Regulation of apoptosis through Caspase-8 and its interacting proteins

정용근 이화여자대학교 세포신호전달연구센터 2000 고사리 세포신호전달 심포지움 Vol. No.2

Caspase-8은 death receptor를 통한 세포죽음에서 upstream initiator caspase로 작용한다. 또한 mitochondria를 통하거나 non-death receptor signaling에서 그 역할이 보고되고 있다. Caspase-8은 대부분 cytosol에 존재하는 protein으로 wild-type을 overexpression 시켰을 때 aggregation되는 경향을 보인다. 반면 active site mutant와 processing defective mutant는 이와 다른 subcellular distribution을 보인다. 이러한 사실은 aggresome 형성을 통하여 caspase-8이 polyglutamic tack disease와 연계될 가능성을 제시하며, Caspase와 결합하는 단백질의 동정을 통하여 세포죽음을 연구하고자 한다. 또한 Caspase-8과 결합하는 Flash의 세포내 발현 pattern은 caspase-8과 비슷한 양상을 보인다. Anti-sense oligonucleotide를 이용한 approach는 Flash가 death receptor를 통한 세포죽음에 관여하고 있음을 보여주며, JB6 세포를 이용한 세포죽음에서 Caspase-8은 Trail과 Fas signaling에 의한 세포 죽음에 critical하다. 이 외에도 yeast two hybrid를 통하여 Caspase-8과 결합하는 단백질을 동정하여 그들이 세포죽음에 관여하는 바를 논하고자 한다.

