KR20170068943A - Method for Detecting ATF4 under Endoplasmic reticulum Stress - Google Patents

Abstract

The present invention relates to a method for detecting an endoplasmic reticulum-stressed ATF4 protein using dsRed expression characteristics derived from Drosophila. More particularly, the present invention relates to a method for providing information on ER stress using a reporter construct comprising a sequence encoding a DsRed gene located in the 5 'UTR region of Drosophila ATF4, ≪ / RTI >

Description

(Method for Detecting ATF4 Under Endoplasmic Reticulum Stress)

The present invention relates to a method for detecting ATF4 protein under endoplasmic reticulum stress using dsRed expression characteristics derived from Drosophila. More specifically, the present invention relates to a method of providing information on ER stress using a reporter structure including a sequence encoding a DsRed gene, which is a fluorescent gene located in the 5 'UTR region of Drosophila ATF4 And compositions therefor.

In the expression of the protein, the gene, which is a design drawing for determining the structure of the protein, is contained in the nucleus. However, since the expression of the protein and the function of the expressed protein function normally, Is an endoplasmic reticulum (ER).

The endoplasmic reticulum is organized to take charge of protein folding, saccharification, transport, etc. In particular, it has an acidified environment suitable for formation of a disulfide bond, which is a typical chemical bond responsible for protein folding. There is a very rich distribution of the chaperone protein, the helping protein, and the calcium ion, which is an auxiliary component of the chaperone. When the genetic information inherent in the messenger RNA is made into amino acid chains with the aid of a ribosome complex, they are transported to the endoplasmic reticulum basically, and the proteins that can function by forming a tertiary structure with the help of the elements in the endoplasmic reticulum listed above . If normal proteins are not processed and unusual proteins such as unfolded or misfolded proteins are produced, they are degraded by a ubiquitin proteasome system in cells such as proteasomes. However, if an error occurs in the ubiquitin-protein degradation system, the abnormal protein accumulates in the ER and induces ER stress.

The ER stress is a term collectively referred to as a cell-level reaction that occurs in all environments that inhibit folding and glycosylation of proteins in the ER and in the environment as described above. Generally, problems arise in the surrounding environment of the cells, and ER stress It is known to be generated. For example, if a problem occurs in the cell's oxidation / reduction system, it can not form a disulfide bond. If the glucose is excessively deficient, the glycation of the protein is not normally performed. If the cell is infected by the virus, The amount of protein produced by using the protein and the amount of protein produced by the host to cope with it goes beyond ordinary levels, so the amount of protein itself acts as a stress.

These ER stresses can cause new diseases or exacerbate conventional diseases. Specific diseases associated with ER stress include neurodegenerative diseases, ischemic diseases, diabetes, and the like. First, in patients with neurodegenerative diseases, protein aggregation occurs due to ER stress. Therefore, intracellular activity is changed in a direction to inhibit it. For example, in patients with Alzheimer's disease, PERK (PKR Like ER The expression of E3 ligase protein Parkin was increased in patients with Parkinson's disease, and ER stress-induced apoptosis was induced by eIF2a (eucaryotic translation initiation factor 2 alpha) . Next, in patients with ischemic diseases, the reduction of oxygen supply generally results in the destruction of the redox-controlling function of the cells, resulting in the generation of ER stress. In the re-supply of oxygen, the oxidative stress- . Finally, when ER stress is generated in the beta cells of the pancreas where the ER structure is developed to produce insulin, the beta cells are killed and the supply of insulin is decreased to cause type 1 diabetes Is known to aggravate diabetes mellitus. In addition, when ER stress is generated in a target cell in which insulin acts, it is known that type 2 diabetes occurs due to the collapse of the intracellular signal transduction pathway by insulin and the slowing of insulin responsiveness.

As a result of these studies, ATF4 (activating transcription factor 4), one of ATF / CREB (activating transcription factor / cyclic AMP response element binding 6 protein) family, has been actively studied to identify the cause of ER stress. . It is known that ATF4 is not expressed properly in most normal cells and is rapidly degraded even after expression and is not detected properly in normal cells.

However, when stress environment factors such as depletion of amino acids, sugars or oxygen are increased, not only the expression of ATF4 is increased but also the expression of ATF4 is inhibited and the level in the cell is increased. It is known that phosphorylation of eIF2α is involved and phosphorylation of eIF2α involves phosphorylation of PERK, GCN2, PKR and HRI.

As such, ATF4 increases the intracellular level when ER stress is generated. Therefore, researches for reducing ER stress by inhibiting ATF4 level have been actively conducted, but there has been no achievement yet.

In particular, it is necessary to confirm the expression characteristics of ATF4 for related studies. Since ATF4 is difficult to detect at the mRNA level, it can be confirmed only as a product by translation. Therefore, There is a problem that can not be done.

