描述線蟲unc-5與eif-3.K在計畫性細胞死亡中的功能

Abstract

在多細胞生物中，計畫性細胞死亡(細胞凋亡)是由基因嚴密調控的路徑，不正常的活化細胞死亡會造成退化性的病徵，然而不當的抑制細胞死亡也會造成腫瘤的生成。在哺乳動物細胞中，廣泛被研究調控細胞凋亡的分子機制主要可以分成內部和外部二個路徑。內部(粒線體)的路徑是藉由內在刺激傳遞至粒線體所引發，而外部(死亡接受器)的路徑則是由死亡的配合基與帶有死亡區域的接受器與轉接器相互作用而活化。就目前為止，還未有外部死亡路徑的調控在線蟲中被報導。根據我們的研究，我們找到一個已知參與在細胞遷移功能且具有死亡區域蛋白的膜接受器─UNC-5。在線蟲之前的研究中，UNC-5/Unc5和他共同的接受器UNC-40/DCC以及其配合基UNC-6(netrin)、UNC-129 (TGF-β)對於指導周圍細胞和生長錐向外移動都是必要的。在我們的研究中發現，當UNC-5失去功能時會造成死細胞數目減少，亦會使活細胞多存活過來。反之，過量表現UNC-5 則會造成大量的細胞死亡，推論UNC-5 可以正向調控細胞死亡。此外，我們也發現UNC-129 (TGF-β)這個分泌型的配合基可以與UNC-5 作用，共同來調控細胞死亡。這也是第一個證據顯示細胞死亡可以透過外來的訊號UNC-129 活化和啟動。進一步的生化與遺傳分析顯示我們另找到一個位於UNC-5下游且直接與UNC-5 作用的蛋白質激酶─SRC-2。我們推論當UNC-5 接受到外來UNC-129 的訊號時，即可將死亡訊息藉由與SRC-2的直接作用而將訊號傳遞下去以啟動促進細胞死亡。執行計畫性細胞死亡最重要的步驟就是活化凋亡蛋白酶(caspases)。在線蟲(C.elegans)中，已知EGL-1(BH3 only)，CED-9 (Bcl-2)，CED-4 (Apaf-1) 和CED-3 caspase 組成了細胞死亡的核心路徑。在此我們另外找到了一個位於CED-3 caspase上游的新細胞死亡調控基因─eif-3.K。當eif-3.K(lf)皆會造成生殖與體細胞屍體數目減少，反之eif-3.K(gf)則能使更多的細胞死亡。利用特定表現的promoter，發現eif-3.K將以細胞自主性(cell-autonomous)的方式來促進細胞死亡。此外，當eif-3.K(lf)可顯著的抑制ced-4大量表現所造成的細胞死亡，卻無法抑制ced-3所造成的細胞死亡，此結果顯示細胞死亡過程中，對於eif-3.K 是有不同的需求。反之，當ced-3(lf) 卻可抑制eif-3.K表現所造成的細胞死亡，這說明eif-3.K 促進細胞死亡時是需要ced-3 且位於ced-3上游來啟動這過程。EIF-3.K 是一個從胚胎到成蟲都可表現在細胞質的高度保留性蛋白，以結構功能上分析顯示，EIF-3.K中具有61個a.a 的WH domain，不但可以參與 protein-DNA/RNA 間的互相作用，對於EIF-3.K 促進細胞死亡的能力上也是必須且必要的。由於人類的eIF3k 亦可部分取代線蟲中eif-3.K 促進細胞死亡的能力，表示由WH domain 所決定之EIF-3.K調控細胞死亡的過程是在不同物種間皆具有高度保守性。

Programmed cell death (apoptosis) is an important developmental process that removes unnecessary or harmful cells and maintains tissue homeostasis, size and shape. Apoptosis in mammals is induced by either intrinsic death stimuli or extrinsic cue through proapoptotic receptors. In C. elegans, however, apoptosis is long thought to be regulated in a cell-autonomous fashion and no external death signal or pro-apoptotic receptor has been identified. We have characterized one death-domain (DD)-containing protein UNC-5, a previously identified guidance receptor mediating axon replusion and cell migration away from extracellular cues UNC-6/netrin and attraction towards UNC-129/TGF-β. Loss-of-function mutations in unc-5 result in a decrease of cell corpses and extra surviving cells, whereas over-expression of unc-5 significantly increases cell corpses. unc-5 likely acts in a cell-autonomous manner to promote apoptosis. In addition, we find that UNC-129, a secreted-form ligand, acts with UNC-5 to promote apoptosis and needs to be secreted for the function. This is the first evidence showing that programmed cell death can be regulated by the external signal UNC-129/TGF-β. Further genetic and biochemical analyses reveal that SRC-2, a nonreceptor tyrosine kinase, interacts with the UNC-5 intracellular region in vitro and acts downstream of unc-5 to mediate apoptosis. We propose that in response to UNC-129, UNC-5 receptor transduces the death signal by interacting with SRC-2 to promote apoptosis. In C. elegans, the core cell death regulators EGL-1(a BH3 domain-containing protein), CED-9 (Bcl-2), and CED-4 (Apaf-1) act in an inhibitory cascade to activate the CED-3 caspase. Here we have identified an additional component eif-3.K (eukaryotic translation initiation factor 3 subunit k) that acts upstream of ced-3 to promote programmed cell death. The loss of eif-3.K reduced cell deaths in both somatic and germ cells, whereas the overexpression of eif-3.K resulted in a slight but significant increase in cell death. Using a cell-specific promoter, we show that eif-3.K promotes cell death in a cell-autonomous manner. In addition, the loss of eif-3.K significantly suppressed cell death-induced through the overexpression of ced-4, but not ced-3, indicating a distinct requirement for eif-3.K in apoptosis. Reciprocally, a loss of ced-3 suppressed cell death induced by the overexpression of eif-3.K. These results indicate that eif-3.K requires ced-3 to promote programmed cell death and that eif-3.K acts upstream of ced-3 to promote this process. The EIF-3.K protein is ubiquitously expressed in embryos and larvae and localizes to the cytoplasm. A structure-function analysis revealed that the 61 amino acid long WH domain of EIF-3.K, potentially involved in protein-DNA/RNA interactions, is both necessary and sufficient for the cell death-promoting activity of EIF-3.K. Because human eIF3k was able to partially substitute for C. elegans eif-3.K in the promotion of cell death, this WH domain-dependent EIF-3.K-mediated cell death process has potentially been conserved throughout evolution.