A reporter system for detecting molecular chaperone-mediated autophagy activity and its application

Abstract

The present invention discloses a reporter system for detecting molecular chaperone-mediated autophagy activity and its application. The reporter system uses the expression vector pLVX-IRES-puro as the basic framework, inserts the target gene CMA reporter sequence after the promoter of the pLVX-IRES-puro, and the CMA reporter sequence comprises a fluorescent protein, an S-Tag1 containing a pentapeptide motif KFERQ connected to the N-terminus of the fluorescent protein, and an S-Tag2 containing a pentapeptide motif KDRVQ connected to the C-terminus of the fluorescent protein; wherein the sequence of the expression vector pLVX-IRES-puro is shown in SEQ ID NO.1. The reporter system has higher expression efficiency and more sensitive detection of CMA activity, and can be used to monitor the activity changes of CMA in the occurrence and development of diseases in real time.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and biofluorescence imaging, and particularly relates to a reporter system for detecting molecular chaperone-mediated autophagy activity and an application thereof. Background Art

[0002] Chaperone-mediated autophagy (CMA) is the only type of selective autophagy in mammalian cells. Its selectivity relies on the recognition and binding of the pentapeptide motif of the target protein by the molecular chaperone protein HSC70, forming an HSC70 / substrate protein complex that translocates to the surface of CMA-active lysosomes. The substrate protein complex then transfers to the tail of the single-span lysosomal-associated membrane protein type 2A (LAMP2A), triggering its assembly into a multimeric molecular transport channel, through which the substrate protein complex enters the lysosomal lumen for degradation. This unique transport mechanism and substrate selectivity underlie CMA's dynamic protein clearance.

[0003] However, existing reporter systems for detecting chaperone-mediated autophagy activity have low expression efficiency and are not sensitive enough to monitor CMA activity, thus limiting their application in CMA-related research. Therefore, a reporter system with higher expression efficiency and more sensitive and accurate detection of CMA activity is urgently needed. Summary of the Invention

[0004] The purpose of the present invention is to overcome the defects of the existing CMA activity reporting system, such as low expression efficiency and insufficient detection sensitivity.

[0005] In order to achieve the above object, the present invention provides a reporter system for detecting molecular chaperone-mediated autophagy activity, wherein the reporter system uses the expression vector pLVX-IRES-puro as the basic framework, and inserts the target gene CMA reporter sequence after the promoter of the pLVX-IRES-puro, wherein the CMA reporter sequence comprises a fluorescent protein, an S-Tag1 containing a pentapeptide motif KFERQ connected to the N-terminus of the fluorescent protein, and an S-Tag2 containing a pentapeptide motif KDRVQ connected to the C-terminus of the fluorescent protein;

[0006] Wherein, the sequence of the expression vector pLVX-IRES-puro is shown as SEQ ID NO.1.

[0007] Preferably, the fluorescent protein comprises any one of EGFP, mCherry, and Dendra2.

[0008] Preferably, the fluorescent protein is Dendra2, and the sequence of Dendra2 is shown in SEQ ID NO.2.

[0009] Preferably, the reporter system further comprises a plurality of rigid linkers, wherein at least one rigid linker is connected between the S-Tag1 containing the pentapeptide motif KFERQ and the fluorescent protein, and at least one rigid linker is connected between the S-Tag2 containing the pentapeptide motif KDRVQ and the fluorescent protein.

[0010] Preferably, the expression vector pLVX-IRES-puro contains at least the replication origin Ori, promoter promoter, internal ribosome entry site sequence IRES and / or RRE, non-fusion resistance marker gene puro, ampicillin antibiotic resistance gene AmpR, retroviral cis-regulatory element HIV-1ψ, and 3'LTR enhancer sequence.

[0011] Preferably, the promoter is a CMV promoter.

[0012] The present invention also provides a recombinant cell line comprising a reporter system for detecting molecular chaperone-mediated autophagy activity as described above.

[0013] Preferably, the host cells of the recombinant cell line include at least any one of mouse and human primary cells, mouse and human tumor cells, and continuous cell lines cultured in vitro.

