Peruzzo Valentina, Tisato Francesco, Porchia Marina, Santini Carlo, Pellei Maura, Traldi Piero
CNR - IENI, Corso Stati Uniti 4, 35127, Padova, Italy.
Scuola di Scienze e Tecnologie, Divisione di Chimica, Università di Camerino, Via S. Agostino 1, I 62032, Camerino, (MC), Italy.
Rapid Commun Mass Spectrom. 2015 Feb 15;29(3):253-62. doi: 10.1002/rcm.7100.
The cytotoxic activity of the copper(I) complex [Cu(thp)4][PF6] (CP) (thp = tris(hydroxymethyl) phosphine) is correlated with its high accumulation in cancer cells. Human copper transporter 1 (hCtr1) has been described as the main trans-membrane protein involved in cellular trafficking of physiological copper. Methionine-rich peptide sequences incorporated in the extracellular domain of hCtr1 play a key role in the cellular internalization of copper. We wish to investigate the interaction of CP with model peptides that mimic the extracellular domain of hCtr1.
The interaction of CP with methionine-rich and methionine-free model peptides has been investigated by electrospray ionization mass spectrometry and the interaction products have been characterized by multiple collisional experiments, using an ion trap mass instrument.
The interaction of CP with selected methionine-rich model peptides, Ac-MMMMPMTFK-NH2 (P1) and Ac-MGMSYMDSK-NH2 (P2), shows that the native copper complex, after sequential loss of phosphines, induces the formation of Cu(P1)(thp) and Cu(P1/P2) adducts reasonably by inclusion of the Cu(I) ion in the peptide framework. Collisionally induced fragmentations (MS(n)) of Cu(P1/P2) give evidence that the metal is coordinated by the thioether-S of two adjacent methionine residues. Interaction of the same peptides with the isostructural complex Ag(thp)4 or AgNO3 yields similar experimental evidence, leading to Ag(P1/P2).
Methionine sequences incorporated in model peptides are crucial for the recruitment of copper from CP. Such a metal-peptide interaction does not take place when methionine-free Ac-NleGNleSYNleDSK-NH2 (P3) is utilized. A mechanism for tumor cell internalization of CP involving: (i) chemically driven sequential loss of phosphines from the native tetrahedral complex, followed by (ii) transfer of Cu(I) to the methionine-rich sequences typical of the hCtr1 transporter, is proposed.
铜（I）配合物[Cu(thp)4][PF6]（CP）（thp = 三（羟甲基）膦）的细胞毒性活性与其在癌细胞中的高积累相关。人类铜转运蛋白1（hCtr1）被描述为参与生理性铜细胞转运的主要跨膜蛋白。hCtr1细胞外结构域中包含的富含甲硫氨酸的肽序列在铜的细胞内化中起关键作用。我们希望研究CP与模拟hCtr1细胞外结构域的模型肽的相互作用。
通过电喷雾电离质谱研究了CP与富含甲硫氨酸和不含甲硫氨酸的模型肽的相互作用，并使用离子阱质谱仪通过多次碰撞实验对相互作用产物进行了表征。
CP与选定的富含甲硫氨酸的模型肽Ac-MMMMPMTFK-NH2（P1）和Ac-MGMSYMDSK-NH2（P2）的相互作用表明，天然铜配合物在依次失去膦后，通过将Cu（I）离子纳入肽骨架中，合理地诱导形成了[Cu(P1)(thp)]（+）和[Cu(P1/P2)]（+）加合物。[Cu(P1/P2)]（+）的碰撞诱导碎裂（MS(n)）表明金属由两个相邻甲硫氨酸残基的硫醚-S配位。相同的肽与同构配合物[Ag(thp)4]（+）或AgNO₃的相互作用产生了类似的实验证据，导致形成[Ag(P1/P2)]（+）。
模型肽中包含的甲硫氨酸序列对于从CP中募集铜至关重要。当使用不含甲硫氨酸的Ac-NleGNleSYNleDSK-NH2（P3）时，不会发生这种金属-肽相互作用。提出了一种CP肿瘤细胞内化机制，包括：（i）化学驱动天然四面体配合物依次失去膦，随后（ii）将Cu（I）转移到hCtr1转运蛋白典型的富含甲硫氨酸的序列中。
