, 1% detergent was prepared (5 mL of detergent was added to 500mL of ultrapure water), and the instrument was cleaned 5 times with a syringe. About 3L of deionized water was prepared, the sample cell was washed 3 times with about 50 times by syringe, and whether the sample cell was washed clean or not in this experiment was important for the results. Next, the sample, Buffer, deionized water was degassed for 10min to remove air bubbles in the liquid. Adding a sample into the instrument, rinsing the reference cell for 2 times by using degassed deionized water, inserting the injector along a right hole aiming at the reference cell, slightly touching the bottom of the reference cell, lifting for about 1mm, slowly pushing in, and sealing the reference cell by using a sealing needle (generally, water needs to be changed once per week, but in order to ensure that the volume of the sample cell is the same as that of the reference cell, the liquid needs to be changed before each use). The sample cell was carefully rinsed 2 times with de-aerated deionized water and the sample was added to the cell by aspiration with a syringe. The empty needle is placed into the handle and placed on the instrument while the syringe range is calibrated. And after the sample loading is finished, starting running on the equipment. Some of the parameters used in this experiment were: 20 drops are added, each drop is 25 mul, and the interval between every two drops is 400 s; 300 μ LVCP (14 μ M), 50 μ L gossypol acetate (700 μ M). After the instrument is operated, sucking out the sample, cleaning according to the instrument cleaning step at the beginning of the experiment, and finally injecting 300 mu L of deionized water into the sample cell and the reference cell for next use.

Protein Thermal stability migration experiment (Protein Thermal Shift)

Mu.g of wild type 6XHis-VCP or truncated mutant protein of VCP was mixed with 5. mu.M gossypol acetate in PCR tubes, respectively, and placed on ice, incubated for 30min in the dark (in vitro assay 50. mu.L: 100mM Tris-HCl pH7.5, 150mM NaCl), and a set of temperature gradients (50/56.8/64.3/72.0/86.6/90 ℃) was set using T100 Thermal Cycler PCR instrument, and the various groups of proteins were heated under the temperature gradients, Thermal Shift reaction conditions (50. mu.L): heating at 25 deg.C for 2min, respectively at 50/56.8/64.3/72/78.6/86.6/90 deg.C for 3min, and cooling to 12 deg.C.

After completion of heating, the mixture was centrifuged at 12000rpm for 2min at 4 ℃ and 30. mu.L of the supernatant was aspirated into a new 1.5mL EP tube, and then 6. mu.L of 6 XLoading buffer was added to the sample, and the sample was boiled at 100 ℃ for 10min, followed by SDS-PAGE polyacrylamide gel electrophoresis and Coomassie blue staining, and the detailed procedure was referred to as Experimental method 3.5.3. The intensity of the strip is obtained by carrying out gray scanning by Image J software, T_mValues were calculated using GraphPad Prism 6 boltzmann fit.

Cell resuscitation

In order to ensure the optimal activity of the cells, the principle of slow freezing and fast dissolving is ensured. The freezing tube taken out of the liquid nitrogen tank is placed in water at 37 DEG CBathing and quickly shaking to melt the powder. The dissolved cell suspension was aspirated by a pipette gun, injected into a 15mL sterile centrifuge tube, mixed with 10 times the volume of fresh medium, centrifuged at 1000rpm for 5min at room temperature, and the supernatant was discarded. The cells were resuspended in 1mL of complete cell culture media and aspirated into a sterile petri dish of appropriate size and placed at 37 ℃ in 5% CO₂And in an aseptic constant-temperature cell culture box, timely replacing a culture medium for subculture after wall adhesion.

