WO2017047846A1 - Antibody specifically binding to arginylated er protein, and use thereof - Google Patents

Abstract

The present invention relates to: an antibody specifically binding to an arginylated ER protein; and a use thereof. Arginylation occurs at the N-terminal of an ER protein by ATE1 R-transferase, the arginylated ER protein, which is a part of the innate immune system inducing cytosolic localization and targeted autophagic vacuoles, is induced into cytosolic dsDNA, the cytosolic dsDNA ATE1-dependently induces the arginylation of an ER protein, the arginylation is stimulated by a misfolded protein in the cytoplasm, and the arginylated ER protein induces the innate immune system by becoming a target as a substrate of an autophagosome inducing autophagic degradation, and thus the antibody specifically binding to an arginylated ER protein, of the present invention, can significantly detect an arginylated ER protein, and thus can be useful in a kit for screening for an immunomodulator, an immunomodulator and a method for screening for an antiviral agent.

Description

 【Specification】

 [Name of invention]

Antibodies that specifically bind to arginated ER proteins _, and uses thereof

 The present invention relates to an antibody that specifically binds to an arginylated protein, an immunomodulator using the antibody, and a virus or bacterial infection diagnosis and a method for screening a therapeutic agent. Background Art

 The body's immune response to foreign antigens can be broadly divided into innate immune response and adaptive immune response. Can be. When the human body is infected with a pathogen, the pathogen must first contact the host in order to cause the disease, and then create an infectious agent, since these pathogens have different surface structures and pathological mechanisms, and thus the defense mechanisms that arise from the host's immune response system. Very diverse

The epithelial surface of the body protects the body from pathogens, prevents colonization and adhesion, and blocks viruses and bacteria entering the cell through specialized cell surface devices that secrete antimicrobial enzymes and peptides. In addition, bacteria, viruses, or parasites that penetrate these barriers encounter macrophages that have surface receptors that can readily bind to various types of pathogens for phagocytosis, which causes an inflammatory reaction. This results in the accumulation of phagocytic neutrophils in the plasma protein and in the place of infection containing complement components that provide humoral intrinsic immunity. Intrinsic immunity is directly involved in infection with the same or other pathogens, leading to first-class host defense through an immediate effect mechanism, which in turn prevents frequent infections of the body. Autophagy is a new type of cell regulatory mechanism, While known natural deaths and necrosis only deal with cell death, they are associated with the death and regeneration of intracellular organelles such as mitochondrial plasma.

 Intracellular organelles have been thought to remain static at normal times, but recent studies have shown that they continue to die and regenerate under normal circumstances. This process destroys the old, degenerative intracellular organelles, providing a nutrient source for regenerating new intracellular organelles. Autophagy protects the entire cell from death through the death and regeneration of these intracellular organelles, and induces the death of the whole cell. The physiological function and clinical significance of endless death and regeneration of intracellular organelles by autophagy have been unclear, but recent researches have suggested the relationship between autophagy and various diseases or aging. In addition, it has been found that autophagy plays an important role in cancer, neurodegenerative diseases, etc., but it is unknown what role autophagy plays in the immune response. A large number of proteins present in the endoplasmic reticulum (ER) are found in the cytosol, but little is known about the posttranslational modi fi cat ion based on the migration from the endoplasmic reticulum to the cell substrate. .

 The N-end rule pathway is a proteolytic system of N-terminal residues that acts as the degron's essential determinant, called N-degron. (proteolytic system). These degrones have specific recognition, such as the family of UBR box-containing proteins known to promote polyubiquitination of proteins and selective proteolysis by proteasomes. It is recognized by the element [N-recognin]. UBR-type

Substrates of N-recognin are arginine (Arg), lysine, Lys, and histidine (N-terminal) positively charged exposed by endoproteolytic cleavage. His) Same type 1, and bulky hydrophobic phenylalanine (phenylalanine, Phe), tyrosine (tyrosine, Tyr), tryptophane (Trp), leucine (Leu) and isoleucine (isoleucine, lie) And type 2 residues. Among them, Arg Tegron is produced by being exposed by being cut by a peptide internal hydrolase (endopeptidase) such as caspase or separase. ER is rich in pro-N-de because the new N 서열 -terminal groups are exposed when the signal sequence of the secreted proteins, which make up about one third of the human protein, is translocation into the ER by a signal peptide hydrolase. Is the source of the gron. In principle, the cleavage can produce potentially labile N-terminus under N-terminal law procedure if exposed to the cytoplasm. Accordingly, the present inventors have made efforts to develop a method for screening immunomodulators, and have produced an antibody that specifically binds to an arginated ER protein, and the ER-existing protein of ATE1 R-transferase using the antibody. N-terminal argination effect of the phosphorus Bip protein, dsDNA as part of the intrinsic immune system of the arginated protein and cytoplasmic positioning by direct confirmation of the effect of inducing autophagic vacuoles to the target, The present invention has been completed by revealing that an antibody that specifically binds to ER protein can be usefully used for immunomodulatory and virus or bacterial infection diagnosis and therapeutic screening methods.

Detailed description of the invention

 [Technical problem]

It is an object of the present invention to provide antibodies and their use which specifically bind to arginylated proteins. [Technical Solution]

 In order to achieve the above object, there is provided an antibody that specifically binds to an arginylated (arginyl at ion) protein.

 The present invention also provides a kit for diagnosing a viral or bacterial infection comprising an antibody that specifically binds to an arginated protein.

 In addition, the present invention provides an immunomodulator screening kit comprising an antibody that specifically binds to an arginated protein.

 In addition, the present invention

 1) treating the test compound in the sample;

 2) provides an immunomodulator screening method comprising the step of measuring the level of arginated protein in the test group treated with the test compound and the control group not treated with the test compound.

 In addition, the present invention

 1) treating the sample with a virus;

 2) treating the test compound to the virus-treated sample;

 3) Provides a virus therapeutic screening method comprising the step of measuring the level and the arginated protein in the experimental group treated with the test compound and virus, and the control treated with the virus. [Favorable effect]

Argination occurs at the N-terminus of the ER protein by ATE1 R-transferase, and the arginated ER protein is autophagi c vacuoles for cytosolic local i zat ion and target. Induced by the cytosolic dsDNA as part of the innate immune system (innate i une system), the cytosolic dsDNA induces argination of the ER protein in an ATE1-dependent manner. Stimulated by the mi sfolding protein, the arginated ER protein is a target of autophagosomes that induces autophagi c degradat ion. And thus induce an innate immune system, the antibody which specifically binds to the arginated ER protein of the present invention can detect the arginated ER protein significantly, and thus, kits for immunomodulator screening, diagnosis of viral or bacterial infections, immunomodulators And virus therapeutic screening methods.

[Brief Description of Drawings]

 FIG. La is a diagram showing sequence alignment of Bip proteins from various species.

 Lb and lc show the results of cycloheximide digestion analysis in 100 nM tappsigargin in the absence (b) or presence (c) for 16 hours.

Figure Id is a diagram showing a result of analysis of ³⁵ S Bip ⁽³⁵ S pulse-chase analysis). FIG. Le is a diagram showing the results of immunoblotting of HeLa cell lines expressing ATE1 isoform.

Π is a total extract of HeLa cell line expressing ATE1 ^1A7A (total extract,

To), the cytosolic fraction (Cyt), and the microsomal fraction (Mic) are classified into cells, followed by cell fraction analysis.

Figure lg is a diagram showing the results of ^{cyclonucleide} digestion analysis of ATE1 ^1A7A -expressing HeLa cell line.

 Figure lh shows the results of immunoblotting of HeLa cell lines expressing Bip-flag-KDEL or Bip-E19V-Hag-KDEL.

Figures li and lj show the results of immunoblotting of HeLa cell lines expressing Ub \ X-Bip-myc / his when ^coexpressed with or without si NA (j) for ATEl ^1A7A (i) or ATE1 .

 2A and 2B show immunoblotting results after transfection of a HeLa cell line with plasmid (a) or poly (dA: dT) dsDNA (b).

2C shows immunoblotting of phosphorylated IRF3 and R-Bip after co-transfection with 0.5 βg / mi poly (dA: dT) or 10 nM siRNA against ATE1 in HeLa cell lines The results are shown.

 2d is a diagram showing the results of immunoblotting after transfection with pcDNA3.1 in the presence or absence of lipofectamine LTX in HeLa cell line.

2e was transfected with a reporter plasmid IgK-IFN β-Luc expressing luciferase from the interferon-β promoter in control and ATEl — ^{/ _} MEFs followed by transfection with poly (dA: dT) dsDNA followed by luciferase reporter analysis. The results are shown.

FIG. 2F shows the luciferase reporter assay after treatment with poly (dA: dT) in combination with ATE1-knockdown or ATE1 ^1A7A overexpression in an IgK-IFN β-Luc-expressing HeLa cell line. to be.

 3A and 3B show RFP signals (red) showing LC3—positive autophagic vacuoles after treatment with poly (dA: dT) in HeLa cell lines stably expressing RFP-GFP-LC3. Compared with the results of immunostaining of R-Bip (A) or p62 (B).

 FIG. 3C shows RFP signals (red) showing LC3-positive autophagic vacuoles following treatment of poly (dA: dT) with ATE1 and Bip siRNAs in HeLa cell lines stably expressing RFP-GFP-LC3 It shows the result of immunostaining of R-Bip compared to the color).

 Figure 4a is a HeLa cell line stably expressing RFP-GFP-LC3

After transfection of plasmids expressing Ub-X-Bip-RFP (X = Arg or Val), X-Bip-RFP fluorescence was compared to GFP-LC3 fluorescence (green) for immunohistochemistry. This figure was confirmed by a chemical test.

 4B shows immunohistochemistry for p62 fluorescence (red) in comparison to X-Bip-RFP fluorescence (green) in HeLa cell lines transiently expressing Ub-X-Bip-GFP (X = Arg or Val). This is confirmed.

4C shows LC3 fluorescence (green) in comparison to _R _ _{B i} pi9-io6_ _YFp fluorescence (red) in HeLa cell line transiently expressing Ub-R-Bip ¹⁹ — ¹⁰⁶ -YFP This is confirmed by immunohistochemistry.

Figure 4d is a ^{Ub-X-Bip 19 - 106} _GFP (X = Arg or Val) in the cell lines HeLa cells transiently expressing the X-Bip ¹⁹ - compared to the ¹⁰⁶ -GFP fluorescence (green) fluorescence emission p62 (red Color) is confirmed by immunohistochemistry.

