Application of CD38 palmitoylation modified interference peptide in tumor treatment

Abstract

The invention relates to a CD38 palmitoylation modified interference peptide. The CD38 palmitoylation modified interference peptide is characterized in that the interference peptide is polypeptide as shown in (a) or (b), wherein (a) has an amino acid sequence as shown in PGCRLSR; and (b) an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acid residues to the amino acid sequence in (a), has a function of interfering CD38 palmitoylation modification and is derived from (a). The invention designs a polypeptide capable of blocking endogenous CD38 palmitoylation, and the expression of CD38 in tumor cells can be depleted, so that the progress of Pano2 tumor is inhibited.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of a CD38 palmitoylated modified interfering peptide in tumor treatment. Background Art

[0002] CD38 is a multifunctional type II transmembrane glycoprotein involved in a variety of physiological and pathological processes, including aging, neurodegenerative diseases, social behavior, and tumorigenesis. As an extracellular enzyme, CD38 has NAD + Hydrolases and ADP - Ribocyclase activity, which converts NAD + It is decomposed into cADPR and ADPR, which bind to intracellular Ca 2+ Mobilization. In addition to its enzymatic function, CD38 also acts as a receptor for CD31 and plays a key role in cell migration, signal transduction, and receptor-mediated adhesion. CD38 is highly expressed in hematological malignancies (such as multiple myeloma), while its expression in non-hematological tissues is relatively low, making it an ideal target for the treatment of hematological malignancies. Currently, multiple CD38 antibodies are in clinical trials, among which daratumumab and isotretinoin have been approved by the US FDA for the treatment of multiple myeloma. Therefore, CD38 is considered an important drug target that deserves further study.

[0003] CD38 expression is significantly upregulated in a variety of cancers, but its regulatory mechanism has not yet been fully clarified. Studies have shown that the expression of CD38 is regulated by multiple factors, such as LXR, NF-κB, STAT and other transcription factors. For example, TNF-α induces the expression of CD38 in human airway smooth muscle cells by activating transcription factors such as NF-κB and AP-1. In addition, in monocytes and chronic lymphocytic leukemia cells, IFN-γ activates the CD38 promoter through IRF-1 and STAT transcription, thereby upregulating CD38 expression. In addition, activation of the LXR signaling pathway can enhance the expression of CD38, which helps host macrophages resist bacterial infection. These findings mainly focus on the transcriptional level regulation of the CD38 gene. However, whether there are other mechanisms such as post-translational modifications that regulate CD38 expression remains to be further studied.

[0004] S-palmitoylation is the only reversible protein lipid modification involving the attachment of a 16-carbon fatty acid to a cysteine ​​residue of a substrate protein via a thioester bond, and this intracellular palmitoylation reaction is mediated by a large family of aspartate-histidine-histidine-cysteine ​​(DHHC or ZDHHC) palmitoyltransferases. Palmitoylation affects cellular function by regulating the structure, stability, subcellular localization, protein-protein interactions, and signaling of substrates. In particular, palmitoyltransferases are essential for the biological functions of many cancer-related proteins, such as RAS, MC1R, PD-L1, and GLUT1. ZDHHC9 promotes the progression of gliomas by promoting the plasma membrane localization of GLUT1, which in turn leads to high levels of glycolysis. In addition, ZDHHC9 can also affect the affinity of HRAS and NRAS on the plasma membrane, thereby controlling tumor development. In addition, the loss of ZDHHC9 significantly slowed the progression of acute lymphoblastic leukemia and pancreatic cancer.

[0005] However, other ways to interfere with CD38 palmitoylation need to be explored in order to achieve the goal of treating tumors. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a CD38 palmitoylated interfering peptide and the application of the interfering peptide in tumor treatment in view of the current status of the prior art.

[0007] The technical solution adopted by the present invention to solve at least one of the above technical problems is: a CD38 palmitoylated interfering peptide, wherein the interfering peptide is a polypeptide as described in (a) or (b) below:

[0008] (a) having the amino acid sequence shown in PGCRLSR;

[0009] (b) The amino acid sequence in (a) is substituted, deleted or added with one or more amino acid residues and is an amino acid sequence derived from (a) that has the function of interfering with the palmitoylation modification of CD38.

[0010] Preferably, the sequence length of (b) is 5 to 12 amino acids.

[0011] Preferably, the interfering peptide further comprises a cell penetrating peptide fused to the (a) or (b) sequence.

[0012] Preferably, the cell penetrating peptide is tat.

[0013] Preferably, the interfering peptide inhibits the progression of Pano2 tumors

[0014] In a second aspect, the present invention provides a composition comprising the interfering peptide according to any one of claims 1 to 4.

[0015] In a third aspect, the present invention provides use of the above-mentioned interfering peptide or the above-mentioned composition in the preparation of a targeted preparation, wherein the targeted preparation is used for targeted interference with palmitoylation modification of CD38.

[0016] In a fourth aspect, the present invention provides use of the above-mentioned interfering peptide or the above-mentioned composition in the preparation of anti-tumor drugs.

[0017] In a fourth aspect, the present invention provides an anti-tumor drug comprising the above-mentioned interfering peptide or the above-mentioned composition.

