Synthetic lethal screening method based on DED1 phase separation defect cell model and application

Abstract

The invention discloses a synthetic lethal screening method based on a DED1 phase separation defect cell model and application, and belongs to the technical field of biotechnology, genetic engineering and drug screening. The DED1 mutant provided by the invention loses phase separation capability through extreme asymmetric RGG motif distribution specificity, but maintains the stress function of protein. A DED1-based phase separation defect cell model constructed by introducing the mutant can be used for identifying complementary cell pathways and researching the action mechanism of abnormal protein aggregation related diseases, and has important application in the aspect of developing drugs or combination therapies for some neurodegenerative diseases.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology, genetic engineering and drug screening technology, and in particular to a synthetic lethal screening method and its application based on a DED1 phase separation defect cell model. Background Technology

[0002] Intracellular liquid-liquid phase separation (LLPS) is a protein aggregation phenomenon that plays a crucial role in cellular stress responses, and its abnormalities are closely related to neurodegenerative diseases and cancer. Due to changes in intermolecular forces, different phase separation states arise: one is reversible liquid-like aggregates formed under certain conditions, such as the formation of stress granules, which participate in the regulation of cellular biochemical responses. This phase separation state can be reversed under conditions such as de-conditioning or metabolic processes like post-translational modifications; the other is the dysregulation of phase-separated aggregates, which further mature into irreversible aggregates composed of pathological fibers—a process that forms the physiological basis of many neurodegenerative diseases. However, therapeutic strategies targeting protein aggregation (such as anti-Aβ drugs for Alzheimer's disease) have not yielded good clinical results, suggesting that protein aggregation may not be the sole pathogenic factor in these diseases, and that other complex stress response pathways exist parallel to it.

[0003] Current research struggles to distinguish the contributions of protein phase separation from other functions, such as protein-protein interactions, limiting our understanding of phase separation-related disease mechanisms and the identification of effective drug targets. Therefore, there is an urgent need to develop novel research tools and methods capable of specifically separating these functions.

[0004] The RNA helicase DED1 in *Saccharomyces cerevisiae* is a homolog of human DDX3 and participates in translation initiation and stress response. Under heat stress, DED1 mediates protein phase separation through its N-terminal and C-terminal RGG motifs, forming stress granules. The mutant reported in this invention, based on its unique loss of phase separation ability while retaining stress function, is an ideal model for elucidating the molecular mechanisms of diseases such as Alzheimer's disease and has significant application value. Summary of the Invention

[0005] The purpose of this invention is to provide a synthetic lethal screening method and its application based on a DED1 phase separation defect cell model, in order to solve the problems existing in the prior art. This invention verifies the significant effect and translational potential of iturin in the prevention and treatment of non-alcoholic fatty liver disease, and provides a new approach for the clinical treatment of non-alcoholic fatty liver disease.

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

[0007] This invention provides a DED1 phase separation defect cell model, wherein the DED1 phase separation defect cell model expresses a DED1 mutant, and the amino acid sequence of the DED1 mutant is shown in SEQ ID NO.1.

[0008] Optionally, the DED1 mutant has the following structural features: its N-terminus contains four RGG motifs (two more than the wild type), and its C-terminus lacks the two RGG motifs originally present at the C-terminus of the wild type DED1 protein;

[0009] The DED1 mutant protein loses its ability to separate into liquid and liquid phases and does not form stress particles under heat stress, but still retains its ability to interact with translation initiation factors.

[0010] Preferably, the nucleotide sequence of the gene encoding the DED1 mutant is shown in SEQ ID NO.2.

[0011] The present invention also provides a DED1 mutant, the amino acid sequence of which is shown in SEQ ID NO.1.

[0012] Preferably, the nucleotide sequence of the gene encoding the DED1 mutant is shown in SEQ ID NO.2.

[0013] The present invention also provides a method for identifying cellular pathways complementary to DED1-mediated phase separation, comprising the following steps:

[0014] The cell model based on DED1 phase separation defect was constructed, and candidate genes were systematically knocked out or interfered with. Synthetic lethal or synthetic pathological phenotypes were detected under stress conditions. Genes that produce synthetic lethality with DED1 mutants were identified. The pathways in which these genes are located are the cell pathways that are complementary to the phase separation function mediated by DED1.

