KR20200013419A - Detection of autophagic body binding protein in vivo - Google Patents

Abstract

The present invention relates to a probe for detecting autophagosome. More specifically, the present invention relates to a probe which specifically detects autophagosome using an LC3-interacting region (LIR) of TP53INP2, which is a protein capable of binding to LC3, and to a use thereof.

Description

Detection method of autophagy binding protein in vivo {Detection of autophagic body binding protein in vivo}

The present invention relates to a method for detecting autophagy binding protein in vivo, and more specifically, LC3 / GABARAP, which is an autophagy binding protein, using a probe including a nuclear localization signal (NLS). It is about checking whether the family and LIR (LC3-interacting region) motifs are combined.

Autophagy plays an important role in cellular functions such as hunger survival, protection from infectious bacteria and regulation of neurodecay, which is an evolutionarily conserved process in all eukaryotic cells from yeast to mammals. appear.

So far, autophagy is known to be divided into three stages: induction / nucleation, membrane elongation / closure, and vesicle trafficking / maturation / fusion / degradation. Initially, the cytoplasm and intracellular organs degenerate into a double-membrane binding structure called autophagosome, and the degraded autopagosome fuses with lysosomes to form autolysosomes. The degenerated material is hydrolyzed and reused. However, the molecular mechanism of action inherent in this process has not been elucidated yet.

Through a variety of genetic and biochemical studies conducted in yeast, fruit flies, and the like, proteins needed to cause autophagy in cells have been identified, and studies to analyze their correlations have been conducted. These studies on autophagy have recently played an important role in genetic analysis of various animal models and diseases. The autophagy process plays an important role in various diseases such as diabetes, metabolic diseases such as hypertension, degenerative brain diseases, infectious diseases, and cancer. This report highlights its importance. Therefore, the method of obtaining information on the autophagy action may play a very important role in the development of disease therapeutics targeting new action points.

As key proteins that function in autophagy, LC3 / GABARAP proteins are known. One type of ATG8 is known in yeast, and six types of mammals are known as LC3A, LC3B, LC3C, GABARAP, GABARAP-L1, and GABATRAP-L2. These proteins are usually in the cytoplasm. When the autophagy is produced, the lipid PE binds to the autophagy membrane. Many proteins then bind to LC3 or GABARAP proteins and position themselves as autophages to function. Proteins that bind to LC3 or GABARAP proteins have LIR (LC3-interacting region) sites and commonly have W / F / Y-X-X-I / L / V sequences. To determine whether the LIR site is actually bound to LC3 or GABARAP protein, GST pulldown (in vitro) and co-immunoprecipitation (in vivo) can be confirmed at present. It has a bad disadvantage. In order to improve this problem, the present inventors have completed the present invention for a method capable of confirming autophagy binding in large quantities in in-vivo.

1. Korea Patent Registration 10-1524426 2. Korean Patent Publication 10-2015-0129166 3. Korea Patent Publication 10-2015-0039894 3. Korea Patent Publication 10-2015-0039894

Lee et al (2017), Development of LC3 / GABARAP sensors containing a LIR and a hydrophobic domain to monitor autophagy, EMBO J, 13; 36 (8): 1100-1116.

An object of the present invention is to determine the binding of the autophagy and LIR motifs directly in vivo, LC3-interacting region (LIR) motif sequence that binds to the LC3 / GABARAP family protein autophagy binding protein; Nuclear position signal sequence; And it provides a probe for autophagy detection comprising the gene of the reporter fluorescent protein.

Another object of the present invention is to provide a method for detecting a new LIR motif having binding to LC3 / GABARAP protein, which is an autophagy binding protein, using the probe.

Another object of the present invention is to provide information on autophagy through the ratio of the expression level of the reporter fluorescent gene bound to the probe and the expression level of the reporter fluorescent gene bound to the LC3 protein present in the cytoplasm To provide.

In order to solve the above problems of the present invention, an object of the present invention is to bind to the LC3 / GABARAP family protein, which is an autophagy binding protein, in order to confirm the binding of the autophagy and LIR motifs directly in vivo. LIR (LC3-interacting region) motif sequence; Nuclear position signal sequence; And it provides a probe for autophagy detection comprising the gene of the reporter fluorescent protein.

The LC3 / GABARAP family protein is, but is not limited to, one or more selected from the group consisting of LC3A, LC3B, LC3C, GABARAP, GABARAP-L1, and GABARAP-L2.

In addition, the LC3-interacting region (LIR) motifs that bind to the LC3 / GABARAP family protein are ATG13, FYCO1, NBR1, Nix / Bnip3L, p62, TP53INP1, TP53INP2 / DOR, ULK1, ULK2, Optineurin, FUNDC1, TBC1D5 LIR1, TBC1D5 LIR2, c-Cbl, β-catenin, Dvl2, MAP15K, Bnip3, ATG4B, Tax1bp1 / T6BP, Stbd1, NDP52, Calreticulin, Clathrin HC, FIP200 and TBC1D25 may be included in the LIR protein selected from the group Certain sequences in the motifs are induced to have binding to LC3 / GABARAP family proteins. Although not limited thereto, the probes prepared in the examples of the present invention were designed to include the LIR motif sequence of the TP53INP2 or Fyco1 protein.

The probe of the present invention comprises a nuclear position signal sequence. “Nuclear localization signal” (NLS) refers to an amino acid sequence that carries a specific substance, such as a protein or nucleic acid, into the cell nucleus, and serves to allow the probe of the present invention to be moved to and positioned in the nucleus. To perform. Although not limited thereto, in one embodiment of the present invention, the nuclear position signal includes the amino acid sequence of SEQ ID NO: 3 (3 × NLS), which is derived from the vector of FIG. 8.

In one embodiment of the present invention, examples of the reporter protein are green fluorescent protein (GFP), modified green fluorescent protein (mGFP), enhanced green fluorescent protein (enhanced green fluorescent protein). EGFP), red fluorescent protein (RFP), modified red fluorescent protein (mRFP) and the like, preferably enhanced green fluorescent protein (EGFP) or modified red fluorescent protein ( mRFP) can be used. By including the reporter protein in the probe of the present invention, it is possible to confirm whether the LC3 / GABARAP family protein is directly bound to the LIR motif while maintaining the cells.

