Proteome analysis of peroxisomes-Function of novel peroxisomal proteins and pathogenesis of peroxisome disorders

过氧化物酶体的蛋白质组分析-新型过氧化物酶体蛋白的功能和过氧化物酶体疾病的发病机制

基本信息

批准号：
14370740
负责人：
IMANAKA Tsuneo
金额：
$ 8.83万
依托单位：
TOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

To understand function of peroxisomes and pathogenesis of peroxisome disorders, we performed proteome analysis of rat liver peroxisomes. Further, we investigated function of novel peroxisomal proteins that we identified. Concerning peroxisome disorders, we analyzed fate of an ABC protein, ALDP that is responsible for adrenoleukodystrophy (ALD) in normal and human ALD fibroblasts. As a result, following new findings were obtained.1.We prepared rat liver peroxiomes by Nycodenz gradient centrifugation and further by immunoisolation using antibody against PMP70. We identified 65 peroxisomal proteins including 5 novel proteins in the peroxisomes by LC/MS/MS analysis after digestion of the protein bands subjected to SDS-PAGE. RAB2, VAP-A, B, which might be involved in peroxisome biogenesis were identified.2.As novel 5 peroxisomal proteins, peroxisomal isozyme of Ion protease, a bi-functional enzyme containing aminoglycoside transferase and acyl-CoA dehydrogenease domains, a homolog to endozepine-related protein, a tumor-related protein with ribonuclease domain, a homolog of CGI-135 which is required organelle fission were identified.3.We cloned the human lon protease cDNA and express Lon protease-His in E.coli. We purified the lon protease and found that the enzyme showed ATP-dependent protease activity against denatured proteins4.We identified 12 peroxin on rat liver peroxisomes. Pex3p was associated with Pex16p and Pex19p and suggested to be important for peroxisomes biogenesis.5.We found most ALDPs with missense mutations were deficient on peroxiomes of human ALD fibroblasts. In addition, the mutant proteins were found to be degraded rapidly after synthesis by proteasomes.Roles of novel peroxisomal proteins are now under investigation.

为了了解过氧化物酶体的功能和过氧化物酶体疾病的发病机理，我们对大鼠肝脏过氧化物酶体进行了蛋白质组分析。此外，我们研究了我们发现的新型过氧化物酶体蛋白的功能。关于过氧化物酶体疾病，我们分析了ABC蛋白ALDP的命运，该ALDP负责正常和人类ALD成纤维细胞中的肾上腺素肌营养不良（ALD）。结果，在获得新的发现后。1。我们通过nycodenz梯度离心制备了大鼠肝过氧组，并使用针对PMP70的抗体进行免疫溶解。我们通过LC/MS/MS分析鉴定了65种过氧化物酶体蛋白在过氧化物酶体中的5种新蛋白，在消化接受SDS-PAGE的蛋白质带后。 Rab2，vap-a，b可能与过氧化物酶体生物发生有关。域，鉴定出需要细胞器裂变的CGI-135的同源物。3。我们将人lon蛋白酶cDNA克隆，并在大肠杆菌中表达lon蛋白酶。我们纯化了LON蛋白酶，发现该酶对抗原蛋白显示ATP依赖性蛋白酶活性。我们在大鼠肝过氧酶体上鉴定了12个过氧蛋白。 PEX3P与PEX16P和PEX19P有关，建议对过氧化物酶体生物发生很重要。5。我们发现大多数具有错义突变的ALDP对人ALD成纤维细胞的过氧组体缺乏。另外，发现突变蛋白在合成后迅速降解。新型过氧化物酶体蛋白的角色正在研究中。