Method and system for detecting function variation in protein phase separation, and use thereof

Abstract

The present invention belongs to the technical field of optical analysis and detection. Provided are a method and system for detecting a function variation in protein phase separation, and the use thereof. The method comprises transferring a first expression vector fused with a fluorescent molecule and a target protein and a second expression vector fused with a fluorescent molecule and paCaMKII into passage cells and exciting the cells, collecting fluorescence intensity values inside and outside phase-separated condensates of a wild-type protein and a target protein and fluorescence intensity values before and after paCaMKII recruitment, and comparing the fluorescence intensity ratios inside and outside the phase-separated condensates of the target protein and the wild-type protein to detect a phase separation variation of the target protein; and taking the percentage of a ratio of fluorescence intensity change values of the target protein and the wild-type protein before and after the paCaMKII recruitment as the efficiency of the target protein for paCaMKII recruitment, and using the spatio-temporal controllability of paCaMKII to detect a functional variation in phase separation of the target protein caused by a disease-associated mutation.

Description

A method, system, and application for detecting variations in protein phase separation function. Technical Field

[0001] This invention belongs to the field of optical analysis and detection technology, specifically relating to a method, system, and application for detecting variations in protein phase separation function. Background Technology

[0002] Protein phase separation refers to the phenomenon where intracellular proteins, acting as solid particles, exhibit droplet-like properties (fluidity, fusion, and dispersion). These proteins can fuse and aggregate to form droplet-like structures that perform specific biological functions. Although lacking a cell membrane, they aggregate to form independent spaces similar to organelles, representing a physical phenomenon based on chemical structure. Protein phase separation variations caused by mutations are closely related to the development of various diseases, including developmental disorders, neurodegenerative diseases, cancer, and some infectious diseases. Detecting protein phase separation variations using bioinformatics methods is of great significance for the study of various life activities. Traditional methods for studying protein phase separation include in vitro droplet formation experiments, gel formation experiments, electron microscopy of fibrils, nuclear magnetic resonance, fibril derivatization, in vitro phase transition measurements, and fluorescence retrieval after photobleaching (FRAP). However, these methods have the following limitations: 1) they are all conducted in vitro, and it is uncertain whether they can actually occur in cells; 2) they cannot detect functional variations in protein phase separation. Therefore, there is an urgent need for methods that can detect functional variations in protein phase separation caused by mutations within living cells, thereby effectively revealing the pathogenesis of related diseases and discovering new treatment strategies.

[0003] International patent application WO 2023 / 108391 A1 discloses a method for detecting protein liquid-phase separation, which enables sub-second timescale detection of protein liquid-phase separation within living neuronal synapses. While this method can detect protein liquid-phase separation processes within living cells, it cannot detect variations in protein liquid-phase separation function.

[0004] Calmodulin-dependent protein kinase II (CaMKII) is a class of protein kinases widely distributed in organisms, whose activity is regulated by calcium ions (Ca²⁺) and calmodulin (CaM). These kinases play crucial roles in cell signal transduction, metabolic regulation, gene expression, and cell growth, differentiation, and apoptosis. For example, when neurons are subjected to high-frequency stimuli, CaMKII is rapidly recruited by influencing the phase separation of synaptic proteins. This spatiotemporal recruitment of CaMKII prompts a rapid initiation of numerous downstream substrate molecules, further inducing long-term synaptic plasticity. Mutations in synaptic proteins during this process lead to phase separation variations, which, depending on their severity, can affect brain nerve cell function to varying degrees. A close relationship exists between alterations in the brain's spatiotemporal recruitment capacity of CaMKII and the phase separation functional variations caused by mutated protein phase separation mutations. The probe Camui, developed using fluorescence resonance transfer (FRLT), utilizes FRLT probes attached before and after CaMKII to infer the initiation state of CaMKII based on FRLT efficiency. CaMKII undergoes structural changes during calcium-calmodulin binding; in the inactive state, the N-terminus and C-terminus of CaMKII are closed; during initiation, the CaMKII structure is open (Takao and Hayashi et al. 2005). However, this method requires external calcium stimulation and calmodulin to regulate CaMKII in living cells. Furthermore, there are no reports of detecting protein phase separation functional variations using CaMKII. Technical issues

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide a method, system and application for detecting protein phase separation functional variations, and to solve the problem that existing methods cannot detect protein phase separation functional variations in living cells. Technical solutions

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] The first aspect of the present invention discloses a method for detecting variations in protein phase separation function, comprising the following steps:

[0008] 1) Transform the first expression vector and the second expression vector into passaged cells and culture them to stimulate the passaged cells, causing the target protein to undergo phase separation;

[0009] The first expression vector is fused with a fluorescent molecule and a target protein, and the second expression vector is fused with a fluorescent molecule and paCaMKII.

