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

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制，我们对大鼠肝脏过氧化物酶体进行了蛋白质组学分析。此外，我们还研究了鉴定出的新型过氧化物酶体蛋白的功能。关于过氧化物酶体疾病，我们分析了正常和人类ALD成纤维细胞中负责肾上腺白质营养不良（ALD）的ABC蛋白ALDP的命运。结果，获得了以下新发现：采用Nycodenz梯度离心法制备大鼠肝脏过氧化物酶，并采用抗PMP70抗体进行免疫分离。经SDS-PAGE酶切蛋白条带后，采用LC/MS/MS分析共鉴定出65种过氧化物酶体蛋白，其中5种为新蛋白。鉴定出可能参与过氧化物酶体生物发生的RAB2、VAP-A、B。作为新的5种过氧化物酶体蛋白，离子蛋白酶的过氧化物酶体同工酶，一种含有氨基糖苷转移酶和酰基辅酶a脱氢酶结构域的双功能酶，一种与内肽相关蛋白的同源物，一种具有核糖核酸酶结构域的肿瘤相关蛋白，一种与细胞器分裂相关的CGI-135的同源物。我们克隆了人lon蛋白酶cDNA，并在大肠杆菌中表达了lon蛋白酶- his。我们纯化了lon蛋白酶，发现该酶对变性蛋白具有atp依赖性蛋白酶活性4。我们在大鼠肝脏过氧化物酶体中鉴定出12种过氧化物。Pex3p与Pex16p和Pex19p相关，被认为对过氧化物酶体的生物发生很重要。我们发现大多数带有错义突变的aldp在人ALD成纤维细胞的过氧化物酶体上存在缺陷。此外，突变蛋白被发现在蛋白酶体合成后被迅速降解。目前正在研究新型过氧化物酶体蛋白的作用。

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.