A polypeptide mediating protein nucleocytoplasmic shuttle and its use

Abstract

The application discloses a polypeptide for mediating protein nucleocytoplasmic shuttle and application thereof, and belongs to the technical field of molecular biology. The amino acid sequence of the polypeptide consists of EAEDEDGLADLLL. The polypeptide is derived from the C-terminal of a monkey face flower NEGAN protein, and has an independent nuclear export signal function. Experiments prove that after the polypeptide is fused to the C-terminal of a nuclear localization transcription factor MvbHLH1 and CmWRKY15, the proteins originally only positioned in the nucleus are changed into nucleocytoplasmic dual localization, which indicates that the polypeptide can give the nucleocytoplasmic shuttle ability to heterologous proteins. The polypeptide provided by the application has the characteristics of short sequence, independent function, wide spectrum application and the like, and provides a new functional module for artificial regulation of protein subcellular localization.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and relates to a polypeptide that mediates nucleocytoplasmic shuttle of a protein and its applications. Background Technology

[0002] In eukaryotic cells, the nuclear membrane divides the nucleus and cytoplasm into two functionally independent compartments: transcription, which is confined to the nucleus, and translation, which takes place in the cytoplasm. Therefore, the subcellular localization of proteins must be precisely regulated. Abnormal localization can lead to loss of function or cellular homeostasis imbalance. Thus, regulating nucleoplasmic shuttle is a key technological node for controlling protein function. The structural basis of nucleoplasmic shuttle is the nuclear pore complex (NPC). Substances with a molecular weight less than 40–60 kDa can diffuse freely, while the transport of larger molecules relies on signal-mediated active transport. Precise regulation of nucleoplasmic shuttle depends on the protein's own nuclear export signal (NES). The NES is a short peptide sequence that mediates the protein's exit from the nucleus by binding to a nuclear export receptor and is the core cis-element for the dynamic regulation of nucleoplasmic shuttle. In plants, the classic NES is the leucine-rich conserved motif LxxxLxxLxL, but numerous non-classical NES sequences also exist. In Arabidopsis thaliana, AtXPO1a and AtXPO1b are core NES receptors with highly redundant functions. AtXPO4 and other receptors can recognize specific NES sequences, such as the NES motif of TOPLESS, to regulate immunity. NES activity is regulated by post-translational modifications: photoactivated kinases phosphorylate the NES-adjacent site of FHY1, blocking its interaction with AtXPO1 and inhibiting nuclear export; immune-activated MAPK kinases also phosphorylate the NES motif of disease resistance transcription factors, promoting the expression of intranuclear disease resistance genes. Furthermore, intranuclear liquid-liquid phase separation can isolate NES sequences, blocking their binding to receptors, and NES-mediated nuclear export can also conversely regulate phase separation, constituting a novel regulatory paradigm for stress responses. Despite significant progress in the study of endogenous NES regulatory mechanisms in plants, there is still a lack of short, functionally independent, and broadly applicable artificial nuclear export regulatory elements. Identifying such NES functional modules for the artificial regulation of subcellular localization is of great significance. Summary of the Invention

[0003] One of the technical problems to be solved by this invention is to provide a polypeptide that mediates protein nucleocytoplasmic shuttle. Another technical problem to be solved by this invention is to provide the application of the aforementioned polypeptide that mediates protein nucleocytoplasmic shuttle.

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0005] A polypeptide that mediates nucleocytoplasmic shuttle of a protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] An isolated nucleic acid molecule encoding the aforementioned polypeptide.

[0007] A recombinant expression vector comprising the aforementioned nucleic acid molecule.

[0008] In some embodiments, the nucleotide sequence is as shown in SEQ ID NO.2.

[0009] A host cell comprising the recombinant expression vector described above.

[0010] A chimeric protein comprising a target protein and the aforementioned polypeptide fused thereto.

[0011] In some embodiments, the target protein is a protein located in the cell nucleus.

[0012] In some embodiments, the target protein is MvbHLH1 or CmWRKY15.

[0013] A method for regulating the subcellular localization of a target protein includes fusing the peptide with the target protein for expression, thereby enabling the target protein to have nucleocytoplasmic shuttle capability or nucleocytoplasmic dual localization capability.