In particular, the reporter protein binding to the LIR and the nuclear position signal coupled to the LC3 / GABARAP family protein present in the cytoplasm can be produced to have a different color, through which the LC3 / GABARAP family protein located in the nucleus and The C / N ratio of the LC3 / GABARAP family protein located in the cytoplasm can provide information about the LIR binding to the LC3 / GABARAP family protein.

In one embodiment of the present invention, the autophagy detection probe is a LIR (LC3-interacting region) region (“LIR (FYCO1)” region of the FYCO1 gene consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2). The LIR site of the TP53INP2 gene (“LIR (TP53INP2)”) consisting of the amino acid sequence; MRFP as a reporter protein; And 3xNLS of SEQ ID NO- may be a structure coupled to, but is not limited thereto, the probe may be represented by LIR (Fy or TP) -mRFP-3xNLS.

In one embodiment of the present invention, the probe is located in the nucleus due to the effect of 3xNLS, can be introduced into the nucleus due to the binding force of the EGFP-LC3 / GABARAP and LIR present in the cytoplasm, and, as a result, By calculating the ratio of reporter protein observed in the nucleus (C / N ratio), it can be used for autovesicle detection.

In addition, the present invention is not limited thereto, but the detection may be performed in-vitro like the GST pulldown-assay or may be performed in-vivo. Conventional detection methods have been analyzed by GST pulldown-assay, but this is difficult to perform in-vivo. However, using the probe and the detection method of the present invention, since the cell can be detected even in the state of holding the cell, it has the advantage of detecting the autoendoplasmic reticulum even in in-vivo.

In addition, the present invention, using the probe or the composition, characterized in that it comprises the step of confirming the expression of autophagy in the cell through the cytoplasm / nucleus ratio (c / n ratio) of the reporter fluorescent protein It provides a way to provide information about child actions.

The present invention solves the problems of the conventional autophagy detection method to enable the detection of autophagy in a more accurate and simple way to obtain and utilize a variety of information in the living body related to the child action.

Figure 1 is a schematic diagram showing the binding of LIR (Fyco1 and TP53INP2) -mRFP-3xNLS and EGFP-mATG8, and mutations thereof. Here, ATG8 is mammalian-derived Atg8 protein, LIR means LC3-binding site, NLS means nuclear position signal (sequence).
Figure 2 shows the results confirmed in vitro that the LIR sites derived from Fyco1 and TP53INP2 and LC3, GABARAPs, respectively. A and C confirm the binding to GFP-labeled LIR by GST pull-down assay. Coprecipitated LIR-GFP protein was analyzed by Western blot. Immobilized GST-fusion protein was confirmed via Coomassie blue stain gel. B and D digitize the results of A and C.
3 is a schematic of mATG mutations and probes containing LIR and LIR mutations derived from Fyco1 in cells.
4 is a result of confirming the position of the probe and mATG mutations including the LIR and LIR mutations derived from Fyco1 in mouse embryonic fibroblasts.
FIG. 5 illustrates the position of FIG. 4 numerically indicated by the C / N ratio according to the intensity of the fluorescent marker. FIG.
Figure 6 is a schematic of mATG8 mutations and probes containing LIR and LIR mutations derived from TP53INP2 in the cell. .
Figure 7 shows the results of confirming the position of the probe and mATG8 mutations including LIR and LIR mutations derived from TP53INP2 in mouse embryonic fibroblasts.
FIG. 8 illustrates the position of FIG. 4 numerically indicated by the C / N ratio according to the intensity of the fluorescent marker. FIG.
Figure 9 shows a cleavage map of the vector derived 3xNLS of the present invention.

Definitions of terms used in the present specification are as follows.

"Autophagy is the process of altering and regulating intracellular components in a lysosomal-dependent manner, eliminating defective large protein complexes and intracellular organelles, which play an important role in cell growth, survival and homeostasis. Autophagy is important for the maintenance of cellular homeostasis through intracellular fluctuations of damaged proteins and organelles. As a result, autophagy can lead to the accumulation of misfolded proteins resulting in neurodegenerative diseases. Occurs (Komatsu et al., (2006), Nature, 441, 880-884).

By "autophagy related disease" is meant a disease that is caused or worsened by the involvement of a child. Although not particularly limited thereto, preferably malignant tumors, neurodegenerative diseases, ischemic diseases, diabetes, pulmonary fibrosis, and the like, and more preferably, malignant tumors such as breast cancer, lung cancer, fibrosarcoma, and colon cancer; Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease; Ischemic diseases such as angina pectoris, myocardiac infarction, cardiac failure, ischemic colitis, ischemic acute renal failure, and acute ischemic stroke; Diabetes, type 1 diabetes, type 2 diabetes, pulmonary fibrosis, and the like.

"Diagnostic" means identifying the presence or characteristic of a pathological condition. In particular, the present invention is useful for providing information on autophagy-related diseases. In the present invention, the increase and decrease characteristics of the autophagy number can be used as a useful indicator (diagnosis marker).

A "diagnosis marker or probe" is a substance that can diagnose autophagy by distinguishing it from other cells. Polypeptides show an increase or decrease in autophagy in the presence of autophagy-related diseases. Or organic biomolecules such as nucleic acids (eg mRNA), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, polysaccharides, etc.) and the like.

"Probe" refers to a nucleic acid fragment, such as RNA or DNA, that corresponds to a few bases to hundreds of bases that can achieve specific binding with mRNA. Probes can be made in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes and the like. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.

"Subject" or "patient" means any single individual in need of treatment, including humans, cattle, dogs, guinea pigs, rabbits, chickens, insects, and the like. Also included are any subjects who participated in clinical research trials showing no disease clinical findings, or subjects who participated in epidemiologic studies or used as controls.

