Fatty acylation modification of PHGDH and its application

Abstract

The present invention provides PHGDH fatty acylation modification and its application. The cysteine ​​residue (C369) at the 369th position of PHGDH undergoes fatty acylation modification, which is jointly regulated by the fatty acyltransferase ZDHHC17 and the de-fatty acylase APT2 of PHGDH. The lack of fatty acylation at the C369 position of PHGDH will lead to a decrease in the catalytic activity of the PHGDH enzyme, providing a new drug target for the treatment of related diseases caused by PHGDH activity disorder.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a cancer biomarker associated with fatty acylation modification of the cysteine ​​residue at position 369 of PHGDH and a use thereof. Background Art

[0002] Phosphoglycerate dehydrogenase (PHGDH) is a key enzyme in serine biosynthesis, mediating the conversion of 3-phosphoglycerate into 3-phosphohydroxypyruvate. PHGDH is highly expressed in about 70% of triple-negative breast cancer, lung cancer, pancreatic cancer and other cancers, and the catalytic activity of PHGDH is crucial to the occurrence, metastasis and drug resistance of cancer, making it an important new target for the development of anticancer drugs.

[0003] Protein fatty acylation can significantly affect the structure of proteins and participate in the regulation of protein transport, localization, conformational changes, and interactions between proteins. Studies have shown that protein fatty acylation disorders are associated with the occurrence and development of various diseases such as cancer. So far, there have been no reports on PHGDH fatty acylation and its biological functions.

[0004] The inventors used protein fatty acylation probes to discover for the first time the fatty acylation modification of the cysteine ​​residue (C369) at position 369 of PHGDH, which is co-regulated by the fatty acyltransferase ZDHHC17 and the de-fatty acylase APT2 of PHGDH, and for the first time clarified that the fatty acylation of C369 of PHGDH is involved in regulating the enzyme activity of PHGDH. These studies will provide a basis for the development of therapeutic drugs and treatment strategies for diseases such as cancer related to PHGDH dysfunction. Summary of the invention

[0005] Based on the above research, the inventors first proposed a cancer biomarker related to the fatty acylation of C369 of PHGDH and its use. The fatty acylation of C369 of PHGDH is regulated by the fatty acyltransferase ZDHHC17 and the de-fatty acylase APT2. The fatty acylation of C369 of PHGDH participates in regulating the enzyme activity of PHGDH. The fatty acylation-defective PHGDH significantly reduces its enzyme activity. This provides a new solution for the diagnosis of clinical cancer at the molecular level and a new drug target for the treatment of cancer.

[0006] The above technical objectives of the present invention are achieved through the following technical solutions: the PHGDH fatty acylation modification and its application, Specifically, the present invention is achieved through the following technical solutions: Fatty acylation modification of PHGDH, wherein the PHGDH is fatty acylated at the cysteine ​​residue at position 369 (C369).

[0007] The fatty acylation at the C369 site of the PHGDH is jointly regulated by the fatty acyltransferase ZDHHC17 and the de-fatty acylase APT2, and the fatty acylation at the C369 site of the PHGDH is further involved in regulating the enzyme activity of PHGDH.

[0008] The fatty acylation modification of the C369 site of PHGDH is used as a biomarker and drug target for cancer.

[0009] The application of the PHGDH fatty acylation modification in the preparation of a product having at least one of the following functions: (1) Molecular diagnosis of cancer; (2) Predict the risk of cancer occurrence and development; (3) Inhibit the growth and metastasis of cancer cells; (4) As drug targets for cancer treatment.

[0010] Compared with the prior art, the present invention has the following beneficial effects: (1) The present inventors confirmed for the first time that PHGDH undergoes fatty acylation modification at the cysteine ​​residue at position 369 (C369).

[0011] (2) The present inventors have proposed for the first time a cancer biomarker associated with fatty acylation modification of the C369 site of PHGDH and its uses, including risk prediction of malignant progression of cancer caused by PHGDH dysfunction.