[0015] The amino acid sequence of the DED1 mutant is shown in SEQ ID NO.1.

[0016] The present invention also provides the application of the DED1 phase separation defect cell model or the DED1 mutant in the preparation of drugs for treating diseases related to abnormal protein aggregation.

[0017] The present invention also provides the application of the DED1 phase separation defect cell model or the DED1 mutant in screening drug targets for diseases related to abnormal protein aggregation.

[0018] The present invention also provides the application of the DED1 phase separation defect cell model or the DED1 mutant in screening drugs for treating diseases related to abnormal protein aggregation.

[0019] The present invention discloses the following technical effects:

[0020] 1. Unique Functional Separation: Achieving specific loss of phase separation ability while maintaining the basic function of the protein. The DED1 mutants with different RGG structures constructed in this invention, wherein the N0C0, N0C2, N0C4, N2C0, N2C2(WT), N2C4, N4C2, and N4C4 structures retain their original phase separation ability, while the DED1-N4C0 structure specifically loses its phase separation ability.

[0021] 2. In this invention, the phase separation and aggregation formed by DED1 will become more pronounced as the stress time increases, and the aggregation can be released as the stress state is relieved.

[0022] 3. Revealing a stress-survival pathway parallel to phase separation: This invention discovered that the deletion of the DED1-N4C0 and PUB1 genes leads to synthetic lethality, proving the existence of a complementary stress pathway. In the temperature sensitivity experiments conducted by combining pub1Δ with different RGG structures in this invention, only the DED1-N4C0 structure and pub1Δ produced synthetic lethality.

[0023] 4. Drug Development Value: This invention reveals a cellular stress response pathway parallel to phase separation, potentially explaining why single-target therapies (such as clearing protein aggregation) have limited therapeutic effects on corresponding diseases. It provides a rare "combination drug" screening model for neurodegenerative diseases and stress states caused by certain abnormal protein aggregation.

[0024] 5. Broad application prospects: DED1 is homologous to the human gene DDX3, which also has phase separation ability. Given the conservation of the RGG structure, the discovery of this invention can be extended to a large number of human protein systems containing the RGG structure. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 Design and expression validation of the DED1-N4C0 mutant; A: Schematic diagram of each mutant; B: Statistical graph of expression level of DED1-N4C0 mutant; C: Schematic diagram of DED1 amino acid sequence;

[0027] Figure 2 N4C0 specifically loses its phase separation ability;

[0028] Figure 3 For the kinetics and reversibility of phase separation;

[0029] Figure 4 This refers to synthetic lethality caused by PUB1 deficiency in a DED1 phase separation defect cell model. Detailed Implementation

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0034] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0035] Example 1: Construction of a DED1-RGG structural rearrangement mutant

[0036] like Figure 1As shown, the wild-type DED1 has two RGG motifs at the N-terminus (positions 51-53, 62-64) and the C-terminus (positions 545, 578). The N4C0 mutant was constructed by eliminating the C-terminal RGG (R545S, R578S) and adding the N-terminal RGG (N44R, N67R) through site-directed PCR mutagenesis. The amino acid sequence of DED1-N4C0 is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0037] Different RGG structures of mutated DED1 were transformed into Saccharomyces cerevisiae using lithium acetate conversion, and expression was induced by galactose. Protein expression was confirmed by RT-PCR and fluorescence microscopy. The results showed that N4C0-GFP was successfully expressed, and the expression level was comparable to that of wild-type N2C2-GFP.

[0038] The RT-PCR verification primers are as follows:

[0039] F: 5'-ACCTCCATTGGACGGATTGT-3' (SEQ ID NO.3);

[0040] R: 5'-GCCATCAAATCTCTGCCGTT-3' (SEQ ID NO. 4).