천연자원으로부터의 천식치료제 개발 연구

이형규 이화여자대학교 세포신호전달연구센터 2009 고사리 세포신호전달 심포지움 Vol. No.11

천식은 증상으로는 기도염증(airway inflammation), 기도과민성(airway hyperresponsivess: AHR), 점막단백 과다분비(mucin hypersecretion)를 면역학적으로는 호산구(eosinophils)의 침윤, Th1 세포수에 비해 Th2 세포수의 증가, 활성화 비만 세포(mast cell)수의 증가를 특징으로 하는 만성기도 염증질환이다. 기도에 백혈구 중에서 과립구(granulocyte)계통인 호산구의 침윤이 특징이며, 호산구는 기도염증과 기관지 수축을 촉진하는 여러 유발물질을 생성하여 천식의 병태/생리에 매우 중요한 역할을 수행하고 있다. 알레르겐은 T 세포를 Th2 세포로의 분화를 일으키며, Th2 세포는 IL-5, GM-CSF, IL-3, IL-13, IL-4 등의 cytokine을 분비함. IL-4는 B 세포에 작용하여 IgE의 생성을 촉진시키며, 비만세포를 활성화시킨다. 천식과 COPD는 모두 호흡곤란, 기침, 가래 등의 기도 질환 증상을 나타내며, 현재까지 질환 자체에 대한 원인치료제는 없다는 공통점이 있으며, 현존하는 천식의 치료에는 염증을 억제하는 조절제(controller)와 호흡곤란증상을 완화시켜주는 완화제(Reliever) 두 가지 뿐으로 아직까지 원인치료제가 없는 상황이다. 특히, 천식관련 총 사망률과 경제부담의 상당부분을 차지하고 있는 중증의 천식(severe persistent)의 대부분은 고용량 스테로이드를 포함한 기존의 최선의 치료법을 받고 있음에도 불구하고, 여전히 천식이 조절되지 않는 문제가 있다. 한국은 빠른 경제성장 이상으로 급속하게 고령화 사회로 옮겨지고 있어서 수명연장에 따른 만성질환이 많아지고 있으며, 특히 천식은 삶의 질을 좌우하는 가장 큰 요인이 되고 있다. 천식은 어린이부터 노인에 이르는 모든 연령층에서 발생하는 난치성 질환으로 환자의 폐기능, 수면, 일상생활 등 생활 전반에 영향을 끼쳐 삶의 질과 생명을 크게 위협하고 있다. 소아의 경우 소아 5명중 1명이 천식환자며, 특히 13-14세 청소년도 10명 중 1명은 천식을 앓고 있으며, 알레르기에 기인된 소아천식 등을 방치하는 경우 학습장애, 집중력 장애, 발육장애를 초래할 수 있으므로 조기치료의 중요성이 강조되고 있다. 또한 COPD로 폐기능이 저하되고 기도가 폐색되면 호흡이 곤란하여 정상적 생활이 곤란하고 증상이 악화되는 심각한 질환임이다. 환자의 수가 증가하는 것보다 더 큰 문제가 될 수 있는 부분은 급증하는 치료비의 부담이다. 연 1-2% 정도의 유병율 증가에 비하여 치료약물 시장의 크기는 연 평균 10%에 달하는 급성장세에 있고, 2013년까지도 연 평균 8%정도의 급성장을 계속할 것으로 전망되고 있다(The asthma and COPD market outlook, Business Insights Ltd. 2005). 이는 천식 및 COPD에 대한 복합치료제 개념의 도입 및 신약의 출현에 따른 것으로, 국내의 자체 기술이 없으면 이와 관련한 국가 사회적인 부담이 더욱 커질 것으로 예상되어, 이에 대한 대책의 마련이 시급한 것으로 파악되고 있다. 천식이 후진국보다 선진국에 많이 발생하는 경향이 있으며 치료에 투입되는 사회적 비용은 에이즈와 결핵으로 인한 비용을 합친 것보다 훨씬 많은 것으로 분석하고 있다. GINA(Global Initiative for Asthma)에서 발표한 국제 치료가이드라인에는 고용량 흡입형 코르티코스테로이드를 포함한 기존의 표준임상 치료법으로 조절되지 않는 중증 알레르기성 천식 환자의 추가요법제로 항-IgE 치료법을 권장하고 있다. 그러나 치료용 항체 형태인 Xolair는 한 싸이클 투약비용이 천만원 정도에 달할 정도로 매우 고가이며, 투약방법이 매우 제한적으로 행하여지기 때문에 시장의 현실과는 거리가 있는 것이 사실이다. 따라서 Xolair와 같이 IgE를 직접 조절할 수 있으면서도 용법이 쉽고 제조 단가 역시 저렴한 약물의 개발이 매우 필요한 현실이다. 2004년 서울대학교 보건대학원에서 보고한 자료에 의하면 천식의 사회적인 비용은 진료비 약 구입비등 직접비용만 한해 9,620억원으로 파악되며, 생산성 손실 비용 등 간접비용은 1조 864억원, 그리고 삶의 질 등 무형의 비용은 2조 664억원 등 전체적으로 4조 1,148억원으로 추산됨(천식의 사회적 비용. 서울대학교 보건대학원 2004년) 조사자료에 의하면 COPD로 인한 사회 경제적 비용도 커 국내의 경우 COPD 환자의 총 진료비는 연평균 1,095억원에 달한다(호흡기학회/COPD 환자 정부 지원 강력촉구. 약업신문 2005년 11월 2일). COPD는 주로 생산성이 높은 연령대에서 나타나는데, COPD 환자의 54%가 45세 이전에 나타나고 있으며 26%는 45세-64세에서 나타나고 있다. 이와 같은 연령대에서 질환이 나타나므로 이 질환이 사회에 미치는 손실은 막대하다. 미국 경제에서 COPD 질환으로 인한 손실은 2001년 328억달러로 추산되며 2008년 550억에 달할 것으로 전망되고 있다(COPD: What Does the Future Have in Store? ⓒ Frost & Sullivan 2002년 6월 24일). 이와 같은 상황에 비춰볼 때 새로운 천식치료제 개발은 수명연장과 함께 건강한 삶을 유지시키는데 중요한 역할을 할 것이며, 사회적 비용도 크게 줄여주게 되어 국가적 부담도 감소하게 될 것이다. 이번 심포지움에서는 식물자원을 소재로 한 천식/만성기관지염 치료제 개발연구에 대한 사례를 소개하고자 한다. 안전성을 입증할 수 있는 천연자원이라면 천식과 같이 만성화되는 질환에서 장기간의 약물투여에 적합한 모델 약물이 될 수가 있으며, 임상적으로 그 효능을 입증할 수 있다면 천식치료제시장에서 새로운 대안이 될 수도 있을 것이다.

