Mitochondrial targeting signal peptide as well as coding gene and application thereof

Abstract

The invention discloses a mitochondrial targeting signal peptide as well as a coding gene and application thereof, and belongs to the technical field of biomedicine. The amino acid sequence of the mitochondrial targeting signal peptide is as shown in SEQ ID NO. 2 or SEQ ID NO. 4. The signal peptide is derived from deep analysis of a PCV2 Cap protein mitochondrial localization signal (MLS) region, and through accurate confirmation of 29th-40th key amino acid regions, the defects that an existing natural signal peptide is lengthy in sequence, unknown in structure and unstable in targeting efficiency and has an off-target hidden danger are effectively overcome. According to the invention, not only is an efficient and safe visualization and positioning means provided for basic research of mitochondria, but also a key technical platform with great potential is provided for transformation medicine application such as gene therapy, protein delivery and pharmaceutical preparations for developing targeted mitochondria.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a mitochondrial targeting signal peptide, its encoding gene, and its applications. Background Technology

[0002] As the energy metabolism center of eukaryotic cells, mitochondria's function is highly dependent on the precise localization and transport of mitochondrial proteins encoded by nuclear genes. It is known that most mitochondrial proteins, when synthesized in the cytoplasm, carry a specific sequence at their N-terminus called a mitochondrial localization signal peptide. This signal peptide can be recognized by receptors on the mitochondrial membrane, guiding the protein precursor across the mitochondrial membrane. After entering the mitochondrial matrix, it is cleaved by specific enzymes, ultimately completing protein maturation and localization. This process involves the unfolding and refolding of the precursor protein, the assistance of molecular chaperones, and the sorting information carried by different fragments of the signal peptide, all of which jointly determine the final sublocalization of the protein in the mitochondria. Therefore, highly efficient and specific mitochondrial localization signal peptides are key molecular tools for achieving targeted delivery of exogenous proteins or therapeutic molecules to the mitochondria.

[0003] However, existing natural mitochondrial targeting signal peptides still face certain limitations in application. First, signal peptides from different sources exhibit varying targeting efficiency and specificity, and some may show unstable targeting or off-target effects due to differences in cell type, physiological state, or the properties of the fusion protein. Second, natural signal peptide sequences are typically long, potentially causing unpredictable effects on the structure and function of the target protein they fuse with, or introducing additional immunogenicity or cytotoxicity risks due to the presence of unnecessary domains. More critically, a systematic and in-depth analysis of the key amino acid sequences or functional domains that determine the targeting function of signal peptides is currently lacking, making it difficult to rationally design and optimize signal peptides for specific applications (such as high-precision organelle tracking and efficient therapeutic molecule delivery). Identifying the core functional sequences from known or newly discovered signal peptides and developing shorter, more efficient, and more stable novel mitochondrial targeting tools based on these sequences is the main challenge currently facing the technology.

[0004] Furthermore, in the field of therapeutic research for mitochondrial-related diseases (such as mitochondrial encephalomyopathy), although gene therapy, protein replacement therapy, or drug delivery strategies targeting mitochondria have shown great potential, their development is severely limited by the lack of efficient and safe delivery vectors. Existing delivery systems often fail to achieve specific accumulation of therapeutic components within mitochondria, leading to limited efficacy or off-target side effects. Therefore, developing a novel mitochondrial-targeting signal peptide with a clearly defined core functional domain, concise sequence, high targeting efficiency, and good biocompatibility is urgently needed and of great significance for promoting innovation in basic mitochondrial biological research tools and overcoming the technical bottlenecks in targeted therapy for mitochondrial diseases. Summary of the Invention

[0005] The purpose of this invention is to provide a mitochondrial targeting signal peptide, its encoding gene, and its applications to address the problems existing in the prior art. This invention provides a novel mitochondrial targeting signal peptide with a short structure and well-defined targeting. By identifying key functional domains at positions 29-40, it overcomes the drawbacks of lengthy and unstable natural signal peptides. Fusion protein systems constructed based on this signal peptide exhibit high targeting efficiency and good biocompatibility, providing an efficient and reliable technical platform for mitochondrial research and targeted disease therapy.

