Polypeptide Fluorescent Probe and Its Application in Detecting Aggregated Proteins in Living Cells

Abstract

The present invention provides a polypeptide fluorescent probe and its application in detecting aggregated proteins in living cells. The structural formula of the fluorescent probe consists of five components: a QB fluorescent small molecule, a cell-penetrating peptide (RRRRRR), a mitochondrial toxic peptide (KLAKLAKKLAKLAK), a CB enzyme cleavage site (GFLG), and an azido peptide. The polypeptide fluorescent probe prepared in this invention can be used to detect aggregated proteins in living cells, resolving the problem that traditional fluorescent probes can only detect aggregated proteins in solution and avoiding the experimental step of fixed cells.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular probe applications, and in particular to a polypeptide fluorescent probe and its application in detecting aggregated proteins in living cells. Background Art

[0002] The structure of a protein is crucial to its function. Abnormal aggregation behavior of proteins may lead to many neurodegenerative diseases, such as Alzheimer's disease, ALS, Parkinson's disease, Huntington's disease, etc.

[0003] The protein aggregation process is very complex, including unfolding, misfolding, the formation of soluble aggregated protein bodies, insoluble aggregated protein bodies, etc. At present, commercial aggregated protein targeting probes (such as amyloid probes such as ThT and Congo red) can usually only identify β-folded highly enriched late amyloid aggregated proteins outside the cell. The commercial intracellular PROTEOSTATR kit cannot penetrate the cell membrane and can only detect aggregated proteins in fixed dead cells. Scientists rarely achieve the detection and analysis of aggregated proteins in living cells. Therefore, if the aggregation behavior of proteins can be detected in living cells, it will help to deepen the understanding of the pathogenesis of the disease.

[0004] Due to the lack of effective solutions for detecting protein aggregation in living cells, people have an incomplete understanding of the occurrence, development and mechanism of protein aggregation behavior in living cells. Therefore, it is of great significance to develop a polypeptide fluorescent probe that can detect the behavior of aggregated proteins in living cells. Summary of the Invention

[0005] Based on this, it is necessary to provide a polypeptide fluorescent probe and its application in detecting aggregated proteins in living cells.

[0006] The present invention adopts the following technical solutions:

[0007] The present invention provides a polypeptide fluorescent probe that can be used to detect aggregated proteins in living cells. Its structure is mainly composed of QB organic fluorescent small molecules, membrane-penetrating peptides, mitochondrial toxic peptides, CB enzyme cleavage site GFLG and azido peptides, and the azido peptide is the mitochondrial complex IV inhibitory part. The specific chemical structure is as follows: Figure 1 shown.

[0008] A method for preparing a polypeptide fluorescent probe that can be used to detect aggregated proteins in living cells comprises the following steps:

[0009] S1, respectively preparing an aqueous solution containing pure polypeptide RKN and a solution containing QB organic fluorescent small molecules, mixing them, adding a catalyst, and stirring the reaction for not less than 12 hours to obtain a crude product of the polypeptide fluorescent probe.

[0010] S2, purifying the crude product of the polypeptide fluorescent probe to obtain the polypeptide fluorescent probe.

[0011] In some embodiments, the reaction molar ratio of the polypeptide RKN and the QB organic fluorescent small molecule is 1:5.

[0012] In some embodiments, the catalyst is selected from a mixture of PyBOP, PPTS, and NMM.

[0013] In some embodiments, the crude product was purified by high performance liquid chromatography, and the process conditions were as follows: a Welchrom C18 chromatographic column was used, and a mobile phase A and a mobile phase B were used for gradient elution at a flow rate of 1 mL / min, wherein the mobile phase A was an aqueous solution containing 0.1% TFA, and the mobile phase B was an acetonitrile solution containing 0.1% TFA.

[0014] The present invention also provides application of the above polypeptide fluorescent probe in detecting aggregated proteins in living cells.

