A cluster luminescence-based method for monitoring the progression of lysozyme amyloid fibril formation

Abstract

The present invention discloses a method for monitoring the amyloid fibrillization process of lysozyme based on cluster luminescence. The method utilizes the property that lysozyme itself can emit fluorescence in an aggregated state, and the intensity of the fluorescence changes with the change of the aggregation state of lysozyme, thereby achieving the purpose of monitoring the amyloid fibrillization process of lysozyme. The method only needs to perform fluorescence detection on the fluorescence of the protein solution that can form amyloid fiber aggregates, without the introduction of exogenous fluorescent probes. The method is simple and has lower cost. The method can fully present the overall process of amyloid fibrillization and has the advantage of high sensitivity, especially high sensitivity to the early stages of amyloid fibrillization.

Description

Technical Field

[0001] The present invention relates to the technical field of luminescent materials, and in particular to a method for monitoring the amyloid fibrillation process of lysozyme based on cluster luminescence. Background Art

[0002] The native conformation of proteins is fundamental to their normal physiological functions. Under certain conditions, proteins can denature and misfold, shifting from their native conformation to a predominantly β-pleated structure. These β-pleated protein sheets aggregate and self-assemble to form insoluble protein fibrils. This process is called amyloid fibrilization, and proteins that undergo this denaturation are collectively referred to as amyloid proteins.

[0003] Studies have shown that amyloid fibril formation consists of three stages: nucleation, growth, and maturation. Generally speaking, during the nucleation stage, some peptide bonds of amyloid proteins break to form polypeptides, and this process is accompanied by the exposure of the hydrophobic domains of the protein. Under the hydrophilic-hydrophobic interaction, the hydrophobic regions approach each other and aggregate to form nuclei. Afterwards, the remaining polypeptides rely on hydrogen bond interactions to assemble onto the formed core, forming a sheet structure dominated by β-folding, and continue to extend. Finally, growth stops, and insoluble protein amyloid long fibers are obtained.

[0004] The entire process of amyloid fibrillation has obvious cytotoxicity. Amyloid fibrillation is closely related to many diseases that threaten human life and health, such as Alzheimer's disease, type 2 diabetes, and lysozyme amyloidosis.

[0005] Lysozyme amyloidosis is an autosomal dominant hereditary disease. Lysozyme amyloid fibrils in human cells become insoluble and deposit in the body, causing damage to the structure and function of organs and ultimately leading to multiple organ failure.

[0006] In recent years, clinical studies have found that oligomers formed in the early stages of amyloid fibril formation are far more cytotoxic than mature amyloid fibrils. Even after removing mature amyloid fibrils with drugs, the disease cannot be cured. Therefore, monitoring the amyloid fibril process, especially its early stages, helps to understand the molecular mechanisms of protein aggregation and the pathogenesis of related diseases, and thus plays a vital role in intervening and treating related diseases.

[0007] Egg white lysozyme, with its 129 amino acids and sequence similar to human lysozyme, is a typical model protein for studying amyloid fibrils. Typically, synthetic chemical fluorescent probes, such as thioflavin T (ThT), are used to detect amyloid fibrils.

[0008] In the past decade, people have discovered that many molecules, including proteins, especially natural macromolecules and polymers, do not emit light in isolation, but emit bright, visible fluorescence in aggregated states. This phenomenon is called clusteroluminescence (CL).

[0009] Cluster luminescence is an essential property of molecules. It shows that even without the addition of traditional fluorescent probes, protein molecules can emit fluorescence in their aggregated state. Moreover, as the aggregation state of the protein molecules changes, their fluorescence also changes accordingly. This provides a new approach for using fluorescence to monitor protein amyloid fibrils. Summary of the Invention

[0010] To address the shortcomings of current technologies, the present invention provides a novel and simple cluster luminescence-based method for monitoring the progression of lysozyme amyloid fibrilization. This cluster luminescence-based method innovatively utilizes the property of lysozyme itself to emit fluorescence when aggregated, and the intensity of this fluorescence changes with changes in the aggregation state of lysozyme, thereby achieving the invention's purpose of monitoring the progression of lysozyme amyloid fibrilization.

