【作者】 王军；

【导师】 肖庚富；

【作者基本信息】 武汉大学 ， 微生物学， 2005， 硕士

【摘要】 肿瘤坏死因子相关的凋亡诱导配体(TNF-related apoptosis-inducing ligand, TRAIL),又称凋亡素-2配体(apoptosis receptor 2 ligand, Apo2L),是1995年发现的肿瘤坏死因子家族的新成员,和其它家族成员一样,同属Ⅱ型跨膜蛋白,由胞浆区(14个aa),跨膜区(26个aa)和胞膜外区(241个aa)组成,胞膜外区有蛋白酶作用位点,可以从外膜上被剪切下来,形成可溶性分子,也可以形成二/三聚体。 到目前为止,TRAIL的五种受体相继被发现:四种膜受体DR4(死亡受体4),DR5(死亡受体5),D_CR1(诱骗受体1),D_CR2(诱骗受体2),和一种可溶性受体OPG。DR4和DR5广泛表达于正常组织细胞和肿瘤细胞,D_CR1或D_CR2表达于正常组织细胞。由于D_CR1和D_CR2的膜外区均有两个与DR4和DR5高度同源的富含半胱氨酸的伪重复序列,能够与TRAIL结合,但D_CR1胞内没有DD(death domain死亡结构域),D_CR2在胞内有一部分DD,与TRAIL结合后,凋亡信号传递中断,正常细胞可以逃逸TRAIL的凋亡诱导作用;但是肿瘤细胞只表达DR4和DR5,所以TRAIL可以选择性地诱导肿瘤细胞凋亡。 对TRAIL的研究,研究者从最初的全长表达,然后不断进行截短,到目前有些研究者表达了TRAIL的114-281氨基酸。我们根据TRAIL的晶体结构分析得知,114—119位氨基酸是高度无序的,且电子密度缺失。另外,130-160氨基酸形成“AA”环,在与受体结合中发挥重要作用,第230位的Cys对其生物活更多还原

【Abstract】 TRAIL(TNF-related apoptosis-inducing ligand) was discovered because of its sequence homology to TNF and FasL, which belongs to type II transmembrane protein as other members of TNF family. Human TRAIL contains 281 amino acids, of which 14 amino acids in N-termini consists of its cytoplasm region, following 26 amino acid makes its transmembrane region and 241 amino acids’ extracellular region. The extracellular region of TRAIL can be proteolytically cleaved from the cell surface and forms a soluble molecule upon cleavage. Like most other TNF family members, This molecule usually forms stable homotrimers or homodimers. In contrast to TNF and FasL, TRAIL mRNA is expressed constitutively in many tissues, which suggests the existence of physiological mechanism that can protect many normal cell types from induction of apoptosis specially by TRAIL.So far, the identification of two signaling receptors of the TNF receptor gene superfamily that bind TRAIL pointed to the mechanism for induction of aoptosis by this ligand: DR4 (death receptor 4) and DR5 (death receptor 5), both contain a conserved death domain motif. In addition to the two DRs, three other receptors bind to TRAIL, and appear to act as ’decoys’: DcRl(TRAIL-R3), DcR2(TRAIL-R4) and OPG . DcR1 and DcR2 have close homology to the extracellular domains of DR4 and DR5 . DcR2 has a truncated, nonfunctional death domain, while DcRl lacktransmembrane and death domains. Both receptors are therefore incapable of trasnsmitting an apoptosis signal . A fifth binding protein ,the soluble TNFR family member osteprotegerin (OPG), binds TRAIL but has lower affinity at physiological temperature .To further investigate the mechanism of how TRAIL inducing apoptosis and the prospect of TRAIL as a clinic drug in cancer therapy, scientists tried to express sTRAIL(41-281) , sTRAIL(95-281) , sTRAIL(101-281) and sTRAIL(114-281) etc. According to the crystallized structure of TRAIL , a TRAIL monomer contains two antiparallel 0 -pleated sheets that form a P sandwich as a core scaffold, and the adjacent subunits in a head- to -tail fashion to form a bell-shaped homotrimer. The most unique feature of TRAIL distinguished from those other TNF family members is an insertion of 12-16 amino acids near the N-terminal cleavage site. The insertion occurs at the ’AA’ loop that was previously known to be important for the receptor binding. The 114-120 amino acids displayed highly disorder and electron densities was lacked. Cys-230 from each subunit at the top portion of the molecule is engaged in intimate van der Waals dispersion interactions with each other in the receptor-binding domain. Mutant of Cys-230 to alanine or serine strongly affected its ability to kill target cells. Binding to its receptors was decreased by at least 200-fold, and the stability of its trimeric structure was reduced. To make sure 114-120 amino acids’ function, The sTRAIL(120-281) was expressed in E. coli. and was purified by Ni-NTA chromatography column and Gel filtration chromatography.As a potential antitumor protein, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has drawn considerable attention. The coding region human soluble TRAIL gene from healthy human peripheral lymphocytes was cloned using RT-PCR and constructed 6XHis-tag vector pET30a-TRAIL(114-281) and pET30a-TRAIL(120-281). After DNA sequencing, The expression of protein was induced by 0.1 mmol/1 IPTG and low temperature (4°C). Soluble protein was purified by Ni-NTA chromatography column. Purified protein was digested with Enterkinase to cut off 6 X His-tag and was purified by Gel filtration chromatography, showing a peak at 60 kD or so. Resluts of SDS-PAGE proved that the 60 kD protein wasTRAIL trimer. The MTT assay showed that TRAIL(114-281) (protein A) and TRAIL(120-281) (protein B) could inhibit the growth of HeLa cell line, at the same time, proein B could inhibit the growth of some types of tumor cell and good relationship of concentration-effect and time-effect. The bioactivity of protein B is weaker obviously than it’s of protein A.更多还原