KR102254728B1 - Composition for Reducing Cell Proliferation Comprising Clast4 - Google Patents

Composition for Reducing Cell Proliferation Comprising Clast4 Download PDF

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KR102254728B1
KR102254728B1 KR1020140186935A KR20140186935A KR102254728B1 KR 102254728 B1 KR102254728 B1 KR 102254728B1 KR 1020140186935 A KR1020140186935 A KR 1020140186935A KR 20140186935 A KR20140186935 A KR 20140186935A KR 102254728 B1 KR102254728 B1 KR 102254728B1
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한승진
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인제대학교 산학협력단
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Abstract

본 발명은 세포 증식을 효과적으로 억제시키는 마우스 유래 단백질인 Clast4의 용도에 관한 것이다. 본 발명의 Clast4는 과발현에 의해 세포 증식을 효과적으로 억제시키는 효과가 있으므로, 암을 포함한 비정상적 세포 증식과 관련된 질환을 치료 및 예방할 수 있는 약학적 조성물로 활용될 수 있다.The present invention relates to the use of Clast4, a mouse-derived protein that effectively inhibits cell proliferation. Since Clast4 of the present invention has an effect of effectively inhibiting cell proliferation by overexpression, it can be used as a pharmaceutical composition capable of treating and preventing diseases related to abnormal cell proliferation, including cancer.

Description

Clast4를 포함하는 세포 증식 억제용 조성물{Composition for Reducing Cell Proliferation Comprising Clast4}Composition for inhibiting cell proliferation comprising Clast4 {Composition for Reducing Cell Proliferation Comprising Clast4}

본 발명은 세포 증식을 효과적으로 억제시키는 마우스 유래 단백질인 Clast4의 용도에 관한 것이다.The present invention relates to the use of Clast4, a mouse-derived protein that effectively inhibits cell proliferation.

진핵생물에서 mRNA의 번역(translation)은 세포증식, 분화, 배 발달 및 난모세포 성숙 등과 같은 세포의 여러 기작들을 수행하기 위해 엄격히 조절된다[Mendez, R. and Richter, J. D. 2001. Translational control by CPEB: a means to the end. Nat Rev Mol Cell Biol 2, 521-529]. 따라서 이러한 단계의 잘못된 조절은 암의 발생과 같은 세포의 기능이상과 밀접하게 관련되어 있다[Audic, Y. and Hartley, R. S. 2004. Post-transcriptional regulation in cancer. Biol Cell 96, 479-498]. 번역은 eIF4F (eukaryotic initiation factor 4F) 복합체의 형성으로부터 시작되며, 여기에 리보솜과 다른 번역 개시인자들이 집합한다[Gingras, A. C ., Raught, B. and Sonenberg, N. 1999. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev Biochem 68, 913-963]. eIF4F 복합체는 eIF4E, eIF4G, 및 eIF4A로 이루어져 있다. eIF4E는 모든 핵-암호화된(nuclear-encoded) mRNAs에 나타나는 5’캡(cap) 구조인, m7GpppN에 결합한다. 스캐폴드 단백질(scaffold protein)인 eIF4G는 eIF4E와 결합하고 ATP-의존적 RNA 헬리카제(helicase)인 eIF4A를 집합시켜, mRNA 2차 구조를 분해한다[Gingras, A. C ., Raught, B. and Sonenberg, N. 1999. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev Biochem 68, 913-963]. eIF4E는 mRNA 내에서 eIF4F 복합체 인자들 중 가장 먼저 번역 개시 복합체에 삽입되는 단백질이므로 조절을 위한 주요 표적이다. eIF4G는 순차적으로 eIF4E와 결합하여 기본적인 번역 개시 복합체를 만들고, 이러한 상호작용은 4E-BP와 같은 인자에 의해 조절된다. 낮은 수준으로 인산화된(hypophosphorylated) 4E-BP는, eIF4G 단백질에서도 발견되는 YXXXXLΦ (Φ는 소수성 잔기임) 모티프를 통해 eIF4E와 결합하기 위해 eIF4G와 경쟁한다[Mader, S., Lee, H., Pause, A. and Sonenberg, N. 1995. The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins. Mol Cell Biol 15, 4990-4997]. mTOR 신호전달 경로를 통한 과인산화(hyperphosphorylation)에 의해 4E-BP는 eIF4E로부터 분리되고, 이는 eIF4G가 eIF4E에 결합하여 번역을 개시하게 한다[Heesom, K. J. and Denton, R. M. 1999. Dissociation of the eukaryotic initiation factor-4E/4E-BP1 complex involves phosphorylation of 4E-BP1 by an mTOR-associated kinase. FEBS Lett 457, 489-493]. 암세포에서 4E-BP의 돌연변이나 조절되지 않은 과인산화는 eIF4E와의 상호작용을 약화시키거나 소실시켜 세포증식의 증가를 유발한다[She, Q. B., et al. 2010. 4E-BP1 is a key effector of the oncogenic activation of the AKT and ERKsignaling pathways that integrates their function in tumors. Cancer Cell 18, 39-51]. 번역 개시는 5’ UTR (untranslated region) 상의 단백질들뿐만 아니라 3’UTR 상의 인자들에 의해서도 조절되는데, CPEB (cytoplasmic polyadenylation element-binding protein)는 3’UTR의 CPE (cytoplasmic polyadenylation element)에 결합한다[Mendez, R. and Richter, J. D. 2001. Translational control by CPEB: a means to the end. Nat Rev Mol Cell Biol 2, 521-529]. CPEB는 PARN (poly(A)-specific ribonuclease)과 상호작용하여 poly(A) tail을 짧게 하거나 Maskin, Neuroguidin 및 eIF4E 운반체(transporter) (4E-T)들과 같은 단백질들 중 하나와 결합하여 eIF4E과 eIF4G의 결합을 방해함으로써 번역 억제에 관여한다[Cao, Q. and Richter, J. D. 2002. Dissolution of the maskin-eIF4E complex by cytoplasmic polyadenylation and poly(A)-binding protein controls cyclin B1 mRNA translation and oocyte maturation. EMBO J 21, 3852-3862]. 번역 개시 신호(예를 들어, 성장인자나 영양소)에 의해, CPEB는 인산화되고, 3’UTR에서 poly(A) tail 신장을 촉진시킨다. poly(A)-결합 단백질(PABP)은 poly(A) tail에 위치하여 5’UTR 상의 eIF4G와의 상호작용에 의해 닫힌-루프(closed-loop) 구조를 형성한다. 이러한 구조는 개시 복합체를 안정화시키고 번역의 개시를 촉진한다[Mendez, R. and Richter, J. D. 2001. Translational control by CPEB: a means to the end. Nat Rev Mol Cell Biol 2, 521-529]. In eukaryotes, the translation of mRNA is tightly regulated to perform several cellular mechanisms such as cell proliferation, differentiation, embryonic development and oocyte maturation [Mendez, R. and Richter, JD 2001. Translational control by CPEB: a means to the end. Nat Rev Mol Cell Biol 2, 521-529]. Therefore, misregulation of this stage is closely related to cell dysfunction, such as the occurrence of cancer [Audic, Y. and Hartley, RS 2004. Post-transcriptional regulation in cancer. Biol Cell 96, 479-498]. Translation begins with the formation of the eIF4F (eukaryotic initiation factor 4F) complex, where ribosomes and other translation initiation factors aggregate [Gingras, A. C., Raught, B. and Sonenberg, N. 1999. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev Biochem 68, 913-963]. The eIF4F complex consists of eIF4E, eIF4G, and eIF4A. eIF4E binds to m7GpppN, a 5'cap structure that appears in all nuclear-encoded mRNAs. The scaffold protein eIF4G binds to eIF4E and aggregates eIF4A, an ATP-dependent RNA helicase, to degrade the secondary structure of the mRNA [Gingras, A. C., Raught, B. and Sonenberg. , N. 1999. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev Biochem 68, 913-963]. eIF4E is the first protein to be inserted into the translation initiation complex among eIF4F complex factors in mRNA, and thus is a major target for regulation. eIF4G sequentially binds to eIF4E to create a basic translation initiation complex, and this interaction is regulated by factors such as 4E-BP. 4E-BP, which is hypophosphorylated to a low level, competes with eIF4G for binding to eIF4E via the YXXXXLΦ (Φ is a hydrophobic residue) motif that is also found in the eIF4G protein [Mader, S., Lee, H., Pause , A. and Sonenberg, N. 1995. The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins. Mol Cell Biol 15, 4990-4997]. 4E-BP is separated from eIF4E by hyperphosphorylation through the mTOR signaling pathway, which causes eIF4G to bind to eIF4E and initiate translation [Heesom, KJ and Denton, RM 1999. Dissociation of the eukaryotic initiation factor] -4E/4E-BP1 complex involves phosphorylation of 4E-BP1 by an mTOR-associated kinase. FEBS Lett 457, 489-493]. Mutation or unregulated hyperphosphorylation of 4E-BP in cancer cells attenuates or eliminates the interaction with eIF4E, leading to increased cell proliferation [She, QB, et al. 2010. 4E-BP1 is a key effector of the oncogenic activation of the AKT and ERKsignaling pathways that integrates their function in tumors. Cancer Cell 18, 39-51]. Translation initiation is regulated not only by proteins on the 5'UTR (untranslated region) but also by factors on the 3'UTR. CPEB (cytoplasmic polyadenylation element-binding protein) binds to the cytoplasmic polyadenylation element (CPE) of 3'UTR [ Mendez, R. and Richter, JD 2001. Translational control by CPEB: a means to the end. Nat Rev Mol Cell Biol 2, 521-529]. CPEB interacts with PARN (poly(A)-specific ribonuclease) to shorten the poly(A) tail or binds to one of the proteins such as Maskin, Neuroguidin and eIF4E transporter (4E-T) to form eIF4E and It is involved in translational inhibition by interfering with the binding of eIF4G [Cao, Q. and Richter, JD 2002. Dissolution of the maskin-eIF4E complex by cytoplasmic polyadenylation and poly(A)-binding protein controls cyclin B1 mRNA translation and oocyte maturation. EMBO J 21, 3852-3862]. By initiating translation signals (eg, growth factors or nutrients), CPEB is phosphorylated and promotes poly(A) tail elongation at 3'UTR. The poly(A)-binding protein (PABP) is located in the poly(A) tail and forms a closed-loop structure by interaction with eIF4G on the 5'UTR. This structure stabilizes the initiation complex and promotes the initiation of translation [Mendez, R. and Richter, JD 2001. Translational control by CPEB: a means to the end. Nat Rev Mol Cell Biol 2, 521-529].

Clast4는 사람 4E-T의 마우스 동족체(homolog)이며[Villaescusa, J. C., et al. 2006. Clast4, the murine homologue of human eIF4E-Transporter, is highly expressed in developing oocytes and post-translationally modified at meiotic maturation. Gene 367, 101-109], eIF4E을 세포질로부터 핵 또는 P-body로 운반하는 역할을 한다[Dostie, J., et al. 2000. A novel shuttling protein, 4E-T, mediates the nuclear import of the mRNA 5' cap-binding protein, eIF4E. EMBO J 19, 3142-3156]. P-body는 PAT1, Dcp1 및 Xrn1과 같은 RNA 분해 관련 단백질들을 일부 포함하고 있기 때문에, P-body에서는 mRNA 붕괴 및/또는 저장이 일어나는 것으로 여겨진다[Mir, M. A., Duran, W. A., Hjelle, B. L., Ye, C. and Panganiban, A. T. 2008. Storage of cellular 5' mRNA caps in P bodies for viral cap-snatching. Proc Natl Acad Sci USA 105, 19294-19299]. 그러나, 마우스 Clast4 단백질의 구체적인 생체 내 조절기작은 아직 보고된 바 없다. Clast4 is a mouse homolog of human 4E-T [Villaescusa, J. C., et al. 2006. Clast4, the murine homologue of human eIF4E-Transporter, is highly expressed in developing oocytes and post-translationally modified at meiotic maturation. Gene 367, 101-109], plays a role in transporting eIF4E from the cytoplasm to the nucleus or P-body [Dostie, J., et al. 2000. A novel shuttling protein, 4E-T, mediates the nuclear import of the mRNA 5'cap-binding protein, eIF4E. EMBO J 19, 3142-3156]. Since the P-body contains some RNA degradation-related proteins such as PAT1, Dcp1 and Xrn1, it is believed that mRNA disruption and/or storage occurs in the P-body [Mir, MA, Duran, WA, Hjelle, BL, Ye , C. and Panganiban, AT 2008. Storage of cellular 5'mRNA caps in P bodies for viral cap-snatching. Proc Natl Acad Sci USA 105, 19294-19299]. However, specific in vivo regulatory mechanisms of the mouse Clast4 protein have not been reported yet.