[0014] The present invention also provides an application of a reporter system for detecting molecular chaperone-mediated autophagy activity as described in any one of the above items, wherein the reporter system for detecting molecular chaperone-mediated autophagy activity is used to monitor changes in CMA activity in real time during pathophysiological processes such as neurodegenerative diseases, retinal degenerative lesions, cancer, and aging.

[0015] Preferably, the reporter system for detecting chaperone-mediated autophagy activity is used to evaluate the intervention effects of drugs and genetic modification on CMA activity.

[0016] Beneficial effects of the present invention:

[0017] (1) Compared with the original reporter system based on the expression vector pPS-CFP2-N, the reporter system of the present invention uses the expression vector pLVX-IREX-puro as the basic skeleton, adds the internal ribosome entry site sequence IRES and RRE sequence, and effectively enhances the translation efficiency of the target protein.

[0018] (2) The original reporter system contains a Kozak motif between the S-Tag connected at the N-terminus and the fluorescent protein Dendra2. Therefore, in the original reporter system, S-Tag-Dendra2 and Dendra2 without S-Tag are expressed independently. Dendra2 without S-Tag cannot exhibit CMA activity, thus reducing the proportion of S-Tag-Dendra2 that can exhibit CMA activity. The Kozak motif in front of the fluorescent protein Dendra2 is removed in this reporter system, thereby reducing the independent expression of Dendra2 and increasing the proportion of S-Tag-Dendra2 protein targeted by CMA and the translation efficiency.

[0019] (3) This reporter system inserts a rigid linker and a CMA-targeted pentapeptide motif at the C-terminus of the fluorescent protein Dendra2, so that the CMA recognition motif can be stably separated from the fluorescent protein Dendra2. The present invention inserts a CMA-targeted pentapeptide motif at the N-terminus and C-terminus, respectively, thereby better exposing the CMA recognition motif at the N-terminus and C-terminus of the fluorescent protein Dendra2, further increasing the efficiency of the fluorescent protein reporter system being recognized by HSC70.

[0020] (4) Since CMA is involved in the regulation of many diseases, this reporter system can be used to monitor CMA activity changes in real time during pathological processes such as neurodegenerative diseases, retinal degeneration, and cancer, as well as physiological processes such as development and aging. It can also be used to evaluate the effectiveness of drug or genetic modification interventions on CMA activity. The structure of this reporter system has been improved compared to traditional reporter systems, with higher expression efficiency and more sensitive detection of CMA activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the structure of the reporter system pLVX-KFERQ-Dendra2-KDRVQ for detecting molecular chaperone-mediated autophagy activity of the present invention.

[0022] Figure 2 Schematic diagram of three CMA reporter sequences, among which CMA reporter2 is an improvement based on CMA reporter1, and CMA reporter3 is an improvement based on CMA reporter2.

[0023] Figure 3 Comparative graphs and statistical graphs of bioluminescence imaging of CMA activity monitoring effects after the human cervical cancer cell line (Hela) was transfected with three CMA reporter systems and then treated with serum starvation, CMA activator, and etoposide.

[0024] Figure 4Comparative graphs and statistical graphs of bioluminescence imaging of CMA activity monitoring effects after three CMA reporter systems were transfected into primary mouse embryonic fibroblasts (MEFs) and then treated with serum starvation, CMA activator, and etoposide. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described below with reference to the accompanying drawings and examples. Experimental materials, experimental instruments, and experimental methods not mentioned in the present invention are all commonly used in the art.

[0026] In recent years, CMA has been increasingly implicated in disease regulation, including aging-related diseases such as neurodegenerative diseases, retinal degeneration, and cancer. Existing reporter systems for detecting CMA activity contain a Kozak motif between the N-terminal S-Tag and the fluorescent protein Dendra2. Consequently, S-Tag-Dendra2 and the fluorescent protein Dendra2 are expressed independently, reducing the proportion of S-Tag-Dendra2 that can exhibit CMA activity and resulting in lower expression efficiency of S-Tag-Dendra2 that can exhibit CMA activity. Therefore, a reporter system capable of more sensitive and accurate detection of CMA activity is crucial for studying changes in CMA activity during disease development.

