PACTS-Assisted Thermal Proteome Profiling for Use in Identifying Peptide-Interacting Proteins

Bioactive peptides play important
roles in various biological processes.
However, the traditional methods for profiling the peptide-interacting
proteins require modifications to the peptide molecules, often leading
to false identifications. We found that the interaction between peptide
ligands and protein receptors induced significant changes in the abundance
of the interacting proteins, which is a signature indicating the interaction
and providing complementary information for use in the classical thermal
proteome profiling (TPP) technique. Herein, we developed a novel Peptide-ligand-induced
Abundance Change of proTeinS (PACTS)-assisted TPP strategy for the
identification of peptide-interacting proteins based on the peptide-ligand-induced
change in protein abundance. The utility and efficacy of this approach
were demonstrated by the identification of the interaction of the
protein 3-phosphoinositide-dependent protein kinase 1 (PDPK1) and
PDPK1-interacting fragment (PIF) pair and by large-scale profiling
of the interacting proteins of PIF. The PACTS-assisted TPP approach
was applied to describe the interactome of amyloid beta (Aβ)
1-42 in THP-1 cells and resulted in the identification of 103 interacting
proteins. Validation experiments indicated that Aβ1-42 interacted
directly with fatty acid synthase and inhibited its enzymatic activity,
providing insights into fatty acid metabolic disorders in Alzheimer’s
disease (AD). Overall, PACTS-assisted TPP is an efficient approach,
and the newly identified Aβ-interacting proteins provide rich
resources for the research on AD.

生物活性肽在多种生物学过程中发挥重要作用。然而，传统的肽相互作用蛋白谱分析方法需要对肽分子进行修饰，这往往会导致假阳性鉴定结果。我们发现，肽配体与蛋白受体之间的相互作用会引发相互作用蛋白的丰度发生显著变化，这一特征可作为相互作用发生的标志，并为经典热蛋白质组分析（thermal proteome profiling, TPP）技术提供补充信息。据此，我们开发了一种全新的肽配体诱导蛋白丰度变化（Peptide-ligand-induced Abundance Change of proTeinS, PACTS）辅助TPP策略，该策略基于肽配体诱导的蛋白丰度变化来鉴定肽相互作用蛋白。通过对3-磷酸肌醇依赖性蛋白激酶1（3-phosphoinositide-dependent protein kinase 1, PDPK1）与PDPK1相互作用片段（PDPK1-interacting fragment, PIF）的配对相互作用进行鉴定，以及对PIF的相互作用蛋白进行大规模谱分析，证实了该方法的实用性与有效性。我们将PACTS辅助TPP策略应用于THP-1细胞中β淀粉样蛋白1-42（amyloid beta, Aβ 1-42）的相互作用组研究，共鉴定得到103种相互作用蛋白。验证实验表明，Aβ1-42可直接与脂肪酸合酶结合并抑制其酶活性，这为阿尔茨海默病（Alzheimer’s disease, AD）中的脂肪酸代谢紊乱研究提供了新的视角。综上，PACTS辅助TPP是一种高效的研究方法，新鉴定得到的Aβ相互作用蛋白为阿尔茨海默病的相关研究提供了丰富的资源。