[0011] The cluster luminescence-based method for monitoring the progression of lysozyme amyloid fibrillation provided by the present invention requires only fluorescence detection of a protein solution capable of forming amyloid fibril aggregates, eliminating the need for exogenous fluorescent probes. This method is simple and cost-effective. Furthermore, the method can fully monitor the nucleation, growth, and maturation stages of amyloid fibrils, demonstrating a particularly high sensitivity in revealing the early stages of lysozyme amyloid fibril formation. Therefore, the method has promising application prospects in the study of amyloid fibril progression, particularly in the early stages of protein aggregation mechanisms and the pathogenesis of related diseases.

[0012] The technical solution adopted in the present invention is as follows:

[0013] A cluster luminescence-based method for monitoring the progress of lysozyme amyloid fibril formation, wherein the method monitors the progress of lysozyme amyloid fibril formation by detecting the autofluorescence intensity of lysozyme.

[0014] The cluster luminescence-based method for monitoring the progression of lysozyme amyloid fibrillation is based on the discovery that lysozyme exhibits cluster luminescence, whereby lysozyme molecules do not emit light in isolation but emit bright, visible fluorescence when aggregated. Therefore, the method utilizes the autofluorescence of lysozyme, whose intensity varies with the aggregation state of the lysozyme, to monitor the progression of lysozyme amyloid fibrillation by detecting the fluorescence intensity in different aggregation states.

[0015] The present invention provides a more specific method for monitoring the process of lysozyme amyloid fibrillation based on cluster luminescence, which comprises the following steps:

[0016] (1) Incubating lysozyme to promote its fibrosis and taking samples at different incubation times;

[0017] (2) centrifuging the sample obtained in step (1), taking the supernatant, and testing its fluorescence spectrum;

[0018] (3) Combined with the fluorescence spectrum obtained in step (2), the kinetic mechanism of lysozyme amyloid fibrillation process was explored.

[0019] Preferably, the lysozyme is hen egg white lysozyme. Hen egg white lysozyme has 129 amino acids, is similar in sequence to human lysozyme, is easy to obtain, and is a typical model protein for studying amyloid fibrillation.

[0020] Preferably, in step (1), the lysozyme is incubated starting from its native conformation. In the method, the lysozyme is incubated starting from its native conformation and its fibrillization is promoted under the incubation environment, so that the overall process of lysozyme amyloid fibrillization can be monitored.

[0021] In a preferred embodiment, the method for incubating lysozyme comprises:

[0022] Lysozyme is added to a buffer solution to obtain a buffer solution containing lysozyme, and the solution is incubated at an incubation temperature. In the buffer solution containing lysozyme, the concentration of lysozyme is 2-30 mg / mL, the concentration of glycine is 0-10 mg / mL, and the concentration of sodium azide is 0-0.2 mg / mL. The pH of the buffer solution containing lysozyme is adjusted to 2-3 using hydrochloric acid. The incubation temperature is 50-70°C. The incubation time is adjusted according to the stage of amyloid fibrillation to be monitored.

[0023] Preferably, in step (2), the wavelength of the excitation light when testing the fluorescence spectrum is 365 nm.

[0024] Preferably, in step (3), the method for exploring the kinetic mechanism of the lysozyme amyloid fibrillation process is specifically to combine the fluorescence spectrum obtained in step (2) to obtain a graph showing the change of fluorescence intensity at a fixed emission wavelength as a function of incubation time. Further preferably, the fixed emission wavelength is in the range of 440-460 nm.

[0025] The method provided by the present invention shows good reliability and stability in monitoring the amyloid fibrillation process of lysozyme, and has good application prospects. In a specific embodiment, the method provided by the present invention is used to monitor the amyloid fibrillation process of lysozyme: the results obtained by the method show that the amyloid fibrillation process of lysozyme goes through three stages: nucleation, growth and maturity, and fully presents the overall process of amyloid fibrillation; the method is compared with the method using the commercial probe ThT, and the monitoring results obtained by the two show the same change trend in the growth and maturity stages of amyloid fibrillation, and the detection sensitivity of the method of the present invention is higher; in the early aggregation stage of amyloid fibrillation, the sensitivity of the method provided by the present invention is much higher than that of the commercial probe ThT, which can prompt early protein denaturation and aggregation; the monitoring results of the method are highly consistent with the fibrosis morphology detected by transmission electron microscopy, further confirming that the object of detection of the present invention is amyloid fibrillation of protein.

