Y染色體上RNA結合模體蛋白與Pim1激酶交互作用之結構表徵

Abstract

肝細胞癌在全世界是高發生率及高死亡率的癌症之一，男性不論年齡皆是罹患肝細胞癌的高危險群，先前的研究資料指出，男性特有之Y染色體上核糖核酸結合模體蛋白 (RBMY) 會於人類和嚙齒動物的肝臟細胞發育初期表現，於成人的肝臟細胞中消失，但RBMY在肝癌組織的表現量會異常上升並與罹患肝細胞癌病人的預後不佳以及五年存活率的下降有所關連。作為一個有生殖細胞特異性的RNA選擇性剪接調控子，RBMY蛋白含有RNA識別模體用來與將被剪接的RNA結合以及四個富含絲胺酸、精胺酸、蘇胺酸和酪胺酸之高重複片段 (SRGY boxes) 負責與剪接因子形成交互作用。目前研究已指出RBMY的活性和功能表現取決於蛋白本身磷酸化的狀態，且我們的合作者藉由人類蛋白質微陣列分析，指出Pim1為最有可能與RBMY交互作用的激酶，然而對Pim1與RBMY之間磷酸化的修飾不甚瞭解，為此，我們透過生化和生物物理的方法深入探討他們之間的交互作用。首先，本實驗室已成功的使用大腸桿菌系統大量表現Pim1激酶，但是全長RBMY表現量不足，因此合成了多段SRGY boxes上序列之RBMY胜肽以進行實驗。等溫滴定量熱法的結果顯示，Pim1在RBMY的SRGY boxes上有多重結合位，而透過體外激酶測定搭配基質輔助雷射脫附游離-飛行式質譜儀 (MALDI-TOF MS)，顯示RBMY上亦有多重磷酸位點。為了發展防止RBMY被磷酸化活化的胜肽抑制劑，我們選其中三條RBMY胜肽在其N端接上細胞穿透胜肽，經證實若給予Hep1細胞RBMY胜肽可有效抑制其轉移活性。最後，我們成功解出RBMY胜肽與Pim1激酶複合體晶體結構，發現胜肽會與Pim1激酶的C端葉產生廣泛的極性交互作用，且從結構的角度分析，Pim1偏好辨認磷酸化位點上游-5及-3位置為鹼性胺基酸 (尤其是精胺酸) 之受質。綜合以上結果，本篇研究呈現了RBMY與Pim1交互作用之細節，且提供了結構資訊給未來在設計胜肽抑制劑時參考。

Hepatocellular carcinoma (HCC) is one of the cancers with high incidence and mortality. Scientists have speculated RNA-Binding Motif on Y chromosome (RBMY) plays an important role in male specific HCC progression. As a germ-cell specific RNA alternative splicing regulator, RBMY contains an RNA recognition motif and four repetitive segments rich in serine, arginine, glycine and tyrosine residues (SRGY boxes) for protein interaction. In previous studies, Provirus Integration in Moloney-1 (Pim1) kinase, a proto-oncogene protein, was identified to interact with RBMY by protein microarray analysis. However, the phosphorylation pattern and binding sites between RBMY and Pim1 kinase remains unclear. To this end, we use a combination of biochemical and biophysical approaches to deeply investigate the interaction between RBMY and Pim1 kinase. Due to the poor expression of full-length RBMY in E. coli, several peptides from SRGY boxes of RBMY were synthesized. The results showed that there are multiple Pim1 kinase binding sites on SRGY boxes of RBMY by using isothermal titration calorimetry (ITC). The in vitro kinase assay was performed to investigate whether the candidate peptides could be phosphorylated by Pim1 kinase. Multiple phosphorylation sites were demonstrated by MALDI-TOF Mass Spectrometry. To develop membrane-permeable peptide inhibitors which prevent the phosphorylation-activation of RBMY by Pim1, three of candidate peptides were conjugated cell penetrating peptide (CPP) to their N-terminus. The binding affinity between CPP-RBMY peptides and Pim1 was relatively the same as that between unconjugated peptides and Pim1. And the presences of these peptides in Hep1 cell could effectively suppress its metastatic activity. Furthermore, two crystal structures of Pim1 in complex with two CPP-RBMY peptides were determined at resolution of 1.98 and 2.17 Å, respectively. RBMY-peptides locate at C-lobe of Pim1 and interact with its αD and αF helix. There is no significant conformational change upon binding of RBMY peptides. Instead, peptides make extensive network polar contacts with Pim1. Complex structures revealed a preference for basic residues upstream of the phosphorylation site, specifically arginine at -5 and -3 positions. These data displayed the insight of interaction between RBMY and Pim1 and could provide structural information for Pim1 peptide inhibitors design.