Cell passage

When cell proliferation reached 90% confluence, passaging was performed. Taking a 10cm sterile petri dish as an example, the original culture medium of the cells was removed, washed gently once with 1mL sterile PBS, discarded, added with 1.5mL trypsin digest to cover all cells sufficiently, and the petri dish was placed at 37 ℃ with 5% CO₂Digesting in an aseptic constant-temperature cell culture box for about 1-5 min (according to the difference of cell adherence, properly adjusting the digestion time of pancreatin, and observing the cell morphology under a microscope to start to become round, stopping digestion). Adding 2mL complete medium culture medium to stop pancreatin digestion, blowing off cells thoroughly and gently, transferring into 15mL sterile centrifuge tube after cells are completely dispersed, centrifuging at 1000rpm at room temperature for 5min, discarding supernatant, culturing and re-suspending cells freshly, transferring cells to sterile culture dish according to appropriate ratio (properly adjusted according to cell size and growth speed), placing at 37 deg.C and 5% CO₂A sterile constant temperature cell culture box.

Cell cryopreservation

Digesting the monolayer cells in the logarithmic phase of growth (the suspension cells are directly transferred to a 15mL sterile centrifuge tube and centrifuged at 1000rpm for 5 mn) by using the trypsin digestion solution, transferring the monolayer cells into a 15mL sterile centrifuge tube, centrifuging at 1000rpm for 5min at room temperature, centrifuging to remove supernatant, resuspending the cells by using the prepared frozen culture solution containing 10% DMSO and 20% fetal calf serum, and adjusting the density to 1 × 10⁷And (3) sucking the cells/mL into a sterile freezing tube, wherein the volume of the cell suspension added into each tube is 1-1.5 mL. Note that the scale of the cells, the cryopreservation time and the operator are marked on the cryopreservation tube; the cell cryopreservation box was placed in a fume hood at room temperature one day in advance, and whether isopropanol in the cryopreservation box was sufficient for use was examined. Placing the cells to be cryopreservedPlacing in a freezing storage box at-80 deg.C, and transferring into a liquid nitrogen tank for storage.

Transient transfection of cells

3.16.1 cell plating

Adherent cells were trypsinized and seeded onto cell culture dishes of appropriate size (6 cm dishes for example) in 5% CO₂And (3) a 37 ℃ sterile constant-temperature cell culture box, wherein the cells are plated for 18 hours, and the next transfection operation can be carried out after the inoculated cells adhere to the wall and the confluency reaches 70-80%.

Plasmid transfected cells

2 sterile 1.5mL EP tubes were selected and labeled A, B, respectively. Adding 200 mu L of serum-free DMEM into the A to incubate 15 mu L of PEI liposome, adding 200 mu L of serum-free DMEM and 5 mu g of plasmid into the B, and uniformly mixing; then A and B are mixed evenly and incubated for 30min at room temperature. And dropwise adding the mixed solution into the cell to be transfected in the step 3.16.1 along the wall of the culture dish, and detecting the expression condition of the transfection plasmid in the cell after 24-48 hours. If a more toxic transfection reagent such as Lipo2000 is used, the complete medium of the cells can be replaced with a new one 6 hours after the end of cell transfection in order to maintain normal cell growth. Whether or not to replace the cells with liquid is determined according to the growth conditions.

Co-immunoprecipitation assay (IP)

HEK293T cell plating and plasmid transfection experiments referring to experiment method 2.4.16, eukaryotic expression plasmids pSG5-Flag-VCP and GFP-LC3 co-transfected cells for 36 hours, cells were treated with gossypol acetate at different concentrations in DMEM complete medium for 12 hours. After the drug treatment, HEK293T cells were digested with trypsin digest, transferred to 1.5mL EP tubes, washed 2 times with 1XPBS, and centrifuged at 1000rpm for 5min at room temperature. The cells were pelleted on ice, 0.8mL of lysate (200 mM Tris-HCl, 50mM NaCl, 1% Triton X-100, protease inhibitor) was added to each tube, gently pipetted 10 times, lysed on ice for 20min, and after the cells were completely lysed, centrifuged at 12000rpm for 15min at 4 ℃ to leave the supernatant. 50ul of each supernatant was taken from each tube as Input, and the remaining samples were added to 1.5mL of EP tubes to which had been added prewashed Flag-beads, placed on a4 ℃ turntable, and incubated for 3 hours. Centrifuging at 4 deg.C for 1min at 2000rpm, and removing the supernatant; the Flag-beads were washed 3 times with a washing solution (200 mM Tris-HCl, 50mM NaCl, 0.5% Triton X-100), centrifuged at 2000rpm and 4 ℃ for 1 min. After the final wash was complete, the whole volume was aspirated, 2 Xloading buffer was added to each EP tube, boiled in a water bath at 100 ℃ for 10min, at 2000rpm, centrifuged at room temperature for 1min, and the supernatant was aspirated into a new 1.5mL EP tube (Do not aspirate into beads). After the sample preparation, the sample was subjected to SDS-PAGE gel electrophoresis. Samples can be stored at-20 ℃.