Leucine Zipper-Mediated Homodimerization of the Pak Kinase Interacting Factor, βPix : Implication for a role in cytoskeletal reorganization

Kim, Seyun,Lee, Seung-Hye,Park, Dongeun 이화여자대학교 세포신호전달연구센터 2001 고사리 세포신호전달 심포지움 Vol. No.3

Pix, a Pak-interacting exchange factor is known to be involved in the regulation of Cdc42/Rac GTPases. The 85-kDa βPix-a protein contains a SH3 domain, the tandem DH and PH domain, a proline-rich region and a GIT1-binding domain. In addition to those domains, βPix-a also contains a putative leucine zipper domain at the carboxyl terminal end. In this study, we demonstrate that the previously identified putative leucine zipper domain mediates the formation of βPix-a homodimers. Using in vitro and in vivo methodologies, we show that deletion of the leucine zipper domain is sufficient to abolish βPix-a homodimerization. In NIH3T3 fibroblast cells, expression of wild type βPix-a induces the formation of membrane ruffles. However, cells expressing the leucine zipper domain-deletion mutant could not form membrane ruffle structures. Moreover, PDGF-mediated cytoskeletal changes were completely blocked by the leucine zipper domain-deletion mutant. The results suggest that the leucine zipper domain enables βPix-a to homodimerize, and homodimerization is essential for βPix-a signaling functions leading to the cytoskeletal reorganization.

Specific cleavage of p62-PKC zeta-Par-4 ternary complex during apoptotic cell death

Shin, Jaekyoon 이화여자대학교 세포신호전달연구센터 2001 고사리 세포신호전달 심포지움 Vol. No.3

Caspases are activated during apoptosis and proteolytically degrade many cytoplasmic proteins, thereby playa central role in the execution of apoptosis. PKC zeta has been recognized as an important mediator of a survival signaling process, and separately reported to associate with p62 or with par-4. Here, we demonstrates that PKC zeta, p62, and par-4 form a ternary complex and that all three molecules are specifically cleaved during apoptotic cell death. The physiological roles of the ternary complex and its individual components are yet understood well. A multiubiquitin chain binding protein p62 seems to play an important role in sequestration of cytosolic proteasome-targeting proteins and, thus, to initiate the formation of cytosolic inclusion bodies. In addition, p62 modulates the NFkB-mediated survival signaling through interactions with PKC zeta, RIP and some TRAF family members. On the other hand, par-4(prostate apoptosis response 4) interacts with PKC zeta and inhibits its enzymatic activity, resulting in apoptosis of several cell lines thus far tested. Moreover, the incresed levels of p62 and par-4 in neurons of Alzheimer disease and ALS patients implies that these proteins are closely related to neuro-degenerative disorders caused by neuronal apoptosis. It has been found that two and one caspase target sites are present in p62 and par-4 respectively and that the major executor of these cleavages is caspase-6. Overexpression of the non-cleavable mutant of p62 increases the viability of cells and simultaneously formes intracytoplasmic inclusions. And the cleavage of par-4 seems to trigger or enhance the apoptotic activity of par-4. In the current presentation, the role of the p62-PKC zeta-par-4 ternary complex in cell death and survival signaling and its relevance to neuro-degeneration will be hypothesized and discussed.

Differential Requirement of Swi/Snf among Different Nuclear Receptors in the Transactivation Function of the Cancer-Amplified Transcription Coactivator ASC-2

Goo, Young-Hwa,Lee, Soo-kyung,Na, Soon-Young,Lee, Jae Woon 이화여자대학교 세포신호전달연구센터 2001 고사리 세포신호전달 심포지움 Vol. No.3

ASC-2 is a recently isolated transcriptional co integrator molecule, which is amplified in human cancers and stimulates transactivation by nuclear receptors, AP-1, NFĸB, SRF, and numerous other transcription factors. ASC-2 contained two nuclear receptor-interaction domains; i.e., the C-terminal LXXLL motif specifically interacting with oxysterol receptors and the N-terminal motif binding a broad range of nuclear receptors. Interestingly, ASC-2 contained a distinct, indirect androgen receptor-interacting domain that involves Rb/Swi/Snf complexes. In addition, we found that the previously defined autonomous transactivation function of ASC-2 also requires Rb/Swi/Snf. Surprisingly, Rb/Swi/Snf was required for ASC-2 to mediate transactivation by androgen and glucocorticoid receptors but not retinoid and thyroid hormone receptors. These results strongly suggest that distinct, ASC-2-containing coactivator complexes are actively assembled in a target factor-specific manner in vivo.

