Pim1激酶在原態和配體結合狀態下的特性研究

Pim1 激酶在細胞的週期調控、增生以及生存等方面都扮演著非常關鍵性的角色。在許多的惡性腫瘤，例如前列腺癌以及多種類型的白血病當中，都有Pim1激酶過度表現的現象，因此Pim1激酶被認為是現今藥物開發中非常重要的標靶蛋白。目前已透過X光結晶繞射的技術來得到許多和抑制物結合的蛋白在活化和不活化狀態下的結構，並利用這些結構來研究抑制物對於蛋白的調控機制。到目前為止，大部分激酶結構的研究是透過X光結晶繞射的技術，只有極少部分是探究激酶在溶液當中的結構和動力學的情形。蛋白質在溶液當中的運動在蛋白活性和功能上一直都扮演相當重要的角色，目前可以透過核磁共振光譜以及其他生物物理的技術來探討。因此，我的實驗目的主要是利用液相核磁共振技術來分析Pim1激酶在脫輔基和與抑制物結合狀態下的特性。為了能夠將核磁共振運用在大分子蛋白的分析，我們將大腸桿菌培養在重水環境當中來表現重組蛋白MBP Pim1，並藉由親和層析法和凝膠層析法純化得到Pim1。完成常用的三共振實驗後，需要透過後續的分析判斷完成Pim1 激酶在序列骨鏈上的判定。另外，我們採用了選擇性同位素標註的方式來提升核磁共振光譜判定的正確性。在核磁共振實驗以外，為了研究Pim1和抑制物結合後的能量變化，我們使用了等量滴定微量熱儀以及式差掃描量熱儀來分析。在等量滴定微量熱儀的結果顯示，Pim1激酶和抑制物的結合均為放熱反應。在式差掃描量熱儀的結果顯示，Pim1 和SGI-1776結合下的熔點溫度比Pim1脫輔基熔點溫度高了10oC，這顯示了在有藥物結合之下，Pim1是比較穩定的。在比較藥物對於蛋白穩定性的影響之外，我們也去比較了野生型Pim1以及其他會致病的突變型的穩定性，從核磁共振的光譜中發現，突變型N82K和L193F相較於野生型來說是較為不穩定的。總結，透過核磁共振和蛋白質動力學的分析結果，對於藥物設計是十分重要的。

關鍵字

Pim1激? ； NMR判定；激?抑制物

並列摘要

Pim1 kinase plays pivotal roles in cell cycle control, proliferation and cell survival. Overexpression of Pim1 has been observed in many malignancies such as prostate cancer and several types of human leukemia [1]. For the reason above, Pim1 is emerging as an important target in drug discovery. X-ray crystallography has revealed various active and inactive conformational states of kinases, which are implicated in their regulation and modulation by inhibitors. However, current structural understanding of kinases is largely based on X-ray crystallographic studies, whereas very little data exist on the conformations and dynamics that kinases adopt in the solution state. Moreover, the important role of protein motions in regulating enzymatic activity and protein function has been demonstrated by NMR spectroscopy along with other biophysical techniques. Thus, the goal of my research project is to characterize Pim1 kinase in apo and inhibitor-bound states by modern solution NMR techniques. To extend the use of NMR to larger systems, the recombinant protein, MBP Pim1, has been produced in deuterated media and then purified with affinity column and gel-filtration. With the standard triple-resonance experiments, the assignment of backbone NMR resonances for Pim1 kinase is still in progress. To confirm spectra assignments, we also adopt several specific isotope labeling strategies. Moreover, in order to investigate the energetic basis of inhibitor binding to Pim1 variants, we have used isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). The results show the interactions of Pim1 and inhibitors are exothermic reactions and the Tm value of Pim1 with SGI-1776 is higher than apo form. It means Pim1 in drug-bound state is more stable than apo form. Besides, we also explore the stability of mutant types N82K, L193F, P311T and WT by NMR. According to the spectra, WT Pim1 is more stable than N82K and L193F. In conclusion, the information from NMR and thermodynamic studies will provide enormous value in Pim1 kinase drug design.

並列關鍵字

Pim1 kinase ； NMR assignment ； kinase inhibitor

參考文獻

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Pim1激酶在原態和配體結合狀態下的特性研究

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