【作者】 陈玮；

【导师】 曹雪涛；

【作者基本信息】 浙江大学 ， 肿瘤学， 2004， 博士

【摘要】 细胞凋亡（apoptosis）是多细胞生物体内高度保守的生物学过程，在个体的生长发育以及存活过程中起着重要作用。研究表明溶酶体（lysosome）参与多种细胞凋亡过程，包括病理状态下的细胞凋亡和生理性细胞凋亡如细胞衰老过程中出现的凋亡。在细胞凋亡进程中，溶酶体膜通透性增高发生于凋亡早期，在线粒体变化之前，并负责进一步触发下游的凋亡信号转导途径，因此溶酶体在细胞凋亡中起着重要作用。本文所研究的新分子来源于骨髓基质细胞。该分子能够通过转位至溶酶体进而促进溶酶体膜通透性增高，来实现其促细胞凋亡功能。结合该分子含有PH和FYVE功能域的结构特征，将其命名为LAPF（lysosome-associated and apoptosis-inducing protein containing PH and FYVE domains）。 一、新蛋白家族Phafins的鉴定和LAPF的序列分析及组织分布 经大规模随机测序，我们从人骨髓基质细胞（BMSC）cDNA文库中分离到一个全长新基因hLAPF，编码279氨基酸的蛋白。该基因已经在GenBank登陆（登陆号为AY037145）。hLAPF的小鼠同源物mLAPF（GenBank登录号NP₀77724）也含有279氨基酸，两者蛋白序列的一致性达90.3％。 经同源比较发现，12个来源于不同物种的未知蛋白与人、鼠LAPF有较高的序列同源性（34～91％）。结构分析预测这14个蛋白均含有一个氨基端的PH结构域和一个羧基端的FYVE结构域，因此将它们归入一个新家族Phafins （protein containing both PH and FYVE domains）。所有成员均含有PH结构域中负责形成高度保守三级空间结构的六个相对保守区域，和FYVE结构域中八个高度保守的半胱氨酸及其核心基序R+HHC+XCG。根据序列同源性和进化分析结果，我们将Phafin家族成员分成浙江大学博士学位论文中文摘要Phafin一1、Phafin一2两个亚家族以及一组未分类的蛋白。 Northem杂交结果显示hLAPF mRNA主要以1 .8 kb的转录形式存在，在心脏和骨骼肌中表达丰度较高，在脑、胸腺、肝、脾、’肾、小肠、胎盘、肺以及外周血淋巴细胞中表达水平较低。mLAPF在成年小鼠组织中的分布与hLAPF基本一致，在心脏和脑组织中呈高表达。hLAPF的细胞分布主要集中在人BMSC、人树突状细胞以及多种造血系肿瘤细胞，实体瘤细胞中则不表达;mLAPF不仅在小鼠BMSC、小鼠树突状细胞以及造血系肿瘤细胞中表达，一些实体瘤细胞如L929细胞中也有一定水平的表达。 观察不同外界刺激如TNFa、LPS、PMA对LAPF表达的影响，发现致凋亡剂量的TNFa分别在刺激后12小时或8小时上调U937细胞中hLAPF或L929细胞中mLAPF的表达，并维持至24小时。与此相一致，hLAPF和mLAPF蛋白表达水平分别在TNF以刺激后36小时和24小时出现增高，并维持至48小时。L队和PMA对LAPF的表达则无影响月NFa呈时间依赖性地诱导LAPF表达增高，提示LAPF可能参与TNFQ诱导的细胞凋亡。二、LAPF的促细胞祠亡功能及其作用机制 为了明确LAPF是否参与细胞凋亡过程，将表达hLAPF或mLAPF真核表达载体瞬时转染飞NFa敏感的L929细胞。结果显示，无需任何凋亡诱导因素的协同，单独过表达hLAPF或mLAPF即可明显触发L929细胞凋亡。LAPF的两种变异体即hLAPF△PH（N端的PH结构域缺失）和hLAPF△FY（C端的FYVE结构域缺失）则不能诱导L929细胞凋亡，表明这两个结构域为LAPF的促凋亡功能所必需。同样，稳定表达hLAPF或mLAPF也能增加L929细胞对hTNFa的敏感性，其中10n留mlh1NFa为诱导L929一hLAPF和L929一mLAPF细胞凋亡的最佳剂量。为了明确L929一LAPF细胞凋亡，我们还分析了其他凋亡指征，包括染色质浓集及边缘化、DNA降解后形成的亚二倍体峰和DNA大片段（50一Zookb），结果进一步证实了L929一LAPF的细胞凋亡。浙江大学博士学位论文中文摘要 我们在共聚焦显微镜下观察细胞凋亡过程中LAPF的亚细胞定位变化。在瞬时转染后24小时，hLAPF一GFP和mLAPF一GFP广泛分布于胞浆与胞核;48小时后则聚集到胞浆的近核周区域，与溶酶体发生部分共定位:60一72小时细胞形态出现收缩、变圆，LAPF与溶酶体几乎完全共定位。而LAPF的两种变异体hLAPF△PH一GFP和hLAPF△FY一GFP均不能与溶酶体发生共定位，提示细胞凋亡过程中LAPF可转位至溶酶体，该转位过程是LAPF两个结构域共同作用的结果。 利用两种亲溶酶体的荧光染料探针，Lyso Tracker和AO，检测凋亡过程中溶酶体的变化。在10 ng/ml hTNFa刺激后12小时，hLAPF一L929细胞的溶酶体膜出现不稳定，48小时溶酶体破坏最严重，而对照细胞无明显变化。与溶酶体膜不稳定相一致，在h1NFa作用12小时的hLAPF一L929细胞浆分离液S一1 00中，可检测到组织蛋白酶D和组织蛋白酶L的释放，随着时间的延长组织蛋白酶的释放愈加明显。同时免疫荧光染色结果进一步证实组织蛋白酶的释放过程。 我们发现泛caspase抑制剂ZVAD一丘Ik并不能抑制hTNFa触发的L929一hLAPF细胞凋亡，而且在该凋亡模型中未检测到casPase一3、8、9的活化，可以推测hTNFa诱导的hL妙F一L929细胞凋亡并不依赖于casPase。然而在hTNTa刺激后24小时，LAPF能诱导线粒体发生一系列变化，包括线更多还原

