Department of Chemistry, Vanderbilt University, Nashville, TN, USA.
Department of Chemistry, Vanderbilt University, Nashville, TN, USA; Center for Structural Biology, Vanderbilt University, Nashville, TN, USA.
Adv Biol Regul. 2023 Dec;90:100987. doi: 10.1016/j.jbior.2023.100987. Epub 2023 Sep 27.
Phosphofructokinase is the central enzyme in glycolysis and constitutes a highly regulated step. The liver isoform (PFKL) compartmentalizes during activation and inhibition in vitro and in vivo, respectively. Compartmentalized PFKL is hypothesized to modulate metabolic flux consistent with its central role as the rate limiting step in glycolysis. PFKL tetramers self-assemble at two interfaces in the monomer (interface 1 and 2), yet how these interfaces contribute to PFKL compartmentalization and drive protein interactions remains unclear. Here, we used site-specific incorporation of noncanonical photocrosslinking amino acids to identify PFKL interactors at interface 1, 2, and the active site. Tandem mass tag-based quantitative interactomics reveals interface 2 as a hotspot for PFKL interactions, particularly with cytoskeletal, glycolytic, and carbohydrate derivative metabolic proteins. Furthermore, PFKL compartmentalization into puncta was observed in human cells using citrate inhibition. Puncta formation attenuated crosslinked protein-protein interactions with the cytoskeleton at interface 2. This result suggests that PFKL compartmentalization sequesters interface 2, but not interface 1, and may modulate associated protein assemblies with the cytoskeleton.
磷酸果糖激酶是糖酵解中的关键酶，也是一个高度调控的步骤。肝同工酶（PFKL）在体外和体内的激活和抑制过程中分别发生区室化。分区化的 PFKL 被假设可以调节代谢通量，与其在糖酵解中作为限速步骤的核心作用一致。PFKL 四聚体在单体的两个界面（界面 1 和 2）上自组装，但这些界面如何促进 PFKL 的区室化并驱动蛋白质相互作用尚不清楚。在这里，我们使用非典型光交联氨基酸的定点掺入来鉴定界面 1、2 和活性位点的 PFKL 相互作用体。基于串联质量标签的定量相互作用组学显示界面 2 是 PFKL 相互作用的热点，特别是与细胞骨架、糖酵解和碳水化合物衍生物代谢蛋白的相互作用。此外，在人类细胞中使用柠檬酸抑制观察到 PFKL 成斑。斑点形成减弱了界面 2 与细胞骨架的交联蛋白-蛋白相互作用。这一结果表明，PFKL 的区室化将界面 2 隔离，但不是界面 1，并且可能调节与细胞骨架相关的蛋白组装。
