Chemical Probe-Based Approach to Delineate the Underlying Mechanism for Nitric Oxide-Mediated Inactivation of Protein Tyrosine Phosphatases in Regulation of Insulin Signaling

歸類在蛋白質酪胺酸磷酸水解酶家族中的一些酵素，已知參與許多生物細胞功能過程間的調控。這類酵素的催化能力是基於具有低解離常數值特性的半胱胺酸，使得酵素在細胞生理環境中可以對受質的酪胺酸磷酸化修飾進行水解作用。蛋白質酪胺酸磷酸水解酶功能的失調會造成異常的酪胺酸磷酸信號，這關係到人類疾病像是癌症與糖尿病的成因。然而，對於標定蛋白質酪胺酸磷酸水解酶來說，現今仍缺乏高效能且可以便利使用的方法。在本論文裡，我們先建立一個新的實驗方法，使用市面上販售會與半胱胺酸其氫硫基反應的探針，在複雜的細胞環境中對蛋白質酪胺酸磷酸水解酶進行標定。基於前述低解離常數值的特性，我們預期酸性的環境條件可能有助於標定反應專一發生在蛋白質酪胺酸磷酸水解酶的半胱胺酸上。的確，即使有大量的BSA與Catalase蛋白質干擾，我們發現ㄧ個帶有PEO修飾的探針在酸鹼值6的條件下還是能夠有效對PTP1B進行標定。我們接著使用此探針來標定與分離細胞內生的蛋白質酪胺酸磷酸水解酶，結果顯示一些表現在Caco-2或是EA.hy926細胞中的蛋白質酪胺酸磷酸水解酶確實可以被分離。在下一個階段的研究裡，我們使用前述的分析技術平台來探討可能參與調控丁酸鈉誘發Caco-2細胞分化的蛋白質酪胺酸磷酸水解酶。結果顯示TC-PTP在Caco-2細胞的分化控制上，可能扮演一個未知的負向調控角色。我們進一步觀察得知，高量表現額外的TC-PTP會妨礙丁酸鈉處理的Caco-2細胞進行分化。最後，我們的分析技術平台應用在蛋白質酪胺酸磷酸水解酶其亞硝基化修飾的特性探討。初步的實驗結果顯示，探針能夠標定有活性的還原態而不是失活性的亞硝基化態PTP1B。接下來，我們檢視由胰島素刺激產生的大量ㄧ氧化氮對於細胞內生的蛋白質酪胺酸磷酸水解酶其調控作用。結果顯示SHP-1、SHP-2與PTP1B其執行酵素催化功能的半胱胺酸可能受到一氧化氮引發的亞硝基化修飾，而更進一步的活性試驗亦證實該酵素的失活。我們亦觀察到胰島素受器以及下游的分子IRS-1與PKB/Akt其酪胺酸的磷酸化修飾增多與一氧化氮有相關性，顯示一氧化氮在調控胰島素的感受性有著關鍵的角色。因此，搭配化學探針所發展的分析平台，我們驗證在胰島素刺激下而產生的ㄧ氧化氮會造成蛋白質酪胺酸磷酸水解酶的失活作用以促進胰島素感受性。Enzymes in the protein tyrosine phosphatase (PTP) superfamily are involved in the regulation of many aspects of biological processes. The catalytic activity of PTPs is mediated by an invariant Cys residue, which has a remarkably low pKa, therefore being able to carry out nucleophilic attack on a substrate, leading to Tyr dephosphorylation. Dysfunction of PTPs results in aberrant tyrosine phosphorylation signaling which has been linked to the etiology of several human diseases, including cancer and diabetes. However, a high efficient and convenient tool for tagging PTPs as a whole is still lacking at the present time. Here, we first established a novel strategy that tags PTPs in a complicated proteome by using a commercial Iodoacetyl-PEO-Biotin probe (the PEO probe) under acidic conditions. We proposed that a low pH condition might facilitate the specificity of labeling reaction towards the active site Cys of PTPs, due to its low pKa character. Indeed, we found that the purified human PTP1B, a prototype of PTP enzymes, could be efficiently alkylated by the PEO probe at as low as pH 6.0 even in a protein mixture with excess amounts of BSA and Catalase. We then applied this probe to tag and isolate endogenous PTPs, and the results show that a number of PTPs expressed in Caco-2 or EA.hy926 cells were appeared in the pull-down fraction. In the second part of our study, the analytic pulldown platform was used to tag endogenous PTPs involved in the regulation of sodium butyrate (NaB)-induced Caco-2 cell differentiation. Our results demonstrated a novel role of TC-PTP as a negative regulator in the control of differentiation in Caco-2 cells. We further observed that a high level of ectopically expressed TC-PTP prevented Caco-2 cells from the entry of differentiation in NaB-treated cells. In the next phase of study, our strategy was applied for the characterization of S-nitrosylation on PTPs. We showed that, not only purified PTP1B but also endogenous PTPs were susceptible to nitric oxide (NO)-mediated S-nitrosylation and inactivation. Our data indicated that multiple cellular PTPs are likely S-nitrosylated at the active site Cys residue concomitantly with a burst of intrinsic NO production. We also observed a critical role of NO in insulin responsiveness, as evidenced by an NO-dependent increase of tyrosine phosphorylation levels of the insulin receptor and its downstream effectors IRS-1 and PKB/AKT. Employing the chemical probe-based approach, we demonstrated that NO mediates the inhibition of insulin receptor PTPs, leading to the enhancement of insulin responsiveness