KR20090119482A - Novel gene encoding programmed cell death inducing protein - Google Patents

Abstract

PURPOSE: A novel protein which induces apoptosis is provided to specifically induce apoptosis in a cell in which Mcl-1L(myeloid cell leukemia-1L) is overexpressed. CONSTITUTION: A composition for apoptosis contains a gene which encodes functionally same polypeptide with polypeptide coded by sequence number 1 or 2. An anticancer composition contains an amino acid sequence of the sequence number 3 which specifically induces apoptosis and functionally same polypeptide with a polypeptide with amino acid sequence of the sequence number 4.

Description

Novel gene encoding programmed cell death inducing protein

The present invention relates to novel proteins that induce cell death.

More specifically, the present invention provides a gene encoding a polypeptide functionally identical to the polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1 or 2 and an apoptosis inducing protein encoded by the gene.

The protein according to the present invention induces apoptosis, and also strongly induces apoptosis specifically in cells overexpressing Mcl-1L (myeloid cell leukemia-1 long).

    Programmed cell death is an important mechanism for cell replacement, tissue remodeling and removal of injured cells (Thomson, C. B., 1995, Science 267, 1456-1462).

The Bcl-2 family protein plays a central role in this apoptosis, and this bcl-2 gene is the breakpoint of the at (14, 18) chromosomal translocation associated with follicular B-cell lymphoma. As an early oncogene (Tsujimoto, Y. et al., 1984, Science 226, 1097-1099).

Overexpression of Bcl-2 protein inhibits apoptosis induced by various causes in vivo and in vitro experiments.

The Mcl-1 protein is an anti-apoptotic Bcl-2 family protein and was initially found as an early inducer during the differentiation of human myeloid leukemia cell lines (Kozopas, KM et al., 1993, Proc. Natl. Acad. Sci. USA 90, 3516-3520). The Mcl-1 protein has a common Bcl-2 homology first domain (BH 1), BH2, BH3 and a C-terminal transmembrane domain (TM) found in other Bcl-2 related proteins, but specifically the N-terminal Proline, glutamic acid, serine, and threonine residues have many PEST sequences.

In addition, mycloid cell leukemia-1 short (Mcl-1S) is a splicing variant of Mcl-1L, which lacks the C-terminal BH1, BH2 and TM domains and performs the opposite function of apoptosis with Mcl-1L. Bae, J. et al., 2000, J. Biol. Chem. 275, 25255-26261).

Misregulation of apoptosis can lead to several diseases, including cancer, autoimmune diseases, neurodegenerative disorders, and viral infections (Thompson CB, Science 1995; 267: 1456). -1462). In particular, dysregulation of Mcl-1 induces a variety of cancers by inducing cell immortalization and cancer cell conversion (tumorigenic conversion), and is known to be closely related to the induction of resistance to anticancer drugs (Warr MR, Shore GC.Curr Mol Med. 2008 Mar; 8 (2): 138-47), and has been shown to be associated with HIV infection and treatment (Grelli S et al., Ann NY Acad Sci. 2006 Dec; 1090: 130-7) It has also been reported to be associated with Rheumatoid arthritis (Liu H et al, J Immunol. 2005 Dec 15; 175 (12): 8337-45).

In addition, recently, gene therapy using apoptosis and anti-apoptosis genes directly or indirectly involved in cancer cell induction and cancer cell maintenance has been studied. However, anticancer drugs used in conventional gene therapy have difficulty in treating cancer cells as well as normal cells by inducing apoptosis.

Recently, the development of an anticancer agent that can effectively distinguish cancer cells from normal cells has been promoted under the concept of “targeted cancer gene therapy.” For example, anti-cell death proteins overexpressed in cancer cells In the case of a protein that specifically reacts to induce apoptosis, developing a gene encoding the anticancer agent has an advantage of obtaining cancer treatment effect without invading normal cells.

That is, the specific apoptosis gene may be a chemical sample for preparing apoptosis inducers commonly used in the field of anticancer drugs, and may be a cancer cell specific apoptosis inducing agent, that is, an anticancer agent, rather than a conventional indiscriminate apoptosis inducing agent. It can provide a chemical framework for manufacturing.

It is an object of the present invention to provide a novel apoptosis inducing protein capable of inducing apoptosis and also inducing cell-specific apoptosis and a gene encoding the same.

The present invention is functionally functional with a polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1 or 2, which induces apoptosis and also strongly induces apoptosis in cells overexpressing Mcl-1L (myeloid cell leukemia-1 long). Genes encoding the same polypeptides are provided.