【Abstract】 Apoptosis is a highly conserved process in multicellular organisms that plays an important role in development and survival. Recent studies have shown that lysosomes participate in apoptosis under certain pathologic condition as well as physiologic situation such as during normal aging. Lysosomal membrane permeabilization (LMP) is an early event in apoptosis, which precedes changes of mitochondria, and is responsible for activation of downstream signal pathway, indicating a dominant role of lysosomes in apoptosis. In the current study, we isolated a novel molecule from human bone marrow stromal cells (BMSC), which could induce apoptosis of L929 cells concomitant with its translocation to lysosomes and thus permeabilization of lysosomal membrane. Given the structural characteristic of containing a PH domain and a FYVE domain, it was designated as LAPF (lysosome-associated and apoptosis-inducing protein containing PH and FYVE domains).Part I Identification of a novel family Phafins and characterization of LAPFA novel full-length gene hLAPF, isolated from a human BMSC cDNA library by large-scale random sequencing, potentially encodes a 279-amino-acid protein. The gene has been deposited to GenBank (GenBank accession Number AY037145). A mouse 279-amino-acid protein (GenBank accession Number NP077724), sharing 90.3 % overallidentity to hLAPF, was also cloned and designated as mouse LAPF (mLAPF).Homology analysis revealed that LAPF shares close similarities (about 34-91 % similarity) to 12 unknown proteins originating from various species. Structural analysis based on multiple alignment showed that all of these proteins were predicted to confain two common structural characteristics; an N-terminal PH domain and a C-terminal FYVE domain. We thus classified these proteins as a novel family, designated as the Phafins (protein containing both PH and FYVE domains). Members are predicted to contain six conserved blocks within the PH domain, which comprise a highly conserved three-dimensional organization, and eight conserved cysteine residues within FYVE domain, two of these residues are part of the core motif R+HHC+XCG. Based on homology comparison and extent of evolutionary conservation, members of the Phafin family can be grouped into two subfamilies: Phafin-1, Phafin-2, and an unclassified group.The mRNA expression of hLAPF in human tissues was examined by Northern blot analysis. An approximately 1.8 kb hLAPF transcript was abundant in heart and skeletal muscle, whereas expression in brain, thymus, spleen, kidney, liver, small intestine, placenta, lung and PBL was weak. mLAPF was also widely expressed in adult mouse tissues, and its expression in heart and brain was strong. hLAPF expression was observed in freshly isolated human BMSC, human dendritic cells (DC) and a variety of leukemia cell lines, but not in solid tumor cell lines. In contrast, mLAPF mRNA could also be detected in some solid tumor cell lines, in addition to leukemia cell lines, mDC and mBMSC.The effects of hTNFa, LPS and PMA treatments on LAPF expression in U937 and L929 cells, which express low levels of LAPF, were examined. Expression of hLAPF mRNA in U937 cells increased 12 h after an apoptosis-triggering dose (50 ng/ml) of hTNFa, and was maintained at high levels until 24 h, whereas mLAPF mRNA levels increased within 8 h in hTNFa-treated L929 cells. In parallel with mRNA expression profiles, protein expression of hLAPF and mLAPF in U937 and L929 cells was induced byhTNFa at 36 h and 24 h respectively, and maintained until at least 48 h. However, neither LPS nor PMA up-regulated LAPF gene expression. The time-dependent increase in LAPF expression in response to hTNFa indicates that LAPF may participate in TNFa-induced apoptosis.PartII LAPF induces apoptosis via a lysosomal-mitochondrial pathwayTo determine whether LAPF is involved in apoptosis, TNFa-sensitive L929 cells were transiently transfected with hLAPF or mLAPF expression vectors. Transient overexpression of hLAPF or mLAPF induced apoptosis of L929 cells, in t更多还原