Thus, the present inventors have found that the level of ATF4 due to the stress of the endoplasmic reticulum can be easily confirmed by the detection of a specific fluorescent reporter, and the present invention has been completed.

1. Lindholm D, Wootz H, Korhonen L. Stress and neurodegenerative diseases. Cell death and differentiation.2006; 13 (3): 385-92. doi: 10.1038 / sj.cdd.4401778 PMID: 16397584. 2. Kakiuchi C, Iwamoto K, Ishiwata M, Bundo M, Kasahara T, Kusumi I, et al. Impaired feedback regulation of XBP1 as a genetic risk factor for bipolar disorder. Nature genetics. 2003; 35 (2): 171-5. doi: 10. 1038 / ng1235 PMID: 12949534.

A main object of the present invention is to provide a composition for detecting ATF4 for providing information on the stress of the endoplasmic reticulum using a fluorescent gene.

It is another object of the present invention to provide a recombinant expression vector containing a fluorescent gene located in the 5 'UTR region of Drosophila ATF4 and a transformed microorganism transformed with the recombinant expression vector.

It is another object of the present invention to provide a system capable of easily and rapidly detecting the intracellular level of ATF4 protein by the stress of the endoplasmic reticulum.

In order to solve the above problems, the present invention provides a composition for detecting ATF4 for providing information on the stress of the endoplasmic reticulum, which comprises a reporter structure comprising a sequence encoding a fluorescent gene located in the ATF4 5'UTR region.

In one embodiment of the present invention, the ATF4 5 'UTR region originates from Drosophila and may be composed of the nucleotide sequence of SEQ ID NO: 1.

In one embodiment of the present invention, the fluorescent gene may be a DsRed gene, and the DsRed gene may be a nucleotide sequence of SEQ ID NO: 2.

In one embodiment of the present invention, the reporter construct may further comprise a tubulin promoter.

In one embodiment of the present invention, the reporter construct may comprise a uORF1 and uORF2 start codon functioning opposite to each other, and the DsRed gene may be fused to uORF2.

Also, the present invention provides a method for providing information on the stress of the endoplasmic reticulum, wherein the composition for detecting ATF4 of the present invention is used, and when the DsRed expression level is increased, the intracellular endoplasmic reticulum stress is also increased.

The present invention relates to a method for easily detecting the level of ATF4 which has been difficult to detect at the level of conventional mRNA, and it is possible to easily measure the level of ATF4 as an endoplasmic stress marker using the present invention, Thereby enabling effective screening of therapeutic agents. Therefore, ATF4-related factors are very useful for finding new therapeutic agents for ER stress disease.

FIG. 1 shows the 5 'UTR structure (A) of Drosophila ATF4-RA mRNA, the schematic diagram (B) of the luciferase reporter construct used in the experiment, and the relative light unit (RLU) will be
Figure 2 is a schematic diagram (A) for the in vivo ATF4 reporter and the experimental results using the reporter.
Figure 3 shows the results of ATF4.50UTR > dsRed expression pattern, which is dependent on ER stress in various organs.
FIG. 4 shows results of tub-ATF4.50UTR> dsRed expression pattern due to malnutrition.
Figure 5 shows the results of the expression of the activated in vivo ATF4 reporter in photoreceptor cells.
FIG. 6 shows the results of tub-ATF4.50UTR> dsRed expression in male regenerating organs.

Representative terms used in the present invention are as follows.

"ER stress" refers collectively to all environmental conditions that inhibit folding and glycation of proteins in the ER, and to cell-level reactions resulting from the environment, which are generated by problems occurring in the environment both inside and outside the cell. In the present invention, the ER stress may be regarded as a major causative symptom that leads to an increased expression of ATF4.

"ER stress disorder" means a disease caused or exacerbated by ER stress. The ER stress disorder may be malignant tumors, neurodegenerative diseases, ischemic diseases, diabetes, pulmonary fibrosis and the like, although not particularly limited thereto. More preferably, the ER stress diseases include breast cancer, lung cancer, fibrosarcoma, Malignant tumors; 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.

"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. The nucleic acid used in the present invention is preferably a CpG-containing nucleic acid such as a CpG island.

"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.

"Primer" refers to an oligonucleotide sequence that hybridizes to a complementary RNA or DNA-targeted polynucleotide and serves as a starting point for the stepwise synthesis of a polynucleotide from a mononucleotide by the action of, for example, the nucleotidyltransferase that occurs in the polymerase chain reaction .

"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.

A "subject" may include all mammals, including humans. An "object" may also include other animals (eg, cattle, sheep, pigs, goats) as well as pets (eg dogs, cats, horses). In the present invention, human beings are preferable.