The present invention also provides an apoptosis inducing protein functionally identical to a polypeptide having an amino acid sequence of SEQ ID NOs: 3 and 4, which induces apoptosis and strongly induces apoptosis in cells where Mcl-1L is overexpressed. do.

The present invention also provides a cell death composition comprising a gene encoding a polypeptide functionally identical to a polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1 or 2, a vector comprising the gene or a microorganism comprising the vector. to provide.

The present invention also provides a composition for apoptosis containing a polypeptide functionally identical to a polypeptide having an amino acid sequence of SEQ ID NOs: 3 and 4, a vector expressing the polypeptide or a microorganism comprising the vector.

In addition, the present invention encodes a polypeptide functionally identical to the polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1 or 2, which induces apoptosis and strongly induces apoptosis specifically in cells overexpressed with Mcl-1L. Provided are an anticancer composition comprising a gene or a protein encoded by the gene.

The protein encoded by the gene according to the present invention induces cell death upon overexpression. Therefore, the cell death composition comprising a protein or a gene encoding the same according to the present invention can be used as a therapeutic agent for diseases related to cell death.

In addition, the protein encoded by the gene according to the present invention binds to Mcl-1L having an anti-apoptotic function and strongly induces apoptosis specifically in cells overexpressing Mcl-1L.

Therefore, the composition comprising a protein or a gene encoding the same according to the present invention can be used as a biomarker of apoptosis or Mcl-1L, and the anticancer composition comprising the protein or a gene encoding the same according to the present invention is normal cells It can effectively treat cancer by inducing apoptosis only in cancer cells overexpressing Mcl-1L without affecting.

The present invention is directed to a gene encoding a polypeptide functionally identical to a polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1 or 2, which induces apoptosis and strongly induces apoptosis in a cell in which Mcl-1L is overexpressed. to provide.

In the present invention, Mcl-1L minor transcripts in human embryonic stem cell lines through conventional PCR methods using primers of Mcl-1L, which are generally thought to help prevent cell death and help cell division in eukaryotic cells. The band was confirmed and its nucleotide sequence was determined to confirm that it is a novel variant of Mcl-1L and provides it as a gene according to the present invention.

The novel gene according to the present invention comprises the nucleotide sequence of SEQ ID NO: 1, as shown in Figure 2, the PEST domain is removed from Mcl-1L and has three specific mutant residues. In the present invention, it is named Mcl-1ES.

In addition, the novel gene according to the present invention includes the nucleotide sequence of SEQ ID NO: 2, which only removes the PEST domain from Mcl-1L, as shown in FIG.

Mcl-1ES according to the present invention is expressed in a variety of cell lines, it was also confirmed that the expression amount is slightly different for each cell line.

The function of Mcl-1ES was found to play a role in inducing apoptosis during intracellular overexpression (see FIG. 4). Therefore, Mcl-1ES and Mcl-1ESM according to the present invention can be used as a prophylactic and therapeutic agent for diseases or abnormalities associated with cell death.

In addition, in the present invention, as confirmed by immunoprecipitation, Mcl-1ES was found to bind with Mcl-1L (see FIG. 3), which was found to inhibit the anti-apoptotic action of Mcl-1L (see FIG. 4). ).

Mcl-1ES acts to induce apoptosis during intracellular overexpression as confirmed above, but apoptosis by Mcl-1ES is increased in Mcl-1L-expressing cells that prevent apoptosis (see FIG. 5). ). This provides the possibility of effectively treating cancer cells by inducing apoptosis specifically to these cancer cells in relation to overexpression of Mcl-1L, which is directly or indirectly involved in cancer cell maintenance and cancer cell induction. That is, using Mcl-1ES according to the present invention, it is possible to develop an anticancer agent that can effectively distinguish between specific cancer cells and normal cells in which Mcl-1L is overexpressed.

The present invention also provides a recombinant vector containing a gene encoding a polypeptide functionally identical to the polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1 or 2 and a microorganism transformed with the recombinant vector.

The present invention also provides an apoptosis inducing protein functionally identical to a polypeptide having an amino acid sequence of SEQ ID NO: 3 or 4 specifically inducing apoptosis in cells overexpressed Mcl-1L.

At this time, the apoptosis inducing protein according to the present invention can be used as a biomarker for detecting Mcl-1L because it specifically binds to Mcl-1L.

