Characterization of the Translationally Controlled Tumor Protein (TCTP) Interactome Reveals Novel Binding Partners in Human Cancer Cells

Translationally
controlled tumor protein (TCTP) is a highly conserved
housekeeping protein present in eukaryotic organisms. It is involved
in regulating many fundamental processes and plays a critical role
in tumor reversion and tumorigenesis. Increasing evidence suggests
that TCTP plays a role in the regulation of cell fate determination
and is a promising therapeutic target for cancer. To decipher the
exact mechanisms by which TCTP functions and how all these functions
are integrated, we analyzed the interactome of TCTP in HeLa cells
by coimmuno­precipitation (IP) and mass spectrometry (MS). A
total of 98 proteins were identified. We confirmed the in vitro and
in vivo association of TCTP with six of the identified binding proteins
using reciprocal IP and bimolecular fluorescence complementation (BiFC)
analysis, respectively. Moreover, TCTP interacted with Y-box-binding
protein 1 (YBX1), and their interaction was localized to the N-terminal
region of TCTP and the 1–129 amino acid (aa) residues of YBX1.
The YBX1 protein plays an important role in cell proliferation, RNA
splicing, DNA repair, drug resistance, and stress response to extracellular
signals. These data suggest that the interaction of TCTP with YBX1
might cooperate or coordinate their functions in the control of diverse
regulatory pathways in cancer cells. Taken together, our results not
only reveal a large number of TCTP-associated proteins that possess
pleiotropic functions, but also provide novel insights into the molecular
mechanisms of TCTP in tumorigenesis.

翻译调控肿瘤蛋白（Translationally controlled tumor protein, TCTP）是真核生物中高度保守的管家蛋白。其参与调控诸多基础生物学过程，并在肿瘤逆转与肿瘤发生中发挥关键作用。越来越多的研究证据表明，TCTP参与细胞命运决定的调控，是极具潜力的癌症治疗靶点。为阐明TCTP发挥功能的具体分子机制，以及其多种功能的整合调控方式，本研究通过免疫共沉淀（coimmunoprecipitation, IP）结合质谱（mass spectrometry, MS）技术，分析了HeLa细胞中TCTP的相互作用组，共鉴定得到98种相互作用蛋白。本研究分别通过反向免疫共沉淀与双分子荧光互补（bimolecular fluorescence complementation, BiFC）实验，验证了TCTP与其中6种鉴定得到的结合蛋白在体外（in vitro）和体内（in vivo）的相互结合关系。此外，研究发现TCTP可与Y盒结合蛋白1（Y-box-binding protein 1, YBX1）发生相互作用，且二者的结合区域分别定位于TCTP的N端区域，以及YBX1的1~129位氨基酸（amino acid, aa）残基区段。YBX1蛋白在细胞增殖、RNA剪接、DNA修复、药物耐受以及细胞对外界信号的应激反应中发挥重要作用。上述结果提示，TCTP与YBX1的相互作用可能协同调控二者的功能，共同参与癌细胞中多种调控通路的运转。综上，本研究不仅鉴定得到大量具备多效性功能的TCTP结合蛋白，同时也为解析TCTP参与肿瘤发生的分子机制提供了全新的研究视角。