[0027] Explanation of terms:

[0028] Internal ribosome entry site (IRES): A cis-acting RNA sequence that mediates the internal entry of the 40S ribosomal subunit on certain eukaryotic and viral messenger RNAs upstream of the translation start codon. While translation of eukaryotic mRNA generally requires a 5' cap to mediate ribosome binding, there are some exceptions in eukaryotic organisms and viruses. For example, some genes have a short RNA sequence (approximately 150-250 bp) at their 5' end that folds into a structure similar to the initiator tRNA, thereby mediating ribosome binding to the RNA and initiating protein translation. This non-translated RNA sequence is called an internal ribosome entry site (IRES), which plays a role in regulating the translation initiation of the target gene and enhancing the efficiency of independent translation of the target gene in overexpression vectors.

[0029] The Rev Response Element (RRE) of HIV is a highly structured RNA segment of approximately 350 nucleotides that is present within the HIV mRNA sequence. In the presence of the HIV-1 accessory protein Rev, HIV-1 mRNA containing the RRE is exported from the nucleus to the cytoplasm for further translation and viral packaging. This element is essential for the efficient transport and packaging of the viral genome into the cytoplasm.

[0030] Because this application has improved the original system, in order to compare whether the improved system has better recognition efficiency than the original system, the requirements for the fluorescence intensity of the fluorescent protein are higher. The fluorescence intensity of the fluorescent protein Dendra2 is higher than that of other fluorescent proteins such as EGFP and mCherry. In addition, the fluorescent protein Dendra2 is a fluorescent protein that can achieve photoconversion. Under normal circumstances, it is in the green light form, but when it is excited by a 405nm ultraviolet light, the fluorescent protein Dendra2 will convert into red light. Therefore, CMA activity can be judged by observing green fluorescent spots. In the case where the background intensity of homogeneous Dendra in the cytoplasm is too high, making it difficult to observe green fluorescent spots, CMA activity can also be judged by observing red fluorescent spots by the photoconversion method, thereby providing two visual methods for quantifying CMA activity. The sequence of the fluorescent protein Dendra2 of this application is shown in SEQ ID NO.2.

[0031] Aiming at the defects of the existing reporting system, this application has been modified and the original reporting system is named CMAreporter1. Figure 2 As shown in the figure, first, the Kozak sequence connecting the N-terminal S-Tag and the fluorescent protein Dendra2 in the original reporter system was removed, and a rigid linker was inserted between the N-terminal S-Tag and the fluorescent protein Dendra2. The resulting reporter system was named CMA reporter2. CMA reporter2 was further modified by inserting a rigid linker and the CMA-targeted pentapeptide motif KDRVQ at the C-terminus of the fluorescent protein Dendra2 to obtain the reporter system CMA reporter3, as shown in the figure. Figure 1As shown, the expression vector pLVX-IRES-puro is used as the basic skeleton, and the target gene CMA reporter sequence is inserted after the promoter of the pLVX-IRES-puro. The CMA reporter sequence comprises a fluorescent protein, an S-Tag1 containing a pentapeptide motif KFERQ connected to the N-terminus of the fluorescent protein, and an S-Tag2 containing a pentapeptide motif KDRVQ connected to the C-terminus of the fluorescent protein. The sequence of the expression vector pLVX-IRES-puro is shown in SEQ ID NO.1, and the reporter system pLVX-KFERQ-Dendra2-KDRVQ is obtained. The sequence of the reporter system pLVX-KFERQ-Dendra2-KDRVQ is shown in SEQ ID NO.3. The expression vector pLVX-IRES-puro contains at least the replication origin Ori, the CMV promoter, an internal ribosome entry site (IRES) sequence and / or an RRE sequence, the non-fusion resistance marker gene puro, the ampicillin antibiotic resistance gene AmpR, the retroviral cis-regulatory element HIV-1ψ, and a 3'LTR enhancer sequence. CMV has a strong ability to initiate transcription of the target gene and enhance translation of the target protein. The sequence of the CMV promoter is shown in SEQ ID NO. 4, the sequence of the internal ribosome entry site (IRES) sequence is shown in SEQ ID NO. 5, the sequence of the RRE sequence is shown in SEQ ID NO. 6, and the sequence of the 3'LTR enhancer is shown in SEQ ID NO. 7.

