KR101883593B1 - Probes for detecting autophagosome derived from FYCO1 and uses thereof - Google Patents

Abstract

The present invention relates to a probe for detecting an autogranular body, and more particularly to a LIR (LC3-interacting region) motif derived from FYCO1 (FYVE and coiled-coil domain containing 1) gene or mutant thereof; The N-terminal hydrophobic domain of ApPDE4 (Aplysia phosphodiesterase 4); And a reporter probe protein site and a use thereof. The present invention solves the problems of various side effects caused by overexpression of LC3, which was previously found in the self-contained animal body detection method, so that it is possible to more accurately and specifically detect an autogranular body, thereby obtaining various information in vivo relating to child action .

Description

(Probe for detecting autophagosome derived from FYCO1 and uses thereof)

The present invention relates to a probe for detecting an autogranular body, and more particularly to a LIR (LC3-interacting region) motif derived from FYCO1 (FYVE and coiled-coil domain containing 1) gene or mutant thereof; The N-terminal hydrophobic domain of ApPDE4 (Aplysia phosphodiesterase 4); And a reporter probe protein site and a use thereof.

The autophagy phenomenon plays an important role in cell function, such as starvation, protection from infectious bacteria, and regulation of neuronal disruption, an evolutionarily conserved process in which all eukaryotic cells, from yeast to mammals appear.

Initially, the cytoplasm and intracellular organs degenerate into a double membrane-bound structure called autophagosome, and the degenerated autophagosome fuses with a lysosome to form an autolysosome, The material degenerated by the material is hydrolyzed and reused. However, since the molecular functional group inherent in this process has not yet been revealed, studies are being actively conducted to identify it

To date, various genetic / biochemical studies on yeast, fruit fly, etc. have identified the proteins necessary for autophagy in the cell, and studies to analyze their correlation . It is known that autophagy is largely divided into three stages: induction / nucleation, membrane elongation / closure, and vesicle trafficking / maturation / fusion / degradation. In addition, the autophagic pathway contains two unique ubiquitin-like junction systems via Atg8PE (phosphatidylethanolamine) or Atg12Atg5, which pathway is associated with the yeast Vps34 homolog class III PI3K (phosphatidylinositol 3-kinase) and Beclin1 Autogrug) with the complex composed of autopragos.

In many types of cells, stimulation of the class I PI3K / AKT signaling pathway by growth factors and cytokines has the effect of inhibiting autophagy. The inhibitory effect is presumably due to the class I PI3K / AKT-dependent activation of the target of rapamycin (TOR) kinase, which downregulates autophagy. For example, in cells treated with PI 3-kinase inhibitors wortmannin and 3-methyladenine (3-MA), autopagosomal precursors do not develop, suggesting that PI3K- Suggesting the importance of inhibiting the activity of the AKT-mTORC1 signaling system. Inhibition or activation of this self-feeding phenomenon has a great influence on not only simple cell activity, but also physiological or pathological changes in vivo including the cells.

Recently, various animal models and genetic analysis of diseases have revealed that autopoiesis process plays an important role in diverse diseases such as diabetes, hypertension, metabolic diseases such as diabetes, degenerative brain disease, infectious diseases, and cancer. .

Therefore, the method of obtaining information on autophagy action may play a very important role in the development of a therapeutic agent targeting a new point of action.

Previously, RFP / GFP-LC3, with GFP or RFP attached to LC3 directly involved in the formation of autophagic bodies, was generally used to label autophagic bodies. However, since LC3 itself is directly involved in the formation of autophagy bodies, various side effects due to LC3 overexpression can not be avoided.

In order to develop a new probe (probe) for detecting an autotrophic corpuscle, the present inventors have developed an LIR (LC3-interacting) probe derived from a mutant FYVE & coiled-coil domain containing 1 end of Aplysia- derived ApPD4 (phosphodiesterase 4) short-form was ligated to the region of the reporter gene, and a reporter fluorescent protein was bound thereto to prepare a probe for detecting an autologous phagocyte, Thereby completing the present invention.

1. Korean Patent No. 10-1524426 2. Korean Patent Publication No. 10-2015-0129166 3. Korean Patent Publication No. 10-2015-0039894

1. Feng Y, He D, Yao Z, Klionsky DJ. The machinery of macroautophagy. Cell Res 2014; 24: 24-41. 2. Pfeifer U. Cellular autophagy and cell atrophy in the rat liver during long-term starvation. A quantitative morphological study with regard to diurnal variations. Virchows Arch B Cell Pathol 1973; 12: 195-211. 3. Klionsky DJ. Autophagy: from phenomenology to molecular understanding in less than a decade. Nat Rev Mol Cell Biol 2007; 8: 931-7.

SUMMARY OF THE INVENTION A principal object of the present invention is to provide a probe for detecting a self contained body.

It is another object of the present invention to provide a composition for detecting an autologous phagocyte containing a probe for detecting an autologous phagocyte and a vector containing the same.

It is still another object of the present invention to provide a method for providing information on the child's action using the probe for detecting the self-contained body.

In order to solve the above-described problems, the present invention provides an LIR (LC3-interacting region) motif derived from FYCO1 (FYVE and coiled-coil domain containing 1); The N-terminal hydrophobic domain of ApPDE4 (Aplysia phosphodiesterase 4); And a probe fluorescent substance detection region.

In one embodiment of the present invention, the N-terminal hydrophobic domain is preferably a N-terminal hydrophobic domain of a short form of ApPDE4 derived from a locus, more preferably a 30 amino acid sequence represented by SEQ ID NO: 1 And most preferably, the amino acid sequence of 20 amino acids represented by SEQ ID NO: 2 is used. The amino acid sequence of SEQ ID NO: 2 corresponds to a core hydrophobic residue in which 10 amino acid residues (RQSGRKSSTD) have been deleted based on the 30 amino acid sequence of SEQ ID NO: 1.

In one embodiment of the present invention, the FYCO1 (FYVE and coiled-coil domain containing 1) gene may be derived from a human. In this case, the LIR motif of the FYCO1 gene may be derived from GenBank accession No. 1. A sequence corresponding to the nucleotide positions 4032 to 4103 of NM_024513 can be used. In one embodiment of the present invention, the LIR motif of FYCO1 may have the amino acid sequence of SEQ ID NO: 3.

In one embodiment of the present invention, the LIR motif of FYCO1 may be derived from a mutation of FYCO1 gene, preferably having the amino acid sequence of SEQ ID NO: 4.

In one embodiment of the present invention, the reporter fluorescent protein is selected from the group consisting of a green fluorescent protein (GFP), a modified green fluorescent protein, an enhanced green fluorescent protein (EGFP), a red fluorescent protein (RFP), a modified red fluorescent protein (mRFP), and most preferably an enhanced green fluorescent protein (EGFP) and a modified red fluorescent protein (mRFP) as reporter proteins.

In one embodiment of the present invention, the probe for detecting an autologous animal includes an N-terminal hydrophobic domain (each of "S (N30)" or "S (N20)" having an amino acid sequence of SEQ ID NO: ); An LIR motif (referred to as "LIR (Fy)") derived from FYCO1 having the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 linked to said domain; And EGFP or mRFP (designated "EGFP / mRFP") linked to the LIR region. An example of a preferred autologous phagocyte detection probe of the present invention is a structure consisting of S (N30) -LIR (Fy) -EGFP / mRFP or S (N20) -LIR (Fy) -EGFP / mRFP, (N20) -LIR (Fy) -EGFP / mRFP.