AHNAK-mediated cell cycle arrest

Bae, Yun Soo,Yang, Eun Young 이화여자대학교 세포신호전달연구센터 2001 고사리 세포신호전달 심포지움 Vol. No.3

It has been reported that AHNAK has an activation activity of PLCγ isozyme in the presence of arachidonic acid. Several lines of evidence indicated that either overexpression or hyper-activation of PLCγ induced cellular transformation. To investigate the roles of AHNAK in cell proliferation, AHNAK-overexpressed cell lines were established. AHNAK-expressed cells showed a growth delay and G1 phase arrest compared to control cells. Overexpression of AHNAK in cells resulted in down-regulation of c-myc and cyclin D2 expression. Furthermore, the expression of CDK inhibitor, p21^(WAF) is up-regulated in the cells. Reciprocal regulation between cyclin D2 and p21^(WAF) may contribute dephosphorylation of retinoblastoma protein(Rb) in AHNAK-overexpressed cells. We studied the effect of arachidonic acid on AHNAK-dependent cell cycle arrest in cells. The result suggested that concerted action of AHNAK and arachidonic acid induces cell cycle progression from. G0/G1 to S phase through PLCγ activation. How molecule mechanisms involving AHNAK, cell cycle-related proteins, PLCγ are integrated into physiological signal that leads for progression of cell cycle remain to be elucidated.

COMP-Ang1 Induces Long-lasting Vascular Enlargement, Blood Flow with Temporal Lymphatic Filopodia in vivo

Cho, Chung-Hyun,Koh, Gou Young,Endothelial Cell Research Group 이화여자대학교 세포신호전달연구센터 2005 고사리 세포신호전달 심포지움 Vol. No.7

Angiopoietin-1(Ang1) is a promising growth factor for therapeutic angiogenesis for ischemic tissues. The cardinal feature of Ang1-induced vascular remodeling in vivo is the enlargement of blood vessels in normal and ischemic tissues. However, little is known about the mechanisms involved in the vascular enlargement process or the specific characteristics of the enlarged vessels. Here, we examined changes in vascular remodeling in mouse trachea by systemic protein and adenoviral delivery of a soluble, stable and potent Ang1 variant, COMP-Ang1. Long-term and sustained exposure to COMP-Ang1 induced long-lasting enlargement of postcapillary venules, terminal arterioles and lymphatics with enhanced blood flow, while short-term exposure induced reversible enlargement of the vessels. COMP-Ang1-induced lymphatic enlargement and filopodia were dependent on circulating COMP-Ang1. The vascular enlargement induced by COMP-Ang1 was a result of endothelial cell proliferation. Auto-amplification of Tie2 expression could be involved in the permanent vascular enlargement induced by COMP-Ang1. Enhanced pericyte recruitment was not a feature of COMP-Ang1-induced vascular enlargement. Thus, long-lasting vascular and lymphatic enlargements could be achieved by long-term and sustained exposure of Ang1 above a certain threshold time and level. Interestingly, the mice received COMP-Ang1 showed promoted wound healing in punched hole injury of ear with enhanced angiogenesis and lymphangiogenesis. Moreover, diabetic mice received COMP-Ang1 showed promoted wound healing in full thickness wound with enhanced blood flow. Therefore, we conclude that COMP-Ang1 could be beneficial growth factor for alleviating ischemic tissues and promoting wound healing.

Protein systems in excitation-contraction (E-C) coupling

Kim, Do Han 이화여자대학교 세포신호전달연구센터 2003 고사리 세포신호전달 심포지움 Vol. No.5

In striated muscles, DHPR senses depolarization of the transverse (T)-tubules, and triggers Ca^(2+) release from SR, and hence muscle contraction. The main SR protein responsible for the Ca^(2+) release from SR is the RyR/Ca^(2+) release channel, whereas the active Ca^(2+) uptake mediated by SERCA, leads to lowing the cytosolic free Ca^(2+) concentration and hence muscle relaxation. The native RyR complex is composed of homo-tetramers of the 565-kDa protein, which is associated with FKBP in a 1:1 molar ratio. We have shown evidence that calcineurin is associated with FKBP-RyR complex and regulates the activity of RyR. The RyR in the junctional SR forms a quaternary protein complex with calsequestrin, triadin and junctin. Calsequestrin is physically anchored to the RyR by triadin and junctin. We have found evidence that the binding site of triadin in the RyR resides in the 2^(nd) transmembrane domain. The anchored calsequestrin may regulate the function of RyR during muscle contraction. Perturbation of the quaternary protein complex by overexpression of junctin leads to cardiac remodling and atrial fibrillation. Junctate is another cardiac SR protein which could regulate the function of the RyR. The overall protein systems for E-C coupling will be discussed.