Experiment of

3.18.1 VCP and LC3 in vitro interaction experiment

Mu.g of 6XHis-VCP protein was incubated with gossypol acetate (in vitro assay 0.5 mL: 50mM Tris-HCl pH7.5, 150mM NaCl, 1% Triton X-100) at different concentrations for 2 hours at 4 ℃ followed by 4. mu.g of GST-LC3 protein and 2 hours at 4 ℃. Each tube draws 100. mu.L of sample as Input. The remaining solution from each tube was aspirated into a new EP tube containing GST-beads that had been pre-washed with lysate and incubated at 4 ℃ for 1 hour. The supernatant was discarded by centrifugation at 4 ℃ and 2000rpm for 1min, washed four times with eluent (50 mM Tris.HCl pH7.5, 150mM NaCl, 1% Triton X-100), discarded completely after the last wash, and then 100. mu.L of 2 Xloading buffer was added and the sample was boiled at 100 ℃ for 10 min. Finally, the supernatant was centrifuged at 2000rpm for 1min at 4 ℃ and used as a Pulldown sample. A suitable volume of sample was taken, loaded onto a 10% protein gel, and run on an SDS-PAGE gel. Electrophoresis of 90V basic layer glue and electrophoresis of 120V separation glue until a target strip is dispersed, transferring the membrane in a Bio-Rad semi-dry membrane transferring instrument, wherein the semi-dry membrane transferring condition of one piece of glue is 0.25mA, 60min (membrane transferring time can be properly adjusted according to the size of protein), after the membrane transferring is finished, placing the piece of glue in 5% of skimmed milk prepared by 1XPBST in a shaking table, and sealing the piece of glue for 1 hour at room temperature. Incubate with specific antibody and shake overnight at 4 ℃. On the next day, the NC membrane was washed with PBST, placed on a room temperature shaker for 5min, washed three times, incubated with the corresponding secondary antibody, placed on a room temperature shaker for 1 hour, washed three times with PBST, subjected to the same antibody condition, exposed to light using an ECL color development kit and a chemiluminescence apparatus, and the results were saved. Another sample of appropriate volume was subjected to SDS-PAGE polyacrylamide gel electrophoresis and stained with Coomassie Brilliant blue, the detailed procedure of which is described in Experimental 3.5.3.

In vitro interaction experiment with P47

Mu.g of 6XHis-VCP protein was incubated with 10. mu.M gossypol acetate and DMSO (in vitro assay 0.5 mL: 50mM Tris-HClpH 7.5, 150mM NaCl, 1% Triton X-100) at 4 ℃ for 2 hours in 1.5mL EP tubes, respectively, followed by addition of 4. mu.g of GST-LC3 or 4. mu.g of GST-P47 protein, respectively, and incubation at 4 ℃ for 2 hours. Each tube draws 100. mu.L of sample as Input. The remaining solution from each tube was aspirated into a new EP tube containing GST-beads that had been pre-washed with lysate and incubated at 4 ℃ for 1 hour. Centrifuging, removing supernatant, washing with eluent four times at 4 deg.C at 2000rpm for 1min, removing supernatant, adding 2 Xsample buffer solution 100 μ L, and decocting at 100 deg.C for 10 min. Finally, the supernatant was centrifuged at 2000rpm for 1min at 4 ℃ and used as a Pulldown sample. SDS-PAGE gel electrophoresis was performed, stained with Coomassie Brilliant blue, and the detailed procedure was described in Experimental 2.4.5.3.