"Treatment" is a method for obtaining beneficial or desired results, including clinical results. To " treating "or" alleviating " a disease, disorder or condition refers to alleviating the condition or disorder or degree of disease or unwanted clinical symptoms or both, It means to extend. Depending on the purpose of the methods described herein, useful or desirable clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms, decrease in the degree of disorder, stabilized (i.e., not worsened) But are not limited to, state of the art, delay or slowing of the disorder progression, improvement or alleviation of the disorder, and remission (partial or complete). Also, treatment does not necessarily occur by the administration of a single dose, but often occurs after administration of a series of doses. Thus, a therapeutically effective amount, an amount sufficient to alleviate, or an amount sufficient to treat a disease, disorder or condition, may be administered in one or more administrations. The present invention refers to the treatment of an eating disorder, preferably anorexia nervosa (AN).

"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.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the detection of ERF stress marker ATF4.

The first response to ER stress is the phosphorylation of eIF2α (eukaryotic translation initiation factor 2 alpha subunit), which reduces translation from mRNA to protein.

eIF2α is responsible for the transport of methionine (Met) as a constituent of the translation initiation complex to the initiation codon AUG of the ribosome. When eIF2? is phosphorylated and inactivated, the binding of Met-tRNA at the initiation site of the ribosome is blocked, reducing the cognitive frequency to the AUG initiation codon and reducing translation from mRNA to protein. Phosphorylation of eIF2α by ERK occurs at the onset of ER stress.

PERK is a type 1 transmembrane serine / threonine kinase located in the membrane of the endoplasmic reticulum. It is inactivated by binding to the endoplasmic reticulum, Bip, in the absence of stress. However, when the number of unfolded proteins in the endoplasmic reticulum increases, the Bip dissociates from PERK and binds to unfolded proteins, which helps them fold normally. Here, the cleaved PERK is oligomerized and then activated by trans-autophosphorylation to phosphorylate eIF2α. As a result, activation of PERK by endoplasmic reticulum stress reduces the synthesis of proteins from mRNA and inhibits the introduction of new proteins into the endoplasmic reticulum

Because of the activity of PERK, eIF2α phosphorylation generally inhibits protein synthesis, but ATF4 translates from mRNA to protein rather. This response acts as a mechanism by which the cells recover from the translational attenuation, which is caused by the stress of the endoplasmic reticulum. In other words, ATF4 activates GADD34 with CHOP corresponding to the lower signaling pathway, and GADD34 allows to form a new translation initiation complex by dephosphorylating eIF2α with PP1 (protein phosphatase 1).

Transcription of the transcription factor ATF4 is up-regulated by eIF2α phosphorylation, which results in an open-reading open-reading frame bypass scanning system, a small open- reading frame. ATF4 is a transcription factor belonging to the CREB / ATF family. ATF4 induces the dephosphorylation of eIF2α through GADD34 induction and activation of PP1. PPlc promotes dephosphorylation of eIF2a. The first step in ER stress response is terminated by recombination of BiP into PERK and dephosphorylation of eIF2a.

Based on such a mechanism, ATF4 is known as a detection index for ER stress, and the present invention relates to a method for detecting the intracellular level of said ATF4.

"ATF4 (activating transcription factor 4)" is a stress protein that is involved in the expression of ATF / CREB (activating transcription factor / cyclic AMP response element binding 6 protein) Means a protein having a molecular weight of about 39 kDa.

The ATF4 is used as a transcription factor that binds to DNA in the form of a dimer. The gene encoding the protein is present on chromosome 22 of human, and the nucleotide sequence of the gene is obtained from a known database such as NCBI's GenBank As an example thereof, GenBank Accession No. 2, pp. NC_000022.10, NC_018933.1, NT_011520.12, and the like, but are not limited thereto.

In particular, ATF4 is difficult to detect at the mRNA level and can be detected as a product by translation. Thus, there is a problem that the ATF4 can not be detected by a method capable of measuring the level of mRNA. However, 'Using a system in which DsRed is fused to the UTR region, the intracellular level of ATF4 can be easily confirmed by detecting the fluorescent reporter.

Therefore, in one aspect, the present invention provides a reporter construct comprising a sequence encoding a DsRed gene located in the 5 'UTR region of Drosophila ATF4; And a composition for detecting ATF4 comprising the same.

The constructs and compositions can be used to identify various levels of intracellular ATF4 and provide various information about the stress of the endoplasmic reticulum.

In the present invention, the ATF4 5'UTR region includes uORF1 and uORF2 start codons which function in opposite to each other, and uORF2 is characterized by a structure in which a reporter gene, for example, a red fluorescent protein (DsRed) gene is fused, In the present invention, the nucleotide sequence of the ATF4 5'UTR region is shown in SEQ ID NO: 1, and the DsRed gene sequence is shown in SEQ ID NO: 2.

The ATF4 and uORF start codon sequences may be obtained from animal-derived, preferably Drosophila known gene sequences. Including wild type, deletions, insertions, non-conservative or conservative substitutions or variants by a combination thereof.