The probe can be very useful for autologous vesicle detection by localization to self-extinguishing (child-acting) vacuoles.

In addition, the present invention provides a composition for detecting an autologous edema containing the probe for detecting an autografted somatic body or a vector containing the probe.

Further, the present invention uses the probe or the composition, and determines that the number of autophagocytic cells and the number of subcultures are increased when the expression level of the reporter fluorescent gene (protein) is increased as compared with that of a normal control group. And provides a method of providing information about the action.

In one embodiment of the present invention, the information on the child action may be related to Huntington's disease.

The present invention solves the problems of various side effects caused by overexpression of LC3, which was previously found in the self-contained animal body detection method, so that it is possible to more accurately and specifically detect an autogranular body, thereby obtaining various information in vivo relating to child action .

Brief Description of the Drawings Fig. 1 is a diagram showing various structures of a probe for detecting an organism of an autologous animal according to the present invention, a schematic view of its domain structure, and localization of RFP-LC3-proaphoretic phagocytes in HEK293T cells.
Figure 2 shows the effective localization of S (N30) -LIR (Fy) -EGFP of RFP-LC3-proton host animals in contrast to S (N30) -EGFP-LIR (Fy) in MEF.
Figure 3 shows the effective localization of S (N30) -LIR (Fy) -EGFP to RFP-LC3-proton self-priming compartments compared to S (N30) -EGFP-LIR (Fy) in MEF during serum starvation.
Figure 4 shows the effective localization of S (N20) -LIR (Fy) -EGFP on RFP-LC3-positive autophagic bodies in WT or atg5 - / - MEFs and cultured cortical neurons.
FIG. 5 shows the effective localization of S (N20) -LIR (Fy) -EGFP for RFP-LC3-proton host animals in WT or atg5 - / - MEFs.
Figure 6 shows a comparison of the efficiency of autophagy detection between S (N20) -LIR (Fy) -EGFP and S (N30) -LIR (Fy) -EGFP in MEF.
Figure 7 shows the effective localization of S (N20) -2xLIR (Fy) -EGFP on RFP-LC3-proton self-assembled monocytes in WT or ATG5 - / - MEFs.
Figure 8 shows the effective localization of S (N20) -LIR (Fy) -EGFP on autogenous somatostatin constructs during the induction of malaria by serum starvation or rapamycin in a child-dependent manner.
Figure 9 shows the effective localization of S (N20) -2xLIR (Fy) -EGFP against autologous vacuole in WT MEFs during the induction of malaria into serum starvation or rapamycin.
Figure 10 shows the effect of S (N20) -LIR (Fy) -EGFP expression on the autologous flux.
Figure 11 monitors the number of autophagic cells of S (N20) -LIR (Fy) -GFP or GFP-LC3-expressing MEFs during serum starvation.
Figure 12 shows that S (N20) -LIR (Fy) - (LH) -LIR (Fy) -LIR expression for autophagy formed in MEFs expressing poly (Q) -extensively mutated hunting tone Represents effective targeting of RFPs.
Figure 13 shows the effect of S (N20) -LIR (Fy) - (-) on the autophagic bodies produced in poly (Q) -extracted mutant hunting tonnage (Nhtt- 150Q) -expressing neurons, rather than Nhtt- Represents effective targeting of RFPs.
Figure 14 shows the detection of autologous membranes by S (N20) -LIR (Fy) -EGFP.
Figure 15 shows the formation of aggregates of RFP-LC3 in atg5 - / - cells.
Figure 16 shows the LIR-dependent localization of S (N20) -LIR (Fyco1) -GFP during the autophagy induction process. The values are the mean ± SEM of three independent experiments. Significant differences were as follows: *** P <0.001 (according to one-way ANOVA and Tukey's multiple-comparison test). The number on the bar indicates the number of cells used in the experiment. Starvation, serum starvation.
Figure 17 shows intracellular localization of RFP-LC3B, mRFP-GABARAP or mRFP-GABARAPL2-positive autophagosomes of S (N20) -LIR (Fyco1) -GFP by autophoresis induction in MEF. The values are the mean ± SEM of three independent experiments. Significant differences were as follows: *** P <0.001 (compared to all other groups according to one-way ANOVA and Tukey's post-hoc test). The number on the bar indicates the number of cells used in the experiment. Starvation, serum starvation.

The terms used in the present invention are defined as follows.

"Autophagy is a lysosomal-dependent process of modulating and modulating intracellular constituents, which plays an important role in cell growth, survival and homeostasis by removing defective macrophage complexes and intracellular organelles Autophagy is important for maintaining cellular homeostasis through intracellular fluctuations of damaged proteins and cell organelles, resulting in the accumulation of misfolded proteins in the event of a decrease in autophagy, resulting in neurodegenerative diseases (Komatsu et al., (2006), Nature, 441, 880-884).

"Self-predisposing-related disease" means a disease caused by or associated with a child's action. But are not limited to, malignant tumors, neurodegenerative diseases, ischemic diseases, diabetes mellitus, pulmonary fibrosis and the like, and more preferably malignant tumors such as breast cancer, lung cancer, fibrosarcoma, colon cancer and the like; Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease; Ischemic diseases such as angina pectoris, myocardial infarction, cardiac failure, ischemic colitis, ischemic acute renal failure, acute ischemic stroke; Diabetes such as type 1 diabetes, type 2 diabetes, and pulmonary fibrosis.

"Diagnosis" means identifying the presence or characteristic of a pathological condition. Among them, the present invention is useful for providing information on auto-predation related diseases. In the present invention, the increase / decrease characteristics of the number of autotrophs can be used as useful indicators (diagnostic markers).

"Diagnostic markers or probes (diagnostic markers or probes) are substances that can distinguish autophagic bodies from other cells and can be used to detect polypeptides that show an increase or decrease in autophagocytosis in the presence of auto- And organic biomolecules such as nucleic acids (e.g., mRNA), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, polysaccharides and the like), and the like.

"Probe" means a nucleic acid fragment such as RNA or DNA corresponding to several bases or several hundreds of bases in a short time, which can achieve specific binding with mRNA. The probe may be prepared in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, or the like. Selection of suitable probes and hybridization conditions may be modified based on those known in the art.

"Subject" or "patient" means any single entity that requires treatment, including human, cow, dog, guinea pig, rabbit, chicken, In addition, any subject who participates in a clinical study test that does not show any disease clinical findings, or who participates in epidemiological studies or used as a control group is included.

"Tissue or cell sample" refers to a collection of similar cells obtained from a subject or tissue of a patient. The source of the tissue or cell sample may be a solid tissue from fresh, frozen and / or preserved organ or tissue sample or biopsy or aspirate; Blood or any blood components; It may be a cell at any point in the pregnancy or development of the subject. Tissue samples can 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 a tissue sample. Includes one or both strands of a double-stranded nucleic acid molecule and includes any fragment or portion of the intact nucleic acid molecule.

"Gene" means any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expression. The gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.

"Protein" also includes fragments, analogs, and derivatives of proteins that retain essentially the same biological activity or function as the reference protein

"Label" or "label " means a compound or composition that facilitates the detection of a reagent, such as a reagent conjugated, conjugated, conjugated, or fused to a nucleic acid probe or antibody. The label may itself be detected (e. G., A radioactive isotope label or a fluorescent label), in the case of an enzyme label, to catalyze the chemical modification of the detectable substrate compound or composition.

"About" means that the reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length is 30, 25, 20, 25, 10, 9, 8, 7, , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.

