Unraveling the interaction between a glycolytic regulator protein EhPpdk and an anaphase promoting complex protein EhApc10: yeast two hybrid screening, in vitro binding assays and molecular simulation study

IF 1.9 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY The Protein Journal Pub Date : 2024-11-01 DOI:10.1007/s10930-024-10238-5
Suchetana Pal, Pinaki Biswas, Raktim Ghosh, Somasri Dam
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Abstract

The anaphase promoting complex (APC or cyclosome) is a major ubiquitin ligase that coordinates mitotic and G1 progression, acting as a major regulator of chromosome segregation. While the human APC contains fourteen subunits, it is yet to be explored in the pathogen Entamoeba histolytica. Our study reveals the existence of a single functional Apc10 homolog in E. histolytica, which acts as a processivity factor of ubiquitin ligase activity in human. A cDNA library generated from HM1:IMSS strain of E. histolytica was screened for interaction partners of EhApc10 in yeast two hybrid study. The novel interactor, a glycolytic enzyme, pyruvate phosphate dikinase (Ppdk) was found to interact with EhApc10 and further validated by in vitro assay. A comprehensive in silico study has emphasized the structural and functional aspects, encompassing physicochemical traits, predictive 3D structure modelling, validation of EhApc10-EhPpdk interaction through molecular docking and simulation. The interplay between a cell cycle protein and a glycolytic enzyme highlights the connection between cellular metabolism and the cell cycle regulatory mechanism. The study serves as the groundwork for future research on the non-mitotic role of APC beyond cell cycle.

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揭示糖酵解调节蛋白EhPpdk与无丝分裂期促进复合蛋白EhApc10之间的相互作用:酵母双杂交筛选、体外结合试验和分子模拟研究。
无丝分裂促进复合体(APC 或 cyclosome)是一种主要的泛素连接酶,可协调有丝分裂和 G1 进程,是染色体分离的主要调节器。人类 APC 含有 14 个亚基,但在病原体 Entamoeba histolytica 中还没有发现。我们的研究揭示了组织溶解实体中存在一个功能性 Apc10 同源物,它是人类泛素连接酶活性的一个过程性因子。在酵母双杂交研究中,从组织溶解酵母的 HM1:IMSS 菌株中产生的 cDNA 文库筛选出了 EhApc10 的相互作用伙伴。研究发现,新的相互作用因子(一种糖酵解酶,丙酮酸磷酸二激酶(Ppdk))与 EhApc10 有相互作用,并通过体外实验进一步验证了这种相互作用。一项全面的硅学研究强调了结构和功能方面,包括理化特性、预测性三维结构建模、通过分子对接和模拟验证 EhApc10-EhPpdk 的相互作用。细胞周期蛋白与糖酵解酶之间的相互作用凸显了细胞代谢与细胞周期调控机制之间的联系。这项研究为今后研究 APC 在细胞周期之外的非有丝分裂作用奠定了基础。
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来源期刊
The Protein Journal
The Protein Journal 生物-生化与分子生物学
CiteScore
5.20
自引率
0.00%
发文量
57
审稿时长
12 months
期刊介绍: The Protein Journal (formerly the Journal of Protein Chemistry) publishes original research work on all aspects of proteins and peptides. These include studies concerned with covalent or three-dimensional structure determination (X-ray, NMR, cryoEM, EPR/ESR, optical methods, etc.), computational aspects of protein structure and function, protein folding and misfolding, assembly, genetics, evolution, proteomics, molecular biology, protein engineering, protein nanotechnology, protein purification and analysis and peptide synthesis, as well as the elucidation and interpretation of the molecular bases of biological activities of proteins and peptides. We accept original research papers, reviews, mini-reviews, hypotheses, opinion papers, and letters to the editor.
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