[0010] 2) Collect fluorescence intensity values ​​of wild-type protein phase-separated condensates and target protein phase-separated condensates before and after paCaMKII recruitment;

[0011] Alternatively, the fluorescence intensity values ​​inside and outside the wild-type protein phase-separated condensate and the target protein phase-separated condensate, as well as the fluorescence intensity values ​​before and after recruiting paCaMKII in the wild-type protein phase-separated condensate and the target protein phase-separated condensate, were collected respectively.

[0012] 3) The percentage of the change in fluorescence intensity before and after the target protein recruits paCaMKII compared with the change in fluorescence intensity before and after the wild-type protein recruits paCaMKII is used as the efficiency of the target protein recruiting paCaMKII, and the variation in the phase separation function of the target protein is detected.

[0013] Alternatively, the phase separation variation of the target protein can be detected by comparing the ratio of fluorescence intensity of the target protein phase separation aggregates in vivo and in vitro with that of the wild-type protein phase separation aggregates in vivo and in vitro; the percentage of the change in fluorescence intensity of the target protein before and after recruiting paCaMKII to that of the wild-type protein before and after recruiting paCaMKII can be used as the efficiency of the target protein recruiting paCaMKII to detect the phase separation functional variation of the target protein; the phase separation variation results of the target protein can be correlated with the efficiency of the target protein recruiting paCaMKII to detect the phase separation functional variation of the target protein.

[0014] Preferably, the target protein is a protein that has phase separation characteristics and can interact with calmodulin-dependent phosphokinase.

[0015] Preferably, the first expression vector and the second expression vector carry different fluorescent molecules, and the fluorescent molecules are all proteins that are not in the 488 excitation band.

[0016] More preferably, the first expression vector is linked to a far-infrared fluorescent protein, and the second expression vector is linked to a red fluorescent protein.

[0017] Preferably, the passaged cells are HEK293T cells or COS7 cells.

[0018] Preferably, the first expression vector and the second expression vector are transferred into passaged cells and cultured for 24-48 h.

[0019] In a second aspect, the present invention discloses the application of the above-described method in the process of detecting the recruitment of calmodulin-dependent phosphokinases by phase separation of mutant proteins under neural activity.

[0020] A third aspect of the present invention discloses the application of the above-described method in screening drugs that can reverse the efficiency of mutated proteins in recruiting calmodulin-dependent phosphokinases.

[0021] A fourth aspect of the present invention discloses a system based on the above method, comprising:

[0022] The phase separation module is used to transfer a first expression vector fused with fluorescent molecules and target proteins, and a second expression vector fused with fluorescent molecules and paCaMKII, into passaged cells. By stimulating the passaged cells, the target protein undergoes phase separation.

[0023] The acquisition module is used to acquire fluorescence intensity values ​​of wild-type protein phase-separated condensates and target protein phase-separated condensates before and after paCaMKII recruitment; or, it is used to acquire fluorescence intensity values ​​inside and outside wild-type protein phase-separated condensates and target protein phase-separated condensates, as well as fluorescence intensity values ​​of wild-type protein phase-separated condensates and target protein phase-separated condensates before and after paCaMKII recruitment.

[0024] The calculation module is used to detect phase separation functional variations of the target protein by using the percentage of the change in fluorescence intensity before and after paCaMKII recruitment by the target protein to the percentage of the change in fluorescence intensity before and after paCaMKII recruitment by the wild-type protein as the efficiency of paCaMKII recruitment by the target protein; or, it is used to detect phase separation variations of the target protein by comparing the ratio of fluorescence intensity before and after paCaMKII recruitment by the target protein to the ratio of fluorescence intensity before and after paCaMKII recruitment by the wild-type protein; it uses the percentage of the change in fluorescence intensity before and after paCaMKII recruitment by the target protein to the percentage of the change in fluorescence intensity before and after paCaMKII recruitment by the wild-type protein as the efficiency of paCaMKII recruitment by the target protein; and it correlates the phase separation variation results of the target protein with the efficiency of paCaMKII recruitment by the target protein to detect phase separation functional variations of the target protein.