[0012] (3) This invention will provide a new basis for the development of therapeutic drugs and treatment strategies for PHGDH-related diseases (especially cancer). BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 :Identification of fatty acylation modification of PHGDH; (A) Structure of lipid probe for detecting protein fatty acylation; (B) Lipid probe metabolic labeling, click chemistry reaction and detection experiments showed that endogenous PHGDH in HEK293T cells can be metabolically labeled by lipid probe and has a preference for 16-carbon palmitic acid, and the control group is DMSO; (C) PHGDH overexpressing Flag tag in HEK293T cells can be metabolically labeled by lipid probe and has a preference for 16-carbon palmitic acid, and the control group is DMSO; (D) After NH2OH treatment of the click chemistry reaction product, the fatty acylation modification of PHGDH protein disappeared, suggesting that the fatty acylation modification occurs on cysteine ​​residues; Figure 2:Analysis of fatty acylation modification sites of PHGDH; (A) N-terminal truncation (PHGDH96-533) and C-terminal truncation (PHGDH1-314) were transfected to detect the lipid modification level; (B) Site-directed mutagenesis combined with lipid probe metabolic labeling experiments showed that the mutation of the cysteine ​​residue at position 369 of PHGDH to serine (C369S) resulted in a significant decrease in the fatty acylation modification of PHGDH; Figure 3 :ZDHHC17 mediates PHGDH fatty acylation modification; (A) Lipid probe metabolic labeling combined with click chemistry reaction detected the effect of overexpressing human ZDHHC17 or ZDHHC23 acyltransferase in HEK293T cells on PHGDH fatty acylation level. The results showed that ZDHHC17 could significantly increase the fatty acylation level of PHGDH; (B) Immunoprecipitation experiments showed that ZDHHC17 could interact with PHGDH, while ZDHHC23 had a weaker interaction with PHGDH; Figure 4 : APT2 mediates the de-fatty acylation of PHGDH; (A) The results of lipid probe metabolic labeling, click chemistry reaction and detection experiments showed that overexpression of APT1 or APT2 in HEK293T cells could significantly reduce the fatty acylation level of PHGDH; (B) Immunoprecipitation experiments showed that APT2 had a strong interaction with PHGDH; Figure 5 :Effect of fatty acylation at the C369 site of PHGDH on the enzyme activity of PHGDH; (A) The intracellular PHGDH enzyme activity detection experiment showed that the pan-ZDHHC acyltransferase inhibitor 2-BP significantly inhibited the enzyme activity of PHGDH; (B) The enzyme activity of the intracellular PHGDH fatty acylation-deficient mutant (C369S) was significantly decreased. DETAILED DESCRIPTION

[0014] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0015] Example 1: Confirmation of fatty acylation modification of PHGDH.

[0016] To identify PHGDH fatty acid acylation, human embryonic kidney HEK293T cells ( Figure 1A), harvest and lyse the cells, take the supernatant after centrifugation, couple biotin through click chemistry reaction, enrich the proteins that undergo click chemistry reaction through streptavidin agarose beads, wash away the unbound proteins, elute the proteins labeled with lipid probe, and detect PHGDH by SDS-PAGE and immunoblotting. The results show that endogenous PHGDH can be labeled with long-chain fatty acid probes and has a preference for 16-carbon palmitic acid ( Figure 1 B). In addition, the human PHGDH expression vector with a Flag tag was transfected into HEK293T cells, and the transfected cells were labeled with different lipid probes. The lipid modification detection results showed that the human PHGDH with a Flag tag also had a preference for 16-carbon palmitic acid ( Figure 1 C).

[0017] To further confirm the lipid modification of PHGDH, we transfected the Flag-PHGDH plasmid with an N-terminal Flag tag into HEK293T cells. 24 hours after the transfection experiment, the cells were labeled with 25 µM lipid probe Alk-C16 and incubated for 9 hours. The cells were harvested and lysed on ice for 30 min, the cell lysate was reacted with azide-Biotin, and the reaction product was reacted with 4% (v / v) NH2OH solution at room temperature for 1 hour. Western blot results showed that Flag-PHGDH overexpressed in HEK293T cells could be metabolically labeled with Alk-C16, and the degree of fatty acylation of PHGDH in the NH2OH-treated group was significantly reduced ( Figure 1 D). This indicates that the fatty acylation modification of PHGDH protein occurs at the cysteine ​​residue.

[0018] Example 2: The fatty acylation modification site of PHGDH was determined to be C369S.

[0019] In order to determine the site of fatty acylation modification of PHGDH, we constructed two truncations of PHGDH: C-terminal truncation (PHGDH1-314) and N-terminal truncation (PHGDH96-533). The full-length (Full length) Flag-PHGDH, Flag-PHGDH1-314, and Flag-PHGDH96-533 plasmids were transfected into HEK293T cells, respectively. The results of fatty acylation modification detection showed that the fatty acylation level of the C-terminal truncation (Flag-PHGDH1-314) was significantly lower than that of the full-length Flag-PHGDH and N-terminal truncation (PHGDH96-533) ( Figure 2 A), indicating that the fatty acylation site of PHGDH is located at the C-terminus.

[0020] To further identify the specific sites of PHGDH fatty acylation modification, we transfected wild-type Flag-PHGDH and mutants (C111S and C369S) into HEK293T cells, respectively, and used Alk-C16 for metabolic labeling. The results of fatty acylation modification detection showed that the fatty acylation level of PHGDH C369S mutant was significantly lower than that of wild-type PHGDH ( Figure 2 A). The above results indicate that Cys369 is the main fatty acylation modification site of PHGDH.

[0021] Example 3: ZDHHC17 mediates fatty acylation modification of PHGDH.

