The role of phospholipase D (PLD) in antiapoptotic process and the molcular mechanism of hAP180 for inhibition of PLD

Abstract

인지질 분해 효소 D (phospholipase D, PLD)는 세포내?외부의 호르몬, 싸이토카인, 성장인자등에 의해 유발되는 항세포사, 세포내 물질이동, 세포분열등에 밀접하게 관여되어 있다. PLD는 phosphatidyl choline을 phosphatidic acid (PA)와 choline으로 가수분해 하는 효소이다. PLD의 산물인 PA는 내세포작용에서 clathrin-coated vesicles에서 clathrin의 조립에 꼭 필요한 물질이며, amphiphysins와 synaptojanin과 AP 180과 같은 단백질들도 vesicle의 구조를 형성하는데 조력하는 단백질이다. 기존연구에서 확인된 결과를 바탕으로 볼 때 주목할 것은 amphiphysins, synaptojanin, AP 180 단백질이 PLD의 활성억제에도 관여한다는 것이다. 본 논문에서는 인간 AP180 (hAP180)을 대상으로 PLD 활성억제에 중요한 구체적 motif를 찾아냈으며 hAP180에 의한 PLD 활성 억제는 암세포의 항암제 민감성을 유도 하였다. 이는 hAP180에 의한 PLD 활성 억제가 암세포의 항암제민감도를 높여 항암제 효력 증강을 시사한다. 이러한 가능성을 확인 하기 위해 첫 번째로 hAP180 유전자를 인간 cDNA pool로 부터 클로닝 하였다. 클로닝된 hAP180을 암세포주에 발현 시킨 결과 phobol-myristate-acetate (PMA)에 의해 증가된 PLD 활성을 억제 하였고 Thr-310에서 Lys-320까지의 11개 아미노산(TVTSPNSTPAK)이 PLD 활성억제에 중요함을 밝혔다. 이를 아미노말단 부분 5개(310-TVTSP-314)와 카르복실말단 6개(315-NSTPAK-320)로 나누어 치리 한 결과 아마노말단 부분의 5개 아미노산이 카르복실말단 6개 아무노산 대비 PLD 활성억제가 매우 유의하였다. 이결과를 토대로 볼 때 hAP180을 구성하는 아미노산 중 310-TVTSP-314 부위가 PLD 활성 억제에 중요한 motif임을 시사한다. 또한, AP180의 C-terminal deletion series와 N-terminal truncated mutant series와 이용한 실험결과에서도 310-TVTSP-314가 PLD 활성억제에 직접 관여하였다. hAP180의 PLD 활성억제는 두 단백질의 결합에 의해 일어남을 시사하는 결과로 PMA에 의한 PLD 활성 증가에 따라 세포안에서 PLD1과 hAP180의 colocalization이 하였다. PLD 활성 억제에 중요한 5개(310-TVTSP-314) 아미노산을 alanine(Ala)으로 각각 단일 아미노산 치환 돌연변이 연구 결과 Thr312, Ser313, Pro314 아미노산이 PLD 활성 억제에 있어 중요한 역할을 갖음이 확인 되었다. 면역침강법을 이용하여 단백질 수준에서의 상호작용을 확인 한 결과에서 Ser313이 Ala으로 치환된 돌연변이의 경우 PLD1과의 결합강도가 매우 낮았다. 이는 Ser313이 PLD1과의 결합에서 매우 중요한 motif임을 시사 한다. 현재까지의 연구를 바탕으로, PLD1의 아미노산 중 hAP180 (특히 310-TVTSP-314 부위)과 결합하는 부위를 탐색할 계획에 있으며, PLD 활성 억제제 개발을 위해 3개 아미노산 (Thr312, Ser313, Pro314)을 표적으로 한 peptido mimetic drug 제작에 착수 할 것이다. 만약 PLD 활성 억제제의 개발이 성공적으로 마무리 될 경우 PLD 매개 항세포사를 조절 하여 항암치료시 암세포의 약제내성 감소, 세포사 증진, 항암제의 효능 및 민감도 증가, 항암제 dose-limit toxicity 감소 등의 효과를 가져 올 수 있을 것으로 사료된다.; The role of phospholipase D (PLD) in antiapoptotic process and the molcular mechanism of hAP180 for inhibition of PLD Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid (PA) and choline. There are at least two PLD isozymes, PLD1 and PLD2. Genetic and pharmacological approaches implicate both PLD isozymes in a diverse range of cellular processes, including receptor signaling, membrane transport control, and actin cytoskeleton reorganization. Several recent studies reported that PLD has a role in signaling pathways that oppose apoptosis and promote cell survival in cancer. In this study, we examined the role of PLD in taxotere-induced apoptosis in stomach cell lines; normal stomach (NSC) and stomach cancer cells (SNU 484). Taxotere treatment resulted in increase of PLD activity. To confirm the role of PLD in taxotere-induced apoptosis, PLDs were transfected into SNU 484 cells. Overexpression of PLD isozymes resulted in inhibition of taxotere-induced apoptotic cell death, evidenced by decreased degradation of chromosomal DNA, and increased cell viability. Concurrently, Bcl-2 expression was upregulated, and taxotere-induced