ATF4 protein and the like can be prepared by PCR using oligonucleotides complementary to the terminal 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 homogenous physiological activity " refers to the overexpression in response to ER stress and the activity of ATF4 involved in such a mechanism.

The present invention is characterized in that a reporter fluorescent protein is fused and expressed in order to detect the intracellular level of the ATF4 protein.

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), enhanced red fluorescent protein (ERFP), a blue fluorescent protein (BFP), an enhanced blue fluorescent protein (EBFP), a yellow fluorescent protein (YFP), an enhanced yellow fluorescent protein (EYFP), a blue fluorescent protein ECFP) and Renila luciferase, red fluorescent protein (DsRed), and the like, but are not limited to the above examples. Preferably, Renila luciferase, a red fluorescent protein (DsRed), and most preferably a red fluorescent protein (DsRed) are used as a reporter protein.

The red fluorescent protein (DsRed) exhibits red fluorescence derived from coral and has a structure similar to that of known fluorescent proteins.

In general, the fluorescent protein has a rigid tertiary structure with a fluorescent chromophore inside the cylindrical structure of the folded β-sheet. However, in the case of the red fluorescent protein (DsRed), four such cylindrical structures are assembled into a single protein to display red fluorescence. Because of the above characteristics, it is very stable and has a higher wavelength excitation and emission value than other fluorescent proteins, so it can be easily confirmed with the naked eye.

The DsRed protein, which exhibits fluorescence throughout the visible wavelength region, that is, a color that allows direct eye selection with only light energy in the atmosphere, is optimally excited at 558 nm, Release. Therefore, it is possible to minimize the problem that the excitation wavelength of another fluorescent protein mainly depends on the ultraviolet light of the low region, that is, the damage of nucleic acid, fatty acid, or amino acid residue which is a main molecule in the living body.

The red fluorescent protein (DsRed) of the present invention also includes the following variants:

In one embodiment of the present invention, the DsRed (Red Fluorescence Protein) gene is fused to the downstream region of Drosophila ATF4.

Therefore, the present invention relates to a reporter construct for ATF4 detection, which comprises a Drosophila ATF4 5'UTR region downstream, preferably a uORF start codon, more preferably uORF2 comprising a red fluorescent protein DsRed gene or a variant thereof To provide a reporter fusion protein. In the specification of the present invention, the sequence encoding DsRed or the fusion protein comprising the same is expressed in terms of a reporter gene, a reporter structure, a reporter protein, and the like.

Further, the present invention relates to a recombinant vector for ATF4 detection for expressing the reporter gene and / or the fusion protein, for providing information on the stress of the endoplasmic reticulum.

The vector may comprise, for example, Drosophila ATF4 5'UTR, uORF, DsRed gene each, and may comprise a sequence encoding the reporter fusion 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.

Preferably, the vector of the invention comprises a tubulin promoter, more preferably said vector can be represented by a cleavage map of Fig. 2A (tub-ATF4.5'UTR > dsRed), but is not limited thereto.

Variants of known sequences of the tubulin promoter may be included within the scope of the present invention. Specifically, the promoter includes a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the known nucleotide sequence .

In another aspect, the present invention relates to a composition for detecting ATF4 for providing information on the stress of the reporter, or the reporter construct comprising the vector.

At this time, the overexpression of dsRed by tub-ATF4.5'UTR> dsRed is checked by measuring gene expression level. 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 nucleic acid sequence may also be modified using a label capable of directly or indirectly providing a detectable signal. Examples of labels include radioactive isotopes, fluorescent molecules, biotin, and the like.

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 reporter fluorescent protein of said transformant. ≪ Desc / Clms Page number 2 >

The amount of reporter fluorescent protein dsRed expressed according to the ATF4 level is determined. The higher the intracellular ATF4 level, the greater the amount of reporter fluorescent protein dsRed expressed. Therefore, the amount of expressed dsRed protein can be quantified to measure the intracellular ATF4 level Based on this, various information related to the ER stress can be obtained

In one embodiment of the present invention, the cell line used for transfection (transfection) is preferably an animal cell, more preferably a mammalian cell line. The medium used for culturing the transfected cell line means a culture medium containing serum in an animal cell culture medium commonly used. 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 dsRed protein in the transfected cell line can be used to confirm the level of ATF4 related to the stress of the endoplasmic reticulum. When the up-regulated level is compared with that of the ATF4, it is determined that the stress of the endoplasmic reticulum is high.

In this regard, the following information can be obtained:

(i) ATF4 reporter translation is based on a mechanism that is stimulated in response to ER stress.

At this time, two uORFs in the ATF4 5'UTR have opposite roles in translation, and Drosophila ATF4 expression is regulated by a mechanism involving these uORFs.

(ii) The reporter construct of the present invention is activated in the presence of ER stress.