[0022] In order to find the acyltransferase for PHGDH fatty acylation, the expression plasmid of human ZDHHC acyltransferase with HA tag was co-transfected with Flag-PHGDH in HEK293T cells and metabolically labeled with Alk-C16. The results of fatty acylation modification detection showed that overexpression of ZDHHC17 could significantly increase the fatty acylation level of PHGDH ( Figure 3 A). In order to detect the interaction between PHGDH and fatty acyl modification enzymes, we conducted immunoprecipitation experiments. We transiently transfected Flag-PHGDH with ZDHHC23 and ZDHHC17 into HEK293T cells, lysed the cells after 48 hours, took the supernatant after centrifugation, added Flag magnetic beads, incubated in a refrigerator at 4°C overnight, washed away non-specifically bound proteins, eluted specifically bound proteins with SDS-PAGE loading buffer, and then performed SDS-PAGE and immunoblotting detection. The results showed that ZDHHC17 can interact with PHGDH, while ZDHHC23 interacts weakly with PHGDH ( Figure 3 B). These results suggest that ZDHHC17 is the major modifying enzyme mediating PHGDH fatty acylation.

[0023] Example 4: APT2 mediates PHGDH de-fatty acylation.

[0024] Potential de-fatty acylases ABHD17A / B / C, APT1 / 2, and LYPLAL1 were co-transfected with Flag-PHGDH in HEK293T cells. The cell lysate was subjected to click reaction, and then the modified proteins were enriched by streptavidin beads. Flag-PHGDH in the pulldown samples was detected by Western blot. The results showed that overexpression of APT1 or APT2 could reduce the level of fatty acylation modification of PHGDH, and APT2 had stronger de-fatty acylation activity ( Figure 4A). In order to detect the interaction between PHGDH and de-fatty acylase, we conducted immunoprecipitation experiments. HA-PHGDH and de-fatty acylase APT1 and APT2 were transiently transfected into HEK293T cells, and the cells were lysed after 48 hours. The supernatant was collected after centrifugation for immunoprecipitation experiments. The results showed that the interaction between APT2 and PHGDH was significantly stronger than that between APT1 and PHGDH ( Figure 4 B). The above results indicate that APT2 is the major de-fatty acylase of PHGDH.

[0025] Example 5: Fatty acylation modification of PHGDH affects its enzyme activity.

[0026] PHGDH is the rate-limiting enzyme in the de novo biosynthesis of serine, and its enzyme activity is essential for its function. Therefore, we want to confirm whether fatty acylation modification affects the enzyme activity of PHGDH. First, HEK293T cells were treated with the pan-ZDHHC inhibitor 2-BP, and the enzyme activity assay was performed. The specific method is as follows: Flag-PHGDH was transfected into HEK293T cells, and 0μM, 10μM, 30μM, and 100μM 2-BP were added 12 h after the transfection experiment. After 24 h, the cells were harvested. The cells were washed twice with pre-cooled PBS and homogenized with pre-cooled PHGDH assay buffer for 30 min. Then, saturated ammonium sulfate was added to precipitate the protein, and the interference of small molecules was removed at the same time, and the sample protein concentration was determined. 4 μL of protein sample was added to a 96-well transparent plate, and the total volume of each sample was adjusted to 25 uL / well with assay buffer. The reaction mixture was added to each sample, positive control, and standard well. The optical density value at a wavelength of 450 nm was immediately measured in the kinetic mode at 37°C. The results showed that 2-BP inhibited the catalytic activity of PHGDH in a concentration-dependent manner. 30 µM 2-BP could significantly inhibit the enzyme activity of PHGDH ( Figure 5 A).

[0027] To determine the effect of fatty acylation on PHGDH enzyme activity, we transfected wild-type PHGDH and PHGDH fatty acylation defective mutant (C369S) into HEK293T cells, harvested the cells 36 hours later, and performed enzyme activity assays as described above. To avoid endogenous PHGDH interfering with enzyme activity assays, we used Flag beads to immunoprecipitate overexpressed Flag-PHGDH from cells and assayed its catalytic activity. Compared with wild-type PHGDH, the enzyme activity of PHGDH fatty acylation defective mutant (C369S) was significantly reduced ( Figure 5B), which is consistent with the results of 2-BP treatment. In summary, inhibiting the fatty acylation modification of cysteine ​​residue 369 of PHGDH can significantly reduce the enzyme activity of PHGDH.

[0028] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. PHGDH fatty acylation modification, characterized in that: The PHGDH undergoes fatty acylation at the cysteine ​​residue at position 369 (C369), which is co-regulated by the fatty acyltransferase ZDHHC17 and the de-fatty acylase APT2.

2. Use of the fatty acylation modification of PHGDH according to claim 1 as a biomarker and target for the occurrence and development of cancer.

3. Use of the PHGDH fatty acylation modification according to claim 1 in the preparation of a product having at least one of the following functions: (1) Molecular diagnosis of cancer; (2) Predict the risk of cancer occurrence and development; (3) Inhibit the growth and metastasis of cancer cells; (4) Provide drug targets for cancer treatment.

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