Throughout this specification, the words " comprising "and" comprising ", unless the context requires otherwise, include the stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

Hereinafter, the present invention will be described in detail.

The present invention relates to a novel probe for detecting an autophagosome, and more particularly, to a probe for detecting an autophagosome including S (N30) -LIR (Fy) -EGFP / mRFP or S (N20) -LIR / mRFP &lt; / RTI &gt; probes, their fabrication and their use.

Self-predation is the process by which cell constituents are removed through lysosomes, and physiologically, autophagy plays a role in balancing cellular organelles and protein production / degradation. Autophagy has been shown to mediate the pathogenesis of disease and participate in the crosstalk of reactive oxygen species (ROS / RNS) during intracellular signaling and protein damage processes.

Self-predation is divided into three categories, depending on how the substrate reaches the lysosome: (i) macroautophagy - a self-predominant self-predation is called self-predation. It is a method of forming an autophagosome. (ii) the element is predominant - the lysosome surrounds and digests the cell construct directly. (iii) Chaperone-mediated pro-chaperone-bound protein complexes bind to the LAMP-2A receptor and take the target protein to lysosomes and degrade

In mammalian cells, a decrease in nutrients inhibits TOR (target of rapamy-cin) and dephosphorylated atg13. Dephosphorylated atg13 binds to atg1 homologue and activates. Activated atg1 is recruited with FIP200. This protein complex elongates the separation membrane and induces autophagosome structure to be completed.

The beclin-1-class III PI3K complex is required for the self-organizing corpuscular structure, and LC3 (light chain 3) -II must be inserted into the membrane of the autogranular bodies. LC3 is a protein similar to ubiquitin and LC3 is post-translationally modified to LC3-II. LC3 is truncated to Atg4 and becomes LC3-I, and then the glycine residue is exposed at the C-terminus. Then, LC3-I and phosphatidylethanolamine (PE) combine to form LC3-II. This process is essential for self-organizing expansions

Typical features of autogenous predation are the formation of autophagic bodies with varying sizes (0.5 - 1.5 μm) depending on the signal, target, and cell type leading to autophagy. More than 35 autophagy-related (Atg) genes have been found through yeast-derived genetic screens that control various processes of autophagy formation.

Therefore, various information on autopatch-related diseases can be obtained by detecting the formed autogenous body, and the present invention relates to such a probe for autogenous body detection.

It is the most traditional way to observe the autogenous phagocytes of each stage that are mature by electron microscopy to see if self-predation is actually happening. Another way is to measure the degradation rate of long-lived proteins by a pulsed-chase method. LC3-based methods have also been widely used to measure self-predation. 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 autopoetic somatosensory membranes is an important step that occurs consistently when autotrophs are formed and the amount of LC3 reflects the relative amount of autologous autologous cells in the cell. The LC3-II protein, when combined with an autophagous body, appears as a small point like a follicle when observed by a cell immuno staining method.

Previously, RFP / GFP-LC3, with GFP or RFP attached to LC3 directly involved in the formation of autophagic bodies, was generally used to label autophagic bodies.

This is due to the fact that RFP is resistant to acidic conditions while GFP is weakened by acidity. LC3, in which GFP and RFP are coupled in series, simultaneously emits GFP and RFP light in self-trapping bodies that are not associated with lysosomes, but when the autolysosome matures, only the RFP is released.

However, since LC3 itself is directly involved in the formation of autophagy bodies, various side effects due to LC3 overexpression can not be avoided.

The present invention relates to a probe for detecting a new self-organizing body and a use thereof, which can solve such a problem.

Therefore, in one aspect, the present invention relates to a probe for detecting an organism containing the above-mentioned components. The probe structure can be used to provide various information on the child's behavior by confirming the intracellular self-engulfing element level.

The probe of the present invention is characterized by containing an LIR motif (LIR [Fy]) of the FYCO1 gene, an N-terminal hydrophobic domain, and a reporter gene.

The FYCO1, N-terminal hydrophobic domain sequence is derived from an animal, preferably FYCO1 from a human known gene sequence and mutants thereof, ApPDE4 is obtained from Aplysia and includes not only wild type but also deletion, , Non-conservative or conservative substitution, or a combination thereof.

The gene coding for the protein or the like can be obtained by PCR using oligonucleotides complementary to the end portion of the nucleotide sequence known in the art and using the genomic DNA or cDNA as a template as a primer, And the like.

The present invention includes both cases where the protein is a functional equivalent. By "functional equivalent" is meant at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% sequence identity with the original amino acid sequence as a result of addition, substitution or deletion of the amino acid Refers to a protein having homology and exhibiting substantially the same physiological activity. &Quot; Substantially homogeneous physiological activity &quot; refers to the activity of each protein involved in the mechanism of action.

(LIR (Fy), RPPDDAVFDIITDEELCQIQESGS) (SEQ ID NO: 3) of FYCO1, known as LC3-, Rab7-, and PI3P-interacting proteins, in the probes of the present invention for monitoring endogenous LC3 located in self- ) Can be used.

In one embodiment of the present invention, the LIR motif of FYCO1 can use the LIR motif (LIR [Fy]) of the FYCO1 gene corresponding to the nucleotide positions 4032 to 4103 of the human FYCO1 gene (GenBank accession No. NM_024513).

In one embodiment of the present invention, the LIR motif may be a mutant of the conserved sequence ((W / F / Y) -XX- (I / L / V)) of the LIR motif of wild type (WT) FYCO1 , Preferably the amino acid sequence of SEQ ID NO: 4 (RPPDDAV F DI A TDEELCQIQESGS).

In particular, in the present invention, by fusing the LIR motif with the N-terminal hydrophobic domain, LC3-positive autophagosomes can be detected more efficiently.

Preferably the N-terminal hydrophobic domain of ApPDE4 (phosphodiesterase 4), more preferably the N-terminal (S (N30)) of the short-form of ApPDE4 and most preferably the hydrophobic residue Based on my amino acid sequence, the core hydrophobic residue S (N20) in which ten amino acid residues (RQSGRKSSTD) are deleted is bound. That is, it is preferable that the domain comprises 30 amino acid sequences represented by SEQ ID NO: 1 or 20 amino acid sequences represented by SEQ ID NO: 2.

These 30 or 20 amino acid residues linked to hydrophobic motifs, LIR, have a significant effect on localization that is effective for LC3-pro-apoptotic bodies.

The FYVE domain in FYCO1 may be associated with PI3P-rich phagophores (preautophagosomal membranes), but a single FYVE domain may be associated with LIR (Fy) for LC3-pro-apoptotic bodies in contrast to S (N30) Lt; RTI ID = 0.0 &gt; cellular &lt; / RTI &gt;

In addition, the present invention is characterized in that a fluorescent protein is fused and expressed as a reporter gene in order to detect the intracellular level of an autogenous animal.

The reporter gene can be expressed in the cell according to its upstream promoter activity and its expression can be confirmed in the cell. Examples of reporter genes include green fluorescent protein (GFP), modified green fluorescent protein, enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), modified red fluorescent protein (AFP), enhanced blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP) ECFP) and Renila luciferase, red fluorescent protein (DsRed), and the like, but are not limited to the above examples. (GFP), a modified green fluorescent protein, an enhanced green fluorescent protein (EGFP), a red fluorescent protein (RFP), a modified red fluorescent protein (mRFP ), And most preferably, enhanced green fluorescent protein (EGFP) and modified red fluorescent protein (mRFP) as reporter proteins.

