利用雙色光調控活體細胞內特異蛋白質的抑制與活化

Two-Color Light Controlled Inhibition-Activation of a Specific Protein Kinase in vitro and in Living Cells

陳子皓(Tzu-Ho Chen)

指導教授 ： 李賢明

共同指導教授 ： 王宗興(Tsung-Shing Wang)

國立臺灣大學/理學院/化學研究所/博士(2020年)

https://doi.org/10.6342/NTU202000383

摘要

本研究論文旨在開發高度選擇性蛋白質化學修飾之方法。此新穎探針設計具有對蛋白質、胺基酸殘基、反應化學等三層面之選擇性，能夠運用於活體細胞內特定蛋白質的化學修飾，並且調控該蛋白質活性。
此蛋白質化學修飾探針基礎為照光驅動，即是利用光激發交聯分子 (photocrosslinker) 與蛋白質之間生成化學鍵結。有別於傳統光親合標記探針 (photoaffinity labeling probe) 所使用之交聯分子須以紫外光激發，且不具備化學選擇性，本文選用釕金屬錯合物 (Ru-TAP complex) 所衍生之探針，為藍光 (波長450奈米) 激發，激發後可選擇性地與蛋白質中較匱乏之色胺酸透過 ＂光誘導電子轉移＂(photo-induced electron transfer) 機制完成鍵結，大幅降低非特異性交聯的可能性。更有甚者，紫外光波段得以運用於觸發額外的光化學反應，進而製備雙色光感探針，以不同波長之光照來修飾並調控標靶蛋白質的活性。
本文選用蛋白質激酶A (cAMP-dependent protein kinase) 測試釕金屬錯合物所衍生之分子探針 (以下簡稱 Probe-1) 實用性。Probe-1 由三個部分組成：第一，對蛋白質激酶A具有特異性但親和力較弱的的胜肽抑制劑 (PKI 14-24)，扮演對蛋白質之選擇性及單一色胺酸鄰近效應 (proximity effect) 的角色；第二，受藍光激發並對色胺酸具有化學選擇性的之釕金屬錯合物作為蛋白質交聯分子；第三，連結於上述兩部分間的鄰位硝基苯化合物連結分子 (2-nitrobenzyl linker)，可接受紫外光刺激而觸發抑制劑與釕金屬錯合物之間共價聯結之光解。當Probe-1與蛋白質激酶A經由抑制劑親合結合時，鄰近於結合位置外的色胺酸 (W196) 便得與搭載於抑制劑上之釕金屬錯合物在藍光激活下進行專一交聯，釕金屬與色胺酸的共價鍵使Probe-1形成"準(pseudo)不可逆”之酵素抑制劑。修飾後的蛋白質激酶A此時活性暫時被抑制。在此等待訊號期間，若以紫外光觸發光解，可使PKI(14-24) 從交聯的Probe-1中釋放，形成可逆性酵素抑制，並恢復大部分蛋白質激酶A之活性。為了證明此探針在蛋白質、胺基酸殘基及反應化學選擇上優異特異性，我們除了在細胞裂解濾液中證明可專一修飾蛋白質激酶A之外，更透過活體細胞測試此方法在複雜環境中的選擇性。我們利用顯微注射的方法將Probe-1導入纖維母細胞內，先後透過藍及紫外雙色光照，壓抑及活化細胞內生性蛋白質激酶A活性，並即時影響下游訊息傳遞鏈，其中受質VASP的磷酸化程度改變，證實了Probe-1在活體細胞內調控蛋白質激酶A籠化及解錮的能力。
Probe-1在蛋白質、胺基酸殘基、反應化學層面之選擇性，及在複雜的活體細胞內多功能性化學修飾目標蛋白之成效，說明此設計具備生物相容性及生物正交性。此雙色光蛋白修飾平台可望能成為廣泛利用之細胞生物學研究工具。

關鍵字

釕金屬錯合物 ； 蛋白質修飾 ； 光親合標記 ； 蛋白激酶活性調控

並列摘要

A selective photoaffinity labeling (PAL) method allowing chemical modification of a protein-of-interest (POI) by conjugating to a designed probe is developed.
The chemoselectivity and residue/protein specificity of this method is high enough that can be applied in the living cells to chemically modify endogenous protein-of-interest for function expansion in a bioorthogonal fashion.
The PAL probe in this study, in contrast to conventional probes that use UV-responsive, non-chemoselective photocrosslinkers, adopted a ruthenium complex that can be excited by 450 nm blue light to chemo-selectively crosslink to the side chain of low-abundant tryptophan through a photo-induced electron transfer (PeT) process. Moreover, this redshifted excitation wavelength used for photocrosslinking thus allows the PAL probe to exploit another wavelength of light, such as UV, to afford a two-color light responsive platform for dual photoreactions on the POI. Residue- and protein-specificity is accomplished through an affinity ligand that draw the probe in close vicinity to a tryptophan near the protein-ligand binding interface.
The protein kinase A (PKA) was chosen as a model POI to examine the highly selective PAL method to demonstrate the versatile functionalities of the ruthenium complex based PAL probe, denoted Probe-1. Probe-1 is composed of a Ru-TAP(phen)-complex (TAP = 1,4,5,8-tetraazaphenanthrene) as photocrosslinker, a peptide inhibitor (PKI 14-24) as PKA affinity ligand, and connected by a UV-labile 2-nitrobenzyl linker. Upon the binding of PKA and Probe-1, blue light illumination at 450 nm generates the photoadduct via a PeT induced covalent bonding between Ru-TAP complex of Probe-1 and the proximal tryptophan residue (W196) near the inhibitor binding site of PKA, resulting in “pseudo irreversible inhibited” PKA (caged form). Subsequent 360 nm light exposure triggers the photolysis of the UV-labile linker, the dissociation of the covalently attached inhibitor moiety makes the PKA “reversibly inhibited” and simultaneously restores most of the PKA activity (uncaged form). To demonstrated the selectivity in the protein-/residue-/chemo-selective levels, not only we demonstrated the PKA-selective PAL in the cell lysate experiments, we also demonstrated the PAL of Probe-1 in living cells to prove its expected feasibility and the bioorthogonality of Probe-1. Microinjection of Probe-1 and PKA into fibroblast cells followed by two-color light stimulation, PKA activity-mediated VASP phosphorylation dynamic change could be successfully shown upon caging and uncaging.
The chemoselective and residue-/protein-specific Probe-1 can minimize the off-target conjugations within the chaotic biological environment such as cytosol, and the two-color light-driven protein caging/uncaging strategy also represents unprecedented photochemical manipulation of native proteins. The platform thus would be particularly powerful for tackling endogenous protein function in living cells.

並列關鍵字

Ruthenium complex ； protein modification ； photoaffinity labeling ； kinase activity manipulation

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