이에 본 발명자들은 Clast4의 인산화, 결합 특성 및 과발현에 의한 효과 등을 실험하여 Clast4의 세포 증식 억제 활성을 발견하고, Clast4를 비정상적 세포 증식에 의한 질환의 치료제로 사용할 수 있음을 확인함으로써 본 발명을 완성하였다.Accordingly, the present inventors completed the present invention by testing the phosphorylation, binding properties, and effects of overexpression of Clast4 to find the cell proliferation inhibitory activity of Clast4 and confirming that Clast4 can be used as a therapeutic agent for diseases caused by abnormal cell proliferation I did.

미국공개특허 2013/0178513A1US Patent Publication 2013/0178513A1

따라서 본 발명의 목적은 Clast4를 유효성분으로 포함하는 세포 증식 억제제를 제공하는 것이다. Accordingly, an object of the present invention is to provide a cell proliferation inhibitor comprising Clast4 as an active ingredient.

또한, 본 발명의 다른 목적은 Clast4를 유효성분으로 포함하는 비정상적 세포 증식 관련 질환의 예방 또는 치료용 약학 조성물을 제공하는 것이다.In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating abnormal cell proliferation-related diseases comprising Clast4 as an active ingredient.

상기와 같은 본 발명의 목적을 달성하기 위하여, 본 발명은 Clast4 단백질, Clast4 단백질을 코딩하는 유전자 또는 상기 유전자를 함유하는 유전자전달체를 포함하는 세포 증식 억제용 조성물을 제공한다.In order to achieve the object of the present invention as described above, the present invention provides a composition for inhibiting cell proliferation comprising a Clast4 protein, a gene encoding the Clast4 protein, or a gene delivery system containing the gene.

본 발명의 일실시예에 있어서, 상기 Clast4 단백질은 서열번호 1로 표시되는 것일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the Clast4 protein may be represented by SEQ ID NO: 1, but is not limited thereto.

본 발명의 일실시예에 있어서, 상기 Clast4 단백질을 코딩하는 유전자는 서열번호 2로 표시되는 것일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the gene encoding the Clast4 protein may be represented by SEQ ID NO: 2, but is not limited thereto.

또한, 본 발명은 Clast4 단백질, Clast4 단백질을 코딩하는 유전자 또는 상기 유전자를 함유하는 유전자전달체를 포함하는 비정상적 세포 증식 관련 질환의 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating abnormal cell proliferation-related diseases comprising a Clast4 protein, a gene encoding the Clast4 protein, or a gene delivery system containing the gene.

본 발명의 일실시예에 있어서, 상기 Clast4 단백질은 서열번호 1로 표시되는 것일 수 있으나, 이에 제한되는 것은 아니다. In one embodiment of the present invention, the Clast4 protein may be represented by SEQ ID NO: 1, but is not limited thereto.

본 발명의 일실시예에 있어서, 상기 Clast4 단백질을 코딩하는 유전자는 서열번호 2로 표시되는 것일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the gene encoding the Clast4 protein may be represented by SEQ ID NO: 2, but is not limited thereto.

본 발명의 일실시예에 있어서, 상기 유전자전달체는 Clast4 단백질을 코딩하는 유전자를 함유하는 재조합 발현벡터일 수 있으나, 이에 제한되는 것은 아니다. In one embodiment of the present invention, the gene delivery system may be a recombinant expression vector containing a gene encoding the Clast4 protein, but is not limited thereto.

본 발명의 일실시예에 있어서, 상기 Clast4 단백질은 eIF4E1 및/또는 CPEB와 결합하여 mRNA의 번역(translation)을 조절하는 것일 수 있다.In one embodiment of the present invention, the Clast4 protein may bind to eIF4E1 and/or CPEB to regulate the translation of mRNA.

본 발명의 일실시예에 있어서, 상기 비정상적 세포 증식 관련 질환은 암, 염증성 질환 또는 이상증식 혈관 질환일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the abnormal cell proliferation-related disease may be cancer, an inflammatory disease, or an abnormal proliferative vascular disease, but is not limited thereto.

본 발명의 일실시예에서 Clast4는 HEK293T 세포에서 과발현되어 세포증식을 효과적으로 억제시켰다. 따라서 본 발명의 Clast4는 암을 포함한 비정상적 세포 증식과 관련된 질환을 치료 및 예방할 수 있는 약학적 조성물로 활용될 수 있다.In one embodiment of the present invention, Clast4 was overexpressed in HEK293T cells to effectively inhibit cell proliferation. Therefore, Clast4 of the present invention can be used as a pharmaceutical composition capable of treating and preventing diseases related to abnormal cell proliferation, including cancer.

도 1은 PKA가 Clast4를 인산화시킨다는 것을 보여준다. (A) 면역침전된 FLAG-Clast4를 5 μCi [γ-32P] ATP와 함께 PKA, Plk1, Aurora A kinase, 및 Cdk1/cyclin B 복합체로 배양하였다. SDS-PAGE 후 인산화 상태 및 Clast4의 발현을 자가방사법(위) 및 FLAG-특이적 항체(아래)로 확인한 결과이다. (B) Clast4-트랜스펙션 HEK293T 세포에 forskolin 및 IBMX을 처리하였다. 면역침전된 Clast4를 항-phospho (Ser/Thr) PKA 기질 항체(위) 및 FLAG-특이적 항체(아래)로 검출하였다. Clast4의 인산화는 10 μM H89의 첨가로 완전히 사라졌다. (C) Clast4-트랜스펙션 세포들을 야생형(WT) 또는 PKA 촉매 서브유닛의 키나아제-사멸 돌연변이(KD)와 함께 공동 트랜스펙션시켰다. 항-phospho (Ser/Thr) PKA 기질 항체를 이용하여 면역침전물에 대해 웨스턴 블랏 분석을 한 결과 Clast4는 WT PKA 과발현에 의해 인산화되었다(위). Clast4 및 PKA 컨스트럭트(constructs)의 발현을 각각 항-FLAG 또는 HA 항체를 사용하여 확인하였다(아래 두 블랏). (D) Clast4의 아미노 말단은 주요한 PKA 인산화 부위이다. FLAG-표지된 전장(full-length) 또는 절단형 Clast4의 모식도(위). 잠재적인 PKA 인산화 부위(S212, RRRNDSYT; T256, S258, RTRRRTASVK; S352, GSRSSSLG)를 나타내었다. PKA 촉매 서브유닛을 이용한 인 비트로(in vitro) 키나아제 어세이 결과 F1 및 F2 단편들이 PKA에 의해 인산화되었다(왼쪽). 면역침전된 단백질들을 FLAG 항체를 사용하여 관찰하였다(오른쪽). (E) 잠재적인 PKA-인산화된 세린 또는 트레오닌 아미노산을 알라닌으로 치환하였다. 돌연변이된 F1 또는 F2 컨스트럭트의 인산화 상태를 자가방사법으로 측정하였다. (F) HEK293T 세포에 트랜스펙션시킨 후 EGFP-표지된 야생형 또는 인산화 부위가 돌연변이된 Clast4의 위치를 확인하였다. Clast4의 P-body로의 위치화는 PKA 억제제의 처리에 의해 증가하였다.
도 2는 Clast4가 eIF4E1s 및 CPEB와 상호작용한다는 것을 보여준다. (A) EGFP-CPEB와 HA-eIF4E1A 또는 HA-eIF4E1B의 공동 트랜스펙션 후에 공동-면역침전으로 두 단백질들 사이의 상호작용을 확인하였다. (B) 야생형 또는 Y29A 돌연변이 중 하나가 트랜스펙션된 뒤 eIF4E1A 또는 eIF4E1B 중 하나를 연속적으로 트랜스펙션시켰다. Clast4 및 eIF4E과의 연관을 공동-면역침전으로 관찰하였다. (C) eIF4E1B는 Clast4의 아미노 말단과 결합하였다. HA-eIF4E1B 및 FLAG-Clast4의 절단형과의 공동 트랜스펙션 후 공동-면역침전물을 HA 항체로 탐지하였다. (D) 잠재적인 PKA 인산화 부위의 인산화는 eIF4E과의 결합에 관여하지 않았다. FLAG-표지된 Clast4 인산화 부위 돌연변이들 중 하나와 HA-표지된 eIF4E1B를 HEK293T 세포에 트랜스펙션시키고, Clast4 및 eIF4E1B의 공동-면역침전을 관찰하였다.
도 3은 Clast4의 과발현이 세포 증식율을 감소시킴을 보여준다. (A) GFP, 4E-BP, 또는 Clast4 컨스트럭트(construct)를 4 × 103 HEK293T 세포에 주입하고, 세포의 수를 지정된 시간에 세었다(***p<0.001). 결과는 3번의 독립된 실험으로 측정하였다. 삽입된 패널은 트랜스펙션된 단백질의 발현을 나타낸다(NS: nonspecific band).Figure 1 shows that PKA phosphorylates Clast4. (A) Immunoprecipitated FLAG-Clast4 was cultured with PKA, Plk1, Aurora A kinase, and Cdk1/cyclin B complex with 5 μCi [γ-32P] ATP. It is the result of confirming the phosphorylation state and expression of Clast4 after SDS-PAGE by autoradiation (top) and FLAG-specific antibody (bottom). (B) Clast4-transfected HEK293T cells were treated with forskolin and IBMX. Immunoprecipitated Clast4 was detected with an anti-phospho (Ser/Thr) PKA substrate antibody (top) and a FLAG-specific antibody (bottom). The phosphorylation of Clast4 completely disappeared with the addition of 10 μM H89. (C) Clast4-transfected cells were co-transfected with wild-type (WT) or kinase-killing mutations (KD) of the PKA catalytic subunit. Western blot analysis of immunoprecipitates using anti-phospho (Ser/Thr) PKA substrate antibody showed that Clast4 was phosphorylated by WT PKA overexpression (top). Expression of Clast4 and PKA constructs was confirmed using anti-FLAG or HA antibodies, respectively (two blots below). (D) The amino terminus of Clast4 is the major PKA phosphorylation site. Schematic diagram of a FLAG-labeled full-length or truncated Clast4 (top). Potential PKA phosphorylation sites (S212, RRRNDSYT; T256, S258, RTRRRTASVK; S352, GSRSSSLG) were shown. In vitro using the PKA catalyst subunit ( in vitro ) As a result of the kinase assay, the F1 and F2 fragments were phosphorylated by PKA (left). Immunoprecipitated proteins were observed using FLAG antibody (right). (E) Potential PKA-phosphorylated serine or threonine amino acid was replaced with alanine. The phosphorylation status of the mutated F1 or F2 construct was measured by autoradiation. (F) After transfection into HEK293T cells, the location of EGFP-labeled wild-type or Clast4 in which the phosphorylation site was mutated was confirmed. The localization of Clast4 to the P-body was increased by treatment with a PKA inhibitor.
Figure 2 shows that Clast4 interacts with eIF4E1s and CPEB. (A) After co-transfection of EGFP-CPEB and HA-eIF4E1A or HA-eIF4E1B, the interaction between the two proteins was confirmed by co-immunoprecipitation. (B) One of the wild-type or Y29A mutations was transfected, followed by successive transfection of either eIF4E1A or eIF4E1B. The association with Clast4 and eIF4E was observed by co-immunoprecipitation. (C) eIF4E1B is bound to the amino terminus of Clast4. After co-transfection with the truncated form of HA-eIF4E1B and FLAG-Clast4, co-immunoprecipitates were detected with HA antibodies. (D) The phosphorylation of the potential PKA phosphorylation site was not involved in binding to eIF4E. One of the FLAG-labeled Clast4 phosphorylation site mutations and HA-labeled eIF4E1B were transfected into HEK293T cells, and co-immunoprecipitation of Clast4 and eIF4E1B was observed.
3 shows that the overexpression of Clast4 reduces the cell proliferation rate. (A) GFP, 4E-BP, or Clast4 construct was injected into 4×10 3 HEK293T cells, and the number of cells was counted at the designated time (***p<0.001). The results were measured in three independent experiments. The inserted panel shows the expression of the transfected protein (NS: nonspecific band).

