KR101694907B1 - Composition for treating mitochondrial dysfunction diseases comprising CTRP1 inhibitor - Google Patents

Abstract

The present invention relates to a composition capable of effectively inhibiting the dynamics of mitochondria by inhibiting the expression or activity of CTRP1 (C1q / TNF-related protein 1). The present invention also relates to a method for screening a therapeutic agent for a disease associated with mitochondrial dysfunction by determining an agent that inhibits the interaction of CTRP1 and visentin as a substance that inhibits mitochondrial division and cleavage.

Description

[0001] The present invention relates to a composition for treating mitochondrial disorder comprising CTRP1 inhibitor,

The present invention relates to a composition capable of effectively inhibiting the dynamics of mitochondria by inhibiting the expression or activity of CTRP1 (C1q / TNF-related protein 1). The present invention also relates to a method for screening a therapeutic agent for a disease associated with mitochondrial dysfunction by determining an agent that inhibits the interaction of CTRP1 and visentin as a substance that inhibits mitochondrial division and cleavage.

Mitochondrial function is involved in energy production, Ca ^{2 +} homeostasis, free radical formation and cell death in the cell. In particular, mitochondrial cleavage modulating activity in mitochondrial dynamics has been implicated in aging, diabetes, and neurological disorders [DC Chan, Mitochondria: dynamic organelles in disease, aging, and development. Cell 125, 1241 (2006); H. Chen, DC Chan, Mitochondrial dynamics-fusion, fission, movement, and mitophagy-in neurodegenerative diseases. Hum . Mol . Genet . 18, 169 (2009); SC Correia et al ., Mitochondrial importance in Alzheimer's, Huntington's and Parkinson's diseases. Adv . Exp . Med . Biol . 724, 205 (2012); Y. Yoon, CA Galloway, BS Jhun, T. Yu, Mitochondrial dynamics in diabetes. Antioxid . Redox Signal . 14, 439 (2011)).

To date, it has been known that mitochondrial cleavage involves Drp1 (dynamin-related protein 1 (known as Dnm1 in yeast) and that mitochondrial fission factor (MFF) is regulated at this Drp1 location. However, the exact mechanism by which Drp1 regulates mitochondrial cleavage is unknown.

Moreover, mitochondria and endoplasmic reticulum are a close interaction and local synthesis, Ca ^{2 +} signaling and mitochondrial cutting. Recently, IFN2 (formin2), which regulates actin synthesis, has been reported to regulate mitochondrial cleavage by interacting with Drp1 at the mitochondrial cleavage site in the endoplasmic reticulum.

On the other hand, CTRP1 has been reported as a secretory protein with metabolic control function similar to adiponectin, and it has been reported that GIF (G protein interacting protein) expresses many in heart and muscle. However, the association between CTRP1 and mitochondrial dynamics is not known.

Korean Patent Laid-Open No. 10-2009-0061727 (June 17, 2009)

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating mitochondrial dystrophy-related diseases, which comprises a substance that inhibits the expression or activity of CTRP1.

It is another object of the present invention to provide a method of treating cancer, which comprises treating a candidate compound with CTRP1 and visentin; And determining whether or not the interaction of CTRP1 and visemen is inhibited in the cell. ≪ Desc / Clms Page number 2 >

It is another object of the present invention to provide a composition for screening therapeutic agents for mitochondrial dysfunction-related diseases, which comprises CTRP1 and visentin.

Mitochondrial dynamics are important for cell homeostasis, death, aging, neurological disorders, and diabetes. Mitochondrial cleavage is involved in the Drp1 gene, but precise truncation control has not yet been reported. In the present invention, it was confirmed that the CTRP1 gene is present in the endoplasmic reticulum and mitochondria, and that this gene interacts with the microtubule and vimentin in the membrane of the endoplasmic reticulum. Furthermore, the present invention has for the first time demonstrated that CTRP1 regulates mitochondrial fluidity through interaction with visentin and regulates mitochondrial cleavage.

Thus, in the present invention, CTRP1 is provided as a novel target for regulating the dynamics of mitochondria. Specifically, it is possible to inhibit mitochondrial division and cleavage by inhibiting the gene expression of CTRP1 or the activity of its protein, thereby effectively preventing, ameliorating or treating diseases caused by dystrophy of mitochondria, such as aging, diabetes or neurological diseases .

Accordingly, in one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of mitochondrial dysfunction-related diseases, comprising a substance that inhibits the expression or activity of CTRP1.

Examples of neuronal diseases include Alzheimer's disease, Huntington's disease, Parkinson's disease and the like. However, if the disease is caused by mitochondrial dysfunction, The present invention is not limited to the above examples and can be included in the scope of the present invention.