[0006] To achieve the above objectives, the present invention provides the following solution: The present invention provides a mitochondrial targeting signal peptide, the amino acid sequence of which is shown in SEQ ID NO.2 or SEQ ID NO.4.

[0007] The present invention also provides a nucleic acid molecule that encodes the mitochondrial targeting signal peptide.

[0008] Optionally, its nucleotide sequence is as shown in SEQ ID NO.8 or SEQ ID NO.10.

[0009] The present invention also provides a fusion protein comprising the aforementioned mitochondrial targeting signal peptide and a target protein attached to its C-terminus.

[0010] Optionally, the target protein may include fluorescent proteins, enzymes, therapeutic proteins, antigenic proteins, and reporter proteins.

[0011] The present invention also provides a recombinant vector comprising the aforementioned nucleic acid molecule or a nucleic acid molecule encoding the aforementioned fusion protein.

[0012] The present invention also provides the application of the mitochondrial-targeting signal peptide, the nucleic acid molecule, the fusion protein, or the recombinant vector in the preparation of a drug delivery system targeting mitochondria.

[0013] The present invention also provides the application of the mitochondrial targeting signal peptide, the nucleic acid molecule, the fusion protein, or the recombinant vector in the preparation of a drug for treating mitochondrial-related diseases.

[0014] The present invention also provides the use of the mitochondrial targeting signal peptide, the nucleic acid molecule, the fusion protein, or the recombinant vector in the preparation of products for locating mitochondria or indicating mitochondrial position.

[0015] The present invention also provides the application of the mitochondrial targeting signal peptide, the nucleic acid molecule, the fusion protein, or the recombinant vector in the preparation of products for studying the interaction between the target protein and mitochondria.

[0016] The present invention discloses the following technical effects: This invention provides a novel mitochondrial-targeting signal peptide with a clear sequence, concise structure, and well-defined mitochondrial targeting function and core functional domain. This signal peptide originates from in-depth analysis of the mitochondrial localization signal (MLS) region of the PCV2 Cap protein, and through precise identification of the key amino acid region at positions 29-40, it effectively avoids the drawbacks of existing natural signal peptides, such as lengthy sequences, unclear structures, unstable targeting efficiency, and off-target risks. The clear definition of the core functional domain lays a clear molecular foundation for constructing a more stable and efficient mitochondrial-targeting tool, overcoming the difficulty of rationally optimizing the design of signal peptides in existing technologies. Experiments have demonstrated that the expression of the fusion protein guided by this core signal peptide has no significant negative impact on cell viability and exhibits excellent biocompatibility.

[0017] This invention not only provides an efficient and safe visualization and localization method for basic research on mitochondria, but also provides a highly promising key technology platform for translational medicine applications such as gene therapy, protein delivery and drug formulation targeting mitochondria, which is expected to promote substantial progress in the diagnosis and treatment of mitochondrial-related diseases. Attached Figure Description

[0018] 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.

[0019] Figure 1 A confocal plot showing the subcellular localization of the mitochondrial localization signal MLS-PCV2 and its three-segment deletion mutant peptides co-transfected with TXN2-DsRed; Figure 2 A confocal plot showing the subcellular localization of the mitochondrial localization signal MLS-PCV2 and its key amino acid mutant peptides co-transfected with TXN2-DsRed; Figure 3 This is a statistical graph showing the cell death rate 48 hours after transfection of empty vectors expressing only EGFP and plasmids expressing the MLS-PCV2-Opt1-EGFP fusion protein into PK15 cells. Detailed Implementation

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] Example 1: Design and confirmation of novel mitochondrial-targeting signal peptides and their coding sequences This embodiment identifies the amino acid sequences and encoding genes of four novel mitochondrial localization signal peptides (MLS) provided by the present invention, and clarifies the key functional domains.

[0026] 1. Signal peptide amino acid sequence design By analyzing the mitochondrial localization domain (amino acids 16-40) of the PCV2 Cap protein, its core targeting sequence and key deletion mutants were designed and validated.

[0027] The novel mitochondrial-targeting signal peptide (MLS-PCV2) sequence is located at positions 16-40 of the PCV2 Cap, and its specific amino acid sequence is shown in Table 1. Five new peptides, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, were obtained by segmental deletion and mutation of key amino acids in the full-length signal peptide (SEQ ID NO.1) for analysis of important functional regions and key amino acids.

