Human igf2bp3 proteins, antibodies and uses thereof

Abstract

The present application relates to the field of physiology and molecular biology, and discloses an m 6 The present application relates to the field of physiology and molecular biology, and discloses an m 6 A binding protein IGF2BP3 ubiquitination and its application in preparation of an anti-tumor drug targeting RNA binding protein. Protein spectrum technology is used to screen the ubiquitination site of IGF2BP3, and the site mutation verifies that the ubiquitination site of IGF2BP3 is K213, which is located in the first KH domain of IGF2BP3. The functional analysis results show that after the mutation of IGF2BP3, the binding of IGF2BP3 with downstream tumor-related genes is weakened, the expression of the downstream tumor-related genes is down-regulated, and the growth of cervical cancer cells is inhibited, which indicates that ubiquitination modification is an important regulation mode affecting the function of IGF2BP3.

Description

Technical Field

[0001] This invention relates to the fields of physiology and molecular biology, specifically to human IGF2BP3, antibodies, and their application in the preparation of antitumor drugs. Background Technology

[0002] RNA-binding proteins play crucial regulatory roles in the RNA lifecycle because they bind to RNA sequences and precisely guide nuclear export, control translation / degradation rates, and regulate transcriptomic localization within the cell—all essential for maintaining cellular homeostasis. Alterations in RNA-binding protein expression or function often lead to abnormal RNA translation and disease, including tumorigenesis. To date, at least 16 families of RNA-binding proteins have been identified as abnormally regulated in tumors. Among them, the highly conserved IGF2BP (insulin-like growth factor 2 mRNA-binding protein) family, including IGF2BP1, IGF2BP2, and IGF2BP3, primarily plays a carcinogenic role in tumorigenesis. IGF2BP1, IGF2BP2, and IGF2BP3 share 59% amino acid sequence homology, with IGF2BP1 and IGF2BP3 sharing an even higher 73%. These family members are characterized by a unique structure consisting of six RNA-binding domains: two RNA recognition motifs (RRMs) in the N-terminal region and four K-homology (KH) domains in the C-terminal region. All four KH domains contribute to RNA binding, ribonucleoprotein particle formation, and intracellular localization. Generally, IGF2BP proteins bind to the 5'-UTR, 3'-UTR, or coding region of target RNAs by recognizing specific RNA motifs. Increasing research shows that members of the IGF2BP family influence tumor cell biological functions and are closely related to tumorigenesis and development. Among them, IGF2BP3 has been found to be highly correlated with different types of tumors and tumor malignancy, making it an important tumor marker. IGF2BP3 overexpression leads to increased IGF2 protein, activating downstream PI3K and MAPK signaling pathways, thereby driving cell proliferation, invasion, and transformation. Furthermore, IGF2BP3 stabilizes the expression of target RNAs, such as CD44 and PDPN, by binding to their 3'-UTR, promoting the formation of invasive pseudopodia and enhancing tumor invasiveness. Recent studies have found that IGF2BP3 is a m 6 A reading protein that recognizes and binds to GG(m) target RNA. 6 The A)C sequence stabilizes the expression of these genes and promotes tumorigenesis, including the MYC gene. This function is mainly determined by the KH domain of IGF2BP3.

[0003] Existing research indicates that the mechanisms regulating IGF2BP3 expression and function are complex. In the cell nucleus, IGF2BP3 transcriptional expression is influenced by two main factors: (i) epigenetic modifications, including DNA methylation and histone acetylation; inhibition of DNA methylation or histone deacetylation upregulates IGF2BP3 mRNA expression; and (ii) transcription factor regulation, including Nanog and NF-κB, which bind to and activate the IGF2BP3 promoter. At the posttranscriptional level, IGF2BP3 is also affected by non-oncogenic microRNAs, which exert their effects by inhibiting the binding of IGF2BP3 to downstream oncogenic RNAs. The function of the IGF2BP3 protein is primarily affected by mTOR or MAPK-mediated phosphorylation, which also influences the translation of its downstream genes. Phosphorylation enhances the binding of IGF2BP3 to the 3'-UTR of the downstream gene IGF2, promoting IGF2 gene expression. Furthermore, the IGF2BP3 protein also undergoes ubiquitination modification. For example, in neuroblastoma, the ubiquitin ligase MKRN2 exerts its tumor suppressor gene function by ubiquitinizing and degrading IGF2BP3, but the site of ubiquitination is unclear. In colorectal cancer, the ubiquitin-specific protease USP11 increases the stability of IGF2BP3 by deubiquitinizing it, thereby promoting tumor growth and metastasis. Therefore, in-depth research into the regulatory mechanism of IGF2BP3 ubiquitination may provide new breakthroughs for cancer treatment.