In vitro interaction experiments with LC3 and mHTT-Q72/HTT-Q25 proteins

Mu.g of 6XHis-VCP protein was incubated with gossypol acetate (0.5 mL in vitro: 50mM Tris-HClpH 7.5, 150mM NaCl, 1% Triton X-100) at different concentrations for 2 hours at 4 ℃ followed by addition of 4. mu.g GST-LC3 protein and 6. mu.g mHTT-Q72/HTT-Q25 protein and further incubation for 2 hours at 4 ℃. Each tube draws 100. mu.L of sample as Input. The remaining solution in each tube was aspirated into a new 1.5mL EP tube containing GST-beads that had been pre-washed with lysate and incubated at 4 ℃ for 1 hour. Centrifuging, removing supernatant, washing with eluent four times at 4 deg.C at 2000rpm for 1min, removing supernatant, adding 2 Xsample buffer solution 100 μ L, and decocting at 100 deg.C for 10 min. Finally, the supernatant was centrifuged at 2000rpm for 1min at 4 ℃ and used as a Pulldown sample. SDS-PAGE gels were run, one gel was stained with Coomassie Brilliant blue, detailed in method 3.5.3, and the other gel was tested with WesternBlot.

In vitro interaction experiment with mHTT-Q72/HTT-Q25 protein

Mu.g of mHTT-Q72/HTT-Q25 protein and 4. mu.g of GST-LC3 protein (in vitro assay 0.5 mL: 50mM Tris-HClpH 7.5, 150mM NaCl, 1% Triton X-100) were incubated in a 10. mu.M gossypol acetate environment (or in an environment of 1/10. mu.g Flag-VCP protein) for 2 hours at 4 ℃ with the DMSO group as a control. Each tube draws 100. mu.L of sample as Input. The remaining solution in each tube was pipetted 350. mu.L each into a new 1.5mL EP tube containing GST-beads which had been prewashed with lysate and incubated for 1 hour at 4 ℃. Centrifuging, removing supernatant, washing with eluent four times at 4 deg.C at 2000rpm for 1min, removing supernatant, adding 2 Xsample buffer solution 100 μ L, and decocting at 100 deg.C for 10 min. Finally, the supernatant was centrifuged at 2000rpm for 1min at 4 ℃ and used as a Pulldown sample. SDS-PAGE is performed, one gel is stained with Coomassie Brilliant blue, the detailed procedure is shown in Experimental 3.5.3, and the other gel is subjected to Western Blot detection.

Protein sample preparation

Using adherent cell sample collection from 24-well plates as an example, cell culture dishes were removed from 5% CO₂And taking out the cell culture medium from a 37 ℃ sterile constant-temperature cell culture box, discarding the cell culture medium in a super clean bench, washing the cell culture medium once with 1X sterile PBS, adding 200uL of 2X sample loading buffer solution into each hole, gently blowing and beating the cell culture medium for a plurality of times, standing the cell culture medium on ice for 20min, fully and uniformly mixing the cell culture medium and the sample by using a pipette, sucking the cell culture medium into a 1.5mL EP tube, boiling the sample for 10min at 100 ℃, and storing the protein sample at-20 ℃ after the preparation is finished.

