李玉梅臺灣大學：生化科學研究所邱昱維Chiou, Yu-WeiYu-WeiChiou2007-11-262018-07-062007-11-262018-07-062007http://ntur.lib.ntu.edu.tw//handle/246246/52761CTP環化水解酶 (GTP cyclohydrolase I, GCH) 是合成輔酶tetrahydrobiopterin (BH4) 的關鍵酵素，GCH突變以致BH4合成不足，降低Tyrosine Hydroxylase活性，進而減少大腦多巴胺的生成，導致大腦基底核對皮質運動區的調控功能降低，骨骼肌群異常拮抗，造成類似巴金森氏症的多巴反應性肌張力不全症 (Dopa-Response Dystonia)。我們實驗發現，較少見的DRD隱性遺傳GCH突變R249S具有正常酵素活性，R249S病人的GCH mRNA表現量也正常，但在細胞中R249S蛋白質表現量不到正常GCH的1/3，推測突變蛋白質的數量減少，可能是致病原因之一。顯性負面調控 (dominant negative, DN) 普遍存在於DRD, 我們同時在細胞內表現顯性突變G201E或L79P會導致正常GCH蛋白表現減少，以致於整體酵素活性降低，形成類似病人身上的發現的DN現象，而溶酶體 (lysosome) 可能參與其中反應。我們隨後發現G201E可能會進到LAMP-1溶酶體中進行分解。在DN相關的實驗中，我們從不具DN現象的細胞中分出一個新發現的蛋白BEC1，主要位在大腦皮質和紋狀體 (striatum) 的神經細胞中，少部分位在小腦的部分Purkinje cell，功能目前未詳，已知BEC1會促進正常GCH蛋白的表現和減少溶酶體中的突變蛋白。最近發現一種小熱休克蛋白 (small heat shock protein sHsp) Hsp27，大量表現似乎會加速突變蛋白的分解。Dopa-responsive dystonia (DRD) is induced by a deficiency of GTP cyclohydrolase I (GCH) and has a postulated autosomal dominant inheritance with a low penetrance. GCH is a key enzyme for biosynthesis of BH4, a cofactor of several monooxygenases. The first part we found an autosomal recessive GCH mutant R24RS possess normal activity in both in vitro and in vivo. It has been proposed the disease may be due to lower mutant protein expression level regulated in a posttranscriptional way. To investigate the mechanism of dominant negative (DN) effect in DRD, we analyzed the biochemical property of two GCH mutants G201E and L79P examined in a cell model. The GCH mutant is unstable and is degraded through lysosomal proteolysis using inhibitor analysis and pulse chase experiment (Part II). Further we found overexpressed G201E was mainly localized in LAMP-1-positive lysosomes and EEA1 early endososomes compartment (Part III). In part IV, a novel protein BEC1 enriched in non-DN cell identified through cDNA subtraction was investigated. We analyzed the distribution of BEC1 and showed that BEC1 protein was prominently expressed in neuronal cells including striatum, cortex and some of the Purkinje cells in cerebellum. The function of BEC1 was still unknown, but it could promote wild-type GCH expression and reduce insoluble mutant protein in lysosomes. We suspect that BEC1 could activate a surveillance mechanism for the insoluble proteins. In Part V, we identified that overexpression of small heat shock protein Hsp27 may accelerate degradation mutant GCH proteins. The role of Hsp27 is discussed.中文摘要……………………………………………………………1 英文摘要..............................................2 Abbreviation………………………………………………………3 CHAPTER 1: INTRODUCTION 1.1 Dystonia……………………………………………4 1.2 Dopa-Responsive Dystonia ………………………4 1.3 Genetic and Gender Features of HPD/DRD……5 1.4 Genes Response to HPD/DRD………………………5 1.5 Biosynthesis of BH4………………………………6 1.6 The Relationships among GCH, BH4, TH, Dopamine and HPD/DRD…6 1.7 Diseases Caused from GCH Mutations……………8 1.8 Characteristics of Human GCH Gene…………8 1.9 Structure of GCH Protein…………………………9 1.10 Recessive GCH Mutant R249S…………………………………10 1.11 Dominant Negative Effect of Mutant GCH…………………11 1.12 Lysosomal Markers………………………………………………11 1.13 Novel Gene in Non-DN Cell……………………………………13 1.14 Molecular chaperones…………………………………………13 1.15 