臺灣博碩士論文加值系統

hnRNP K 隸屬於hnRNP 蛋白家族，已知可以與核酸和蛋白質交互作用，並參與在許多細胞的基因調控，包含利用影響核染色質的重新組合、轉錄、基因接合，以及轉譯的方式來調控基因表現。此外，研究也顯示hnRNP K受到許多後轉譯的修飾包含磷酸化、泛素化、SUMO 蛋白質修飾化以及精胺酸甲基化，且精胺酸甲基化目前也被研究認為可以影響蛋白質之間的交互作用。在這些後轉譯的修飾中，精胺酸甲基化主要發生在hnRNP K的KI功能區，但是，精胺酸甲基化所影響的細胞功能目前並不清楚。使人感到有趣的是，KI功能區上具有精胺酸/甘胺酸/甘胺酸 (RGG repeat)的重複序列和具有與SH3功能區交互作用的脯胺酸聚集(proline-rich)序列，這些特有的序列(KI功能區)是一個具有演化特性的區域，並且此區域在脊椎動物至哺乳類動物中具有高度保留的胺基酸序列，但是，在演化較早期的物種並未發現KI功能區，這樣的現象，暗示著KI功能區是一個演化後期新增的功能區，可能使hnRNP K新增一些生物性的功能，例如:蛋白質交互作用。此外，這些精胺酸甲基化的胺基酸也都相當靠近蛋白質交互作用區域，極有可能甲基化也會影響蛋白質交互作用。我的研究著重於研究hnRNP K甲基化所造成和影響的生物功能。我利用重組的KI功能區片段來觀察其甲基化的情形，並研究其甲基化與其他後轉譯修飾之間是否會相互影響而造成生物功能的改變，另外也同時探討此區域之甲基化與蛋白質交互作用的調控關係。我已利用重組蛋白KI功能區片段去找出許多可能與hnRNP K交互作用的蛋白質，目前，也進一步探討其間可能被甲基化調控的蛋白質交互作用。同時，也正在建立一個共同表現KI功能區和甲基轉移脢的系統，以利加速研究KI功能區甲基化與他後轉譯的修飾、蛋白質交互作用的關係。

Heterogeneous nuclear ribonucleoprotein K (hnRNP K), a member of hnRNP complex, can interact with RNA, DNA, and various proteins to participate in the regulation of chromatin remodeling, transcription, splicing and translation. Several post-translational modifications of hnRNP K such as phosphorylation, ubiquitination, sumoylation and arginine methylation have been reported and may account for its diverse interactions. Among these, arginine methylations locate in the K-interactive (KI) domain of hnRNP K but their functional details remain unclear. Interestingly, the KI domain in which contains many arginine/glycine/glycine (RGG) repeats and proline-rich regions responsible for SH3-domain binding is evolutionarily conserved between X. laevis and mammals but not in fly, nematode and yeast, suggesting that KI domain has emerged later in evolution and allows K protein to gain new function like protein-protein interaction. In addition, the fact that all arginine methylation sites reside closely to the region of several known protein-protein interaction suggests the potential role of these methylation to regulate protein interactions of hnRNP K. My thesis study mainly focused on the functional roles of hnRNP K methylation by using a recombinant KI domain to probe the potential cross-talk between methylation and other protein modifications as well as protein-protein interaction. Currently, I have generated several tagged KI domain and identified their interacting proteins. Whether these interactions were regulated by arginine methylation is under study. Furthermore, a co-expressed system of KI and methyltransferase has been explored to facilitate the future study of cross-talk between arginine methylation and other hnRNP K modifications.

Table of contents......................................i
List of figures........................................iii
List of abbreviation...................................v
Chineses abstract......................................1
Abstract...............................................3
Introduction...........................................5
Materials and Methods..................................10
Materals...............................................10
Apparatus..............................................11
Methods................................................12
construction of plasmids...............................12
purification of proteins...............................14
in vitro methylation for fluorographic analysis........16
pull down assay........................................17
in solution digestion..................................18
in gel digestion.......................................18
mass-spectrometric analysis............................19
analysis of hnRNP K by two-dimensional gel electrophoresis .......................................19
Results................................................21
Construction and purification of KI domain.............21
In vitro methylation of KI domain......................21

Identification of methylation sites in KI domain.......22
Verification of KI methylation sites by comparison between Trx-KI domain and arginine mutants during in vitro methylation............................................23
Identification of hnRNP K and KI domain interacting proteins...............................................24
Direct interaction of hnRNP K with p32 and mature form p32 through KI domain......................................24
Functional characterization of KI domain interactomes by cluster of orthologous groups in eukaryote (KOG).......25
Profiling of hnRNP K and KI domain interacting proteins by two-dimensional gel electrophoresis....................25
Discussion.............................................27
References.............................................33
Figures................................................40
Table..................................................56

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