In a variety of tissues, including malpighian tubules, fat bodies, midgut, dislocation, salivary glands, and brain, dsRed expression under ER stress caused by chemicals is increased. In particular, it was confirmed in one embodiment that there is a clear induction of tub-ATF4.50UTR > dsRed in salivary glands. In addition, during normal development, levels of tub-ATF4.50UTR> dsRed are increased in the presence of ER stress, such as dislocation,

That is, ER stress activates the ATF4 reporter. This allows the ATF4 reporter to detect both the stresses caused by the intrinsic and extrinsic sources, indicating that this response is tissue dependent.

(iii) The reporter construct of the present invention corresponds to a nutritional deprivation that causes ER stress.

 That is, it is activated to a high level in intestines when cultivated in a limited feeding, suggesting that the reporter of the present invention can provide information on energy metabolism. However, ER stress-activated RNase, a marker for the pathway mediated by IRE1, which triggers mRNA splicing of xbp1, does not appear to correspond to malnutrition.

(iv) ATF4 reporter is active in Drosophila photoreceptors.

That is, ATF4 translation is promoted during normal photoreceptor development, which can also provide information about such photoreceptor development.

(v) ATF4 reporters are highly expressed in adult male reproductive organs.

In one embodiment, DsRed was observed in the area of the testicular and testicular duct as well as the accessory gland and appendix. That is, UPR signaling can provide information on the activity in an organ having high protein secretion activity.

Thus, the present invention includes all uses that provide a variety of information on ER stress by identifying ATF4 intracellular levels using a reporter construct comprising a sequence encoding the DsRed gene located in the ATF4 5'UTR region.

That is, since the level of ATF4 due to the stress of the endoplasmic reticulum can be easily confirmed by the detection of a specific fluorescent reporter, it can be used as an application for diagnosing the degree of endoplasmic reticulum stress. Further, it can be used as a factor relating to ATF4, It will be useful to find out.

<Examples>

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.

Materials and methods

structure

All Drosophila reporters were subcloned into the pCasper4 vector. To express the reporters, the tubulin promoter and SV40 3'UTR were subcloned into NotI / KpnI and PstI sites, respectively. The 5'UTR sequence of Drosophila ATF4-RA was obtained by RT-PCR from y, w larvae, and dsRed. The T4 sequence was PCR amplified from pPelican plasmid. The dsRed.T4 has a much shorter maturation time compared to dsRed (RFP) (half-time < 43 min), without significant green emission and improved solubility.

The dsRed ORF was fused to 3 'of the ATF4 5'UTR after three nucleotides (ACC) (dsRed.T4 is now referred to as dsRed) and the dsRed AUG codon was matched to the original position of the ATF4 start codon. This sequence was first subcloned into pBluescript-SK (-) and then transferred to pCasper4 vector.

5 ' RACE

Total RNA was extracted with TRIzol reagent (Ambion, USA) according to protocol. 5'RACE was performed according to the Invitrogen's kit manual (Invitrogen, USA). Briefly, the first stand primer (GSP1) was annealed to the mRNA of 5'ATF4-luc and the mRNA was copied into cDNA with SUPERSCRIPTI RT.

After dCTP and TdT purified cDNAs were labeled, dC-labeled cDNA was amplified using AAP (abridged anchor primer) and GSP2 primers, and primary PCR products were amplified using AUAP (abridged universal amplification primer) and GSP3 primers And amplified again. The secondary PCR products were analyzed by electrophoresis using 1% agarose gel and extracted using the High Pure PCR Product Purification Kit (Roche). The extracted PCR products were cloned and sequenced using pLPS-B Blunt topo vector (ELPis biotech, Korea). The transcriptional initiation site was determined as the 3 ' first nucleotide for adapter sequences that were ligated 5 ' to the mRNA transcript. The primers used were: GSP1 primer 5'ATTATAAATGTCGTTCG-3 ', GSP2 primer 5'CTGCAACTCCGATAAATAAC-3', GSP3 primer 5'GCATACGACGATTCTGTGAT-3 '.

Paris genetics Fly genetics )

The genes are expressed in Drosophila through standard Gal4 / UAS systems. The following flies are known in the art: gmr-gal4, uas-Rh-1G69D / Cyo, tub-gal4, uasxbp1-EGFP / Cyo, uas-Aβ, uas-MJD-tr-Q78. UAS-lacZ was obtained from Blooming Stone Stock Center.

Immunohistochemical analysis

All fluorescence images were obtained with a Zeiss LSM710 confocal microscope using a x20 or x40 objective. The following antibodies were used: rabbit anti-dsRed antibody (1:50 from Clontech or 1: 500 from Dr. SW Kang), rabbit anti-GFP (1: 2000, Molecular Probes, catalog no. A6455) (1: 500, Developmental Studies Hybridoma Bank, N2 7A1, University of Iowa, USA), monoclonal anti-rhodopsin 1 - Paloidine (1: 200; Molecular Probes).