본 발명은 비정상적 세포 증식과 관련된 질환을 치료 및 예방할 수 있는 약학적 조성물로서 마우스 유래 Clast4 단백질의 신규한 용도를 제공한다는 점에 특징이 있다. The present invention is characterized in that it provides a novel use of the mouse-derived Clast4 protein as a pharmaceutical composition capable of treating and preventing diseases associated with abnormal cell proliferation.

eIF4E는 번역 개시 과정에서 중심조절자 역할을 하는데, eIF4E와 eIF4G의 결합이 mRNA의 번역을 촉발하기 때문에, 여러 단백질들이 이 결합을 저해함으로써 번역과정을 조절한다. 사람 4E-T는 eIF4E 결합단백질 중의 하나로, 결합한 mRNA의 번역을 저해할 뿐만 아니라, eIF4E를 processing body (P-body)로 이동시키는 기능을 가지고 있다. Clast4는 이러한 사람의 4E-T와 상동성을 가지는 마우스 유래 단백질로 번역 조절에 중요한 기능을 할 것으로 추측되지만, 그 특징은 아직 잘 알려져 있지 않다. eIF4E acts as a central regulator in the translation initiation process. Since the binding of eIF4E and eIF4G triggers the translation of mRNA, several proteins regulate the translation process by inhibiting this binding. Human 4E-T is one of the eIF4E binding proteins, and has the function of not only inhibiting the translation of bound mRNA, but also moving eIF4E to the processing body (P-body). Clast4 is a mouse-derived protein that has homology with human 4E-T and is expected to play an important function in translational regulation, but its characteristics are not yet well known.

본 발명자들은 Clast4의 인산화된 상태와 eIF4E와의 결합력, Clast4 과발현시 세포증식의 변화에 대한 특징을 관찰하였다. 그 결과, Clast4는 PKA에 의해 생체 내(in vivo)에서 아미노 말단의 몇몇 잔기가 인산화되는 것으로 확인되었으나, PKA에 의해 인산화된 Clast4는 eIF4E와의 결합력이나 Clast4의 세포 내 위치(localization)에는 큰 변화가 없었다(도 1). 또한, Clast4는 eIF4E1 및 CPEB와 결합하며, Clast4의 보존된 eIF4E 결합 서열인 YXXXXLΦ가 eIF4E1A와의 결합에서는 중요하지만 eIF4E1B와의 결합에서는 큰 영향이 없는 것으로 관찰되었다(도 2). 또한, Clast4를 과발현하였을 때 종래 잘 알려져 있던 eIF4E 조절자인 4E-BP와 유사한 수준으로 세포의 증식이 감소되는 것을 확인하였다(도 3). 이러한 결과들은 Clast4가 세포 내에서 전반적인 번역 조절에 관여하고 있다는 것을 시사하며, 비정상적 세포 증식과 관련된 질환을 치료 및 예방할 수 있는 약학적 조성물로 사용될 수 있음을 보여준다.The present inventors observed the characteristics of the phosphorylated state of Clast4, the binding ability to eIF4E, and the change in cell proliferation upon overexpression of Clast4. As a result, Clast4 is in vivo (in In vivo ), it was confirmed that some residues at the amino terminal were phosphorylated, but Clast4 phosphorylated by PKA had no significant change in the binding strength to eIF4E or the intracellular localization of Clast4 (FIG. 1). In addition, Clast4 binds to eIF4E1 and CPEB, and YXXXXLΦ, the conserved eIF4E binding sequence of Clast4, is important in binding to eIF4E1A, but it was observed that there is no significant effect in binding to eIF4E1B (Fig. In addition, when Clast4 was overexpressed, it was confirmed that cell proliferation was reduced to a level similar to that of 4E-BP, a conventionally well-known eIF4E modulator (FIG. 3). These results suggest that Clast4 is involved in overall translational regulation within cells, and shows that it can be used as a pharmaceutical composition that can treat and prevent diseases related to abnormal cell proliferation.

본 발명에서 '비정상적 세포 증식 관련 질환'이란 세포의 비정상적 증식으로 인해 야기되는 질환을 의미하며, 예를 들어 암, 염증성 질환, 이상증식 혈관 질환 등이 있으나 이에 제한되는 것은 아니다. 본 발명의 조성물이 적용될 수 있는 상기 암의 예로는 위암, 폐암, 유방암, 대장암, 간암, 뇌암, 난소암, 전립선암, 후두암, 췌장암, 신장암, 방광암, 신경내분비 암, 부신암, 결장암, 자궁경부암, 골암, 피부암, 갑상선암 등이 있으나 이에 제한되는 것은 아니다. 본 발명의 조성물이 적용될 수 있는 상기 '염증성 질환'은 염증을 유발하는 모든 질환을 포함하며, 예를 들어 퇴행성 관절염, 크론병, 뇌막염, 염증성 위궤양, 건선, 아토피, 비염, 결막염, 천식, 피부염, 염증성 장질환, 염증성 위궤양, 사구체신염, 염증성 피부 질환 등이 포함되지만, 이에 제한되는 것은 아니다. 본 발명의 조성물이 적용될 수 있는 상기 '이상증식 혈관 질환'은 혈관 세포의 과도한 증식에 의해 야기되는 질환으로, 동맥경화증, 혈관염증질환, 천식, 협착증, 해면상혈관종, 수포질환, 혈관 기형, 맥관염, 유전성 출혈성 모세혈관확장증(HHT), 폐혈증, 미숙아 망막증, 켈로이드성 반흔, 황반변성, 당뇨병성 망막증, 염증성 장 및 치주 질환, 복수, 복막 유착, 자궁내막증, 자궁 출혈, 관절염, 자가면역질환 등 다양한 질환을 포함하나, 이에 제한되는 것은 아니다.In the present invention, "abnormal cell proliferation related disease" means a disease caused by abnormal proliferation of cells, and examples include cancer, inflammatory disease, abnormal proliferative vascular disease, but are not limited thereto. Examples of the cancers to which the composition of the present invention can be applied include gastric cancer, lung cancer, breast cancer, colon cancer, liver cancer, brain cancer, ovarian cancer, prostate cancer, laryngeal cancer, pancreatic cancer, kidney cancer, bladder cancer, neuroendocrine cancer, adrenal cancer, colon cancer, Cervical cancer, bone cancer, skin cancer, thyroid cancer, and the like, but are not limited thereto. The'inflammatory disease' to which the composition of the present invention can be applied includes all diseases that cause inflammation, for example, degenerative arthritis, Crohn's disease, meningitis, inflammatory gastric ulcer, psoriasis, atopy, rhinitis, conjunctivitis, asthma, dermatitis, Inflammatory bowel disease, inflammatory gastric ulcer, glomerulonephritis, inflammatory skin disease, etc. The'abnormal proliferative vascular disease' to which the composition of the present invention can be applied is a disease caused by excessive proliferation of vascular cells, atherosclerosis, vascular inflammatory disease, asthma, stenosis, cavernous hemangioma, blister disease, vascular malformation, vasculitis , Hereditary hemorrhagic telangiectasia (HHT), sepsis, retinopathy of prematurity, keloid scar, macular degeneration, diabetic retinopathy, inflammatory bowel and periodontal disease, ascites, peritoneal adhesion, endometriosis, uterine bleeding, arthritis, autoimmune diseases, etc. Including, but not limited to disease.

상기와 같은 비정상적 세포 증식 관련 질환의 치료 효과를 나타내는 본 발명에 따른 실험결과들을 토대로, 본 발명은 Clast4 단백질, Clast4 단백질을 코딩하는 유전자 또는 상기 유전자를 함유하는 유전자전달체를 포함하는 비정상적 세포 증식 관련 질환의 예방 및 치료용 약학 조성물을 제공할 수 있다. Based on the experimental results according to the present invention showing the therapeutic effect of such abnormal cell proliferation-related diseases, the present invention provides an abnormal cell proliferation-related disease including a gene encoding Clast4 protein, a Clast4 protein, or a gene delivery system containing the gene. It can provide a pharmaceutical composition for the prevention and treatment of.

본 발명의 일실시예에 있어서, 상기 유전자전달체는 Clast4 유전자가 도입되는 세포 또는 생물체 내에서 Clast4를 발현시키거나 그 발현수준을 증가시키기 위한 재조합 발현벡터일 수 있다. 상기 ‘발현벡터’는 발현벡터의 전사를 위해 삽입된 부위와 이에 작동가능하게 연결된 Clast4를 암호화하는 부위를 포함하는 선형 또는 원형의 DNA 또는 RNA 분자이다. 상기 발현벡터의 전사를 위해 삽입된 부위에는 프로모터 및 종료 암호 서열이 포함된다. 상기 ‘프로모터’는 RNA 폴리머라제가 결합하고 mRNA 합성이 개시되는 유전자 부분을 의미한다. 상기 발현벡터는 하나 이상의 복제 개시점, 선별 마커, 증강자(enhancer), 폴리아데닐화 신호 등이 포함될 수 있다. 일반적으로 발현벡터는 플라스미드 또는 바이러스 DNA로부터 유도되는 것들이 사용되나, RNA 바이러스 벡터(레트로바이러스 벡터, 렌티바이러스 벡터 등)도 포함될 수 있으며, 발현 목적에 따라 다양한 발현벡터를 이용할 수 있다. 또한, 발현벡터에 삽입되는 외래 유전자의 크기, 염기서열 등도 공지의 기술에 따라 다양하게 변화시킬 수 있다.
In one embodiment of the present invention, the gene delivery system may be a recombinant expression vector for expressing Clast4 or increasing its expression level in cells or organisms into which the Clast4 gene is introduced. The'expression vector' is a linear or circular DNA or RNA molecule comprising a site inserted for transcription of the expression vector and a site encoding Clast4 operably linked thereto. The site inserted for transcription of the expression vector includes a promoter and a termination coding sequence. The'promoter' refers to a portion of a gene where RNA polymerase binds and mRNA synthesis starts. The expression vector may include one or more replication initiation points, selection markers, enhancers, polyadenylation signals, and the like. In general, expression vectors are those derived from plasmid or viral DNA, but RNA viral vectors (retroviral vectors, lentiviral vectors, etc.) may also be included, and various expression vectors may be used depending on the purpose of expression. In addition, the size and nucleotide sequence of the foreign gene inserted into the expression vector can be variously changed according to known techniques.

또한, 본 발명에 따른 약학 조성물은 하나 이상의 약학적으로 허용되는 담체, 부형제 또는 희석제를 포함할 수 있다. 상기에서 '약학적으로 허용되는' 이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장장애, 현기증과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 말한다. 상기 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 또한, 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다.In addition, the pharmaceutical composition according to the present invention may include one or more pharmaceutically acceptable carriers, excipients, or diluents. In the above, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not usually cause allergic reactions such as gastrointestinal disorders or dizziness or similar reactions when administered to humans. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils. In addition, fillers, anti-aggregating agents, lubricants, wetting agents, flavoring agents, emulsifying agents, and preservatives may additionally be included.

또한, 본 발명의 조성물은 포유동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 공지된 방법을 사용하여 제형화될 수 있다. 제형은 분말, 과립, 정제, 에멀젼, 시럽, 에어로졸, 연질 또는 경질 젤라틴 캅셀, 멸균 주사용액, 멸균 분말의 형태일 수 있다.In addition, the compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulation may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powders.

본 발명에 따른 조성물은 경구, 경피, 피하, 정맥 또는 근육을 포함한 여러 경로를 통해 투여될 수 있으며, 활성 성분의 투여량은 투여 경로, 환자의 연령, 성별, 체중 및 환자의 중증도 등의 여러 인자에 따라 적절히 선택될 수 있다. 또한, 본 발명의 약학 조성물은 비정상적 세포 증식 관련 질환의 증상을 예방, 개선 또는 치료하는 효과를 가지는 공지의 화합물과 병행하여 투여할 수 있다.
The composition according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient is a number of factors such as the route of administration, the patient's age, sex, weight, and the patient's severity. It can be appropriately selected according to. In addition, the pharmaceutical composition of the present invention can be administered in combination with a known compound having an effect of preventing, ameliorating or treating symptoms of abnormal cell proliferation-related diseases.

이하 본 발명을 실시예에 의하여 더욱 상세하게 설명한다. 하기 실시예는 단지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 국한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail by examples. The following examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited to these examples.

