In-vitro phosphorylation activation method of E3 ubiquitin ligase Smurf1
Through the mixing of FGFR1 and Smurf1 in a specific buffer, the in vitro phosphorylation activation of Smurf1 is achieved, which solves the shortcomings of Smurf1 activation methods in the prior art, provides stable and scientific research methods, and verifies the function of key phosphorylation sites.
Patent Information
- Application Number
- CN202510444903.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-10
- Publication Date
- 2025-07-11
AI Technical Summary
The lack of effective in vitro methods in the prior art to activate the phosphorylation process of the E3 ubiquitin ligase Smurf1, which has affected the study and understanding of the interaction between Smurf1 and substrate proteins.
FGFR1 was used to mix the fibroblast growth factor receptor FGFR1 with E3 ubiquitin ligase Smurf1 in a specific buffer to activate Smurf1 by phosphorylation, and in vitro activation of Smurf1 was achieved in cells using fusion proteins and gene expression vectors.
The stable in vitro phosphorylation activation of Smurf1 was achieved, ensuring the scientificity and stability of intermolecular interactions, providing new experimental methods for studying the function of Smurf1, and verifying that key phosphorylation sites do not affect protein interactions.
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Figure CN120290509A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of molecular biology, and particularly to an in vitro phosphorylation activation method for E3 ubiquitin ligase Smurf1. Background Art
[0002] Proteins are important components that make up cells, tissues, organs, and organisms, and are also the basic molecules that execute biological life activities. Proteins are the material basis of life, the basic organic matter that constitutes cells, the main bearers of life activities, and substances that are closely related to life and various forms of life activities. In the human body, there are thousands of types of proteins, and different proteins perform different functions, but they are all formed by more than 20 essential amino acids in different forms. These proteins are not static in the human body, but are in a dynamic balance of continuous degradation and synthesis to maintain life activities. Therefore, maintaining normal protein metabolism in cells is crucial for the normal functioning of life activities. So far, intracellular proteins are mainly degraded through two pathways, one is the lysosomal degradation pathway, and the other is the ubiquitin-mediated proteasomal degradation pathway.
[0003] The ubiquitin-proteasome system controls the degradation of the vast majority of proteins in eukaryotic cells and plays an irreplaceable role in protein homeostasis. The ubiquitin-proteasome regulates many basic cellular processes, such as the cell cycle, DNA damage repair, membrane trafficking, signal transduction, etc. Dysfunction of this system can lead to various diseases, including malignancies such as cancer, cardiovascular diseases, and neurodegenerative diseases. Therefore, the ubiquitin-proteasome system is considered a potential target for treating many diseases. In recent years, significant progress has also been made in proteasome inhibitors, which have extended the lives of thousands of multiple myeloma patients. Because the ubiquitin-regulated protein degradation process is very important, on October 6, 2004, the Royal Swedish Academy of Sciences announced that the 2004 Nobel Prize in Chemistry was awarded to American scientist Irwin Rose, Israeli scientists Aaron Ciechanover and Avram Hershko for their discovery of the ubiquitin-regulated protein degradation process.