 5A and 5B show the results of immunoblotting with FK antibody after treatment with geldanamycin (a) or various stress sources (b) in HeLa cell lines:

 A23187: calcium carrier (calcium i onophor e);

 CCCP: carbonyl cyanide m-chlorophenylhydrazone (m— ch 1 oropheny 1 hydr azone) [prot onophore (H + ionophore) and mitocon H laa Anticoupler of oxidative phosphorylated ion;

 BTZ: bortezomib; And

 BafAl: bafilomycin A1.

 Figure 5c is a diagram showing the correlation between arginization and ubiquitination in DNA induced endogenous immune response.

 5d is a diagram showing the results of immunoblotting of HeLa cell lines expressing a mutant in which Ub-HA or Lys is converted to Arg and ubiquitination is terminated.

 Figure 5e is a HeLa cell line converting 1 i / u poly (dA: dT) dsDNA well, and then, immunostaining of FK2, R-Bip or p62 antibody.

 6 shows sequence alignment of CRT (a), PDI (b), GRP94 (c), and Erdj5 (d). ,

FIG. 7A shows a step-diluted anti—R-Bip antibody and an immobilized 11-mer R-Bip peptide (R-Bip-peptide) floating on the N-terminal end of R-Bip in 96-well plate. And then incubated with an anti-goat secondary antibody bound to horseradish peroxide, and then the amount of R-Bip antibody binding to the immobilized R-Bip peptide was determined by the OD value at 450 nm of the secondary antibody. Based on the determination, the peptide competition assay was performed. Bip-peptide as a control, 10-mer peptides were used which stood at the N-terminal portion of the non-arginated Bip.

 7B is a diagram illustrating peptide competition assays after anti-R-Bip antibodies were cultured alone or in combination with R-Bip-peptides or Bip-peptides.

 FIG. 7C shows peptide competition assays using R-CRT-peptide and CRT-peptides, respectively, which accentuate the N-terminal portion of arginated or non-arginated CRTs.

 FIG. 7D is a diagram showing peptide competition analysis using R-PDI-peptide and PDI-peptide, each of which is bound to the N-terminal portion of PDI, which is either arginated or unarginated.

 FIG. 7E is a dot blot analysis of anti-R-Bip antibodies against peptides floating on the N-terminal region of unarginated (E-peptide) or arginated (R-peptide) Bip The results are shown.

 8A shows a schematic of the ATE1 gene and its spliced isoform.

8B is a diagram showing the results of immunoblotting over time of R-Bip, Bip and LC3 in HeLa cell lines transiently expressing ATE1 ^1A7A or treated with ^{cyclonucleosides} .

 8C is a diagram illustrating argination of Bip after ATE1-knockdown in HeLa cell lines expressing ^^.

Figure 8d is a diagram showing the results of immunoblotting after transfection of plasmid expressing ATE1 ^1A7A in HeLa cell line.

 FIG. 8E shows the full length compared to Ub-X—Bip—myc / his, which is cleavage ((: 01; ^ 11310 101131 cleavage) by Ub hydrolases at the Ub-Bi junction. It is a figure which shows schematic diagram of Bip.

 Figures 9a and 9b is a diagram showing the results of immunoblotting after infecting the HeLa cell line lipofectamine LTX alone or various DNA forms.

9C expresses siRNA # 204 and NHK-GFP for ATE1 in HeLa cell lines. After co-transfection with the plasmid, the cells were classified into cell substrates and immunoblot is shown.

 9d is a diagram showing the results of immunostaining of R-Bip after co-transfection with a mixture of NHK-GFP plasmid and ATE1 siRNA # 204 in HeLa cell line, compared to NHK-GFP fluorescence.

10 shows immunostaining of R—Bip compared to when using antibodies against KDEL sequences after 200 nM Topsigargin was incubated for 6 hours in the absence or presence of HEK293 cell line (2.5 × 10 ⁵ / well) It is a figure which shows the analysis result.

 Figure 11a is a diagram showing the result of performing immunoblot after transfection with HeLa cell line with poly (dA: dT) dsDNA or 5 'ppp-dsRNA.

FIG. Lib shows NF-κΒ β p50 and immunostaining results in HeLa cell lines (3 × 10 ⁶ / well) treated with 0.5 / well poly (dA: dT) dsDNA for 16 hours.

 11C is a diagram showing the results of immunoblotting after transfection with HeLa cell line with ATE1 siRNA.

 Figure lid shows the results of immunoblotting of R-Bip and LC3 after transfection with HeLa cell line with poly (dA: dT).

Figure lie shows control and ATE1 ^{_ /} — MEF transfected with IgK-IFN β-Luc reporter plasmid expressing luciferase from the interferon-β promoter, followed by 18 hours after transfection with poly (dA: dT) dsDNA. , dual luciferase activity - a diagram showing a result of measurement using a luciferase reporter assay systems (dual-Luci f erase reporter assay system, Promega), and a Wallac Victor ² (Wal lac VICTOR ^2, Perkin Elmer).

 12A is compared with RFP signal (red) showing LC3-positive autophagy after treatment with poly (dA: dT) dsDNA or 5′ppp dsRNA in HeLa cell line stably expressing RFP-GFP-LC3 Shows the results of immunostaining of R-Bip.

Figure 12b is a diagram showing the immunostaining results of R-Bip and p62 after treatment with poly (dA: dT) dsDNA or 5 'ppp dsRNA in HeLa cell line. Figure 13 shows a schematic of the Bip protein expressed using the Ub fusion technique:

 a: full length and Bip protein;

 b: Ub-X-Bip-GFP [X-Arg, Glu (circular (nat) cleaved by Ub hydrolase in the Gly76 residue of the Ub moiety producing the reference Ub and X-Bip-GFP ive), or Val]; and

c: Reference Ub and X-Bip ^19. Ub-X-Bip ¹⁹ — ¹⁰⁶ -GFP [X-Arg cleaved by Ub hydrolase to the Gly76 residue of the Ub moiety producing ¹⁰⁶ -GFP. , Glu (native, or Val).

 Figure 14a is a HeLa cell line stably expressing YFP-LC3

Ub-R— Bip ¹⁹ — ¹⁰⁶ -GFP was transfected, and then R-Bi _P ¹⁹¹⁰⁶ -RFP fluorescence Cred) was compared by YFP-LC3 fluorescence (green) by immunohistochemistry. It is confirmed figure.

FIG. 14B shows p62 (red) immune tissue compared to X-Bipl9-106-GFP fluorescence (green) in MEF transiently expressing Ub-X-Bip ^{19 "106} -RFP (X = Arg or Val) This figure was confirmed by a chemical test.

Figure 15a is a diagram showing the results of immunoblotting over time after the heat shock (heat shock) at 42 ^° C treated HeLa cell line.

 Figure 15b is a diagram showing the results of immunoblotting with polyubiquitin chain-specific FK antibody after treatment with HeLa cell line with various stress sources:

 A23187: calcium ionophore;

CCCP: carbonyl cyanide m-chlorophenylhydrazone [protonophore (H ⁺ ionophore) and oxidative phosphorylation in mitochondria -^ Uncoupler];

 BTZ: bortezomib; And

BafAl: bafilomycin A1. FIG. 15C is a diagram showing the result of performing the immunoblotting after transfection with a plasmid expressing CL1-YFP or its control group in the HeLa cell line (left). Fig. 1 shows immunoprecipitation results with GFP antibodies capable of binding to both CL1-YFP and EGFP (right).

 FIG. 15D shows polyubiquitination using FK2 antibody capable of binding to both polyubiquitin conjugates and free Ubs after treatment with poly (dA: dT) dsDNA in He cell lines, as compared to R-Bip and p62 Figure shows the immunostaining results of the protein. [Best form for implementation of the invention]

 Hereinafter, the present invention will be described in detail. The present invention provides an antibody that specifically binds to an arginylat ionized protein. .

 The protein is preferably an ER (Endopl aside Ret iculum) protein,

More preferably, it is any one selected from the group consisting of BIP, CRT and PE) I, wherein the BIP protein has an amino acid sequence as set out in SEQ ID NO: 1, and the CRT protein has an amino acid sequence as set out in SEQ ID NO: 2 Most preferably, the PDI protein has an amino acid sequence as set forth in SEQ ID NO: 3, but is not limited thereto.

Preferably, the N ᅳ terminal Glul9 of the BIP protein, the N-terminal Glul8 of the CRT protein, or the Ν-terminal Aspl8 of the ΡΕ ⁾ ί protein are arginated, and the arginated protein is ATE1 R-transferase. More preferably, the N-terminal Glul9 of the BIP protein, the N-terminal Glul8 of the CRT protein, or the N-terminal Aspl8 of the PDI protein are induced through argination or an endogenous immune response to the double stranded DNA (dsDNA). It doesn't work.

