| 摘要: | 近幾年年來，科學家對蛋白質酵素研究，已經不在僅僅只著重於蛋白功能的探討，同時也將研究的領域延伸至蛋白質的三度空間結構分析。在決定蛋白質或是核甘酸原子大小結構時，雖然已有許許多多不同方式得到蛋白質三度空間結構，例如:NMR 或是Cryo-EM 等，然而利用X-ray光源對蛋白質結晶分析，依然是當前決定蛋白結構方法之中最有用且有效的一種方法。因此，結合蛋白質結構與蛋白質功能性的研究，儼然已成為現在蛋白酵素研究上的主要的趨勢。本實驗室為蛋白結構與功能實驗室，成立的最主要目的是想藉由生化實驗的結果來篩選一些有趣蛋白酵素突變態或是一些結構尚未被定出蛋白質酵素，利用X-ray Crystallography的方法決定出蛋白突變態或是新蛋白酵素的結構，以利後續科學的研究。在初期，本實驗室主要會與其他實驗室或其他學校合作，將一些已被研究多時純種蛋白酵素或是突變態蛋白酵素，加以高度純化且培養其蛋白質晶體，最後利用同步輻射的高能X-ray光源來獲得其演色光點，再運用高速電腦運算方式，最後將蛋白酵素三度空間立體結構解析出來。本實驗將繼續與中國醫藥大學中國醫學研究所萬磊副教授合作，主要是想解析生長激素受體Growth Hormone Receptor (GHR)在細胞膜內(cytoplasm)中的蛋白結構，同時此一膜內相關局部突變受體蛋白三度空間結構也希望藉由X-ray Crystallography方式將其解析出來。再此同時突變態的一些生化性質也會一併被探討，例如透過2D膠的實驗來探討純種與突變種之間對其作用的蛋白質種類有哪些不同或是比較純種與突變態之間對一些特定蛋白的結合能力的差異⋯等等。由於萬磊副教授實驗室發現生長激素受體 c.1319基因段突變G>T (Cys/Phe)會影響到生長激素缺乏病童(GHD)對生長激素治療上的效果。綜合上述的發現，GHR c.1319的上游基因突變是與生長激素取代療法有著很大的相關性。GHR c.1319的上游基因突變，也連帶影響到下游受體蛋白結構。如果能夠透過受體結構上的改變的分析，就更能清楚地了解受體的磷酸化方式與反應機制，進而找尋出對於生長激素缺乏病童更加有效的治療方法。 In recent years, enzyme research has not only focused on protein functional studies, but has increasingly extended into protein structural analyses. X-ray crystallography still remains the most useful way to visualize proteins and nucleic acids at atomic resolution. Through study of proteins at the atomic level, structural mechanisms can be elucidated. Nowadays, an important trend in enzyme research is to combine the protein functional and the structural study with structural analyses using X-ray crystallography. Basically, the protein structural determination and functional study are major goals for our lab. Based on biochemical studies, some interesting proteins or mutants will be investigated and then their structures are trying to be obtained via methods of the protein crystallography, X-ray diffractions and computer calculation. At early stage for our lab establishing, we will be continuing to keep collaboration with the lab of the Associate Professor Lei Wan in China Medical University in Tai-Chung. Recently, his published a paper showing that possible influences of single nucleotide polymorphisms (SNPs) on first-year growth velocity in response to growth hormone (GH) therapy in Growth Hormone deficiency (GHD) children. Their lab recruited a total of 154 GHD prepubertal children who had undergone GH therapy for 1 y. To exclude the possibility that the genotype/allele variants influenced the height of GHD patients, they studied the same gene polymorphisms in 208 familial short stature (FSS) patients and 100 normal control individuals. In the present study, the first-year growth velocities of GHD patients treated with GH were measured and then compared with the allelic frequencies of various SNP of genes involved in the GH insulin-like growth factor-I (IGF-I) axis. Only c.1319 G>T Cys/Phe of the GH receptor (GHR) gene showed significant correlation with first-year growth velocity (p=0.02). However, the genetic frequency of the c.1319 G>T polymorphism of GHD did not correlate with FSS and normal controls. Therefore, the c.1319 G>T polymorphism does not influence the height of individuals but can affect the therapeutic efficacy of GH in GHD patients. Moreover, the GHR c.1319 T allele showed higher transcriptional activity and stronger signal transducers and activators of transcription (STAT)-5 Tyr694 phosphorylation. Based on these findings, we conclude that the GHRc.1319 T allele is associated with the therapeutic efficacy of GH replacement therapy. In order to investigate differences of the structural region in cytoplasm between wild type GHR and GHR c.1319 G>T mutant or understanding structural interactions of the receptor with negative or positive regulators, X-ray crystallography method is the major method for the determination of those relative proteins. The protein structural information may lead us own more detail knowledge about interface interactions between the protein/receptor or the molecule/receptor. Meanwhile, further biochemical studies will also be measured on the GHR c.1319 G>T Cys/Phe mutant, e.g. determination of the GHR cytoplasmic domain (wild type) interacted proteins by 2D gel electrophoresis and mass spectrometry; determination of the differences in the binding proteins between mutant (c. 1319G>T Cys/Phe) and wild type GHR….etc. The aim of our research is to analyze both results of the protein structural information and biochemistry study so that we might provide a new pathway for the treatment of GHD, Growth Hormone deficiency, patients 研究期間：9908 ~ 10007 |