"Tissue or cell sample" means a collection of similar cells obtained from a tissue of a subject or patient. Sources of tissue or cell samples may include solid tissue from fresh, frozen and / or preserved organ or tissue samples or biopsies or aspirates; Blood or any blood component; Cells at any time of pregnancy or development in the subject. Tissue samples may also be primary or cultured cells or cell lines.

"Nucleic acid" is meant to include any DNA or RNA, such as chromosomes, mitochondria, viruses and / or bacterial nucleic acids present in tissue samples. One or both strands of a double-stranded nucleic acid molecule and any fragment or portion of an intact nucleic acid molecule.

"Gene" means any nucleic acid sequence or portion thereof that has a functional role in protein coding or transcription or in the regulation of other gene expression. The gene may consist of any nucleic acid encoding a functional protein or only a portion of a nucleic acid encoding or expressing a protein. Nucleic acid sequences can include gene abnormalities in exons, introns, initiation or termination regions, promoter sequences, other regulatory sequences, or unique sequences adjacent to genes.

A "protein" is also intended to include fragments, analogs and derivatives of a protein that retain essentially the same biological activity or function as the reference protein.

"Transformation, transfection or transfection" refers to the process by which extracellular DNA enters a host cell in the presence or absence of an accompanying substance. A “transfected cell” refers to a cell in which extracellular DNA is introduced into the cell and has extracellular DNA. DNA can be introduced into cells so that nucleic acids can be inserted into chromosomes or replicated into extrachromosomal material. Cells into which external DNA is introduced are called "transformers."

The term "vector" refers to any nucleic acid comprising a competent nucleotide sequence that is inserted into a host cell, recombined with and inserted into the host cell genome, or spontaneously replicates as an episome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and the like.

By "label" or "label" is meant a compound or composition that directly or indirectly facilitates detection of a reagent conjugated to, or fused to, conjugated to, or fused to a reagent, such as a nucleic acid probe or antibody. The label may itself be detected (eg, a radioisotope label or fluorescent label) or, in the case of an enzyme label, may catalyze the chemical modification of the detectable substrate compound or composition.

"About" means 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4 for reference quantities, levels, values, numbers, frequencies, percentages, dimensions, sizes, quantities, weights, or lengths. , Amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length, varying by about 3, 2 or 1%.

Throughout this specification, the terms “comprises” and “comprising”, unless otherwise indicated in the context, include any given step or element, or group of steps or elements, but any other step or element, or step or element It should be understood that this group is not excluded.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated concretely.

The present invention relates to a new probe for detecting autophagosomes, and more specifically to autologous phagocytosis using the LIR site and nuclear position signal sequence (NLS), a protein capable of binding to LC3. Specific detection probes and uses thereof.

Autophagy is a process in which cellular constructs are removed through lysosomes. Physiologically, autophagy plays a role in balancing organelles and proteins. Autophagy has been shown to mediate the development of disease and to be involved in the crosstalk of free radicals and free radicals (ROS / RNS) that occur during intracellular signaling and protein damage.

Autophagy is classified into three categories depending on how the substrate reaches lysosomes: (i) Macroautophagy-Commonly referred to as autophagy is called autophagy. It is a method of forming autophagosomes. (ii) Desporation-Lysosomes directly surround and digest cellular constructs. (iii) Chaperone-mediated autophagy-chaperone-bound protein complexes bind to LAMP-2A receptors, bringing target proteins to lysosomes for degradation

In mammalian cells, decreased nutrients inhibit the target of rapamy-cin (TOR), which results in dephosphorylated atg13. Dephosphorylated atg13 binds to and activates atg1 homologue. Activated atg1 recruits to FIP200. This protein complex extends the membrane to induce the completion of the autophagosome structure.

In order to achieve autophagy structure, beclin-1-class III PI3K complex is required and LC3 (light chain 3) -II must be inserted into the membrane of autophagy. LC3 is a protein similar to ubiquitin and LC3 is post translational modifications to LC3-II. LC3 is truncated by Atg4 to LC3-I and exposes glycine residues at the C terminus. After LC3-I and PE (phosphatydilethanolamine) is combined to form LC3-II. This process is essential for autophagy expansion

A typical feature of autophagy is the formation of autophagosomes of varying sizes (0.5-1.5 um) depending on the signal, target, and cell type inducing autophagy. Yeast genetic screens have discovered more than 35 autophagy-related (Atg) genes that regulate the various processes of autophagy formation.

Therefore, it is possible to obtain various information on autophagy-related diseases by detecting the formed autophagy, and the present invention relates to a probe for detecting such autophagy.

To determine if autophagy is actually taking place, the most traditional method is to observe the autophagy at each stage of maturity with an electron microscope. Another method is to measure the rate of degradation of long-lived proteins using the PULSE-CHASE method. Methods based on LC3 have also been widely used to measure autophagy. LC3 is converted to LC3-II in combination with PE. LC3-I and II can be distinguished by differences in mobility in electrophoresis. The incorporation of LC3-II into the autophagy membranes consistently occurs when autophages are formed and the amount of LC3 reflects the relative amount of intracellular autophages. When combined with autophagy, LC3-II proteins appear as tiny dots, like vesicles, when observed by cell immunostaining.

In the past, RFP / GFP-LC3 with GFP or RFP attached to LC3 directly involved in autophagy was generally used to label autophagy. This uses the principle that RFP is resistant to acidic conditions while GFP is weakened in acidic conditions. LC3, which is a combination of GFP and RFP, emits GFP and RFP light simultaneously in lysosomal non-lysosomal autophagy, but only when RFC is matured.

However, since LC3 itself is directly involved in autophagy formation, various side effects due to LC3 overexpression were inevitable.

In addition, the above method is a method that can be performed only in-vitro, and there is a problem that it is cumbersome to be used for diagnosis or detection of disease using autophagy.

The present invention relates to novel autophagosome detection probes and their use, including LC3-interacting region (LIR) sites and nuclear position signal (NLS) sequences of certain proteins, which can solve these problems.