activation of procaspase-3 was inhibited after PLDs transfection. However, when PLD was selectively inhibited by specific siRNA-PLD1 or -PLD2, taxotere-induced apoptosis was exacerbated in SNU484 cells. On top of this, PA--the product of PLDs, also resulted in upregulation of Bcl-2 in SNU484. Although PA-induced Bcl-2 expression was blocked by mepacrine, an inhibitor of phospholipase A2 (PLA2), increased Bcl-2 expression by PA was not abrogated by propranolol, an inhibitor of PA phospholyhydrolase (PAP). Taken together, PLD1 and PLD2 are closely related with Bcl-2 expression together with PLA2, but not with PAP, during taxotere-induced apoptosis in SNU484 cells. The role of phospholipase D (PLD) in antiapoptotic process and the molcular mechanism of hAP180 for inhibition of PLD Phospholipase D (PLD) hydrolyses phosphatidyl choline to phosphatidic acid (PA) and choline. PLD is involved in many physiological and structural functions of the cell, including anti-apoptotic signal, vesicle trafficking, mitogenesis and so on, which are controlled by hormone, cytokines, and growth factors. Above all studies reported that PLD acts as a survival factor against death signal in several cancer cells. This study tried to elucidate inhibitory mechanisms of PLD activity. According to previous studies, clathrin assembly proteins such as amphiphysins, synaptojanin, and AP180 can inhibit PLD activity. AP180 is localized to clathrin-coated vesicles budding from presynaptic plasma membrane. To begin the investigation where is a critical motif in AP180, which is responsible for binding with PLD and inhibition of PLD activity, at first, human AP180 (hAP180) gene was cloned from human cDNA library, and inhibition of phobol-12-myristate 13-acetate (PMA) induced PLD activity by hAP180 transfection was detected. Moreover Bcl-2 expression induced by PA treatment or PLD1 overexpression was inhibited by AP180 transfection, but not by overexpression of PLD2. The results from hAP180 deletion series study, the 11ea amino acids from Pro-310 to Lys-320 were important residues for binding with PLD1 in KATOIII cells. These eleven amino acids also involved in not only inhibition of PLD activity, but inhibition of PLD1-induced rescued survival rate during taxotere- and etoposide-induced apoptosis. Among these amino acids, five peptides (Pro31- Pro314) in C-terminal part were shown as a critical region for the inhibition of PLD activity through treatment of the synthetic penta peptide into both of KATOIII and SiHa cells. From the point mutants study, Thr312, Ser313, and Pro314 on hAP180 are critical amino acids for the inhibition of PLD activity. Moreover, Ser313 is the most important amino acid for the inhibition of PLD activity. This result is further suppoted by PLD assay and immunoprecipitation. Taken together, these results promise for the possibility of developing peptido-mimetic drug which can inhibit PLD activity. If these amino acids sequences will be applied to synthesis of a therapeutic peptido-mimetic drug, PLD-related survival signal and cancer development will be under control.