Electrophoresis and antibody incubation

Carefully adding the prepared protein sample into a sample loading hole of SDS-PAGE gel with proper concentration by using a microsyringe (the protein gel with proper concentration is selected according to the size of the protein to be detected, the optimal separation range of 6% of the gel is 200-400 KD, the optimal separation range of 8% of the gel is 100-200 KD, the optimal separation range of 10% of the gel is 30-100 KD, and the optimal separation range of 15% of the gel is less than 30 KD), firstly carrying out constant-voltage electrophoresis for 30min by using 90V, lowering the voltage to 120V, continuing the constant-voltage electrophoresis until bromophenol blue completely runs out of the separation gel, and finishing the protein electrophoresis. Proteins were then transferred from SDS-PAGE gels to nitrocellulose membranes (NC membranes) using a Bio-Rad semi-dry transfer membrane instrument. Firstly, completely soaking SDS-PAGE gel, filter paper and an NC membrane in a membrane transferring buffer solution, and sequentially placing 2 layers of filter paper, the NC membrane, the SDS-PAGE gel and two layers of filter paper in the membrane transferring buffer solution from bottom to top. The film transfer conditions are as follows: constant flow (0.25 mA/gel) for 1 hour (membrane-rotating time can be adjusted properly according to the size of protein). Transferring the NC membrane after membrane transfer into ponceau staining solution, prejudging the condition of protein transfer, then placing the NC membrane into 5% skimmed milk solution prepared by 1XPBST, and sealing on a shaking table for 1 hour at room temperature. Pouring off the confining liquid, cleaning the milk residues on the NC membrane by 1XPBST, respectively adding the specific antibodies into 5% BSA solution according to the recommended use proportion of the specific antibodies, completely soaking the NC membrane in the prepared specific antibody solution, and placing the NC membrane in a shaking table for incubation at 4 ℃ overnight. The next day, the NC membrane was washed with 1XPBST buffer solution 3 times, and placed on a room temperature shaking table for 5min each time, and then according to the recommended use ratio of the second antibody, the second antibody was added into 5% skimmed milk solution, added onto the NC membrane, and placed on a shaking table for incubation for 1 hour at room temperature after the NC membrane was completely soaked in the second antibody. Washed three times with PBST, with the same antibody. And (5) after the NC membrane is washed, preparing for color development, and detecting a protein band.

Color reaction

Protein bands were detected using an ECL color kit and a ChemiScope 3400 Mini chemiluminescent imaging system. According to the ECL color reaction steps, the ratio of 1:1, uniformly mixing the color developing solutions A and B in equal volume, covering the mixture on an NC membrane, incubating at room temperature for 1min, and placing the NC membrane in a chemical exposure instrument for exposure and development to obtain a chemical color developing picture. And storing the result. Note that the NC film cannot dry off during the WesternBlot process, otherwise the experimental results are affected.

Trypsin enzymolysis test

Trypsin lysis system 50 μ L (20 mM Tris-HCl, pH7.5, 80mM NaCl, 2.5mM MgCl)₂1mM DTT, 10% glycerol 6XHis-VCP protein (10. mu.g) were incubated with different small molecule compounds at 4 ℃ for 1 hour, DMSO was used as a control, trypsin (Promega V5111, 50. mu.g/mL) was added, and incubation was carried out in a 37 ℃ incubator for 1 hour. Add 10. mu.L of 6 Xloading buffer, boil at 100 ℃ for 10min, stop the reaction. Loading the enzymolysis product to 10% SDS-PAGE protein gel, carrying out electrophoresis, and detecting enzymolysis bands by Coomassie brilliant blue staining, wherein the steps are detailed in an experimental method 3.5.3.

Cell viability assay

5000 cells of Q47 were seeded into 96-well plate cell culture plates every empty on the first dayAdd 190. mu.L of medium, 5% CO₂And a constant-temperature cell culture box at 37 ℃. The next day, after the cells were completely attached to the wall, 10 μ L of small-molecule gossypol acetate (final concentration from high to low, maximum concentration of 50 μ M, two-fold gradient dilution of 9 concentrations, DMSO as control group, 3 multiple wells per well) was added to each well, and the wells were placed in 5% CO₂And a constant-temperature cell culture box at 37 ℃. After 72 hours, 20. mu.L of 0.1% AlamarBlue solution was added to each well, incubated in a cell incubator for 4 hours, and the absorbance at 590 nm was measured on an EnSpire multifunctional microplate reader, and finally data analysis was performed using GraphPad Prism 6.