[0028] Table 1. Mitochondrial localization signals and their deletion mutant protein sequences 2. Design and synthesis of coding genes To facilitate gene manipulation and expression, corresponding coding DNA sequences were designed for the four signal peptides mentioned above. To ensure successful expression and translation of the inserted sequence and EGFP protein into the fusion protein, a start codon "ATG" was introduced at the 5' end. The sequences were obtained through chemical synthesis and are detailed in Table 2.

[0029] Table 2. Mitochondrial localization signal DNA sequences Example 2 Construction of mitochondrial-targeting fusion protein expression plasmid This embodiment describes how to fuse the signal peptide coding sequence from Example 1 with a reporter gene (EGFP) and clone it into an expression vector to construct a plasmid that can be used for expression in eukaryotic cells.

[0030] Primer design and site-specific PCR deletion: To construct the fusion expression plasmids of five deletion mutants MLS-PCV2-Opt1, MLS-PCV2-Opt2, MLS-PCV2-Opt3, MLS-PCV2-ΔCore and MLS-PCV2-mutVYR, five pairs of specific primers were designed (Table 3). The precise deletion of the target fragment was achieved by overlapping extension PCR using the plasmid PCV2-MLS (Wanting Yu, Mitochondrial Localization Signal of Porcine Circovirus Type 2 Capsid Protein Plays a Critical Role in Cap-Induced Apoptosis, Vet Sci. 2021 Nov 10;8(11):272. doi:10.3390 / vetsci8110272) as a template.

[0031] Table 3 Primer sequences used to construct deletion mutant plasmids Fragment ligation: After PCR products were recovered by agarose gel electrophoresis, they were digested with Dpn I enzyme to remove the template plasmid. A "signal peptide-EGFP" fusion expression cassette was constructed using the upgraded Hieff Clone® Universal II One Step Cloning Kit for homologous recombination cloning.

[0032] Transformation and Identification: The ligation product was transformed into DH5α competent cells and plated on LB agar plates containing 100 μg / mL ampicillin. Single colonies were picked, colony PCR was performed, and the colonies were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Six correct plasmids were obtained: pMLS-PCV2-EGFP (control), pMLS-PCV2-Opt1-EGFP, pMLS-PCV2-Opt2-EGFP, pMLS-PCV2-Opt3-EGFP, pMLS-PCV2-ΔCore-EGFP, and pMLS-PCV2-mutVYR-EGFP.

[0033] Example 3: Cellular verification of mitochondrial targeting function and identification of key functional domains This embodiment uses confocal microscopy to verify the targeting efficiency of the signal peptide and identify the key amino acid regions that affect the targeting function.

[0034] 1. Cell Culture and Transfection Cells: PK15 cells, cultured in DMEM containing 10% FBS.

[0035] Plating: Cells were seeded into 12-well plates containing sterile cell spreaders (18 mm) at a density of 1 × 10⁻⁶. 5 One hole / hole.

[0036] Co-transfection: When cell confluence reaches 70%-85%, perform transfection at a ratio of 1 μg plasmid to 2 μL transfection reagent per well. Before transfection, discard the cell supernatant in the 12-well plate and wash the cell surface 2-3 times with PBS buffer. Add 1 mL of sterile DMEM (free of FBS and antibiotics) to each well of the 12-well plate and incubate for 0.5-1 h.

[0037] For example, transfection is performed using one well of a 12-well plate: Place two 1.5 mL sterile centrifuge tubes on a test tube rack, add 75 μL of DMEM basal medium to each tube, and label them tube A and tube B, respectively. Add 1 μg of the corresponding plasmid to tube A and 2 μL of liposome transfection reagent to tube B. Gently tap tubes A and B, then add the liquid from tube B to tube A, mix the liquids in the same way, and incubate at room temperature for 25-30 min.

[0038] Remove the 12-well plate, use a pipette to draw up the mixture, and slowly add it dropwise into the wells of the 12-well plate in a circular motion. Gently shake the cell culture plate back and forth and side to side 20 times each, and then place the cell culture plate in a cell culture incubator. After incubation for 6 hours, change the medium, discard the original medium, add 1 mL of DMEM complete medium containing 2% FBS to each well, and then return the plate to the cell culture incubator.