[0014] The use of the polypeptide, the nucleic acid molecule, the recombinant expression vector, or the chimeric protein in any of the following:

[0015] (a) Preparation of transgenic plants or plant breeding;

[0016] (b) Regulating the synthesis of plant secondary metabolites;

[0017] (c) Constructing molecular switches related to plant stress or disease resistance;

[0018] (d) Reduce the off-target effects of gene editing tools;

[0019] (e) Treatment of diseases associated with abnormal activation of transcription factors.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention marks the first identification and isolation of a 13-amino acid nucleocytoplasmic shuttle peptide from the C-terminus of the monkey face flower (NEGAN) protein, its amino acid sequence shown in SEQ ID NO: 1. Experiments confirmed that this peptide possesses independent nuclear output signaling activity: after being fused to the C-terminus of transcription factors MvbHLH1 and CmWRKY15, which are originally localized only in the nucleus, both chimeric proteins exhibited significant nucleocytoplasmic dual localization characteristics, indicating that this peptide can confer nucleocytoplasmic shuttle capability to heterologous proteins. This function is independent of the overall structure of the source protein and is not limited by the type or species of the receptor protein, possessing significant advantages such as functional independence, broad applicability, short sequence, and ease of manipulation.

[0022] The polypeptides described in this invention can serve as universal subcellular localization regulatory elements, with wide applications in plant molecular breeding and next-generation precision medicine. For example, fusing them to nuclear localization transcription factors involved in the synthesis of secondary metabolites can break down the spatial barriers between nuclear transcriptional regulation and cytoplasmic synthesis pathways, increasing the accumulation of target products; fusing them to disease resistance-related transcription factors can construct molecular switches triggered by pathogen infection signals, achieving spatiotemporal specific regulation of disease resistance traits; fusing them to gene editing tools can reduce off-target risks by regulating the nuclear residence time of Cas9 proteins; fusing them to oncogenes can "expel" them from the nucleus, preventing their transcription of downstream genes and ultimately inducing apoptosis in cancer cells; fusing them to… Key regulatory proteins in the pathway (e.g.) It locks it in the cytoplasm and inhibits The nuclear translocation and activation of this peptide can quell the inflammatory response at its source. The identification of this peptide provides a novel functional module and technical means for the artificial regulation of protein subcellular localization. Attached Figure Description

[0023] Figure 1 A schematic diagram of the C-terminus of the NEGAN protein truncated and a subcellular localization diagram of the truncated protein;

[0024] Figure 2 A schematic diagram of the structure of MvbHLH1 fused with the NEGAN protein NES motif and the results of subcellular localization analysis; B. Schematic diagram of the structure of MvbHLH1 and the chimeric fusion protein; YFP is the fluorescence channel of the target protein, NLS is the nuclear labeling channel, DIC is the differential interference difference bright field imaging, Merge is the multi-channel merged image, and the number in the lower right corner is the number of positive cells / total number of observed cells;

[0025] Figure 3 A. Schematic diagram of the structure of CmWRKY15 fused with the NEGAN NES motif and subcellular localization analysis results; B. Schematic diagram of the structure of CmWRKY15 and the chimeric fusion protein; C. Subcellular localization results; YFP is the fluorescent channel of the target protein, NLS is the nuclear labeling channel, DIC is differential interference bright field imaging, Merge is the multi-channel merged image, and the number in the lower right corner is the number of positive cells / total number of observed cells. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, or are performed according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0027] Example 1

[0028] NEGAN gene cloning and identification of the C-terminal nuclear output signal (NES) core motif of the protein.

[0029] 0.1 g of flower bud tissue (approximately 5 mm in length) from the Mimulus lemwisii-LF10 strain was rapidly placed in a pre-cooled mortar, flash-frozen in liquid nitrogen, and ground into powder. Total RNA was extracted according to the kit instructions, and RNA integrity was assessed by 1% agarose gel electrophoresis. Following the procedures outlined in the Evo M-MLV reverse transcription kit (Aikerui Biotechnology, China), the total RNA was reverse transcribed to synthesize cDNA. The product was sealed and stored at -20 °C as a template for subsequent gene cloning. The NEGAN sequence information (gene number: ML3G226600.1) was obtained from the *Heliotropium indicum* reference genome number (http: / / mimubase.org / ). Specific primers (F: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCatggaaaacacacctgtaggtgt (SEQ ID NO.5); R: GGGGACCACTTTGTACAAGAAAGCTGGGTCatttggccccagtaggcc (SEQ ID NO.6)) were designed using SnapGene® 3.2.1 software. Using the obtained cDNA as a template, full-length PCR amplification of the target gene was performed using high-fidelity DNA polymerase, and the products were detected by 1% agarose gel electrophoresis. The target band was recovered, cloned into the pDONR207 initiation vector, and transformed into *E. coli* DH5α. Competent cells. Positive clones were selected and preliminarily identified by colony-linked PCR, followed by plasmid extraction and sequencing. The CDS sequence of NEGAN was obtained as shown in SEQ ID NO.3, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.4. The correctly sequenced entry vector was used for LR recombination to recombinate the full-length NEGAN coding sequence into the plant expression vector pEarleyGate101 (carrying a YFP fluorescent tag), obtaining the wild-type recombinant vector 35S::YFP.