<< 실시예Example 1> 1>

실험재료 및 방법Experimental materials and methods

<1-1> 실험재료<1-1> Experimental materials

항-4E-T 및 항-phospho (Ser/Thr) PKA 기질(substrate) 항체를 Bethyl (Montgomery, AL) 및 Cell Signaling (Danvers, CO)사에서 각각 구입하였다. FLAG 에피토프 항체 및 다른 화합물들은 Sigma Aldrich (St. Louis, MO)사에서 구입하였다. 올리고뉴클레오티드 합성 및 DNA 시퀀싱은 Cosmo Genetech (Seoul, Korea)에서 수행하였다. eIF4E1B 유전자는 마우스 난소로부터 RT-PCR을 이용하여 분리하였으며, 사용된 프라이머는 다음과 같다.Anti-4E-T and anti-phospho (Ser/Thr) PKA substrate antibodies were purchased from Bethyl (Montgomery, AL) and Cell Signaling (Danvers, CO), respectively. FLAG epitope antibodies and other compounds were purchased from Sigma Aldrich (St. Louis, MO). Oligonucleotide synthesis and DNA sequencing were performed at Cosmo Genetech (Seoul, Korea). The eIF4E1B gene was isolated from mouse ovary using RT-PCR, and the primers used are as follows.

- Forward primer: TAGCCCGGGGTACCATGAACAAAGTTGAGGGTGGAGGGC (서열번호 3) -Forward primer: TAGCCCGGGGTACCATGAACAAAGTTGAGGGTGGAGGGC (SEQ ID NO: 3)

- Reverse primer: TTAACGCGGCCGCCACCACAAACTTGTTTGCTAAG (서열번호 4)
-Reverse primer: TTAACGCGGCCGCCACCACAAACTTGTTTGCTAAG (SEQ ID NO: 4)

<1-2> <1-2> Clast4Clast4 돌연변이의 제작 Creation of mutations

Open Biosystems에서 얻은 Clast4 cDNA (MMM1013-7512592, Clone ID: 5359290)를 pcDNA 3.1 topo (Invitrogen Corp., Carlsbad, CA) 또는 pCMV Tag2 (Clontech, Mountain View, CA) 벡터에 클로닝하였다. Clast4의 위치(localization)를 확인하기 위하여, GFP 유전자를 Clast4-pcDNA 3.1 컨스트럭트(construct)에 삽입시켜 Clast4-GFP 융합 유전자를 만들었다. 제한효소 처리나 PCR 반응을 통해 Clast4의 절단형(truncated forms)을 만들었다(도 1D). Clast4의 비-인산화형(non-phosphorylated form)을 만들기 위해, QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA)를 사용하여 지정된 세린 또는 트레오닌 부위(Ser212, Thr256, Ser258, Ser352)에 돌연변이를 유발하였다.
Clast4 cDNA (MMM1013-7512592, Clone ID: 5359290) obtained from Open Biosystems was cloned into pcDNA 3.1 topo (Invitrogen Corp., Carlsbad, CA) or pCMV Tag2 (Clontech, Mountain View, CA) vector. In order to confirm the localization of Clast4, the GFP gene was inserted into the Clast4-pcDNA 3.1 construct to create a Clast4-GFP fusion gene. Truncated forms of Clast4 were made through restriction enzyme treatment or PCR reaction (Fig. 1D). To create a non-phosphorylated form of Clast4, mutations were made to the designated serine or threonine sites (Ser212, Thr256, Ser258, Ser352) using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Triggered.

<1-3> 면역침전 및 인 비트로 <1-3> Immunoprecipitation and in vitro 키나아제Kinase 어세이( Assay( inin vitrovitro kinasekinase assayassay ))

60% confluence에서 HEK293T 세포를 JetPEI (Polyplus-transfection Inc., NY)로 트랜스펙션시키고, 36시간 후 용해 버퍼(50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% NP40, 1 mM EDTA, 1 μg/ml leupeptin, 1 mM aprotinin, 1 mM PMSF, 1 mM sodium orthovadate, 1 mM NaF)로 수득하였다. 수득한 세포 용해물을 4℃에서 항-FLAG 항체로 배양한 후 protein G sepharose (GE Healthcare Bio-Sciences, Piscataway, NJ)로 침전시켰다. 면역침전물을 용해 버퍼 및 키나아제 버퍼(25 mM Tris-HCl pH 7.5, 5 mM β-glycophosphate, 0.2 mM dithiothreitol, 1 mM sodium orthovadate, 1 mM magnesium chloride)로 연속하여 세 번 세척하였다. 그 후 5 유닛의 PKA 촉매 서브유닛(catalytic subunit) (Promega, Madison, Wisconsin), Plk1, Aurora A 키나아제(SignalChem, Richmond, CA) 및 Cdk1 각각을 포함하거나 포함하지 않는, 250μM ATP 및 10 μCi/ml [γ-32P] ATP (3000 Ci/mmol, PerkinElmer)를 포함한 키나아제 버퍼로 면역침전물을 37℃에서 30분간 배양하였다. 상기 시료를 8% SDS-PAGE 겔에 녹이고 PVDF 막으로 이동시켰다. 방사성 표지된 Clast4를 X-선 필름(Kodak, Rochester, NY)이나 phosphor image analyzer (Typhoon 9400, GE Healthcare Bio-Sciences)로 검출하였다.
HEK293T cells were transfected with JetPEI (Polyplus-transfection Inc., NY) at 60% confluence, and after 36 hours lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% NP40, 1 mM EDTA, 1 μg/ml leupeptin, 1 mM aprotinin, 1 mM PMSF, 1 mM sodium orthovadate, 1 mM NaF). The obtained cell lysate was incubated with an anti-FLAG antibody at 4° C. and then precipitated with protein G sepharose (GE Healthcare Bio-Sciences, Piscataway, NJ). The immunoprecipitates were washed three times in succession with lysis buffer and kinase buffer (25 mM Tris-HCl pH 7.5, 5 mM β-glycophosphate, 0.2 mM dithiothreitol, 1 mM sodium orthovadate, 1 mM magnesium chloride). Then 5 units of PKA catalytic subunit (Promega, Madison, Wisconsin), Plk1, Aurora A kinase (SignalChem, Richmond, CA) and 250 μM ATP and 10 μCi/ml, each with or without Cdk1 [γ-32P] The immunoprecipitates were incubated at 37°C for 30 minutes with a kinase buffer containing ATP (3000 Ci/mmol, PerkinElmer). The sample was dissolved on an 8% SDS-PAGE gel and transferred to a PVDF membrane. Radiolabeled Clast4 was detected with an X-ray film (Kodak, Rochester, NY) or a phosphor image analyzer (Typhoon 9400, GE Healthcare Bio-Sciences).

<1-4> 생체 내(<1-4> In vivo ( inin vivovivo ) 인산화 실험) Phosphorylation experiment

생체 내 인산화 실험을 위하여, Clast4-트랜스펙션 세포들을 0.2% FBS가 첨가된 DMEM에서 16시간 동안 배양하였다. 생체 내 PKA를 활성화하기 위하여, 10 μM H89와 함께 또는 단독으로, 10 μM Forskolin 및 0.1 mM IBMX을 30분간 처리하였다. 면역침전된 Clast4는 항-phospho (Ser/Thr) PKA 기질 항체로 SDS-PAGE 및 PVDF막을 이용하여 검출하였다.
For in vivo phosphorylation experiments, Clast4-transfected cells were cultured in DMEM supplemented with 0.2% FBS for 16 hours. In order to activate PKA in vivo, 10 μM Forskolin and 0.1 mM IBMX were treated with 10 μM H89 or alone for 30 minutes. Immunoprecipitated Clast4 was detected using an anti-phospho (Ser/Thr) PKA substrate antibody using SDS-PAGE and PVDF membranes.

<1-5> 공동 면역침전<1-5> Joint immunoprecipitation

HA-표지된 eIF4E1B-트랜스펙션 세포들을 60 mm 접시에 나누고, FLAG-표지된 Clast4 단편들을 연속해서 트랜스펙션시켰다. FLAG 항체로 면역침전시킨 후 HA 항체로 웨스턴 블랏팅을 수행하였다.
HA-labeled eIF4E1B-transfected cells were divided into 60 mm dishes, and FLAG-labeled Clast4 fragments were successively transfected. After immunoprecipitation with FLAG antibody, Western blotting was performed with HA antibody.

<1-6> 면역세포화학(<1-6> Immunocytochemistry ( ImmunocytochemistryImmunocytochemistry ))

HEK293T 세포를 poly-L-lysine-코팅된 슬라이드 글라스 위에서 배양하고 GFP-표지된 Clast4로 트랜스펙션시켰다. 16시간의 혈청 기아(serum starvation) 상태 후, H89의 존재 및 부존재 하에 forskolin 및 IBMX을 처리하였다. 상기 세포를 PBS에서 1시간 동안 4% 포름알데히드에 고정시키고 블락킹 버퍼(PBS 내 1% BSA, 0.3% Triton-X-100)로 침투시켰다. 블락킹 버퍼 안에 Dcp1 항체를 2시간 동안 첨가하고 CY3-결합된 항-토끼(PA43004, GE Healthcare Bio-Sciences) 항체를 처리하였다. 상기 시료를 공초점 레이저-스캐닝 현미경(LSM 510 META, Carl Zeiss, Germany)으로 관찰하였다.
HEK293T cells were cultured on poly-L-lysine-coated slide glass and transfected with GFP-labeled Clast4. After 16 hours of serum starvation, forskolin and IBMX were treated in the presence and absence of H89. The cells were fixed in 4% formaldehyde in PBS for 1 hour and infiltrated with blocking buffer (1% BSA in PBS, 0.3% Triton-X-100). Dcp1 antibody was added in blocking buffer for 2 hours, and CY3-conjugated anti-rabbit (PA43004, GE Healthcare Bio-Sciences) antibody was treated. The sample was observed with a confocal laser-scanning microscope (LSM 510 META, Carl Zeiss, Germany).

<1-7> 증식 실험<1-7> Proliferation experiment

GFP, Clast4, 또는 4E-BP-트랜스펙션된 세포들(2,000/웰)을 96-웰 배양 플레이트에 넣고, 20 μl의 MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, PBS 내 0.5 mg/ml)를 정해진 시간 간격으로 첨가하여 살아 있는 세포들의 수를 확인하였다. 37℃에서 4시간 동안 배양한 후, 생성된 포르마잔(formazan)을 MTT 어세이 용액(이소프로판올 내 4 mM HCL, 0.1% NP40)에 녹였다. 착색된 용액의 흡광도를 마이크로플레이트 리더(MQX 200R, BioTek Instruments, Winooski, VT, USA)를 사용하여 620 nm의 참조 필터로 590 nm에서 측정함으로써 정량화하였다.
GFP, Clast4, or 4E-BP-transfected cells (2,000/well) were placed in a 96-well culture plate, and 20 μl of MTT (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide, 0.5 mg/ml in PBS) was added at predetermined time intervals to check the number of living cells. After incubation at 37° C. for 4 hours, the produced formazan was dissolved in MTT assay solution (4 mM HCL in isopropanol, 0.1% NP40). The absorbance of the colored solution was quantified by measuring at 590 nm with a reference filter of 620 nm using a microplate reader (MQX 200R, BioTek Instruments, Winooski, VT, USA).