Therefore, the present invention provides, as a preferred embodiment, a probe structure for detecting an autosorbing entity having a structure of S (N30) -LIR (Fy) -EGFP / mRFP or S (N20) -LIR (Fy) -EGFP / And most preferably provides an S (N20) -LIR (Fy) -EGFP / mRFP probe.

In particular, the probes of the present invention are effectively located in self-extinguishing (child-acting) vacuoles and are thus very useful as self-contained sensor cells.

The present invention also relates to a recombinant vector for the detection of an autologous phagocyte for providing information on the sub-phagocyte containing the probe for detecting an autologous phagocyte.

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

A "vector" refers to an expression vector capable of expressing a desired protein in an appropriate host cell, including an essential regulatory element operably linked to the expression of the gene insert.

The vector may be a plasmid, phage particle or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. Because the plasmid is the most commonly used form of the current vector, the terms "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. (B) an antibiotic resistance gene which allows the host cell transformed with the plasmid vector to be selected, and (c) a plasmid vector which is capable of selecting a host cell transformed with the plasmid vector. ) Contains a restriction enzyme cleavage site into which a foreign DNA fragment can be inserted. Although there is no suitable restriction enzyme cleavage site, the use of a synthetic oligonucleotide adapter or a linker according to a conventional method can easily ligate the vector and the foreign DNA

By "operably linked" is meant that when a gene is linked downstream of the promoter sequence of the present invention, the gene is linked in a form capable of expression, and in order to achieve the above object, any sequence may be further included have. Examples of the arbitrary sequence include an operator sequence and an in frame sequence. In addition to the gene of the present invention, a cis element such as an enhancer, a splice signal, a splicing signal, a poly A addition signal, a selection marker, a ribosome binding sequence (SD sequence), and the like. Examples of selectable markers include, but are not limited to, chloramphenicol resistance genes, ampicillin resistance genes, dihydrofolate reductase, neomycin resistance genes and the like, but there are limited additional components to be operably linked by the above examples It is not.

In another aspect, the present invention relates to a composition for the detection of autoradiographic bodies and information on self-contained body detection and autonomic activity, comprising the probe or the vector.

At this time, the expression level of the reporter fluorescent protein is checked to detect an autogranular body. The expression level measurement includes both measuring mRNA or its protein expression level.

RT-PCR, competitive RT-PCR, real-time RT-PCR, and RNase protection assay (RPA) were used for the measurement of mRNA expression levels. RNase protection assay, Northern blotting, DNA chip, and the like.

At this time, the primers used can initiate DNA synthesis in the presence of reagents for the polymerization reaction (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at the appropriate buffer solution and temperature. The primer of the present invention is a sense and antisense nucleic acid having 7 to 50 nucleotide sequences for each marker gene-specific primer. Primers can incorporate additional features that do not alter the primer's basic properties 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.

Methods for this analysis include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket, But are not limited to, immunoelectrophoresis, immunohistochemistry, immunoprecipitation assays, complement fixation assays, fluorescence activated cell sorters (FACS), protein chips, and the like. .

The present invention also provides a cell transformed with the recombinant vector.

The transforming method may be performed by a method known in the art such as, but not limited to, heat shock method, calcium phosphate precipitation method, electroporation method, gene gun, But are not limited to, methods of introducing the vector of the present invention by means of the above-mentioned examples, such as silicate carbide whiskers, sonication, sedimentation using PEG (polyethylenglycol), and natural introduction methods.

Also, the present invention provides a method for producing a recombinant vector, comprising the steps of: transforming a target cell with the recombinant vector; And quantifying the amount of the reporter fluorescent protein of the transformant.

The amount of reporter fluorescent protein expressed by the autogenous phagocytosis level is determined. The higher the level of intracellular autophagocytosis, the greater the amount of reporter fluorescent protein expressed. Therefore, quantifying the amount of fluorescent protein expressed will result in an intracellular self- Can be measured and based on this, various information related to the child's action can be obtained

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

In the present invention, the expression level of the fluorescent protein in the transfected cell line can be used to confirm the level of the phagocytosis of the child's action related organism, and when it is measured as up-regulation by comparison analysis, it is judged that the child action is high .

Since the self-fertilized corpuscular probe of the present invention can be effectively used to detect autocorrelation bodies, it can be used to provide information on an auto-predation-related disease.

Recently, an important role of self-feeding has been revealed in various diseases and pathological phenomena such as neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, cancer, diabetes, inflammatory reaction and the like.

Child behavior is known to inhibit neuronal cell death through cytoprotective effects in certain neurodegenerative diseases. Accumulation of protein aggregates disrupts normal neuronal function and is cytotoxic. Child behavior is associated with the degradation of abnormal proteins, which may occur in neurodegenerative diseases, such as: Extension of glutamine oligos in Huntington's protein (huntington, Htt) causing Huntington's disease and α-synuclein leading to Parkinson's disease, tau protein associated with frontal dementia, mutation of Cu-ZnSOD in Lou Gehrig's disease are examples. In Atg7 deficient mice, neuronal dysfunction and apoptosis occur. Child action not only eliminates this protein aggregation but also protects the cells by maintaining homeostasis of neurons. It has also been reported that the autophagic vacuole accumulates in brain tissue of neurodegenerative disease patients. The mutation of presenilin-1 protein in Alzheimer's disease inhibits maturation and lysosomal degradation of autophagic ER and loss of presenilin-1 protein interferes with acidification of autophagic ER

In addition, childhood is also involved in cancer disease.

Childhood plays two roles in tumorigenesis. The first is the induction of apoptosis as a tumor suppressor, and the second is the inhibition of apoptosis in the tumorigenesis mechanism. The main causes of tumorigenesis are DNA damage and mutations resulting from factors such as radioactivity, oxidative stress, and aging. Mitochondrial DNA is more susceptible to damage due to the lack of recovery mechanisms and protection by histone proteins. Mitochondria are a byproduct of electron transport system activity and can produce large amounts of reactive oxygen, which can damage mitochondrial DNA and cause oxidation of the inner and outer membranes. In addition, mitochondrial dysfunction accumulates more active oxygen, resulting in oxidative stress of cells and damage to other mitochondria. Childhood action inhibits carcinogenesis by eliminating damaged mitochondria and alleviating oxidative stress. On the other hand, in tumor formation, the child action can survive cancer cells by removing damaged proteins and cell organelles in limited nutrient and hypoxic state and in radioactivity exposure.

In addition, the child action is also involved in diabetes mellitus.

Diabetes is caused by dysfunction of pancreatic β-cells and insulin resistance. The dysfunction of mitochondria caused by oxidative stress plays an important role in the development of diabetes, and the damaged mitochondria are eliminated by child action. Atg7 - / - mice show a sharp decrease in pancreatic beta cells and thus a decrease in insulin secretion. In addition, Atg7 mutant mice exhibit hypoglycemia and hyperglycemia. Following dysfunction of the pancreatic β-cells, dysfunction of the child's function is associated with insulin resistance. ER stress causes insulin resistance by weakening the cell membrane expression of the insulin receptor. Insulin resistance cyclically inhibits the function of the kidneys, which ultimately leads to the death of pancreatic β cells by interfering with the degradation of cellular organelles such as proteins and mitochondria. Therefore, child action is an important mechanism for pancreatic β cell function and cell homeostasis and may affect insulin sensitivity. In the case of aging, the activity of child action is thought to be closely related to aging since it is related to performance within the party.

In addition, the child action is also involved in cardiovascular diseases.

