KR100788493B1 - Method for the screening factors affecting the formation of the death-inducing signaling complex in a test tube - Google Patents

Abstract

A method for forming a death-inducing signaling complex(DISC) is provided to reproduce the formation of the DISC in a test tube by producing a Fas-DD(Fas death domain) protein, which is difficult to be produced. And a method for searching materials controlling the formation of the DISC is provided to be utilized for developing anti-cancer agents and therapeutic agents of degenerative nerve diseases. A water-soluble Fas-DD protein is described as SEQ ID : NO. 2. To form a DISC, the Fas-DD protein, a refolded procaspase-8 obtained by removing contaminated protein from procaspase-8(gene: NM_001228) through centrifugation, dialyzing it with a protein solution and acetic acid and then refolding it in a buffer solution consisting of 50-150 mM hepes and 1-10 mM DTT dithiothreitol, and GB1-FADD(B1 Ig binding domain of protein G-FADD) fused protein are mixed in a test tube. A method for searching a DISC forming control material comprises a step of adding a material to be searched into the test tube containing the constituents of the DISC to identify the existence of the procaspase-8 activity.

Description

Method for the screening factors affecting the formation of the death-inducing signaling complex in a test tube

Figure 1 shows the production process and cleavage map of the recombinant vector pETc8 for procaspase-8 expression.

Figure 2 shows the production process and cleavage map of the recombinant vector pETddm for the expression of soluble Pars-DD (Fas death domain) protein.

Figure 3 shows the production process and cleavage map of the recombinant vector pETgfadd for GB1-FADD fusion protein expression.

Figure 4 shows that the procaspase-8 is activated by forming a DISC in vitro. Activated caspase-8 cleaved the fluorescent substrate and measured fluorescence. It can be seen that activated caspase-8 increases when all of the DISC constituent proteins (Fas death domain, FADD, procaspase-8) are added (○) as compared to when only procaspase-8 (●) is present. When the Pars-DD lpr mutant protein was added instead of the Pars-DD (▲), the same level of activation was shown as when only procaspase-8 and FADD were present without the pars-DD (□). The addition of pars-DD and procaspase-8 was the same as the presence of only procaspase-8. This suggests that procaspase-8 forms DISC together with pars-DD and FADD in vitro, and that inhibition of procaspase-8 by DISC formation is inhibited when interaction is inhibited.

5 shows the results of exploring the regulation of DISC formation by compounds. Compounds KYS00595 (▲) and KSW30193 (●) inhibited activity as compared to the absence of compound (○) at 100 μM concentration. In particular, KSW30193 was found to have almost no activity.

6 is to investigate the degree of inhibition of DISC formation according to the concentration of KSW30193. KSW30193 was found to inhibit about 50% of the activation of procaspase-8 by DISC formation at about 1 nM.

Figure 7 examined whether the compound or compound dissolved DMSO directly affects the caspase-8 activity. It can be seen that there is no effect on the commercially available caspase-8 activity (1 M) at a water concentration of 100 μM (▲) and DMSO 2% (●).

The present invention relates to a method for screening apoptosis complex-forming regulators in vitro, and more particularly, to produce an activatable procaspase-8 and to produce apoptosis protein, water-soluble PAS-DD (Fas death domain). Formation of a death-inducing signaling complex (DISC) in vitro by mixing a protein with a Fas-associated via death domain (FADD), and apoptosis by confirming the activation of procaspase-8 by DISC formation Related biochemical studies, as well as the discovery of DISC formation regulators that can be used to develop disease treatments.