[0015] Specifically, the above-mentioned polypeptide fluorescent probe can be used for mitochondrial imaging in living cells under a pH 6.5-7.4 environment.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The structure of the peptide fluorescent probe consists of five parts: QB organic fluorescent small molecule, used for image tracing and response to aggregated proteins; membrane-penetrating peptide (RRRRRR), which helps the probe enter the cell membrane; mitochondrial toxic peptide (KLAKLAKKLAKLAK) that can form an a-helix to destroy the mitochondrial membrane and cause mitochondrial damage; CB enzyme cleavage site (GFLG) that cuts the peptide into two parts to exert its effect; azido peptide is a mitochondrial complex IV inhibitor, thereby inhibiting the mitochondrial oxidative respiratory chain. After being cleaved by enzymes, the two peptides act on the mitochondria separately to cause mitochondrial damage. The use of this peptide fluorescent probe can realize the detection of aggregated proteins in the mitochondria of living cells, solving the problem that traditional fluorescent probes can only detect aggregated proteins in solution and avoiding the process of fixing cells.

[0018] (2) The method for preparing the fluorescent probe for aggregated proteins in mitochondria of living cells of the present invention has simple process and low cost.

[0019] (3) The polypeptide fluorescent probe provided by the present invention exhibits obvious molecular rotor luminescence phenomenon, avoids the interference of background fluorescence, and realizes the detection of aggregated proteins in living cells, which has great application prospects in the field of biomedical imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1The chemical structure of a peptide fluorescent probe used to detect aggregated proteins in mitochondria of living cells.

[0021] Figure 2 for Figure 1 Synthesis route of peptide fluorescent probes.

[0022] Figure 3 for Figure 1 Mass spectrometry characterization of the peptide fluorescent probe.

[0023] Figure 4 for Figure 1 Experimental diagram of using peptide fluorescent probe to detect changes in fluorescence intensity in aggregated protein solution.

[0024] Figure 5 for Figure 1 Confocal micrograph of MG132-induced breast cancer MCF-7 cells stained with the peptide fluorescent probe.

[0025] Figure 6 for Figure 1 Confocal micrograph of staurosporine-induced breast cancer MCF-7 cells stained with the peptide fluorescent probe. DETAILED DESCRIPTION

[0026] The present invention will be further described in detail below with reference to specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0027] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0028] Example 1

[0029] This embodiment provides a polypeptide fluorescent probe for detecting aggregated proteins in mitochondria of living cells, and its chemical structure is as follows: Figure 1 shown.

[0030] The structure of the peptide fluorescent probe consists of the following five parts:

[0031] 1) QB organic fluorescent small molecules for image tracking and response to aggregated proteins;

[0032] 2) cell-penetrating peptide (RRRRRR), which helps to introduce the probe into the cell membrane;

[0033] 3) Mitochondrial toxic peptide (KLAKLAKKLAKLAK), which can form a helix to destroy the mitochondrial membrane and cause mitochondrial damage;

[0034] 4) CB cleavage site (GFLG), which cuts the polypeptide into two parts;

[0035] 5) Azido peptide is a mitochondrial complex IV inhibitor, thereby inhibiting the mitochondrial oxidative respiratory chain. After being cleaved by enzymes, the two peptides act on the mitochondria separately, causing mitochondrial damage.

[0036] This polypeptide fluorescent probe can detect aggregated proteins in living cells, solving the problem that traditional probes can only detect the behavior of aggregated proteins in solution, and can avoid the process of fixing cells.

[0037] This example further describes the preparation method of the polypeptide fluorescent probe. The synthetic route is shown in Figure 2 , including the following steps:

[0038] S1, pure polypeptide RKN (commissioned to Gill Biochemical Company for synthesis) was dissolved in water to prepare a polypeptide RKN aqueous solution.

[0039] The QB organic fluorescent small molecule (synthesized according to the method in the paper Hashoul, D, Yavin, et al. Single point mutation detection in living cancer cells by far-red emitting PNA-FIT probes[J]. Chemical communications, 2016) was dissolved in DMF to prepare a QB solution.