The arginated protein is induced by a mi sfolded cytoplasmic protein and co-located with autophagic vacuoles Argination of the N-terminal Glul8 of the BIP protein, the N-terminal Glul8 of the CRT protein, or the N-terminal Aspl8 of the PDI protein, which forms a puncta, results in autophagy N-degron to deliver the protein to the autophagosome. It is preferred to act as, but is not limited thereto. In a specific embodiment of the present invention, the present inventors, Bip (NCBI Reference Sequence: NP_005338) (SEQ ID NO: 1), CRT (NCBI Reference Sequence: NP_004334) (SEQ ID NO: 2), PDI ((NCBI Reference Sequence: NP_000909) A peptide polynucleotide sequence corresponding to the N-terminal sequence of the arginated mature protein, wherein the rabbit polyclonal antibody specific for the arginated form (R-Bip, R-CRT, R-PDI) of (SEQ ID NO: 3) is REEEDKKEDVGC (SEQ ID NO: 4), REPAVYFKEQ (SEQ ID NO: 5), and RDAPEEEDHVL (SEQ ID NO: 6), respectively prepared, and then the specificity of the antibody was confirmed, the antibody of the present invention is more arginine than the non-arginated peptide It has been shown to bind more favorably to the normalized form (R-Bip, R-CRT, R-PDI) peptide (see Figure 7.) Antibodies of the present invention are immunoglobulin molecules immunologically having a specific antigen and a semiungol To include, specifically to the antigen Means a protein molecule that acts as a receptor, and includes polyclonal antibodies, monoclonal antibodies, whole antibodies and antibody fragments, and also chimeric antibodies (eg, humanized murine antibodies) and heterologous bonds. Includes forms produced by genetic engineering such as antibodies (eg, bispecific antibodies) Full antibodies are structures having two full length light chains and two full length heavy chains, each light chain The total antibody comprises IgA, IgD, IgE, IgM and IgG, and IgG is a subtype and includes IgGl, IgG2, IgG3 and IgG4. A fragment having a function, and includes Fab, Fab 'F (ab') 2, Fv, etc. The Fab is a variable region of the light and heavy chains, a constant region of the light chain and a first constant region of the chain (CH1). Domain) Has a single antigen-binding site in the structure. Fab 'is at least one cysteine at the C terminus of the heavy chain CH1 domain It differs from Fab in that it has a hinge region containing residues. F (ab ') 2 antibodies are produced by disulfide bonds of cysteine residues in the hinge region of Fab'. Fv (var iable fragment) means a minimum antibody fragment having only the heavy chain variable region and light chain variable region. Double-chain Fv (dsFv) is a disulfide bond is linked to the heavy chain variable region and light chain variable region, and short-chain Fv (scFV) is generally covalently linked to the variable region of the heavy chain and light and variable regions through a peptide linker. Such antibody fragments can be obtained using proteolytic enzymes (e.g., the entire antibody can be restricted to papain and Fabs can be obtained and pepsin can be used to obtain F (ab ') 2 fragments). Can be produced through genetic recombination technology. The present invention also provides a kit for diagnosing a virus or a bacterial infection comprising an antibody that specifically binds to an arginylat ionized protein. The present invention also provides an immunomodulator comprising an antibody specifically binding to an arginylat ionized protein, and a kit for screening a virus or a bacterial infection therapeutic agent.

 The protein is preferably an ER (Endoplasmic Ret iculum) protein, more preferably any one selected from the group consisting of BIP, CRT, and PDI, wherein the BIP protein has an amino acid sequence as set forth in SEQ ID NO: 1, The CRT protein has an amino acid sequence set forth in SEQ ID NO: 2, and the PDI protein most preferably has an amino acid sequence set forth in SEQ ID NO: 3, but is not limited thereto.

The arginated protein is preferably N-terminal Glul9 of the BIP protein, N-terminal Glul8 of the CRT protein, or N-terminal Aspl8 of the PDI protein is arginated by arginine _: The arginated protein is BIP by ATE1 R-transferase. N-terminal Glul9 of the protein, N-terminal Glul & of the CRT protein, or N-terminal Aspl8 of the PDI protein are more preferably induced through argination or endogenous immune response to double-stranded DNA (dsDNA). Do not. The arginated protein is induced by a misfolded cytoplasmic protein, forms a cytoplasmic point (puncta) colocated with autophagic vacuoles, and the N-terminal Glul9, CRT protein of BIP protein. Argination of the N-terminal Gplul8 or the N-terminal Aspl8 of the PDI protein preferably acts as an autophagy N-degron for delivery of the protein to the autophagosome.

In a specific embodiment of the present invention, the inventors performed analysis of putative N-degrons in ER (endoplasmic reticulum), and when analyzing the P1 'residues of the signal peptide cleavage site, approximately 27% ( 132 of 498) exposed N-terminal residues in an unstable state, confirming that they were significantly lower compared to the existing N-Degron code, which included 13 of 20 amino acids (SMSriram). et al. Nature Rev. Mo 1 Cell Biol. 12, 735 (2011)). As a result of reviewing various literatures, most of the N-end ^' rule substrates identified so far are N-terminal Arg or pro-N-degrons (Asn, Gin, Cys, Asp, Glu) N-terminal Arg).

 In addition, a cycloheximide digestion assay was performed for the turnover of Bip, a Glul9 exposed chaperone, which assists in translocation and folding of newborn polypeptides. It was confirmed that it may be related to other degradation pathways not related to theasome (see FIGS. Lb to Id).

In addition, the physiological substrate of ^argination confirmed that ATE1 ^1A7A was a major R-transferase for Bip, CRT and PDI (see FIGS. Le and 8b to 8d). About 70% of the R-Bip produced by ATE1 ^1A7A was ^detected from the soluble fraction (see Figure If), while the non- ^arginated (lumen's) Bip was markedly short-lived, while the (cytoplasmic) R -Bip was relatively long in life (Figure lg), which confirmed that Bip of lumen was metabolized to some extent through retrotranslocation and argination (see Figure lg). In addition, ᅳ ^1A7A overexpression readily induced argination of recombinant Bip exposed to N-terminal Ghil9, as a result of confirming whether Bip argination occurs in N-terminal GluW. It was confirmed that ATE1 ^1A7A mediated ^argination was inhibited while being stabilized by (see FIGS. Lh to lj). Thus, it was confirmed that Bip was arginated by ATE1 R-transferase at its N-terminal Glul9 in vivo.

 In addition, the inventors found that Bip argination was not induced to detectable levels even by 6 hours of ER stress, which indicates that the main role of R-Bip is not to remove the final misfolded ER protein, but outside the ER. It was confirmed that it participates in the process of the process (see FIG. 8B).

 In addition, as a result of confirming the stress that induces argination, argination of ER protein is induced through an intrinsic immune response to double-stranded DNA (dsDNA) (see FIGS. 2A, 9A and 9B), and induced by double-stranded DNA. It was confirmed that ATE1 plays a role in Bip argination (see FIGS. 9C and 9D). Also, unlike double-stranded DNA (dsDNA), double-stranded RNA (dsR A) has a relatively inducible activity for inducing argination. Weak spots were identified (see FIG. 11A). These results suggest that cytoplasmic dsDNA triggers retrotranslocat ion and ATE-1 dependent argination of several Ca ++-coupled ER chaperones. In addition, the inventors also found that argination of Bip, PDI, and CRT is strongly induced by cytoplasmic poly (dA: dT) dsDNA, an immunostimulatory reagent that expresses viral dsDNAs (see FIGS. 2B, 11A-11C). It was confirmed that ATE1-dependent argination of ER presence plays an important role in cytoplasmic intrinsic immune response to dsDNA.

In addition, the present inventors confirmed whether R-Bip plays a role in the autophagy pathway during DNA-induced endogenous immune response, and cytoplasmic dsDNA induces both Bip argination and autophagy. R-Bip is delivered to autophagy fear, suggesting that R-Bip plays a role in autophagy degradation of cellular components (const i tuents) during dsDNA triggered intrinsic immune response. (See FIG. 3 and FIG. 12).

 In addition, the present inventors confirmed that N-terminal Arg acts as an autophagy N-degron necessary for Bip to be targeted to autophagosomes, and thus argination targets Bip and other cytoplasmic ER proteins to autophagy. It was confirmed that autophagy produced N-degron (see FIGS. 4 and 14).

 In addition, the inventors confirmed whether argination correlated with misfolded cytoplasmic protein. As a result, argination of ER protein is induced by misfolded cytoplasmic protein. Therefore, arginated ER chaperone is metabolized with misfolded cytoplasmic protein. (turnover), it was confirmed (see Fig. 5 and 15).

 In addition, the present inventors have investigated the role of R-Bip in autophagy clearance of misfolded cytoplasmic protein. As a result of investigating the colocalization between R-Bip, ubiquitinated protein, and autophagosome, R-Bip plays a role in autophagy clearance, confirming that its N-terminal Arg is through its ability to act as trans-acting N-degron for i sfolded protein cargo (FIG. 5E). And FIG. 15D).

Therefore, the ER protein of the present invention generates argination at the N-terminus by ATE1 R-transferase, and the arginated ER protein is cytosolic (local cytosol) and autophagy to the target (autophagi c). As part of the innate immune system that induces vacuoles, the cytosolic dsDNA is induced, and the cytosolic dsDNA induces argination of the ER protein in an ATE1-dependent manner, which is misfolded in the cytoplasm. Stimulated by (mi sfolding) protein, the arginated ER protein is targeted as a substrate of autophagosomes that induce autophagi c degradat ion, inducing an innate immune system, Antibodies that specifically bind to arginated ER proteins can detect arginated ER proteins significantly, thus infecting viruses or bacteria Stage and can be useful for immunomodulation and theta, viral or bacterial infection treatment kit for screening. Kits of the present invention may include one or more other component compositions, solutions or devices suitable for analytical methods, as well as antibodies that selectively recognize arginated ER proteins for measuring expression levels of arginated ER proteins. . Specifically, the arginated ER of the present invention. Kits for measuring the expression level of a protein may include a substrate, a suitable buffer, a secondary antibody labeled with a chromophore or a fluorophore, a chromogenic substrate and the like for immunological detection of the antibody. The substrate may be a nitrocell membrane film, a 96 well plate synthesized with a polyvinyl resin, a 96 well plate synthesized with a polystyrene resin, a slide glass made of glass, and the like. Alkaline phosphatase (alkal ine phosphatase) can be used, the fluorescent material can be used FITC, RITC, etc., the color substrate is ABTS (2, 2 '-azino-bis (3-ethylbenzothiazoline-6-6) Sulfonic acid)) or 0PD (o-phenylenediamine), TMB (tetramethyl benzidine) can be used.

 The kit is preferably, but not limited to, i) measuring the amount by detecting fluorescence by fluorescence label attached to the antibody, i) sur face plasmon resonance (SPR) method or SPRK sur face pl asmon resonance imaging (ii) method; . The antibody may be prepared by an antibody production method known to those skilled in the art for the epitope or antigen of the present invention.

 The kits of the present invention can be used to measure arginated ER proteins derived through endogenous immune response to cytoplasmic double stranded DNA (dsDNA). The kit not only enables the diagnosis of viral or bacterial infections of the practitioner and the screening of immunomodulators and viral or bacterial infections by measuring arginated ER proteins, but also by monitoring the individual's response to treatment. It is possible to change the treatment depending on the result.