[0004] The inventors of this application previously used proteomic profiling to screen and discover that IGF2BP3 is a substrate molecule of the ubiquitin ligase Parkin. Parkin is a 465-amino acid protein encoded by the tumor suppressor gene PARK2, consisting of an N-terminal ubiquitin-like domain (Ubl), a cysteine-rich RING0 domain, two C-terminal RING domains (RING1, RING2), and an "in-between RING (IBR)" domain separating the two C-terminal RING domains. Similar to many proteins containing circular domains, Parkin has E3 ubiquitin ligase function, regulating cellular biological functions, including mitochondrial homeostasis, anti-oxidative stress, and mitophagy, by ubiquitinizing different protein molecules, thereby preventing disease. During mitophagy, the protein kinase PINK1 on the outer mitochondrial membrane interacts with the E3 ubiquitin ligase Parkin through ubiquitin phosphorylation (p-Ser65-Ub), recruiting it to the mitochondrial surface. This promotes the ubiquitination of outer mitochondrial membrane proteins, which are then recognized by autophagy adaptor proteins and recruited into autophagic vesicles, initiating mitophagy. Parkin mediates mitophagy to clear damaged mitochondria, reduce the production of reactive oxygen species (ROS), and prevent oxidative stress damage to cells. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this application focuses on the ubiquitination modification of the IGF2BP3 protein. By screening and identifying ubiquitination sites in the IGF2BP3 protein, and then verifying the impact of ubiquitination modification on the expression and function of the IGF2BP3 protein through mutation experiments, this application helps to further clarify the molecular mechanism regulating the RNA-binding protein IGF2BP3 and its role and importance in tumorigenesis. It provides new targets, ideas, and experimental methods for cervical cancer treatment, and also provides experimental evidence for the development of new targeted drugs for cervical cancer.

[0006] This application provides a human IGF2BP3 protein, wherein the human IGF2BP3 protein is ubiquitinated at the K213 site.

[0007] The application of human IGF2BP3 protein in the preparation of antitumor drugs targeting RNA-binding proteins, wherein the human IGF2BP3 protein is ubiquitinated at the K213 site.

[0008] This application also provides a human IGF2BP3 protein, wherein the K213 site of the human IGF2BP3 protein is mutated.

[0009] Furthermore, a KR mutation occurs at the K213 site.

[0010] The application of human IGF2BP3 protein in the preparation of antitumor drugs targeting RNA-binding proteins, wherein the human IGF2BP3 protein is ubiquitinated at the K213 site.

[0011] The application of human IGF2BP3 protein in the preparation of antitumor drugs targeting RNA-binding proteins, wherein the human IGF2BP3 protein has a mutation at the K213 site, and the K213 site has a KR mutation.

[0012] The application of human IGF2BP3 protein in the preparation of targeted drugs for the treatment of cervical cancer, wherein the human IGF2BP3 protein is ubiquitinated at the K213 site.

[0013] The application of human IGF2BP3 protein in the preparation of targeted drugs for the treatment of cervical cancer, wherein the human IGF2BP3 protein has a mutation at the K213 site, and the K213 site has a KR mutation.

[0014] This application also provides an antibody that specifically binds to human IGF2BP3 protein, wherein the human IGF2BP3 protein is ubiquitinated at site K213. This means that the antibody cannot bind to human IGF2BP3 protein that is not ubiquitinated at site K213.

[0015] This application also provides an antibody that specifically binds to human IGF2BP3 protein, wherein the human IGF2BP3 protein has a mutation at the K213 site, specifically a KR mutation at the K213 site. This means that the antibody cannot bind to human IGF2BP3 protein without the KR mutation at the K213 site.

[0016] This application also provides a short peptide with the amino acid sequence LLVPTQFVGAIIGKEGATIR, wherein the amino acid residue K in the amino acid sequence is ubiquitinated.