The nuclear localization signal (NLS) peptide includes a sequence related to the NLS function as a minimum sequence and may further include other sequences. As used herein, the term “nuclear localizationsignal” refers to an amino acid sequence that serves to transport a specific substance, such as a protein or nucleic acid, into the cell nucleus. It may be prepared by a method commonly used in the art.

In one embodiment of the present invention, the nuclear localization signal (NLS) peptide may be derived from a human cell, and more specifically, may include the peptide sequence of SEQ ID NO.

In one aspect, the present invention relates to a probe for autophagosome detection. The probe construct can be used to identify intracellular phagocytosis levels and provide a variety of information about the progeny.

In one embodiment, a probe of the present invention may include a LC3-interacting region (LIR) region of a Tumor Protein P53 Inducible Nuclear Protein 2 (TP53INP2) or FYVE1 (FYVE And Coiled-Coil Domain Containing 1) gene; 3 × NLS Peptides; And reporter fluorescent protein.

The TP53INP2 and FYCO1 peptide sequences may be obtained from an amino acid sequence expressed from an animal, preferably human known gene sequence information, and the NLS peptide may be derived from human cells. Such genes or proteins include variants by wild type as well as deletions, insertions, non-conservative or conservative substitutions or combinations thereof.

The gene encoding the protein, etc., by referring to the nucleotide sequence known in the art, deletes the oligonucleotide complementary to its terminal portion, and then uses the primer as a primer and genomic DNA or cDNA of a specific cell as a template. It can be obtained by performing.

The present invention includes both functional equivalents of the proteins. "Functional equivalent" means at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% sequence with the original amino acid sequence as a result of the addition, substitution, or deletion of the amino acid It has the same identity and refers to the protein which shows substantially homogeneous physiological activity. 'Substantially homogeneous physiological activity' refers to the activity of each protein involved in the mechanism of child interaction.

Functional LIRs are found in various proteins. Some of them are autophagy receptors, while others are substrates of autophagy. Importantly, more and more autophagy regulatory proteins interact with Atg8 / LC3 / GABARAP, such as Rab guanosine triphosphatase (GTPase) -activating proteins (GAPs), Atg3, Atg4B, and elements of the Atg1 / ULK1 complex. It turns out to be.

In one embodiment of the present invention, LC3-positive autophagosomes activity is more easily achieved by combining LC3-interacting region (LIR) regions of the TP53INP2 gene with 3xNLS, a nuclear position signal sequence, among various LIR motifs. You can check it.

The LIR (LC3-interacting region) region, for example, is expressed by a nucleotide sequence derived from the TP53INP2 gene or FYCO1 gene, it may have an amino acid sequence consisting of SEQ ID NO: 1 or SEQ ID NO: 2. In particular, the LC3-interacting region (LIR) region of TP53INP2 may include tryptophan-leucine-isoleucine-isoleucine (Trp-Leu-Ile-Ile; WLII), and the LIR region of FYCO1 is phenylalanine-aspartic acid-isoleucine. -Isoleucine (Phe-Asp-Ile-Ile; FDII). The amino acid sequence includes a wild type, and includes variants by deletion, insertion, non-conservative or conservative substitution, or a combination thereof.

In addition, the present invention can be expressed by fusing fluorescent proteins as reporter genes to detect intracellular levels of autophagosomes. The reporter fluorescent protein gene may be expressed in accordance with the promoter activity of the upstream (upstream) of the gene can be confirmed whether the expression in the cell. Examples of reporter fluorescent proteins include green fluorescent protein (GFP), modified green fluorescent protein, enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), modified red fluorescence Protein (mRFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), indigo fluorescent protein (CFP), and enhanced indigo fluorescent protein (ECFP) and Renila luciferase, red fluorescent protein (DsRed), and the like, but are not limited to the examples above. Preferably, green fluorescent protein (GFP), modified green fluorescent protein, enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), modified red fluorescent protein (mRFP) ), Most preferably enhanced green fluorescent protein (EGFP) and modified red fluorescent protein (mRFP) are used as reporter proteins.

In another aspect, the present invention relates to a recombinant vector for detecting autophagosomes for providing information on child action, containing the probe for detecting autophagosomes.

The vector may include, for example, each of the genes contained in the probe or may include a sequence encoding a recombinant protein to which they are fused.

The "vector" refers to a gene construct which is an expression vector capable of expressing a protein of interest in a suitable host cell, and which contains essential regulatory elements operably linked to express the gene insert. Vectors can be plasmids, phage particles or simply potential genomic inserts. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are the most commonly used form of current vectors, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. Plasmid vectors include (a) an initiation point for replication to efficiently replicate hundreds of plasmid vectors per host cell, (b) antibiotic resistance genes to allow selection of host cells transformed with the plasmid vector, and (c It has a structure that includes restriction enzyme cleavage site where foreign DNA fragments can be inserted. Although no suitable restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods can facilitate ligation of the vector and foreign DNA.

The "operably linked" means that when the gene is linked downstream of the promoter sequence of the present invention, it is linked in a form capable of expression of the gene, and further include any sequence to achieve the above object. Can be. Examples of the arbitrary sequences include an operator sequence, a sequence for in frame, and the like, as well as a cis element such as an enhancer and a splicing signal in addition to the gene of the present invention. (splicing signal), poly A addition signal (poly A addition signal), a selection marker (selection marker), a ribosome binding sequence (ribosome binding sequence, SD sequence) and the like may be further included. As an example of a selection marker, chloramphenicol resistance gene, ampicillin resistance gene, dihydrofolate reductase, neomycin resistance gene, etc. may be used, but additional components for operably linked by the above examples are limited. It is not.

In another aspect, the present invention relates to a composition for autophagosome detection, including the probe or the vector, for autophagosome detection and providing information on child activity.

In one embodiment of the present invention, the detection may be performed by measuring the expression level of the reporter fluorescent protein. The expression level measurement includes all of the mRNA or protein expression levels thereof. Analytical methods for measuring mRNA expression levels include reverse transcriptase (RT-PCR), competitive reverse transcriptase (RT) PCR, real time reverse transcriptase (Realtime RT-PCR), and RNase protection assay (RPA; RNase protection assay, Northern blotting, DNA chip, and the like, but are not limited thereto.