[0032] The improved reporter system CMA reporter3 structure is pLVX-KFERQ-Dendra2-KDRVQ. Both KFERQ and KDRVQ can be replaced by other pentapeptide motifs similar to KFERQ, as this motif is not unique. The CMA recognizes a certain arrangement of amino acids in the pentapeptide motif, among which glutamine Q is required. At either end of the motif, the other four amino acids are composed of the following three types: one or two negatively charged amino acids (aspartic acid D or glutamic acid E), one or two positively charged amino acids (lysine K or arginine R), and one or two hydrophobic amino acids (phenylalanine F, isoleucine I, leucine L, valine V).

[0033] The three reporter systems, CMA reporter1, CMA reporter2, and CMA reporter3, were tested for their CMA activity under conditions known to activate CMA, such as serum starvation, DNA damage stimulation, and CMA activators.

[0034] Experimental plasmids and cells :

[0035] pSPAX2 (nucleotide sequence as shown in SEQ ID NO.8), PMD2.G (nucleotide sequence as shown in SEQ ID NO.9), CMA reporter 1 (nucleotide sequence as shown in SEQ ID NO.10), CMA reporter 2 (nucleotide sequence as shown in SEQ ID NO.11), CMA reporter3 (nucleotide sequence as shown in SEQ ID NO.12), 293T cells (derived from the Chinese Academy of Sciences), human cervical cancer cell line (derived from the Chinese Academy of Sciences), and mouse primary embryonic fibroblasts (obtained from fresh lung tissue isolated from mice).

[0036] Experimental methods :

[0037] pSPAX2, PMD2.G, CMA reporter 1 / CMA reporter2 / CMAreporter3 were transfected into 293T cells at a ratio of 2:1:3 using a three-plasmid transfection system. Lentiviral supernatants were collected 48 hours after transfection. Human cervical cancer cell lines (Hela) and primary mouse embryonic fibroblasts (MEF) in good condition were prepared in advance and evenly plated into cell culture dishes for overnight culture. After 18-24 hours of culture, the cell culture medium (containing 8ug / ml polybrene) was replaced, and CMA reporter 1, CMA reporter 2, CMA reporter 3, and viral supernatant were added to Hela and MEF cells, respectively, to obtain cell lines stably expressing the above-mentioned reporter system.

[0038] Cell lines expressing different reporter systems were subjected to serum starvation, CA (CMA activator, sourced from Selleck, CAS#S6797), and etoposide (DNA damage stimuli). Serum starvation was performed for 16 hours. CA activators were treated with 10 μM CA for 24 hours. DNA damage stimuli were treated with 10 μM etoposide (sourced from MCE, CAS#HY-13629) for 24 hours. CMA activity was monitored using bioluminescence imaging after treatment.

[0039] Experimental results :

[0040] like Figure 3The figure shows the changes in CMA activity in three CMA reporter systems observed in human cervical cancer cell lines. According to the quantitative statistical results, under conditions of CMA activation such as serum starvation, CA (CMA activator), and Etoposide (DNA damage), compared with the original CMA reporter system CMA reporter1, each step of the transformation in this application significantly increased the number of Dendra2 fluorescent protein spots appearing in the cells, that is, CMA reporter3>CMA reporter2>CMA reporter1. The experimental results show that the reporter system for detecting molecular chaperone-mediated autophagy activity provided by this application significantly improves the sensitivity of CMA activity detection.