For normal cardiovascular function, the child's action is necessarily a necessary cellular mechanism or abnormal child action can cause hypertension and heart failure. One of myocardial diseases, danon disease, is caused by a deficiency of LAMP-2 protein, which is involved in the association of autophagic endoplasmic reticulum with lysosomes. In the cardiovascular disease model, the childhood effects of cell protection or cell death depend on pathological conditions. In coronary artery thrombosis, cardiac myocytes cause childbearing. In the transverse aortic constriction (TAC) model, deficiency of Atg5 protein increases cell death, whereas deficiency of Beclin1 protein protects cell from deterioration of cardiac 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 - associated gene, causes mitochondrial - specific malfunctions in the heart through its interaction with p62.

In addition, the child action is also involved in the inflammatory reaction.

Child action is an important component of the innate immune response and plays a variety of functions in the adaptive immune response. Child action limits the growth of pathogens that have invaded through the pathway of autolytic solubilization. Bacteria, viruses, parasites, etc. are targets of child action. Child action enables the discovery of specific single-stranded RNA viruses. In this process, virus replication mediators transferred into endosomes are recognized by pathogen pattern-recognition receptors (PRR) and TLR7. Childhood is also involved in the production of proinflammatory cytokines. In the plasmacytoid dendritic cell (pDC), a child is a necessary mechanism for the generation of type 1 interferon (IFN) . However, embryonic fibroblasts from ATG5 deficient mice have been shown to stimulate the production of type 1 interferon for infection with a single-stranded RNA virus, suggesting that the action of the child suppresses the production of cytokines. Thus, child behavior plays two opposing roles in the inflammatory and antiinflammatory responses in congenital immune responses.

As such, since the child action is involved in a mechanism for progressing to various diseases such as neurodegenerative diseases and cancer, diabetes, inflammation reaction, etc., the related information can be obtained and provided by using the self-contained body detection probe of the present invention.

Thus, the present invention can be used to identify subcellular levels of autophagy bodies using structures such as S (N30) -LIR (Fy) -EGFP / mRFP or S (N20) -LIR &Lt; RTI ID = 0.0 &gt; and / or &lt; / RTI &gt;

That is, since the level of the autoporosis body can be easily confirmed through the detection of a specific fluorescent reporter, it can be effectively used for the purpose of related to the detection of an autogranular body, and it will be useful for discovering a new therapeutic agent for a child-related disease .

< Example >

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

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

Unless otherwise defined, 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 of testing the present invention, the preferred materials and methods are described herein.

Object and method

1. DNA structure

Plasmids were prepared from pEGFP-C3 or pEGFP-N3 backbones.

(LIR [Fy]) of the FYCO1 gene corresponding to the nucleotide position 4032-4103 of human FYCO1 (GenBank accession No. NM_024513) or the p62 gene corresponding to the nucleotide position 1050-1127 of human p62 (GenBank accession No. NM_003900) LIR motifs (LIR [p62]) were generated by primer extension using synthetic primers and subcloned into the pEGFP-C3 or pEGFP-N3 vector (or spectral variants monomeric at the HindIII-KpnI site) red fluorescent protein (mRFP) or cyan fluorescent protein (CFP)), EGFP-LIR and LIR-EGFP, respectively.

In order to generate S (N30) -LIR (Fy) -EGFP and EGFP-LIR (Fy) -LIR (Fy), S (N30) and LIR (Fy) amplified by PCR were substituted with pEGFP- LIR (Fy) and pEGFP-C3-LIR (Fy) at HindIII-HindIII sites.

C3-LIR (Fy) or pEGFP-C3-LIR (Fy) as templates, respectively, to generate S (N30) -EGFP-LIR (Fy) and S (N30) -EGFP- LIR (Fy) and EGFP-LIR (Fy) -LIR (Fy) were amplified using LIR (Fy) -LIR (Fy) and XbaI- And subcloned into the KpnI site.

S (N30) -S (N30) -EGFP-LIR (p62) and EGFP-LIR (p62) were amplified using pEGFP-C3-LIR mRFP &lt; / RTI &gt; at the XbaI-ApaI site.

Nucleotide positions 4241 to 4498 of human EEA1 (GenBank accession No. NM_003566) amplified using GFP-EEA1 wt (Addgene # 42307) as a template to generate pcDNA3.1-FYVE-EGFP-LIR (Fy) FYVE motif (of EEA1 gene) was inserted into the pcDNA3.1-S (N30) -EGFP-LIR (Fy) vector at the position of NheI-XbaI.

In addition, the amplified S (N20) and LIR (Fy) were inserted into the pEGFP-N3-LIR (Fy) vector at the HindIII-HindIII site and the KpnI-BamHI site, respectively.

(Fy) -LIR (Fy) at the HindIII-HindIII site to generate amplified S (N20) to generate pEGFP-N3-S (N20) -LIR Respectively. (N20) -mRFP amplified using pcDNA3.1 (+) - S (N30) -mRFP as template to produce pEGFP-C3-S (N20) -EGFP-LIR (Fy) EGFP was subcloned into the NheI-XhoI site into the pEGFP-C3-LIR (Fy) -LIR (Fy) vector. Using different fluorescence wavelengths, EGFP was replaced by CFP or mRFP.

2. Cell culture, transfection, drug treatment and cell imaging

HEK293T cells or MEFs were cultured in a humidified atmosphere containing 5% (v / v) CO ₂ in cell culture medium (Dulbecco's modified Eagles medium [DMEM]: 10% [v / v] FBS and penicillin / streptomycin supplementation) Lt; / RTI &gt; Primary neuronal cell cultures were performed.

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

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

To induce autophagy, HeLa cells, WT MEFs, atg5 - / - MEFs, or cultured neurons were incubated with 10 nM rapamycin in the presence or absence of 10 mM NH ₄ Cl for 4 h Lt; / RTI &gt;

3. Immunocytochemical analysis

To detect endogenous LC3, cells were rinsed with PBS and fixed with 4% paraformaldehyde for 10 minutes. Cells were permeabilized with 0.1% Triton X-100 for 5 minutes and blocked with 3% bovine serum albumin (BSA) for 1 hour at room temperature.

Primary antibodies (anti-LC3, BML, 1: 500, # PM036) were added to the blocking solution or overnight at 4 ° C. The cells were washed three times with 1 × PBS, washed and incubated with CY3-anti-rabbit secondary antibodies for 1 hour at room temperature (Jackson Laboratory, 1: 500) and then washed three times with 1 × PBS .

The cells were mounted on glass slides and analyzed by LSM700 confocal microscopy (Carl Zeiss).

4. Immunogold electron microscopy.

WT or atg5 - / - MEFs transfected with Lipofectamine 2000 were fixed in PBS containing 0.1% glutaraldehyde, 4% formaldehyde, and 3.5% sucrose at 4 ° C for 1 h. The cells were separated from the culture dish using a cell scraper and pelleted by centrifugation at 2000 x g for 2 min at 4 [deg.] C.

 The cell pellets were resuspended in warm agar (1% in PBS) and centrifuged again. After three washes in PBS, the agar-embedded cell pellets were dehydrated by ethanol and propylene oxide solution steps and embedded in Epon 812 mixture.

For immuno-gold labeling experiments, the ultra-thin (80-nm) portion of the Formvar-coated gold grid was floated on a freshly prepared 3% sodium meta-iodate drop for 30 minutes. The immunogold labeling process consists of the manufacturer's modified protocol (British Biocell International). After etching and washing, a 50-μL dropwise lattice of Solution A (PBS, pH 8.2, 4% normal goat serum, 1% BSA, 0.1% Tween 20, and 0.1% sodium azide) was placed and incubated for 30 minutes Respectively.