[0030] Using the correctly sequenced full-length entry vector pDONR207-NEGAN as a template, truncated primers were designed. The truncated primer design strategy is as follows: a stop codon (TAA, TGA, or TAG) and an attB recombinant sequence are introduced after the target truncation site, so that the protein encoded by the amplified product terminates translation prematurely at the specified site.

[0031] Δ1 truncated form: The reverse primer is designed to introduce a stop codon after the codon encoding the 231st amino acid, and the truncated fragment encoding the 1st to 231st amino acids is amplified.

[0032] Δ2 truncated form: The reverse primer design introduces a stop codon after the codon encoding the 244th amino acid, amplifying the truncated fragment encoding the 1st to 244th amino acids.

[0033] After the truncated fragments were recovered, cloned into the pDONR207 vector, and transformed into E. coli, positive clones were selected for Sanger sequencing verification to ensure that the truncation boundaries were accurate and that no unexpected mutations were introduced. The correctly sequenced truncated entry vector was then constructed into the pEarleyGate101 vector via LR recombination to obtain the truncated mutant expression vectors 35S::NEGAN-Δ1-YFP (deleted from position 231 onwards) and 35S::NEGAN-Δ2-YFP (deleted from position 244 onwards).

[0034] The correctly identified wild-type recombinant expression vector (35S::YFP), Δ1 truncated vector (35S::NEGAN-Δ1-YFP), and Δ2 truncated vector (35S:NEGAN-Δ2-YFP) were transformed into GV3101 competent cells using the heat shock method. Positive Agrobacterium strains were obtained through antibiotic selection. Single colonies of positive Agrobacterium were picked and inoculated into solutions containing 50 mg / L... -1 Kanamycin with 50 mg·L -1 In rifampicin-resistant YEB liquid medium, incubate overnight at 28 ℃ and 200 rpm in a constant temperature shaker until the bacterial culture OD reaches zero. 600 =1.0. Centrifuge at 4000 rpm for 10 min at room temperature, discard the supernatant and collect the bacterial pellet, resuspend the bacterial cells in the suspension, and adjust the OD of the bacterial culture. 600 YFP fluorescence signal distribution was prepared by incubating at 0.8°C in the dark for 3 h, and then injected into the back of tobacco leaves. The infected tobacco leaf tissue was collected, and the distribution of YFP fluorescence signals was observed using a laser confocal microscope. Images were taken and saved for subcellular localization analysis.

[0035] The results show that ( Figure 1The fluorescence signal of the Δ1 (Δ1-231 aa) mutant protein was completely concentrated in the nucleus, losing its ability to localize in the cytoplasm; while the Δ2 (Δ1-244 aa) protein exhibited the same nucleoplasmic dual localization characteristics as the wild type, without losing its ability to localize in the cytoplasm. This indicates that the key NES region at the C-terminus of the NEGAN protein that regulates nucleoplasmic shuttle is between amino acids 232 and 244 (EAEDEDGLADLLL (SEQ ID NO.1)).

[0036] Example 2

[0037] NES motifs mediate the nuclear export of the nuclear-localized transcription factor MvbHLH1.

[0038] To determine whether the NEGAN nucleocytoplasmic shuttle NES amino acid motif possesses universality in regulating the subcellular localization of other transcription factors, MvbHLH1 (nuclear localization, sequence shown in SEQ ID NO.7), which is involved in plant anthocyanin synthesis, was selected for the experiment. Based on the 13 core amino acid sites related to nucleocytoplasmic localization identified in NEGAN in Example 1, a terminal addition strategy was used to fuse them to the C-terminus of the MvbHLH1 protein to construct a chimeric mutant: MvbHLH1-NN.

[0039] The correctly sequenced pDONR207-MvbHLH1-NN introductory vector was directionally recombined into the pEarleyGate104 expression vector (carrying a YFP fluorescent tag) via an LR Clonase II-mediated recombination reaction. The recombinant product was then transformed into DH5α. Subsequently, the culture was plated on LB agar with kanamycin and incubated upside down at 37°C for 12–16 h. After antibiotic screening, bacterial PCR, and sequencing verification, 35S::Chimera-MvbHLH1 was obtained. 1-628 -NEGAN 232-244 -YFP expression vector. Agrobacterium GV3101 competent cells were transformed using the freeze-thaw method. Positive strains were selected after resistance screening and PCR identification. Wild-type 35S::MvbHLH1::YFP vector was used as a control, and Agrobacterium was transformed and injected into tobacco leaves. Changes in the subcellular localization pattern of the fusion protein were observed using laser confocal scanning microscopy.