[0041] like Figure 4 The figure shows the changes in CMA activity observed in three CMA reporter systems in primary mouse embryonic fibroblasts. According to the quantitative statistical results, under conditions of CMA activation such as serum starvation, CA (CMA activator), and Etoposide (DNA damage), each step of the modification in this application significantly increased the number of Dendra2 fluorescent protein spots appearing in the cells compared to the original CMA reporter system CMA reporter1, that is, CMA reporter3>CMA reporter2>CMA reporter1. The experimental results show that the reporter system for detecting molecular chaperone-mediated autophagy activity provided by this application significantly improves the sensitivity of CMA activity detection.

[0042] The present invention provides a recombinant cell line comprising the above-mentioned reporter system for detecting molecular chaperone-mediated autophagy activity. The host cells of the recombinant cell line include mouse and human primary cells such as hematopoietic stem cells, mesenchymal stem cells, T cells, B cells, macrophages, nerve cells, fibroblasts, etc., tumor cells include mouse and human lung cancer cells, liver cancer cells, colorectal cancer cells, pancreatic cancer cells, cervical cancer cells, ovarian cancer cells, leukemia cells, melanoma cells, etc., and in vitro cultured continuous cell lines such as human embryonic kidney cells, mouse and human microglia, mouse monocytes and macrophages, human hepatic stellate cells, etc.

[0043] This reporter system for detecting molecular chaperone-mediated autophagy activity can be used to monitor changes in CMA activity in real time during pathophysiological processes such as neurodegenerative diseases, retinal degeneration, cancer, development, and aging, as well as to evaluate the intervention effects of drugs or genetic modification on CMA activity.

[0044] In summary, the Kozak sequence connecting the N-terminal S-tag and Dendra2 in the original reporter system was removed; a rigid linker was inserted between the N-terminal S-tag and Dendra2; and a rigid linker and the CMA-targeting pentapeptide motif, KDRVQ, were inserted at the C-terminus of Dendra2, resulting in a new CMA activity reporter system. This system further improves translation efficiency and increases the efficiency of fluorescent protein recognition by HSC70. This reporter system can be used to monitor changes in CMA activity in real time, study changes in CMA activity during disease development, and evaluate the effectiveness of drug interventions on CMA activity, possessing significant practical application value.

[0045] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A reporter system for detecting molecular chaperone-mediated autophagy activity, characterized in that: The reporter system uses the expression vector pLVX-IRES-puro as the basic skeleton, and inserts the target gene CMA reporter sequence after the promoter of the pLVX-IRES-puro. The CMA reporter sequence comprises a fluorescent protein, an S-Tag1 containing a pentapeptide motif KFERQ connected to the N-terminus of the fluorescent protein, and an S-Tag2 containing a pentapeptide motif KDRVQ connected to the C-terminus of the fluorescent protein, thereby obtaining the reporter system pLVX-KFERQ-Dendra2-KDRVQ. The sequence of the reporter system pLVX-KFERQ-Dendra2-KDRVQ is shown in SEQ ID NO.

3. Wherein, the sequence of the expression vector pLVX-IRES-puro is shown as SEQ ID NO.1; The reporter system further comprises a plurality of rigid linkers, wherein at least one rigid linker is connected between the S-Tag1 containing the pentapeptide motif KFERQ and the fluorescent protein, and at least one rigid linker is connected between the S-Tag2 containing the pentapeptide motif KDRVQ and the fluorescent protein.

2. A recombinant cell line comprising the reporter system for detecting molecular chaperone-mediated autophagy activity according to claim 1.

3. The recombinant cell line according to claim 2, wherein The host cells of the recombinant cell line include at least any one of mouse and human primary cells, mouse and human tumor cells, and continuous cell lines cultured in vitro.

4. A use of the reporter system for detecting molecular chaperone-mediated autophagy activity according to claim 1, characterized in that: The reporter system for detecting molecular chaperone-mediated autophagy activity is used to monitor changes in CMA activity in real time during the pathophysiological processes of neurodegenerative diseases, retinal degenerative lesions, cancer, development, and aging.

5. The use according to claim 4, characterized in that The reporter system for detecting molecular chaperone-mediated autophagy activity is used to evaluate the intervention effect of drugs or genetic modification on CMA activity.

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