To generate the guinea pig anti-ATF4 antibody, the full length of the ATF4 coding sequence was subcloned into the XhoI and NotI sites in pET14b (Novagen). The resulting ~50 kDa His-tagged recombinant protein was purified to generate polyclonal antibodies. The antiserum was then affinity purified for the same epitope.

Nutritional Restriction Nutrient restriction )

The hatched larvae were grown in an apple juice plate at 25 ° C with active yeast paste. The larvae were collected 47 to 49 hours after the egg laying (AEL) and stored at 25 ° C for 5 h in standard confectionery (5.9% w / v Glucose, 6.6% Cornmeal, 1.2% Baker's Yeast, 0.7% Agar in water) With nutrient limited (NR) medium (5% Sucrose, 1% Agar in PBS). Moved

feeding  analysis( Feeding assay )

Larvae were collected from 47 to 49 h AEL. The larvae were starved for 4 h and then fed to Schneider's Drosophila medium (Gibco) for 5 h at 25 ° C with 10 μg / ml tunicamycin, 1 μM tapsigargin and 5 mM DTT. All compounds were purchased from Sigma-Aldrich.

RT - PCR

For cDNA synthesis, 1 μg RNA was transcribed using SuperScript First-Strand synthesis kit (Invitrogen, USA). PCR amplification was performed for 25 cycles using taq polymerase (Roche).

The primers used were: Luciferase forward primer; 5 'CTCGCATGCCAGAGATCCTA-3', Luciferase reverse primer; 5'AAGGCTCCT CAGAAACAGCT-3 ', rp49 forward primer; 5 'AGATCGTGAAGAAGCGCACCAAG-3', rp49 reverse primer; 5 'CACCAGGAACTTCTTGAATCCGG-3' Rp49 was used as a housekeeping control to normalize the amount of cDNA between each sample. Results were expressed as mRNA relative expression detected in control samples.

Luciferase  analysis

The 5'UTR DNA of Drosophila ATF4-RA was obtained by RT-PCR from y, w larvae and subcloned into pGL3-basic vector (Promega, USA) and labeled with 5'ATF4-luc.

Mutation constructs (5'ATF4.uORF1AUA-luc, 5'ATF4.uORF2AUA-luc, and 5'ATF4.uORF1 ^AUA uORF2 ^AUA -luc) were isolated using the QuickChange site-defined mutagenesis kit (Stratagene) Respectively.

Mutant DNA sequences were diversified by DNA sequencing. Drosophila S2 cells were co-infected transiently with Effectene (Qiagen) with wild type or mutant reporter plasmid plus pRL (Renilla luc). After 96 hours, luciferase activity was measured using a DUAL-GIO luciferase assay system (Promega, USA). Firefly luciferase activity was normalized to Renilla luciferase activity.

Example  One : Drosophila ATF4  Identification of expression control mechanism

Previous studies have shown that Drosophila ATF4 protein levels are increased in response to ER stress caused by misexpression of mutant Rhosopsin-1. Thus, in this experiment, the ER stress-dependent ATF4 induction mechanism was observed in Drosophila .

In mammals, ATF4 expression is modulated in response to eIF2a phosphorylation by a mechanism involving two uORFs (upstream open reading frames) at the 5'UTR.

As shown in FIG. 1A, Drosophila ATF4-RA also had two uORFs at the 5'UTR.

To examine the role of these two uORFs in ATF4 expression control, reporter constructs were constructed with the tubulin alpha 1 promoter, which induces the expression of chimeric transcripts containing ATF4 5'UTR fused to the luciferase coding sequence. The construct was designed to determine the effect of the 5'UTR on translation of the luciferase reporter coding sequence

To evaluate the role of uORFs, additional constructs with uORF initiation codons mutated to AUA were constructed. The reporter start site of the reporter was analyzed by 5'RACE and sequencing.

The luciferase reporter has two uORFs in the 5'UTR. These plasmids were transfected into cultured S2 cells and luciferase activity induced by those with wild-type 5'UTR, or uORF1 ^AUA , or uORF2 ^AUA mutated 5'UTR was measured (Fig. 1B).

DTT treatment increased firefly luciferase activity in cells transfected with the wild-type luciferase reporter (5'ATF4-luc), which means that ATF4 reporter translation is stimulated in response to ER stress.

Mutations in uORF1 (5'ATF4.uORF1 ^AUA- luc) cause a significant reduction in luciferase activity in S2 cells. However, when uORF2 mutated, there was an increase in luciferase activity, which was independent of ER stress (Fig. 1C).

In addition, if both uORF1 and uORF2 mutate, the effect of uORFs is lost. The level of mRNA from the reporter was similar under all conditions.