In the present invention, the mRNA or protein of the CTRP1 gene can be identified in the known gene database. For example, the nucleic acid sequence of the human CTRP1 gene can be found in Genbank Accession No. NM_030968.3, and the amino acid sequence of the protein encoded thereby can be found in Genbank Accession No. NP_112230.

In the present invention, the substance that inhibits the expression of CTRP1 includes siRNA, shRNA, or antisense nucleic acid capable of specifically binding to mRNA of CTRP1.

As used herein, the term "siRNA" means a short double-stranded RNA capable of inducing RNAi (RNA interference) through cleavage of a specific mRNA. The siRNA consists of a sense RNA strand having a sequence homologous to the mRNA of the target gene and an antisense RNA strand having a complementary sequence. Since siRNA can inhibit expression of a target gene, it can be provided as an efficient gene knockdown method or as a method of gene therapy.

The siRNA is not limited to a complete pair of double-stranded RNA portions that are paired with each other, but is paired by a mismatch (the corresponding base is not complementary), a bulge (no base corresponding to one chain) May be included. The total length is 10 to 100 bases, preferably 15 to 80 bases, more preferably 20 to 70 bases. The siRNA terminal structure is capable of blunt or cohesive termini as long as it can inhibit the expression of the target gene by the RNAi effect. The adhesive end structure can be a structure having a 3 'end protruding structure and a 5' end protruding structure. The number of protruding bases is not limited. The siRNA may be a small RNA (for example, a natural RNA molecule such as a tRNA, a rRNA, or a viral RNA, or an artificial RNA molecule) at a protruding portion at one end within a range that can maintain the effect of suppressing the expression of a target gene, . ≪ / RTI > The siRNA end structure does not need to have a truncation structure on both sides, and may be a step-loop structure in which the terminal region of the double-stranded RNA is connected by linker RNA.

The siRNA used in the present invention may be a complete form having polynucleotide pairing itself, that is, a form in which siRNA is directly synthesized in a test tube and introduced into a cell through two transformation processes, Single chain oligonucleotide fragments and their reverse-phase trefoil can be derived from single-stranded polynucleotides separated by a spacer, for example, a form of siRNA expression vector or PCR-derived siRNA prepared so that the siRNA is expressed in a cell The expression cassette may be introduced into the cell through a transformation or infection process. The determination of how to prepare siRNAs and introduce them into cells or animals may depend on the objective and the cellular biological function of the target gene product.

As used herein, the term "shRNA" is intended to overcome the disadvantages of high cost biosynthetic cost of siRNA, short term maintenance of RNA interference effect due to low cell transfection efficiency, The siRNA can be expressed by introducing it into a cell using a virus and plasmid expression vector system. The shRNA is converted into an siRNA having a correct structure by the siRNA processing enzyme (Dicer or Rnase Ⅲ) Is widely known.

As used herein, the term "antisense nucleic acid" means DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, and refers to a complementary sequence in mRNA, . ≪ / RTI > The antisense sequence of the present invention means a DNA or RNA sequence complementary to mRNA of CTRP1 and capable of binding to its mRNA and can be used for translation of mRNA of CTRP1, translocation into the cytoplasm, maturation or any other overall biological It is possible to inhibit the essential activity for the function. The length of the antisense nucleic acid is 6 to 100 bases, preferably 8 to 60 bases, more preferably 10 to 40 bases.

In the case of antisense RNA, it can be synthesized in vitro in a conventional manner and administered in vivo, or the antisense RNA can be synthesized in vivo. One example of the synthesis of antisense RNA in vitro is the use of RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow the antisense RNA to be transcribed using a vector in which the origin of the recognition site (MCS) is in the opposite direction. Such antisense RNAs are preferably made such that translation stop codons are present in the sequence so that they are not translated into the peptide sequence.

The composition of the present invention may contain one or more of siRNA, shRNA, or antisense nucleic acid of CTRP1, as well as agents that promote the influx of siRNA, shRNA, or antisense nucleic acid molecules, such as liposomes or cholesterol, May be combined with one kind of lipophilic carrier among a plurality of sterols including the above. The antisense nucleic acid may also be conjugated to a peptide that is absorbed by the cell. Examples of useful peptides include peptide hormones, antigens or antibodies, and peptide toxins.

In the present invention, the substance that inhibits the activity of CTRP1 includes an antibody capable of specifically binding to the protein of CTRP1.

Such antibodies include both monoclonal antibodies and chimeric, humanized and human antibodies thereto, as well as novel antibodies as well as antibodies known in the art. As long as the antibody specifically binds to CTRP1, it comprises a functional fragment of an antibody molecule as well as a complete form having the full length of two heavy chains and two light chains. The functional fragment of the molecule of the antibody refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') ₂ and Fv.