[0040] The results show that ( Figure 2 MvbHLH1 is specifically enriched only in the nucleus, with no visible fluorescent signal in the cytoplasm, and does not possess nucleoplasmic shuttling ability. In contrast, the chimeric protein Chimera-MvbHLH1, which incorporates 13 amino acids from the NEGAN protein, is a different case. 1-628 ::NEGAN 232-244Its YFP fluorescence signal was distributed in both the nucleus and cytoplasm, showing a significant difference from the control group. Experimental results confirmed that the 13-amino acid sequence EAEDEDDLADLLL identified at the C-terminus of the NEGAN protein can independently mediate the protein's nuclear export process, transforming it from nuclear localization to nucleocytoplasmic dual localization, thus enabling nucleocytoplasmic shuttling.

[0041] Example 3

[0042] NES motif mediates nuclear export of the heterologous transcription factor CmWRKY15.

[0043] To determine whether the NEGAN nucleocytoplasmic shuttle NES amino acid motif possesses universality in regulating the subcellular localization of other transcription factors, CmWRKY15 (nuclear localization, sequence shown in SEQ ID NO. 8), which is unrelated to anthocyanin synthesis, was selected. Based on the 13 core amino acid sites related to nucleocytoplasmic localization identified in NEGAN in Example 1, a terminal addition strategy was used to fuse them to the C-terminus of the CmWRKY15 protein to construct a chimeric mutant: CmWRKY15-NN.

[0044] The correctly sequenced pDONR207-CmWRKY15-NN introductory vector was directionally recombined into the pEarleyGate104 expression vector (carrying a YFP fluorescent tag) via an LR Clonase II-mediated recombination reaction. The recombinant product was then transformed into DH5α. Subsequently, the culture was plated on LB agar with kanamycin and incubated upside down at 37°C for 12–16 h. After antibiotic screening, bacterial PCR, and sequencing verification, 35S::Chimera-CmWRKY15 was obtained. 1-269 ::NEGAN 232-244 -YFP expression vector. This vector was transformed into Agrobacterium GV3101 competent cells using the freeze-thaw method. Positive strains were selected after resistance screening and PCR identification. Wild-type 35S::CmWRKY15-YFP vector was used as a control, and Agrobacterium was transformed and injected into tobacco leaves. Changes in the subcellular localization pattern of the fusion protein were observed using laser confocal scanning microscopy.

[0045] The results are as follows Figure 3 As shown, the fluorescence signal of CmWRKY15 is specifically enriched only in the cell nucleus, completely co-localizing with the NLS nuclear labeling signal. No visible fluorescence signal is observed in the cytoplasm, confirming that CmWRKY15 is a nuclear-localized protein without nucleoplasmic shuttling ability. However, the chimeric protein Chimera-CmWRKY15, which incorporates a 13-amino acid core motif from a NEGAN protein, is different. 1-269 ::NEGAN 232-244Its fluorescence signal is clearly distributed not only in the cell nucleus, but also exhibits a wide and uniform fluorescence signal in the cytoplasm. The observed field of view shows a stable nucleoplasmic dual localization phenotype, which is significantly different from the nuclear localization of CmWRKY15. This experimental result further confirms that the 13-amino acid sequence EAEEDEDGLADLLL identified from the C-terminus of the NEGAN protein is a broad-spectrum functional NES motif with independent nuclear export function.

[0046] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. A polypeptide that mediates nuclear and cytoplasmic shuttling, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. An isolated nucleic acid molecule, characterized in that, Encoding the polypeptide of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

2.

4. A recombinant expression vector, characterized in that, It includes the nucleic acid molecule as described in claim 2 or 3.

5. A host cell, characterized in that, It includes the recombinant expression vector as described in claim 4.

6. A chimeric protein, characterized in that, The invention comprises a target protein and a polypeptide of claim 1 fused thereto, wherein the polypeptide of claim 1 is fused to the C-terminus of the target protein; the target protein is MvbHLH1 or CmWRKY15, the amino acid sequence of MvbHLH1 is shown in SEQ ID NO.7, and the amino acid sequence of CmWRKY15 is shown in SEQ ID NO.

8.

7. A method for regulating the subcellular localization of target proteins, characterized in that, The polypeptide of claim 1 is fused to the C-terminus of a target protein, thereby enabling the target protein to have nucleocytoplasmic dual localization capability. The target protein is MvbHLH1 or CmWRKY15, the amino acid sequence of MvbHLH1 is shown in SEQ ID NO.7, and the amino acid sequence of CmWRKY15 is shown in SEQ ID NO.8.

Images