[0004] The E3 ubiquitin ligase Smurf1 (SMAD ubiquitination regulatory factor 1) is an important enzyme belonging to the HECT-type E3 ubiquitin ligase family. Smurf1 mainly mediates the ubiquitination process in cells to regulate the degradation of target proteins, and thus participates in various cellular processes, such as cell signaling, cell cycle control, and cell migration. The core of its function lies in attaching ubiquitin molecules to target proteins, marking them for degradation by the proteasome. Smurf1 plays a crucial role in the TGF-β signaling pathway. TGF-β is an important cytokine that regulates processes such as cell growth, differentiation, and death. Smurf1 regulates the strength and duration of this signaling pathway by ubiquitinating and degrading some receptors and effector molecules related to TGF-β signaling. For example, Smurf1 can inhibit the overactivation of TGF-β receptors and Smad proteins by ubiquitination, maintaining the balance of intracellular signals. In addition, Smurf1 is also involved in regulating biological processes such as muscle development, nervous system formation, and immune responses. In oncology, the function of Smurf1 is considered a double-edged sword. In some cases, it may promote the growth and metastasis of cancer cells by promoting the degradation of tumor suppressors. However, in other cases, the function of Smurf1 may also inhibit cancer progression. Therefore, studying the specific mechanism of Smurf1 and its role in different cellular environments is crucial for understanding its biological function and developing new therapeutic strategies. Currently, there are more and more studies on Smurf1. To efficiently explore the interaction between Smurf1 and substrate proteins, it is crucial to construct a stable in vitro ubiquitination system. Currently, some studies have used co-expressed protein kinases and substrate proteins in Escherichia coli to enhance the activity of the produced proteins. Therefore, it is crucial to find protein kinases specific to the substrate, and the key phosphorylation sites need to be clarified to identify which mutants cannot be used as application cases of this method. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to provide an in vitro phosphorylation activation method for the E3 ubiquitin ligase Smurf1.
[0006] The technical solution of the present invention is: the application of fibroblast growth factor receptor FGFR1 in the in vitro phosphorylation activation of the E3 ubiquitin ligase Smurf1.
[0007] Further, the method for in vitro phosphorylation activation is: mixing FGFR1 and Smurf1 in the FGFR1 assay buffer to phosphorylate and activate Smurf1.
[0008] Furthermore, the composition of the FGFR1 assay buffer is: 10 mM HEPES, pH 7.5, 10 mM MnCl2, 150 mM NaCl, 5 mM DTT, and 0.01% Triton X-100.
[0009] A fusion protein, which comprises two functional proteins, namely fibroblast growth factor receptor FGFR1 and E3 ubiquitin ligase Smurf1.
[0010] Furthermore, the fibroblast growth factor receptor FGFR1 and the E3 ubiquitin ligase Smurf1 are linked by a linker peptide.
[0011] A gene encoding the above-mentioned fusion protein.
[0012] An expression vector containing the above-mentioned gene.
[0013] A cell containing the above-mentioned expression vector.
[0014] This application provides an experimental method for in vitro phosphorylation activation of Smurf1 through a systematic multi-step analysis process, and screens out the key targets for Smurf1 phosphorylation activation. The specific beneficial effects include:
[0015] 1. Innovative method for in vitro activation of E3 ubiquitin ligase: Based on molecular biology, FGFR1 in the human body is used to achieve in vitro phosphorylation activation of E3 ubiquitin ligase.
[0016] 2. Precise analysis of molecular mechanism: By using the method of single-site mutation screening one by one, the key phosphorylation site for FGFR1 to activate Smurf1 is screened out, and it is proved that this site does not affect protein-protein interaction.
[0017] 3. Comprehensive in vivo experiments: The relevant molecular mechanisms are verified in cells, and it is proved that in vitro FGFR1 activation of Smurf1 does not change the function of Smurf1 itself, which can be used for related mechanism research.
[0018] Compared with the prior art, the present invention has the following beneficial effects:
[0019] The present invention provides a systematic and scientific method for in vitro activation of Smurf1 activity. Through multi-step refined analysis, the stability and scientificity of the molecular interactions used are ensured, providing a new idea for the construction of a highly active in vitro ubiquitination system. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 It is the experimental basis of the present invention. The applicant found that rFGFR1 can phosphorylate and activate E3 ubiquitin ligase Smurf1 in vitro, and we preliminarily predicted the important phosphorylation targets therein.
[0021] Figure 2 In the implementation of the present invention, a point mutant protein of Smurf1 was constructed, and in vitro ubiquitination experiments were used to prove that Y69, Y251, and Y387 might be potential sites for rFGFR1-mediated phosphorylation of Smurf1.
[0022] Figure 3 The key experiment of the present invention proved that intracellular Y69F, Y251F, and Y387F would affect the ubiquitination activity of Smurf1, and rFGFR1 could activate the ubiquitination activity of all Smurf1 mutants except the Y69F mutant.
[0023] Figure 4 It is for Figure 3 statistical explanation.