At this time, the primers used may initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffers and temperatures. The primers of the present invention are sense and antisense nucleic acids having 7 to 50 nucleotide sequences as primers specific for each marker gene. Primers can incorporate additional features that do not change the basic properties of the primers that serve as a starting point for DNA synthesis. The primers can be chemically synthesized using other well known methods and can be modified using many means known in the art.

The protein expression level can be determined by using an antibody that specifically binds to the protein of the gene. "Antibody" means a specific protein molecule directed against an antigenic site. Polyclonal antibodies, monoclonal antibodies and recombinant antibodies. Analytical methods for this purpose include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, and rockets. Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorter (FACS), protein chip, etc. .

In another aspect, the present invention provides a cell transformed with the recombinant vector. The transformation method is a method known in the art, such as, but not limited to, heat shock method (heat shock method, calcium phosphate precipitation method, electroporation, gene gun, silicone Although there are carbide carbide whiskers, sonication, and precipitation using polyethylene (polyethylenglycol), a natural introduction method, etc., the method of introducing the vector of the present invention is not limited by the above examples.

In addition, the present invention comprises the steps of transforming the target cell with the recombinant vector; And it provides a method for providing information on the child action by identifying the autophagosome level comprising the step of quantifying the C / N ratio of the reporter fluorescent protein of the transformant.

The C / N ratio has the following meaning. The amount of reporter fluorescent protein expressed depends on the level of autophagosome, and the higher the level of autophagosome in the cell, the greater the amount of reporter fluorescent protein expressed. In particular, in the present invention, the autophagy-associated protein LC3 / GABARAP is mutated so that it does not participate in autophagy, but exists in the cytoplasm, and the LC3 / GABARAP and the LIR sites of the probe located in the nucleus bind to each other. LC3 / GABARAP was allowed to enter the nucleus. By measuring the ratio of fluorescent markers of LC3 / GABARAP identified in the cytoplasm and nucleus in various ways, various information related to the progeny can be obtained.

In one embodiment of the present invention, the cell line used for transfection (transformation) is preferably an animal cell, more preferably a mammalian cell line. The medium that can be used in the present invention may be any medium conventionally used for culturing animal cells, for example, Eagles' MEM (Eagle's minimum essential medium, Eagle, H. Science 130: 432 (1959)), α -MEM (Stanner, CP et al., Nat. New Biol. 230: 52 (1971)), Iscove's MEM (Iscove, N. et al., J. Exp. Med. 147: 923 (1978)), 199 medium (Morgan et al., Proc. Soc. Exp. Bio. Med., 73: 1 (1950)), CMRL 1066, RPMI 1640 (Moore et al., J. Amer. Med. Assoc. 199: 519 (1967) ), F12 (Ham, Proc. Natl. Acad. Sci. USA 53: 288 (1965)), F10 (Ham, RG Exp. Cell Res. 29: 515 (1963)), DMEM (Dulbecco's modification of Eagle's medium, Dulbecco , R. et al., Virology 8: 396 (1959)), mixtures of DMEM and F12 (Barnes, D. et al., Anal. Biochem. 102: 255 (1980)), Way-mouth's MB752 / 1 (Waymouth , CJ Natl. Cancer Inst. 22: 1003 (1959)), McCoy's 5A (McCoy, TA, et al., Proc. Soc.Exp. Biol. Med. 100: 115 (1959)) and the MCDB series (Ham, RG et al., In Vitro 14:11 (1978)) and the like.

In the present invention, by comparing the cytoplasmic and nuclear expression ratios (C / N ratio) of the fluorescent protein in the transfected cell line can determine the level of autophagosomes related to associative phagocytosis, when the ratio is determined to be low, the progeny is measured We judge by many.

As described above, when the autophagosome probe of the present invention is used, autophagosome detection can be effectively performed, and thus the present invention can be used to provide information on autophagy-related diseases.

Recently, the important role of autophagy has been revealed in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease and various diseases and pathological phenomena such as cancer, diabetes and inflammatory reactions. It is known that progeny inhibits apoptosis of neurons through cytoprotective function in certain neurodegenerative diseases. Accumulation of protein aggregates interferes with normal neuronal function and is cytotoxic. Offspring is associated with the breakdown of abnormal proteins that can occur in neurodegenerative diseases. Examples include the expansion of glutamine multimers in Huntington's protein (Huntington's), which causes Huntington's disease, and the α-synuclein (induced Parkinson's disease), the tau protein associated with prefrontal dementia, and the Cu-ZnSOD mutation in Lou Gehrig's disease. In mice lacking Atg7, neuronal dysfunction and apoptosis occur. Progeny protects cells by maintaining neuronal homeostasis as well as eliminating protein aggregation. In addition, autophagic vacuole has been reported to accumulate in brain tissue of neurodegenerative diseases. In Alzheimer's disease, mutations in the presenilin-1 protein inhibit the maturation of autophagy vesicles and their resolution through lysosomes, and the loss of the presenilin-1 protein interferes with the protein degradation by preventing the acidification of autophagy vesicles.

The child action is also involved in cancer diseases. Offspring play two roles in tumorigenesis. The first is induction of apoptosis as tumor suppressor, and the second is inhibition of apoptosis in tumor formation mechanism. The major causes of tumor formation are DNA damage and mutations resulting from factors such as radiation, oxidative stress, and aging. Mitochondrial DNA is more susceptible to damage due to its repair mechanism and lack of protection by histone proteins. Mitochondria are a by-product of electron transport system activity, which can generate large amounts of free radicals, and their lack of control of these free radicals can damage mitochondrial DNA and lead to oxidation of the inner and outer membranes. Mitochondrial dysfunction also accumulates more free radicals, resulting in cellular oxidative stress and other mitochondrial damage. The child action suppresses carcinogenesis by removing damaged mitochondria and relieving oxidative stress. On the other hand, the progeny in tumorigenesis can survive cancer cells by removing damaged proteins and organelles in limited nutrition, hypoxia and radiation exposure.