Experiment of fruit fly

3.22.1 fruit fly model

(1) Nervous system drive trainelav-GAL4Obtained from the seed center of Drosophila Indiana BlumeUAS-full- HTT-128Q(HTT expressing full length human and containing 128Q crossed with GAL4 line);

(2) will be provided withpUAST-Exon1-HTT-72QInjecting the carrier into embryo of w1118 Drosophila to produceUAS-Exon1-HTT- 72QA drosophila strain. After hybridization with GAL4 strain, human HTT fragment with 72Q at the N-terminus was expressed and its expression was verified by homogeneous time-resolved fluorescence (HTRF) and Western Blot.

Behavior experiment

Before the experiment is started, the fruit flies need to be maintained, the fruit flies in the culture tubes are moved into new culture tubes about four to five days, and the new culture tubes are placed in a constant-temperature culture box at 25 ℃ for maintenance. By femaleelav-GAL4Fruit flies and malesUAS-full-HTT-16Q，UAS-full-HTT-128Q，UAS-Exon1-HTT-25Q，UAS-HTT-Exon1-72QFruit fly hybridization (hybridization combination is 6 female and 4 male), placing in a constant temperature incubator at 25 ℃, obtaining offspring after about 10 days, selecting virgins on the same day, placing in 15mL tubes containing clean food, and using 15 tubes as a group for standby. The food was prepared according to standard methods, 400. mu.L of food per tube was dried, and then liquid compound was added to the surface of the food, which was placed on a clean bench to ventilate overnight. (food is dissolved in microwave oven, added into centrifuge tube, and compound is added after food is solidifiedThe fruit flies can be raised after being fully infiltrated into the food, and the food and the medicine are changed every two days). Selecting virgins on the next day, putting the virgins into a culture medium added with the medicine for culture, knocking the tubes at the same time every day from the next day, and keeping the room temperature consistent. Knocking once every other day, recording the fruit fly climbing pipe condition (counting the number of fruit flies climbing to the height of 10cm in 15 seconds), and recording the survival number of the fruit flies in each group of experiments. And (4) calculating and analyzing a climbing pipe curve and a fruit fly survival curve (the period is about 2 weeks). Three biological replicates were made.

Drosophila head HTT protein content level test experiment

The drosophila 6 days after drug feeding was sacrificed (drug feeding was started from the next day of virgins), the head was removed under a dissecting mirror, lysate (1 XPBS +1% TritonX-100, proteasome inhibitor was added during lysis), the head was sonicated, 12000g, 10min, and the supernatant was retained. And (3) detecting the concentration by using a BCA protein quantitative kit, adding a certain amount of protein into each well in a 384-well plate, and adding an HTT corresponding antibody pair to ensure that the volume ratio of the antibody pair to the protein sample is 3: 2, 4 ℃ overnight. The next day, the 384 well plates were placed in a plate reader, excited with a laser, and the HTRF signal was measured.

Immunofluorescence confocal assay

3.23.1 immunofluorescence experiment after treatment of HeLa cells with gossypol acetate

Inoculation 2 × 10 on day one⁴HeLa cells into 24-well plate cell culture plates with coverslips placed in advance, 5% CO₂And a constant-temperature cell culture box at 37 ℃. The next day, after the cells were fully adherent, the medium was discarded, 1XPBS was washed 1 time, 4% PFA was fixed at room temperature for 10min, the post-permeabilization solution was punched at room temperature for 10min, and 1XPBS was washed 3 times for 5min each time. Add 500. mu.L of 5% BSA solution into each well, block for 30min at room temperature, formulate primary antibody with 5% BSA according to the recommended use ratio of the corresponding specific primary antibody, add the formulated primary antibody into 24-well plate, completely soak the slide in the primary antibody, and place in refrigerator at 4 ℃ overnight. The next day, the slides were washed 3 times with PBST, then co-incubated with the corresponding fluorescent secondary antibody (protected from light) for 1 hour at room temperature, and washed 3 times with PBST, under the same conditions. The mounting medium was dropped onto the slide (7. mu.L of mounting medium per slide) and carefully inverted onto the mounting mediumOn top, no cavitation is generated, which would otherwise affect the viewing field. After the mounting, drying in the dark, placing in a specimen box, storing at 4 ℃, and analyzing data pictures after detecting results by a fluorescence confocal microscope.