[0018] Compared with the prior art, the advantages of the present invention are:

[0019] The present invention describes for the first time that CD38 is palmitoylated by ZDHHC9 at the Cys16 site, and this post-translational modification contributes to the stability of CD38 in tumor cells;

[0020] At the same time, the present invention designs a polypeptide that can block endogenous CD38 palmitoylation and deplete the expression of CD38 in tumor cells, thereby inhibiting the progression of Pano2 tumors;

[0021] These findings provide additional information on the homeostatic mechanisms of CD38 and may help target CD38 palmitoylation as a new strategy for cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a result diagram of the dynamic regulation of CD38 protein expression by CD38 palmitoylation in Example 1 of the present invention; wherein,

[0023] Figure 1 A is the immunoblot image of CD38 expression in Pano2, KPC and LLC cells after incubation with different concentrations of 2-BP for 24 hours;

[0024] Figure 1 B is the immunoblot image of CD38 expression in Pano2, KPC and LLC cells after incubation with different concentrations of Palmostatin B for 24 hours;

[0025] Figure 1 C is the immunoblot of CD38 expression after 2-BP (100 μM) and Palmostatin B (10 μM) were incubated with Pano2 cells for different time periods;

[0026] Figure 1 D is the immunofluorescence image of CD38 expression in Pano2 cells after Pano2 cells were incubated with 100 μM 2-BP, 10 μM palmitic acid B and 10 μM ML348 for 24 hours, CD38 is in red, cell nuclei are in blue, scale bar is 10 μm;

[0027] Figure 1 E is Figure 1 Statistical graph of immunofluorescence intensity in D;

[0028] Figure 1 F is a schematic diagram of the experimental process of ABE;

[0029] Figure 1 G is the result of ABE experiment to detect the palmitoylation level of exogenous CD38 in HEK293T cells;

[0030] Figure 1 H is Figure 1 Statistical graph of palmitoylation levels in G;

[0031] Figure 1 I is the result of ABE experiment analysis showing the palmitoylation level of CD38 in HEK293T cells after incubation with 2-BP (100 μM);

[0032] Figure 1 J is Figure 1 Statistical graph of palmitoylation levels in I;

[0033] Figure 1 K is the result of ABE experiment to detect the palmitoylation level of endogenous CD38 in HEK293T cells;

[0034] Figure 1 L is Figure 1 Statistical graph of palmitoylation levels in K;

[0035] Figure 2 This is a result diagram of the verification of the palmitoylation site of CD38 in Example 2 of the present invention; wherein

[0036] Figure 2 A is a comparison of some amino acid sequences of CD38 from various species;

[0037] Figure 2 B is a schematic diagram of the structure of the CD38 molecule. The structural domain of CD38 includes three parts: intracellular, transmembrane and extracellular;

[0038] Figure 2 C is a schematic diagram of the topological model of CD38 palmitoylation;

[0039] Figure 2 D is the palmitoylation level results of CD38 WT, WT+2BP and C16S in HEK293T cells as shown in the ABE experiment analysis;

[0040] Figure 2 E is an immunoblot of CD38 expression in Pano2 cells treated with 2-BP and CHX for different time periods;

[0041] Figure 2 F is the immunoblot of CD38 expression in CD38-WT and CD38-C16S B16F10 cells pretreated with CHX;

[0042] Figure 2 G is the immunoblot of CD38 expression after CD38-C16S B16F10 cells were treated with MG132;

[0043] Figure 2 H is Figure 2 Quantitative graph of CD38 protein content in F;

[0044] Figure 2 I Figure 2 Quantitative graph of CD38 protein content in G;

[0045] Figure 2 J is the result of CD38 ubiquitination level detection;

[0046] Figure 3 This is a diagram showing the verification result that ZDHHC9 is a palmitoylation modification enzyme of CD38 in Example 3 of the present invention; wherein,

[0047] Figure 3 A is an immunoblot image of CD38 expression after transfection of different ZDHHC enzyme siRNA in HEK293T cells;

[0048] Figure 3 B is the immunoblot image and related protein quantification image of CD38 expression after transfection of two independent siRNAs targeting ZDHHC9 in HEK293T cells;

[0049] Figure 3 C is an immunoblot of CD38 expression in HEK293T cells transfected with different concentrations of ZDHHC9 vector with Flag tag;

[0050] Figure 3 D is a graph showing the results of the co-immunoprecipitation (co-IP) experiment;

[0051] Figure 3 E is an immunoblot image of the endogenous expression of CD38 protein in Pano2 cells with stable knockdown of ZDHHC9 and the results of QPCR analysis of CD38 mRNA levels;

[0052] Figure 3 F is the immunoblot image of immunoprecipitated endogenous CD38 protein in ZDHHC9 knockdown cells and the result of palmitoylation analysis (ABE);

[0053] Figure 3G is a representative immunofluorescence image of CD38 staining of shZDHHC9 cells and shControl cells and a quantitative analysis of CD38 fluorescence intensity; in the figure, blue is the cell nucleus labeled with DAPI, red is CD38 staining, and the scale bar is 20 μm;

[0054] Figure 3 H is the co-localization immunofluorescence image and statistical analysis of CD38 and ZDHHC9 in Pano2 cells; scale bar is 50 μm;

[0055] Figure 4 This is a graph showing the results of the experimental peptide CPPtat-S1 inhibiting CD38 palmitoylation and protein expression in Example 4 of the present invention; wherein,

[0056] Figure 4 A is a result diagram of the experimental peptide (CPPtat-S1) and the control peptide (CPPtat-S1 control) designed according to the sequence map of mouse CD38 polypeptide;