In addition, child action is also involved in diabetes disease. Diabetes is caused by dysfunction of pancreatic β cells and insulin resistance. Mitochondrial dysfunction caused by oxidative stress plays an important role in the development of diabetes, and damaged mitochondria are eliminated by child action. Atg7-/-mice show a sharp decrease in pancreatic β cells and thus a decrease in insulin secretion. Atg7 mutant mice also exhibit hypoinsulinemia and hyperglycemia. In addition to pancreatic β-cell dysfunction, child dysfunction is associated with insulin resistance. The vesicles stress weakens the cell membrane expression of the insulin receptor and causes insulin resistance. Insulin resistance cyclically inhibits child activity and eventually disrupts the degradation of organelles such as dysfunctional proteins and mitochondria, leading to the death of pancreatic β cells. As a result, progeny is an important mechanism for pancreatic β-cell function and cell homeostasis and may affect insulin sensitivity. As aging is associated with glucose tolerance, it is thought that the activity of the offspring is closely related to aging.

In addition, child action is also involved in cardiovascular disease. Childhood mechanisms are essential for normal cardiovascular function, or abnormal child interactions can cause hypertension and heart failure. Danon disease, one of myocardial diseases, is caused by a deficiency of LAMP-2 protein, which is involved in the binding of autophagy to lysosomes. In a disease model associated with cardiovascular disease, the offspring have the effect of cytoprotection or apoptosis depending on pathological conditions. In coronary artery thrombosis, cardiomyocytes have a child action. In the transverse aortic constriction (TAC) model, the deficiency of Atg5 protein increases apoptosis, but the deficiency of Beclin1 protein protects cells from deterioration of heart function. In other words, Beclin1 interacts with Bcell lymphoma 2 (Bcl-2) protein to inhibit anti-apoptotic effects and induce apoptosis. Parkin, known as a Parkinson's disease gene, causes mitochondrial-specific progeny in the heart through its interaction with p62.

In addition, offspring are also involved in inflammatory responses. Offspring is an important component of the innate immune response and plays a variety of functions in the adaptive immune response. The progeny limit the growth of invading pathogens through the autophagolytic pathway. Bacteria, viruses, and parasites are targets of offspring. Progeny enable the search for specific single-stranded RNA viruses. Virus replication mediators migrated into the endosomes in this process are recognized by pathogen pattern-recognition receptors (PRRs) and TLR7. In addition, gestation is associated with the production of proinflammatory cytokines. In plasmacytoid dendritic cells (pDCs), gestation is a mechanism required for the production of infection-initiated type I interferon (IFN). . Embryonic fibroblasts of ATG5 deficient mice, however, promoted the production of type 1 interferon against infection with single-stranded RNA viruses, demonstrating that progeny inhibits the production of cytokines. Thus, the offspring play two opposite roles in pre-inflammatory and anti-inflammatory responses in innate immune responses.

As such, the child action is involved in neurodegenerative diseases and mechanisms that progress to various diseases such as cancer, diabetes, inflammatory reactions, etc. Therefore, the autophagosome detection probe of the present invention may be used to obtain and provide relevant information.

As such, the present invention uses the LIR (Fy or TP) -mRFP-3xNLS construct to identify the C / N ratio, which is the ratio of autophagy in the cytoplasm and nucleus, to provide a variety of information on child interaction. It includes.

In other words, the level of autophagy can be easily identified through the detection of a specific fluorescent reporter, so that it can be effectively used for autophagy detection, and further, it can be useful for discovering new therapeutic agents for diseases related to child function. .

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Unless otherwise indicated, nucleic acids are recorded in a 5 '→ 3' orientation from left to right. The numerical ranges listed in the specification include numbers defining the ranges, and include each integer or any non-integer fraction within the defined ranges.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing the invention, the preferred materials and methods are described herein.

Experimental Example

1. Cell Culture, Transfection, Drug Treatment and Cells Imaging

HEK293T cells or MEFs were moistened at 37 ° C. with 5% (v / v) CO ₂ in cell culture medium (Dulbecco's modified Eagle's medium [DMEM]: 10% [v / v] FBS and penicillin / streptomycin supplement). Grown in. Primary neuronal cultures were performed.

For serum starvation, HeLa cells or MEFs were incubated for 24 h in cell culture medium without FBS. The cells were transfected with DNA constructs using either calcium phosphate method or Lipofectamine 2000 (Life Technologies, Carlsbad, Calif., USA) and incubated for 24 hours.

Fluorescence images were obtained by confocal laser-scanning microscope (Radiance 2000, Zeiss) and analyzed by NIH Image J software (National Institutes of Health, Bethesda, MD, USA).

To induce autophagy, HeLa cells, WT MEFs, atg5-/-MEFs, or cultured neurons with 10 nM rapamycin for 4 hours in the presence or absence of 10 mM NH ₄ Cl depending on cell type and experiment Incubated for

2. With NLS LIR  Fusion effect

2- 1. LIR  Motif use

To generate new probes for monitoring endogenous LC3 located in autophagy, LIR motifs of proteins that bind well known LC3 were used.