A method of injecting a substance that inhibits the expression or activity of CTRP1 into cells can be implemented in various forms such as a liposome or a method using a vector system as a genetic engineering technique. Alternatively, a preparation in the form of a pharmaceutical preparation and administration into the body in a conventional manner may be used.

When the composition of the present invention is used as a pharmaceutical composition, the content of the active ingredient in the composition may be appropriately adjusted according to the symptom of the disease, the progress of the symptom, the condition of the patient and the like, %, Preferably 0.001 to 50% by weight, based on the total weight of the composition. The content ratio is a value based on the dried amount from which the solvent is removed.

The pharmaceutical composition may further comprise suitable carriers, excipients and diluents conventionally used in the production of pharmaceutical compositions. It may be formulated into tablets, capsules, tablets, capsules, suspensions, emulsions, syrups, An external preparation, a suppository or a sterilized injection solution, and the like.

When the pharmaceutical composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may include at least one excipient and / or lubricant. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

The preferred dosage of the pharmaceutical composition will vary depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. For a more preferable effect, the dose of the composition of the present invention is preferably 0.1 mg / kg to 100 mg / kg per day based on the active ingredient, but is not limited thereto. The administration may be carried out once a day or divided into several doses. The compositions of the present invention may be administered to a mammal, including a human, in various ways. All modes of administration may be expected, for example, by oral, intravenous, intramuscular, subcutaneous injection, and the like. The pharmaceutical dosage form of the composition of the present invention may be used in the form of a pharmaceutically acceptable salt of the active ingredient, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable combination.

Meanwhile, in the present invention, for the first time, it has been found that CTRP1 plays an important role in controlling the dynamics of mitochondria with vimentin. Therefore, a substance that regulates the dynamics of mitochondria can be screened using the interaction between CTRP1 and visemen. For example, a substance that inhibits the interaction between CTRP1 and vimentin can be screened to provide therapeutic candidates for diseases caused by dystrophy of mitochondria, such as senescence, diabetes or neurological diseases.

Accordingly, in another aspect, the present invention relates to a composition for screening a therapeutic agent for mitochondrial dysfunction-related diseases, comprising CTRP1 and visentin.

The present invention also relates to a method of treating cancer, comprising the steps of: treating a candidate compound to CTRP1 and visentin; And determining whether the interaction of CTRP1 and vimentin in the cell is inhibited. ≪ Desc / Clms Page number 2 > The present invention relates to a method for screening for a therapeutic agent for mitochondrial dysmotility related diseases.

The screening method of the present invention comprises treating a candidate compound with CTRP1 and visentin, wherein the candidate compound is treated with a cell expressing CTRP1 and vimentin or with a mixture of isolated CTRP1 and isolated visentin Candidate compounds can be treated directly.

In the present invention, the mRNA or protein of the vimentin gene can be confirmed by its sequence information in a known gene database. For example, the nucleic acid sequence of the human vimentin gene can be found in Genbank Accession No. NM_003380.3, and the amino acid sequence of the protein encoded thereby can be found in Genbank Accession No. NP_003371.2.

The term "candidate compound" in the present invention means a candidate substance which is expected to inhibit the interaction of CTRP1 and visentin. Such candidate substances may include not only a single compound such as an organic or inorganic compound but also a complex of a protein, a carbohydrate, a nucleic acid molecule (RNA, DNA, etc.) and a polymer compound such as lipid and a plurality of compounds.

The term "treatment" in the present invention means not only the candidate compound directly contacts with any one or more of CTRP1 and visentene, but also the fact that the substance affects the cell membrane and the signal generated from the cell membrane is at least one of CTRP1 and visentin And the like. Thus, in the present invention, the candidate compound includes not only substances that are permeable to cell membranes but also substances that are impermeable to cell membranes. At this time, the candidate compound is treated within the effective amount range. The term "effective amount" means an amount sufficient to induce the reaction. Since the accurate result can not be obtained at an effective amount or less, it is preferable to measure the inhibitory ability within an effective amount range.

When the candidate compound is treated to cells expressing CTRP1 and visentin,

(a) treating the candidate compound with CTRP1 and cells expressing vimentin; And (b) determining whether the interaction of CTRP1 and vimentin in the cell is inhibited. ≪ Desc / Clms Page number 2 >

The cells expressing CTRP1 and vimentin may be cells originally comprising CTRP1 and visemen, or intentionally prepared by transfecting cells with vectors expressing CTRP1 and vimentin. Also, in the case of CTRP1 and vimentin-expressing vectors, CTRP1 and vimentin may be expressed in one vector, respectively, because they contain different promoters for CTRP1 and vimentin, and may express CTRP1-expressing vectors and vimentin Vector may be prepared by co-transfection.