Cells kill themselves by growing, damaging, aging, or harmful cells. This is called apoptosis. Such apoptosis is associated with various diseases and is known to occur by various external stimuli such as ultraviolet rays and viruses. Apoptosis is largely divided into ligand-induced (extrinsic pathway) and intracellular signaling (intrinsic pathway). These two pathways occur independently or in conjunction with each other. Among these, Fas-mediated apoptosis is a representative pathway of apoptosis induced by ligand. In the case of apoptosis induced by the pars ligand, the pars and FADD bind, thereby binding and activating FADD to procaspase-8, a protease [Muzio et al. (1996) Cell 85, 817-827; Yang et al (1998) Mol. Cell 1, 319-325. Other ligands, such as TNF-α and TRAIL, also bind to the TNF receptor, TRAIL receptor, and the like, thereby inducing apoptosis by activating procaspase-8 by attracting FADD. In this case, FADD has two domains, which bind to DD of various receptors by using a death domain (DD), and procaspase-8 by using a death effector domain (DED). It is called a death-inducing signaling complex. However, apoptosis induced by TNF-α binds to a TNF receptor, a protein called TRADD (TNFR1-associated death domain protein), which in turn binds to FADD to activate procaspase-8 to induce apoptosis. Known. CFLIP, vFLIP, etc. are present as proteins that inhibit DISC formation, and ALPS (Eberstadt at al (1997) Nat Struc Biol 4, 983-985) is expressed by inhibition of DISC formation due to mutations in proteins involved in DISC formation. Is known as a disease [Fadeel & Orrenius (2005) J Intern Med, 258, 479-517]. So far, there have been results showing that procaspase-8 is activated with Na-citrate by producing procaspase-8 without DED [Boatright et al (2004) Biochem J, 382,651-657], but without DED Because it is a protein, it cannot interact with FADD and cannot reproduce DISC formation.

In the present invention, it was possible to reproduce the formation of DISC in vitro by producing procaspase-8 including DED and producing Fas-DD, which had been difficult to produce. This invention can be applied to the development of anti-cancer drugs, neurodegenerative diseases and the like by analyzing the effects on the DISC formation of various disease-related proteins, as well as the substances that control the formation of DISC.

Accordingly, the present inventors have made efforts to solve the above problems, and as a result, the production of procaspase-8 including DED, and also produced a water-soluble Fas-DD protein that was difficult to produce a lot of DISC in vitro The present invention was made possible by developing a method for searching for substances that control DISC formation, as well as analyzing the effects on DISC formation of various disease-related proteins.

Accordingly, an object of the present invention is to produce procaspase-8, a water-soluble Fas-DD protein including DED, and to reproduce DISC using the same, thereby applying it to research on the mechanism of apoptosis and to develop an effective therapeutic agent.

In particular, many attempts to develop therapeutic agents to date have been made by activating enzymes and inhibiting activities essential to cell activity. In the present invention, only the activity of caspase-8, which is involved in cell death, is inhibited. It is intended to be applied to the development of therapeutics.

The present invention is characterized by a method for refolding procaspase-8 and a method for forming apoptosis complex (DISC) in vitro due to the production of water-soluble Pas-DD (DD) protein.

In addition, the present invention is a mixture of procaspase-8, a water-soluble pars-DD protein and GB1-FADD (B1 Ig binding domain of protein G-FADD) fusion protein to form apoptosis complex (DISC) in vitro and mixed It is another feature of the method to search for apoptosis complex (DISC) formation regulator by adding a substance to be searched to check the activity of procaspase-8.

The present invention will be described in more detail as follows.

The present invention introduces a human procaspase-8 gene [NM_001228] into the NdeI and BamHI restriction sites of the pET15b [Novagen, USA] protein expression vector to produce an activatable procaspase-8 as an inclusion body. Expression vector pETc8 was prepared. Procaspase-8 is produced from the vector and then refolded to produce procaspase-8 which is activated.

The present inventors first developed a method for refolding procaspase-8, and thus, it was possible to produce procaspase-8 that was activated.

That is, after removing the contaminated protein by centrifugation of the procaspase-8 produced from the procaspase-8 expression vector, and then dialyzed with protein solution and acetic acid, the protein solution mixed with distilled water is 50 ~ 150 mM Hepes ( Hepatic) and 1-10 mM dithiothreitol (DTT) buffer can be refolded at pH 7.0-8.5 to obtain activated procaspase-8 having a purity of 95% or more. At this time, if the composition of the buffer solution is out of the above range there is a problem that the yield of the refolding is low, which is not preferable.

In addition, the present invention produces a water-soluble pars-DD protein through the mutation of pars-DD, wherein the human-derived pars-DD gene (amino acid 202-309) (numbering except for a signal peptide) [NM_000043] Induced mutation of amino acid sequence 245th glutamic acid and 247th lysine residue, and expression of the induced gene ( Fas-DDdm ) [SEQ ID NO: 1] by BamHI and XhoI restriction enzyme site of pET28a [Novagen, USA] protein expression vector Vector pETddm was prepared.