The kit of the present invention may include a first acquisition reagent (capture reagent) that binds to the arginated ER protein and a second acquisition drug that does not bind to the first acquisition reagent. The first acquisition reagent is an antibody or an antibody that specifically binds to a metal chelate, preferably an arginated ER protein, and the second acquisition reagent is a conjugate labeled with a chromophore, a fluorescent substance, a radioisotope, or a colloid. ) Is a secondary antibody. The chromophore can be peroxidase, alkaline phosphatase or acid phosphatase (e.g. horseradi sh peroxidase) and if it is fluorescent, Oresincarboxylic acid (FCA), Fluorescein isothiocyanate (FITC), Fluorescein thiourea (Fffl),

7-aceoxycoumarin-3-yl, fluorescein-5-yl, fluorescein-6-yl, 2 ', 7'-dichlorofluorescein-5-yl,

 2 '， 7′-dichlorofluorescein-6-yl ， dihydropenttramethylosamine ᅳ 4-yl ，

Tetramethylrodamine-6-yl, tetramethylrodamine-6-yl,

4, 4-difluoro-5, 7-dimethyl-4-bora-3a, 4adiaza-S-indacene 3-ethyl or 4, 4-difluoro-5, diphenyl-4-bora -3a, 4a-daza-s—indacene-3 ᅳ ethyl is possible. When a sample obtained from an individual is brought into contact with a primary acquisition reagent, preferably an antibody that specifically binds to an arginated ER protein, the sample may be diluted to a suitable degree prior to contact with the antibody and the antibody may be washed or separated from the complex. It can be fixed on a solid to facilitate subsequent steps. The solid phase can be glass or plastic such as a micro crot iter plate, rod, bead or mi crobead. The antibody may also be bound to a probe substrate or protein chip. The sample may be placed in a constant temperature with the antibody and washed to determine the antibody-marker complex. This is done by placing the washed mixture with a secondary acquisition reagent, preferably a secondary antibody. Determination of the amount or presence of the antibody-marker complex can be accomplished through fluorescence, luminescence, chemi luminescence, absorbance, reflection or transmission. In addition to the above method, the protein marker in the sample can be detected by performing an indirect assay method such as competition or inhibition reaction analysis with a monoclonal antibody that binds to another epitope of the protein marker. In addition, the present invention

 1) treating the test compound in the sample;

 2) provides an immunomodulator screening method comprising the step of measuring the level of arginated protein in the test group treated with the test compound and the control group not treated with the test compound.

 In addition, the present invention

 1) treating the sample with a virus;

 2) treating the test compound to the virus-treated sample;

 3) Provides a method for screening a virus therapeutic agent comprising the step of selecting a test compound to reduce the level of arginated protein in the test compound and the virus-treated experimental group, and the virus-treated control group.

 The sample of step 1) is preferably one or more selected from the group consisting of isolated whole blood, serum, plasma, saliva, urine, sputum, lymph, cerebrospinal fluid and intercellular fluid, but not always limited thereto.

 The arginated protein is preferably arginated by N-terminal Glul9 of BIP protein, N-terminal Glul8 of CRT protein or N-terminal Aspl8 of PDI protein, and the level of arginated protein is specific to arginated protein. It is preferable to measure using an antibody which binds suitably.

The ER protein of the present invention generates argination at the N-terminus by ATE1 R-transferase, and the arginated ER protein induces cytosolic local izat ion and autophagic vacuoles to the target. Induced by the cytosolic dsDNA as part of the innate i une system, the cytosolic dsDNA induces argination of ER protein in an ATE1-dependent manner, and the argination is misfolded in the cytoplasm. stimulated by sfolding proteins, the arginated ER protein is targeted as an autophagosome substrate that induces autophagic degradat ions and induces an innate immune system. ， Arginated When the expression level of the protein is decreased, the test compound may be used as an immunomodulator, or after the virus and the test compound are treated simultaneously, the test compound which reduces the arginated protein level may be used as a virus therapeutic agent. By measuring the expression level of the arginated protein can be usefully used for screening immunomodulators or viral therapeutics. In addition, the screening method ¾ of the present invention can be screened for immunomodulators or viral therapeutic agents by processing the antibody binding to the arginated protein and then searching for the amount of bound antibody.

 As a method for detecting the amount of the bound antibody, it is preferable to use a high-throughput screening (HTS) system, which includes a fluorescence method performed by detecting fluorescence by fluorescence labeling, and a plasmon resonance change on the surface without labeling the fluorescence material. Surreal plasmon resonance (SPR) method for real-time measurement or SPRK surface plasmon resonance imaging (SPR) method for imaging and confirming the SPR system is preferably used, but is not limited thereto.

 The fluorescence method is a method of labeling an antibody binding to a specific antibody to the arginated protein marker with a fluorescent substance using a fluorescence scanner program and spotting the signal by spotting. . The fluorescent material is Cy3, Cy5, poly L-lysine-fluorescein sothiocyanat (FITC), rhodamine-B-isothiocyanate (rhodamine-B soisocyanate, ITC) ), Preferably any one selected from the group consisting of rhodamine, but is not limited thereto.

 Unlike the fluorescence method, the SPR system can analyze the binding degree of the antibody in real time without the need to label the sample with a fluorescent material. In the case of SPRI, multiple samples can be analyzed simultaneously using a microalignment method.

In addition, the method of the present invention is a substrate that will not react with the enzyme, Proteins and the like can include washes or eluents that can be removed and retain only bound arginated protein markers. Samples used for analysis include biological samples that can be distinguished from normal conditions such as serum, urine and tear saliva. Preferably from biological liquid samples such as blood, serum, plasma. Samples may be prepared to increase the detection sensitivity of arginated protein markers. For example, serum samples obtained from patients may be anion exchange chromatography, affinity chromatography, si ze exclusion chromatography, and: Liquid _. It can be pretreated using a method such as chromatography, sequential al extract ion or gel electrophoresis.

 In addition, the levels of arginated protein in the test compound-treated test group and the test compound-untreated control group, or the test compound and the virus-treated test group, and the virus-treated control group, showed the levels of arginated protein in Western blot. , ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunity staining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, and the like. Through the analytical methods, it is possible to compare the amount of antigen-antibody complex formation in the normal control group with the amount of antigen-antibody complex formation in the control group, and to determine a substance that reduces the expression level of the arginated protein. And viral therapeutics.

As used herein, the term “antigen-antibody complex” means a combination of an arginated protein and an antibody specific thereto, and the amount of the antigen-antibody complex formed quantitatively through the magnitude of the signal of a detection ion label. It can be measured by Such detection labels may be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, niicropart icles, redox molecules, and radioisotopes, but are not necessarily limited thereto. When enzymes are used as detection labels, the available enzymes include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase or alkaline phosphatase and acetylcholinese. Terases, glucose oxidases, nucleokinase and GDPase, RNase, glucose oxidase Luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate art aminotransferase, phosphphenolpyruvate decarboxylase, β-latamases, and the like. Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, 0-phthalaldehyde, fluorescaman, and the like. Ligands include, but are not limited to, biotin derivatives. Luminescent materials include, but are not limited to, acridinium ester, luciferin, luciferase, and the like. Microparticles include, but are not limited to, colloidal gold and colored latex spouts. Redox molecules include ferrocene, ruthenium complex, barologen, quinone, Ti ion, Cs ion, diimide, 1,4'benzoquinone, hydroquinone, K ₄ W (CN) ₈ , [Os (bpy) ₃ ] ^{2 +} , [RU (bpy) ₃ ] ²⁺ , [M0 (CN) ₈ ] ^4-, and the like. Radioactive isotopes ， ¹⁴ C,

₃₂ p, 35 _S; 3 ₁ , 51 _Cr; 57 _CO; 58 _CO; 59ρ _{6; 90γ} , 125J; 131 j; 186 _Re and the like.

 Protein expression level measurement is preferably by using an ELISA method. ELISA is a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support, attached to a solid support Direct sandwich ELISA using another labeled antibody that recognizes the antigen in the antibody-antigen complex, a label that recognizes the antibody after reacting with another antibody that recognizes the antigen in the antibody-antigen complex attached to the solid support Various ELISA methods include indirect sandwich ELISA using secondary antibodies. More preferably, the antibody is attached to a solid support and the sample is reacted, followed by the attachment of a labeled antibody that recognizes the antigen of the antigen-antibody complex to enzymatic color development or to an antibody that recognizes the antigen of the antigen-antibody complex. Detected by sandwich ELISA method by attaching labeled secondary antibody and enzymatic color development.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples. However, the following Examples and Experimental Examples merely illustrate the present invention, but the scope of the present invention is not limited by the following Examples and Experimental Examples.

Example 1 Preparation of Plasmid and Other Reagents

 Plasmids encoding the ATE1 R-transferase subtype isoiorms were prepared by the method described in Kwon-1999 (Y. T. Kwon et al, Mo 1. Cell. Biol. 19, 823 (1999)). In addition, the polymerase chain reaction (PCR) was used to construct a Bip-flag-KDEL fragment to construct a full-length Bip-flag-KDEL-encoded plasmid in which the C-terminal KDEL ER residual signal was replaced with flag-KDEL. Was amplified from pENTR / hBipc (Invi trogen) and inserted into Hindlll / BamHI-KDEL of vector pcDNA3.0.

 In addition, Glul9-to-Val point mutations in Bip were generated using the QuickChange XL method (Stratagene). Ub-X-Bip-GFP and

Plasmids encoding Ub-X-Bipl9—106—GFP were constructed at the BamHI / Hindlll position of pEGFP-N3 and amplified by PCR. In addition, Bip fragments were amplified from pENTR / hBipc (Invitrogen) using PCR to construct cDNA encoding full-length Bip-myc / his, and inserted into vector pcDNA3.1-myc / his of the EcoRV-XhoI region. It was.

 In addition, plasmids encoding Ub-X-Bip-myc / his were constructed in the BamHI / Xbal region of pcDNA3.1-myc / his using PCR amplification. The nucleotide sequence of the plasmid thus constructed was confirmed by sequencing.

 In addition, the following plasmids are commercially available. Were used: N3-EGFP (Addgene, 1 in cat # 608), Ub-HA (Addgene, 17608: pRK5-HA-ubiquit in-WT), Ub-mt-HA (Addgene, 17603: pRK5-HA-ubiquitin- K0), YFP-CLl (Addgene, 11950), Ig-IFN β-LucCAddgene, 14886), leni 1 lar-luc (Pr omega, E2231, pRL-SV40 vector).