<< 실시예Example 2> 2>

PKAPKA 에 의한 On by Clast4Clast4 의 인산화Phosphorylation of

난모세포의 성숙 과정 동안 Clast4가 키나아제에 의해 인산화된다는 사실이 알려져 있으므로[Villaescusa, J. C., et al. 2006. Clast4, the murine homologue of human eIF4E-Transporter, is highly expressed in developing oocytes and post-translationally modified at meiotic maturation. Gene 367, 101-109], 본 발명자들은 세포주기의 진행 과정에서 Clast4를 인산화시키는 키나아제를 찾기 위하여 세포주기 조절과 관련된 여러 키나아제로 인 비트로(in vitro) 키나아제 어세이를 수행하였다. 그 결과 실험에 사용한 키나아제 중 PKA가 다른 키나아제(즉, polo-like kinase 1 (plk1), Aurora A kinase, 및 cyclin-dependent kinase 1 (Cdk1))에 비해 더욱 강하게 Clast4를 인산화시키는 것을 확인하였다(도 1A). 사람 4E-T는 P-body로 운반되고, 효모에서 PKA는 P-body 형성과 관련된 것으로 알려져 있기 때문에[Tudisca, V., et al. 2012. PKA isoforms coordinate mRNA fate during nutrient starvation. J Cell Sci 125, 5221-5232], 본 발명자들은 Clast4 인산화에 있어서 PKA의 역할에 주목하였다. 생체 내에서 Clast4의 인산화를 확인하기 위하여, PKA를 아데니릴 사이클레이즈 활성인자(adenylyl cyclase activator)인 forskolin 및 포스포디에스터레이즈 저해인자(phosphodiesterase inhibitor)인 IBMX로 활성화시켰다. Clast4의 면역침전 후, 인산화된 보존된 부위(RXXpS/T)를 확인할 수 있는 항-phospho (Ser/Thr) PKA 기질 항체로 Clast4의 인산화 상태를 측정하였다. Clast4는 생체 내에서 PKA의 활성화에 의해 인산화되었고, 이러한 효과는 PKA 억제인자인 H89의 처리에 의해 완전히 사라졌다(도 1B). 추가로, 세포 내 Clast4의 인산화 상태는 키나아제-사멸형(kinase-dead form)이 아닌 PKA 촉매 서브유닛(catalytic subunit)의 과발현에 의해 증가하였다(도 1C). 이러한 결과들은 Clast4가 세포 내에서 그 자체로 PKA 기질이라는 것을 보여준다.It is known that Clast4 is phosphorylated by kinase during the oocyte maturation process [Villaescusa, J. C., et al. 2006. Clast4, the murine homologue of human eIF4E-Transporter, is highly expressed in developing oocytes and post-translationally modified at meiotic maturation. Gene 367, 101-109], the present inventors performed an in vitro kinase assay with several kinases related to cell cycle regulation in order to find a kinase that phosphorylates Clast4 in the course of the cell cycle. As a result, it was confirmed that PKA phosphorylates Clast4 more strongly than other kinases (ie, polo-like kinase 1 (plk1), Aurora A kinase, and cyclin-dependent kinase 1 (Cdk1)) among the kinases used in the experiment (Fig. 1A). Since human 4E-T is transported to the P-body, and in yeast, PKA is known to be involved in P-body formation [Tudisca, V., et al. 2012. PKA isoforms coordinate mRNA fate during nutrient starvation. J Cell Sci 125, 5221-5232], the present inventors noted the role of PKA in Clast4 phosphorylation. To confirm the phosphorylation of Clast4 in vivo, PKA was activated with forskolin, an adenylyl cyclase activator, and IBMX, a phosphodiesterase inhibitor. After immunoprecipitation of Clast4, the phosphorylation state of Clast4 was measured with an anti-phospho (Ser/Thr) PKA substrate antibody capable of identifying the phosphorylated conserved site (RXXpS/T). Clast4 was phosphorylated by the activation of PKA in vivo, and this effect was completely eliminated by treatment with the PKA inhibitor H89 (Fig. 1B). In addition, the phosphorylation state of Clast4 in the cell was increased by overexpression of the PKA catalytic subunit, not the kinase-dead form (FIG. 1C). These results show that Clast4 is itself a PKA substrate in cells.

웹-기반 프로그램(http://scansite.mit.edu/ 및 http://www.phosphosite.org/homeAction.do)을 사용하여, 잠재적인 PKA 인산화 부위인 S212, S256, S258 및 S352 부위를 찾았다. Clast4에서 PKA 인산화 부위를 찾기 위하여 Clast4의 여러 절단형(truncated forms)을 만들었다. F1 및 F2 단편은 [γ-32P] ATP-함유 반응 버퍼에서 PKA 촉매 서브유닛에 의해 인산화되었으나, F3 또는 F4는 인산화되지 않았으며(도 1D), 이는 Clast4의 아미노-말단 절반이 주된 PKA 인산화 부위를 함유한다는 것을 의미한다. F1 및 F2 단편의 인산화된 잔기들을 결정하기 위해, F1의 S212 및 F2의 T256, S258 및 S352를 비-인산화된 아미노산 잔기인 알라닌으로 돌연변이시키고, 인 비트로(in vitro) 인산화를 수행하였다(도 1E). [γ-32P]의 F1 단편에의 삽입은 S212A의 돌연변이에 의해 감소하였다. F2 단편에서, 신호의 강도는 단일 아미노산 돌연변이에 의해 크게 감소하지 않았으나, 모든 잠재적인 인산화 부위의 돌연변이에 의해 크게 감소하였다. 이러한 결과들은 Clast4의 여러 부위가 PKA에 의해 인산화될 수 있다는 것을 강하게 시사한다. Using a web-based program (http://scansite.mit.edu/ and http://www.phosphosite.org/homeAction.do), potential PKA phosphorylation sites S212, S256, S258 and S352 sites were found. . Several truncated forms of Clast4 were created to find the PKA phosphorylation site in Clast4. The F1 and F2 fragments were phosphorylated by the PKA catalytic subunit in the [γ-32P] ATP-containing reaction buffer, but neither F3 nor F4 was phosphorylated (Fig. 1D), which is the amino-terminal half of Clast4 being the main PKA phosphorylation site. It means that it contains. To determine the phosphorylated residues of the F1 and F2 fragments, S212 of F1 and T256, S258 and S352 of F2 were mutated to alanine, a non-phosphorylated amino acid residue, and phosphorylation was performed in vitro (Fig. 1E. ). The insertion of [γ-32P] into the F1 fragment was reduced by mutation of S212A. In the F2 fragment, the intensity of the signal was not significantly reduced by a single amino acid mutation, but by mutations in all potential phosphorylation sites. These results strongly suggest that several sites of Clast4 can be phosphorylated by PKA.

사람 4E-T의 고갈은 P-body의 크기를 크게 감소시키며[Ferraiuolo, M. A., et al. 2005. A role for the eIF4Ebinding protein 4E-T in P-body formation and mRNA decay. J cell biol 170, 913-924], 산화적 스트레스는 JNK (c-Jun N-terminal kinase)에 의해 여러 부위의 사람 4E-T 인산화를 촉진시켜 더 큰 P-body의 형성을 야기한다는 사실이 보고되었다[Cargnello, M., et al. 2012. Phosphorylation of the eukaryotic translation initiation factor 4E-transporter (4E-T) by c-Jun N-terminal kinase promotes stress-dependent P-body assembly. Mol Cell Biol 32, 4572-4584]. 추가로 효모에서 PKA 촉매 서브유닛 Tpk는 지수적으로 증가하는 세포들에서 번역 개시인자인 Pab1 및 Rps3와 관련이 있고, Rpg1 및 eIF4G1 단백질을 조절하여 cap-의존적 번역을 조절하며, 글루코스 고갈(starvation)은 Tpk와 개시인자들 사이의 상호작용을 없애 그것의 P-body 내 축적을 야기한다[Tudisca, V., et al. 2012. PKA isoforms coordinate mRNA fate during nutrient starvation. J Cell Sci 125, 5221-5232]. 따라서 본 발명자들은 GFP-융합 야생형 또는 인산화 부위 돌연변이 Clast4를 이용하여 PKA 인산화가 Clast4의 P-body 위치화(localization)에 미치는 영향을 조사하였다. HEK293T 세포에 트랜스펙션 시킨 후, Clast4는 P-body와 세포질에서 발견되었다(도 1F). PKA 억제제(H89)의 처리로 P-body 크기가 증가하였으나, Clast4 상의 PKA 부위의 돌연변이는 그것의 위치화나 P-body의 크기에 영향을 미치지 않았다(도 1F).
Depletion of human 4E-T significantly reduces the size of the P-body [Ferraiuolo, MA, et al. 2005. A role for the eIF4Ebinding protein 4E-T in P-body formation and mRNA decay. J cell biol 170, 913-924], reported that oxidative stress promotes the phosphorylation of human 4E-T at various sites by JNK (c-Jun N-terminal kinase), leading to the formation of a larger P-body. [Cargnello, M., et al. 2012. Phosphorylation of the eukaryotic translation initiation factor 4E-transporter (4E-T) by c-Jun N-terminal kinase promotes stress-dependent P-body assembly. Mol Cell Biol 32, 4572-4584]. In addition, in yeast, the PKA catalytic subunit Tpk is associated with the translation initiating factors Pab1 and Rps3 in exponentially increasing cells, regulates cap-dependent translation by regulating Rpg1 and eIF4G1 proteins, and glucose starvation. Eliminates the interaction between Tpk and initiating factors, causing its accumulation in the P-body [Tudisca, V., et al. 2012. PKA isoforms coordinate mRNA fate during nutrient starvation. J Cell Sci 125, 5221-5232]. Therefore, the present inventors investigated the effect of PKA phosphorylation on the P-body localization of Clast4 using GFP-fused wild type or phosphorylation site mutant Clast4. After transfection into HEK293T cells, Clast4 was found in P-body and cytoplasm (FIG. 1F). Treatment of the PKA inhibitor (H89) increased the size of the P-body, but mutations in the PKA site on Clast4 did not affect its localization or the size of the P-body (FIG. 1F).

<< 실시예Example 3> 3>

eIF4E1eIF4E1 및 And CPEBCPEB 와 Wow Clast4Clast4 의 상호작용Interaction of

사람 4E-T는 또 다른 eIF4E-결합 단백질인 Maskin과 유사한 역할을 하여, Cap 구조의 eIF4E와 mRNA의 3’UTR 상의 CPEB를 연결시켜 번역의 개시를 조절한다. 하지만, 포유류에서 Clast4와 CPEB 및 eIF4E의 상호작용은 보고된 바 없으므로, 본 발명자들은 이들의 결합 특성을 조사하였고, 공동-면역침전 결과 Clast4는 CPEB, eIF4E1A, 및 eIF4E1B와 결합할 수 있었다(도 2A). eIF4E1A는 eIF4E-결합 모티프인 YXXXXLΦ를 가진 단백질과 결합할 수 있으므로, Clast4의 대응되는 티로신 잔기를 알라닌으로 치환하고, Clast4 및 eIF4E1A 또는 eIF4E1B와의 결합 특이성을 관찰하였다. Clast4와 eIF4E1A 사이의 결합 친화력(binding affinity)은 위 돌연변이에 의해 급격히 감소하였으나, eIF4E1B에 대한 결합 친화력은 크게 감소하지 않았다(도 2B). 이러한 결과들은 Clast4가 다른 방식의 eIF4E-결합 모티프 의존성에 의해 eIF4E1들과 결합할 수 있다는 것을 보여준다. Clast4의 여러 절단형(truncated form)들과의 공동-면역침전 결과 Clast4의 아미노 말단이 eIF4E1B의 결합 부위로 확인되었다(도 2C). Clast4가 eIF4E1s 및 CPEB와 결합할 수 있다는 사실을 고려하면, Clast4는 mRNA 번역 조절에 관여할 수 있다는 것을 알 수 있다. Human 4E-T plays a similar role to Maskin, another eIF4E-binding protein, and regulates the initiation of translation by linking eIF4E of the Cap structure with CPEB on the 3'UTR of mRNA. However, since the interaction of Clast4 with CPEB and eIF4E in mammals has not been reported, the present inventors investigated their binding properties, and as a result of co-immunoprecipitation, Clast4 was able to bind CPEB, eIF4E1A, and eIF4E1B (Fig. 2A). ). Since eIF4E1A can bind to a protein having the eIF4E-binding motif YXXXXLΦ, the corresponding tyrosine residue of Clast4 was substituted with alanine, and the binding specificity with Clast4 and eIF4E1A or eIF4E1B was observed. The binding affinity between Clast4 and eIF4E1A was rapidly decreased by the above mutation, but the binding affinity to eIF4E1B was not significantly decreased (FIG. 2B). These results show that Clast4 can bind to eIF4E1s by different ways of eIF4E-binding motif dependence. As a result of co-immunoprecipitation with various truncated forms of Clast4, the amino terminus of Clast4 was identified as the binding site of eIF4E1B (FIG. 2C). Considering the fact that Clast4 can bind eIF4E1s and CPEB, it can be seen that Clast4 may be involved in the regulation of mRNA translation.