[0026] Compared with the prior art, the main advantages of the present invention include at least:

[0027] 1) The method provided by the present invention only requires fluorescence detection of a protein solution capable of forming amyloid fibril aggregates, which is simple and cost-effective. In particular, the present invention does not require the introduction of exogenous fluorescent probes and only utilizes the cluster luminescence of the amyloid protein itself for monitoring, thus preventing interference with the amyloid fibrillization process of lysozyme.

[0028] 2) The method provided by the present invention demonstrates good reliability and stability in monitoring the amyloid fibrillization process of lysozyme. Furthermore, the method can explore the kinetic mechanism of lysozyme amyloid fibrillization, fully present the overall process of amyloid fibrillization, and possesses the advantage of high sensitivity.

[0029] 3) The method provided by the present invention is highly sensitive to the early stages of amyloid fibrillation, suggesting early protein denaturation and aggregation. It has good application prospects in the study of the process of amyloid fibrillation, especially in the study of the aggregation mechanism of protein molecules in the early stages of fibrillation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The UV-visible absorption spectra of egg white lysozyme solutions with different concentrations;

[0031] Figure 2 This is the photoluminescence spectrum of 20 mg / mL hen egg white lysozyme solution;

[0032] Figure 3 Electron micrographs of mature amyloid proteins measured at 8 hours, 2 days, and 7 days;

[0033] Figure 4 The fluorescence spectra of the samples at different incubation times measured in Example 2;

[0034] Figure 5 This is a kinetic diagram of the amyloid fibrillation process of lysozyme measured in Example 2;

[0035] Figure 6 The fluorescence spectra of the samples at different incubation times measured in Example 3;

[0036] Figure 7 This is a kinetic diagram of the early lysozyme amyloid fibrillation process measured in Example 3. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with the accompanying drawings and specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The operating methods in the following examples where no specific conditions are specified are generally performed under conventional conditions or as recommended by the manufacturer.

[0038] Example 1

[0039] In this example, lysozyme was incubated to promote its fibrillization, and samples of the overall process of lysozyme amyloid fibrillization were obtained by incubation. The UV-visible absorption spectra, photoluminescence spectra, and transmission electron microscopy images of the samples obtained at different incubation times were tested.

[0040] The method for incubating lysozyme in this example comprises adding lysozyme to a buffer solution comprising glycine, sodium azide, and deionized water to obtain a buffer solution containing lysozyme, adjusting the pH to 2-3 with hydrochloric acid, and then incubating at an incubation temperature. The lysozyme used in this example is hen egg white lysozyme.

[0041] The specific process includes the following:

[0042] 1.1 Buffer solution preparation:

[0043] Place 50 mL of deionized water in a 100 mL double-necked round-bottom flask, add 5 mg of sodium azide and 50 mg of glycine. Finally, add 1.0 g of egg white lysozyme and shake well to a concentration of 20 mg / mL. Adjust the pH to 2.2 with 1 M hydrochloric acid.

[0044] 1.2 Incubation and sampling:

[0045] The prepared solution was sealed and heated in an oil bath at 65°C for 7 days. 4 mL of the solution was taken out at the 8th hour. Thereafter, 4 mL of the solution was taken out every 24 hours and stored in a refrigerator at 4°C.

[0046] 1.3 Sample processing and testing:

[0047] After 7 days, all solutions were centrifuged at 12,000 r / min for 20 minutes, and the supernatant was collected. Photoluminescence spectra and transmission electron microscopy images were taken. The egg white lysozyme concentration in the supernatant was calibrated using UV-visible absorption spectroscopy to confirm that samples collected at different times had the same concentration.

[0048] Figure 1 UV-Vis absorption spectra of egg white lysozyme solutions at different concentrations are provided. Because protein gelation occurs during incubation, some protein is lost during centrifugation. Therefore, the UV-Vis absorption spectra of the supernatants removed at different times in the examples were tested, and the concentrations were brought to a consistent level by adding a small amount of buffer solution. When the concentrations were consistent, the UV-Vis absorption spectra of the solutions should overlap.

