Blue light-controlled protein degradation system based on KFERQ sequence and its construction method and application

Abstract

The present invention belongs to the field of biophysics technology and specifically relates to a KFERQ sequence-based blue-light-controlled protein degradation system, its construction method, and application. The blue-light-controlled protein degradation system includes a blue-light control unit and a targeting unit; the blue-light control unit is formed by the recombination of the KFERQ sequence and the LOV2 sequence; and the targeting unit is a protein targeting sequence. The blue-light-controlled protein degradation system of the present invention provides a blue-light-controlled protein degradation system by recombination of the KFERQ sequence, the LOV2 sequence, and an endogenous protein targeting sequence, and by utilizing the light-sensitive regulatory properties of the LOV2 domain, offering a new strategy for endogenous protein degradation.

Description

Technical Field

[0001] The present invention belongs to the technical field of biophysics, and in particular relates to a blue light-controlled protein degradation system based on a KFERQ sequence, a construction method thereof, and an application thereof. Background Art

[0002] Targeted degradation of overexpressed or abnormally activated proteins within cells is a promising approach for treating human tumors. Common degradation pathways that have been discovered to regulate some intracellular proteins include the proteasome and lysosome pathways. Generally speaking, the proteasome eliminates short-lived and soluble misfolded proteins through the ubiquitin-proteasome system (UPS). In contrast, the lysosome is responsible for degrading long-lived proteins, insoluble protein aggregates, and even entire organelles. Lysosomes are the primary degradation compartment of the cell, receiving their degraded materials through endocytosis, phagocytosis, or autophagy.

[0003] To date, three autophagy-related pathways have been described in higher eukaryotes: macroautophagy, chaperone-mediated autophagy (CMA), and microautophagy. In CMA, specific proteins are recognized by Hsc70, which, together with other chaperones and co-chaperones, guides them to the surface of the lysosome. After binding to LAMP-2A, the target protein is transferred to the lysosomal membrane and degraded. Both autophagy and the ubiquitin-proteasome system can degrade substrates through ubiquitination. Several methods for targeting specific protein degradation based on cell self-degradation methods have been gradually invented, mainly including PROTAC (PROteolysis Targeting Chimeras), LYTAC (Lysosome-Targeting Chimaera), abTAC (Antibody-based PROTAC), and AUTOTAC (AUTOphagy-TArgeting Chimera) targeted degradation strategies. Therefore, it is of great significance to develop a widely applicable light-controlled system based on a stable protein degradation strategy to achieve the effect of switching endogenous protein degradation. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a blue light-controlled protein degradation system based on the KFERQ sequence, and its construction method and application. The blue light-controlled protein degradation system recombines the KFERQ sequence, LOV2 sequence and endogenous protein targeting sequence, and utilizes the light-sensitive regulatory characteristics of the LOV2 domain to provide a protein degradation system that can be controlled by blue light, providing a new strategy for endogenous protein degradation.

[0005] In microautophagy, lysosomes directly engulf autophagic cargo and cause its degradation. In CMA, proteins are selected by a molecular chaperone (Hsc70), localized to lysosomes, and directly degraded across the lysosomal membrane. CMA has two unique functions: CMA only degrades certain proteins but not organelles; the formation of autophagosomes is unnecessary in CMA. According to previous studies, as long as the KFERQ sequence is exposed, non-CMA substrates attached to the KFERQ sequence can also obey CMA. Therefore, in the present invention, we choose to use CMA and the microautophagy-lysosome pathway as the degradation strategy of the present invention to degrade endogenous proteins.