The water-soluble pars-DD protein represented by SEQ ID NO: 2 was produced from the vector, and the procaspase-8 and the produced FADD were mixed to confirm DISC formation in vitro. This was shown by measuring caspase-8 activity that procaspase-8 is activated. However, it was shown that DISC formation was reproduced in vitro by observing the inhibition of procaspase-8 activation when Fas-DDdm induced mutations known to inhibit DISC formation and inhibit cell death. Reproduction of DISC formation shows that the mechanism of ligand-mediated apoptosis, which plays an important role in apoptosis, can be used to search for various compounds that regulate it.

In addition, the present invention is a mixture of procaspase-8, a water-soluble pars-DD protein and GB1-FADD (B1 Ig binding domain of protein G-FADD) fusion protein to form apoptosis complex (DISC) in vitro and mixed It is another feature of the method to search for apoptosis complex (DISC) formation regulator by adding a substance to be searched to check the activity of procaspase-8.

The formation of apoptosis complex (DISC) is activated and the caspase-8 is activated as caspase-8. The effect of caspase on the presence or absence of the substance to be examined is measured to investigate the effect on the formation of DISC. That is, when the caspase activity is increased by addition, it is a substance for inducing DISC formation, and when the activity is inhibited, it is a substance for inhibiting DISC formation.

Therefore, the present invention was confirmed that the production of procaspase-8, apoptosis complex formed upon apoptosis through FADD can be activated in vitro by reproducing the cell death complex. Until now, the development of many therapeutic agents centered on activated enzymes such as caspase-8 and inhibited the activity of enzymes essential for intracellular activity. The ability to search for substances that modulate formation has the effect of being applicable to the development of therapeutic agents for diseases known to occur due to abnormalities in these signals. In addition, it provides an effective method for studying the mechanism and regulation of apoptosis signaling, which is still controversial.

Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited to these Examples.

Example 1 Preparation of Procaspase-8

(1) Preparation of protein expression vector

To mass express procaspase-8 (amino acids 1-479) in Escherichia coli, a human procaspase-8 gene [NM_001228] was introduced into the NdeI and BamHI restriction sites of the pET15b [Novagen, USA] protein expression vector. Expression vector pETc8 was prepared (see FIG. 1).

(2) production of protein

0.7)]에 이를 때까지 기른 후 0.5 mM의 이소프로필 β-D-티오갈락토피라노시드(isopropyl β-D-thiogalactopyranoside)를 첨가하였고, 이후 37 ℃에서 3시간 동안 길러 단백질을 대량 발현시켜 생산하였다.In order to express the protein of interest using the protein expression vector pETc8 completed in the above example, these expression vectors were transformed into E. coli BL21 (DE3). The transformed Escherichia coli BL21 (DE3) was logarithmic phase (OD600) in LB broth to which 100 μg / ml Ampicillin was added at 37 ° C.

0.7)], and then 0.5 mM isopropyl β-D-thiogalactopyranoside was added, followed by mass expression of the protein for 3 hours at 37 ° C. It was.

(3) refolding and separation of proteins

E. coli cells produced using 1 liter of medium were collected by centrifugation, dissolved in 40 ml of buffer (10 mM Tris, 1 mM EDTA, pH 8.0), and the cells were crushed. Then, the cells were crushed at 2,520 rpm at 4 ° C. The supernatant was discarded by centrifugation for 30 minutes. The contaminants were re-dissolved in the buffer and precipitated contaminated protein three times by centrifugation at 2,560 rpm for 30 minutes. The precipitate was dissolved in a buffer containing 6M guanidine HCl (100 mM Tris, 5 mM DTT, pH 8.0), dissolved in ice for 1 hour, and the undissolved precipitate was removed by centrifugation at 14000 rpm for 30 minutes. The recovered protein solution was mixed with the same volume of glacial acetic acid, dialyzed for about 12 hours using 2 liters of 50% acetic acid, and then mixed with 10 times of secondary distilled water. The resulting protein solution was mixed with twice the refolding buffer (100 mM Hepes, 5 mM DTT, pH 8.0) and then refolded by adjusting the pH to 7.3. The refolded protein was concentrated using ultrafiltration, and the purity was confirmed by electrophoresis to produce procaspase-8 having a purity of 95% or more.

