二號吡咯啉-5-羧酸鹽還原酶之表達與功能性分析

Abstract

吡咯啉-5-羧酸鹽還原酶(Pyrroline-5-carboxylate reductase, PYCR)普遍被認為是管家酵素，可與NAD(P)H共同催化脯氨酸的生成。在斑馬魚有三種PYCR同源蛋白，分別是Pycr1a、Pycr1b與Pycr3。分別由Pycr1a/Pycr1b 和 Pycr3兩條路徑來催化脯氨酸的生成。Pycr1a與Pycr1b位於粒線體內而Pycr3位於細胞質中。研究顯示Pycr1a與Pycr1b可保護細胞抵禦氧化傷害，而Pycr1a與Pycr1b基因突變皆會影響粒線體的功能，進而造成早衰症及畸形小頭症等相關疾病，而本論文之目的乃探討Pycr1b在斑馬魚發育過程中之基因及蛋白質表現及分布。在此論文中，我以反轉錄聚合酶鏈式反應(RT-PCR)發現pycr1b基因從胚胎早期發育階段與成魚所有組織中皆有高度表現。而在原位雜合(in situ hybridization)實驗中我觀測到pycr1b基因在腦部及其特定區域有高度表達。我以Pycr1b C端約一半的序列製作了一個GST融合重組蛋白，並以之為抗原製作了一支抗體。利用西方墨點法，此Pycr1b抗體可以專一的辨識到一條約34 kDa的斑馬魚粒線體蛋白，而在一天大的斑馬魚胚胎粒線體蛋白中Pycr1b的條帶在Pycr1b (-/-)基因剔除魚比野生型的魚明顯減弱。此證明了此Pycr1b (-/-)突變魚是Pycr1b基因缺失突變魚，後續可更進一步用以檢測Pycr1b功能並進行其他實驗。

Pyrroline-5-carboxylate reductase (PYCR) is a housekeeping enzyme catalyzing the reduction of delta (1)-pyrroline-5-carboxylate (P5C) to proline with NAD(P)H as a cofactor. In zebrafish, there are three PYCR family genes, including pycr1a, pycr1b and pycr3, to catalyze the production of proline in two separate reactions by Pycr1a/Pycr1b or Pycr3. Pycr1a and Pycr1b are located in mitochondria and Pycr3 is a cytosolic protein. Pycr1a and Pycr1b can protect cells from overt oxidative stress. Mutations in the pycr1a or pycr1b gene can alter mitochondrial function and result in progeria and microcephaly-related diseases. The objective of this thesis is to understand the spatial and temporal expression of pycr1b gene during development in zebrafish. Using RT-PCR, I found that the Pycr1b is highly expressed from early developmental stages and different adult tissues. Embryos or larval zebrafish were fixed at designated stages and subjected to whole-mount in situ hybridization analysis against pycr1b. I found that pycr1b was highly expressed in the brain. To carry out the project, I also generated and characterized a Pycr1b antibody specifically recognizing a GST-fusion protein containing the C-terminal half of zebrafish Pycr1b. By Western blotting, the generated Pycr1b antibody can recognized a ~ 34 kDa protein from a mitochondria fraction of zebrafish embryos. I further observed that the Pycr1b band was weaker in pycr1b (-/-) than that of wildtype 1-day old zebrafish embryos. This suggests that the pycr1b (-/-) mutant is a null mutant which can be used for Pycr1b functional assay. The Pycr1b antibody may also be used to further analyze protein expression pattern and other functional test for Pycr1b.