[0006] At the molecular level, LOV2 domains are a subset of the larger family of environmental sensing domains known as PAS (Period ARNTSingleminded) domains. All of these domains are approximately 110 amino acid residues long and adopt a mixed α / β protein fold, with several α-helices positioned on one side of an antiparallel β-sheet. Many PAS domains participate in protein-protein interactions, often modulating the strength of these interactions through the presence of small, internally bound cofactors or ligands in environmentally sensitive concentrations or configurations. LOV2 domains achieve their photosensitizing function through specific binding to a flavin chromophore. Photochemically triggered free radical chemistry leads to the specific formation of a new covalent adduct between the gamma sulfur atom of a conserved cysteine residue (Cys 450 in the widely studied LOV2 domain of Avena sativaphototrophin 1, commonly referred to as "AsLOV2") and the C4a position of the flavin pentytetrazine ring. This modification allows for analysis in visible absorption spectroscopy by eliminating significant blue absorption around 450 nm while increasing near-ultraviolet absorption around 390 nm. Coupled with the concomitant protonation of the adjacent N5 position, this change effectively serves as a photochemical trigger for a series of subsequent structural transformations. Most importantly, this also includes reversing the hydrogen bonding activity of the conserved Gln residue in the LOVβ sheet (Gln 513 in AsLOV2), converting it from donating hydrogen bonds to accepting hydrogen bonds in the deprotonated N5, which, combined with other structural modifications, triggers a wider range of allosteric changes. A large number of studies have shown that circular arrangements are a powerful method for evolving new genetically encoded fluorescent probes and biocatalysts. Due to the small size of LOV2 and the spatial proximity of its N and C termini, it is an ideal candidate for circular arrangement. Therefore, the inventors applied an existing design of a cyclically permuted LOV2 that can provide a new cage surface while maintaining compatibility with existing LOV2-based synthetic equipment.

[0007] At the same time, non-CMA substrates fused to the KFERQ sequence can obey the CMA rules when exposed to KFERQ, rapidly degrading the targeted protein. Therefore, to achieve the above objectives, the present invention provides a blue-light-controlled protein degradation system based on the KFERQ sequence, comprising a blue-light control unit and a targeting unit; the blue-light control unit is recombined from the KFERQ sequence and the LOV2 sequence; and the targeting unit is a protein targeting sequence.

[0008] In the present technical solution, the blue light control unit recombination sequence can be a KFERQ-LOV2 sequence or a LOV2-KFERQ sequence.

[0009] Furthermore, in the above technical solution, the blue light control unit is a KFERQ-LOV2 (KL) recombinant sequence.

[0010] Furthermore, in the above technical solution, the LOV2 sequence is an eLOV2 or cpLOV2 sequence; the protein targeting sequence is an endogenous protein sequence, which is any one of the EGFP sequence overexpressed in the cell, the endogenous AKT sequence, and the endogenous pAKT sequence.

[0011] The present invention also provides a method for constructing a blue light-controlled protein degradation system based on the KFERQ sequence, comprising the following steps:

[0012] (1) Obtaining the LOV2 sequence: The validated eLOV2 or cpLOV2 was used as the LOV2 sequence;

[0013] (2) Construction of recombinant plasmid: Using FV185 as a plasmid vector, the eLOV2 or cpLOV2 sequence was first recombined with the KEFRQ sequence to obtain the KL or LOV2-KFERQ (LK) recombinant sequence, and then the protein targeting sequence was inserted at the N-terminus or C-terminus to obtain the recombinant plasmid.

[0014] Furthermore, in the above technical solution, the recombinant plasmid includes a protein targeting sequence-LK or KL-protein targeting sequence.

[0015] Preferably, the recombinant plasmid includes a KL-protein targeting sequence.

[0016] The present invention also provides an application of a blue light-controlled protein degradation system in degrading exogenously overexpressed EGFP and degrading endogenous cancer-promoting proteins.

[0017] Furthermore, in the above technical solution, when applied to degrade exogenously overexpressed EGFP, the plasmid containing the blue light-controlled protein degradation system with the protein targeting sequence EGFP is transfected into the recipient cells for 6-18 hours, and then cultured under light conditions for 24 hours to detect the degradation of EGFP.

[0018] Furthermore, in the above technical solution, when applied to the degradation of endogenous cancer-promoting proteins, the plasmid of the blue light-controlled protein degradation system with the protein targeting sequence 2xAKTin is transfected into the recipient cells for 6-18 hours, and then cultured under light conditions for 24 hours to detect the degradation of AKT and pAKT.

[0019] Furthermore, in the above technical solution, the illumination conditions are: blue light, wavelength of 470nm, power of 4mW / cm 2 .

[0020] The present invention has the following beneficial effects:

[0021] The present invention provides a protein degradation system that can be controlled by blue light by recombining the KFERQ sequence, LOV2 sequence and endogenous protein targeting sequence and utilizing the light-sensitive regulation characteristics of the LOV2 domain, thereby providing a new strategy for endogenous protein degradation.

[0022] The present invention has determined through experiments that the KFERQ sequence placed at the N-terminus of the recombinant polypeptide has higher Hsc70 recognition and binding efficiency, providing a good template for the insertion and replacement of other regulatory elements.