[0039] 2. Cell fixation, staining and observation 24 hours after transfection, discard the culture medium in the wells and wash the cells three times with phosphate-buffered saline (PBS), then remove the PBS. Add 400 μL of 4% paraformaldehyde cell fixation solution to each well and incubate at room temperature for 15 min. Then wash three times with PBS buffer. Add 400 μL of 1:1000 diluted 4',6-diamidinyl-2-phenylindole (DAPI) to each well for nuclear staining and incubate at room temperature in the dark for 15 min. Remove the DAPI and add 1 mL of PBS buffer to each well. Prepare and label the adhesion slides accordingly. Add a small drop of antifluorescence quencher to the center of each slide. Tilt the 12-well plate at 45°, pick up the cell smears with tweezers, and blot off excess liquid with a paper towel. Place the smears on the adhesion slides with antifluorescence quencher and mount them with colorless, quick-drying clear nail polish. The slide was observed and photographed under a confocal fluorescence microscope, and then stored in a refrigerator at 4°C in the dark.

[0040] 3. Results Analysis Fluorescence localization results as follows Figure 1 As shown, the four signal peptide-EGFP fusion proteins designed in this invention exhibit significant differences in subcellular localization. Both the full-length signal peptide fusion protein of the pMLS-EGFP control group and the fusion protein lacking amino acids 16-22 (pMLS-PCV2-Opt1-EGFP group) showed highly efficient mitochondrial co-localization: their green fluorescence highly overlapped with the red fluorescence of the mitochondrial marker protein TXN2-DsRed, mainly concentrated in the mitochondrial structures in the cytoplasm, with only a small amount distributed in the nucleus, indicating that these two groups of signal peptides have complete mitochondrial targeting function.

[0041] In contrast, the fusion protein lacking amino acids 23-28 (pMLS-PCV2-Opt2-EGFP group) showed a decrease in mitochondrial localization efficiency, manifested as weakened mitochondrial colocalization signal and relatively enhanced intranuclear fluorescence signal, suggesting that the loss of amino acids in this region may affect the localization specificity or efficiency of the signal peptide.

[0042] Crucially, the localization pattern of the fusion protein lacking amino acids 29-40 (pMLS-PCV2-ΔCore-EGFP group) was fundamentally altered: its green fluorescence was diffusely distributed in the nucleus and cytoplasm, showing no significant co-localization with the mitochondrial labeling signal, and no punctate aggregation was observed in the nucleolus. This result contrasts sharply with the localization pattern of the control group, strongly demonstrating that the region of amino acids 29-40 in the PCV2 MLS sequence (sequence "VHPRHRYRWRRK", SEQ ID NO. 23) is a key structural domain indispensable for maintaining its mitochondrial targeting function, and the deletion of this region will lead to the complete loss of localization ability of the signal peptide.

[0043] To further elucidate the functional residues within this critical region, a systematic mutation analysis was performed on several conserved sites. This included the mutation of the cholesterol-binding motif VxxxxYxxR to AxxxxAxxA, resulting in MLS-PCV2-mut-VYR (sequence: RRRPWLAHPRHRARWARK, SEQ ID NO. 6); the mutation of the positively charged basic amino acid clusters (32R, 34R, and 36R) and (38R, 39R, and 40K) to A, resulting in MLS-PCV2-Opt3 sequences RRRPWLVHPAHAYAWRRK (SEQ ID NO. 4) and MLS-PCV2-mut-RRK sequences RRRPWLVHPRHRYRWAAA (SEQ ID NO. 24); and the mutation of aromatic amino acids (27W and 37W) to A, resulting in the sequence MLS-PCV2-mut-WW (sequence: RRRPALVHPRHRYRARRK, SEQ ID NO. 24). NO.25), and histidine residues (30H and 33H) to obtain MLS-PCV2-mut-HH (sequence RRRPWLVAPRARYRWRRK, SEQ ID NO.26), the results showed that cholesterol binding sites and arginine play a decisive role in mitochondrial localization.