[0057] Figure 4 B is the result of palmitoylation analysis (ABE) after Pano2 cells were treated with CPPtat-S1 control peptide or CPPtat-S1 polypeptide;

[0058] Figure 4 C is the immunoblot of CD38 after Pano2 cells were treated with different concentrations of CPPtat-S1 control peptide or CPPtat-S1 peptide for 24 hours;

[0059] Figure 4 D is the QPCR analysis result of CD38 after Pano2 cells were treated with different concentrations of CPPtat-S1 control peptide or CPPtat-S1 polypeptide for 24 hours;

[0060] Figure 4 E is a result diagram showing the effect of CPPtat-S1 control peptide or CPPtat-S1 polypeptide treatment on the proliferation of Pano2 cells using CCK8 cell proliferation assay;

[0061] Figure 4 F is the result of using a scratch test to detect the effect of CPPtat-S1 control peptide or CPPtat-S1 polypeptide treatment on the migration of Pano2 cells;

[0062] Figure 4 G is a result diagram of the experimental peptide (CPPtat-S2) and the control peptide (CPPtat-S2control) designed according to the sequence map of human CD38 polypeptide;

[0063] Figure 4H is the immunoblot of CD38 after MIAPaCa-2 cells were treated with different concentrations of the experimental peptide (CPPtat-S2);

[0064] Figure 5 This is a result diagram of CD38 expression promoting the malignant progression of Pano2 pancreatic cancer in Example 5 of the present invention; wherein,

[0065] Figure 5 A is the result of QPCR verification of the knockdown efficiency of CD38 at the transcriptional and protein levels in Pano2 cells;

[0066] Figure 5 B is the result of Western blot verification of the knockdown efficiency of CD38 at the transcriptional and protein levels in Pano2 cells;

[0067] Figure 5 C is the result of flow cytometry analysis to verify the knockdown efficiency of CD38 at the transcriptional and protein levels in Pano2 cells;

[0068] Figure 5 D is Figure 5 Fluorescence intensity statistics of flow cytometry analysis in C;

[0069] Figure 5 E is a graph showing the results of CCK-8 experiments on control (shControl) Pano2 cells and CD38 knockdown (shCD38) Pano2 cells;

[0070] Figure 5 F is a graph showing the results of the clone formation experiment of control (shControl) Pano2 cells and CD38 knockdown (shCD38) Pano2 cells;

[0071] Figure 5 G is a graph showing the results of in vivo growth of control (shControl) Pano2 cells and CD38 knockdown (shCD38) Pano2 cells;

[0072] Figure 5 H is Figure 5 Weight statistics of tumor size in G;

[0073] Figure 6 This is a result diagram of the CPPtat-S1 polypeptide inhibiting CD38 palmitoylation and slowing down the growth of Pano2 pancreatic cancer in Example 5 of the present invention; wherein

[0074] Figure 6 A is a schematic diagram of the mouse treatment regimen;

[0075] Figure 6 B is a graph showing the changes in tumor volume after subcutaneous injection of Pano2 cells in mice;

[0076] Figure 6 C is a photograph of the tumor at the end of treatment;

[0077] Figure 6 D is Figure 6 Statistical graph of tumor volume in C;

[0078] Figure 6 E is a representative image of HE staining and immunohistochemical staining (Ki67, Tunel, CD38) obtained after partial tumor tissue was removed and embedded in paraffin after peptide treatment; scale bar is 50 μm;

[0079] Figure 6 F is Figure 6 Statistical graph of the number of Ki67-positive cells in E;

[0080] Figure 6 G is Figure 6 Statistical graph of the number of Tunel-positive cells in E. DETAILED DESCRIPTION

[0081] The present invention is further described in detail below with reference to the accompanying drawings.

[0082] The general experimental methods involved in the following examples are as follows:

[0083] (1) Cell culture and transfection

[0084] Mouse Lewis lung cancer LLC cells, human colorectal cancer HCT116 cells, mouse pancreatic cancer Pano2 cells, human pancreatic cancer BxPC-3 and MIAPaCa-2 cell lines were purchased from the American Type Culture Collection (ATCC). The KPC cancer cell line derived from KrasG12D / +;Trp53R172H / +;Pdx1-Cre mice was purchased from Shanghai Model Organisms Technology Co., Ltd. All cell lines were tested and confirmed to be free of mycoplasma contamination. Pano2 and BxPC-3 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS, Excellbio) and 1% penicillin / streptomycin (P / S, Beyotime). LLC, MIAPaCa-2, HCT116, KPC, and HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and 1% penicillin / streptomycin. All cells were cultured in a 5% CO2, 37°C incubator.

[0085] In the transfection experiment, first, cells were inoculated in a culture plate, and transfection was started when the cell density reached 50-60%. Next, 2 μg of plasmid (Qingke, China) and 4 μl of transfection reagent (Qingke, China) were mixed with 250 μl of opti-MEM medium (Gibco, USA). Then, the plasmid mixture was mixed with the transfection reagent mixture and incubated at room temperature for 20 minutes, and finally the complex was added to the cells and cultured for 6-8 hours. After transfection, the culture medium was replaced with complete normal medium and cultured for 48 hours.