In addition, NHK-GFP plasmid was obtained by Dr. It was donated by Nobuko Hosokawa (Kyoto University, Japan). In addition, the following other reagents were commercially available: thapsigarginCSigma, T9033), digitonin (Sigma, D141), bafilomycin AKSigma, B1793), geldanamycin (Sigma, G3381), MG132 (Calbiochem, 474790), poly (dA dT) (Invivogen), 5ppp-RNA (I nv i vogen), salmon sperm DNA (Sigma, D1626) RNAiMaxClnvitrogen, 13778), Lipofectamine 2000 (Invitrogen, 11668), Lipofectamine LTX (Invi trogen, 15338), protein A / G plus agarose bead (Santa Cruz, sc2003), car bony 1 cyanide ch 1 or opheny 1 hydr azone (S i gma, C2759), A23187 (Sigma, C7522), bortezomib (BTZ, Selleckchem, S1013), hydrogen peroxide (Sigma , H1009), Dual-luci ferase reporter assay system (Pr omega, E1910).

Example 2 Preparation of Antibody

 Antibodies used in the present invention used those commercially available as follows:

 Rabbit monoclonal anti-Bip (Cell Signaling, cat. # 3177, 1: 1000), rabbit polyclonal anti-GFP (Abchem, ab290, 1: 3000), mouse monoclonal ant i-LC3 (Santa Cruz sc271625, 1: 1000), rabbit polyclonal ant i-GAPDH (Santa Cruz, sc25778, 1: 2000), mouse monoclonal anti-flag (Sigma, fl804, 1: 3000), rabbit monoclonal ant i-ATEK Santa Cruz, sc-271220 , 1: 500), rabbit monoclonal ant i-IRF_3 (Cel 1 Signaling, 11904P, 1: 500), rabbit anti-phospho-IRF— 3 (Ser396) (Cel 1 Signaling, 4947S, 1: 500), mouse single Clone ant i -CHOP (Cel 1 Signaling, 2895S, 1: 200), mouse monoclonal anti-calreticulin / CRT (BD Biosciences, 612136, 1: 5000) ᅳ mouse monoclonal anti-polyubiquitylated FK2 clone (Mi 11 ipore, cat # 04-263), rabbit monoclonal anti-PDKCell Signaling, 2446S, 1: 1000).

 Secondary antibodies were also used as commercially available as follows:

alexa f luor 488 goat ant i -rabbit IgGdnvitrogen, A11034, 1: 200), alexa f luor 555 donkey ant i -goat IgGdnvitrogen, A21432, 1: 200), alexa f luor 555 goat ant i -mouse IgGdnvitrogen, A21424, 1 : 200), ant i -rabbit IgG-HRP (Cell Signaling, 7074, 1: 10000), ant i-mouse IgG-HR (Cel l Signaling, 7076, 1: 10000).

Example 3 Preparation of Antibodies Against Arginated Bip, CRT, PI) I

 Argination of Bip (NCBI Reference Sequence: NP_005338) (SEQ ID NO: 1), CRT (NCBI Reference Sequence: NP_004334) (SEQ ID NO: 2), PDI ((NCBI Reference Sequence: NP—000909) (SEQ ID NO: 3) Rabbit polyclonal antibodies specific for the modified forms (R-Bip, R-CRT, R-PDI) correspond to the N-terminal sequence of the arginated mature protein through a custom service of AbFront ier Inc. (Seoul, Korea). Peptide sequences REEEDKKEDVGC (SEQ ID NO: 4), REPAVYFKEQ (SEQ ID NO: 5), and RDAPEEEDHVL (SEQ ID NO: 6) were prepared.

 Specifically, immunization was performed with the peptides in rabbits and boosted at intervals of three weeks with incomplete Freunds adjuvant (IFA). After IgG purification using immobilized Protein A, the antibody was subjected to two steps of affinity chromatography. First, the antibody was negatively purified using non-arginated peptads, EEED KEDVGC (SEQ ID NO: 7), EPAVYFKEQ (SEQ ID NO: 8), or DAPEEEDHVLC SEQ ID NO: 9, to remove nonspecific IgG. Arginated peptides REEEDKKEDVGC (SEQ ID NO: 4), REPAVYFKEQ (SEQ ID NO: 5) or RDAPEEEDHVL (SEQ ID NO: 6) by eluting the antibody at better f low-through from first affinity chromatography in 0.1 M citric acid Positively purified using

Example 4 Identification of Antibody Specifically Binding to Arginated Bip, CRT, and PDI

The <Example 3> and arginine stylized Bip, CRT, PE) I a specific antibody is arginine Chemistry ^{"immunoblotting} for the cell culture, where induction stressor is not present, or present to combine with about made in putting Analysis was used to confirm the specificity of the purified antibody. Specificity was independently determined using peptide binding assays. Specifically, peptides for Bip, CRT and PDI were each diluted to 2 mg / ml in coating buffer (0.1 M carbonate pH 9.6) and incubated at 4 ^° C overnight to immobilize onto 96-wel l plates. Next, incubation at 37 ° C. for 1 hour in blocking buff A (2% skim milk in TBS-T). After washing with TBS-T, the immobilized peptide was incubated at 37 ^° C for 2 hours with blocking buffer containing primary antibodies against Bip CRT or PDI in the presence or absence of competing peptides and then in horseradi sh peroxide. Incubated with conjugated goat ant i -rabbi t secondary antibody. Binding of antibodies to immobilized peptides was assessed according to optical density (OD) at 450 nm.

As a result, it was confirmed that the level of R-Bip is lower than or at the boundary of detection limit in various cell lines such as HeLa cells, HEK293 cells, and mouse fibroblasts. The same analysis with a Bip antibody against luminal Bip (Cel l signal ing, cat # 3177) revealed high levels of const i tut ive signals, which resulted in the detection of unarginated subpopulations. It confirmed that it was a signal to express. In addition, as a result of poly (dA: dT) dsDNA treatment, the level of R-Bip increased significantly (determined to increase by -100 times by density measurement method), but the Bip level of the lumen remained unchanged or only slightly changed. This indicates that the ant iR-Bip antibody does not recognize non-arginated Bip in the lumen. Similar analyzes showed that the ant iR-CRT antibody had a high specificity for R-CRT (FIG. 7). In contrast, immunoblotting assays with ant i-PDI antibodies detected persistent (const i tut ive) levels of R-PDI, but the cells were treated with arginated PDT when treated with poly (dA: dT) dsDNA. There was a marked increase in the proportion of non-arginated PDI. Whether the basic R-PDI signal exhibits endogenous R-PDI or cross reactivity to arginated PE ⁾ I has not yet been identified. These results were confirmed in a peptide ide binding-binding assay, in which the inventors found that the R-Bip peptide (REEEDKKEDVGC) (SEQ ID NO: 4), R-CRT, in which the antibodies were immobilized on a plate The ability to bind to peptide (REPAVYFKEQ) (SEQ ID NO: 5) was measured. Immunoblotting Consistent with the results of the assay, this binding assay also showed that the anti-R-Bip antibody binds more favorably to the i 隱 obilized R-Bip peptide than the non-arginated Bip peptad. In contrast to anti-R-Bip antibodies, anti-R-CRT and anti-PDI antibodies were found to show less affinity for arginated species.

Example 5 Subcellular Fractionation

 Cells were trypsinized to obtain cytoplasmic proteins, followed by centrifugation at 1,500 × g to obtain pellets. Plasma membranes of harvested cells were dissolved in lysis buffer (110 mM KOAc, 25 mM K-HEPES, pH 7.2, 2.5 mM Mg (0Ac) 2, 1) with glyconside extracted from 0.01% digitonin and Digitalis purpurea. permeabilization using mM EGTA). Cells containing bulk ER and digittonin permeated cells were centrifuged at 1,000 X g for 5 minutes to obtain pellets. After centrifugation, the resulting upper layer was centrifuged again at 15,000 X g, whereby digittonin permeated. Cytoplasmic fractions containing soluble cytoplasmic protein and autophagy fears that emerged out of the ized cells were obtained. Another method of obtaining cytoplasmic protein is to inflate cells with a storage lysis buffer (10 mM KOAc, 10 mM KHEPES, pH 7.2, 1.5 mM Mg (0Ac) 2) and then use a Dounce homogenizer. To tear the plasma membrane. After adjusting the buffer to 0.25 M sucrose, the cell suspension was centrifuged at 1,000 X g for 10 minutes to discard unbroken cells and nuclei, and then centrifuged at 12,000 X g for 15 minutes to remove mitochondria. The resulting conflict was ultracentrifuged at 100,000 × g for 1 hour to separate the soluble cytoplasmic protein from the microsomes showing the porous structure containing fragments of ER.

Example 6 Cell Culture, Immunoblotting and Immunoprecipitation

HeLa, PC3, HEK293 cells as well as + / + and ATE1-/-MEFs were incubated in 5% C02 incubator with DMEM (GIBCO) supplemented with 10% FBS (GIBCO). For immunoblotting NETN lysis buffer (lOOmMNaCl, 20mMTris— HCl, pH 8.0, O.SmMEDTA, 0.5% Nonidet P-40) or RIPA buffer (50mMTris-HCl, pH8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate Cells were lysed using 0.1% SDS). Total cell lysates were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Cell lysates were incubated with primary antibodies for 4 hours at 4 ^° C. for immunoprecipitation followed by incubation with protein A / G agarose bead (Santa Cruz). Proteins bound to antibody-bead complexes were washed with cold NETN burr and separated by SDS-PAGE for immunoprecipitation analysis.