[0040] The polypeptide RKN aqueous solution and the QB solution were mixed, a catalyst was added, and an amide reaction was carried out. The stirring time during the reaction was not less than 12 hours to obtain a crude product of the polypeptide fluorescent probe.

[0041] S2. The crude product prepared in step S1 is purified by high-performance liquid chromatography to obtain a peptide fluorescent probe. The high-performance liquid chromatography column used in this section is a Welchrom C18 column (5 μm, 250×4.6 mm). Gradient elution is performed at a flow rate of 1 mL / min using mobile phases A and B. Mobile phase A is an aqueous solution containing 0.1% TFA, and mobile phase B is an acetonitrile solution containing 0.1% TFA. The elution procedure is as follows:

[0042] Time (min) The proportion of mobile phase B 0 20％ 10 40％ 20 90％ 30 100％

[0043] Specifically, in this embodiment, RKN (348 mg, 0.12 mmol), QB (200 mg, 0.58 mmol), PyBOP (benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, 75 mg, 0.144 μmol), PPTS (pyridine p-toluenesulfonate, 30 mg, 0.12 μmol), NMM (N-methylmorpholine, 48 mg, 0.48 μmol), N, N-dimethylformamide (DMF) 2 mL, and PBS buffer 2 mL were added to a 100 mL round-bottom flask. Under nitrogen atmosphere, the reaction was stirred at room temperature for 24 h to obtain a crude product, which was purified by high performance liquid chromatography and freeze-dried to obtain 20 mg of the product polypeptide fluorescent probe (QRKN) with a yield of 5.18%.

[0044] The prepared peptide fluorescent probe (QRKN) was characterized by high-resolution mass spectrometry, and the structure of the probe was confirmed. Figure 3 As shown:

[0045] HRMS(ESI)m / z:[M+3H] 3+ , found: 1104.69; [M+4H] 4+ , found: 828.77; [M+5H] 5+ , found: 663.22; [M+6H] 6+ , found: 552.85. [M+7H] 7+ , measured value: 474.02.

[0046] Example 2

[0047] This example provides a fluorescence study using a peptide fluorescent probe (QRKN) to detect the aggregation of BSA model proteins induced under different conditions. The specific steps are as follows:

[0048] QRKN was dissolved in DMSO to prepare a peptide fluorescent probe solution containing 1 mM QRKN (referred to as "QRKN mother solution").

[0049] BSA was dissolved in water to prepare a model protein solution containing 1 M BSA (referred to as "BSA mother solution").

[0050] Ten microliters of QRKN stock solution and 50 microliters of BSA stock solution were taken, and 940 microliters of water were added to prepare a mixed solution with a final volume of 1 mL (containing 5 μM QRKN and 50 mM BSA protein). The effect of increasing temperature (298K, 303K, 308K, 313K, 318K, 323K, 328K, 333K, 338K, 343K) on the fluorescence intensity during the detection of aggregated proteins was investigated.

[0051] The fluorescence intensity of QRKN at different temperatures was tested. Figure 4 As shown in Figure A, the temperature between 300K and 350K induces protein aggregation. As the temperature increases, the fluorescence increases, indicating that the degree of protein aggregation increases.

[0052] This example also tests the effect of the mixed solution on the fluorescence intensity during the detection of aggregated proteins within the time period of 0-1800 s.

[0053] The fluorescence intensity of QRKN was tested at different times, and the statistical results are as follows: Figure 4 As shown in Figure B, the test results of protein aggregation induced from 0s to 1800s show that as time goes on, the detected fluorescence increases, indicating that the degree of protein aggregation increases.