[0025] A fifth aspect of the present invention discloses a method for constructing a phenotypic database for detecting disease mutations related to target proteins. According to the above method, the relationship between each disease-related target protein mutation and the phase separation functional variation of the target protein is obtained, and a phenotypic database for detecting disease mutations related to target proteins is established.

[0026] A sixth aspect of the present invention discloses a phenotypic database obtained by the above-described construction method. Beneficial effects

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] This invention provides a method for detecting functional variations in protein phase separation. A photoactivated calmodulin-dependent phosphokinase (paCaMKII) fused with a fluorescent molecule is selected as a second expression vector. Photostimulation activates CaMKII, eliminating the need for additional calcium stimulation and calmodulin activation. A first expression vector is selected, fused with the fluorescent molecule and the target protein. Utilizing the spatiotemporal controllability of paCaMKII, the target protein phases and spatiotemporally recruits paCaMKII upon photostimulation. The first and second expression vectors are transfected into passaged cells for photoactivation, enabling detection within live cells. The efficiency of wild-type protein recruiting paCaMKII is used as a control to calculate the efficiency of the target protein recruiting paCaMKII. Based on changes in the target protein's paCaMKII recruitment efficiency, functional variations caused by phase separation variations in the target protein are detected, thus enabling the detection of functional variations caused by phase separation variations within live cells. Furthermore, the alteration of the condensation capacity of phase-separating molecules will be assessed by calculating the fluorescence partition coefficient of the target protein (the ratio of fluorescence intensity inside and outside the phase-separating condensate), thus detecting protein phase-separation variations. By correlating protein phase-separation variations with the efficiency of the target protein in recruiting paCaMKII, the functional variations caused by the target protein's phase-separation variations can be detected more precisely. This method links the spatiotemporal recruitment capacity of paCaMKII with protein functional variations, enabling the quantification of functional changes caused by disease-related mutant protein phase-separation variations. It can be used to establish a quantifiable phenotypic database of disease-related mutations of target proteins, comparing phenotypes caused by different mutations. It can also be applied to rapidly screen for disease-related mutations that alter protein phase-separation properties and to screen for drugs that can reverse the ability to recruit paCaMKII, thereby improving functional abnormalities caused by mutant protein phase-separation variations.

[0029] Furthermore, tandemly attaching fluorescent molecules of different colors to the target protein and paCaMKII respectively can improve the contrast of quantification recruitment efficiency and enhance the accuracy of detecting protein phase separation. The selected fluorescent molecules are proteins with excitation bands other than 488 nm, which prevents the fluorescent molecules from activating paCaMKII while simultaneously imaging the protein. Attached Figure Description

[0030] Figure 1 is a schematic flowchart of the method for detecting protein phase separation functional variations of the present invention; wherein, A is the target protein of phase separation and its mutant first expression vector, B is the process of transferring the first expression vector and the second expression vector into passaged cells to excite paCaMKII, C is the acquisition of microscopic imaging data using a fluorescence microscope, and D is the detection of the change in paCaMKII recruitment efficiency caused by the mutation.

[0031] Figure 2 is a flowchart of detecting phase separation function variations of a target protein by utilizing the efficiency of recruiting paCaMKII by the target protein.

[0032] Figure 3 shows the phase separation variation results associated with the target protein and the efficiency of the target protein in recruiting paCaMKII, and is a flowchart for detecting phase separation functional variations of the target protein.

[0033] Figure 4 shows the plasmid map of the first expression vector fused with fluorescent molecules and SHANK3 FL;

[0034] Figure 5 shows the plasmid map of the second expression vector fused with fluorescent molecules and paCaMKII;

[0035] Figure 6 shows the results of detecting the phase separation function variation of SHANK3-Ex21-InsG3680 using the method of detecting protein phase separation function variation; where A is the fluorescence result of the first and second expression vectors before blue light excitation, B is the fluorescence result of the first and second expression vectors after blue light excitation, C is the result of paCaMKII being recruited into the SHANK3-Ex21-InsG3680 phase separation condensate, D is the partition coefficient of SHANK3 FL and SHANK3-Ex21-InsG3680 phase separation condensate, E is the efficiency of SHANK3 FL and SHANK3-Ex21-InsG3680 phase separation condensate in recruiting paCaMKII, and F is the linear relationship between the partition coefficient and the paCaMKII recruitment efficiency. Embodiments of the present invention