Autophagic flow assay

2×10⁴HeLa cells stably expressing mCherry-EGFP-LC3 were seeded in a coverslipped 24-well plate in 5% CO₂And a 37 ℃ cell constant-temperature incubator is used for absorbing the culture medium after the cells adhere to the wall the next day, washing 1 time by using 1XPBS, fixedly punching the holes for 10min by using cold methanol, and washing 3 times by using 1XPBS for 5min each time. Sealing the sealed tablet, drying in the dark, placing in a specimen box, storing in the dark at 4 ℃, and analyzing data pictures after detecting results by a fluorescence confocal microscope.

Co-localization experiment of intracellular LC3 and LAMP1

2×10⁴HeLa cells were seeded in 24-well cell culture dishes previously covered with coverslips and placed in 5% CO₂And a constant-temperature incubator at 37 ℃, after the cells adhere to the wall the next day, the culture medium is sucked off, 1XPBS is washed for 1 time, 4% PFA is fixed for 10min at room temperature, a permeabilizing solution is added for 10min at room temperature, and 1XPBS is washed for 3 times for 5min each time. Add 500. mu.l PBST-formulated 5% BSA to each well, block for 30min at room temperature, and incubate with the corresponding primary antibody overnight. The next day, PBST washs the slide, room temperature 5min, washs 3 times, with corresponding fluorescence second antibody co-incubation (light-resistant), room temperature 1 hour, 1XPBST washs 3 times, condition is same anti, seals the piece with the piece, and the light-resistant is dried, keeps in 4 ℃ of specimen box light-resistant, waits to fluorescence confocal microscope test result, data picture analysis.

Experiment on non-denatured glue

The purified prokaryotic protein 6XHis-VCP (10. mu.g) was incubated with gossypol acetate (1/10/20. mu.M) at various concentrations in 50. mu.l buffer (200 mM Tris-HCl, 50mM NaCl) at 4 ℃ for 1 hour, and after the incubation, Native-PAGE was performed. The Native-PAGE electrophoresis does not need adding a deforming agent such as SDS and the like. The biological macromolecules in the process can keep the natural shape and the charge number of the biological macromolecules, and the proteins are separated according to different electrophoretic migration rates of different proteins and the action of gel molecular sieves. Native-PAGE electrophoresis is basically the same as SDS-PAGE electrophoresis in operation, but the preparation of reagents does not need to add a denaturant, samples are maintained at 4 ℃ in the experiment process, ice blocks can be added in the BN-PAGE process, and the result is influenced by the overhigh standing temperature.

Gene knock-down assay

3.25.1 cell plating

After trypsinizing the cells, the cells are plated on a cell culture dish of suitable size (6-well plate for example) and the density of the plated cells is 40-50% in about 18 hours, which allows the next transfection procedure.

Gene knock-down transfection

2 sterile RNasefree 1.5mL EP tubes were labeled A and B, respectively. Respectively adding 200 mu L of serum-free and double-antibody-free DMEM into the A tube and adding 6 mu L of lipofectamine RNAiMAX into the A tube, and uniformly mixing; add 10. mu.L siRNA (10. mu.M) to tube B, mix well, let stand at room temperature for 5 minutes, mix A and B and incubate for 25 min. And adding the mixed solution into the cell culture dish to be transfected in the step, and after 48-72 hours, collecting the lysate for HTRF experiment or SDS-PAGE electrophoresis to detect the protein knockdown condition. In this process, it was ensured that the consumable material of RNasefree was used, and siRNA was prevented from being degraded and not functioning.

Homogeneous time-resolved fluorescence (HTRF) assay for detecting HTT protein levels

The cells were lysed with lysis buffer (1 XPBS containing 0.4% Triton X-100 and protease inhibitors) and tested with the corresponding antibodies, and BCA was used to measure the total protein concentration in all samples to ensure consistent protein levels in the samples added. Detection of different protein concentrations or cell numbers ensures that the fluorescence signal is in the linear range, with the blank sample as background signal.