Example 7 Protein degradation assays

To Phelps cells achieved 80% confluence for track analysis were as 100 nM thapsigargin treated for 6 hours to induce the transcription of ^{Bip, 35 S methionine / cysteine.} (5 S Express protein labeling mix; Perkin_Elmer) '12 minutes to Phelps cover manufactured in the following, cyclo hex polyimide presence of trace extracts, immunoprecipitation, NuPAGE 10% -Bis-Tris SDS -PAGE (Invitrogen), self-radiation recording process (autoradiography), and ³ ¾ amount (quantitation) (Molecular Imager FX of System, Bio-Rad, Hercules, CA) was performed as described. 8W confluent cells were treated with 10 ug / ml cyclonucleamide for cyclonucleamide follow-up digestion analysis. At the indicated time point, the cells were lysed on ice for 30 minutes with RIPA buffer containing a protease inhibitor cocktail and centrifuged at 15,000 g for 20 minutes. After centrifugation, immunoblotting was performed on the 10 _U g protein in the upper layer. Example 8 RNA Interference Analysis

Cells in 6-well plates (1 × lOVwell) were transfected with 100 pmol siRNA using RNAi Max reagent (Invitrogen). Using predesigned Silencer Select siRNAs (Invitrogen), ATE1 using the following sequences Knowdown:

 siATEl # 202 (HSS117-202-sense, GAUUCCUUUGCAGUUCACCCUUGGA (SEQ ID NO: 10); HSS117-202-anti sense, UCCMGGGUGAACUGCAAAGGAAUC (SEQ ID NO: 11));

 siATEl # 203 (HSS117-203-sense, GAGAGCCAUCAUGCCUUACGGUGUU (SEQ ID NO: 12); HSS117-203-ant i sense, AACACCGUAAGGCAUGAUGGCUCUC (SEQ ID NO: 13)); and siATEl # 204 (HSS117-204-sense, ACCCACCAUCUUCAUCUUC : 14); HSS117-204-anti sense, UUUGGUGGAAACAAAGAUGGUGGGU (SEQ ID NO .: 15)).

Example 9 Immunohistochemistry

 Immunohistochemistry for arginated forms of proteins present in the ER in cultured cells was performed using standard procedures (Tasaki et al-, Annu, Rev. Biochem. I, 261 (2012)).

 Specifically, . Cells were washed twice with PBS, fixed for 10 minutes at room temperature with 4% paraformaldehyde, treated with 0.25% Triton X-100 for 15 minutes and incubated in blocking solution (3% BSA in PBS). Next, after incubation with the primary antibody was incubated with Alexa Fluor-488-labeled goat ant i -rabbit IgG (Invitrogen). Confocal ¾ images were acquired with a confocal laser scanning microscope 510 Meta (Zeiss) and analyzed using Zeiss LSM Image Browser (ver. 4.2.0.121).

Cells grown on coverslips were transfected with one poly (dA: dT) (lg / ml) to determine the intracellular location of the recombinant protein. After 16 hours of transfection, cells were fixed with 4% paraformaldehyde for 0.5 hours at silver and washed twice with PBS, 0.1 with blocking solution (1% normal goat serum and 0. Triton X-100). Incubated for 1 hour in phosphate-buffered saline (PBS), followed by primary antibody followed by secondary antibody. Fluorescence using two ImageJ (l) plugins, colocalization finder (developed by Chris istophe Laummonerie and Jerome Mut-terer) and JACoP (Just Another Co-local izat ion Plugin, 2) Co-localization of the images was evaluated.

Example 10 Luciferase Reporter Assay

 Cells were transfected with either the IgK-IFNp-Luc reporter plasmid (Addgene) or the Renillar-Luc control plasmid. The cells were simultaneously transfected with poly (dA: dT) dsDNA and pcDNA-ATEl-3. After 18 hours of transfection, luciferase activity was measured using a Dual-lucif erase reporter assay system (Promega) and a VICTOR 1420 mulit i label counter (Perkin-Elmer). The experiment was repeated at least three times. Error bars indicate standard error of the mean of three independent experiments.

Experimental Example 1 Analysis of N-degrons Presumed in ERCendoplasmic Reticulum

 The N-end rule pathway is known as a selective proteolytic system that targets short-lived proteins released into the cytoplasm after translation (SMSriram et al., Nature Rev. Mo I. Cell. Biol. 12,735 (2011)). However, proteins in the secretory pathway corresponding to about one-third of human proteome are exposed to new N-terminal residues at the same time as the signal peptide is cleaved by the signal peptide peptidase and enters the ER at the same time as the translation. ER is an abundant source of N-degron.

 Thus, the inventors have found that a subset of proteins present in the N-degron ER is retrotranslocated with intact folding and cytoplasmic N-terminus until metabolized by the proteolytic machinery. Targeted by law component, it was confirmed that it participates in various processes outside ER.

Specifically. To assess the overall frequency at which putative N-terminal degradation determinants are produced in lumens, an Apercu database containing 498 human proteins entering the ER was used (M. Scott et al. Bio informatics 20, 937 (2004). As a result, as shown in Tables 1 to 4, when analyzing the P1 'residues of the signal peptide cleavage site, it was confirmed that approximately 27% (132 of 498) exposed N-terminal residues in an unstable state. This was found to be significantly lower compared to the existing N-degron code which included 13 of 20 amino acids (SMSriram et al. Nature Rev. Mo 1 Cell Biol. 12, 735 (2011)) (Table 1 to Table 4).

 As a result of reviewing various documents, most of the N-end rule substrates identified so far are N-terminal Arg or pro-N-degrons (Asn, Gin, Cys, Asp, Glu) -Produces terminal Arg).

TABLE 1

 E -rcsiding proteins that carry arginylyation- | 5ermissive destabilizing N-fcrminals residues. Asp and Giu.

TABLE 2 ER-rcsiding proteins carrying tertiary destabilizing N-tcrminals residues (Asn, Gin. And Cys) whose modifications can generate argin lation-permissive residues (Asp, Glu, and oxidized Cys).

 [protein name INCBI Access I'N-tormlnat sequenced

 SS 4 NP 006271 AA ASLGAtALLtLSSLSRCSAEACLEPQlTPSYYTTSOAVlSTETWtVEISLTC ^ NR QNW ^

 00103β

 PCS 5 NP 006191 GWGSRCCCPGRLOLLCVtALLGGCLL ^ C TRVYTNHWAVKIAGGFPEAN lASKYGPINlG GST2 NP 002404 MAGNSlLLAAVStLSACTOSyFALQVG ARUCY VTPPAVTGSPeFE VFRAQQNCVEFYPfFIIT

KDEL2 M IFRLTGDLSHLAAtVfLLLKfWKT SCAGISGKSQLLFALVmKYLDLFTSFISLYNTSM hypothetical protein

 UGT2B7 NP 001065 ^ VKWTSViLLlQLSFCFSSGNCGKVLNWAAEYSHVVMNI T! LDELlQRGHENnVUASSASILFD

ICE NP 003860 MRtH L ARLSAVACGLLLLLVRGQGQDSASPIRTTHTCMVLGSLVHVKGA AGVQTFtGIPFA

VP EB3

 VLCS-H2 NP 036386

pregnancy specific bea -elycopro

toin NP 008836 IVteTLSAPPCTQRiKWKGLLLTASLLNFWNLP AQVTIEAEPTKVSEGKOVLLLVHNLPQNLTG

P OC NP 006219 MKViLCDLL SLFSSVFSSCQ DCLTCQEKLHPAtDSFDLEVCILECEe VFPSPLWTPCTKV

Misugumtn 23 NP 076997 P ^ SStSSPWG HVPKAtL ^^ VAL! LLHSALAQS ROPAPPGQQK EAPVOVLTQtGRSV GT immunoglobulin J chain NP 653247 MKNHLLFWGVlAVRKAVHVKAOEDERlVLVDNKCKCA ITSRHR ^ SEDPNEm RNlRR

HRC NP 002143 MGHH PWLHASVLWAGVASLLtPPAMTQQtRGOGLGFRN 4STGVAGLSEEASAEL HHL

G6PE NP 004276 MWNMUVAMCLAlXOCLOAQELQOHVSIIt GATGDLAKKYUWQdLFOLrLDEAGRGHSFSFH

ERO! -L beta NP 063944 SQOV RAGAGQGVAAAyQLL \ nVSFLRS \ ^^ AQVTGVLOOGLCDlD $ tONPNTYK (FPK (K tQ

CRAAP NP 057526 MVFLPLKWSI ^ IMSFLLSSLLALL STPSWCQSTEASP RSDGTPFPWNKI LPE IPVMYDL

CASQ1 NP 001222 MGPRA GLRLALLLLLVLGtPKSGVOGQEGLDFPEYDGVDRVlN ^^

car oxypeptidase E precursor NP 001864 MAG GGSALU ^ LCGALAAdGWLLGAEAQEPGAPAAGMR RLQQEDGiSFEYHRYPEL EA amytoid beta (A4) precursor-tike pr

 NP 005157

otein 1 tGPASPAARGLSR PGQPPLPLLLPLLLLLL AQPAIGSUVGGSPGAAEAPGSAQVAGLCG LT

3BH2 MGWSCLVTGAGGLLGQRIVRLLVEEKeLKElRALOKAFRPELREeFSKLQ

TABLE 3

TABLE 4

Human ER-rcsiding proteins whose arginylation- ennissive residues (Asp and Glu) are not cvolutionariiy conserved.

Also, experimentally check if there is something that goes through argination. To reduce the list to 25 Asp / Ghi-proteins, one with two arginating substrates (Asp and Glu), then narrowed down to 11 Asp / Glu-proteins that have evolutionarily retained L-Arg. . The important thing is, 11

Asp / Glu-proteins include molecules such as peron (Bip, GRP94, calreticulin (CRT)) and oxidoreductases (protein disulfide isomerases (PDI) and ErdJ5) and ¾ as the major Ca ++ — binding folding factors. Abundance (FIG. La, FIG. 6 and Table 3).

All three proteins (Bip, PDI, CRT) that we chose to elucidate below are biomarkers _. It has been shown to be arginated in vivo, suggesting that other substances present in the ER may also undergo N-end rule modification.

Experimental Example 2 Confirmation of Turnover of Bip, a Glul9 Exposed Chaperone

Some ER chaperones involved in the holding cycle are known as unfolded protein reactions (UPR: Some of the unfolded proteins response is transcriptionally induced to remove misfolded proteins by ER-associated protein degradation (ERAD) (RRKopito, Cell 88, 427 (1997)). However, despite extensive research on ER protein quality control, the metabolic fate of chaperone itself is still poorly understood.

 Therefore, the present inventors confirmed the turnover of Bp, a Glul9-exposed chaperone that assists translocation and folding, with a cycloheximide digestion assay.