[0054] Example 3

[0055] This embodiment provides a method for detecting aggregated proteins in living cells using a polypeptide fluorescent probe (QRKN), comprising the following steps:

[0056] Breast cancer MCF-7 cells (commercially available) were cultured in a DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0057] Then breast cancer MCF-7 cells were cultured at 1×10 5 Density passaged into glass-bottomed cell culture dishes containing Dulbecco's Modified Eagle Medium (DMEM). After 24 hours of culture, the cells were washed twice with PBS buffer (pH = 7.4). 0.5 μg / mL MG132 drug was then added to the glass-bottomed cell culture dish to induce intracellular protein aggregation by interfering with the protein degradation pathway. The cells were incubated in a 37°C, 5% carbon dioxide incubator for 1 hour. A 5 μM aqueous solution of the peptide fluorescent probe was then added and incubated in the incubator for 4 hours. After the culture was completed, the culture dish was removed, the supernatant was removed, and the cells were gently washed twice with PBS buffer and immersed in DMEM culture medium before optical imaging. The culture dish was then subjected to fluorescence confocal imaging.

[0058] The cell fluorescence imaging instrument used was a Zeiss LSM 880 confocal microscope with an excitation wavelength of 543 nm. The imaging test results are shown in the figure. Figure 5 As shown in FIG1 , the polypeptide fluorescent probe prepared in Example 1 indeed has an excellent intracellular aggregated protein imaging effect.

[0059] Example 4

[0060] This example provides a method for detecting aggregated proteins in living cells. The experimental method and steps are basically the same as those in Example 3, with the only difference being that 10 μM staurosporine is used instead of 0.5 μg / mL MG132.

[0061] The cell fluorescence imaging instrument used was a Zeiss LSM 880 confocal microscope with an excitation wavelength of 543 nm. The imaging test results are shown in the figure. Figure 6 As shown: It can be seen that the polypeptide fluorescent probe prepared in Example 1 does have an excellent intracellular aggregated protein imaging effect.

[0062] It can be seen from this that the polypeptide fluorescent probe of the present invention is applied to imaging of intracellular aggregated proteins in breast cancer cells. The fluorescent probe exhibits excellent performance in imaging aggregated proteins in cells, and can detect the occurrence of aggregated protein behavior in living pathological cells induced by MG132 and staurosporine.

[0063] It is important to note that the above embodiments are intended only to further illustrate and describe the technical solutions of the present invention and are not intended to further limit the technical solutions of the present invention. The methods of the present invention are merely preferred implementations and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A polypeptide fluorescent probe having the structural formula:

2. The method for preparing the polypeptide fluorescent probe according to claim 1, characterized in that: The steps include: S1, separately preparing an aqueous solution containing the polypeptide RKN and a solution containing the QB organic fluorescent small molecule, mixing the aqueous solution containing the polypeptide RKN and the solution containing the QB organic fluorescent small molecule, adding a catalyst, and stirring the reaction for at least 12 hours to obtain a crude product of the polypeptide fluorescent probe; S2, purifying the crude product of the polypeptide fluorescent probe to obtain the polypeptide fluorescent probe.

3. The method for preparing a polypeptide fluorescent probe according to claim 2, characterized in that: The reaction molar ratio of the polypeptide RKN and the QB organic fluorescent small molecule is (1-1.5): (5-6).

4. The method for preparing a polypeptide fluorescent probe according to claim 2, wherein: The catalyst is selected from a mixture of PyBOP, PPTS and NMM.

5. The method for preparing a polypeptide fluorescent probe according to any one of claims 2 to 4, characterized in that: The crude product was purified by high performance liquid chromatography.

6. The method for preparing a polypeptide fluorescent probe according to claim 5, characterized in that: The process conditions for purifying the crude product by high performance liquid chromatography are as follows: a Welchrom C18 chromatographic column is used, and a mobile phase A and a mobile phase B are used for gradient elution at a flow rate of 1 mL / min, wherein the mobile phase A is an aqueous solution containing 0.1% TFA, and the mobile phase B is an acetonitrile solution containing 0.1% TFA.

7. Use of the polypeptide fluorescent probe according to claim 1 in the preparation of a product for detecting aggregated proteins in living cells.

8. The use according to claim 7, characterized in that The polypeptide fluorescent probe can be applied to fluorescent imaging of aggregated proteins in living cells under a pH range of 6.5 to 7.

4.

9. The use according to claim 7, characterized in that The living cells are cancer cells.

10. The use according to claim 9, characterized in that The living cells are breast cancer cells.

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