These results are consistent with the idea that uORF1 and uORF2 have an opposite role in the translation of the major ORFs and showed that the translational control mechanism of ATF4 is conserved between Drosophila and mammals. That is, Drosophila ATF4 expression is regulated by a mechanism involving uORF.

Example  2 : in vivo ATF4  Development of translational activity reporter

To investigate how ATF4 expression is regulated during normal development as well as ER stress, a modified in vivo ATF4 reporter from ATF4 luciferase reporter was developed. The dsRed gene under control of the ATF4 5'UTR was located (Figure 2A).

The mutant Rhodopsin-1 (Rh-1G69D) was misexpressed in the larval eye imaginal discs via the gmr-gal4 driver to determine if the in vivo ATF4 reporter responded to ER stress.

Under these conditions, dsRed reporter induction was observed in larval eye discs expressing Rh-1G69D (Fig. 2D). Although ATF4 antibody staining was not sensitive enough to detect endogenous ATF4 expression in normally developed tissues, it was possible to detect endogenous ATF4 protein induction by Rh-1G69D misexpression (Fig. 2E), and a pattern of dsRed reporter induction Respectively.

Next, we tested whether other aggregation prone proteins could activate the ATF4 translation reporter.

Of these, MJD-tr-Q78 and Aβ were tested. Expression of MJDtr-Q78 through the gmr-gal4 driver in larval eye adult discs caused a severe eye ablation phenotype, but not to a detectable level of the ATF4 reporter (not shown). In contrast, dsRed induction was observed, albeit at a reduced level, when the ATF4 reporter was co-expressed with A [beta], the peptide that makes up the Alzheimer's disease (Fig. 2C).

These results suggest a newly developed in vivo ATF4 reporter corresponding to some stress-inducible proteins in vivo.

Example  3: ER  Stress and in vivo ATF4  Expression Reporter Relationship

tub-ATF4.5'UTR> dsRed maintains inactivity in the stress-free larval eye discs, while malpighian tubules, fat bodies, midgut, dislocations, salivary glands, In the various tissues involved, this reporter activity, which is the inherent stress implicated during normal development, was detected (Figures 3A, 3E, 3I, 3M and 3Q).

To characterize the ATF4 translation profile independently in response to in vivo ER stress, we raised a second larval with ER stress caused by chemicals. 10 μg / mL of tunicamycin, 1 μM of thapsigargin and 5 mM of DTT for 5 h, and the larvae were dissected and stained with anti-dsRed antibody.

Under these conditions, the expression of tub-ATF4.5'UTR > dsRed increased widely. In particular, there was a clear induction of tub-ATF4.50UTR> dsRed in salivary glands (FIG. 3E-H).

In addition, increased levels of tub-ATF4.50UTR > dsRed were observed in the displaced, middle, and lobes with ER stress (Fig. 3I-3P) as the larvae were nurtured. However, there was no noticeable change in dsRed expression in larval brain and spindle organs (Figs. 3A-D and 3Q-T).

In addition, in vivo ATF4 reporter was identified in Drosophila S2 cells. This reporter also responded to ER stress resulting in compound, DTT, tunicamycin, tapsigargin in Drosophila S2 cells.

These results indicate that ER stress activates the ATF4 expression reporter in vivo. That is, ATF4 reporters can detect both stresses caused by intrinsic and extrinsic sources, indicating that this response is tissue-dependent.

Example  4: lack of nutrition in vivo ATF4  Expression Reporter Relationship

ATF4 translation is activated in response to a number of other stress conditions, including amino acid deficiency. To test whether the in vivo ATF4 reporter was activated in response to malnutrition, the second larvae were grown for 18 hours under standard feeding or nutritionally restricted conditions (amino acid deficient).

A reproducible pattern of dsRed expression was found in the optic lobe and brain stem at standard feeding.

Double labeling was performed with an anti-repo (glial marker) antibody that was not co-located with the ATF4 reporter (Fig. 4A). Although the tub-ATF4.5'UTR > dsRed reporter was activated in the brain, there was no significant change in the induction of dsRed between standard feeding and limited feeding (Figs. 4A and 4B).

However, the tub-ATF4.5'UTR > dsRed reporter was activated at high levels in the intestine when the larvae were raised in limited feeding compared to standard feeding (Figs. 4C and 4D). Furthermore, in larvae, dsRed expression was significantly induced in dislocations, lipids, and spindle organs (Fig. 4E-J).

Since ATF4 is one of the three transcription factors mediating UPR, the present inventors sought to determine whether other UPR pathways also correspond to malnutrition. The pathway mediated by the ER stress activated RNase, IRE1, which triggers mRNA splicing of the transcription factor, xbp1, was specifically tested. Activation of this pathway can be detected through the xbp1-EGFP reporter, where EGFP is expressed in a frame only ER-stress triggers the splicing of xbp1 mRNA. EGFP expression did not vary significantly depending on nutritional restrictions.