mRNA Transport on the ER Subdomains in Rice Endosperm Cells

Choi, Sang-Bong 이화여자대학교 세포신호전달연구센터 2003 고사리 세포신호전달 심포지움 Vol. No.5

Rice synthesizes and accumulates two major classes of storage proteins, prolamines and glutelins, which are stored in different subcellular compartments of the secretory pathway. Prolamines are deposited directly as spherical protein granules within the ER lumen, whereas glutelins are transported from the ER to the Golgi where they are eventually packaged in protein storage vacuoles. It has been previously reported that prolamine and glutelin RNAs asymmetrically distributed within the cell. Here, an evidence will be presented that prolamine RNAs are enriched on the specific ER subdomain as an RNA signal dependent manner. To identify the cis-acting element required for the prolamine RNA targeting, transgenic rice plants expressing the modified prolamine RNAs were generated and used for in situ RT -PCR. Fluorescently-labeled signals from the transgenic lines were detected and compared with those from wild-type rice seed sections. Results showed that the transport and targeting of prolamine RNAs to the protein body ER requires specific RNA signal determinants although the presence of an initiation codon is essential. One of the RNA signal determinants is located within the 3' untranslated region and a second one resides on the upstream of coding sequences. Prolamine RNA movement was visualized using GFP-RNA interaction. Prolamine and glutelin RNAs are transported as a particles ranging in size from 0.1-0.5 um in diameter. These granules move at speeds up to 0.3-0.5 um/sec, and anchored to PB presumably mediated by cytoskeletal components. Further efforts are being made to identify the trans-acting factors that bind to prolamine RNA sequence.

Application of Proteomic Tools to Studies on Ubiquitin-dependent Proteolysis

Hwang, Ji-Won,Min, Kyoeng-Woo,Hwang, Yong-Pil,Park, Yoon,Lee, Jong-Sik,Yoon, Jong-Bok 이화여자대학교 세포신호전달연구센터 2004 고사리 세포신호전달 심포지움 Vol. No.6

The cullin-containing E3 ubiquitin ligases play an important role in regulating the abundance of key proteins involved in cellular processes such as cell cycle and c)10kine signaling. They have multisubunit modular structures in which substrate recognition and the catalysis of ubiquitination are carried out by distinct polypeptides. In a search for proteins involved in regulation of cullin-containing E3 ubiquitin ligases we immunopurified CUL4B-containing complex from HeLa cells and identified TIP120A as an associated protein by mass spectrometry. Immunoprecipitation of cullins revealed that all cullins tested specifically interacted with TIP120A. Reciprocal immunoaffinity purification of TIP120A confirmed the stable interaction of TIP120A with cullin family proteins. TIP120A formed a complex with CUL1 and Rbx1, but interfered with the binding of Skpl and F-box proteins to CUL1. TIP120A greatly reduced the ubiquitination of phosphorylated I kappa B alpha by SCF^(β-TrCP) ubiquitin ligase. These results suggest that TIP120A functions as a negative regulator of SCF E3 ubiquitin ligases and may modulate other cullin ligases in a similar fashion. In addition, we show that TIP120A binds to the unneddylated CUL1 but not the neddylated one. The association of TIP120A with CUL1 requires both the N-terminal stalk and the C-terminal globular domain of CUL1. TIP120A efficiently inhibits neddylation of CUL1 but does not affect substrate-independent ubiquitination by CUL1/Rbx1, implying that it blocks the access of Nedd8 to the conjugation site but does not interfere with the interaction of the E2 with Rbx1. Our data suggest that the association/dissociation of TIP120A coupled to neddylation/deneddylation of CUL1 may play an important role in assembly and disassembly of SCF ubiquitin ligases.