Further, a gene encoding an apoptosis inducing protein according to the present invention, a vector comprising the same and a microorganism transformed with the vector or an apoptosis inducing protein according to the present invention, a vector expressing the same and a microorganism transformed with the vector The composition comprising is available as an apoptosis agent and an anticancer agent that induces apoptosis specifically in cells overexpressing Mcl-1L.

Through the following examples will be described the present invention in more detail. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

< Example  1> Mcl -One ES clonning

1 x PCR reaction solution, 2.5 mM MgCl ₂ , 250 μM dNTP, 2.5 U recombinant Taq polymerase, 1 μM each in cDNA of human embryonic stem cell line CHA3 (CHA Stem Cell Institute, Pochon CHA University, Seoul, Korea) PCR was performed through primers (pcMcl-1L 5'-AGTGGATCCATGTTTGGCCTCAAAAGAAAC (SEQ ID NO: 5) and pcMcl-1L 3'-CTAGAATTCTTACAGTAAGGCTATCTT (SEQ ID NO: 6)) to obtain a PCR minor product of about 600 bp, and pcDNA3 (Invitrogen, Carlsbad, CA; Restriction enzymes BamHl and EcoRl of the multiple cloning site of Cat # A-150228) were cloned into the treated plasmid to obtain pc FlagMcl- 1 ES (see FIG. 6A).

In addition, the pcFLAGMcl-1ES plasmid DNA was treated with BamH1, Not1 restriction enzyme to extract DNA of about 600 bp and pCMV-Myc (Clontech, Mountain View, CA) was treated with Bgl2, Not1 restriction enzyme. DNA of approximately 3700 bp was extracted to ligation the two DNAs and subcloned the pCMV-Myc plasmid to obtain pCMV-Myc Mcl-1ES (see FIG. 6B).

After sequencing through the sanger method, the nucleotide sequences were compared and analyzed through Mcl-1L and blast search to identify new variants and named Mcl-1ES and Mcl-1ESM (SEQ ID NOs: 1 and 2, SEQ ID NO: 3 and 4 and FIG. 2)

< Example  2> Mcl -One ES Expression Study of

In order to confirm whether Mcl-1ES identified in Example 1 is expressed in cell lines other than human embryonic stem cells, CHA3 (CHA Stem Cell Institute, Pochon CHA University, Seoul 135-081, Korea), KM1214 (Korea Cell Line Bank) (KCLB) Cat # 80014), DU-145 (KCLB Cat # 30081), NCL-H596 (KCLB Cat # 90596), AGS (KCLB Cat # 21739), and A172 (KCLB Cat # 21620) in various cell lines cDNA was obtained and PCR was performed using the pcFlagMcl-1L 5 'primer and pcMcl-1L 3' primer described in Example 1, and the expression pattern is shown in FIG.

As a result, it was confirmed that the expression pattern of Mcl-1ES is expressed in various cells and there is a difference in the degree of expression. (See Fig. 1)

< Example  3> Mcl -One ES  of Binding protein

To confirm Mcl-1L and Mcl-1ES binding, a 293T cell line (ATCC; 293T Cat # CRL-11268 ™) was used. 3 × 10 ⁶ pcMcl cultured with being held the Welfect-EX Total 6㎍ pc FlagMcl -1 ES 293T obtained in Example 1 by using the transfection kit of -1L (Bae, J. et al. , (2000) J Biol.Chem. 275, 25255-25261), and protein was sampled using NP-40 lysis buffer (50 mM Tris-HCl at pH 8.0, 0.15 M NaCl, and 1% NP-40). Immunoprecipitation using binding of Protein-G-Agarose (Upstate, Charlottesville, VA) through antibody of anti-Mcl (Santa Cruz Biotechnology: sc-819) and anti-Flag M2 antibodies (Sigma: F1804) The binding of Mcl-1L and Mcl-1ES was confirmed (see FIG. 3).

< Example  4> MCl -One ES  Induction of Apoptosis

To confirm apoptosis-inducing function of Mcl-1ES, 293T cells were cultured at 1 × 10 ⁶ using Welfect-EX transfection kit and transfected with total 3㎍ pcFlagMcl- 1 ES alone or with pcMcl-1L . Observation of the death pattern was confirmed to induce apoptosis when Mcl-1ES alone overexpression (see FIG. 4).

In addition, when Mcl-1L is overexpressed together, it was confirmed that Mcl-1ES induced apoptosis more strongly than when overexpressed alone (see FIG. 4).