4E-BP와 eIF4E의 결합은 mTOR 신호전달 경로에 의한 인산화에 의존적이며, 체세포 분열에서 과-인산화된(hyper-phosphorylated) 사람 4E-T는 eIF4E에 대해 약한 친화력을 가진다[Pyronnet, S., et al. 2001. Suppression of cap-dependent translation in mitosis. Genes Dev 15, 2083-2093]. Clast4는 마우스 난모세포 성숙 과정의 MI 및 MII기에서 알 수 없는 키나아제에 의해 인산화된다[Villaescusa, J. C., et al. 2006. Clast4, the murine homologue of human eIF4E-Transporter, is highly expressed in developing oocytes and post-translationally modified at meiotic maturation. Gene 367, 101-109]. 따라서, Clast4 인산화가 eIF4E와의 결합 특성에 미치는 영향을 관찰하였다. 공동-면역침전 결과, Clast4의 잠재적인 PKA 인산화 부위의 돌연변이는 Clast4와 eIF4E 사이의 결합 능력에 영향을 미치지 않았다(도 2D). 그러나, Aurora A 키나아제, Plk1, Greatwall 키나아제, 및 MAP 키나아제와 같은 여러 키나아제가 난모세포 성숙의 MIMII 전이(transition) 과정 동안 활성화되었기 때문에, 이러한 키나아제들은 Clast4 인산화에 관여할 수 있다. 실제로, PKA, Plk1, Aurora A 키나아제, 및 cdc2/cyclin B를 포함하는 여러 키나아제들은 적어도 인 비트로(in vitro)에서 Clast4를 인산화할 수 있다(도 1).
Binding of 4E-BP and eIF4E is dependent on phosphorylation by the mTOR signaling pathway, and hyper-phosphorylated human 4E-T in somatic cell division has a weak affinity for eIF4E [Pyronnet, S., et al. al. 2001. Suppression of cap-dependent translation in mitosis. Genes Dev 15, 2083-2093]. Clast4 is phosphorylated by an unknown kinase in the MI and MII stages of mouse oocyte maturation [Villaescusa, JC, et al. 2006. Clast4, the murine homologue of human eIF4E-Transporter, is highly expressed in developing oocytes and post-translationally modified at meiotic maturation. Gene 367, 101-109]. Therefore, the effect of Clast4 phosphorylation on the binding properties with eIF4E was observed. As a result of co-immunoprecipitation, mutations in the potential PKA phosphorylation site of Clast4 did not affect the binding capacity between Clast4 and eIF4E (Fig. 2D). However, since several kinases such as Aurora A kinase, Plk1, Greatwall kinase, and MAP kinase were activated during the MIMII transition of oocyte maturation, these kinases may be involved in Clast4 phosphorylation. Indeed, several kinases including PKA, Plk1, Aurora A kinase, and cdc2/cyclin B are capable of phosphorylating Clast4 at least in vitro (FIG. 1).

<< 실시예Example 4> 4>

Clast4Clast4 의 과발현에 의한 세포증식 지연 효과Effect of delaying cell proliferation by overexpression of

4E-BP는 eIF4E에 결합하여 번역 개시를 방해하고, 4E-BP의 발현 수준은 발암 활성과 관련이 있기 때문에[She, Q. B., et al. 2010. 4E-BP1 is a key effector of the oncogenic activation of the AKT and ERK signaling pathways that integrates their function in tumors. Cancer Cell 18, 39-51], 본 발명자들은 Clast4의 과발현이 세포증식에 영향을 미치는지를 확인하였다. 4E-BP의 과발현은 GFP 과발현에 비해 세포증식을 정상 성장의 30%까지 감소시켰으며, Clast4의 과발현 역시 세포증식을 4E-BP와 유사한 수준으로 감소시켰다(도 3). 이러한 결과는 Clast4가 4E-BP와 유사한 mRNA 번역 조절인자로 기능한다는 것을 보여준다.
4E-BP binds to eIF4E and interferes with translation initiation, and the expression level of 4E-BP is associated with carcinogenic activity [She, QB, et al. 2010. 4E-BP1 is a key effector of the oncogenic activation of the AKT and ERK signaling pathways that integrates their function in tumors. Cancer Cell 18, 39-51], the present inventors confirmed whether overexpression of Clast4 affects cell proliferation. Overexpression of 4E-BP reduced cell proliferation to 30% of normal growth compared to overexpression of GFP, and overexpression of Clast4 also reduced cell proliferation to a level similar to that of 4E-BP (FIG. 3). These results show that Clast4 functions as an mRNA translation regulator similar to 4E-BP.

이제까지 본 발명에 대하여 그 바람직한 실시 예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.
So far, the present invention has been looked at around its preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the above description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