LIR protein LIR motif Targeted protein ATG13 GGSSGNTHDD F VM I DFKPAFSKDDI LC3 (A, B, C), GABARAP, -L1, -L2 FYCO1 RPPDDAV F DI I TDEELCQIQESGS LC3 (A, B, C), GABARAP, -L1, -L2 NBR1 QSQSSASSED Y II I LPECFDTSRPL LC3 (A, B, C), GABARAP, -L1, -L2 Nix / Bnip3L LPPPAGLNSSW VE L PMNSSNGNDNG LC3 (A, B, C), GABARAP, -L1, -L2 p62 EGRPEEQMESDNCSGGDDD W TH L SSK LC3 (A, B, C), GABARAP, -L1, -L2 TP53INP1 PEFNEKEDDE W IL V DFIDTCTGFSA LC3 (A, B, C), GABARAP, -L1, -L2 TP53INP2 / DOR FVSEEDEVDG W LI I DLPDSYAAPPS LC3 (A, B, C), GABARAP, -L1, -L2 ULK1 SKDSSCDTDD F VM V PAQFPGDLVAE LC3 (A, B, C), GABARAP, -L1, -L2 ULK2 SKNSSCDTDD F VL V PHNISSDHSCD LC3 (A, B, C), GABARAP, -L1, -L2 Optineurin LNSSGSSEDS F VE I RMAEGEAEGSV LC3 (A, B), GABARAP, -L1, -L2 FUNDC1 PQDYESDDDS Y EV L DLTEYARRHQW LC3 (A, B), GABARAP, -L2 TBC1D5 LIR1 EGTFNSYRKE W EE L FVNNNYLATIR LC3 (A, B), GABARAP-L1 TBC1D5 LIR2 PDDDSSKDSG F TI V SPLDI LC3 (A, B), GABARAP-L1 c-Cbl DISNASSSFGW LS L DGDPTTNVTEG LC3B β-catenin VVKLLHPPSHW PL I KATVGLIRNLA LC3B Dvl2 ESGLEVRDRM W LK I TIPNAFLGSDV LC3B, GABARAP MAP15K LSVPEYRSRV Y QM I LECGGSSGTSR LC3B, GABARAP-L1 Bnip3 GMQEESLQGS W VE L HFSNNGNGGSV LC3B, GABARAP-L2 ATG4B MDAATLT Y DT L RFAEFEDFPET LC3 (B, C) Tax1bp1 / T6BP TMEDEGNSD M LV V TTKAGLLELKIE LC3 (B, C), GABARAP Stbd1 NSQDRVDHEE W EM V PRHSSWGDVGV LC3 (B, C), GABARAP, -L1, -L2 NDP52 QFRPENEED I LV V TTQGEVEEIEQH LC3C Calreticulin QVESGSLEDD W DF L PPKKIKDPDAS GABARAP Clathrin hc YAKKVGYTPD W IF L LRNVMRISPDQ GABARAP FIP200 PDSIDAHTFD F ET I PHPNIEQTIHQ GABARAP, -L1 TBC1D25 PSEDSPLLED W DI I SPKDVIGSDVL GABARAP, -L1, -L2 AtNBR1 SVDALCGVSE W DP I LEELQEMGFCD AtATG8 family members DmATG1B SCSSHEDSDD F VL V PKNLPEDQRQG DmATG8A PfAtg3 VHDLKIVDND W LL P SYEEDNNPTDI PfAtg8 ScAtg1 LVSDRSFERE Y VV V EKKSVEVNSLA ScAtg8 ScAtg19 DGDDNEKALT W EE L ScAtg8 ScAtg3 EEQELDGVGD W ED L QDDIDDSLRVD ScAtg8 ScAtg32 HNVNDSISGS and QA I QPLDLGASFIP ScAtg8 ScAtg34 SGSTLSRPFTW EE I ScAtg8

In particular, FYCO1-derived or TP53INP2-derived LIR motifs out of the 34 LIR motifs were used in the experiments to determine if the LIR motif could detect LC3-positive autophagy during induction of child interactions. That is, LIR (Fyco1) -mRFP-3xNLS and LIR (TP53INP2) -mRFP-3xNLS using the LIR sites of FYCO1 and TP53INP2 prepared above were compared with detection candidates.

The interaction of LC3 and GABARAP with LIR sequences of the present invention was confirmed in vitro. In order to confirm the binding of LC3 and GABARAP to GFP-labeled LIR by GST pull-down assay, Western blot confirmed the binding of LC3 and GABARAP and GFP-labeled LIR fused to GST immobilized on gel. All. As a result, as shown in Figs. 2A and 2C, it was confirmed that the LIR sequence showed a band by binding to the LC3 domain or the GABARAP domain. B and D digitize the results of A and C.

From the results of B and D, it is possible to confirm the species having more specificity in each LIR sequence among the LC3 family, LC3A and LC3B have a high binding strength for the LIR sequence derived from Fyco1, GATE16 is relatively relative to LIR derived from TP53INP2 It was confirmed that it has a high binding force.

Two LIR motifs used in embodiments of the present invention LIR protein LIR motif amino acid sequence SEQ ID NO: FYCO1 RPPDDAV F DI I TDEELCQIQESGS SEQ ID NO: 1 TP53INP2 FVSEEDEVDG W LI I DLPDSYAAPPS SEQ ID NO: 2

2-2. Fusion of nuclear signal sequence and LIR motif

The LC3 family, an autophagy-related protein of the present invention, was confirmed whether the LIR motif coupled with the nuclear position signal sequence could actually induce influx into the nucleus.

For this purpose, the nucleotide sequence encoding the LIR sequence (SEQ ID NO: 1) was isolated from the FYCO1 gene, labeled with mRFP, and the nuclear position signal sequence (SEQ ID NO: 3) was fused to finally LIR (Fyco1) -mRFP-. 3 × NLS was formed.

In addition, to confirm the binding of the LIR and LC3 family, in the LIR sequence, a mutation in which the last I (Ile) was replaced with A (Alanine) in the conserved site "Phe-Asp-Ile-Ile; FDII" Induced.

In the same manner, the nucleotide sequence encoding the LIR sequence (SEQ ID NO: 2) is separated from the TP53INP2 gene, and the fluorescence protein and nuclear position signal sequence are fused to form LIR (TP53INP2) -mRFP-3xNLS, and in LIR derived from TP53INP2. Mutations in which the W (Trp) and the fourth I (Ile) of the conserved site "Trp-Leu-Ile-Ile; WLII" were replaced with A (Alanine), respectively, were induced.

The probe manufactured as described above was modeled in FIGS. 3 and 6.

3. In-nuclear localization of LIR-mRFP-3xNLS

Using the probe prepared in Example 1, an experiment was performed to confirm whether the interaction between the LIR sequence and the LC3 family protein induces nuclear influx of the LC3 family protein.