[0023] The blue light-controlled degradation system containing KEFRQ-cpLOV2 (KL) designed in the present invention can target endogenous and exogenous proteins, and can significantly reduce the levels of overexpressed EGFP and endogenous AKT and pAKT in cells, thereby achieving significant degradation of intracellular EGFP and endogenous cancer-promoting proteins, while effectively inhibiting cell proliferation activity, providing a basis for cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the composition of the recombinant plasmid of the present invention;

[0025] Figure 2 Schematic diagram of the action mechanism of KL-EGFP in cells of the present invention;

[0026] Figure 3 470nm, 4mW / cm 2 Green fluorescence changes after KL-EGFP degradation regulated by blue light;

[0027] Figure 4 This is a graph showing the changes in green fluorescence content and intensity after KL-EGFP degradation regulated by blue light in the present invention;

[0028] Figure 5 This is a graph showing the EGFP protein level after KL-EGFP degradation regulated by blue light in the present invention;

[0029] Figure 6 Schematic diagram of the mechanism of action of KL-GFP nanobody targeted degradation of EGFP in the present invention;

[0030] Figure 7 This is a graph showing the level and intensity changes of the intracellular overexpressed EGFP detected by fluorescence microscopy of the present invention by blue light regulation of KL-GFP nanobody;

[0031] Figure 8 This is a graph showing the level and intensity changes of KL-GFP nanobody regulated by blue light to degrade overexpressed EGFP in cells using flow cytometry.

[0032] Figure 9This is a graph showing the changes in the level and intensity of overexpressed EGFP in cells regulated by KL-GFP nanobody by western blot detection of blue light;

[0033] Figure 10 This is a graph showing the changes in intracellular AKT and pAKT levels after blue light regulation by KL-2xAKTin of the present invention;

[0034] Figure 11 This is a graph showing the proliferation changes of U251 cells after KL-2xAKTin knocked down AKT and pAKT in the CCK8 experiment of the present invention. DETAILED DESCRIPTION

[0035] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The raw materials involved in the following examples, unless otherwise specified, are all common commercial products and can be purchased from the market. All methods not specifically described are carried out according to conventional methods in the art.

[0036] Lentiviral plasmid vector FV185 was purchased from Tianjin Sheweis Biotechnology Co., Ltd.;

[0037] The present invention is described in further detail below in conjunction with the embodiments:

[0038] Example 1: Construction of recombinant plasmid

[0039] Using the lentiviral plasmid vector FV185 as a vector, inverse amplification PCR was used to obtain the backbone with appropriate primers. The validated eLOV2 and cpLOV2 sequences were then synthesized by Bioengineering based on homologous sequences. The nucleotide sequences of eLOV2 and cpLOV2 are shown in Seq_1 and Seq_2, respectively. These sequences were then recombined with the KEFRQ sequence (nucleotide sequence shown in Seq_3) to obtain KL (KEFRQ-eLOV2, KEFRQ-cpLOV2) or LK (eLOV2-KEFRQ, cpLOV2-KEFRQ) recombinant sequences. The resulting KL / LK recombinant sequences were then inserted into the EGFP sequence (EGFP sequence from LentiCRISPR v2-EGFP, Addgene #167188, nucleotide sequence shown in Seq_4) at the N- or C-terminus to generate recombinant plasmids containing the above sequences. The recombinant plasmid can be KEFRQ-eLOV2-EGFP, KEFRQ-cpLOV2-EGFP, EGFP-eLOV2-KEFRQ, EGFP-cpLOV2-KEFRQ sequence, wherein the amino acid sequences of EGFP-eLOV2-KEFRQ (EGFP-eLOV2-K) and KEFRQ-cpLOV2-EGFP (K-cpLOV2-EGFP) are as shown in Seq_7 and Seq_8, respectively.

[0040] The Q in the KFERQ sequence of the recombinant plasmid was mutated to A to obtain the mK sequence, resulting in the recombinant plasmids KEFRA-eLOV2-EGFP, KEFRA-cpLOV2-EGFP, EGFP-eLOV2-KEFRA, and EGFP-cpLOV2-KEFRA. The amino acid sequences of EGFP-eLOV2-KEFRA (EGFP-eLOV2-mK) and KEFRA-cpLOV2-EGFP (mK-cpLOV2-EGFP) are shown in Seq_9 and Seq_10, respectively.