[0041] The constructed mutant strain was placed in YPDA medium and cultured overnight in a shaker at 30°C. The cells were harvested the next day, washed twice with PBS, and then transferred to GAL medium for induction at 0h, 1h, 2h, and 3h. After induction, total RNA was extracted from the *Saccharomyces cerevisiae* cells. The obtained total RNA was reverse transcribed according to the reverse transcriptase instructions to obtain cDNA, which was then used as a template for RT-qPCR. Actin was used as an internal reference gene. The difference in amplification Ct values ​​between the target gene and the internal reference gene in the sample group and the control group was calculated. The relative transcription level was calculated using the gene 2- ΔΔCt The expression fold was calculated using this method to analyze and compare the expression levels of DED1 with different RGG structures.

[0042] SEQ ID NO.1 (DED1-N4C0 amino acid sequence):

[0043] MAELSEQVQNLSINDNNENGYVPPHLRGKPRSARNNSSNYNNNRGGYNGGRGGGSFFSNNRRGGYGRGGFFGGNNGGSRSNGRSGGRWIDGKHVPAPRNEKAEIAIFGVPEDPNFQSSGINFDNYDDIPVDASGKDVPEPITEFTSPPLDGLLLENIKLARFTKPTPVQKYSVPIVANGRDLMACAQTGSGKTGGFLFPVLSESFKTGPSPQPESQGSFYQRKAYPTAVIMAPTRELATQIFDEAKKFTYRSWVKACVVYGGSPIGNQLREIERGCDLLVATPGRLNDLLERGKISLANVKYLVLDEADRMLDMGFEPQIRHIVEDCDMTPVGERQTLMFSATFPADIQHLARDFLSDYIFLSVGRVGSTSENITQKVLYVENQDKKSALLDLLSASTDGLTLIFVETKRMADQLTDFLIMQNFRATAIHGDRTQSERERALAAFRSGAATLLVATAVAARGLDIPNVTHVINYDLPSDVDDYVHRIGRTGRAGNTGLATAFFNSENSNIVKGLHEILTEANQEVPSFLKDAMMSAPGSRSNSRSGGFGRNNNRDYRKAGGASAGGWGSSRSRDNSFSGGSGWGSDSKSSGWGNSGGSNNSSWW;

[0044] SEQ ID NO.2 (DED1-N4C0 nucleotide sequence):

[0045]

[0046] Example 2: N4CO specifically loses its phase separation ability

[0047] The constructed bacterial strain was placed in YPDA medium and cultured overnight in a shaker at 30°C. The cells were collected by centrifugation, washed twice with PBS, and then transferred to GAL medium for induction for 3-4 hours. OD values ​​were measured after induction. 600 Based on the measured results, 10 samples were collected from different mutants. 7 A bacterial cell was placed under a 40°C heat stress for 2 hours, and the phase separation phenomenon was observed under an upright fluorescence microscope.

[0048] The results are as follows Figure 2 As shown, all eight mutants except N4C0 formed stress granules, including N0C0 (0 RGGs) and N0C4 (4 RGGs, but distributed at the C-terminus), which has the same polarity. This suggests that phase separation capability may be mainly determined by the number and spatial symmetry of the N-terminal RGGs. Figure 2 As shown.

[0049] Kinetic analysis of the DED1 mutant, which exhibits phase separation, revealed that its aggregation degree increased significantly with prolonged stress time, and this aggregation was reversible; the stress particles disappeared upon removal of the stress state. Figure 3 ).

[0050] Example 3: Synthetic lethal effects of N4C0 and PUB1 gene deletion

[0051] Pub1 is an RNA-binding protein involved in the regulation of mRNA stability and stress granule assembly.

[0052] 1. Construct a Saccharomyces cerevisiae mutant strain, and use homologous recombination to knock out the PUB1 gene to construct the pub1Δ strain, and then overexpress DED1 containing different RGG structures.

[0053] Primers used for the construction and validation of pub1Δ strain:

[0054] P1: 5'-GCCTTCGCTTCTTTTTTGTTGTAATTTTTC-3' (SEQ ID NO.5);

[0055] P2: 5'-CGAACAAAGAGTAGATGTGGTAATCTTC-3' (SEQ ID NO. 6);

[0056] P3: 5'-CCACATCTACTCTTTGTTCGATTCCAGACATGGAGGCCCAGAATACC-3' (SEQ IDNO.7);

[0057] P4: 5'-CTTTTTGTTTCATTCCACTTTTCTTCATAATATCAGTATAGCGACCAGCATTCAC-3' (SEQ ID NO. 8);

[0058] P5: 5'-CGCTATACTGATATTATGAAGAAAAGTGGAATG-3' (SEQ ID NO.9);

[0059] P6: 5'-GACGAAGGAAGGAAATAAGACACAG-3' (SEQ ID NO. 10).