[0086] (2) Western blotting experiment

[0087] The cells were lysed on ice for 10 minutes with RIPA lysis buffer (Bimake, USA) containing 1% protease inhibitor (Bimake, USA) and 1% phosphatase inhibitor (Bimake, USA), and the cells were washed with PBS before lysis. Then, the cell lysate was centrifuged at 12,000 rpm at 4 ° C for 10 minutes, and the supernatant was taken to determine the protein concentration using the BCA protein quantification kit (Bimake, China). After determining the protein concentration, 5x SDS-PAGE loading buffer (Bimake, China) was added to the cell lysate, and the mixture was boiled at 95 ° C for 8 minutes. Next, equal amounts of protein samples were separated by SDS-PAGE gel and transferred to nitrocellulose filter membranes (HATF00010, Millipore). The membrane was placed on a shaker and blocked with 1% BSA at room temperature for 1 hour and 30 minutes. After blocking, the primary antibody was incubated with the membrane at 4 ° C overnight. The next day, the corresponding secondary antibody was added and further incubated with the membrane for 1 hour and 30 minutes at room temperature. Finally, the protein bands on the membrane were developed using ECL reagent and ChemiDoc XRS+ imager (Bio-Rad Laboratories Inc, USA).

[0088] (3) Immunostaining

[0089] The slides were pre-placed in the cell culture plates and the cells were then seeded on the slides. When the cells reached the appropriate confluence, the culture medium was aspirated and the cells were washed with cold sterile PBS and then fixed with 4% paraformaldehyde (PFA) at room temperature for 15 minutes. The slides were washed three times with PBS for 5 minutes each. The cells were blocked in blocking solution (2% BSA (Biyuntian, China) and 0.5% TritonX-100 (Biyuntian, China)) for 1 hour at room temperature and incubated with the primary antibody overnight at 4°C. Subsequently, the cells were rewarmed for 30 minutes and washed three times with cold PBS for 5 minutes each. After washing, the cells were incubated with the secondary antibody at room temperature for 1 hour in a light-proof box. Finally, the nuclei were stained with an anti-fluorescence quenching sealing solution containing DAPI (Biyuntian, China) for 5 minutes. Images were acquired using a Leica DM6B microscope or a Zeiss LSM 880NLO confocal microscope.

[0090] (4) Real-time fluorescence quantitative PCR

[0091] Total RNA was extracted from cells using RNAprep Pure Micro Kit (Tiangen Biochemical Technology, China) according to the supplier's recommendations. The concentration and quality of RNA were measured by NanoDrop 2000 (Thermo Fisher Scientific). Subsequently, cDNA was synthesized using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Novozyme, China) according to the manufacturer's instructions. The brief process was as follows: first, genomic DNA was further removed from RNA, and then the reverse transcription reaction system was configured for reverse transcription reaction. The total reaction mixture was 20 μL. The relative expression level of mRNA was determined using the ABI Quantstudio3 system (ABI, California, USA). The thermal cycle program used was: pre-denaturation at 95°C for 3 minutes, followed by 40 cycles of two-step PCR, heating at 95°C for 10 seconds, followed by heating at 55°C for 30 seconds, and finally extension at 65°C to 95°C for 5 seconds. The final results were calculated using the 2-ΔΔCt method, with GAPDH as the internal reference.

[0092] (5) Cell proliferation assay

[0093] After the cells were digested with 0.25% trypsin (New Cyme, Suzhou, China), they were seeded in 96-well plates at a density of 2000 cells per well, and 100 μL of culture medium was added to each well. According to the specified time point, the growth medium was discarded, washed three times with PBS, and then 100 μL of fresh culture medium containing 10 μL of cell counting kit-8 (CCK-8) (Dojindo, Japan) was added to each well. The 96-well plates were cultured in a dark incubator containing 5% CO2 and 37°C for 2 hours. Subsequently, the optical density (OD) was measured at a wavelength of 450 nm using a Spectra Max M5 microplate reader (Molecular Devices, Sunnyvale, USA).

[0094] (6) Cell scratch test

[0095] The cell scratch test was performed using μ-dish (IBIDI, Germany) according to the manufacturer's instructions. The brief process is as follows: μ-dish was placed in a 6-well plate in advance. The cells were digested with 0.25% trypsin and then plated at 2×10 ^5 The cells were seeded in serum-free medium at a concentration of 10 cells / well. When the cells almost covered the bottom of the well, the μ-dish was carefully removed, the old medium was discarded, and the cells were washed twice with PBS. Subsequently, the cells were cultured in serum-free medium containing the corresponding drugs at 37°C and 5% CO2 for 0 hours and 24 hours respectively. An inverted microscope (Leica DMi8, Wetzlar, Germany) was used to take pictures of each well, and the degree of cell migration was evaluated using ImageJ software.

[0096] (7) Cell cloning experiment

[0097] In brief, Pano2 shControl cells and shCD38 cells were digested with 0.25% trypsin and counted using a cell counting plate, respectively. The cells were then seeded in 6-well plates (500 cells per well) and cultured in normal culture medium containing 2 μg / mL puromycin. The culture medium was changed every two days. After 14 days, the cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 30 minutes and stained with 1% crystal violet solution (Sangon Biotech, China) for 20 minutes. The corresponding cell culture wells were then photographed using a camera, and the images were processed using Adobe Illustrator (v.2020).

