老鼠肝臟轉錄因數AGP/EBP結構與功能之研究

Abstract

α1-acid glycoprotein是肝臟為應付緊急生理需求-發炎，高燒等症狀時會快速產生之蛋白質之一。因此研究此基因之起動子(promoter)與其調節子(regulatory factor)，AGP/EBP,之間的作用關係，將有助於吾人明白基因轉錄的調控機制。 本論文首先篩選(screen)出大老鼠的AGP/EBP基因，定出基因序列(sequence)，共1333個鹼基對(bps)，可轉訊 297個胺基酸，並且和小老鼠的序列比較，發現只有一個胺基酸不同，再和其他調節子(如：C/EBP)做基因與胺基酸的序則比較，發現在其羧基端有basic amino acid以及leucine zippe特殊結構，初步將之歸入C/EBP家族之一員。 接著將此調節子基因接入質體(pET-plasmid)再轉染大腸菌使其在其中表現增殖，再經適當的純化步驟得到一個合成蛋白(recombinant protein)。將此recombinant protein及nuclear extract分別做gel retardation與 DNase I footprinting分析，得知此調節子(AGP/EBP)具備有接在起動子(promoter)上某些特殊核酸接合區(DNA－binding motif)-C,D,E之潛力。 因此本論文的研究轉入兩個方面來探討：一者將此調節子(AGP/EBP)胺基端做適當刪減(deletion)，接入表現質體(reporter plasmid)利用同時轉染(cotransfection)及CAT assay初步區分其transcription activation domain；再各利用起動子之不同變異種(mutants)，以及多元化(oligomerized)之特殊接合區C,D,E,以同時轉染(cotransfection)方式來探討起動子－AGP/EBP在不同的核酸接合區－C,D,E,所扮演之角色是同雙子或異雙子。

AGP/EBP is a liver enriched transcription factor that specifically binds to the cis-elements in the promoter region of α1-acid glycoprotein (AGP) gene. Since α1- acid glycoprotein is one of the major acute phase proteins found in liver, studying the interaction between the AGP promoter and its transactivator, AGP/EBP, will give us some information about the regulatory mechanism of gene expression. In this thesis, the gene coding rat AGP/EBP has been cloned and sequenced. Its full length cDNA has l333 bps and encodes a polypeptide of 297 amino acids, which contains a potential leucine zipper and a conserved basic domain; Meanwhile, compared with the mouse AGP/EBP, we found that they were almost identical, except that rat AGP/EBP has one more amino acid. As AGP/EBP shares a high degree of homology in theleucine zipper and basic amino acids region with C/EBP and NF-IL6, it belongs to theleucine zipper superfamily of proteins. But the sequences of AGP/EBP and C/EBP are completely different ouside these two domains. Constructing the AGP/EBP DNA fragment to pET plasmid, we got the recombinant protein, reAGP/EBP-FL. From EMSA and DNase I footprinting data, we can conclude that the recombinant AGP/EBP binds the cis-elements-C,D,E, regions of the promoter of AGP gene in a sequence-specific manner. Therefore, we will go on the study in the following directions: First, we want to delineate the structure/function relationship of AGP/EBP. A series of N-terminal deleted mutants were created and tested for their functions in vivo using AGP promoter/chloramphenicol acetyl transferase reporter gene assay; Second, using some mutants of the promoter of AGP gene and oligomerized-C,D,E/pCAT, we try to know the delicate interaction between the C, D, E motifs and AGP/EBP, which may act as a homodimer or heterodimer.