As a result, as shown in FIG. Lb, cycloheximide decomposition analysis and the results showed that the half-life (t ₀ — ₈ , hr) of Bip was 2-5 hours in the absence of thapsigargin ( Degrees lb). In addition, as shown in FIG. Lc, Bip is clearly present on the flux of proteolysis even after it is transcriptionally activated by an ER stressor tapsygin that induces unfolding protein reaction (UPR). Confirmed. Next, the pulsed cells were treated for 12 minutes with ³⁵ S-Met / Cys, and then pulse-chase analysis was performed, followed by immunoprecipitation over time. Short half-life was determined. Availability ³⁵ S-Met / Cys labeled the half-life of _{Bip (t 0 - 60, min} ) is ~ was 25-45 minutes (Fig. Id). Proteasome inhibition (MG132) has a half-life in the trace-was increased to (t _{0 60,} min) significantly without an increase in pulse labeling (labeling) indicating the zero-time level of the metabolic stability of the (zero time level) of which Bip It was confirmed that metabolism (turnover) may be associated with other degradation pathways not associated with proteasomes.

Experimental Example 3 Confirmation of Physiological Substrate of Argination by ΑΊΈ1 R-transferase As an Arginylat Ion-permissive PI ′ Residue of Human Bip

Ghil9, Mouse (Glu), Platypus (Ornithorhynchus anat.inus) (Asp), Taeniopygia guttata (Asp), African Claw (Xenopus laevis) (Asp), Zebrafish (Danio rer io) (Glu), Drosophila (Drosophi la melanogaster) (Glu), It is well conserved in other species, such as Caenorhabditis elegans (Glu) (Fig. La, Fig. 6 and Table 3). While it is possible for ER proteins to be artificially exposed to cytoplasmic R-transferases during preparation for extraction, consistently, mass spectroscopy of the total protein isolated from 2D-SDS PAGE showed arginine of Bip and PDI. Normalized forms have been identified (RGHu et al., J. Biol. Chem. 282,8237 (2006)).

Construction of Antibodies to <3-l> Arginated Bip, CRT, PI) I

 We used a peptide (REEEDKKEDVGC) (SEQ ID NO: 4) that complements the R-Bip N-terminal residue to determine whether Bip is an in vivo substrate for argination in Glul9. The antibody R-Bip was designed to recognize the arginated form of Bip (R-Bip) but not to recognize the non-arginated form, and then confirmed the specificity of the antibody.

 As a result, it was confirmed that the anti-R-Bip antibody binds more cyclically to the immobilized R-Bip peptide than the non-arginated Bip peptide.

<3-2> Confirmation of R-Bip Generation through ATE1 Subtype Overexpression

The inventors of the immunoblotting analysis of various cell lines showed no R-Bip signal, which confirmed that Bip argination may be induced by stress. Thus, the inventors determined whether R-Bip could be generated by overexpressing the ATE1 subtype, and in previous studies, ATE1 was found to produce four subtypes with a size of 60 kDa and optionally spliced. ^Were : ΑΤΕ1 ^1Α7Α (ΑΤΕ1-3), ΑΤΕ1 ^1Α7Β / ΑΤΕ1-4, ATE1 ^1B7A / ATE1-1, ATEl ^1B7B / ATEl-2 (YTKwon et al, 19, 823 (1999)) (FIG. 8A).

Thus, plasmids encoding ATEl R-transferase isoforms were prepared by the method described in Kwon-1999 (YTKwon et al, Mol. Cell. Biol. 19, 823 (1999)), plasmids expressing each of the four ATE1 subtypes were transfected into a HeLa cell line, followed by immunoblotting, and confirmed in vivo argination. In addition, ^{immunoblotting} was ^performed in the HeLa cell line expressing ATE1 ^1A7A and treated with cyclonucleosides, or the HeLa cell line treated with ^siATEl by the same method.

As a result, as shown in FIGS. Le and 8b to 8d, it was confirmed that ATE1 ^1A7A is the major R-transferase for Bip compared to ATE1 ^1B7A and ATE1 ^1B7B . (FIG. Le, and FIGS. 8B-8D).

In addition, after transfecting ATE1 ^1A7A into the HeLa cell line, the cells were classified into whole extracts, cell substrates, and microsomes, and then ^subjected to cell fraction analysis. As shown in FIG. If, ATE1 ^1A7A from the soluble fraction of the cytoplasm. About 70% of the generated R-Bip was found to be detected (Figure If).

In addition, as a result of cycloheximide digestion analysis of ATE1 ^1A7A expressing cells, as shown in FIG. ^Lg , ^unarginated (lumen) Bip has a short lifespan, whereas (cytoplasmic) R-Bip It has a relatively long lifespan (Fig. Lg), which confirms that the lumen Bip is metabolized to some degree through retrotranslocat ion and argination (Fig. Lg).

Experimental Example 4 Confirmation of Bip Argination at N-Terminal Glul9

 We confirmed whether Bip argination occurs at the N-terminal Gulul9.

 Specifically, the Bip-f lag-KDEL or Bip-E19V-f lag-KDEL prepared above was transfected into a HeLa cell line, followed by immunoblotting.

As a result, as shown in FIG. Lh, ATE1 ^1A7A overexpression was easily induced argination of recombinant Bip exposed the N-terminal Glul9, Glul9 to Val variants stabilized by the N-end rule, ATE1 ^1A7A mediated ^argination It was confirmed that it was suppressed (Figure lh). In addition, to further elucidate Bip argination in the cytoplasm, the inventors expressed Ub-Glu-Bip-myc / his, wherein the C-terminal Gly76 of ubiquitin (Ub) was fused with Glul9 of mature Bip. Subsequently (FIG. 8E), in the same manner, ATE1 ^1A7A overexpressed in HeLa cell line expressing ^Ub —Glu- ^BipB myc / his, followed by ^{immunoblotting} . On the other hand, in the Ub fusion technique, the new Ub-Glu-Bip-myc / his from ribosomes is detoxified and split into Ub and Glu-Bip-myc / his by deubiquitase (Tasaki et Ά \., Annu). J? Ev.Bioch n. S, 261 (2012)).

As a result, as shown in FIG. Li, ^argination of Glu-Bip-myc / his was confirmed to be accelerated by expressing ATE1 ^1A7A (FIG. Li). In addition, the inventors confirmed the argination after knocking down ATE1 using RNA interference.

 As a result, as shown in FIG. Lj, argination of Glu—Bip-myc / his was confirmed to be reduced by ATEl-knockdown, and in contrast, Ub-Val-Bip- with Glu-to-Val variants. myc / his was not readily arginated. Ub-Arg-Bip-myc / his, a permanently arginated variant, was found to produce R-Bip signals essentially regardless of ATE1 overexpression or knockdown (FIG. Li, lg).

 Based on these results, Bip itself has its own N-terminus in vivo.

Glul9 was confirmed to be arginated by ATE1 R-transferase.

Experimental Example 5 Argination Confirmation of PDI and CRT by ATE1

Part of the CRT was found to exist in the arginating form in the cytoplasm (MBDecca et al., J. Biol. Che. 282,8237 (2007)). To extend this finding, RDAPEEEDHVL (SEQ ID NO: 6) and REPAVYFKEQ (SEQ ID NO: 5) an antibody specific for the PDI and the CRT and the arginine type that was manufactured using the ^■ (Fig. 7). Unlike Bip, it can be analyzed in various cell lines. Sufficient amounts of R-PDI and R-CRTs were produced continuously (FIGS. 2B, 8D, 9A, and lla), confirming that arginated ER proteins have some distinct physiological function in the cytoplasm. . Even so, it was both confirmed that arginine screen by more ATE1 ^1A7 ^ 1 ATE1 ^1B7A and ATE1 ^1B7B more efficiently (Fig. 8d).

 Based on these results, it was confirmed that the N-end rule pathway may have a broad role in the regulation of ER protein.

Experimental Example 6 Confirmation of Arginine Proliferation of ER Protein through Endogenous Immune Response to Cytoplasmic Double-stranded DNA

 As confirmed in Experimental Example 3, Bip argination was not induced to a detectable level even by 6 hours of ER stress (FIG. 8b), which is the main role of R-Bip finally folding. It was confirmed that the ER protein did not remove the ER protein, but participated in a process outside the ER (FIG.

 In addition, lipofectamine LTX alone or in various DNA forms (salmon sperm DNA (Salmi.Sp.DNA), GFP-fused mouse α—ant trypsin nul l Hong-ong (mNHK-GFP), Ubiquitin fusion at the R-GFP C_terminus (Ub-R-GFP), spontaneously misfolded substrate model (YFP-CLl), mutant antitrepsin protein well (ATZ)) was then transfected, followed by immunoblotting. It was.

As a result, as shown in FIGS. 2A, 9A, and 9B, the argination of Bip, PDI, and CRT was examined when argination induced stress was induced when cells were transiently transfected with various dsDNA molecules. It was confirmed that the degree is triggered in various ways (Figs. 2a, 9a and 9b). In addition, as shown in FIG. 9C, analysis of the transfection reagent showed that argination of Bip, PDI, and CRT was triggered by dsDNA in the cytoplasm rather than in the extracellular space (FIGS. 9C, lines ² and 3). .

In addition, heLa cell lines expressing siRNA #RNA and NHK-GFP for ATE1. After coplasmotyping together the plasmids, the cell substrates were fractionated, and RA interference analysis was performed.

 As a result, as shown in FIGS. 9C and 9D, it was confirmed that ATE1 plays a role in DNA-induced Bip argination (FIG. 9C and FIG. 9D). In addition, the cell fraction analysis, it was confirmed that most of the DNA-induced R-Bip is soluble in the cytoplasm (Fig. 9c).

 In addition, unlike double-stranded DNA (dsDNA), double-stranded RNA (dsRNA) was found to have a relatively weak activity inducing argination (Fig. 11a). From the above results, it was confirmed that the cytoplasmic dsDNA triggered the retrotranslocat ion and ATE-1 dissociative argination of various Ca ++-bound ER saperons.

Cytoplasmic dsDNA can be a signal of infection by DNA-containing microorganisms, such as viruses, thus eliciting endogenous immune responses, including phosphorylation of the transcription factor IRF3 and interferon-β synthesis induced by IRF3. (Hou-2011). Consistently FIG Bip, PDI, arginine screen of CRT is in the cytoplasm immunity enhancement reagent expressing viral dsDNAs poly (dA: dT) were strongly induced by dsDNA _(. Fig. 2b, Fig. 11a to Fig. 11c). In addition, ATEl-knockdown using RNA interference strongly inhibited IRF3 phosphorylation induced by DNA (FIG. 2C). As a result of the dual-luci ferase reporter assay in which the interferon-β promoter induces luciferase expression, ATE1-/-mouse embryoni cf ibroblasts (MEFs) (Kwon-2002) Induced impaired inter feron-β production was shown to be impaired (FIGS. 2E, 2F and lie). Thus, it was confirmed that ATE1-dependent argination of ER presence plays an important role in the cytoplasm's intrinsic immune response to dsDNA.

