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

为了了解过氧酶体的功能和过氧酶体疾病的发病机制，我们对大鼠肝脏进行了蛋白质组学分析。此外，我们还研究了我们鉴定的新的过氧化体蛋白的功能。关于过氧化物酶体疾病，我们分析了一种ABC蛋白ALDP的命运，这种蛋白导致了正常和人类ALD成纤维细胞的肾上腺脑白质营养不良(ALD)。1.采用Nycodenz梯度离心法制备大鼠肝脏过氧化物体，并用抗PMP70抗体进行免疫分离。通过LC/MS/MS分析鉴定了65个过氧化体蛋白，其中包括5个新的蛋白。鉴定了可能参与过氧体生物发生的RAB2、VAP-A、B等新的5种过氧体蛋白。2.克隆了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--离子蛋白的过氧体同工酶，该酶与含有核糖核酸酶结构域的肿瘤相关蛋白内皮齐平相关蛋白同源物，以及需要细胞器分裂的CGI-135同源物。我们对该酶进行了纯化，发现该酶对变性蛋白具有依赖于ATP的酶活性。Pex3p与Pex16p和Pex19p相关，提示Pex16p和Pex19p在过氧化物体的生物发生中起重要作用。5.我们发现大多数错义突变的ALDPs在人ALD成纤维细胞的过氧化物体上是缺陷的。此外，突变的蛋白质在被蛋白酶合成后迅速降解。新的过氧体蛋白质的角色目前正在研究中。

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.