< Example  5> MCl -One ES  Apoptosis-inducing aspect ( viability )

In order to observe the change in apoptosis due to the binding of Mcl-1ES and MCl-1L confirmed in Example 3 was performed (viability) experiment.

Specifically, 293T cells were cultured in 3.5 X 10 ⁴ and total 100 ng of DNA (Mcl-1ES coding plasmid, Mcl-1L + 1ES coding plasmid, and Mcl-1ES All (Mcl-1ESM) coding plasmid, respectively) was dose-dependent. Transfection was performed using a Welfect-EX transfection kit, and transfection was performed with 10% GFP plasmid (CLONTECH). After 24 hours, only cells containing GFP were counted to determine cell death.

As a result, as shown in Figure 5, when Mcl-1ES is expressed with Mcl-1L, the degree of apoptosis was stronger.

1 is an electrophoretic picture showing that Mcl-1ES is expressed in various cells.

2 is a diagram showing the structure of Mcl-1ES and Mcl-1ESM compared to Mcl-1L.

3 is an enzyme immunoprecipitation (IP) photograph showing that the proteins of Mcl-1L and Mcl-1ES bind.

4 shows that Mcl-1ES induces apoptosis in 293T cells, and when Mcl-1L is expressed together, it shows a stronger induction of apoptosis.

5 is a viability graph showing that Mcl-1ES strongly induces apoptosis when expressed with Mcl-1L.

Figure 6a is a restriction map of pcFlagMcl-1ES in accordance with the present invention, Figure 6b is a restriction map of pcCMV-Myc Mcl-1ES in accordance with the present invention.