These results show that the in vivo ATF4 reporter responds to nutritional deprivation. That is, ATF4 is specifically activated by nutritional restriction, suggesting that it may play an essential role in energy metabolism.

Example  5: in vivo ATF4  Expression reporter Photoreceptor  activation

Previous studies have shown that the UPR sensor xbp1-EGFP is active in photoreceptors during the late pupa development. To determine if the ATF4 branch of signaling was similarly activated in photoreceptor cells, ATF4 receptor activity was examined during the pupal and adult stages.

At the beginning of the pupa (37 h: APF after pupa formation), ATF4 reporter activity could be detected in photoreceptor cells (Fig. 5A). This reporter activity was maintained at 48 h APF, but the pattern of expression was altered (Fig. 5B). At 96 hours of puparium, dsRed was still expressed in photoreceptor cells. In the adult retina, we found that the dsRed reporter was expressed, but not in the photoreceptor (Fig. 5D), similar to the reported xbp1-EGFP pattern overlapping Homothorax-positive lattice cells.

These results indicate that ATF4 translation is promoted during normal photoreceptor development. In other words, the in vivo ATF4 reporter is active in Drosophila photoreceptors.

Example  6: ATF4  Reporter High expression  domain

Previous studies on xbp1-EGFP and xbp1p> dsRed, xbp1 receptors in Drosophila have shown that the xbp1 pathway of UPR is active in non-female adult male reproductive organs.

Specifically, the xbp1-EGFP reporter was detected in a restricted region of the testis and subspecies. On the other hand, xbp1p> dsRed was not expressed in the testes themselves, but in the appendix and accessory glands.

To test whether the ATF4 reporter was active in male regenerating acid organs, the adult regenerating organs were dissected and stained with anti-dsRed antibody.

As a result, as can be seen from Fig. 6, DsRed was observed in a limited area of the testicular duct and the testicular duct as well as the accessory duct and appendix. That is, the ATF4 reporter was highly expressed in adult male reproductive organs.

These results are consistent with the fact that UPR signaling is active in organs with high protein secretion activity.

<110> Industry Academic Cooperation Foundation of Ulsan University <120> Method for Detecting ATF4 under Endoplasmic reticulum Stress <130> PN1507-199 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 304 <212> DNA <213> ATF4 5'UTR gene sequence <400> 1 actgattcca acactcatta ccgttccaaa tgaaacgcaa aaaggttcaa ttgttatttt 60 ctctcacgat ttcggcattt atattaacct aaagatggct ctatagtttg cgcgcaagca 120 aaaacaagca gcacctaggt gcccaaacac aacaaaatga aaacgtataa aaatcaaaac 180 gccatcatgg cattttcaca acaacaaaac acaccgcgaa aacaaccatt ctcattcttc 240 ggaccggatt tcatactctc aaaacaaaac atatctttga cccctgattt gaactctgga 300 TACC 304 <210> 2 <211> 678 <212> DNA <213> DsRed gene sequence <400> 2 atggcctcct ccgaggacgt catcaaggag ttcatgcgct tcaaggtgcg catggagggc 60 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc 120 acccagaccg ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc 180 ctgtcccccc agttccagta cggctccaag gtgtacgtga agcaccccgc cgacatcccc 240 gactacaaga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag 300 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggctc cttcatctac 360 aaggtgaagt tcatcggcgt gaacttcccc tccgacggcc ccgtaatgca gaagaagact 420 atgggctggg aggcctccac cgagcgcctg tacccccgcg acggcgtgct gaagggcgag 480 atccacaagg ccctgaagct gaaggacggc ggccactacc tggtggagtt caagtccatc 540 tacatggcca agaagcccgt gcagctgccc ggctactact acgtggactc caagctggac 600 atcacctccc acaacgagga ctacaccatc gtggagcagt acgagcgcgc cgagggccgc 660 caccacctgt tcctgtag 678

Claims (6)

A reporter construct comprising a sequence encoding a fluorescent gene located in the ATF4 5'UTR region. The method according to claim 1,
Wherein the ATF4 5'UTR region is derived from Drosophila and consists of the nucleotide sequence of SEQ ID NO: 1. The method according to claim 1,
Wherein the fluorescent gene is a DsRed gene and the DsRed gene is a nucleotide sequence of SEQ ID NO: 2. 2. The composition for detecting ATF4 according to claim 1, wherein the fluorescent gene is a DsRed gene. The method according to claim 1,
Wherein the reporter construct further comprises a tubulin promoter. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt; The method according to claim 1,
Wherein the reporter construct comprises a uORF1 and uORF2 start codon functioning in opposition to each other and wherein the DsRed gene is fused to uORF2. Use of the composition for detecting ATF4 according to any one of claims 1 to 5,
And that the intracellular endoplasmic reticulum stress is also increased when the DsRed expression level is increased.

Images