[0017] The antibodies mentioned in this application may be polyclonal antibodies, monoclonal antibodies, or antibody fragments that retain their antigen-binding properties after being treated by chemical means or enzyme digestion.

[0018] The ubiquitin ligase Parkin mediates the formation of mitochondrial autophagic vesicles by ubiquitinizing mitochondrial membrane proteins. The inventors of this application used proteomics to screen for a novel substrate molecule of Parkin, IGF2BP3, a m... 6 A-binding protein. Because IGF2BP3 protein ubiquitination has not been clearly reported, there are no commercially available ubiquitination antibodies for IGF2BP3. This application first used in vivo and in vitro ubiquitination methods to determine that Parkin induces IGF2BP3 protein ubiquitination. Ubiquitination modification weakens the stability of IGF2BP3, while proteasome MG132 inhibitors can stabilize IGF2BP3 expression. Figure 1 Therefore, IGF2BP3 exists in a ubiquitinated state and can be degraded by ubiquitination.

[0019] IGF2BP3 is regulated by multiple factors, including gene expression, epigenetics, post-transcriptional and post-translational levels, with ubiquitination modification rarely reported. The inventors of this application screened for ubiquitination sites in IGF2BP3 using proteomics and verified this by KR point mutation (from lysine (K) to arginine (R), confirming that the ubiquitination site of IGF2BP3 is K213, located in its first KH domain, and constructed a KR mutant plasmid. In vivo ubiquitination experiments showed that compared to wild-type IGF2BP3 protein, the mutant IGF2BP3 protein exhibited significantly reduced ubiquitination levels and weakened Parkin's degradation effect on the protein. Figure 2 ).

[0020] IGF2BP3 is an m 6A reading protein stabilizes the expression of downstream genes closely related to tumorigenesis. Therefore, the inventors of this application overexpressed a mutant IGF2BP3 protein in human cervical cancer cells. The results showed that, compared with wild-type IGF2BP3 protein, the binding of mutant IGF2BP3 to downstream oncogenes was significantly reduced, and correspondingly, the expression level of its downstream oncogenes also decreased. Figure 3 The results suggest that IGF2BP3 mutations weaken the RNA stability of downstream genes. Furthermore, the inventors of this application analyzed the effect of ubiquitinated IGF2BP3 on the growth of cervical cancer cells. The results showed that a KR (lysine to arginine) mutation at the K213 site of the IGF2BP3 protein (i.e., a lysine to arginine mutation at site 213) significantly inhibited cell colony formation, suggesting that IGF2BP3 ubiquitination modification affects tumor growth. These experimental results confirm that ubiquitination modification of the RNA-binding protein IGF2BP3 affects its pro-cancer function, providing experimental evidence for the development of anti-tumor drugs targeting RNA-binding proteins. Attached Figure Description

[0021] Figure 1 Parkin ubiquitination degrades IGF2BP3: (A) HEK293 cells were first overexpressed with Flag-Parkin, and protein samples were prepared after lysis. Immunoprecipitation was performed using FLAG affinity purification gel, and the products after FLAG antibody precipitation were analyzed by proteomic analysis. The peptide scores and coverage of Parkin and IGF2BP3 proteins are shown in the figure; (B) HEK293 cells were first overexpressed with Flag-Parkin and GFP-IGF2BP3, and protein samples were prepared after lysis. Immunoprecipitation was performed using FLAG affinity purification gel, and the GFP-IGF2BP3 protein level was detected by Western blot; (C) HeLa-229 and MS751 cells were transfected with Flag-Parkin, treated with or without the proteasome inhibitor MG132 (10 μM), cells were collected and lysed, and the IGF2BP3 protein level was detected by Western blot; (D) HeLa cells were first transfected with GFP-Parkin, and after membrane permeabilization and fixation, they were co-incubated with anti-IGF2BP3 antibody and then subjected to Alexa Fluor. (E) In vivo ubiquitination experiment. HEK293 cells were first co-transfected with Flag-Parkin, GFP-IGF2BP3 and HA-ubiquitin, then treated with MG132 (10 μM) for 8 hours. Cells were collected and lysed. Protein samples were precipitated with GFP-Trap Agarose and the ubiquitin protein level was detected by Western blot. (F) In vitro ubiquitination experiment. The reaction system included ubiquitin ligase GST-Parkin 0.5 μg, substrate protein GST-IGF2BP3 0.4 μg, E1 0.5 μg, E2 0.5 μg and ubiquitin 5 μg. After incubation at 37℃ for 3 hours, the ubiquitin protein level was detected by Western blot.