[0098] (8) Immunohistochemistry

[0099] At the end of the treatment, the experimental mice were euthanized. The tumors were removed and fixed with 4% paraformaldehyde (PFA). Subsequently, the tumors were embedded in paraffin and cut into paraffin sections with a thickness of 5 microns. The paraffin sections were dewaxed in xylene and rehydrated in graded concentrations of ethanol. The sections were washed three times with distilled water for 5 minutes each. The sections were then boiled in sodium citrate buffer to restore the antigens. After the sections were removed, they were cooled to room temperature and washed three times with PBS for 5 minutes each. Next, the sections were treated with 3% hydrogen peroxide at room temperature for 15 minutes to inhibit the activity of endogenous peroxidase. After washing, the sections were blocked in blocking buffer (3% BSA) for 30 minutes. After gently shaking to remove the blocking solution, the sections were incubated with the primary antibody and placed flat in a wet box at 4°C overnight. The next day, the sections were incubated with biotin-labeled secondary antibodies for 2 hours and incubated with HRP (horseradish peroxidase)-labeled streptavidin for another 1 hour. Then, the DAB kit (Saiweier, Wuhan) was used for signal detection according to the supplier's recommendations. Finally, the sections were imaged using a Leica DM6B microscope or a Zeiss LSM 880NLO confocal microscope.

[0100] (9) Mouse xenograft model

[0101] C57BL / 6 mice (6-8 weeks old, male) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. All experimental mice were anesthetized with 1.25% Avertin (20 mg / kg; Sigma, USA). Then, Pano2 cells were digested with 0.25% trypsin and centrifuged at 1000 rpm for 4 minutes at 4°C. After removing the supernatant, the cells were resuspended in cold sterile PBS. 5×10 ^6 Pano2 cells were carefully injected into the right lumbar region of the mice. Ten days after tumor inoculation, the mice were randomly divided into three groups (n=5 in each group): control group, CPPtat-S1 control group, and CPPtat-S1 group. Two days later, the mice were subcutaneously injected with CPPtat-S1 control (10 mg / kg) and CPPtat-S1 (10 mg / kg). The body weight of the mice was measured every two days. At the same time, the tumor volume was monitored and calculated using the formula (1 / 2×length×width ^2 After treatment, the tumors were removed for further experiments. At the same time, the tumors were weighed and photographed.

[0102] (10) Acyl-biotin exchange experiment

[0103] In short, the HA-tagged CD38 construct was transfected into HEK293T cells. After two days, the cells were lysed in a rotator at 4 ° C for 2 hours using NP-40 lysis buffer containing a protease inhibitor cocktail (EDTA-free, 100X in DMSO) and 50mM NEM (N-ethylmaleimide). After centrifugation (4 ° C, 10,000rpm, 10 minutes), the supernatant was collected. The obtained supernatant was incubated with HA beads (Biyuntian, China) at room temperature for 2 hours to purify HA-CD38. After purification, the beads were suspended in a lysis buffer of pH 7.2 and divided into two groups: -HAM (hydroxylamine) group (without HAM solution) and +HAM group (with HAM solution). Next, the two groups were rotated and incubated at room temperature for 1 hour. The beads were washed three times with lysis buffers of pH 7.2 and pH 6.2, respectively. Subsequently, the beads were incubated with 2 μM Biotin-BMCC (Thermo Fisher Scientific Inc.) at 4 °C for 1 h and then washed with lysis buffer at pH 6.2. Finally, the beads were resuspended in 1x SDS-PAGE loading buffer (Biyuntian, China) and heated at 95 °C for 10 min. Finally, the palmitoylation level of CD38 was detected by immunoprecipitation and detected using streptavidin-HRP.

[0104] (11) Co-immunoprecipitation experiment

[0105] Cells were lysed on ice for 10 min with NP-40 buffer (Biyuntian, China) containing 1% protease inhibitors, and the lysate was centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was retained to avoid adsorption to the bottom of the tube. The primary antibody was added together with the corresponding supernatant and incubated overnight at 4°C with rotation. The next day, according to the instructions, protein A / G agarose beads (Thermo Fisher Scientific) were first washed twice with TBST, and then the supernatant containing antibodies was added and incubated for another 4 h at room temperature. Subsequently, the beads were collected using a magnetic rack (Thermo Fisher Scientific) and resuspended in 50 μL of 1xSDS-PAGE loading buffer. Finally, the beads were heated at 95°C for 10 min, and the loading buffer was collected for Western blotting.

[0106] Example 1 Palmitoylation regulates the expression of CD38 protein in tumor cells

[0107] The transcriptional regulation of CD38 and its functional significance have been extensively studied. However, it remains unclear how CD38 protein is regulated by post-translational modifications. https: / / swisspalm.org / ) database mass spectrometry study found that CD38 was identified as a potential target for palmitoylation modification. Palmitoylation is a lipid modification that can regulate cellular localization and precise protein interactions of many membrane proteins.

[0108] To investigate whether CD38 can be palmitoylated, we first used different doses of 2-bromopalmitic acid (2-BP, a typical broad-spectrum palmitoyltransferase inhibitor) to study its effect on CD38 protein expression in different tumor cells (such as Pano2 pancreatic cancer, KPC pancreatic cancer, and LLC lung cancer); then treated Pano2, KPC, and LLC cells with different doses of Palmostatin B (an acyl protein thioesterase 1 and 2 (APT-1, APT-2) inhibitor) to explore the effect of Palmostatin B on CD38 protein expression; at the same time, we investigated the effect of specific concentrations of 2-BP and Palmostatin B treatment of Pano2 for different periods of time on CD38 protein expression. Figure 1 As shown in A, after 24 hours of 2-BP treatment, we observed a significant decrease in the CD38 protein level in each tumor cell. Figure 1 As shown in B, after 24 hours of Palmostatin B treatment, the expression of CD38 in Pano2, KPC and LLC cells increased significantly. In addition, treatment of Pano2 cells with 2-BP or Palmostatin B reduced or increased the expression of CD38 protein, respectively, in a time-dependent manner ( Figure 1 C).