[0041] The KEFRQ-cpLOV2 recombinant sequence was inserted into the GFP nanobody (nucleotide sequence as shown in Seq_5) or 2xAKTin (nucleotide sequence as shown in Seq_6) sequence at the C-terminus to obtain plasmids containing the KEFRQ-cpLOV2-GFP nanobody (KL-G) sequence and the KEFRQ-cpLOV2-2xAKTin (KL-A) sequence, whose amino acid sequences are shown in Seq_11 and Seq_12, respectively. The Q in the above recombinant sequence KFERQ was mutated to A to obtain the KEFRA-cpLOV2-GFP nanobody (mKL-G) sequence and the KEFRA-cpLOV2-2xAKTin (mKL-A) sequence, whose amino acid sequences are shown in Seq_13 and Seq_14, respectively. Among them, the schematic diagram of some recombinant plasmids is shown in Figure 1 shown.

[0042] Example 2: Blue light regulation of KL-EGFP to target and degrade its own EGFP

[0043] When human embryonic kidney cells HEK-293T were cultured in DMEM to 80% confluence, the plasmid constructed in Example 1 was divided into 6 groups, group 1 was transfected with EGFP-eLOV2-KFERA (EGFP-eLOV2-mK) plasmid, groups 2 and 3 were transfected with EGFP-eLOV2-KFERQ (EGFP-eLOV2-K) plasmid, group 4 was transfected with KFERA-cpLOV2-EGFP (mK-cpLOV2-EGFP) plasmid, and groups 5 and 6 were transfected with KFERQ-cpLOV2-EGFP (K-cpLOV2-EGFP) plasmid. The medium was changed 6-18 hours after transfection, and then cultured in a combination of dark and blue light environments. Among them, the 293T cells in groups 1, 2, 4 and 5 were placed in a 470 nm wavelength, 4 mW / cm 2 The other two groups were cultured in the dark. Figure 2 As shown, after culturing for 24 h, the degradation of EGFP was observed using a fluorescence microscope. Figure 3Then the green fluorescence content and intensity changes were measured using the FITC channel of the flow cytometer. Each group of experiments was repeated three times or more. The results are shown in the figure below. Figure 4 The above results all show that the protein degradation system recombined with LK and EGFP can show good EGFP degradation ability under blue light, and the degradation efficiency of KL-EGFP is higher. 2 Blue light can be used to regulate the degradation of KL-EGFP, and KFERQ has a more robust degradation effect when it is at the N-terminus; after the Q in the KFERQ sequence is mutated to A, the resulting recombinant plasmid has basically no degradation effect.

[0044] The 293T cells transfected with the KL system in the above 6 groups were lysed using a cell lysis buffer containing a protease inhibitor (PMSF), and the total protein was extracted. The EGFP protein level was detected by Western blotting. The results are as follows: Figure 5 The results also show that KL-EGFP recombinant protein can more significantly degrade EGFP under certain conditions of blue light irradiation. At the same time, the degradation effect of KFERQ-cpLOV2-EGFP plasmid transfection is better than that of EGFP-eLOV2-KFERQ plasmid transfection, indicating that KL-EGFP is significantly degraded after blue light regulation.

[0045] Example 3: Blue light regulated KL-G targeted degradation of EGFP

[0046] Camelid species have unusual heavy chain immunoglobulin antibodies that lack light chains and contain a single antigen-binding variable domain (V H H). This V H The H domain is also called a nanobody, and nanobodies that specifically recognize green fluorescent protein (GFP) (GFP nanobody) have been widely used.

[0047] To further demonstrate the feasibility of this protein degradation system, the KL system was recombined with a nanobody that can bind to EGFP. Based on the grouping experience of Example 2, 293T cells overexpressing EGFP were directly divided into three groups and transfected with the KL-G or mKL-G plasmid obtained in Example 1: Group 1 was transfected with mKL-G plasmid, and Groups 2 and 3 were transfected with KL-G plasmid. Groups 1 and 3 were exposed to 470 nm wavelength and 4 mW / cm 2 The degradation mechanism of the two groups was as follows: Figure 6 After culturing for 24 h, the content and intensity of EGFP were detected by fluorescence microscopy, flow cytometry and Western blotting. The results were as follows: Figure 7-9The results showed that KL-G could significantly degrade intracellular EGFP under the regulation of specific blue light wavelength and energy, while mKL-G had little effect on intracellular EGFP degradation.