[0036] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0037] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0038] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0039] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0040] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0041] This invention provides a method for detecting variations in protein phase separation function, as shown in Figures 1 and 2, comprising the following steps:

[0042] I. Construction

[0043] 1. Construct a first expression vector that integrates a fluorescent molecule and a target protein;

[0044] The target protein is a protein with phase separation characteristics and capable of interacting with CaMKII. Preferably, the target protein is SynGAP protein, SAPAP protein, or SHANK3 protein. The fluorescent molecule is a far-infrared fluorescent protein.

[0045] 2. Construct a second expression vector incorporating a fluorescent molecule and a photoactivatable calmodulin-dependent phosphokinase (paCaMKII);

[0046] The first expression vector and the second expression vector carry different fluorescent molecules, and the fluorescent molecules are all proteins that are not excited in the 488 excitation band; the fluorescent molecules are red fluorescent proteins.

[0047] II. Measurement

[0048] The first expression vector and the second expression vector were transferred into passaged cells at a mass ratio of 1:1 and cultured for 24-48 h. The passaged cells with the first expression vector and the second expression vector were excited by blue light to induce paCaMKII to be recruited into the phase-separated condensate of the target protein. At the same time, wild-type protein was used as a control. The fluorescence intensity values ​​of wild-type protein phase-separated condensate and target protein phase-separated condensate before and after paCaMKII recruitment were collected.

[0049] The passaged cells include, but are not limited to, HEK293T cells or COS7 cells.

[0050] [Revised from Rule 26, December 2024] III. Calculation

[0051] [Corrected according to Rule 26, December 23, 2024] [Deleted]

[0052] (Formula 1)

[0053] The efficiency of the target protein in recruiting paCaMKII is calculated according to Formula 1. Based on the efficiency of the target protein in recruiting paCaMKII, functional variations caused by phase separation variations in the target protein are detected.

[0054] This invention provides a method for detecting variations in protein phase separation function, as shown in Figures 1 and 3, comprising the following steps:

[0055] I. Construction

[0056] Construct a first expression vector fused with a fluorescent molecule and the target protein, and a second expression vector fused with a fluorescent molecule and paCaMKII. For specific methods, please refer to step one above.

[0057] II. Measurement

[0058] The first expression vector and the second expression vector were transferred into passaged cells at a mass ratio of 1:1 and cultured for 24-48 h. The passaged cells containing the first and second expression vectors were excited by blue light to induce paCaMKII to be recruited into the phase-separated condensate of the target protein. At the same time, wild-type protein was used as a control. The fluorescence intensity values ​​inside and outside the phase-separated condensate of wild-type protein and the phase-separated condensate of target protein were collected, as well as the fluorescence intensity values ​​before and after the recruitment of paCaMKII in the phase-separated condensate of wild-type protein and the phase-separated condensate of target protein.

[0059] The passaged cells include, but are not limited to, HEK293T cells or COS7 cells.

[0060] III. Calculation

[0061] [Revised from Rule 26 to 23.12.2024] 1. Calculate the partition coefficient of the target protein phase-separated condensate:

[0062] [Corrected according to Rule 26, December 23, 2024] [Deleted]

[0063] According to Formula 2, the partition coefficients of wild-type protein phase-separated condensates and target protein phase-separated condensates are calculated respectively to quantify the phase-separation ability of wild-type protein and target protein and detect the phase-separation variation of target protein.

[0064] [Revised according to Rule 26, December 2024] 2. Calculate the efficiency of the target protein in recruiting paCaMKII:

[0065] [Corrected according to Rule 26, December 23, 2024] [Deleted]

[0066] Calculate the efficiency of the target protein in recruiting paCaMKII according to Formula 1.

[0067] 3. Detecting functional variations caused by phase separation variations in target proteins:

[0068] The phase separation variation results of the target protein were correlated with the efficiency of the target protein in recruiting paCaMKII, thus probing the functional variations caused by the phase separation variation of the target protein.

[0069] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0070] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications in the art, unless otherwise stated.