[0109] To further identify the depalmitoylation enzyme that affects CD38 protein levels, we used ML348 and ML349 (ML348 and ML349 correspond to APT1 and APT2 inhibitors, respectively) to detect their effects on CD38 expression in tumor cells. We observed that the expression of CD38 protein increased after treatment with ML348 but not ML349 (not shown in the figure), indicating that APT1 may be a depalmitoylation enzyme for CD38. Similarly, after treating HCT116 cells with ML348 at different time points, we found that pharmacological inhibition of APT1 activity could time-dependently increase CD38 expression. The effects of 2-BP, Palmostatin B and ML348 on CD38 protein expression in Pano2 cells were further verified by immunofluorescence staining, which was consistent with the results of immunoblotting ( Figure 1 D and Figure 1 E). Collectively, these results suggest that CD38 protein levels in tumor cells are regulated by S-palmitoylation.

[0110] To verify whether CD38 is directly regulated by S-palmitoylation, we performed an acyl-biotin exchange (ABE) experiment, which is a commonly used tool for detecting S-palmitoylation. Figure 1 As shown in F, briefly, the free cysteine ​​thiol groups of the protein were first blocked using N-ethylmaleimide (NEM), followed by depalmitoylation using hydroxylamine (HAM), since HAM can break the thioester bond connecting palmitic acid to cysteine. Finally, the palmitoylated cysteine ​​was labeled with biotin, and then S-palmitoylation was detected by immunoblotting. Figure 1 As shown in G and 1H, we used HA magnetic beads to pull down exogenous CD38 protein from cell lysate and performed ABE detection. The results again showed that CD38 was palmitoylated. Since protein palmitoylation is considered to be enzymatically reversible, the addition of 2-BP significantly inhibited the palmitoylation level of exogenous CD38 in HEK293T cells ( Figure 1 I and Figure 1 J). To verify whether endogenous CD38 can also be palmitoylated, we used anti-CD38 antibody to perform co-immunoprecipitation of CD38 protein, and then used ABE assay to detect the palmitoylation level of endogenous CD38. The results showed that endogenous CD38 also has palmitoylation ( Figure 1 K and Figure 1 L).

[0111] Altogether, these results suggest that palmitoylation is responsible for regulating CD38 protein expression in tumor cells.

[0112] Example 2 Palmitoylation at Cys16 promotes proteasomal degradation of CD38

[0113] To further identify the potential palmitoylation modification site of CD38, we used UniProt Web tool to analyze the sequence alignment of CD38 homologous proteins in different species and found that the amino acid sequence of CD38 in multiple species has a highly conserved cysteine ​​at position 16 ( Figure 2 A) In addition, using the CSS-palm 4.0 tool, cysteine ​​at position 16 was predicted to be a highly likely palmitoylation site in both human and mouse sequences. Figure 2 B and 2C show the structure of CD38: including a short N-terminal cytoplasmic domain, a transmembrane domain, and a longer C-terminal extracellular domain. The position of the cysteine ​​residue at position 16 of CD38 is similar to the palmitoylation site of typical membrane proteins. Based on the above analysis, we speculate that the cysteine ​​residue at position 16 of CD38 is a potential palmitoylation site.

[0114] To determine the palmitoylation site of CD38, we constructed a CD38 C16S mutant with a C-terminal HA tag by performing a specific site mutation at cysteine ​​16. Wild-type CD38 and the C16S mutant were then transfected into HEK293T cells, and the changes in palmitoylation levels were detected by ABE assay. It is noteworthy that compared with the control group, the CD38 palmitoylation level of the C16S mutant was reduced by more than 90% (the ratio of CD38 palmitoylation level to INPUT CD38 level) ( Figure 2 D). Similarly, 2-BP treatment can also inhibit the palmitoylation level of CD38 ( Figure 2 D).

[0115] Palmitoylation modification is considered to be essential for the plasma membrane localization of many membrane-associated proteins, so we introduced exogenously expressed EGFP-CD38 and EGFP-CD38 C16S plasmids to detect the subcellular localization of CD38 in cells. The Cys16 mutation of CD38 caused the CD38 protein to separate from the plasma membrane and move to the cytoplasm, while the wild-type CD38 was mainly localized on the plasma membrane. These results indicate that Cys16 is the major palmitoylation site at the N-terminus of CD38.

[0116] Next, we studied whether palmitoylation regulates the expression of CD38 in tumor cells by affecting its stability. In the Cycloheximide experiment, cells were first inoculated in culture plates, and chloramphenicol-X (Cycloheximide, CHX) was added to the cell culture medium when the cell density reached 70-80%, and then cells were collected at different time points (such as 0min, 15min, 30min, 1h, etc.) for Western blotting experiments. Compared with DMSO treatment, 2-BP treatment accelerated the turnover rate of CD38 and promoted the degradation of CD38 ( Figure 2 E).