Rabbit GFP monoclonal antibody (Cell Signaling Technology, Danvers, MA cat.) Diluted in solution B (same as Solution A except 1% normal goat serum) in a humid chamber at room temperature for 3 hours. # 2956), and rinsed in solution B. The cells were washed twice with PBS,

The lattice was reacted with 15-nm-gold-conjugated goat anti-rabbit IgG secondary antibody diluted in solution A. Controls for the specificity of GFP immunogold labeling include 1) omission of the primary antibody and 2) replacement of the primary antibody with a preimmune serum.

After washing in PBS and deionized water, the lattice was contrast dyed with uranyl acetate (7 min) and lead satrate (2 min) and observed using a Tecnai G2 Spirit electron microscope (FEI, Hillsboro, OR, USA) .

Example  One : ApPDE4 And the fusion effect with the N-terminal hydrophobic domain of

1-1. LIR  Using motifs

The LIR motif of the well-known LC3-binding protein was used to generate a novel probe for monitoring endogenous LC3 located in the autolysed corpus.

Using LIR motifs of p62 (LIR (p62), EGRPEEQMESDNCSGGDDDTHTHSSK) or LIR motifs of FYCO1 (LIR (Fy), RPPDDAVFDIITDEELCQIQESGS) known as LC3-, Rab7- and PI3P-interacting proteins, the LIR motif Lt; RTI ID = 0.0 &gt; LC3-positive &lt; / RTI &gt; autophagous bodies during induction

EGFP was fused to the N-terminus of the LIR motif from FYCO1 or p62 to generate EGFP-LIR (Fy) or EGFP-LIR (p62), respectively (FIG.

LC3 was expressed in HEK293T cells alone (cultured for 24 h) either with EGFP-LIR (Fy) or EGFP-LIR (p62) with RFP-LC3 alone or with RFP-LC3 and then the cells were treated with rapamycin (10 nM, Lysosomotrophic reagent during inhibition of lysosomal degradation with NH ₄ Cl (10 mM, 4 h), a common inducer of childhood: maximizing their accumulation, For easy detection).

As a result, RFP-LC3 was located in the autolytic vacuole in cells treated with rapamycin and NH ₄ Cl, whereas control cells showed almost no RFP-LC3-positive autoantigen in the absence of the child-induced signal.

On the other hand, EGFP-LIR (Fy) or EGFP-LIR (p62) none rapamycin and NH ₄ Cl LC3- detected in the treated cells was not self-position predators body, the expression pattern was most prevalent (Fig. 1C ).

These results demonstrate that the LIR motifs themselves are insufficient in stable association with LC3 in autophagy bodies.

1-2. The hydrophobic domain and LIR  The fusion of motifs

N-terminal hydrophobic domains are important in plasma membrane targeting. The N-terminal hydrophobic domain itself is not sufficient for membrane targeting, and additional domains such as the basic-rich domain and UCR1-2 are required for localization to the plasma membrane.

We therefore further determined whether the additional N-terminal hydrophobic domain from the ApPDE4 short-form could induce subcellular localization of EGFP-LIR (Fy) or EGFP-LIR (p62) Respectively.

In addition, since the FYVE domain in FYCO1 may be associated with PI3P-rich phagophores (preautophagosomal membranes), the PI3P-binding FYVE domain from EEA1, together with the LIR motif, And whether or not it could lead.

To this end, the N-terminal hydrophobic region (N-terminal region consisting of 30 hydrophobic amino acid residues in S (N30)) is fused to the N-terminus of EGFP-LIR resulting in S (N30) EGFP-LIR (Fy) or S (N30) -EGFP-LIR (p62) was also produced by the addition of FYVE-EGFP-LIR Were compared.

As a result, as, rapamycin and NH S (N30) -EGFP-LIR (Fy) in HeLa cells treated with ₄ Cl is most LC3- RFP-positive woman was located predators body shown in 1C, while the S (N30) -EGFP-LIR (p62) was less efficiently located in the RFP-LC3-pro-apoptotic host.

 As a control, S (N30) -mRFP was not located in the GFP-LC3-pro-apoptotic body. These results suggest the suitability of the LIR motif (LIR (Fy)) fused with the hydrophobic domain as a sensor for self-trapping bodies.

In addition, a single FYVE domain did not affect the cellular localization of EGFP-LIR (Fy) to RFP-LC3-proton host animals in contrast to in S (N30) composed of hydrophobic residues (FIG. 1C). In addition, the data were effectively located on RFP-LC3-pro-apoptotic bodies when FYCO1-derived EGFP-LIR (Fy) was fused with the hydrophobic domain (not in the membrane itself).

That is, when fused with the N-terminal hydrophobic domain of ApPDE4, it was confirmed that EGFP-LIR from p62 non-FYCO1 was located more efficiently in RFP-LC3-positive autophagosomes.

Therefore, such a recombinant protein may be provided as a new self-contained body sensor.

Example  2 : LIR ( Fy ) Adjacent to the motif S ( N30 ) Hydrophobic motif

A hydrophobic domain adjacent to the N-terminus of the LIR (Fy) motif fused to EGFP in WT MEFs is more effectively located in the RFP-LC3-proton host.

Since FYCO1-derived LIR motifs are more suitable for detection of endogenous LC3-positive autophagic endoplasmic reticulum during child-induced induction with rapamycin (as opposed to p62-derived), we used LIR (Fy) .

In order to test whether the position of the LIR (Fy) motif near S (N30) can promote subcellular localization in intrinsic LC3 in autoradiographic bodies, let S (N30) be LIR (Fy) -EGFP Terminal, resulting in the formation of S (N30) -LIR (Fy) -EGFP (Figure 2A).

(N30) -EGFP-LIR (Fy) -GFP-LIR (Fy) -EGFP to compare the localization of S (N30) -EGFP-LIR (Fy) and S or S (N30) -LIR (Fy) and expressing -EGFP in WT MEFs, the cells _{NH 4 Cl (10 mM, 4} h; Figure 2) and with or without incubation with rapamycin or (10 nM, 4 h).

It was also incubated during serum starvation (for 24 h; FIG. 3).

As shown in Figures 2B-E, S (N30) -LIR (Fy) -EGFP during the induction of the action on rapamycin induced RFP-LC3-positive proliferative responses to S (N30) -EGFP- More effectively located in the endoplasmic reticulum.

According to these results, although S (N30) -EGFP-LIR (Fy) is located in autophagic bodies, their localization to cytoplasm is more prevalent than in S (N30) -EGFP-LIR , Suggesting that the S (N30) hydrophobic motif adjacent to the LIR (Fy) motif may be more effective in LC3 recognition and induce localization in the membrane.

In order to identify the minimal essential regions within the hydrophobic residues of S (N30) we have formed deletion mutant constructs of S (N30).

Based on the amino acid sequence in the hydrophobic residue of S (N30), 10 amino acid residues (RQSGRKSSTD) deletions were determined because the core hydrophobic residues were located in region S (N30).

When S (N20) -LIR (Fy) -EGFP is expressed during childhood induction with rapamycin (FIGS. 4A and B) or during serum starvation in WT MEFs (FIG. 5), this is specific for RFP-LC3- Respectively. The latter was accumulated more strongly as a result of the inhibition of lysosomal degradation by WT MEFs NH4Cl, but not by atg5 - / - MEFs lacking malnutrition.