Example 2: Production of Pars-DD and FADD

(1) Production of Water Soluble Pars-DD Protein

Fas is a membrane receptor and when combined with the Fas ligand, Pas-DD in the cytoplasmic C-terminus binds to FADD, and FADD binds to procaspase-8 to form a so-called DISC, which activates apoptosis signals. do. Many researchers have tried to elucidate the structural biological mechanisms of DISC formation, but have failed because they cannot produce pars-DD as water-soluble. In order to solve this problem, the present invention induced the mutation of amino acid sequence 245th glutamic acid and 247th lysine residue of human-derived pars-DD gene (amino acid 202-309) [NM_000043]. The expression vector pETddm was prepared by transferring the gene ( Fas-DDdm ) from which the two amino acid residues were substituted with alanine (E245A, K247A) to the BamHI and XhoI restriction sites of the pET28a [Novagen, USA] protein expression vector. [See FIG. 2]. The expression vector pETddm was expressed as in Example 1, and the produced protein Fas-DDdm was separated using a nickel-nitrilotriacetic acid-agarose (Ni-NTA) affinity column [Qiagen, USA], and thrombin. By removing histidine-tags, it was possible to produce Pas-DDdm (Fas death domain double mutant), a water-soluble protein with a solubility of over 50 mg / ml with a final 95% purity. The gene was observed to be apoptosis due to mass expression during transfection of Hela cells similar to wild type, indicating no change in function. In addition, the protein was produced in a similar manner by further inducing V238N mutations known to inhibit apoptosis as mutations observed in lymphatic hyperplasia (lymphoproliferative syndrome).

(2) production of FADD

FADD has DD and DED, so it binds with Pars-DD through DD, and procaspase-8 is a mediator protein that signals apoptosis by binding through DED. In the present invention, FADD was produced by fusing the protein G1 (B1 Ig binding domain of protein G-FADD) to the N-terminus of FADD. The expression vector pETgfadd was prepared by introducing the FADD gene [NM_003824] into the BamHI and XhoI restriction enzyme sites of the pET-30aGB1 protein expression vector (see FIG. 3). The expression vector pETgfadd was expressed in GB1-FADD protein as in Example 1, and the produced protein was separated using a nickel nitrilotriacetic acid-agarose (Ni-NTA) affinity column [Qiagen, USA].

Experimental Example 1: Activation of Procaspase-8 by DISC Reproduction in Vitro

DISC generally refers to a complex of Pas, FADD, Procaspase-8, which, when formed, cleaves Procaspase-8 and activates Caspase-8. In the present invention, it was investigated whether procaspase-8 is activated by caspase-8 by producing the cytoplasmic site (Fas-DD) of pars and mixing it with a constituent protein forming a DISC complex. Caspase-8 activity was determined in 90 μl of reaction buffer (20 mM HEPES, pH 7.4, 2 mM EDTA, 0.1% CHAPS, 5) containing 1 μM Pars-DDdm, 5 μM GB1-FADD, 3 μM procaspase-8. mM DTT, 5% sucrose) After 10 minutes at 25 ° C., 10 μl of 2 mM Ac-IETD-AFC (N-acetyl-Ile-Glu-Thr-Asp-AFC (7-amino-4-trifluoromethyl coumarin) )) Was added to track the reaction. Activity was quantified by measuring the fluorescence at 535 nm (excitation 405 nm) at 25 ° C using an fMAX spectrofluorometer, cleaved from the substrate by Caspase-8.

As shown in FIG. 4, it can be seen that activated Caspase-8 increases significantly with time when all the DISC components of PAS-DDdm, GB1-FADD, and procaspase-8 (○) are present. However, when only caspase-8 was present (●), activated caspase-8 could not be observed under experimental conditions, and when activating GB1-FADD to procaspase-8 (□), slight activation was observed. Could be observed. This is thought to be activated by the effect of increasing the concentration of procaspase-8 by binding GB1-FADD and procaspase-8 through DED. On the other hand, when water-soluble pars-DDdm, which is known to have no interaction, was added to procaspase-8 (◆), it showed little activation as in the presence of only procaspase-8. However, when pars-DDdm (V238A), the lpr mutant protein of pars-DD (▲), is mixed with GB1-FADD, procaspase-8, the activation of caspase-8 is similar to when no water-soluble pars-DDdm is present. It can be seen that less. This shows that mutations in Pars-DD can't effectively activate procaspase-8 without binding to FADD, which is an in vitro experiment showing the abnormal cell death found in lpr mutations. This is because basic DISCs are formed in vitro, activating procaspase-8 to caspase-8, and if caspag ligand induces oligomerization of pars, it is more effective to induce binding between basic DISCs. It is thought that -8 will be activated.