[0049] Figure 2 The photoluminescence spectrum of a 20 mg / mL hen egg white lysozyme solution is given, with an excitation wavelength of 365 nm and an emission wavelength of 440-460 nm.

[0050] Figure 3 Transmission electron micrographs of amyloid fibers measured at the 8th hour, the second day, and the seventh day (from top to bottom) during the amyloid fibrillation process are shown, with magnifications of 80,000, 80,000, and 20,000 times, respectively. It can be seen that in this embodiment, at the 8th hour, lysozyme was in a fragmented nucleation state; on the 2nd day, lysozyme formed short rod-shaped aggregates, initially showing fiber assembly; and on the 7th day, complete and ordered amyloid fibers were formed. This indicates that in this embodiment, a sample of the overall process of lysozyme amyloid fibrillation was obtained by incubating lysozyme for 7 days, that is, a fibrosis sample including three stages: nucleation phase, growth phase, and maturation phase.

[0051] Example 2

[0052] In this example, the amyloid protein fibrillization process was monitored on the samples of the overall lysozyme amyloid fibrillization process obtained in Example 1. Two monitoring methods were used, and the fluorescence spectra and fiber growth kinetics diagrams obtained by the two monitoring methods were compared.

[0053] The monitoring methods are: a) a method for monitoring the amyloid fibrillation process of lysozyme based on cluster luminescence provided by the present invention; b) a method for monitoring the amyloid fibrillation process of lysozyme using the commercial probe ThT.

[0054] Specifically include the following:

[0055] 2.1 Experimental Group:

[0056] The experimental groups included a test group and a control group. The test group used the cluster luminescence-based method provided by the present invention to monitor the progression of lysozyme amyloid fibrillation, where the measured fluorescence came only from the protein solution and was named CL. The control group used a commercial probe ThT to monitor the progression of lysozyme amyloid fibrillation, named ThT.

[0057] 2.2 Experimental samples:

[0058] The experimental sample is the sample of the overall process of lysozyme amyloid fibrillation obtained in Example 1.

[0059] 2.3 Experimental methods:

[0060] The fluorescence intensity of samples from the control and experimental groups was measured at different incubation times, and the fibrosis morphology of the samples was examined using transmission electron microscopy. The fluorescence spectrum of the experimental group was tested at an excitation wavelength of 365 nm. The control group had the commercially available probe ThT added to the experimental samples. The fluorescence was derived from ThT at a concentration of 40 μM. To avoid internal filtration effects caused by high protein concentrations, the experimental samples were diluted 25-fold with a buffered solvent, and the fluorescence spectrum was tested at an excitation wavelength of 440 nm.

[0061] 2.4 Experimental results and analysis:

[0062] The fluorescence spectra of the samples at different incubation times are shown in Figure 4 The kinetic diagram of the amyloid fibrillation process of lysozyme obtained by combining the obtained fluorescence spectrum is shown in Figure 5 .

[0063] Figure 4 The fluorescence spectra of the samples at different incubation times measured in Example 2 are given. A and B correspond to the fluorescence spectra of the control group and the test group, respectively. Figure 4 It can be seen that the maximum fluorescence emission wavelength of the ThT probe is at 488 nm, while the maximum fluorescence emission wavelength of CL is at 448 nm.

[0064] Figure 5 The kinetic diagram of the amyloid fibrillization process of lysozyme, obtained by combining the obtained fluorescence spectrum, is a plot of the change in fluorescence intensity at a fixed emission wavelength versus incubation time. The fluorescence intensity of the ThT curve is the fluorescence intensity at an emission wavelength of 488 nm, and the fluorescence intensity of the CL curve is the fluorescence intensity at an emission wavelength of 448 nm.

[0065] Kinetic diagram analyzing the process of lysozyme amyloid fibril formation:

[0066] from Figure 5As can be seen, throughout the amyloid fibril formation process, the fluorescence intensity of the commercial probe ThT remained unchanged from day 0 to day 1, then increased significantly on day 2, reaching a maximum on day 4, and then fluctuated steadily. The increased ThT fluorescence intensity indicates an increase in the protein's β-sheet conformation. Therefore, according to the commonly used definition method, days 0 to 1 of the incubation process can be defined as the nucleation phase, days 1 to 4 as the growth phase, and days 4 to 7 as the maturation phase.