The cells include all cells of animal origin such as human or bovine, goat, pig, mouse, rabbit, hamster, zodiac, guinea pig, etc. Primary cells, secondary cells, immortalized cells and the like can be used. In addition, cells that have been engineered to stably or transiently overexpress CTRP1 and / or Vimentin in a cell using a recombinant vector comprising CTRP1 and / or a non-mentin gene can be used.

Furthermore, the detection of a substance that inhibits the interaction of CTRP1 and visentin can be carried out in vivo using not only cell level but also experimental animals such as mice, rabbits, hamsters, stomachs, guinea pigs and the like.

In the step (b) of the present invention, whether the interaction of CTRP1 and visemen is inhibited can be confirmed by applying techniques commonly used in the art to determine whether the protein-protein interactions or protein-compound interactions occur have. For example, high throughput screening (HTS) using immunological assays, fluorescence assays, yeast two-hybrid, detection of phage display peptide clones binding to CTRP1 or visentin, natural products and chemical libraries, , A drug-hitting HTS or a screening method using cell-based screening, etc., but the present invention is not limited thereto.

In one preferred embodiment, an antibody specific for CTRP1 or visentin can be used to detect whether the interaction of CTRP1 and visemen is inhibited. For example, the amount of the formed antigen-antibody complex formed can be compared with the amount of the antigen-antibody complex formed in the control group not treated with the candidate substance to determine the inhibition. Absolute or relative differences in the amount of antigen-antibody complex formation can be ascertained by molecular biology or histological analysis including, but not limited to, immunostaining, immunoprecipitation and immunostaining.

In the detection method using the antigen-antibody reaction, the formation amount of the antigen-antibody complex can be quantitatively measured through the signal label of the detection label. In the present invention, the term "detection label" is to be detected by means of spectroscopic, photochemical, biochemical, immunochemical, chemical or other physicochemical means ≪ / RTI > Such detection labels include, but are not limited to, enzymes, minerals, ligands, luminescent materials, microparticles, redox molecules, and radioactive isotopes.

In another preferred embodiment, it is possible to detect whether the interaction of CTRP1 and visimentin is inhibited by the signal intensity of the reporter protein after the CTRP1 or visiment is ligated with the reporter protein, respectively.

The reporter protein may be a luciferase, a chloramphenicol acetyltransferase, a beta-glucuronidase, a beta-galactosidase, an alkaline phosphatase, or a fluorescent protein, wherein the fluorescent protein is a green fluorescent a fluorescent protein, a red fluorescent protein, a blue fluorescent protein, a yellow fluorescent protein, a cyan fluorescent protein, an enhanced green fluorescent protein, , An enhanced red fluorescent protein, an enhanced blue fluorescent protein, an enhanced yellow fluorescent protein, or an enhanced blue fluorescent protein.

In order to express the reporter protein in the form of CTRP1 and vimentin linked to each other, a gene encoding CTRP1 and an expression vector in which a reporter gene is operably linked, and a gene encoding vimentin and an expression vector in which a reporter gene is operatively linked Can be co-transfected into cells.

When the reporter protein is a fluorescent protein, fluorescence intensity of the reporter protein can be measured using a fluorescence microscope to confirm whether the interaction of CTRP1 and visentin is inhibited. When a luminescent enzyme such as luciferase is used as a reporter protein, Lt; RTI ID = 0.0 > luciferase < / RTI > activity assay or luciferase luminescence inhibits the interaction of CTRP1 and vimentin. In addition, an enzyme capable of converting a substrate into a coloring product capable of emitting detectable light such as chloramphenicol acetyltransferase, beta-glucuronidase, beta-galactosidase, alkaline phosphatase and the like is used as the reporter protein It can be confirmed whether the interaction of CTRP1 and visentin is inhibited by an optical signal obtained by an enzyme reaction using a chromogenic substrate.

On the other hand, when the candidate compound is treated to a mixture of isolated CTRP1 and isolated visentin,

(a ') treating the candidate compound with a mixture of isolated CTRP1 and isolated visentin; And (b ') determining whether the interaction of CTRP1 and vimentin is inhibited. The present invention also provides a method of screening for a therapeutic agent for mitochondrial dysfunction related disease.

In the present invention, the term "isolated" means a protein expressed and isolated in a cell, and is separable by applying common separation techniques known in the art. A method for separating and purifying a protein expressed in a cell is not particularly limited. Isolated CTRP1 and isolated visentin can react directly with the candidate compound in vitro, not in the cell.

The method for confirming whether the interaction of CTRP1 and visentin is inhibited is as described above.