Hsp27………………………………………………………………14 CHAPTER 2: MATERIALS AND METHODS 2.1 Cell Culture…………………………………………16 2.2 Plasmids, Primers and Oligonucleotides………16 2.3 Transfection………………………………………………………18 2.4 Northern Blot………………………………………19 2.5 Frozen Sections for Central Nerve System of Mouse……20 2.6 Immunohistochemistry……………………………………………21 2.7 Western Blot………………………………………………………22 2.8 Transformation of Competent E. coli Cells…23 2.9 pMAL Protein Fusion and Purification…………23 2.10 GCH Activity Assay……………………………………………25 2.11 Preparation of Polyclonal Antibody…………30 2.12 Pulse-Chase………………………………………………………31 2.13 Protease Inhibitors……………………………………………31 2.14 PCR-Select cDNA Subtraction…………………………………32 CHAPTER 3: RESULTS 3.1 HPD/DRD Induced by a Recessive GCH Mutation 3.1.1 R249S mutant protein expressed in E. coli had normal enzyme activity.....33 3.1.2 R249S mutant protein expression level was low in eukaryotic cells……33 3.2 Dominant Negative Mechanisms of Mutant GCH Protein 3.2.1 Comparison the Enzyme Activities in the Mutant and Wild-Type GCH…34 3.2.2 E. coli–Expressed G201E Protein Contains Very Low Enzyme Activity……34 3.2.3 G201E Down Regulates the Activity of Wild-Type GCH in Co-Transfect…35 3.2.4 G201E Decreases the Expression of Wild-Type GCH in Protein Level………35 3.2.5 G201E and Wild-Type GCH Proteins Form Hetero-Complex in vivo………35 3.2.6 G201E is Labile to Lysosomal Proteases…………………36 3.2.7 Effect of Other Dominant Mutation in GCH………………36 3.3 Destination of Mutant GCH Protein G201E 3.3.1 Most of G201E proteins are located in vacuole-like structures………………37 3.3.2 G201E is localized with LAMP-1 but not LAMP-2A………………………38 3.4 Identification of a Novel Gene Involved in Dominant Negative 3.4.1 A novel gene preferentially expressed in non-DN cells encodes a 559AA polypeptide………………………………………39 3.4.2 Conserved domain and sequence analysis………………39 3.4.3 R13 (BEC1) RNA is enriched in human heart and muscle40 3.4.4 R13 (BEC1) protein is enriched in brain………………41 3.4.5 Distribution of BEC1 protein in central nervous system (CNS) of mouse…41 3.4.6 BEC1 promotes wild-type GCH protein expression but reduce mutant GCH…42 3.4.7 BEC1 decreases mutant GCH containing particles………43 3.5 Hsp27 Facilitate Degradation of Mutant GCH Protein 3.5.1 Phospho-Hsp27 changed the appearance of GCH-II from foci to homogenously distribution in cytosol…………………44 3.5.2 Phospho-Hsp27 increased the expression of GCH-II in soluble fraction………44 3.5.3 Overexpression of Hsp27 reduced the expression of G201E protein………45 CHAPTER 4: DISCUSSION 4.1 The Lower Protein Expression Level of Recessive GCH Mutant R249S….46 4.2 Reduction of Wild-type GCH Protein by Co-expression of Dominant GCH Mutant…………………………………………………47 4.3 Fate of Dominant GCH Mutant G201E…………………………48 4.4 Regulation on Protein Expression by Novel Protein BEC1…50 4.5 Hsp27 Modulates the Expression of GCH Protein………52 4.6 Perspectives………………………………………………………54 FIGURES AND LEGENDS…………………………………………………56 APPENDEX………………………………………………………………80 REFERENCE………………………………………………………………872540055 bytesapplication/pdfen-US肌肉張力不全症GTP環化水解酶BEC1Hsp27顯性負面調控溶酶體DRDGCHdominant negativelysosomeotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/52761/1/ntu-96-D87242001-1.pdf