LC3-positive autoantibodies to co-localization of GFP sensors between cells expressing S (N20) -LIR (Fy) -EGFP and cells expressing S (N30) -LIR No significant difference was observed (FIG. 6).

These results indicate that the long 20 amino acid residues linked to the hydrophobic motif, LIR, are sufficient for effective localization to the LC3-pro-apoptotic body (Figure 4).

Next, to determine whether the duplication of LIR affects the efficiency of subcellular localization of S (N20) -LIR (Fy) -EGFP, two LIR motifs were substituted for C (N20) -Terminus and the N-terminus of EGFP; The formed structure was named S (N20) -2xLIR (Fy) -EGFP.

Successful expression of S (N20) -LIR (Fy) -2xLIR (Fy) -EGFP with RFP-LC3 and child action was induced in rapamycin from WT MEFs.

As a result, S (N20) -2xLIR (Fy) -EGFP in the case of S (N20) -LIR (Fy) -EGFP (containing a single LIR motif) in the cytosol in WT MEFs other than atg5 - / - MEFs Lt; RTI ID = 0.0 &gt; RFP-LC3-positive &lt; / RTI &gt;

These results suggest that when a single LIR motif is combined with a hydrophobic motif (S (N20), it is more efficient and sufficient for autophagic target targeting through association with LC3).

In addition, in order to test whether S (N20) -LIR (Fy) -EGFP detects LC3-positive autophagic bodies in post-mitotic neurons, S (N20) Or atg5 - / - neurons with RFP-LC3.

The neurons were then incubated with rapamycin during inhibition of lysosomal degradation with NH4Cl.

As a result, as shown in FIGS. 4C and D, S (N20) -LIR (Fy) -EGFP was mostly located in RFP-LC3-proaphoretic predators in WT neurons other than atg5 - / -.

Example  3: S ( N20 ) - LIR ( Fy ) - Of EGFP Self-extinguishing  In vacuo Positioning

LF (Fy) -EGFP was found to be more sensitive than WT MEFs and atg5- / LF without expression of exogenous RFP-LC3, as S (N20) -LIR (Fy) -EGFP could be used as a sensor candidate. - MEFs to determine if endogenous LC3 could be specifically detected in autologous predators induced by serum starvation or rapamycin.

We confirmed that S (N20) -LIR (Fy) -EGFP is expressed in either WT or atg5 - / - MEFs, which show dominant subpopulation by serum starvation or rapamycin (10 nM, 4 h).

As shown in FIGS. 8A and B, S (N20) -LIR (Fy) -EGFP was effectively located in the self-extinguishing vacuole in WT rather than atg5 - / - MEFs.

The S (N20) -LIR (Fy) -EGFP accumulates more strongly during the inhibition of lysosomal degradation by NH4Cl, indicating that any exogenous RFP-LC3 Means that the produced LC3-proton can effectively detect predators.

Comparison of S (N20) -LIR (Fy) -EGFP and S (N20) -2xLIR (Fy) -LIR (Fy) -EGFP for the cellular localization of endogenous LC3-progenitor predators in WT MEFs , There was no significant difference (Fig. 9); These results were consistent with the data shown in FIG.

On the other hand, in order to directly test whether S (N20) -LIR (Fy) -EGFP is located in the digestive vacuole, immunocytochemistry is performed using an anti-LC3 antibody; Immuno-gold-electron microscopy analysis was performed using anti-GFP antibodies in S (N20) -LIR (Fy) -EGFP-expressing WT or atg5 - / - MEFs

As a result, as shown in Fig. 8C, S (N20) -LIR (Fy) -EGFP was found to be the most abundantly detected by the anti-LC3 antibody induced by the franking activity from the WT, Lt; RTI ID = 0.0 &gt; LC3-positive &lt; / RTI &gt;

Similarly, in WT cells expressing the GFP-sensor (S (N20) -LIR (Fy) -EGFP), immunoglobulin-labeled GFP is predominantly found in autophagic somatic membrane and autophagic bodies ), Immunoglobulin-labeled GFP was also occasionally detected in the cytoplasm (Fig. 8D [ac]). In contrast, GFP labeled with gold particles was detected only in the cytoplasm in atg5 - / - MEF (Fig. 8D [d]).

These data clearly show that S (N20) -LIR (Fy) -EGFP is preferentially localized in the digestive vacuoles in MEFs during child-induced induction, and S (N20) -LIR Of the new self-organism sensor.

Thus, it was found that S (N20) -LIR (Fy) -EGFP is effectively located in autophagic vacuole during induction of the child's action from serotyping or rapamycin in WT, but not atg5 - / - MEFs.

Example  4 : Self-extinguishing To the flux  effect

S (N20) -LIR (Fy) -EGFP reflects the pathological process of autogenous somatogenesis / regulation without affecting self-extinguishing flux during child-induced induction.

Although the S (N20) -LIR (Fy) -EGFP is located in the endogenous LC3-protonic self-extinguishing vacuole (FIG. 8), this recombinant protein is expressed by the expression of exogenous LIR motifs It can affect the autophagic flux and affect its function.

Therefore, it was tested whether S (N20) -LIR (Fy) -EGFP expression could affect self-extinguishing flux in WT MEFs.

To measure self-extinguishing flux, the level of LC3-II in cells expressing S (N20) -LIR (Fy) -EGFP in the presence or absence of NH4Cl was quantified by Western blot analysis.

As shown in FIGS. 10A and B, MEFs expressing S (N20) -LIR (Fy) -EGFP showed similar induction as in control cells, indicating that expression of S (N20) -LIR It does not affect the self-extinguishing flux.

As another assay to determine the self-extinguishing flux in living cells, fluorescence-based assays including tandem monomeric RFP-GFP-tagged LC3 were used.

In this analysis, co-localization of GFP and RFP fluorescence refers to a compartment that is not fused with lysosomes such as autophagic bodies; This allows simultaneous evaluation of the flux through the self-extinguishing compartment without any pharmacological treatment. Therefore, self-extinguishing fluxes were analyzed in S (N20) -LIR (Fy) -CFP-expressing MEFs by measuring the efficiency of co-localization of RFP and GFP signals using Image J software.

As shown in Fig. 10C, S (N20) -LIR (Fy) -CFP was located in both GFP- and RFP-LC3-positive self-extinguishing vacuoles. Co-localization efficiencies were similar in S (N20) -LIR (Fy) -ECFP and ECFP expressing MEFs (FIG. 10 D and E).

LC3-EGFP or S (N20) -LIR (Fy) -EGFP to compare S (N20) -LIR (Fy) -EGFP and LC3-EGFP as sensors for the detection of pathologic LC3- The number of autologous phagocytes was counted every 3 hours after starvation in the expressing MEFs.

Interestingly, as shown in Fig. 11, GFP-LC3-positive progenitors were readily detectable in MEFs expressing GFP-LC3-GFP at baseline (without serum starvation), while S (N20) -LIR (Fy) -EGFP positive autophagocytes were not detected. However, the number of autophagy bodies in MEFs expressing S (N20) -LIR (Fy) -EGFP gradually increased after serum starvation. In the MEFs expressing S (N20) -LIR (Fy) -EGFP, the number of autoregulated cells was increased similarly to that of LC3-EGFP-expressing MEFs (FIG. 11).

These data suggest that S (N20) -LIR (Fy) -EGFP can be used as a sensor for endogenous LC3-positive autophagic bodies without self-extinguishing flux effects.