The agent for inhibiting the interaction of CTRP1 and visentin obtained by the screening method of the present invention can treat various diseases caused by mitochondrial dynamics by inhibiting mitochondrial cleavage during mitochondrial division.

In the present invention, CTRP1 can be used as a target for treating various diseases caused by mitochondrial dynamics, and further, the interaction candidate between CTRP1 and non-mentin can be used to screen therapeutic candidates of the diseases.

FIG. 1 shows the result of examining the position of CTRP1 in MEF cells through immunostaining. The red color shows anti-CTRP1, the green color shows anti-calreticolin, and the blue color shows anti-COX4 antibody (Scale bar = 10 μm).
FIG. 2 shows electron microscopic observation of MEF cells treated with anti-CTRP1 antibody (red arrow) linked with 10 nano gold particles. ER indicates the endoplasmic reticulum, and Mit indicates the mitochondria (Scale bar = 200 nm).
FIG. 3 shows the results of detection of CTRP1, tubulin, carnexin, ACSL4 and COX4 using Western blotting after separating cell organelles of MEF cells by subcellular fractionation (subcellular fractionation). ER is an endoplasmic reticulum, Mit is a mitochondrial fraction, MAM is a mitochondria associated ER membrane fraction, and P-Mit is a pure-mitochondrial fraction.
FIG. 4 shows the results of detection of CTRP1 and calreticulin in the pellet (P) and supernatant (S) fractions after treating buffer, Na ₂ CO ₃ and tryptone X-100, respectively, .
FIG. 5 shows the results of detection of the presence of CTRP1 and calreticulin in the case where trypsin was treated with the ER and the case where the ER was isolated from the MEF cell, respectively.
FIG. 6 shows the CTRP1 ^{fl / fl} And Cre ⁺ CTRP1 ^{fl / fl} The cells were stained with MitoTracker (Scale bar = 10 μm).
Figure 7 is the result of quantifying the length of the mitochondria in the results of Figure 6 (n = 256 to 427 mitochondria; Error bars, data representing mean ± SD; * p <0.001).
Figure 8 shows the CTRP1 ^{fl / fl} And Cre ⁺ CTRP1 ^{fl / fl} The cells are stained with anti-Drp1 antibody (green) and MitoTracker (red), respectively (Scale bar = 10 μm).
Figure 9 shows the results of quantifying Drp1 puncta per mitochondrial length in the results of Figure 8 (n = 67-88 mitochondria; Error bars, data representing mean ± SD).
FIG. 10 shows the results of treatment of control siRNA, CTRP1 siRNA or Drp1 siRNA with U2OS cells and staining with anti-Drp1 antibody (green) and MitoTracker (red), respectively (Scale bar = 10 .mu.m).
Figure 11 shows the results of quantifying the length of mitochondria in the results of Figure 10 (n = 185 to 278 mitochondria; Error bars, data representing mean ± standard deviation; * p <0.001).
Figure 12 shows the results of quantifying Drp1 puncta per mitochondrial length in the results of Figure 10 (n = 45 to 63 mitochondria; Error bars, data representing mean ± SD; *, p <0.001).
FIG. 13 shows the result of anti-Flag-bead pull down assay after transfection of 293T cells with Flag-vector (lane 1) or CTRP1 (lane 2) with Flag attached thereto. The precipitated proteins were separated by SDS-PAGE and detected by silver staining. Vimentin and tubulin were identified by mass spectrometry (arrow head). An asterisk (*) indicates an immunoglobulin G heavy chain.
Fig. 14 shows the interaction of endogenous CTRP1 and visentin, showing the results of detection of whole cell lysates of 293T cells by immuno-precipitation with a control or anti-CTRP1 antibody followed by antibody. 293T cells were co-transfected with Flag-CTRP1 and Myc-his-Vimentin, and whole cell lysates were immunoprecipitated with anti-Flagbad and immunoblotted with anti-Myc antibodies. An asterisk (*) indicates an immunoglobulin G heavy chain.
Fig. 15 shows the result of staining U2OS cells with anti-CTRP1 and anti-Vientin cells (Scale bar = 10 mu m).
Figure 16 shows the results of transfecting U2OS cells with siRNA specific for vimentin or treating with okadaic acid (1 μM) or calyculin (5 μM) inhibiting vimentin synthesis and staining with MitoTracker .
Figure 17 shows the results of quantitating the length of the mitochondria in the results of Figure 16 (n = 258 to 296 mitochondria; Error bars, data representing mean ± standard deviation).
Figure 18 shows the results of transfecting U2OS cells with siRNA specific for non-mentin and then staining with anti-Drp1 antibody (green) and MitoTracker (red) (Scale bar = 10 μm).
FIG. 19 shows a time-lapse image of USOS cells transfected with mitochondria, green fluorescence (GFP) coupled with red fluorescence (REP) and CTRP1 (Scale bar = 5 μm).
FIG. 20 shows a time-lapse image of USOS cells transfected with mitochondria linked with red fluorescence (REP) and vimentin linked with green fluorescence (GFP) (Scale bar = 5 μm).
Figure 21 shows the results of overlapping mitochondrial time-lapse images of USOS cells transfected with control siRNA, vimentin siRNA or CTRP1 siRNA. Each color is the result of confirming mitochondrial fluidity change at each time (Scale bar = 5 μm).
Figure 22 shows the result of overlapping mitochondrial time-lapse images in U2OS cells. Arrows indicate mitochondria during cleavage (Scale bar = 5 μm).
Figure 23 shows a time-lapse image of USOS cells transfected with mitochondria coupled with red fluorescence (REP) and vimentin coupled with green fluorescence (GFP) (Scale bar = 5 μm).
Figure 24 shows the time-lapse (time) of USOS cells transfected with mitochondria linked by red fluorescence (REP), visemantin coupled with green fluorescence (GFP), CTRP1 or ER -lapse) Represents an image. Arrows indicate mitochondrial cleavage sites and arrowheads indicate mitochondrial cleavage sites (Scale bar = 5 μm).
Figure 25 shows a percentage (n = 31) in which colocalization of CTRP1, vimentin and ER occurs at the mitochondrial cleavage site.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. Mouse making