<110> Inje University Industry-Academic Cooperation <120> Composition for Reducing Cell Proliferation Comprising Clast4 <130> PN1412-372 <160> 4 <170> KopatentIn 2.0 <210> 1 <211> 983 <212> PRT <213> Artificial Sequence <220> <223> Clast4 protein <400> 1 Met Glu Lys Ser Val Ala Glu Thr Glu Asn Gly Asp Ala Phe Leu Glu 1 5 10 15 Leu Lys Lys Leu Pro Thr Ser Lys Ser Pro His Arg Tyr Thr Lys Glu 20 25 30 Glu Leu Leu Asp Ile Lys Glu Arg Pro Tyr Ser Lys Gln Arg Pro Ser 35 40 45 Cys Leu Ser Glu Lys Tyr Asp Ser Asp Gly Val Trp Asp Pro Glu Lys 50 55 60 Trp His Ala Ser Leu Tyr Pro Ala Ser Gly Arg Ser Ser Pro Val Glu 65 70 75 80 Ser Leu Lys Lys Glu Ser Glu Ser Asp Arg Pro Ser Leu Val Arg Arg 85 90 95 Ile Ala Asp Pro Arg Glu Arg Val Lys Glu Asp Asp Leu Asp Val Val 100 105 110 Leu Ser Pro Gln Arg Arg Ser Phe Gly Gly Gly Cys His Val Thr Ala 115 120 125 Ala Val Ser Ser Arg Arg Ser Gly Ser Pro Leu Glu Lys Asp Ser Asp 130 135 140 Gly Leu Arg Leu Leu Gly Gly Arg Arg Ile Gly Ser Gly Arg Ile Ile 145 150 155 160 Ser Ala Arg Ala Phe Glu Lys Asp His Arg Leu Ser Asp Lys Asp Leu 165 170 175 Arg Asp Leu Arg Asp Arg Asp Arg Glu Arg Asp Tyr Lys Asp Lys Arg 180 185 190 Phe Arg Arg Glu Phe Gly Asp Ser Lys Arg Val Phe Gly Glu Arg Arg 195 200 205 Arg Asn Asp Ser Tyr Thr Glu Glu Glu Pro Glu Trp Phe Ser Ala Gly 210 215 220 Pro Thr Ser Gln Ser Glu Thr Ile Glu Leu Thr Gly Phe Asp Asp Lys 225 230 235 240 Ile Leu Glu Glu Asp His Lys Gly Arg Lys Arg Thr Arg Arg Arg Thr 245 250 255 Ala Ser Val Lys Glu Gly Ile Val Glu Cys Asn Gly Gly Val Ala Glu 260 265 270 Glu Asp Glu Val Glu Val Ile Leu Ala Gln Glu Pro Ser Ala Asp Gln 275 280 285 Glu Val Pro Arg Asp Val Ile Leu Pro Glu Gln Ser Pro Gly Glu Phe 290 295 300 Asp Phe Asn Glu Phe Phe Asn Leu Asp Lys Val Pro Cys Leu Ala Ser 305 310 315 320 Met Ile Glu Asp Val Leu Gly Glu Gly Ser Val Ser Ala Ser Arg Phe 325 330 335 Ser Arg Trp Phe Ser Asn Pro Ser Arg Ser Gly Ser Arg Ser Ser Ser 340 345 350 Leu Gly Ser Thr Pro His Glu Glu Leu Glu Arg Leu Ala Gly Leu Glu 355 360 365 Gln Ala Val Leu Ser Pro Gly Gln Asn Ser Gly Asn Tyr Phe Ala Pro 370 375 380 Ile Pro Ser Glu Asp His Ala Glu Asn Lys Val Asp Ile Leu Glu Met 385 390 395 400 Leu Gln Lys Ala Lys Val Asp Leu Lys Pro Leu Leu Ser Ser Leu Ser 405 410 415 Ala Asn Lys Glu Lys Leu Lys Glu Ser Ser His Ser Gly Val Val Leu 420 425 430 Ser Val Glu Glu Val Glu Ala Gly Leu Lys Gly Leu Lys Val Asp Gln 435 440 445 Gln Met Lys Asn Ser Thr Pro Phe Met Ala Glu His Leu Glu Glu Thr 450 455 460 Leu Ser Ala Ala Ser Ser Asn Arg Gln Leu Lys Lys Asp Gly Asp Met 465 470 475 480 Thr Ala Phe Asn Lys Leu Val Asn Thr Met Lys Ala Ser Gly Thr Leu 485 490 495 Pro Thr Gln Pro Lys Val Ser Arg Asn Val Glu Ser His Leu Leu Ala 500 505 510 Pro Ala Glu Ile Pro Gly Gln Pro Val Ser Lys Asn Ile Leu Gln Glu 515 520 525 Leu Leu Gly Gln Pro Val Gln Arg Pro Ala Ser Ser Asn Leu Leu Ser 530 535 540 Gly Leu Met Gly Ser Leu Glu Ala Thr Ala Ser Leu Leu Ser Gln Arg 545 550 555 560 Ala Pro Ser Pro Pro Met Ser Gln Val Phe Arg Thr Gln Ala Ala Ser 565 570 575 Ala Asp Tyr Leu His Pro Arg Ile Pro Ser Pro Ile Gly Phe Pro Ser 580 585 590 Gly Pro Gln Gln Leu Leu Gly Asp Pro Phe Gln Gly Met Arg Lys Pro 595 600 605 Met Ser Pro Val Ser Ala Gln Met Ser Gln Leu Glu Leu Gln Gln Ala 610 615 620 Ala Leu Glu Gly Leu Ala Leu Pro His Asp Leu Ala Val Gln Thr Ala 625 630 635 640 Pro Phe Tyr Gln Pro Gly Phe Ser Lys Pro Gln Val Asp Arg Thr Arg 645 650 655 Asp Gly Leu Arg Asn Arg Gln Gln Arg Met Ser Lys Ser Pro Ala Pro 660 665 670 Met His Gly Gly Asn Ser Ser Ser Pro Ala Pro Ala Ala Ser Ile Thr 675 680 685 Ser Met Leu Ser Pro Ser Phe Thr Pro Thr Ser Val Ile Arg Lys Met 690 695 700 Tyr Glu Ser Arg Glu Lys Thr Lys Glu Glu Met Ala Pro Gly Met Val 705 710 715 720 Val Pro Gly Asp Gly Lys Glu Asp Thr Gln Lys Thr Ser Glu Glu Asn 725 730 735 Leu Leu Ser Ser Asn Pro Ile Pro Asn Thr Asp Gln Asp Ser Ser Thr 740 745 750 Thr Asn Pro Lys Leu Ser Thr Leu Gln Arg Ser Ser Cys Ser Thr Pro 755 760 765 Leu Ser Gln Thr Ser Arg Tyr Thr Lys Glu Gln Asp Tyr Arg Pro Lys 770 775 780 Thr Ala Gly Arg Lys Thr Pro Thr Leu Ala Ser Pro Val Pro Gly Thr 785 790 795 800 Pro Phe Leu Arg Pro Thr His Gln Val Pro Leu Val Pro His Val Pro 805 810 815 Ile Val Arg Pro Ala His Gln Leu His Pro Gly Leu Val Gln Arg Leu 820 825 830 Ile Ala Gln Gly Val His Pro Gln His Leu Pro Ser Leu Leu Gln Ala 835 840 845 Gly Val Leu Pro Pro Gly Ile Asp Met Ala Pro Leu Gln Gly Leu Ser 850 855 860 Gly Pro Leu Leu Gly Gln Pro Leu Tyr Pro Leu Val Ser Ala Ala Ser 865 870 875 880 His Pro Leu Leu Asn Pro Arg Pro Gly Thr Pro Leu His Leu Ala Val 885 890 895 Met Gln Gln Gln Leu Gln Arg Ser Val Leu His Pro Pro Gly Ser Ser 900 905 910 Ser Gln Ala Ala Ala Ile Ser Val Gln Thr Pro Gln Asn Val Pro Ser 915 920 925 Arg Ser Gly Met Pro His Met His Ser Gln Leu Glu His Arg Thr Ser 930 935 940 Gln Arg Ser Ser Ser Pro Val Gly Leu Ala Lys Trp Phe Gly Ser Asp 945 950 955 960 Val Leu Gln Gln Pro Leu Pro Ser Met Pro Thr Lys Val Ile Ser Val 965 970 975 Asp Glu Leu Glu Tyr Arg Gln 980 <210> 2 <211> 3792 <212> DNA <213> Artificial Sequence <220> <223> Clast4 coding gene <400> 2 ttcttccgga ggagcctcta gcccggggcc tgggtgaggg gccaggggtt cgttggagga 60 gtggagtttc ccagaggagg cccgcaccgg ccctccctcg ccgcggacgc ccctcacatg 120 gccacaggtg tggccggctg agagaggtgc tctccggccg gttccccagc ccctctcgct 180 cgggtaataa ggggcagctg gtgccatcca ttgacaagtg acaggcactt tccagcagcc 240 cgacttgccg gatcggcagc ccaaccagtc ctacctctac tctactccgg gaaacttcac 300 caccaccgcc gccaggtgca aaaatattgt acaccaagaa gccatggaga aaagtgtggc 360 tgaaacagaa aatggagatg ctttccttga gctgaagaaa ctgcccacct ccaaatcccc 420 ccatcgctat acgaaagagg agctcttgga tataaaagaa cgtccatact ccaagcagag 480 gccatcatgc ctctctgaaa aatatgacag tgatggtgtc tgggaccctg agaagtggca 540 tgcctctctc tacccagcct cggggcggag ctcaccagtg gaaagtctga agaaagagtc 600 agaatccgat cgaccttctc tggtgcgcag aatagcagat ccacgagagc gagtgaaaga 660 agatgactta gatgttgttc tcagcccaca gagacgaagc tttggaggtg gctgccatgt 720 gacagctgct gttagctccc gacgatctgg cagtccattg gagaaagaca gtgatggact 780 tcgcctgctt ggtggacgaa gaattggcag tggaaggata atttctgccc gggcctttga 840 aaaggatcac cgtctcagtg ataaggacct gcgagacttg agagaccgag acagagagag 900 ggactacaag gacaaacgtt tcaggaggga gtttggagat agtaaacgtg tctttggtga 960 gcgtagaaga aacgattcat acactgagga agaaccagaa tggttttcag ctggacccac 1020 cagtcagtct gaaaccatag agctgactgg ctttgatgat aagattctag aggaggatca 1080 caaaggcaga aaaagaacaa ggcgacggac tgcctctgtg aaggaaggca tagtggagtg 1140 caatggaggg gtggctgaag aggacgaggt ggaagtcatt ctggcccagg agccctctgc 1200 tgatcaggaa gttccaaggg atgtcatcct acctgagcag tccccaggag aatttgactt 1260 taatgaattc tttaaccttg ataaggtgcc atgcttggct tcgatgatag aagatgttct 1320 gggggaagga tcggtctctg ccagccgctt cagtaggtgg ttctctaacc caagccggtc 1380 agggagcaga tccagcagcc ttggatccac accccatgaa gaacttgaga gactggcagg 1440 tttggagcaa gccgtcctct ctcctggaca gaactcgggg aattattttg ctcctatacc 1500 atcggaagac catgctgaaa ataaagtgga cattttagaa atgctacaga aagccaaagt 1560 agatttaaaa cctcttcttt ctagcctttc tgcaaataaa gaaaaactta aggagagctc 1620 acattcaggg gttgtacttt cagtggaaga ggtagaagca gggctgaagg gcttgaaagt 1680 ggaccagcaa atgaagaatt caactccctt catggcagaa catttagagg agaccctgag 1740 tgctgcgagt agcaatagac agctcaagaa agatggagat atgactgcat tcaacaagct 1800 agtgaacacc atgaaggcaa gtggaacttt gcctacccag cccaaagtca gtcgaaacgt 1860 tgaaagtcac ttattggccc ctgctgagat tccaggccag cctgtttcta aaaacatcct 1920 acaggaactt ctgggtcaac cggttcagag acccgcttct tccaatcttc tcagtggcct 1980 tatggggagc ttggaggcta ctgcatcttt attgagccaa agagcgccct ctcctccaat 2040 gtcacaggta tttcgaactc aagcagcctc agcagactac cttcatccaa ggataccatc 2100 accaataggt ttcccttcag gaccccagca actacttgga gatccattcc aaggcatgcg 2160 caagcccatg agtcctgtct cagcccagat gagccagcta gaattgcagc aagctgcttt 2220 ggaggggctg gccctaccac atgaccttgc agtccagaca gcacccttct accagcctgg 2280 ttttagcaaa ccacaagtgg acagaaccag agatgggctc agaaacaggc aacagcgaat 2340 gtcaaagtca ccagcaccca tgcatggtgg aaattcctct tccccagctc ctgctgcctc 2400 catcacaagc atgctttctc cttccttcac ccctacctca gtgatccgga agatgtatga 2460 aagcagagag aaaaccaagg aggaaatggc acctggaatg gtggttcctg gtgatggtaa 2520 agaggatact cagaagacca gcgaagagaa cctgctgtca tccaatccca tacccaatac 2580 tgatcaagac tcttctacga caaatcctaa gctttcaaca ttacagcggt cttcatgttc 2640 caccccactc tcccagacca gccgttatac caaagaacaa gattatcgac ctaaaacagc 2700 aggaaggaaa acacctacct tggcatcccc agtcccagga acaccttttc tccgccctac 2760 ccaccaagtt ccccttgtcc ctcatgttcc tattgttcga cctgcacacc agcttcaccc 2820 aggattagtc caaaggttga tagcccaagg ggtacatcca cagcatcttc caagtttgct 2880 ccaagctggt gtgcttcctc ctgggataga catggctcct ttacaggggc tatctggccc 2940 actcctgggt caacccttgt accctttggt ctctgctgct agtcaccctc tcctaaaccc 3000 tcgtcctggg acccctctgc atttggcggt gatgcagcag cagctacagc gctcagttct 3060 gcatcctcca ggctctagtt cccaggcagc agctatcagc gttcagactc ctcagaatgt 3120 acccagccgg tcgggcatgc cccacatgca ctcccagctg gagcatcgta ccagccaaag 3180 gagcagctcc cctgtgggcc ttgccaaatg gtttggctca gatgtgctac agcagcctct 3240 gccctccatg cccaccaaag tcatcagtgt agatgaactg gaatatagac agtgaagagg 3300 gcaggctggc tcacccatac ctggacctgt ggtgacaccc tggtcatgac tctcattccc 3360 tctttgtaat gggcttttac attggagcac actatgtgaa gatgtttagg ggatccacat 3420 acctaccatg atctacatta tgacagaagg ctgttaaatc gaatgaacct acatggttca 3480 aatacaaggg atacaagatt gtcagtcctg gaagtctttc ttttataaaa tatgtgaatg 3540 aagtgttggt gtcttctaga ggtgacacct aagggttctg aaaaaataaa atgtatagac 3600 ccttatgtac agacctgtgt ataaactttt gtacatacaa atagggtagc tttttttgaa 3660 cttatacata cagctgtaca taaagtaact atcagttagg cttgtgtcaa ctgtttggat 3720 ttttttcact tgaatatttg ggactttttc ttttggttta ttaaaagtta catatgccac 3780 gtgtgtgaac ga 3792 <210> 3 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> eIF4E1B forward primer <400> 3 tagcccgggg taccatgaac aaagttgagg gtggagggc 39 <210> 4 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> eIF4E1B reverse primer <400> 4 ttaacgcggc cgccaccaca aacttgtttg ctaag 35 <110> Inje University Industry-Academic Cooperation <120> Composition for Reducing Cell Proliferation Comprising Clast4 <130> PN1412-372 <160> 4 <170> KopatentIn 2.0 <210> 1 <211> 983 <212> PRT <213> Artificial Sequence <220> <223> Clast4 protein <400> 1 Met Glu Lys Ser Val Ala Glu Thr Glu Asn Gly Asp Ala Phe Leu Glu 1 5 10 15 Leu Lys Lys Leu Pro Thr Ser Lys Ser Pro His Arg Tyr Thr Lys Glu 20 25 30 Glu Leu Leu Asp Ile Lys Glu Arg Pro Tyr Ser Lys Gln Arg Pro Ser 35 40 45 Cys Leu Ser Glu Lys Tyr Asp Ser Asp Gly Val Trp Asp Pro Glu Lys 50 55 60 Trp His Ala Ser Leu Tyr Pro Ala Ser Gly Arg Ser Ser Pro Val Glu 65 70 75 80 Ser Leu Lys Lys Glu Ser Glu Ser Asp Arg Pro Ser Leu Val Arg Arg 85 90 95 Ile Ala Asp Pro Arg Glu Arg Val Lys Glu Asp Asp Leu Asp Val Val 100 105 110 Leu Ser Pro Gln Arg Arg Ser Phe Gly Gly Gly Cys His Val Thr Ala 115 120 125 Ala Val Ser Ser Arg Arg Ser Gly Ser Pro Leu Glu Lys Asp Ser Asp 130 135 140 Gly Leu Arg Leu Leu Gly Gly Arg Arg Ile Gly Ser Gly Arg Ile Ile 145 150 155 160 Ser Ala Arg Ala Phe Glu Lys Asp His Arg Leu Ser Asp Lys Asp Leu 165 170 175 Arg Asp Leu Arg Asp Arg Asp Arg Glu Arg Asp Tyr Lys Asp Lys Arg 180 185 190 Phe Arg Arg Glu Phe Gly Asp Ser Lys Arg Val Phe Gly Glu Arg Arg 195 200 205 Arg Asn Asp Ser Tyr Thr Glu Glu Glu Pro Glu Trp Phe Ser Ala Gly 210 215 220 Pro Thr Ser Gln Ser Glu Thr Ile Glu Leu Thr Gly Phe Asp Asp Lys 225 230 235 240 Ile Leu Glu Glu Asp His Lys Gly Arg Lys Arg Thr Arg Arg Arg Thr 245 250 255 Ala Ser Val Lys Glu Gly Ile Val Glu Cys Asn Gly Gly Val Ala Glu 260 265 270 Glu Asp Glu Val Glu Val Ile Leu Ala Gln Glu Pro Ser Ala Asp Gln 275 280 285 Glu Val Pro Arg Asp Val Ile Leu Pro Glu Gln Ser Pro Gly Glu Phe 290 295 300 Asp Phe Asn Glu Phe Phe Asn Leu Asp Lys Val Pro Cys Leu Ala Ser 305 310 315 320 Met Ile Glu Asp Val Leu Gly Glu Gly Ser Val Ser Ala Ser Arg Phe 325 330 335 Ser Arg Trp Phe Ser Asn Pro Ser Arg Ser Gly Ser Arg Ser Ser Ser 340 345 350 Leu Gly Ser Thr Pro His Glu Glu Leu Glu Arg Leu Ala Gly Leu Glu 355 360 365 Gln Ala Val Leu Ser Pro Gly Gln Asn Ser Gly Asn Tyr Phe Ala Pro 370 375 380 Ile Pro Ser Glu Asp His Ala Glu Asn Lys Val Asp Ile Leu Glu Met 385 390 395 400 Leu Gln Lys Ala Lys Val Asp Leu Lys Pro Leu Leu Ser Ser Leu Ser 405 410 415 Ala Asn Lys Glu Lys Leu Lys Glu Ser Ser His Ser Gly Val Val Leu 420 425 430 Ser Val Glu Glu Val Glu Ala Gly Leu Lys Gly Leu Lys Val Asp Gln 435 440 445 Gln Met Lys Asn Ser Thr Pro Phe Met Ala Glu His Leu Glu Glu Thr 450 455 460 Leu Ser Ala Ala Ser Ser Asn Arg Gln Leu Lys Lys Asp Gly Asp Met 465 470 475 480 Thr Ala Phe Asn Lys Leu Val Asn Thr Met Lys Ala Ser Gly Thr Leu 485 490 495 Pro Thr Gln Pro Lys Val Ser Arg Asn Val Glu Ser His Leu Leu Ala 500 505 510 Pro Ala Glu Ile Pro Gly Gln Pro Val Ser Lys Asn Ile Leu Gln Glu 515 520 525 Leu Leu Gly Gln Pro Val Gln Arg Pro Ala Ser Ser Asn Leu Leu Ser 530 535 540 Gly Leu Met Gly Ser Leu Glu Ala Thr Ala Ser Leu Leu Ser Gln Arg 545 550 555 560 Ala Pro Ser Pro Pro Met Ser Gln Val Phe Arg Thr Gln Ala Ala Ser 565 570 575 Ala Asp Tyr Leu His Pro Arg Ile Pro Ser Pro Ile Gly Phe Pro Ser 580 585 590 Gly Pro Gln Gln Leu Leu Gly Asp Pro Phe Gln Gly Met Arg Lys Pro 595 600 605 Met Ser Pro Val Ser Ala Gln Met Ser Gln Leu Glu Leu Gln Gln Ala 610 615 620 Ala Leu Glu Gly Leu Ala Leu Pro His Asp Leu Ala Val Gln Thr Ala 625 630 635 640 Pro Phe Tyr Gln Pro Gly Phe Ser Lys Pro Gln Val Asp Arg Thr Arg 645 650 655 Asp Gly Leu Arg Asn Arg Gln Gln Arg Met Ser Lys Ser Pro Ala Pro 660 665 670 Met His Gly Gly Asn Ser Ser Ser Pro Ala Pro Ala Ala Ser Ile Thr 675 680 685 Ser Met Leu Ser Pro Ser Phe Thr Pro Thr Ser Val Ile Arg Lys Met 690 695 700 Tyr Glu Ser Arg Glu Lys Thr Lys Glu Glu Met Ala Pro Gly Met Val 705 710 715 720 Val Pro Gly Asp Gly Lys Glu Asp Thr Gln Lys Thr Ser Glu Glu Asn 725 730 735 Leu Leu Ser Ser Asn Pro Ile Pro Asn Thr Asp Gln Asp Ser Ser Thr 740 745 750 Thr Asn Pro Lys Leu Ser Thr Leu Gln Arg Ser Ser Cys Ser Thr Pro 755 760 765 Leu Ser Gln Thr Ser Arg Tyr Thr Lys Glu Gln Asp Tyr Arg Pro Lys 770 775 780 Thr Ala Gly Arg Lys Thr Pro Thr Leu Ala Ser Pro Val Pro Gly Thr 785 790 795 800 Pro Phe Leu Arg Pro Thr His Gln Val Pro Leu Val Pro His Val Pro 805 810 815 Ile Val Arg Pro Ala His Gln Leu His Pro Gly Leu Val Gln Arg Leu 820 825 830 Ile Ala Gln Gly Val His Pro Gln His Leu Pro Ser Leu Leu Gln Ala 835 840 845 Gly Val Leu Pro Pro Gly Ile Asp Met Ala Pro Leu Gln Gly Leu Ser 850 855 860 Gly Pro Leu Leu Gly Gln Pro Leu Tyr Pro Leu Val Ser Ala Ala Ser 865 870 875 880 His Pro Leu Leu Asn Pro Arg Pro Gly Thr Pro Leu His Leu Ala Val 885 890 895 Met Gln Gln Gln Leu Gln Arg Ser Val Leu His Pro Pro Gly Ser Ser 900 905 910 Ser Gln Ala Ala Ala Ile Ser Val Gln Thr Pro Gln Asn Val Pro Ser 915 920 925 Arg Ser Gly Met Pro His Met His Ser Gln Leu Glu His Arg Thr Ser 930 935 940 Gln Arg Ser Ser Ser Pro Val Gly Leu Ala Lys Trp Phe Gly Ser Asp 945 950 955 960 Val Leu Gln Gln Pro Leu Pro Ser Met Pro Thr Lys Val Ile Ser Val 965 970 975 Asp Glu Leu Glu Tyr Arg Gln 980 <210> 2 <211> 3792 <212> DNA <213> Artificial Sequence <220> <223> Clast4 coding gene <400> 2 ttcttccgga ggagcctcta gcccggggcc tgggtgaggg gccaggggtt cgttggagga 60 gtggagtttc ccagaggagg cccgcaccgg ccctccctcg ccgcggacgc ccctcacatg 120 gccacaggtg tggccggctg agagaggtgc tctccggccg gttccccagc ccctctcgct 180 cgggtaataa ggggcagctg gtgccatcca ttgacaagtg acaggcactt tccagcagcc 240 cgacttgccg gatcggcagc ccaaccagtc ctacctctac tctactccgg gaaacttcac 300 caccaccgcc gccaggtgca aaaatattgt acaccaagaa gccatggaga aaagtgtggc 360 tgaaacagaa aatggagatg ctttccttga gctgaagaaa ctgcccacct ccaaatcccc 420 ccatcgctat acgaaagagg agctcttgga tataaaagaa cgtccatact ccaagcagag 480 gccatcatgc ctctctgaaa aatatgacag tgatggtgtc tgggaccctg agaagtggca 540 tgcctctctc tacccagcct cggggcggag ctcaccagtg gaaagtctga agaaagagtc 600 agaatccgat cgaccttctc tggtgcgcag aatagcagat ccacgagagc gagtgaaaga 660 agatgactta gatgttgttc tcagcccaca gagacgaagc tttggaggtg gctgccatgt 720 gacagctgct gttagctccc gacgatctgg cagtccattg gagaaagaca gtgatggact 780 tcgcctgctt ggtggacgaa gaattggcag tggaaggata atttctgccc gggcctttga 840 aaaggatcac cgtctcagtg ataaggacct gcgagacttg agagaccgag acagagagag 900 ggactacaag gacaaacgtt tcaggaggga gtttggagat agtaaacgtg tctttggtga 960 gcgtagaaga aacgattcat acactgagga agaaccagaa tggttttcag ctggacccac 1020 cagtcagtct gaaaccatag agctgactgg ctttgatgat aagattctag aggaggatca 1080 caaaggcaga aaaagaacaa ggcgacggac tgcctctgtg aaggaaggca tagtggagtg 1140 caatggaggg gtggctgaag aggacgaggt ggaagtcatt ctggcccagg agccctctgc 1200 tgatcaggaa gttccaaggg atgtcatcct acctgagcag tccccaggag aatttgactt 1260 taatgaattc tttaaccttg ataaggtgcc atgcttggct tcgatgatag aagatgttct 1320 gggggaagga tcggtctctg ccagccgctt cagtaggtgg ttctctaacc caagccggtc 1380 agggagcaga tccagcagcc ttggatccac accccatgaa gaacttgaga gactggcagg 1440 tttggagcaa gccgtcctct ctcctggaca gaactcgggg aattattttg ctcctatacc 1500 atcggaagac catgctgaaa ataaagtgga cattttagaa atgctacaga aagccaaagt 1560 agatttaaaa cctcttcttt ctagcctttc tgcaaataaa gaaaaactta aggagagctc 1620 acattcaggg gttgtacttt cagtggaaga ggtagaagca gggctgaagg gcttgaaagt 1680 ggaccagcaa atgaagaatt caactccctt catggcagaa catttagagg agaccctgag 1740 tgctgcgagt agcaatagac agctcaagaa agatggagat atgactgcat tcaacaagct 1800 agtgaacacc atgaaggcaa gtggaacttt gcctacccag cccaaagtca gtcgaaacgt 1860 tgaaagtcac ttattggccc ctgctgagat tccaggccag cctgtttcta aaaacatcct 1920 acaggaactt ctgggtcaac cggttcagag acccgcttct tccaatcttc tcagtggcct 1980 tatggggagc ttggaggcta ctgcatcttt attgagccaa agagcgccct ctcctccaat 2040 gtcacaggta tttcgaactc aagcagcctc agcagactac cttcatccaa ggataccatc 2100 accaataggt ttcccttcag gaccccagca actacttgga gatccattcc aaggcatgcg 2160 caagcccatg agtcctgtct cagcccagat gagccagcta gaattgcagc aagctgcttt 2220 ggaggggctg gccctaccac atgaccttgc agtccagaca gcacccttct accagcctgg 2280 ttttagcaaa ccacaagtgg acagaaccag agatgggctc agaaacaggc aacagcgaat 2340 gtcaaagtca ccagcaccca tgcatggtgg aaattcctct tccccagctc ctgctgcctc 2400 catcacaagc atgctttctc cttccttcac ccctacctca gtgatccgga agatgtatga 2460 aagcagagag aaaaccaagg aggaaatggc acctggaatg gtggttcctg gtgatggtaa 2520 agaggatact cagaagacca gcgaagagaa cctgctgtca tccaatccca tacccaatac 2580 tgatcaagac tcttctacga caaatcctaa gctttcaaca ttacagcggt cttcatgttc 2640 caccccactc tcccagacca gccgttatac caaagaacaa gattatcgac ctaaaacagc 2700 aggaaggaaa acacctacct tggcatcccc agtcccagga acaccttttc tccgccctac 2760 ccaccaagtt ccccttgtcc ctcatgttcc tattgttcga cctgcacacc agcttcaccc 2820 aggattagtc caaaggttga tagcccaagg ggtacatcca cagcatcttc caagtttgct 2880 ccaagctggt gtgcttcctc ctgggataga catggctcct ttacaggggc tatctggccc 2940 actcctgggt caacccttgt accctttggt ctctgctgct agtcaccctc tcctaaaccc 3000 tcgtcctggg acccctctgc atttggcggt gatgcagcag cagctacagc gctcagttct 3060 gcatcctcca ggctctagtt cccaggcagc agctatcagc gttcagactc ctcagaatgt 3120 acccagccgg tcgggcatgc cccacatgca ctcccagctg gagcatcgta ccagccaaag 3180 gagcagctcc cctgtgggcc ttgccaaatg gtttggctca gatgtgctac agcagcctct 3240 gccctccatg cccaccaaag tcatcagtgt agatgaactg gaatatagac agtgaagagg 3300 gcaggctggc tcacccatac ctggacctgt ggtgacaccc tggtcatgac tctcattccc 3360 tctttgtaat gggcttttac attggagcac actatgtgaa gatgtttagg ggatccacat 3420 acctaccatg atctacatta tgacagaagg ctgttaaatc gaatgaacct acatggttca 3480 aatacaaggg atacaagatt gtcagtcctg gaagtctttc ttttataaaa tatgtgaatg 3540 aagtgttggt gtcttctaga ggtgacacct aagggttctg aaaaaataaa atgtatagac 3600 ccttatgtac agacctgtgt ataaactttt gtacatacaa atagggtagc tttttttgaa 3660 cttatacata cagctgtaca taaagtaact atcagttagg cttgtgtcaa ctgtttggat 3720 ttttttcact tgaatatttg ggactttttc ttttggttta ttaaaagtta catatgccac 3780 gtgtgtgaac ga 3792 <210> 3 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> eIF4E1B forward primer <400> 3 tagcccgggg taccatgaac aaagttgagg gtggagggc 39 <210> 4 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> eIF4E1B reverse primer <400> 4 ttaacgcggc cgccaccaca aacttgtttg ctaag 35

