Amblyomin-X是一种Kunitz型FXa抑制剂，通过对Amblyomma sculptum蜱唾液腺的转录组分析鉴定出来。这种蛋白质由两个大小相等的结构域组成，可诱导不同肿瘤细胞系的凋亡，促进肿瘤生长的回归和转移的减少。为了研究Amblyomin-X的N-末端（N-ter）和C-末端（C-ter）结构特性和功能作用，我们通过固相肽合成法合成了它们，并解决了N-ter结构域的X射线晶体学结构，证实了它的Kunitz型标志，并研究了它们的生物学特性。我们在这里展示了C-ter结构域负责肿瘤细胞对Amblyomin-X的摄取，并强调了该结构域传递细胞内载荷的能力，即将低细胞摄取效率分子(p15)与C-ter结构域耦合后，内部检测强度得到显著增强。相反，Amblyomin-X的N-ter Kunitz结构域不能穿过细胞膜，但当将其微注入细胞或与TAT细胞穿透肽融合时，它与肿瘤细胞的细胞毒性相关。此外，我们确定了最小长度的C-末端结构域F2C，能够进入SK-MEL-28细胞，并引发动力蛋白链基因表达调控，动力蛋白是参与Amblyomin-X摄取和细胞内转运的分子马达。Copyright © 2023 Morais, Ciccone, Stura, Alvarez-Flores, Mourier, Driessche, Sciani, Iqbal, Kalil, Pereira, Marques-Porto, Cunegundes, Juliano, Servent and Chudzinski-Tavassi.

Amblyomin-X is a Kunitz-type FXa inhibitor identified through the transcriptome analysis of the salivary gland from Amblyomma sculptum tick. This protein consists of two domains of equivalent size, triggers apoptosis in different tumor cell lines, and promotes regression of tumor growth, and reduction of metastasis. To study the structural properties and functional roles of the N-terminal (N-ter) and C-terminal (C-ter) domains of Amblyomin-X, we synthesized them by solid-phase peptide synthesis, solved the X-Ray crystallographic structure of the N-ter domain, confirming its Kunitz-type signature, and studied their biological properties. We show here that the C-ter domain is responsible for the uptake of Amblyomin-X by tumor cells and highlight the ability of this domain to deliver intracellular cargo by the strong enhancement of the intracellular detection of molecules with low cellular-uptake efficiency (p15) after their coupling with the C-ter domain. In contrast, the N-ter Kunitz domain of Amblyomin-X is not capable of crossing through the cell membrane but is associated with tumor cell cytotoxicity when it is microinjected into the cells or fused to TAT cell-penetrating peptide. Additionally, we identify the minimum length C-terminal domain named F2C able to enter in the SK-MEL-28 cells and induces dynein chains gene expression modulation, a molecular motor that plays a role in the uptake and intracellular trafficking of Amblyomin-X.Copyright © 2023 Morais, Ciccone, Stura, Alvarez-Flores, Mourier, Driessche, Sciani, Iqbal, Kalil, Pereira, Marques-Porto, Cunegundes, Juliano, Servent and Chudzinski-Tavassi.