Experimental Example 7 Confirmation of (Colocalization) Cytoplasmic Puncta Formation Co-located with Autophagic Vacuoles of Arginated Bip We have found that the cytosolic poly (dA: dT) not only synthesizes LC3, but also induces LC3-I, which is an inactive form of LC3, to be converted into LC3-I I, where LC3-I I is a lipidated form that can be conjugated with autophagy bilayer structures (Figure lid). Autophagy induction was similarly observed for other types of dsDNA, suggesting a general role for autophagy in endogenous immune response to DNA-containing microorganisms.

 Accordingly, we examined whether R-Bip plays a role in the autophagy pathway during DNA-induced endogenous immune response. Indeed, R-Bip immunostaining in dsDNA-treated HeLa cells revealed cytoplasmic puncta with a diameter of 0.1-1, which was similar in size to the autophagy fear (FIGS. 3A and 12A). ). Co-staining analysis showed that the DNA-induced R-Bip and LC3 positive autophagy points (puncta) were in the same position (FIG. 3A). RNA interference analysis of ATE1 and Bip confirmed that the LC3 point (puncta) and the R-Bip point (puncta) exist at the same position and in the ATE1-dependent formation (FIG. 3C). Autophagy fear and R-Bip point are co-located using co-staining with autophagy adapter P62 / SQTSM, which mediates delivery of misfolded cytoplasmic proteins to autophagosomes It was further confirmed that it was present in (FIG. 3B and FIG. 12B). Thus, it was confirmed that cytoplasmic dsDNA induced both argination and autophagy of Bip and then R-Bip was transferred to autophagy fear, which was observed during dsDNA triggered intrinsic immune response and cell composition (const i tuent s). R-Bip plays a role in autophagy digestion.

Experimental Example 8 Confirmation of Action as N-degron at the N-Terminal of the R-Bip

In the N-end rule pathway, the N-terminal Arg of short-lived proteins acts as a major N-degron that is recognized and bound by N-recognins for proteasome degradation. This N-end rule recognition is essential for targeting substrates to proteasomes via polyubiquitination. In view of the role of N-terminal Arg in Ub dependent selective proteolysis, the present inventors have found that N-terminal Arg confirmed that Bip acts as an autophagy N-degron required for Bip targeting to autophagosomes.

Specifically, the plasmid expressing Ub-X-Bip-GFP (X = Arg, Glu, or Val) which is divided into Ub and X-Bip-GFP while simultaneously decoding HeLa cells stably expressing RFP-GFP-LC3 Transfection with (FIG. 13B). Co-localization analysis of X-Bip-RFP and LC3 showed that Arg-Bip-GFP was readily delivered to autophagosomes (FIG. 4A). In sharp contrast, Arg-to-Val mutations negated the colocalization of such autophagy fears (FIG. 4A). As an independent assay testing the activity of N-terminal Arg as autophagy N-degron, co-staining of Arg-Bip-GFP and p62 was performed. Val-Bip-GFP staining appeared dispersed, while Arg-Bip-GFP formed a cytoplasmic spot (puncta) that faithfully colocalized with p62 (puncta) (FIG. 4B). Compared with Arg-Bip—GFP, Glu-Bip-GFP formed a limited degree of R-Bip positive point (puncta), which was confirmed to be due to partial argination. However, the R-Bip positive point (puncta) from Ub-Glu-Bip-GFP was in the same position without exception as the p62 positive point (puncta) (FIG. 4B). In order to further identify N-terminal Arg as a general autophagy delivery determinant, Ub-X-Bip ^{19 106} ᅳ GFP (X = Glu, Arg, or Val) was constructed, where Ub's C-terminal Gly76 was bound to ATPase and substrate. Fusion with N-terminal 88 residue-Bip fragment without domain (FIG. 13C). N-end rule dependency colocalization was similarly observed in Ub-X-Bip ¹⁹ — ¹⁰⁶ -GFP (FIGS. 4C, 4D and 14). Therefore, it was confirmed that argination produced autophagy N-degron, which targets even Bip and other cytoplasmic ER proteins.

Experimental Example 9 Induction of Argination of ER Protein by Mi-folded Cytoplasmic Protein

Lumen's Bip is a substrate recognition r ognin that finally binds / absorbs the misfolded ER protein and delivers its cargo to the ERAD machinery. To identify molecules that interact with arginated forms of ER saperon, Whether argination correlates with misfolding cytoplasmic protein.

 In fact, HeLa cells treated with the Hsp90 inhibitor geldanamycin (known to promote the formation of misfolded proteins) showed increased argination for Bip ᅳ CRT, PI) I (FIG. 5A). Similar correlations were also seen when these cells were treated with heat shock to produce misfolded proteins (FIG. 15A). Subsequently, the screening to identify stressors that induce argination in response to the question of whether argination correlates with an increase in ubiquitination has shown that argination of Bip, CRT, and PDI has been associated with prolonged periods of time (e.g., , 16 hours) was strongly correlated with proteasome inhibition, the direct result of which is increased ubiquitination of misfolded proteins as with other protein substrates (FIGS. 5B and 15B). In addition, the correlation between arginization and ubiquitination was observed among DNA-induced endogenous immune reactions (FIG. 5C), suggesting that triggering argination of ER protein in DNA-induced endogenous immune reactions may be an increase in ubiquitination. do. Finally, it was examined whether argination was inhibited when ubiquitination was disrupted. HeLa cells expressing mutated Ub (where all seven Lys residues changed to Arg to interfere with Ub chain extension) contained a significantly reduced amount of R-Bip (FIG. 5D). Therefore, it was confirmed that arginated ER chaperone is involved in the turnover of misfolded cytoplasmic protein.

Example 8 Colocalization of R-Bip and Ubiquitinated Proteins in Autophagy

Some of the misfolded / damaged cytoplasmic proteins are not good substrates for proteasomes, one of which is due to their prone properties. Recent studies have shown that the proteasome resistant substrates are directed to autophagy by autophagy adapters such as p62. Until now, the substrate is autophagy It is not known whether specific degrons are required for delivery (such as N-degrons for Ub ^' ligase), and how autophagy adapters selectively recognize / bind autophagy protein substrates rather than normal proteasome substrates. It is a state. To determine the role of R-Bip in autophagy clearance of misfolded cytoplasmic proteins, colocalization between R-Bip, ubiquitinated protein and autophagosome was investigated.

 Specifically, when HeLa cells treated with poly (dA: dT) dsDNA were stained with Ub specific FK2 antibody, this staining revealed a puncta-like structure located identically to autophagy fear positive for LC3 and p62. 5e and 15d, a ubiquitination protein subpopulation destined for autophagy degradation was shown. Further analysis showed that autophagy fears positive for both Ub and LC3 / p62 were located with the R-Bip point (puncta) without exception (FIGS. 5E and 15D). These results suggest that R-Bip is associated with ubiquitinated proteins in the process of autophagy degradation. Thus, the question arises whether R-Bip interacts with misfolded proteins. Immunoprecipitation assays showed that R-Bip interacts directly or indirectly with CL1-YFP, a model substrate undergoing spontaneous mi sfolding (FIG. 15C). Thus, R-Bip plays a role in autophagy clearance of misfolded cytoplasmic proteins, which has the ability of its N-terminal Arg to act as a trans-acting N-degron for mi sfolded protein cargoes. It was confirmed through the.

Claims

[Range of request]

 [Claim 1]

 An antibody that specifically binds to an arginylat ionized protein.

[Claim 2]

The antibody of claim 1, wherein the protein is any one selected from the group consisting of BIP, CRT, and PE ⁾ I.

[Claim 3]

 According to claim 2, wherein the BIP protein has an amino acid sequence of SEQ ID NO: 1, the CRT protein has an amino acid sequence of SEQ ID NO: 2, the PDI protein has an amino acid sequence of SEQ ID NO: 3 Antibody characterized by the above-mentioned.

[Claim 4]

 The antibody of claim 2, wherein the BIP protein is N-terminal Glul9, the CRT protein is N-terminal Glul8, or the PDI protein is N-terminal Aspl8.

[Claim 5]

 According to claim 1, wherein the arginated protein is characterized in that the N- terminal Glul9 of the BIP protein, the N- terminal Glul8 of the CRT protein or the N- terminal Aspl8 of the PDI protein is arginated by ATE1 R-transferase Antibody.

[Claim 6]

The antibody of claim 1, wherein the arginated protein is derived through intrinsic immune response to double-stranded DNA (dsDNA).

[Claim 7]

 The antibody of claim 1, wherein the arginated protein forms a cytoplasmic spot (puncta) located with autophagic vacuoles.

[Claim 8]

 The antibody according to claim 1, wherein the N-terminal Gkil9 of BIP protein, the N-terminal Glul8 of CRT protein, or the N-terminal AsplS of PDI protein is arginated with autophagy N-degron.

[Claim 9]

 The antibody of claim 1, wherein the arginated protein is induced by a misfolded cytoplasmic protein.

[Claim 10]

 Virus or bacterial infection diagnostic kit comprising the antibody of claim 1.

[Claim 11]

 An immunomodulator screening kit comprising the antibody of claim 1.

[Claim 12]

 1) treating the test compound in the sample;

 2) An immunomodulator screening method comprising the step of measuring the level of arginated protein in the experimental group treated with the test compound and the control group not treated with the test compound.

[Claim 13]

According to claim 12, The step 1) and the sample is separated whole blood, serum, plasma, saliva Urine, sputum, lymph, cerebrospinal fluid, and intercellular fluid.

[Claim 14]

 The method of claim 12, wherein the step 2) and the arginated protein is BIP protein

N′-terminal Glul9, N-terminal Glul8 of CRT protein or N-terminal Aspl8 of PDI protein is arginated.

[Claim 15]

 The method of claim 12, wherein the level of arginated protein of step 2) is measured using an antibody that specifically binds to the arginated protein.

[Claim 16]

 1) treating the sample with a virus;

 2) treating the test compound to the virus-treated sample;

 3) A method for screening a viral therapeutic agent, comprising the step of selecting a test compound that reduces the level of arginated protein in a test compound and a virus-treated experimental group, and a virus-treated control group.