[0022] Figure 2 Proteomic analysis of IGF2BP3 ubiquitination sites: (A) HEK293 cells were first transfected with Flag-Parkin, GFP-IGF2BP3, and HA-ubiquitin, then treated with MG132 (10 μM) for 8 hours. After lysis, protein samples were prepared and immunoprecipitated using GFP-Trap Agarose. The precipitated protein samples were purified by SDS-PAGE gel, stained with Coomassie Brilliant Blue, and IGF2BP3 bands were excised. After digestion of the gel strips, IGF2BP3 ubiquitination sites were screened by LC-MS. The proteomic sequence of the ubiquitinated peptide is shown as LLVPTQFVGAIIGKEGATIR; (B) Domain diagram of the IGF2BP3 protein. The protein contains two RNA recognition motifs and four KH domains, with the K213 ubiquitination site located in the first KH domain; (C) HEK293 cells were first co-transfected with Flag-Parkin, wild-type or mutant GFP-IGF2BP3 and HA-ubiquitin, then treated with MG132 (10 μM) for 8 hours, the cells were collected and lysed, the protein samples were precipitated with GFP-Trap Agarose, and the ubiquitin protein level was detected by Western blot; (D) HeLa cells were transfected with wild-type or mutant GFP-IGF2BP3 and Flag-Parkin, respectively, the cells were collected and lysed, and the IGF2BP3 protein level was detected by Western blot.

[0023] Figure 3IGF2BP3 ubiquitination affects the expression of its downstream genes and tumor cell growth: (A) HeLa-Parkin stable cell lines were first transfected with wild-type or mutant IGF2BP3, and intracellular mRNA was collected and extracted. After being denatured by heat, it was added to Hybond-N + After being subjected to UV crosslinking and BSA blocking on the membrane, it is then combined with m 6 (A) Antibody incubation was performed, and Western blot was used to detect RNA methylation levels. Methylene blue staining was used to show the amount of RNA loaded. (B) HEK293 cells were first co-transfected with Flag-Parkin, wild-type, or mutant GFP-IGF2BP3. After cross-linking the protein-RNA complex, separating and lysing the nuclear precipitate, and fragmenting the chromatin, RNA-immunoprecipitation was performed using GFP-Trap Agarose. Finally, the IGF2BP3-bound RNA was purified after immunoprecipitation. Real-time PCR was used to analyze the changes in IGF2BP3 binding to downstream genes' RNA. (C) HeLa-Parkin stable cell lines were first transfected with wild-type or mutant IGF2BP3, and cellular RNA was collected and extracted. After reverse transcription, the mRNA levels of downstream genes of IGF2BP3 protein were analyzed by Real-time PCR. (D) Colony formation assay was used to analyze changes in tumor cell growth and proliferation. HeLa-Parkin stable cell lines were transfected with wild-type or mutant IGF2BP3. After two weeks of cell growth, the cells were fixed with methanol, stained with crystal violet, and photographed to record the resulting clonal colonies. Detailed Implementation

[0024] Example 1: Ubiquitin ligase Parkin induces ubiquitination of IGF2BP3 protein

[0025] The ubiquitin ligase Parkin mediates the formation of mitochondrial autophagic vesicles by ubiquitinizing mitochondrial membrane proteins. Firstly, the inventors of this application confirmed the interaction between Parkin and IGF2BP3 through immunoprecipitation experiments. Figure 1 (A-1B) This conclusion is consistent with the results of proteomic analysis revealing the immunoprecipitation complex of Parkin. Secondly, overexpression of Parkin in cervical cancer cells (HeLa) reduced IGF2BP3 protein expression, while the reduction in IGF2BP3 protein was reversed after treatment with a proteasome inhibitor. Figure 1 C-1D suggests the possibility of Parkin ubiquitination degrading IGF2BP3. Finally, both in vivo and in vitro ubiquitination experiments confirmed that Parkin directly induces ubiquitination of the IGF2BP3 protein. Figure 1 E-1F). Therefore, IGF2BP3 exists in a ubiquitinated state and can be degraded by ubiquitination.