[0071] The mutation (G3680) in the SHANK3 gene, where the guanine nucleotide (G) is inserted into exon 21 at position 3680, is a genetic variant associated with autism spectrum disorder and severe intellectual disability (Durand et al., 2007). In this paper, this mutated protein is named SHANK3-Ex21-InsG3680. The following explanation uses the intrasynaptic mutated SHANK3 protein (SHANK3-Ex21-InsG3680) as an example to further illustrate this method. It should be understood that any mutant protein other than the mutated SHANK3 protein that exhibits phase-separation characteristics and can interact with CaMKII can be analyzed using the following method to detect protein phase-separation functional variations.

[0072] I. Construction

[0073] 1. Construct a first expression vector that integrates fluorescent molecules and target proteins.

[0074] Using wild-type SHANK3 as a control protein, far-infrared fluorescent protein iRFP 670 was ligated to the N-terminus of the full-length SHANK3 (SHANK3 FL) to obtain the fusion gene SHANK3 FL-iRFP 670. The fusion gene SHANK3 FL-iRFP 670 was inserted into the multiple cloning site of the pcDNA3.1+ expression vector. DNA sequencing and restriction enzyme digestion were used to verify that the fusion gene SHANK3 FL-iRFP 670 was correctly inserted into the multiple cloning site of the pcDNA3.1+ expression vector, resulting in the first expression vector fused with the fluorescent molecule and SHANK3 FL (Figure 4).

[0075] Using SHANK3-Ex21-InsG3680 as the target protein, the far-infrared fluorescent protein iRFP 670 was ligated to the N-terminus of SHANK3-Ex21-InsG3680 to obtain the fusion gene SHANK3-Ex21-InsG3680-iRFP 670. The fusion gene SHANK3-Ex21-InsG3680-iRFP 670 was inserted into the multiple cloning site of the pcDNA3.1+ expression vector. DNA sequencing and restriction enzyme digestion were used to verify that the fusion gene SHANK3-Ex21-InsG3680-iRFP 670 was correctly inserted into the multiple cloning site of the pcDNA3.1+ expression vector, resulting in the first expression vector fused with the fluorescent molecule and SHANK3-Ex21-InsG3680.

[0076] 2. Construct a second expression vector incorporating a fluorescent molecule and paCaMKII.

[0077] The red fluorescent protein mCherry was ligated to the N-terminus of paCaMKII (purchased from Addgene; Catalog #: 165431) to obtain the fusion gene paCaMKII-mCherry. The fusion gene paCaMKII-mCherry was inserted into the multiple cloning site of the pcDNA3.1+ expression vector. DNA sequencing and restriction enzyme digestion were used to verify that the fusion gene paCaMKII-mCherry was correctly inserted into the multiple cloning site of the pcDNA3.1+ expression vector, resulting in a second expression vector fused with the fluorescent molecule and paCaMKII (Figure 5).

[0078] II. Measurement

[0079] HEK293T cells were seeded into 35 mm MatTek glass-bottom imaging dishes (Mat Tek P35G-0-10-C) for direct imaging. When the cell density reached 70%–90%, 3 μg of a first expression vector fused with a fluorescent molecule and SHANK3 FL and 3 μg of a second expression vector fused with a fluorescent molecule and paCaMKII were transfected into HEK293T cells using Lipofectamine 2000 transfection reagent (ThemoFisher 11668030). Simultaneously, 3 μg of a first expression vector fused with a fluorescent molecule and SHANK3-Ex21-InsG3680 and 3 μg of a second expression vector fused with a fluorescent molecule and paCaMKII were transfected into another group of HEK293T cells using Lipofectamine 2000 transfection reagent. All cell groups were cultured for 48 h.

[0080] Using a Zeiss LSM 900 confocal laser scanning microscope (CLSM), images of the two groups of cells cultured for 48 h were taken. Plan-Apochromat 63 was used for imaging. / 1.4 Oil DIC M27 objective lens, xy field 512 With 512 pixels and a pixel size of 70 nm, the excitation wavelength of the first carrier was set to 647 nm, and the excitation wavelength of the second carrier was set to 561 nm. The fluorescence intensities of SHANK3 FL, SHANK3-Ex21-InsG3680, and paCaMKII were collected. The fluorescence results of SHANK3 FL and paCaMKII before blue light excitation are shown in Figure 6A.