[0067] The cluster luminescence (CL) fluorescence intensity obtained by the method provided by the present invention increases significantly between 0 and 1 day, continues to increase and reaches a maximum between 2 and 4 days, and then fluctuates steadily, indicating that fibrosis is complete. In addition, the increase in cluster luminescence intensity indicates that the protein has denatured and aggregated.

[0068] First, the experimental group and the control group showed the same upward trend in the growth and maturation stages of amyloid fibrils, indicating that the method of the present invention can indeed prove the formation of amyloid fibers without relying on external fluorescent probes, and the operation is simpler and the cost is lower. Secondly, it can be found that the detection sensitivity of the method of the present invention is higher, especially on the first day, the fluorescence intensity is increased to 2 times that of the 0th day, while ThT does not change, which preliminarily indicates that this monitoring method is more sensitive to the early aggregation of amyloid fibrils. Thirdly, the monitoring results of the method are highly consistent with the fibrosis morphology detected by transmission electron microscopy, further confirming that the object of monitoring of the present invention is protein amyloid fibrillation.

[0069] Example 3

[0070] This embodiment is for monitoring the early stage of amyloid fibrosis, which specifically includes the following process:

[0071] 3.1 Buffer solution preparation:

[0072] Place 50 mL of deionized water in a 100 mL double-necked round-bottom flask, adjust the pH to 2.2 with 1 M hydrochloric acid, add 5 mg of sodium azide and 50 mg of glycine. Finally, add 1.0 g of hen egg white lysozyme and shake well to a concentration of 20 mg / mL.

[0073] 3.2 Incubation and sampling:

[0074] The prepared solution was heated in an oil bath at 65°C for 24 hours, 4 mL of the solution was taken out at 2, 4, 6, 8, 10, 12 and 24 hours, and stored in a refrigerator at 4°C.

[0075] 3.3 Sample processing and testing:

[0076] The resulting solution was centrifuged at 12,000 r / min for 20 minutes, and the supernatant was collected and tested for photoluminescence spectroscopy. The egg white lysozyme concentration in the supernatant was calibrated using the UV-visible absorption spectrum provided in Example 1 to confirm that samples collected at different times had the same concentration.

[0077] 3.4 Monitoring the progression of early amyloid fibrillation

[0078] Two monitoring methods were used, and the fluorescence spectra and fiber growth kinetics obtained by the two monitoring methods were compared.

[0079] The monitoring methods are: a) a method for monitoring the amyloid fibrillation process of lysozyme based on cluster luminescence provided by the present invention; b) a method for monitoring the amyloid fibrillation process of lysozyme using the commercial probe ThT.

[0080] Specifically include the following:

[0081] 3.4.1 Experimental group:

[0082] The experimental groups included a test group and a control group. The test group used the cluster luminescence-based method provided by the present invention to monitor the progression of lysozyme amyloid fibrillation, where the measured fluorescence came only from the protein solution and was named CL. The control group used a commercial probe ThT to monitor the progression of lysozyme amyloid fibrillation, named ThT.

[0083] 3.4.2 Experimental samples:

[0084] The experimental samples were samples of the entire process of lysozyme amyloid fibrillation obtained in Example 3, that is, samples obtained at 2, 4, 6, 8, 10, 12 and 24 hours in 24 hours.

[0085] 3.4.3 Experimental methods:

[0086] The fluorescence intensity of samples from the control and experimental groups was measured at different incubation times, and the fibrosis morphology of the samples was examined using transmission electron microscopy. The fluorescence spectrum of the experimental group was tested at an excitation wavelength of 365 nm. The control group had the commercially available probe ThT added to the experimental samples. The fluorescence was derived from ThT at a concentration of 40 μM. To avoid internal filtration effects caused by high protein concentrations, the experimental samples were diluted 25-fold with a buffered solvent, and the fluorescence spectrum was tested at an excitation wavelength of 440 nm.