Behavioral experiments in mice

Mice were housed in the SPF-grade animal laboratory at the laboratory animal center of the university of Compound Dan, at room temperature 21-23 ℃ with light-dark cycles every 12 hours. APP/PS1 mice of the C57 strain, 20 weeks old, were randomly divided into control and administration groups of 8 mice each. During the experiment, 15mg/kg of gossypol acetate is injected into the abdominal cavity. Dosing was done every two days for 10 weeks. After the administration is finished, the mice are subjected to behavioral tests including open field tests and new and old object identification tests by a small animal experiment platform. And (5) after the experiment is finished, analyzing and processing data. Mice were purchased from sanderian corporation, kyoto, zhongkou.

Note in fig. 1: the inhibition efficiency of 9 compounds with higher inhibition efficiency on the activity of VCP enzyme. Wherein the inhibition efficiency of the gossypol acetate on the activity of VCP enzyme reaches 80-90%. The inhibitory efficiencies of the 9 compounds were: 88.99% of gossypol acetate, 87.61% of salvianolic acid A, 79.14% of myricetin, 78.79% of gossypol, 78.79% of wedelolactone, 73.74% of baicalein, 73.64% of salvianolic acid C, 72.5% of catechin gallate and 70% of phloretin.

。

Sequence listing

<110> university of Compound Dan

Application of gossypol acetate in preparation of medicines for treating neurodegenerative diseases

<130>001

<160>16

<170>SIPOSequenceListing 1.0

<210>1

<211>70

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

taagaaggag atataccatg gattacaagg atgacgatga caagagccag atggcttctg 60

gagccgattc 70

<210>2

<211>46

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

ttgtcgacgg agctcgaatt cttagccata caggtcatca tcattg 46

<210>3

<211>41

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

acgataaggt acctaggatc catggcttct ggagccgatt c 41

<210>4

<211>46

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

tgctggatat ctgcagaatt cttagccata caggtcatca tcattg 46

<210>5

<211>41

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

cagcaaatgg gtcgcggatc catggcttct ggagccgatt c 41

<210>6

<211>45

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

ttgtcgacgg agctcgaatt cctgatgggt tactctggct caagg 45

<210>7

<211>41

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

cagcaaatgg gtcgcggatc catggcttct ggagccgatt c 41

<210>8

<211>40

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

ttgtcgacgg agctcgaatt cctccccttc gcagtggatc 40

<210>9

<211>43

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

cagcaaatgg gtcgcggatc cggcctagag gatgtcaaac gtg 43

<210>10

<211>46

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

ttgtcgacgg agctcgaatt cttagccata caggtcatca tcattg 46

<210>11

<211>42

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

cagcaaatgg gtcgcggatc ctgcaggaag cagctagctc ag 42

<210>12

<211>44

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

ttgtcgacgg agctcgaatt ctgatgggtt actctggctc aagg 44

<210>13

<211>44

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

acagcaaatg ggtcgggatc ccctatcaaa cgagaggatg agga 44

<210>14

<211>46

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

ttgtcgacgg agctcgaatt cttagccata caggtcatca tcattg 46

<210>15

<211>39

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

ttccaggggc ccctgggatc catggcggcg gagcgacag 39

<210>16

<211>43

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>16

ctcgagtcga cccgggaatt cttatgttaa ccgctgcacg atg 43

Claims (3)

1. Use of gossypol acetate in preparing medicine for treating neurodegenerative diseases is provided.

2. The use of claim 1, wherein the neurodegenerative disease is huntington's disease.

3. Use according to claim 2, comprising:

(1) the gossypol acetate is specifically combined with VCP, so that the interaction between VCP and LC3 is improved;

(2) the gossypol acetate is specifically combined with VCP, so that the interaction between VCP and mHTT is improved;

(3) the gossypol acetate is specifically combined with VCP, so that the formation of VCP, LC3 and mHTT complexes is promoted, and then toxic protein mHTT is degraded by autophagosomes;

(4) gossypol acetate improved the mHTT protein-induced disease phenotype in both the huntington cell model and the drosophila disease model.

Images