Example  5: Application in the Huntington model of cellular disease

To determine whether fluorescence sensors based on S (N20) -LIR (Fy) -EGFP can also detect autophagic bodies generated in cellular disease models, the association of Huntington's disease (HD) Cellular poly (Q) model was selected

The N-terminus of the poly (Q) -extracted (150Q) mutant Huntington was fused with EGFP (150Q-Nhtt-EGFP) or 16Q-Nhtt-EGFP to evaluate the new child effect sensor in the cellular disease model, Or atg5 - / - MEFs for 24 to 48 hours.

As shown in FIG. 12A, 150Q-Nhtt-EGFP-expressing WT MEFs showed small or large poly (Q) aggregates and S (N20) -LIR (Fy) -RFP-positive self-peptic vacuoles.

A portion of the poly (Q) aggregate was accumulated in S (N20) -LIR (Fy) -RFP-positive self-extinguishing vacuole (Fig. 12). However, atg5 - / - MEFs showed more accumulation of poly (Q) aggregates and poly (Q) aggregates, which are much larger than WT MEFs without any S (N20) -LIR (Fy) -RFP- 12).

In addition, it was confirmed that S (N20) -LIR (Fy) -RFP is located in the endogenous LC3-proaphoretic host body in the cultured neurons expressing 150Q-Nhtt-EGFP (FIG. That is, the S (N20) -LIR (Fy) -RFP of the present invention can detect poly (Q) -extended-mutant-Huntington-expressing MEFs and autoligenic bodies accumulated in neurons.

And, the sensor of the present invention was used to clearly observe the child membrane that can enclose the RFP-LC3-positive vacuole (Fig. 14). Second, according to our results (Supplementary Figure 9), overexpressed RFP-LC3 sometimes induced LC3-positive aggregates in atg5 - / - cells, but the probes of the present invention showed visible agglutination in atg5 - / - cells (Fig. 15). This means that the exogenous LC3-proton can be an indicator of specific detection of phagocytosis.

Example  6: mutation FYCO1 LIR  Motif LIR ( Fy ))

The present inventors prepared a probe of the present invention having a wild-type FYCO LIR motif having the amino acid sequence of SEQ ID NO: 3 and a mutant FYCO LIR motif having the amino acid sequence of SEQ ID NO: 4, and found that the loci in the autophagy induction process (localization) was observed. The fabrication and testing of the above probes are the same as those of the above embodiments.

Constructs including wild-type LIR (S (N20) -LIR (Fyco1) -GFP) or mutant LIR (S (N20) -LIR (Fyco1) _m -GFP) It has been selected. HyD-LIR (Fy) in the _m -GFP, conservative sequence of LIR motifs FYCO1 ((W / F / Y ) -XX- (I / L / V)) to point to the alanine-mutants were (FYCO1: AV F DI I TD -> AV F DI A TD). For induction of autophagy, the cells were subjected to serum deprivation for 8 hours.

Confocal microscopy revealed that the probes of the invention with wild-type or mutant LIR inserted in wild-type MEFs were localized in RFP-LC3-positive autophagic vacuoles during the autophagy induction process (Fig. 16A). 16B shows the number of S (N20) -LIR (Fyco1) _{WT / m-} GFP-positive dots per cell and FIG. 16C shows the fluorescence intensity of the sensor (probe) containing the wild type or mutant LIR / Cyoxsol ratio. The results show that the sensor (probe) using the LIR region of FYCO1 positions to the autophagosome depending on the binding function with the LC3 of the LIR region.

Example 7 Measurement of Detection Effect of Probes According to Types of Autophagosomes

There are six LC3 families (LC3A, LC3B, LC3C, GABARAP, GABARAPL1, and GABARAPL2) in mammals while there is one LC3 (ATG8) in yeast.

17 (A) is a confocal microscope photograph showing intracellular localization of S (N20) -LIR (Fyco1) -GFP structure in MEF following induction of autophagic action. S (N20) -LIR (Fyco1) -GFP construct RFP-LC3, mRFP-GABARAP or mRFP-GABARAPL2 and transfection (co-transfected) and, 24 hours later, by the cells in 10 mM NH ₄ Cl lysosomal with Inhibition conditions were incubated with 10 nM rapamycin for 4 h. Scale bar, 10 μm. 17B shows the ratio of the sensor (probe) -buffer RFP-LC3, mRFP-GABARAP or mRFP-GABARAPL2 and FIG. 17C shows the ratio of the fluorescence of S (N20) -LIR (Fyco1) -GFP The ratio of intensity of Autophagosome / Cyoxsol.

The above results show that the sensor of the present invention (probe) detects LC3B-positive autophagosome better than GABARAP, GABARAPL2-positive autophagosome in autophagosome.

It can be seen from the above results that the novel sensor S (N20) -LIR (Fy) -RFP of the present invention can be used for the detection of LC3-positive autophagic bodies that are produced under pathological conditions in a cellular disease model.

Based on these results, it is possible to detect LC3-positive autophagic bodies easily and effectively because of the excellent effect of the self-phagocyte detection method using the probe of the present invention. Based on this, it is possible to detect the LC3- Will be highly utilized.

<110> Industry Academic Cooperation Foundation of Kyungpook National University          Institute for Industry-Academy Cooperation of Hannam University          SNU R & DB Foundation <120> Probes for detecting autophagosome derived from FYCO1 and uses          the <130> PN1701-063 <150> KR 16/0011365 <151> 2016-01-29 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> N30 peptide sequence <400> 1 Met Gln Lys Leu Asn Phe Leu Ser Pro Leu Phe Asn Lys Asn Gly Lys   1 5 10 15 Asn Arg Asp Gly Arg Gln Ser Gly Arg Lys Ser Ser Thr Asp              20 25 30 <210> 2 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> N20 peptide sequence <400> 2 Met Gln Lys Leu Asn Phe Leu Ser Pro Leu Phe Asn Lys Asn Gly Lys   1 5 10 15 Asn Arg Asp Gly              20 <210> 3 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> LIR motif of FYCO1 <400> 3 Arg Pro Pro Asp Asp Ala Val Phe Asp Ile Ile Thr Asp Glu Glu Leu   1 5 10 15 Cys Gln Ile Gln Glu Ser Gly Ser              20 <210> 4 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Mutant of LIR motif of FYCO1 <400> 4 Arg Pro Pro Asp Asp Ala Val Phe Asp Ile Ala Thr Asp Glu Glu Leu   1 5 10 15 Cys Gln Ile Gln Glu Ser Gly Ser              20

Claims (10)

An LC3-interacting region (LIR) motif derived from FYCO1 (FYVE and coiled-coil domain containing 1) consisting of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4; An N-terminal hydrophobic domain of ApPDE4 (Aplysia phosphodiesterase 4) consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; And a reporter fluorescent protein site. delete delete The method according to claim 1,
Wherein the reporter fluorescent protein is an enhanced green fluorescent protein (EGFP) or a modified red fluorescent protein (mRFP). The method according to claim 1,
Wherein the FYCO1 is derived from a human. The method according to claim 1,
Wherein the probe comprises an N-terminal hydrophobic domain having the amino acid sequence of SEQ ID NO: 2; An LIR motif derived from FYCO1 having the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 linked to said domain; And EGFP or mRFP linked to the LIR region. The method according to claim 1,
Wherein the probe is localized in autophagic vacuoles. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; A composition for detecting an autologous animal comprising the probe for the detection of an autologous animal of claim 1 or a vector expressing the same. The method according to any one of claims 1 to 8, wherein the expression of the reporter fluorescent protein is increased as compared with the normal control, Delivery method. 10. The method of claim 9,
Wherein the information relates to Huntington ' s disease.

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