[0044] (1) After the cholesterol-binding motif VxxxxYxxR is mutated to AxxxxAxxA (as shown in SEQ ID NO.6), the localization pattern of the fusion protein is fundamentally changed: the green fluorescence is mainly concentrated in the cell nucleus and there is no significant co-localization with the mitochondrial marker, indicating that the motif is essential for maintaining the mitochondrial targeting function and its structural integrity cannot be destroyed.

[0045] (2) The C-terminal basic amino acid cluster 32 R, 34 R, and 36 After the R mutation to alanine (as shown in SEQ ID NO.4), the fusion protein was highly concentrated in the cytoplasm and well co-localized with mitochondrial markers, with extremely low nuclear and nucleolar signals. This result indicates that... 32 R, 34 R, and 36 R primarily mediates non-specific nuclear localization; its deletion not only does not affect mitochondrial targeting but also significantly enhances localization specificity, thereby yielding mitochondrial targeting signal peptide variants with greater application potential. Figure 2 ).

[0046] Example 4: Cellular safety assessment of mitochondrial-targeting fusion protein This embodiment evaluates the effect of expressing the mitochondrial-targeting fusion protein of the present invention on host cell viability.

[0047] 1. Experimental Grouping Experimental group: transfected with pMLS-PCV2-Opt1 plasmid (expressing MLS-PCV2-Opt1-EGFP fusion protein).

[0048] Control group: transfected with pcDNA3.1-EGFP empty vector plasmid (expressing only EGFP).

[0049] 2. Experimental Methods PK15 cells at 5×10 4 The plasmids were seeded at a density of 1 μg / well in 12-well plates, with 3 replicates. After 24 hours, the plasmids of the experimental and control groups were transfected (1 μg / well) according to the method in Example 3. After 6 hours of transfection, the medium was replaced with complete medium containing 10% FBS, and the culture was continued for 48 hours.

[0050] 3. Cell viability detection Collect the culture supernatant from each well and digest adherent cells with trypsin. Combine the supernatant with the cells, and count the dead cells and total cells separately. Calculate the cell mortality rate 48 hours after plasmid transfection into PK15 cells.

[0051] The results are as follows Figure 3 As shown, there was no statistically significant difference in cell death rate between the experimental group (pMLS-PCV2-Opt1-EGFP) and the control group (pcDNA3.1-EGFP). This indicates that the fusion protein expressed under the guidance of the signal peptide of this invention, when overexpressed in cells, did not produce detectable toxic effects on the normal proliferation activity of PK15 cells, demonstrating good biocompatibility.

[0052] 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 mitochondrial-targeting signaling peptide, characterized in that, Its amino acid sequence is shown in SEQ ID NO.2 or SEQ ID NO.

4.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the mitochondrial targeting signal peptide of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.8 or SEQ ID NO.

10.

4. A fusion protein, characterized in that, The fusion protein includes the mitochondrial targeting signal peptide as described in claim 1, and the target protein attached to its C-terminus.

5. The fusion protein according to claim 4, characterized in that, The target proteins include fluorescent proteins, enzymes, therapeutic proteins, antigenic proteins, and reporter proteins.

6. A recombinant vector, characterized in that, A nucleic acid molecule comprising the nucleic acid molecule of claim 2 or 3 or encoding the fusion protein of claim 4 or 5.

7. The use of the mitochondrial-targeting signal peptide of claim 1, the nucleic acid molecule of claim 2 or 3, the fusion protein of claim 4 or 5, or the recombinant vector of claim 6 in the preparation of a drug delivery system targeting mitochondria.

8. The use of the mitochondrial-targeting signal peptide of claim 1, the nucleic acid molecule of claim 2 or 3, the fusion protein of claim 4 or 5, or the recombinant vector of claim 6 in the preparation of a medicament for treating mitochondrial-related diseases.

9. The use of the mitochondrial targeting signal peptide of claim 1, the nucleic acid molecule of claim 2 or 3, the fusion protein of claim 4 or 5, or the recombinant vector of claim 6 in the preparation of a product for locating mitochondria or indicating mitochondrial position.

10. The use of the mitochondrial targeting signal peptide of claim 1, the nucleic acid molecule of claim 2 or 3, the fusion protein of claim 4 or 5, or the recombinant vector of claim 6 in the preparation of a product for studying the interaction between a target protein and mitochondria.

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