[0117] To further verify the results, we detected the expression level of CD38 in five common tumor cell types (such as 4T1, Pano2, LLC, B16F10 and MC38) and found that the expression level of CD38 in B16F10 cells was the lowest. Subsequently, we used lentiviral vectors to stably express wild-type HA-CD38 and C16S mutants in B16F10 cells. The degradation rate and stability of CD38 protein were then studied using CHX (cycloheximide) experiments. After the addition of cycloheximide (CHX), the expression of CD38 was observed at different time points. The results of the CHX experiment showed that the C16S mutation significantly reduced the stability of the CD38 protein and accelerated its degradation ( Figure 2 F and Figure 2H). Palmitoylation can affect the degradation process of proteins and thus regulate protein stability. Next, we investigated whether palmitoylation of CD38 affects its stability by affecting the ubiquitin proteasome pathway. Interestingly, the use of the proteasome inhibitor MG132 was able to almost completely rescue the decrease in CD38 protein levels caused by the C16S mutation, indicating that palmitoylation can prevent the degradation of CD38 protein by inhibiting the proteasome pathway ( Figure 2 G and Figure 2 I). In addition, CD38-WT and CD38-C16S mutant cells were treated with MG132, and then the CD38 expression and ubiquitination levels were detected by immunoblotting. Compared with CD38 wild-type cells, the ubiquitination level of CD38 in C16S mutant cells was significantly increased ( Figure 2 J). Collectively, these results suggest that palmitoylation of CD38 is essential for CD38 protein stability and prevents CD38 degradation by inhibiting the ubiquitin-proteasome pathway.

[0118] Example 3 ZDHHC9-mediated CD38 palmitoylation maintains protein stability in tumor cells

[0119] Twenty-three members of the palmitoyltransferase family have been found in mammals. In order to identify the palmitoyltransferase responsible for CD38 palmitoylation, we screened all members using specific small interfering RNA (siRNA). Since CD38 is mainly distributed on the cell membrane, only a small amount of CD38 is localized in the cytoplasm. In general, spatial co-localization is often observed between the substrate and the corresponding palmitoyltransferase; therefore, we first excluded those palmitoyltransferases associated with CD38 that are localized in the nucleus. Next, we extended the analysis to the remaining enzymes, including ZDHHC2, ZDHHC3, ZDHHC5, ZDHHC7, ZDHHC9, ZDHHC11, ZDHHC13, ZDHHC14, ZDHHC17, ZDHHC18, and ZDHHC21. According to previous literature reports, we selected the corresponding siRNA sequences. By using siRNA targeting technology to silence the above enzymes respectively, western blotting results showed that when ZDHHC9 was knocked out in HEK293T cells, the CD38 protein level was significantly decreased ( Figure 3 A). To confirm that this phenomenon was not caused by off-target effects of siRNA, we used two independent and non-overlapping siRNA sequences for the experiment. Consistent with the results, silencing ZDHHC9 using another independent siRNA (siZDHHC9#2) also significantly reduced CD38 protein levels ( Figure 3B). Next, we used plasmid transfection to transfect ZDHHC9 plasmid into HEK293T cells. We found that as the expression of ZDHHC9 increased, the expression of CD38 protein increased significantly ( Figure 3 C). In addition, since palmitoyltransferases have been shown to interact with certain substrates, we hypothesized that ZDHHC9 mediates palmitoylation of CD38 by directly interacting with it. As expected, to examine the interaction between endogenously expressed ZDHHC9 and CD38 in HEK293T cells, co-immunoprecipitation (Co-IP) experiments showed that endogenous ZDHHC9 indeed interacted with CD38 ( Figure 3 D).

[0120] To further validate our findings, we used a lentiviral-mediated shRNA approach to silence endogenous ZDHHC9 expression in Pano2 tumor cells. Quantitative PCR (QPCR) and western blotting results confirmed that ZDHHC9 knockout was successful. It should be noted that knocking out ZDHHC9 only reduced the protein level of CD38 without affecting its mRNA level ( Figure 3 E), indicating that ZDHHC9 regulates CD38 protein levels at the post-translational level. In addition, ABE experiments confirmed that after knocking out ZDHHC9, the palmitoylation level of CD38 was significantly reduced ( Figure 3 F). At the same time, immunofluorescence experiments showed that the level of CD38 was significantly downregulated in ZDHHC9 knockout tumor cells ( Figure 3 G). In addition, endogenously expressed ZDHHC9 exhibited subcellular colocalization with CD38 in Pano2 cells ( Figure 3 H). Next, we speculated that ZDHHC9-mediated palmitoylation of CD38 could stabilize its expression in tumor cells. We quantitatively measured intracellular CD38 protein levels after cycloheximide (CHX) treatment of ZDHHC9-knockout Pano2 cells and control cells for 0, 3, 6, and 12 hours. The results showed that the degradation rate of CD38 in Pano2 cells was significantly increased after ZDHHC9 knockout. Therefore, these results indicate that ZDHHC9 is the main enzyme for CD38 palmitoylation and maintains high levels of CD38 expression in tumor cells by affecting its protein stability.