CTRP1 ^{fl / fl} The mice were purchased from the KOMP (KnockOut Mouse Project, USA) by purchasing a CTRP1 conditional knockout construct, injected into mouse stem cells (E14 clone) by electric shock, and microinjected at the mouse blastocyst stage to obtain CTRP1 knockout (conditional knockout) mice. Exon 2 of CTRP1 gene was designed to be deleted when cre recombinase acts.

Mouse genotyping primer information is shown in Table 1.

primer order SEQ ID NO: loxP site primer LR-5'-ACT GAT GGC GAG CTC AGA CC -3 ' One Genomic primer DF - 5'-GCA CAC CTG TAT ACC AGACA -3 ' 2 Genomic primer DF2 - 5'-GCA CAC CTG TAT ACC AGACE -3 ' 3 Genomic primer DR-5'-GAG GGA GAG AAG AAA GCC TA -3 ' 4

Example  2. Cell culture and Transactions and  medication

MEF, 293T, Hela and U2OS cells were cultured in DMEM (Dulbecco's Modified Eagles Medium) medium containing 10% FBS. Transfection was performed by transfecting cells with a specific plasmid and siRNA using lipofectamine 2000 (Invitrogen) Respectively. DMSO (dimethyl sulfoxide) was added to the culture medium for 1 hour, and the cells were treated for 1 hour with 10 μM norcodazole (Sigma), 1 μM okada (OKA; Sigma) or 5 nM caliculin- Lt; / RTI &gt;

The siRNA information used is shown in [Table 2].

primer order SEQ ID NO: CTRP1 siRNA (h) (Santa Cruz Biotechnology) target sequence A 5'-CAGTTGAGACTCTGCTTAAtt -3 ' 5 target sequence B 5'-GAAACTACC TCTGGCTTAAtt -3 ' 6 target sequence C 5'-GAAGTACACGTCCCAATCAtt -3 ' 7 Drp1 siRNA (Santa Cruz Biotechnology) 목표 시퀀스 5'-AACGCAGAGCAGCGGAAAGAGTT-3 ' 8 Vimentin siRNA (Santa Cruz Biotechnology) 목표 시퀀스 5'-ATGGAAGAGAACTTTGCCG-3 ' 9 Silencer Negative Control siRNA (Ambion)

Example  3. Cell organelle separation

The cells were washed with PBS (Ca ^{2 +} and Mg ^{2 +} free) for isolation of cells in the MEF or U2OS cells, and then placed in isolation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EGTA and 250 mM sucrose) After lysis, the nucleus and the remaining cells were removed with a centrifuge at 800 g for 10 minutes, and only the supernatant was separated and separated at a rate of 8000 g. The resulting precipitate corresponds to the crude mitochondrial fraction, and the precipitate obtained by separating the supernatant at a rate of 100,000 g corresponds to the LM fraction. The crude mitochondrial fraction was placed in a 30% Percoll density gradient in isolation buffer (IB) and centrifuged at 95,000 g for 30 min. The upper bands of the two bands corresponded to mitochondria associated ER membrane (MAM) fraction and mitochondrial fraction .