[0024] Figure 5 The key result of the present invention is that Y69F is the most critical site for rFGFR1-mediated phosphorylation activation of Smurf1.
[0025] Figure 6 The verification of the present invention is that the Y69F mutation does not cause the above phenomenon by reducing the binding of Smurf1 to the substrate protein, and the reason for the above phenomenon is that rFGFR1 cannot phosphorylate and activate Smurf1. Detailed implementation method
[0026] Unless otherwise specified, the experimental methods in the following examples are all conventional methods. Unless otherwise specified, the test materials used in the following examples are all purchased from commercial channels.
[0027] Proteins and reagents:
[0028] Recombinant FGFR1 (rFGFR1) was purchased from Abcam (product number ab290111), GST-PTEN (purchased from Abnova, product number: H00005728-Q01) (500 ng / μL), E1 enzyme: His-Ube1 (purchased from Sigma-Aldrich, product number: 23-021-M) (50 ng / μL), E2 enzyme: GST-UbcH5c (purchased from Boston Biochem, product number: E2-625-100) (200 ng / μL), ubiquitin (Ub): PET-15-Ub (protein expressed by the corresponding plasmid) (500 ng / μL), ATP: 10 mmol / L (purchased from Sigma-Aldrich, product number: 11140965001),
[0029] Example 1 Prediction of key phosphorylation sites for Smurf1 activation and preparation of mutants
[0030] The key phosphorylation sites activated by Smurf1 were predicted using https: / / www.phosphosite.org, and 9 potential phosphorylation sites including Y66, Y69, Y113, Y251, Y297, Y348, Y387, Y414 and Y561 were screened.( Figure 1 )
[0031] All mutant constructs of SMURF 1 were generated using the Q5 site-directed mutagenesis kit (NEB) (performed according to the kit instructions), and all mutations were confirmed by sequencing. His-Flag-Smurf1 protein was purified through the Escherichia coli expression system (using Ni-NTA affinity chromatography and a Flag antibody affinity column) to obtain His-Flag-tagged Smurf1 wild type and Y66F, Y69F, Y113F, Y251F, Y297F, Y387F, Y414F and Y561F mutants.
[0032] Recombinant FGFR1 (rFGFR1) in the active form was purchased from Abcam (product number ab290111) and stored at -80 °C. 1 μg of His-Flag-Smurf1 wild type was mixed with 0.5 μg of active rFGFR1 in FGFR1 assay buffer (10 mM HEPES pH 7.5, 10 mM MnCl2, 150 mM NaCl, 5 mM DTT, 0.01% Triton X-100). In the control group, Smurf1 without rFGFR1 was incubated alone.
[0033] Incubate at 37 °C for 1 hour, and then terminate the reaction on ice. Proteins were separated using 12% SDS-PAGE and analyzed by Western blot after transferring to the membrane. Primary antibody: anti-phosphotyrosine antibody (anti-p-Tyr-100, Cell Signaling Technology, product number #9411, diluted 1:1000); secondary antibody: HRP-labeled goat anti-rabbit IgG (1:5000).
[0034] The results showed that Smurf1 in the rFGFR1 group showed a significant phosphorylation band at approximately 90 kDa (see Figure 1 ), and there was no signal in the control group, confirming that rFGFR1 can phosphorylate Smurf1 in vitro.
[0035] Example 2: Functional verification of the key phosphorylation site Y69 of Smurf1
[0036] Mix 1 μg of Smurf1 wild type or Smurf1 mutant with 0.5 μg of GST-PTEN (substrate), 0.2 μg of HA-Ub (Sigma), 50 nM of E1 enzyme (UBA1), 100 nM of E2 enzyme (UbcH5c), 2 mM of ATP, and 0.5 μg of rFGFR1. The control group is the wild type group without rFGFR1.
[0037] Incubate at 37 °C for 3 hours. Enrich PTEN by GST Pull-down, and detect the ubiquitination level using an anti-HA antibody (Covance, catalog number MMS-101P, 1:2000) after SDS-PAGE.