[0048] Example 4: Blue light regulates KL-A to target and degrade AKT

[0049] AKTin is a short peptide that can specifically bind to AKT, and 2xAKTin has been proven to have a higher AKT binding efficiency. After connecting the C-terminus of the KL recombinant sequence to the 2xAKTin sequence, a recombinant plasmid was obtained. The recombinant KL-2xAKTin or mKL-2xAKTin plasmid, packaging structure and vector structure were transiently co-transfected into 293T cells at a ratio of 2:1.5:1 to package the recombinant lentivirus. The medium was changed 6-18 hours after transfection, and the 293T cell culture medium was collected after culturing for another 24-48 hours. After centrifugation at 4°C and 1000g for 10 minutes, it was filtered through a 0.22μm filter membrane to infect U251 glioma cells. After 2-7 days of high-concentration puromycin screening (5μg / mL), it was replaced with a maintenance concentration (1μg / mL) of puromycin and continued to be cultured. After culturing for 24 hours under dark and blue light conditions, total protein was extracted, and changes in intracellular AKT1 and pAKT protein levels were detected by western blotting. The results are as follows Figure 10 As shown in the results, it can be seen that after the blue light treatment, both AKT and pAKT in the KL-A group were effectively targeted for degradation.

[0050] The U251 cells stably overexpressing KL-A were divided into two groups for CCK8 experiments to verify the changes in cell growth after AKT1 knockdown: 2000 cells per well, three replicates per group, were plated in a 96-well plate. After 24 hours, they adhered to the wall. CCK8 reagent was added at 0, 1, 2, 3, 4, and 5 days to detect cell viability and content. The results are shown in Figure 2. Figure 11 As shown in the results, cell proliferation experiments conducted on various tumor cells further confirmed that the protein degradation system can effectively inhibit cell proliferation activity after degrading endogenous AKT and pAKT.

[0051] In summary, the blue light-controlled protein degradation system of the present invention can significantly reduce the levels of overexpressed EGFP and endogenous AKT and pAKT in cells by recombining the KFERQ sequence, LOV2 sequence and endogenous protein targeting sequence, and by utilizing the light-sensitive regulatory properties of the LOV2 domain. In particular, KFERQ has a more robust degradation efficiency when it is at the N-terminus. This study provides a protein degradation system that can be controlled by blue light, which has a significant degradation effect on intracellular EGFP and endogenous cancer-promoting proteins, and can effectively inhibit the proliferation activity of cells, providing a basis for cancer treatment.

[0052] Finally, it should be emphasized that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A blue light-controlled protein degradation system based on KFERQ sequence, characterized in that: The blue light controlled protein degradation system includes a blue light control unit and a targeting unit; the blue light control unit is recombined from a KFERQ sequence and a LOV2 sequence; the targeting unit is a protein targeting sequence; The KFERQ sequence is encoded by the nucleotide sequence of Seq_3; The LOV2 sequence is a cpLOV2 sequence; the cpLOV2 sequence is encoded by the nucleotide sequence of Seq_2; The blue light control unit is a KFERQ-LOV2 (KL) recombinant sequence.

2. The blue light-controlled protein degradation system based on KFERQ sequence according to claim 1, characterized in that: The protein targeting sequence is GFP nanobody or AKTin or 2×AKTin; the nucleotide sequence of the 2×AKTin is shown in Seq_6.

3. The method for constructing a blue light-controlled protein degradation system based on KFERQ sequence according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) Obtaining the LOV2 sequence: The validated cpLOV2 was used as the LOV2 sequence; (2) Construction of recombinant plasmid: Using FV185 as a plasmid vector, the cpLOV2 sequence and the KEFRQ sequence were first recombined to obtain the KL recombinant sequence, and then the protein targeting sequence was inserted into the C-terminus to obtain the recombinant plasmid.

4. The method for constructing a blue light-controlled protein degradation system based on KFERQ sequence according to claim 3, characterized in that: The recombinant plasmid includes a KL-protein targeting sequence.

5. Use of the blue light-controlled protein degradation system according to any one of claims 1 to 2 in the preparation of products for degrading exogenously overexpressed EGFP or degrading endogenous cancer-promoting proteins.

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