[0081] A short-duration blue light exciter (1% 488 nm laser, single-frame pulse: 5.03 s) was used to excite paCaMKII, inducing its recruitment into either the SHANK3 FL or SHANK3-Ex21-InsG3680 phase-separated condensates. Fluorescence intensities inside and outside the SHANK3 FL and SHANK3-Ex21-InsG3680 phase-separated condensates were collected, as well as before and after paCaMKII recruitment. The fluorescence results of SHANK3 FL and paCaMKII after blue light excitation are shown in Figure 6B.

[0082] The fluorescence results before and after blue light excitation show that SHANK3 FL expression in HEK293T cells undergoes phase separation, while paCaMKII is uniformly distributed in HEK293T cells.

[0083] III. Calculation

[0084] 1. Using ImageJ software, the convolutional neural network method StarDist (https: / / github.com / stardist / stardist) was used to detect and segment paCaMKII in SHANK3 FL phase-separated condensates and paCaMKII in SHANK3-Ex21-InsG3680 phase-separated condensates.

[0085] The segmentation results of the SHANK3 FL phase-separated condensate are shown in Figure 6C. When paCaMKII is recruited into the SHANK3 FL phase-separated condensate, colocalization leads to the formation of orange colocalization of red and green fluorescent proteins.

[0086] 2. Calculate the phase separation ability of the target protein: Referring to Formula 2, the partition coefficient of the corresponding protein phase separation aggregate is obtained by calculating the fluorescence ratio inside and outside the SHANK3 FL phase separation aggregate and the SHANK3-Ex21-InsG3680 phase separation aggregate, respectively.

[0087] The calculation results are shown in Figure 6D. The partition coefficient of the SHANK3 FL phase-separating condensate is 14, and the partition coefficient of the SHANK3-Ex21-InsG3680 phase-separating condensate is 4. This result indicates that SHANK3 FL significantly loses its phase-separating ability after mutation, and its molecules are evenly distributed within the cell, proving that the phase-separating ability of the target protein has changed.

[0088] 3. Calculate the efficiency of the target protein in recruiting paCaMKII: Referring to Formula 1, with the efficiency of SHANK3 FL in recruiting paCaMKII as 100%, calculate the efficiency of SHANK3-Ex21-InsG3680 in recruiting paCaMKII.

[0089] The calculation results are shown in Figure 6E. SHANK3-Ex21-InsG3680 recruits paCaMKII with an efficiency of <5%, which is at least 95% lower than that of SHANK3 FL. This result indicates that SHANK3 FL exhibits abnormal paCaMKII recruitment after mutation, suggesting that abnormal paCaMKII recruitment efficiency reflects phase separation dysfunction. This result is consistent with the association between SHANK3-Ex21-InsG3680 and autism spectrum disorder with severe intellectual disability symptoms.

[0090] 4. Investigate the relationship between the phase separation ability of the target protein, the recruitment efficiency of paCaMKII, and functional variation: As shown in Figure 6F, with the recruitment efficiency of the target protein of paCaMKII as the x-axis and the partition coefficient of the phase separation condensate of the target protein as the y-axis, the linear regression equation of the paCaMKII recruitment efficiency and the partition coefficient is obtained by linear regression analysis: Y = 12.34X + 2.502.

[0091] The linear regression equation showed that the phase-separating ability of the target protein was linearly correlated with the paCaMKII recruitment efficiency. A higher partition coefficient correlated with a higher efficiency in recruiting paCaMKII, while a lower partition coefficient correlated with a relatively lower efficiency. These results indicate that both the phase-separating ability of the target protein and the efficiency in recruiting paCaMKII are related to variations in protein phase-separating function.

[0092] In summary, the phase-separation ability of a target protein affects the efficiency of its recruitment of paCaMKII. Based on the efficiency of paCaMKII recruitment, disease-induced protein phase-separation variations and abnormal protein phase-separation function can be detected.