Claims (9)

삭제delete 삭제delete 삭제delete Clast4의 단백질 또는 Clast4 단백질을 코딩하는 유전자를 유효성분으로 포함하는 난소암 예방 또는 치료용 약학 조성물. A pharmaceutical composition for preventing or treating ovarian cancer comprising a Clast4 protein or a gene encoding a Clast4 protein as an active ingredient. 제4항에 있어서,
상기 Clast4 단백질은 서열번호 1로 표시되는 것을 특징으로 하는 약학 조성물.The method of claim 4,
The Clast4 protein is a pharmaceutical composition, characterized in that represented by SEQ ID NO: 1. 제4항에 있어서,
상기 Clast4 단백질을 코딩하는 유전자는 서열번호 2로 표시되는 것을 특징으로 하는 약학 조성물.The method of claim 4,
A pharmaceutical composition, characterized in that the gene encoding the Clast4 protein is represented by SEQ ID NO: 2. 제4항에 있어서,
상기 Clast4 단백질을 코딩하는 유전자는 상기 유전자가 세포 내에서 발현되도록 프로모터와 작동가능하게 연결된 발현벡터 내에 함유된 형태인 것을 특징으로 하는 약학 조성물.The method of claim 4,
A pharmaceutical composition, characterized in that the gene encoding the Clast4 protein is contained in an expression vector operably linked to a promoter so that the gene is expressed in cells. 제4항에 있어서,
상기 Clast4 단백질은 eIF4E1 및 CPEB와 결합하여 mRNA의 번역(translation)을 조절하는 것을 특징으로 하는 약학 조성물.The method of claim 4,
The Clast4 protein binds to eIF4E1 and CPEB to regulate mRNA translation. 삭제delete
KR1020140186935A 2014-12-23 2014-12-23 Composition for Reducing Cell Proliferation Comprising Clast4 KR102254728B1 (en)

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