The LC3 family proteins did not enter the autophagy but stayed in the cytoplasm, causing mutations. The C-terminal glycine (Gly) residues of the LC3 family (LC3 / GABARAP) labeled with EGFP were replaced with alanine (Ala) residues.

4, 5, 7 and 8, according to the affinity for LIR of each LC3 family, the result of confirming the LC3 family flowing into the nucleus from the cytoplasm (Fig. 4 and 7) and the amount thereof It is shown numerically and graphically (FIGS. 5 and 8). 4 and 7, in the case of EGFP, it was widely distributed throughout the cytoplasm, and when expressed with the NLS-coupled LIR probe, it was directly confirmed that the EGFP was introduced into the nucleus. As discussed above, the affinity for binding was slightly different for each LC3 family, but it was confirmed that most of the binding was introduced into the nucleus.

Based on these results, the autophagosome detection method using the probe of the present invention is a method using the interaction of LC3 / GABARAP and LIR motifs of autophagosomes directly in cells, in particular, binding of LC3 / GABARAP and LIR motifs in in-vivo This can be useful as a way to check sex.

<110> KYUNGPOOK NATIONAL UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Detection of autophagic body binding protein in vivo <130> PN1709-358 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 25 <212> PRT <213> Unknown <220> <223> TP53INP2 protein LIR sequence <400> 1 Phe Val Ser Glu Glu Asp Glu Val Asp Gly Trp Leu Ile Ile Asp Leu   1 5 10 15 Pro Asp Ser Tyr Ala Ala Pro Pro Ser              20 25 <210> 2 <211> 23 <212> PRT <213> Unknown <220> <223> Fyco1 protein LIR sequence <400> 2 Arg Pro Asp Asp Ala Val Phe Asp Ile Ile Thr Asp Glu Glu Leu Cys   1 5 10 15 Gln Ile Gln Glu Ser Gly Ser              20 <210> 3 <211> 23 <212> PRT <213> Unknown <220> <223> 3xNLS sequence <400> 3 Asp Pro Lys Lys Lys Arg Lys Val Asp Pro Lys Lys Lys Lys Arg Lys Val   1 5 10 15 Asp Pro Lys Lys Lys Arg Lys              20

Claims (17)

An LC3-interacting region (LIR) motif sequence that binds an LC3 / GABARAP family protein; Nuclear position signal sequence; And a probe for autophagy detection comprising a gene of a reporter fluorescent protein. The method of claim 1,
The LC3 / GABARAP family protein is at least one selected from the group consisting of LC3A, LC3B, LC3C, GABARAP, GABARAP-L1 and GABARAP-L2, autophagy probes for detection. The method of claim 1,
LIR motifs binding to the LC3 / GABARAP family protein are ATG13, FYCO1, NBR1, Nix / Bnip3L, p62, TP53INP1, TP53INP2 / DOR, ULK1, ULK2, Optineurin, FUNDC1, TBC1D5 LIR1, TBC1D5 LIR2, c-C-C-C Probe for autophagy detection characterized in that the sequence included in the LIR protein selected from the group consisting of catenin, Dvl2, MAP15K, Bnip3, ATG4B, Tax1bp1 / T6BP, Stbd1, NDP52, Calreticulin, Clathrin HC, FIP200 and TBC1D25. The method of claim 1,
The LIR motif that binds to the LC3 / GABARAP family protein comprises a LIR motif sequence of TP53INP2 or Fyco1 protein. The method of claim 4, wherein
Probe for autophagosome detection, characterized in that the LIR motif sequence of the TP53INP2 protein comprises the amino acid sequence of SEQ ID NO: 1. The method of claim 4, wherein
Probe for autophagosome detection, characterized in that the LIR motif sequence of the Fyco1 protein comprises the amino acid sequence of SEQ ID NO: 2. The method of claim 4, wherein
Probe for detecting autophagosome, characterized in that the LIR motif of the TP53INP2 and FYCO1 protein is derived from human cells. The method of claim 5,
The LIR (LC3-interacting region) region of the TP53INP2 gene includes tryptophan-leucine-isoleucine-isoleucine (Trp-Leu-Ile-Ile; WLII). The method of claim 6,
The LIR (LC3-interacting region) region of the FYCO1 gene includes a phenylalanine-aspartic acid-isoleucine-isoleucine (Phe-Asp-Ile-Ile; FDII) probe for detecting autophagosome. The method of claim 1,
The nuclear position signal sequence is a probe for autophagosome detection, characterized in that it comprises the amino acid sequence of SEQ ID NO: 3. The method of claim 1,
The reporter fluorescent protein may be a green fluorescent protein (GFP), a modified green fluorescent protein, an enhanced green fluorescent protein (EGFP), a red fluorescent protein (RFP), or a modified red fluorescent protein. (mRFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), indigo fluorescent protein (CFP), and enhanced indigo fluorescent protein ( ECFP) and Renilla luciferase (Renila luciferase) and red fluorescent protein (DsRed) at least one selected from the group comprising a probe for autophagosome detection.
The method of claim 11,
The reporter fluorescent protein is a modified red fluorescent protein (mRFP) characterized in that the autophagosome detection probe. The method of claim 1,
The probe may comprise a L3-Lactate (LIR) motif sequence of SEQ ID NO: 1 or SEQ ID NO: 2; The nuclear position signal sequence of SEQ ID 3; And an mRFP coupled to the LIR motif. The method of claim 1,
The probe is a probe for autophagosome detection, characterized in that located in the nucleus. A composition for detecting autophagosome comprising the probe for detecting autophagosome of claim 1 or a vector containing the same. An expression level in the nucleus of the reporter fluorescent gene bound to the probe of claim 1; And
A method for providing information on autophagy, comprising measuring the C / N ratio, which is the ratio of the expression level of the reporter fluorescent gene bound to the LC3 protein present in the cytoplasm. The method of claim 16,
The reporter fluorescence gene coupled to the probe of claim 1 and the reporter fluorescence gene coupled with the LC3 protein present in the cytoplasm comprises different colors of fluorescence, providing information about autophagy.

Images