Endothelial KSR2 regulated by genetic variation protects against atherosclerosis through AMPKα1 stabilization

<b>Rationale:</b> The single nucleotide polymorphism (SNP) rs11830157 within the scaffold protein kinase suppressor of Ras 2 (<i>KSR2</i>) locus is strongly associated with the incidence of coronary artery disease (CAD), yet its functional role remains undefined. This study aimed to investigate the potential impact of rs11830157 polymorphism on atherosclerosis and to elucidate the underlying molecular mechanisms. Methods<b>:</b> Dual-luciferase reporter assays, chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assays (EMSA), and CRISPR/Cas9 gene-editing techniques were used to investigate the regulatory role of the SNP rs11830157. To assess the role of <i>KSR2</i> in atherosclerosis, we utilized global <i>KSR2</i> knockout mice fed a high-fat diet ad libitum, pair-fed global <i>KSR2</i> and <i>Apoe</i> (Apolipoprotein E) double knockout mice, and mice with endothelial-specific <i>KSR2</i> overexpression mediated by AAV9-<i>ICAM2</i>. Results<b>:</b> Genetic analyses identified SNP rs12822146, in linkage disequilibrium with rs11830157 and located within an endothelial enhancer, as a regulator of <i>KSR2</i> expression via differential binding of the transcriptional repressor <i>XBP1s</i>. <i>KSR2</i> expression was significantly reduced in endothelial cells within atherosclerotic plaques in both humans and mice. Using multiple <i>KSR2</i> gene-edited mouse models, we demonstrated that endothelial <i>KSR2</i> protects against atherosclerosis by suppressing inflammation and apoptosis. Mechanistic studies revealed that <i>KSR2</i> competes with <i>CRBN</i> for binding to the K52 site of <i>AMPKα1</i>, inhibiting <i>CRL4A^CRBN</i> E3 ubiquitin ligase complex-mediated K48-linked polyubiquitination and proteasomal degradation of <i>AMPKα1</i>. The subsequently activated <i>AMPK</i> signaling pathway maintains glycolytic balance in endothelial cells, ultimately exerting anti-inflammatory and anti-apoptotic effects. Conclusions<b>:</b> Our findings provide the first comprehensive molecular explanation of the rs12822146-<i>KSR2</i>-atherosclerosis axis, with important implications for both primary prevention and secondary treatment of CAD.