[0121] Example 4 The polypeptide inhibits the palmitoylation of CD38 by blocking the interaction between ZDHHC9 and CD38

[0122] We designed a competitive peptide with the amino acid sequence of PGCRLSR, which can interfere with the interaction between ZDHHC9 and CD38, thereby specifically inhibiting the palmitoylation of CD38. First, we designed an experimental peptide containing the palmitoylation site of CD38 at the Cys16 site and a control peptide with a mutated palmitoylation site. To improve the intracellular trafficking efficiency of the peptide, we fused a cell-penetrating peptide (CPP) with the experimental peptide (CPPtat-S1) and the control peptide (CPPtat-S1 control) ( Figure 4 A). Next, we used the ABE method to detect the palmitoylation level of CD38 after incubation of cells with different peptides. Obviously, CPPtat-S1 peptide (non-CPPtat-S1 control peptide) significantly reduced the palmitoylation of CD38 ( Figure 4 B). In addition, we noticed that CPPtat-S1 control peptide did not affect the expression of CD38, while after CPPtat-S1 treatment, the expression of CD38 decreased in a dose-dependent manner ( Figure 4 C). It is noteworthy that CPPtat-S1 only altered CD38 protein levels but did not affect its transcriptional activity ( Figure 4 D). The same effect was also verified in LLC cells. In addition, CPPtat-S1 peptide significantly inhibited the growth and migration of pancreatic cancer cells ( Figure 4 Considering that the amino acid sequences encoded by human and mouse CD38 at the Cys16 site are different, we further designed a competitive peptide targeting the palmitoylation site of human CD38: CPPtat-S2 ( Figure 4 G). Next, we evaluated the effect of CPPtat-S2 on CD38 expression in MIAPaCa-2 cells derived from human pancreatic cancer. We observed that the CPPtat-S2 peptide targeting the human CD38 sequence also inhibited the expression of CD38 protein ( Figure 4 H).

[0123] Example 5 CD38 palmitoylated polypeptide inhibits Pano2 tumor progression

[0124] To further validate the oncogenic role of CD38 in pancreatic cancer, we used the Pano2 cell pancreatic cancer model. This is a typical cold immune tumor that is resistant to emerging immune checkpoint blockade therapies such as PD-L1 inhibitors. We used QPCR, Western blot, and flow cytometry analysis to confirm the knockdown efficiency of CD38 at the transcriptional and protein levels ( Figure 5A~5D). Next, we selected the stable clones with the highest CD38 knockdown efficiency for subsequent studies. The proliferation ability of tumor cells in vitro was evaluated by CCK-8 and clone formation assays. Figure 5 As shown in E and 5F, the proliferation of Pano2 cells with CD38 knockdown was significantly reduced compared with the control group. Next, to evaluate the growth of tumors in vivo, we subcutaneously inoculated Pano2 cells with the control group shControl or shCD38 into immunocompetent C57BL / 6 mice. The results showed that CD38 deficiency significantly inhibited the growth of Pano2 tumors in vivo, suggesting that CD38 expression may promote the malignant progression of Pano2 pancreatic cancer ( Figure 5 G and 5H).

[0125] Next, we tested whether specific inhibition of CD38 palmitoylation by competitive peptides could delay the growth of Pano2 tumors. First, we established a subcutaneous pancreatic cancer model in mice. Briefly, 5×10 ^6 Pano2 cells were injected subcutaneously into the right flank of immunocompetent C57BL / 6 mice, and tumor growth was monitored every 2 to 3 days. 3 We randomly divided mice into three groups and treated them with Vehicle, CPPtat-S1 control peptide, and CPPtat-S1 peptide, respectively. CPPtat-S1 peptide was administered to Pano2 tumor-bearing mice by intratumoral injection every two days. Finally, tumors were collected 2 days after the last treatment ( Figure 6 In particular, we observed that the growth rate (volume and weight) of Pano2 tumors in mice treated with CPPtat-S1 was significantly lower than that in the control group ( Figure 6 B~6D). Immunohistochemistry (IHC) staining showed that the number of Ki67-positive cells decreased after CPPtat-S1 treatment compared with the control group, while the number of apoptotic cells did not change significantly ( Figure 6 E-6G). In addition, peptide treatment did not cause changes in body weight compared to the initial body weight of mice.

[0126] Collectively, these results indicate that our designed CPPtat-S1 peptide effectively inhibits the progression of Pano2 tumors by inhibiting the palmitoylation of CD38.

Claims

1. A CD38 palmitoylated interfering peptide, characterized in that: The interfering peptide is a polypeptide as described in (a) or (b) below: (a) having the amino acid sequence shown in PGCRLSR; (b) The amino acid sequence in (a) is substituted, deleted or added with one or more amino acid residues and is an amino acid sequence derived from (a) that has the function of interfering with the palmitoylation modification of CD38.

2. The interfering peptide according to claim 1, characterized in that: The sequence length of (b) is 5 to 12 amino acids.

3. The interfering peptide according to claim 1, characterized in that: The interfering peptide also includes a cell penetrating peptide fused to the (a) or (b) sequence.

4. The interfering peptide according to claim 3, characterized in that: The cell penetrating peptide is tat.

5. A composition, characterized in that Contains the interfering peptide according to any one of claims 1 to 4.

6. Use of the interfering peptide according to any one of claims 1 to 4 or the composition according to claim 5 in the preparation of a targeted preparation, characterized in that: The targeted agent is used for targeted interference with palmitoylation modification of CD38.

7. Use of the interfering peptide according to any one of claims 1 to 4 or the composition according to claim 5 in the preparation of anti-tumor drugs.

8. An anti-tumor drug, characterized in that: Contains the interfering peptide according to any one of claims 1 to 4 or the composition according to claim 5.

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