Example  4. Immunoprecipitation and Western Blat

For immunoprecipitation experiments, the plasmid DNA was transfected 24 hrs before and placed in whole-cell extract buffer (10 mM HEPES, pH 7.9; 400 mM NaCl; 0.1 mM EDTA; 5 mM glycerol; 1 mM DTT and protease inhibitors) Cells were lysed. Cell lysates were treated with TEG reaction buffer (20 mM Tris-HCl at pH 7.4, 1 mM EDTA, 10% glycerol, 1 mM DTT, and 150 μg / (Sigma) or Protein A agarose (Upstate Biotech, Lake Placid, NY) for 3 hours or overnight. After washing with TEG reaction buffer containing 0.1% Triton-X 100, proteins were separated from the beads and analyzed by Western blotting.

For western blotting, the cells were lysed with whole-cell extract buffer or homogenizer, followed by size separation by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred to a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ) .

The primary antibodies used were CTRP1 (R & D systems), Drp1 (BD bioscience), Bip, calreticulin, calnexin, kinectin1, prohibitin, ACSL4 Antibodies against Cruz Biotechnology, Tubulin, Vimentin, Flag (Sigma), Myc and COX4 (Abcam) were used as the secondary antibodies, anti-rabbit IgG conjugated with fluorescein, - mouse IgG, anti-goat IgG (invitrogen) was used. Anti-rabbit, anti-mouse and anti-goat antibodies were used in combination with horseradish peroxidase (Zymed Laboratories) as the secondary antibody of Western blot. Protein and antibody complexes were identified using the ECL Plus system (Amersham Biosciences).

Example  5. Immunostaining

Cells were fixed with PBS (4% formaldehyde) for 20 min at room temperature, washed with PBS and permeabilized with PBS (0.1% Triton X-100) for 15 min. The primary antibody was treated for 1 to 3 hours, washed with PBS, treated with secondary antibody for 1 hour, and mounted using Vectashield (VECTOR).

Example  6. Real-time observation microscope Confocal  microscope

Fluorescently labeled signals were observed in living cells using a spinning disc confocal system (A1C; Nikon) with a 60x NA 1.4 oil objective lens using a 5% CO ₂ by LCI chamber (cham- lide TC, LCI) at 37 ° C. Immobilized cells were observed with a 63x oil objective (NA 1.4) lens using a laser scanning confocal microscope (LSM780; Carl Zeiss). Images were acquired using Photoshop (Adobe), IMARIS (Bitplane AG), or ImageJ (National Institutes of Health) Program.

Experiment result

CTRP1 knockout mouse was constructed to study CTRP1 function and it was confirmed that the absence of CTRP1 gene did not result in normal early embryogenesis. This shows that CTRP1 can have important functions at the cellular level.

Specifically, in the present invention, the position of CTRP1 in the cell was examined first. As a result, CTRP1 was partially located in mitochondria and ER (FIG. 1). The cells treated with CTRP1 harboring 5 nano and 10 nano gold particles were confirmed by electron microscopy to be present between the endoplasmic reticulum and the mitochondria (FIG. 2). Cell organelle separation experiments revealed that CTRP1 is present in the cytoplasm and the endoplasmic reticulum (Fig. 3). Trypsin treatment of the isolated endoplasmic reticulum revealed that CTRP1 was an endoplasmic reticulum membrane protein (FIGS. 4 and 5). This suggests that CTRP1 is an endoplasmic reticulum membrane protein present between the endoplasmic reticulum and the mitochondria.

CTRP1 conditional knockout mouse CTRP1 conditonal knockout mouse embryonic fibroblasts (MEFs) were constructed to characterize intracellular CTRP1 cells. When CTRP1 conditional MEF cells were treated with Cre lentivirus to inhibit CTRP1 expression or to treat CTRP1 expression by treating CTRP1 siRNA with U2OS cells and to examine changes in endoplasmic reticulum and mitochondrial morphology, morphology of mitochondria was observed (Figs. 6 and 10) . Instead, the shape of the endoplasmic reticulum and Golgi body was unchanged. It was confirmed that the average length of mitochondria was significantly increased compared with the control group (Figs. 7 and 11). This shows that CTRP1 can be involved in mitochondrial cleavage. Drp1 is an important protein for mitochondrial cleavage. Problems with the expression of Drp1 protein and the location of mitochondria cause problems with mitochondrial cleavage control, resulting in long mitochondria. The location of MFF involved in the localization of Drp1 and Drp1 in CTRP1 expression-inhibited cells was observed, but it was confirmed that there was no difference from the control group (FIGS. 8, 9, 10 and 12). These results suggest that CTRP1 may be involved in mitochondrial cleavage independently of Drp1-mediated mitochondrial cleavage.