Experimental Example 2: Screening for Compounds That Inhibit DISC Formation

When the compound to be searched was added to the conditions shown in Experimental Example 1, it was observed that the compound interferes with the formation of DISC, and by using this, it is possible to search for a compound that affects DISC formation, that is, cell death. . In order to search for a compound for inhibiting DISC formation, 2 μl (final concentration 100 μM) of the compound to be searched was added to the reaction solution, and activity inhibition was examined as shown in FIG. 5. 5 shows examples of KYS00595 and KSW30193 among compounds on the KIST compound database as a compound to be searched. KSW30193 (●) inhibited the activation of caspase-8 by DISC formation compared to the control group (○), but only 30% of KYS00595 (▲). As a result of investigating the degree of inhibition according to the concentration using the compound of KSW30193, as shown in Figure 6 it can be seen that KSW30193 has an IC ₅₀ value of about 1 nM. FIG. 7 shows a control (○) experiment on commercially available caspase-8 (1 μM) to investigate whether compound KSW30193 or DMSO (dimethyl sulfoxide) in which the compound is dissolved directly affects the activity of caspase-8. The effect was investigated by adding compound KSW30193 (100 μM, ▲) and DMSO (●) up to 2%. As a result, the compound and DMSO showed no direct effect on the activity of caspase-8, and it was found that compound KSW30193 is a compound that affects DISC formation. These compounds are thought to be able to be developed as a therapeutic agent for degenerative neuropathy and hepatitis as a compound that inhibits DISC formation.

As described above, the present invention has been shown to be able to activate procaspase-8 by producing procaspase-8 and reproducing in vitro apoptosis complexes formed upon apoptosis through FADD. Until now, the development of many therapeutic agents centered on activated enzymes such as caspase-8 and inhibited the activity of enzymes essential for intracellular activity. The ability to search for substances that modulate formation has the effect of being applicable to the development of therapeutic agents for diseases known to occur due to abnormalities in these signals. In addition, it provides an effective method for studying the mechanism and regulation of apoptosis signaling, which is still controversial.

Attach sequence list electronic file  

Claims (5)

delete A water soluble FAS death domain protein represented by SEQ ID NO: 2. A water soluble pars-DD protein of claim 2; Centrifugation of procaspase-8 [gene: NM_001228] to remove contaminated protein, followed by dialysis with protein solution and acetic acid, and then the protein solution mixed with distilled water was mixed with 50 to 150 mM Hepes and 1 to 10 mM. Refolded procaspase-8 obtained by refolding a buffer consisting of dithiothreitol (DTT) at pH 7.0-8.5; And A method of forming an apoptosis complex (DISC) by mixing a GB1-FADD (B1 Ig binding domain of protein G-FADD) fusion protein in vitro. A component for forming apoptosis complex (DISC) A water soluble pars-DD protein of claim 2; Centrifugation of procaspase-8 [gene: NM_001228] to remove contaminated protein, followed by dialysis with protein solution and acetic acid, and then the protein solution mixed with distilled water was mixed with 50 to 150 mM Hepes and 1 to 10 mM. Refolded procaspase-8 obtained by refolding a buffer consisting of dithiothreitol (DTT) at pH 7.0-8.5; And GB1-FADD (B1 Ig binding domain of protein G-FADD) fusion protein; Method of detecting apoptosis complex (DISC) formation regulator characterized in that the mixing in vitro and adding a substance to be explored therein to check the activity of procaspase-8. 5. The method according to claim 4, wherein apoptosis complex (DISC) is formed to activate procaspase-8 as caspase-8, which is an inducer of DISC formation if the caspase activity is increased due to the addition of a substance to be explored. If this is inhibited, the discovery method characterized in that the DISC formation inhibitor.

Images