<110> College of Medicine Pochon CHA University Industry-Academic Cooperation Foundation <120> Novel gene encoding programmed cell death inducing protein <130> P08-106 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 594 <212> DNA <213> Homo sapiens <400> 1 atgtttggcc tcaaaagaaa cgcggtaatc ggactcaacc tctactgtgg gggggccggc 60 ttgggggccg gcagcggcgg cgccacccgc ccgggagggc gacttttggc caccggcgcc 120 aaggacacaa agccaatggg caggtctggg gccaccagca ggaaggcgct ggagacctta 180 cgacgggttg gggatggcgt gcagcgcaac cacgagacgg ccttccaagg catgcttcgg 240 agactggaca tcaaaaacga agacgatgtg aaatcgttgt ctcgagtgat gatccatgtt 300 ttcagcgacg gcgtaacaaa ctggggcagg attgtgactc tcatttcttt tggtgccttt 360 gtggctaaac acttgaagac cataaaccaa gaaagctgca tcgaaccatt agcagaaagt 420 atcacagacg ttctcgtaag gacaaaacgg gactggctag ttaaacaaag aggctgggat 480 gggtttgtgg agttcttcca agtagaggac ctagaaggtg gcatcaggaa tgtgctgctg 540 gcttttgcag gtgttgctgg agtaggagct ggtttggcat atctaaaaag atag 594 <210> 2 <211> 595 <212> DNA <213> Homo sapiens <400> 2 atgtttggcc tcaaaagaaa cgcggtaatc ggactcaacc tctactgtgg gggggccggc 60 ttgggggccg gcagcggcgg cgccacccgc ccgggagggc gacttttggc caccggcgcc 120 aaggacacaa agccaatggg caggtctggg gccaccagca ggaaggcgct ggagacctta 180 cgacgggttg gggatggcgt gcagcgcaac cacgagacgg ccttccaagg catgcttcgg 240 aaactggaca tcaaaaacga agacgatgtg aaatcgttgt ctcgagtgat gatccatgtt 300 ttcagcgacg gcgtaacaaa ctggggcagg attgtgactc tcatttcttt tggtgccttt 360 gtggctaaac acttgaagac cataaaccaa gaaagctgca tcgaaccatt agcagaaagt 420 atcacagacg ttctcgtaag gacaaaacgg gactggctag ttaaacaaag aggctgggat 480 gggtttgtgg agttcttcca tgtagaggac ctagaaggtg gcatcaggaa tgtgctgctg 540 gcttttgcag gtgttgctgg agtaggagct ggtttggcat atctaataaa gatag 595 <210> 3 <211> 197 <212> PRT <213> Homo sapiens <400> 3 Met Phe Gly Leu Lys Arg Asn Ala Val Ile Gly Leu Asn Leu Tyr Cys   1 5 10 15 Gly Gly Ala Gly Leu Gly Ala Gly Ser Gly Gly Ala Thr Arg Pro Gly              20 25 30 Gly Arg Leu Leu Ala Thr Gly Ala Lys Asp Thr Lys Pro Met Gly Arg          35 40 45 Ser Gly Ala Thr Ser Arg Lys Ala Leu Glu Thr Leu Arg Arg Val Gly      50 55 60 Asp Gly Val Gln Arg Asn His Glu Thr Ala Phe Gln Gly Met Leu Arg  65 70 75 80 Arg Leu Asp Ile Lys Asn Glu Asp Asp Val Lys Ser Leu Ser Arg Val                  85 90 95 Met Ile His Val Phe Ser Asp Gly Val Thr Asn Trp Gly Arg Ile Val             100 105 110 Thr Leu Ile Ser Phe Gly Ala Phe Val Ala Lys His Leu Lys Thr Ile         115 120 125 Asn Gln Glu Ser Cys Ile Glu Pro Leu Ala Glu Ser Ile Thr Asp Val     130 135 140 Leu Val Arg Thr Lys Arg Asp Trp Leu Val Lys Gln Arg Gly Trp Asp 145 150 155 160 Gly Phe Val Glu Phe Phe Gln Val Glu Asp Leu Glu Gly Gly Ile Arg                 165 170 175 Asn Val Leu Leu Ala Phe Ala Gly Val Ala Gly Val Gly Ala Gly Leu             180 185 190 Ala Tyr Leu Lys Arg         195 <210> 4 <211> 197 <212> PRT <213> Homo sapiens <400> 4 Met Phe Gly Leu Lys Arg Asn Ala Val Ile Gly Leu Asn Leu Tyr Cys   1 5 10 15 Gly Gly Ala Gly Leu Gly Ala Gly Ser Gly Gly Ala Thr Arg Pro Gly              20 25 30 Gly Arg Leu Leu Ala Thr Gly Ala Lys Asp Thr Lys Pro Met Gly Arg          35 40 45 Ser Gly Ala Thr Ser Arg Lys Ala Leu Glu Thr Leu Arg Arg Val Gly      50 55 60 Asp Gly Val Gln Arg Asn His Glu Thr Ala Phe Gln Gly Met Leu Arg  65 70 75 80 Lys Leu Asp Ile Lys Asn Glu Asp Asp Val Lys Ser Leu Ser Arg Val                  85 90 95 Met Ile His Val Phe Ser Asp Gly Val Thr Asn Trp Gly Arg Ile Val             100 105 110 Thr Leu Ile Ser Phe Gly Ala Phe Val Ala Lys His Leu Lys Thr Ile         115 120 125 Asn Gln Glu Ser Cys Ile Glu Pro Leu Ala Glu Ser Ile Thr Asp Val     130 135 140 Leu Val Arg Thr Lys Arg Asp Trp Leu Val Lys Gln Arg Gly Trp Asp 145 150 155 160 Gly Phe Val Glu Phe Phe His Val Glu Asp Leu Glu Gly Gly Ile Arg                 165 170 175 Asn Val Leu Leu Ala Phe Ala Gly Val Ala Gly Val Gly Ala Gly Leu             180 185 190 Ala Tyr Leu Ile Arg         195 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 agtggatcca tgtttggcct caaaagaaac 30 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ctagaattct tacagtaagg ctatctt 27  

Claims (11)

A gene encoding a polypeptide functionally identical to the polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1. A gene encoding a polypeptide functionally identical to the polypeptide encoded by SEQ ID NO: 2. Cell death composition containing at least one of the gene of claim 1 and the gene of claim 2. A polypeptide functionally identical to a polypeptide having an amino acid sequence of SEQ ID NO. A polypeptide functionally identical to a polypeptide having an amino acid sequence of SEQ ID NO. A composition for cell death containing at least one of the polypeptide of claim 3 and the polypeptide of claim 4. An anticancer composition comprising a gene encoding a polypeptide functionally identical to a polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 2 that specifically induces apoptosis in a cell overexpressing Mcl-1L. An anticancer agent composition comprising a polypeptide functionally identical to a polypeptide having an amino acid sequence of SEQ ID NO: 3 or an amino acid sequence of SEQ ID NO: 4 specifically inducing apoptosis in a cell in which Mcl-1L is overexpressed. A recombinant vector containing at least one of the gene of claim 1 and the gene of claim 2. A microorganism transformed with a recombinant vector containing at least one of the gene of claim 1 and the gene of claim 2. An anticancer agent composition comprising the recombinant vector of claim 9 or the microorganism of claim 10.

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