[0093] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for detecting a functional variation in a protein phase separation, comprising, Includes the following steps: 1) Transform the first expression vector and the second expression vector into passaged cells and culture them to stimulate the passaged cells, causing the target protein to undergo phase separation; The first expression vector is fused with a fluorescent molecule and a target protein, and the second expression vector is fused with a fluorescent molecule and paCaMKII. 2) Collect fluorescence intensity values ​​of wild-type protein phase-separated condensates and target protein phase-separated condensates before and after recruiting paCaMKII; Alternatively, the fluorescence intensity values ​​inside and outside the wild-type protein phase-separated condensate and the target protein phase-separated condensate, as well as the fluorescence intensity values ​​before and after recruiting paCaMKII in the wild-type protein phase-separated condensate and the target protein phase-separated condensate, were collected respectively. 3) The percentage of the change in fluorescence intensity before and after the target protein recruits paCaMKII compared with the change in fluorescence intensity before and after the wild-type protein recruits paCaMKII is used as the efficiency of the target protein recruiting paCaMKII, and the variation in the phase separation function of the target protein is detected. Alternatively, the phase separation variation of the target protein can be detected by comparing the ratio of fluorescence intensity of the target protein phase separation aggregates in vivo and in vitro with that of the wild-type protein phase separation aggregates in vivo and in vitro; the percentage of the change in fluorescence intensity of the target protein before and after recruiting paCaMKII to that of the wild-type protein before and after recruiting paCaMKII can be used as the efficiency of the target protein recruiting paCaMKII to detect the phase separation functional variation of the target protein; the phase separation variation results of the target protein can be correlated with the efficiency of the target protein recruiting paCaMKII to detect the phase separation functional variation of the target protein.

2. The method of claim 1, wherein the protein functional variant is a protein that is involved in phase separation. The target protein is a protein that has phase separation characteristics and can interact with calmodulin-dependent phosphokinase.

3. The method of claim 1, wherein the functional variation is a protein phase separation. The first expression vector and the second expression vector each carry different fluorescent molecules, and all of the fluorescent molecules are proteins that are not in the 488 excitation band.

4. The method of claim 3, wherein the protein is a functional variant of a protein that phase separates. The first expression vector is linked to a far-infrared fluorescent protein, and the second expression vector is linked to a red fluorescent protein.

5. The method of claim 1, wherein the functional variation is a protein phase separation. The first and second expression vectors were transferred into passaged cells and cultured for 24–48 h.

6. The application of the method according to any one of claims 1 to 5 in the process of detecting the recruitment of calmodulin-dependent phosphokinases under neural activity during the phase separation of mutant proteins.

7. The use of the method according to any one of claims 1 to 5 in screening for drugs that can reverse the efficiency of mutated proteins in recruiting calmodulin-dependent phosphokinases.

8. System based on the method according to any one of claims 1 to 5, characterized in that include: The phase separation module is used to transfer a first expression vector fused with fluorescent molecules and target proteins, and a second expression vector fused with fluorescent molecules and paCaMKII, into passaged cells. By stimulating the passaged cells, the target protein undergoes phase separation. The acquisition module is used to acquire fluorescence intensity values ​​of wild-type protein phase-separated condensates and target protein phase-separated condensates before and after recruitment of paCaMKII; Alternatively, it can be used to collect fluorescence intensity values ​​inside and outside wild-type protein phase-separated condensates and target protein phase-separated condensates, as well as fluorescence intensity values ​​before and after recruitment of paCaMKII in wild-type protein phase-separated condensates and target protein phase-separated condensates. The calculation module is used to measure the efficiency of the target protein in recruiting paCaMKII as the percentage of the change in fluorescence intensity before and after the target protein recruits paCaMKII to that before and after the wild-type protein recruits paCaMKII, and to detect the variation in the phase separation function of the target protein. Alternatively, it can be used to compare the fluorescence intensity ratio of target protein phase separation aggregates inside and outside the cell with that of wild-type protein phase separation aggregates to detect phase separation variations of the target protein. The percentage of the change in fluorescence intensity before and after the target protein recruits paCaMKII compared to the change in fluorescence intensity before and after the wild-type protein recruits paCaMKII is used as the efficiency of the target protein recruiting paCaMKII, and the variation in the phase separation function of the target protein is detected. The phase separation variation of the target protein was correlated with the efficiency of the target protein in recruiting paCaMKII, thus detecting phase separation functional variation of the target protein.

9. A method for constructing a phenotype database for detecting a mutation associated with a target protein-related disease, characterized by, According to any one of claims 1 to 5, the method obtains the relationship between each disease-related target protein mutation and the target protein phase separation function variation, and establishes a phenotypic database for detecting disease mutations related to target proteins.

10. The phenotypic database obtained by the construction method of claim 9.

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