[0026] Example 2: Screening and confirmation that the ubiquitination site of IGF2BP3 protein is K213

[0027] Based on Example 1, to further clarify the molecular mechanism by which Parkin induces ubiquitination of IGF2BP3 protein, the inventors of this application analyzed the ubiquitination sites of IGF2BP3 protein using proteomics. The results showed that the peptide sequence undergoing ubiquitination modification was LLVPTQFVGAIIGKEGATIR ​​( Figure 2 A). Sequence alignment revealed that the ubiquitination site of the IGF2BP3 protein is K213, located on its first KH domain. Figure 2 B). After inducing a KR (lysine to arginine) mutation at the K213 site of the IGF2BP3 protein, Parkin significantly reduced the level of IGF2BP3 protein ubiquitination, while Parkin significantly reduced the degradation of mutant IGF2BP3 protein (2C-2D), confirming that the ubiquitination site of the IGF2BP3 protein is K213.

[0028] Furthermore, by conjugating the ubiquitinated peptide sequence LLVPTQFVGAIIGKEGATIR ​​with carrier proteins such as BSA and KLH, antibodies specifically recognizing IGF2BP3 protein with ubiquitination at the K213 site can be prepared by immunizing mice, rats, and rabbits with the conjugation product. Additionally, for mutant IGF2BP3 protein with a KR mutation at the K213 site, LLVPTQFVGAIIGREGATIR ​​can also be conjugated with carrier proteins such as BSA and KLH, and antibodies specifically recognizing mutant IGF2BP3 protein with a KR mutation at the K213 site can be prepared by immunizing mice, rats, and rabbits with the conjugation product.

[0029] The prepared antibody can be used to qualitatively and quantitatively detect human IGF2BP3 protein with ubiquitination at the K213 site and mutant human IGF2BP3 protein with KR mutation at the K213 site.

[0030] Example 3: Ubiquitination modification affects the function of IGF2BP3 protein.

[0031] IGF2BP3 is an m 6 A reading protein that recognizes and binds to GG(m) target RNA. 6 A)C sequence, thereby stabilizing the expression of these genes. Dot blot analysis results showed that mutations at the IGF2BP3 ubiquitination site reduced intracellular RNA methylation levels ( Figure 3A). The inventors of this application further analyzed the ability of IGF2BP3 to bind to target RNA through RNA immunoprecipitation experiments, and the results showed ( Figure 3 B) The ubiquitination site mutation significantly weakens the RNA binding of IGF2BP3 to downstream oncogenes such as MYC, TK1, and FSCN. The inventors of this application also used Real-time PCR to detect changes in intracellular IGF2BP3 downstream gene mRNA levels after the ubiquitination site mutation, and the results showed ( Figure 3 C) Mutations at the IGF2BP3 ubiquitination site significantly downregulated the expression of downstream oncogenes such as MYC, TK1, FSCN, and MARCRSL1. These results all indicate that IGF2BP3 protein ubiquitination modification helps enhance its regulation of downstream oncogene expression. Finally, colony formation assays confirmed that mutations at the IGF2BP3 ubiquitination site indeed inhibited tumor cell growth and proliferation. Figure 3 D) indicates that ubiquitination is an important regulatory mechanism affecting the function of IGF2BP3 protein. sequence list <110> Zhejiang Provincial People's Hospital <120> Human IGF2BP3 protein, antibody and its application <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> PRT <213> Artificial Sequence <400> 1 Leu Leu Val Pro Thr Gln Phe Val Gly Ala Ile Ile Gly Lys Glu Gly 1 5 10 15 Ala Thr Ile Arg 20 <210> 2 <211> 20 <212> PRT <213> Artificial Sequence <400> 2 Leu Leu Val Pro Thr Gln Phe Val Gly Ala Ile Ile Gly Arg Glu Gly 1 5 10 15 Ala Thr Ile Arg 20

Claims

1. Human IGF2BP3 protein, characterized in that, The human IGF2BP3 protein has a full-length sequence of 579 amino acids, including a short peptide with the amino acid sequence LLVPTQFVGAIIGKEGATIR; the human IGF2BP3 protein has a mutation from lysine to arginine at the K213 site.

2. The use of the human IGF2BP3 protein as described in claim 1 in the preparation of a targeted drug for the treatment of cervical cancer.

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