[0038] The results are as Figure 2 shown. The ubiquitination level of PTEN in the Y69F mutant is significantly reduced, indicating that Y69 is the key site for rFGFR1 to activate Smurf1.
[0039] Example 3: Verification of phosphorylation dependence at the Y69 site in cells
[0040] Transfect LN229 glioma cells (ATCC) with the HA-Ub plasmid (Lipofectamine 3000, Thermo Fisher). After 24 hours, treat with 10 μM MG132 (proteasome inhibitor) for 12 hours. Lyse the cells by sonication, mix the cell lysate with 1 μg of His-Flag-Smurf1 WT or mutant, and incubate at 37 °C for 6 hours. Perform immunoprecipitation (IP) using anti-PTEN antibody-conjugated magnetic beads (Thermo Fisher), and detect the ubiquitination level by Western blot (anti-HA antibody).
[0041] The results show that the ubiquitination level of PTEN in the Y69F mutant group is lower than that in the wild type ([ Figure 3 、 Figure 4 ), and rFGFR1 can significantly enhance the ubiquitination activity of all Smurf1 except Y69F, confirming that Y69 phosphorylation is crucial for Smurf1 activity.
[0042] Example 4: The Y69 site is the site for rFGFR1-mediated phosphorylation of Smurf1
[0043] 1 μg of the WT, Y66F, Y69F, Y113F, Y251F, Y297F, Y348F, Y387F, Y414F, and Y561F mutants of His-Flag-Smurf1 were mixed with 0.5 μg of active rFGFR1 in FGFR1 assay buffer (10 mM HEPES pH 7.5, 10 mM MnCl2, 150 mM NaCl, 5 mM DTT, 0.01% Triton X-100). As a control, all Smurf1 wild-type and mutants without rFGFR1 were incubated separately.
[0044] Incubate at 37 °C for 1 hour, and then terminate the reaction on ice. Separate the proteins using 12% SDS-PAGE and analyze by Western blot after transferring the membrane. Primary antibody: anti-phosphotyrosine antibody (anti-p-Tyr-100, Cell Signaling Technology, catalog number #9411, diluted 1:1000); secondary antibody: HRP-labeled goat anti-rabbit IgG (1:5000).
[0045] The results showed that Y69 is the only key phosphorylation site for rFGFR1-mediated Smurf1 activation ( Figure 5 ).
[0046] Example 5: Pull-down verification of the binding of Smurf1 to the substrate
[0047] The wild-type of Smurf1 and the Y69F mutant were incubated with GST-PTEN in binding buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% NP-40) for 2 hours. Enrich the GST-PTEN complex using glutathione agarose beads (GE Healthcare), and detect the binding amount of Smurf1 by anti-Flag antibody after SDS-PAGE.
[0048] The binding ability of the Y69F mutant to PTEN was not different from that of the wild-type ( Figure 6 ), indicating that phosphorylation does not affect the interaction between Smurf1 and the substrate.
Claims
1. Use of fibroblast growth factor receptor FGFR1 in in vitro phosphorylation activation of E3 ubiquitin ligase Smurf1.
2. The application according to claim 1, characterized in that, The method for in vitro phosphorylation activation is as follows: Mix FGFR1 and Smurf1 in an FGFR1 assay buffer to phosphorylate and activate Smurf1.
3. The application according to claim 2, wherein The composition of the FGFR1 assay buffer is: 10 mM HEPES, pH 7.5, 10 mM MnCl2, 150 mM NaCl, 5 mM DTT, and 0.01% Triton X-100.
4. A fusion protein, characterized in that, The fusion protein comprises two functional proteins, fibroblast growth factor receptor FGFR1 and E3 ubiquitin ligase Smurf1.
5. The fusion protein according to claim 4, wherein The fibroblast growth factor receptor FGFR1 and the E3 ubiquitin ligase Smurf1 are linked by a linker peptide.
6. A gene encoding the fusion protein according to claim 4.
7. An expression vector containing the gene according to claim 6.
8. A cell containing the expression vector according to claim 7.