[0087] 3.4.4 Experimental results and analysis:

[0088] The fluorescence spectra of the samples at different incubation times are shown in Figure 6 The kinetic diagram of the early lysozyme amyloid fibrillation process obtained by combining the obtained fluorescence spectrum is shown in Figure 7.

[0089] Figure 6 A and B correspond to the fluorescence spectra of the control group and the experimental group in Example 2. It can be seen that the maximum fluorescence emission wavelength of the ThT probe is at 488 nm, while the maximum emission wavelength of CL is at 448 nm.

[0090] Figure 7 The kinetic diagram of the amyloid fibrillization process of lysozyme, obtained by combining the obtained fluorescence spectrum, is a plot of the change in fluorescence intensity at a fixed emission wavelength versus incubation time. The fluorescence intensity of the ThT curve is the fluorescence intensity at an emission wavelength of 488 nm, and the fluorescence intensity of the CL curve is the fluorescence intensity at an emission wavelength of 448 nm.

[0091] Kinetic diagram analyzing the early stages of lysozyme amyloid fibril formation:

[0092] Depend on Figure 7 It can be seen that in the early stages of amyloid fibrillation, especially within 12 hours, the fluorescence intensity of the commercial probe ThT hardly changes, while the intensity of CL described in the present invention does not change significantly within 0-4 hours, but begins to gradually increase after 6 hours. Moreover, at the 24th hour, the fluorescence intensity of ThT is only 1.3 times the initial level, while the intensity of CL is 2 times the initial level. The increase in fluorescence intensity indicates that the protein has denatured and aggregated. Obviously, the method described in the present invention is much more sensitive to early changes in amyloid fibrillation than the commercial ThT probe, can indicate the early aggregation of lysozyme, and has broad application prospects.

[0093] In addition, it should be understood that after reading the above description of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for monitoring the process of lysozyme amyloid fibrillation based on cluster luminescence, characterized in that: The method monitors the lysozyme amyloid fibrillation process by detecting the autofluorescence intensity of lysozyme at an emission wavelength range of 440-460 nm.

2. The method for monitoring the process of lysozyme amyloid fibrillation based on cluster luminescence according to claim 1, characterized in that: The method comprises the following steps: (1) Incubate lysozyme to promote its fibrillation and take samples at different incubation times; (2) Centrifuging the sample obtained in step (1), taking the supernatant, and testing its fluorescence spectrum; (3) Combined with the fluorescence spectrum obtained in step (2), the kinetic mechanism of lysozyme amyloid fibrillation process was explored.

3. The method for monitoring the process of lysozyme amyloid fibrillation based on cluster luminescence according to claim 1 or 2, characterized in that: The lysozyme is hen egg white lysozyme.

4. The method for monitoring the process of lysozyme amyloid fibrillation based on cluster luminescence according to claim 2, characterized in that: In step (1), the lysozyme is incubated starting from its native conformation.

5. The method for monitoring the process of lysozyme amyloid fibrillation based on cluster luminescence according to claim 2, characterized in that: In step (1), the method of incubating lysozyme comprises: adding lysozyme to the buffer solution to obtain a buffer solution containing lysozyme, and incubating at an incubation temperature; In the buffer solution containing lysozyme, the concentration of lysozyme is 2-30 mg / mL, the concentration of glycine is 0-10 mg / mL, and the concentration of sodium azide is 0-0.2 mg / mL; The pH of the buffer solution containing lysozyme is adjusted to 2-3 with hydrochloric acid; The incubation temperature is 50-70°C.

6. The method for monitoring the process of lysozyme amyloid fibrillation based on cluster luminescence according to claim 2, characterized in that: In step (2), the wavelength of the excitation light when testing the fluorescence spectrum is 365 nm.

7. The method for monitoring the process of lysozyme amyloid fibrillation based on cluster luminescence according to claim 6, characterized in that: In step (3), the method for exploring the kinetic mechanism of the lysozyme amyloid fibrillation process is specifically to combine the fluorescence spectrum obtained in step (2) to obtain a graph of the change of fluorescence intensity at a fixed emission wavelength with incubation time.

8. The method for monitoring the process of lysozyme amyloid fibrillation based on cluster luminescence according to claim 7, characterized in that: The fixed emission wavelength ranges from 440 to 460 nm.

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