[0060] Using the primers described above, upstream and downstream homologous arms of PUB1 and fragments such as kanMx were amplified. Using cDNA as a template, the upstream homologous arm of PUB1 was amplified using primers p1 and p2, and the downstream homologous arm of Pub1 was amplified using primers p5 and p6. Using pgsku plasmid as a template, kanMx was amplified using primers p3 and p4. The three fragments were then ligated using overlap PCR. The recovered PCR product was then used to construct a *Saccharomyces cerevisiae* mutant strain via lithium acetate chemical transformation. Similarly, using lithium acetate transformation, the DED1 plasmid was transformed into the constructed pub1Δ mutant strain to obtain the pub1Δ+DED1 strain.

[0061] 2. The method for detecting heat stress survival is as follows:

[0062] (1) Place the strain constructed above in 5 mL of YPDA and incubate overnight in a shaker at 30°C and 220 r / min;

[0063] (2) Collect the bacterial cells, wash them twice with PBS, transfer them to GAL medium, and incubate for 3-4 h;

[0064] (3) Measure the OD of the induced bacterial culture. 600 Press 10 8 Each bacterial cell was taken and the bacterial solution was transferred to an EP tube and centrifuged at 4℃ and 4500 r / min for 2 min.

[0065] (4) Discard the supernatant in the EP tube, add 1 mL of pre-chilled PBS to suspend the bacterial cells, and dilute the bacterial cells to 10⁻¹⁰ with pre-chilled PBS. 7 / mL, 10 6 / mL, 10 5 / mL, 10 4 / mL;

[0066] (5) Spot the sample on a YPDA plate and incubate it at 30℃ and 40℃ for 48 h respectively. Observe the growth of yeast colonies and take pictures to record the results.

[0067] Yeast growth was observed by spotting samples at 10-fold serial dilutions, such as... Figure 4 As shown, overexpression of N4C0 and PUB1 deficiency resulted in synthetic lethality under 40°C stress conditions, while other RGG structures did not exhibit synthetic lethality.

[0068] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A cell model based on DED1 phase separation defects, characterized in that, The DED1 mutant was expressed in the DED1 phase separation defect cell model, and the amino acid sequence of the DED1 mutant is shown in SEQ ID NO.

1.

2. The cell model based on DED1 phase separation defects as described in claim 1, characterized in that, The nucleotide sequence of the gene encoding the DED1 mutant is shown in SEQ ID NO.

2.

3. A DED1 mutant, characterized in that, The amino acid sequence of the DED1 mutant is shown in SEQ ID NO.

1.

4. The DED1 mutant as described in claim 3, characterized in that, The nucleotide sequence of the gene encoding the DED1 mutant is shown in SEQ ID NO.

2.

5. A method for identifying cellular pathways complementary to DED1-mediated phase separation, characterized in that, Includes the following steps: Construct the DED1 phase separation defect cell model as described in claim 1, systematically knock out or interfere with candidate genes, detect synthetic lethal or synthetic pathological phenotypes under stress conditions, identify genes that produce synthetic lethality with DED1 mutants, and the pathways in which the genes are located are cellular pathways that are complementary to the phase separation function mediated by DED1. The amino acid sequence of the DED1 mutant is shown in SEQ ID NO.

1.

6. The use of the DED1 phase separation defect cell model as described in claim 1 or the DED1 mutant as described in claim 3 in the preparation of a medicament for treating diseases related to abnormal protein aggregation.

7. The application of the DED1 phase separation defect cell model as described in claim 1 or the DED1 mutant as described in claim 3 in screening drug targets for diseases related to abnormal protein aggregation.

8. The application of the DED1 phase separation defect cell model as described in claim 1 or the DED1 mutant as described in claim 3 in screening drugs for treating diseases related to abnormal protein aggregation.

Images