鈣離子如何活化切割拓樸異構酶的蛋白酶之研究

Abstract

時間與空間上的嚴格控制對鈣蛋白酶來調控它的蛋白質水解活性功能是很重要的。這裡，我們證實位於細胞質中的鈣蛋白酶2可以切割水解位於細胞核的人類拓樸異構酶1（hTOP1）及拓樸異構酶2 beta (hTOP2 beta)，而拓樸異構酶2 alpha (hTOP2 alpha)並不會被水解切割,此現象可能透過Ca2+所誘導的鈣蛋白酶2進入細胞核而發生。此由以下的證據支持：(一)細胞處理Ca2+或Ca2+離子載體(ionophores)會造成在細胞核內的hTOP1及hTOP2 beta蛋白質被快速切割水解。 （二）兩種Ca2+螯合物都能有效地阻止ionomycin (一種Ca2 +離子載體)所引起的hTOP1及hTOP2 beta的蛋白質切割；而且添加Ca2+可以直接活化細胞萃取物中蛋白酶切割hTOP1與hTOP2 beta的現象；都支持細胞內的鈣離子濃度[Ca2 +]i對於hTOP1與hTOP2 beta蛋白質的切割是必要的。（三）進一步，我們利用重組蛋白質，實驗顯示鈣蛋白酶2 比鈣蛋白酶 1具有更高的效率去切割hTOP1蛋白質。(四)與以上概念一致，我們推論鈣蛋白酶2可能是主要Ca2 +活化切割hTOP1的蛋白酶之一，因為在降低鈣蛋白酶2表現(si-Capn2)的細胞中hTOP1對於ionomycin 誘導的蛋白質切割有高的抗性。(五)另外，我們的實驗也發現hTOP2 beta與hTOP1均是鈣蛋白酶2的新受質，且hTOP2 beta似乎較hTOP1更容易在相同濃度的ionomycin處理下被切割。(六)鈣蛋白酶2所切割hTOP1蛋白質的位置位於hTOP1的N端(N-terminus)第158及183個胺基酸賴胺酸(K158 and K183)上，而切割後的大片斷hTOP1(hTOP1tr)蛋白質則具有更強解螺旋 (relaxation)的能力。（七）此外，hTOP1tr蛋白質依然保有可以和DNA及喜樹鹼(camptothesin，CPT)形成可切割複合體（TOP1 cleavable complex，TOP1cc），並具有與核仁蛋白質nucleolin的蛋白交互作用能力。（八）細胞處理ionomycin後，鈣蛋白酶 2的活化似乎可以保護細胞免於CPT所引起的細胞毒殺害作用。總結,我們的結果為細胞質中的鈣蛋白酶2如何切割細胞核內蛋白質提供了良好的說明與實驗證據支持：在被Ca2+活化後的鈣蛋白酶2會藉由細胞質-核穿梭運輸，讓鈣蛋白酶 2 從細胞質到細胞核從而接觸到並切割其核內受質。

Crucial to calpain function is the tight regulation of its proteolytic activity, which is temporally and spatially controlled. Here, we demonstrated that the cytoplasm-located calpain 2 cleaves human nuclear topoisomerases I (hTOP1) and II beta (hTOP2 beta) but not II alpha (hTOP2 alpha) possibly through the Ca2+-induced nuclear translocation of active proteases. This is supported by the followings: (I) Treatments of cells with Ca2+ or Ca2+ ionophores caused a rapid proteolytic cleavage of hTOP1 and 2 beta in the nucleus. (II) Elevated intracelluar [Ca2+] is responsible for this hTOP1 and 2 beta proteolysis event as suggested by the observations that two Ca2+ chelators could both effectively block the ionomycin-induced cleavage of hTOP1 and 2 beta and addition of Ca2+ in the in vitro protease activation assay caused hTOP1 and 2 beta proteolysis. (III) Using recombinant proteins, our in vitro experiments showed that calpain 2 cleaved hTOP1 more efficient than calpain 1 did.(IV) Consistent with above notion that calpain 2 as a main protease responsible for Ca2+-activated proteolysis of hTOP1, hTOP1 proteins in calpain 2-knockdown (si-Capn2) cells were resistant to the ionomycin-induced proteolysis. (V) Similar to hTOP1, hTOP2 beta has also been identified as a novel substrate for calpain 2. In addition, Ca2+-activated calpain 2 appeared to cleave hTOP2 beta more completed than hTOP1 in the same dose of ionomycin treatment. (VI) The calpain 2 cleavage sites of hTOP1 were mapped at its N’-terminus K158 and K183 and the resulting hTOP1tr exhibited an enhanced relaxation activity. (VII) In addition, the hTOP1tr proteins remained the abilities to form the hTOP1-DNA-camptothecin (CPT) cleavable complex (hTOP1cc) and to interact with nucleolin proteins. (VIII) Ionomycin treatment caused a calpain 2-dependent protection of cells from cytotoxic killing by CPT. In sum, our results provided a good support for the regulation of calpain in the proteolytic cleavage of nuclear proteins via a cytoplasmic-to-nuclear trafficking of calpain 2.