The CTRP1 binding factor was examined to analyze the expression pattern of mitochondrial prolongation in CTRP1 expression-inhibited cells. The protein bound to CTRP1 with Flag was separated into acrylamide gel, silver stained and analyzed with megamass to show that microtubule and vimentin bind to CTRP1 (Fig. 13). Immunoprecipitation experiments demonstrated that CTRP1 and vimentin bind (Figure 14). Vimentin is a type III intermediate filament that has been reported to be involved in mitochondrial mobility and morphology dynamics. Long-term mitochondria such as CTRP1 can be observed in cells inhibiting vimentin expression, and similarly long mitochondria were observed when okadaic acid and calyculin, which inhibit vimentin synthesis, were treated (Fig. 16, 17). It was also confirmed that the mitochondrial position of Drp1 did not change as in CTRP1 (Fig. 18).

In order to investigate the interaction between CTRP1 and vimentin in mitochondrial dynamics, CTRP1 and Vimentin, in which GFP (green fluorescence), RFP (red fluorescence) or BFP (blue fluorescence) We observed the role of CTRP1 and visentin. CTRP1 is located at the site of mitochondrial cleavage, and visemen encapsulates where mitochondrial cleavage occurs when mitochondrial cleavage occurs (FIGS. 19 and 20). Vimentin is involved in mitochondrial fluidity, and mitochondrial fluidity increases when inhibiting vimentin expression, and CTRP1 also increases mitochondrial fluidity (FIG. 21). This indicates that CTRP1 and vimentin can play a role in fixing mitochondria. It can be seen that mitochondria in which cleavage occurs compared to mitochondria in which cleavage does not occur has a somewhat reduced fluidity (Fig. 22). Vimentin is involved in the mitochondrial cleavage process once again, through the phenomenon surrounding mitochondrial cleavage at the time of mitochondrial cleavage (Fig. 23). Recent reports have reported that IFN2 protein in the endoplasmic reticulum regulates mitochondrial cleavage by participating in the location of Drp1 when mitochondrial cleavage occurs through actin synthesis. So far, the mitochondrial cleavage process has been known to involve Drp1 protein, but the process is not yet known in detail, and many processes are expected to exist. Therefore, it has been demonstrated that there is a process in which mitochondria cleavage is regulated by the action of CTRP1 and vimentin in addition to mitochondrial cleavage by actin synthesis by Drp1 and IFN2.

Vimentin interacting with CTRP1 has been implicated in a variety of diseases, particularly Alzheimer's disease and Huntington's Disse, which are related to mitochondrial fluidity. This suggests that CTRP1 may be involved in or interacting with vimentin or may be important for neurological diseases.

<110> Ewha University - Industry Collaboration Foundation <120> Composition for treating mitochondrial dysfunction diseases          comprising CTRP1 inhibitor <130> DPP20140104KR <160> 9 <170> Kopatentin 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> loxP site primer <400> 1 actgatggcg agctcagacc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Genomic primer <400> 2 gcacacctgt ataccagaca 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Genomic primer <400> 3 gcacacctgt ataccagaca 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Genomic primer <400> 4 gagggagaga agaaagccta 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> target sequence A of CTRP1 siRNA <400> 5 cagttgagac tctgcttaat t 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> target sequence B of CTRP1 siRNA <400> 6 gaaactacct ctggcttaat t 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> target sequence C of CTRP1 siRNA <400> 7 gaagtacacg tcccaatcat t 21 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> target sequence of Drp1 siRNA <400> 8 aacgcagagc agcggaaaga gtt 23 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> target sequence of Vimentin siRNA <400> 9 atggaagaga actttgccg 19

Claims (11)

delete delete delete Treating the candidate compound expected to inhibit the interaction of CTRP1 and visentin with CTRP1 and vimentin;
Confirming whether the interaction of CTRP1 and visentin is inhibited; And
And determining that the candidate compound inhibits mitochondrial cleavage or cleavage if it interferes with the interaction.
A method for screening for a mitotic or cleavage inhibitor of mitochondria.
delete 5. The method of claim 4, wherein the interaction of CTRP1 and visemen is detected using an antibody specific for CTRP1 or vimentin.
7. The method of claim 6, wherein the interaction of CTRP1 and visemen is detected by immuno-staining, immunoprecipitation, or immunostaining.
5. The method of claim 4, wherein the CTRP1 and the non-human are each a reporter protein.
9. The method of claim 8, wherein the interaction of CTRP1 and visentin is detected by the signal magnitude of the reporter protein.
CTRP1 and visentin,
Treating the candidate compound expected to inhibit the interaction of CTRP1 and visemen with CTRP1 and visentin and determining whether the interaction of CTRP1 and visemain is inhibited and then determining whether the candidate compound inhibits the interaction Lt; RTI ID = 0.0 &gt; mitochondria &lt; / RTI &gt;
A composition for screening for mitochondrial cleavage or cleavage inhibitors. delete

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